text:
Tanshinone IIA sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by abnormal inflammation, persistent and progressive lung function decline. The anti-inflammatory actions of tanshinone IIA, which is the most important active component from Chinese herbal medicine Danshen, have been well studied. However, it remains unknown whether sodium tanshinone IIA sulfonate (STS) protects against the development of COPD. Here we found that STS inhalation (5 mg/kg, 30 min per session, twice a day) significantly attenuated lung function decline, airspace enlargement, mucus production, bronchial collagen deposition, inflammatory responses and oxidative stress caused by cigarette smoke (CS) and lipopolysaccharide (LPS) exposures in mice. Moreover, treatment with STS (10 μg/ml) reduced CS extract (CSE)-induced IL-6 and IL-8 secretion in human bronchial epithelial (16HBE) cells. The anti-inflammatory actions of STS were associated with inhibition of ERK1/2 and NF-κB activations. Interestingly, STS inhibited CS-induced reduction of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse lungs and in 16HBE cells. Treatment with a specific CFTR inhibitor CFTR-Inh172 augmented CSE-induced ERK1/2 and NF-κB-dependent inflammatory responses, but abolished the inhibitory action of STS on IL-6 and IL-8 secretion in 16HBE cells. These results demonstrate that CS-induced COPD and down-regulation of CFTR are prevented by STS.

Sodium tanshinone IIA sulfonate (STS), a water-soluble substance, derives from tanshinone IIA (TIIA). TIIA is the main pharmacological component from Chinese herbal medicine Danshen, the dried root of Saviamiltiorrhiza. Danshen has been widely used in many countries including China, Japan and the United States to treat cardiovascular and cerebrovascular diseases with few adverse effects 9 . In recent years, STS and TIIA have been found to be anti-inflammatory in experimental lung and cardiac diseases [10][11][12] . In these experiments, STS or TIIA was administered via intraperitoneal injection. However, intraperitoneal injection may bring poor treatment compliance. Furthermore, excess STS will be administered intraperitoneally so as to reach effective concentration in lungs, which inevitably limits its clinical use. More importantly, it is not known whether STS attenuates CS-induced inflammatory responses and subsequent COPD.
In this study, we evaluated effects of STS inhalation on the development of COPD in mice exposed to CS. We also investigated the mechanisms underlying the protective roles of STS on lung inflammatory and injurious responses to CS in vivo mouse lungs and in vitro lung epithelial cells. To the best of our knowledge, this is the first report regarding the effects of STS inhalation on CS-induced COPD and underlying mechanisms.

STS protects against CS-induced reduction of CFTR in 16HBE cells and mouse lungs. Sustained
exposure to CS or CSE has been shown to reduce CFTR expression. As expected, CSE exposure reduced CFTR protein in 16HBE cells (Fig. 7A). STS treatment attenuated the reduction of CFTR protein caused by CSE incubation (CSE group: 48.93 ± 4.35%, CSE + STS group: 84.18 ± 9.55%, percentage of the CTL group, Fig. 7A). Similarly, STS administration attenuated the inhibitory effect of CS on CFTR protein abundance in mouse lungs, determined by Western blotting (CS group: 39.88 ± 9.85%, CS + STS group: 71.20 ± 8.53%, percentage of the CTL group, Fig. 7B) and immunohistochemistry (Fig. 7C). These results indicate STS protects against CS-induced reduction of CFTR level.

Discussion
Danshen and its derivative products including STS have been applied to treat cardiovascular and cerebrovascular diseases both in Asian and Western countries 9 . Our study provided evidence that a long-term aerosol inhalation of STS reduced lung physiological and pathological manifestations in mice exposed to CS. This was associated with the reduction of CS-induced oxidative stress and inflammatory responses in vivo in mice and in vitro in cells. This is corroborated by another finding showing that STS is effective in clinical treatment of pulmonary hypertension, a common complication of COPD 13 . LPS helps to induce emphysema in shorter time than pure CS exposure (couples of months vs 6-8 months) 14 . We used both LPS and CS exposures in mice, and treated them with STS. STS was described as an antioxidant to reduce oxidative stress and inflammatory responses, which is associated with its function such as scavenging oxygen free radicals, preventing lipid peroxidation 15 , inhibiting low density lipoprotein oxidation 16 , increasing Zn superoxide dismutase (SOD) activity as well as mRNA and protein expression 17 , activating the Nrf2 pathway 18 and inhibiting NF-κB and MAPK signaling pathways 19 . In the present study, we found that STS treatment significantly reduced lung inflammatory responses to CS exposure. This may be due to inhibition of NF-κB and MAPK signaling pathway, which is shown in our and previous studies 19 . A broad spectrum of anti-inflammatory drugs, including inhibitors of the pro-inflammatory enzymes PDE4, Janus kinases, NF-κB kinase, p38 mitogen-activated protein kinase, and PI3 kinase-γ and -δ, were developed for COPD treatment, but side effects after oral administration limited their clinical application 20 . Unlike significant advances in the development of long-acting bronchodilators, it has been proved difficult to find safe and effective anti-inflammatory treatments for COPD 20 . As a medicine used in traditional Chinese medicine, side effects and safety of Danshen and Danshen products have been fully evaluated in clinical practice 9 . Indeed, we found that increases of organ and body indices were attenuated by STS treatment, suggesting the dose of STS (5 mg/kg, 30 min per session, twice per day) here to treat COPD did not cause a detectable toxic effect. Based on our findings, STS is a potential drug for preventing COPD, which could be conveniently delivered by aerosol inhalation.
Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel, is expressed mainly in exocrine tissues. In lung, loss of CFTR function results in mucociliary clearance defect, airway surface liquid depletion, chronic bacterial infection and airway chronic inflammation 21 . CS-mediated CFTR dysfunction results in inflammation and oxidative stress, and both are involved in the pathogenesis of COPD emphysema 22 . Therefore, pharmacological approaches aimed at enhancing CFTR expression and/or function could be potentially beneficial to COPD.
In response to CS exposure, level of CFTR was reduced in mouse lungs and in lung epithelial cells. Recent studies showed CFTR gene expression in human airway epithelial cells was regulated by oxidative stress 23,24 . Current findings and others showed that STS protected against oxidative stress 25 . These findings suggest that STS protects against CFTR reduction through reducing oxidative stress. GSH, one of the most important antioxidants against free radicals in lungs, affords protection against oxidative damage on the alveolar epithelial fluid interface 26 . Compared with mice without STS treatment, higher ratio of free GSH to GSSH was found in lung tissue from COPD model mice treated with STS, which means a better cellular stability against oxidative damage 27 . These findings suggest that there is an interaction between CFTR and oxidative stress, which is disrupted by CS exposure. Mucus hypersecretion, an important factor of predicting the morbidity and mortality in COPD, is contributed by CS-induced acquired CFTR dysfunction, while it can be markedly improved by the CFTR potentiator in vitro 28 . Our preliminary studies have shown that STS inhalation down-regulates Muc5AC and Muc5B levels in BALF of mice with COPD. This was in agreement with the finding that STS inhalation reduced CS-induced goblet cell hypertrophy and hyperplasia of the airway epithelium in COPD mice. The pathogenesis of airway mucus hypersecretion induced by CS, which is complicated and incompletely understood, may involve inflammation, oxidative stress, signal transduction and decreased expression of CFTR 29 . Treatments should counteract each factor contributing to airway mucus hypersecretion. Our study shows that treatment with aerosolized STS twice daily (5 mg/kg) is associated with significant improvements in mucus clearance. The mechanism of benefit is thought to be attributed to inflammation and oxidative stress inhibition, as well as CFTR expression maintenance.
MAPK signaling pathways regulate pro-inflammatory mediator IL-8 expression in cystic fibrosis lung epithelial cell lines 30 . Inhibition of CFTR function enhanced inflammatory responses to CS by activation of p38/ ERK MAPK or NF-κB pathway in airway bronchial epithelial cells 31,32 . This is consistent with our findings that pharmacological inhibition of CFTR augments CS-induced IL-6 and IL-8 expression as well as MAPK and NF-κB activation, despite CFTR inhibition by CFTR-Inh172 (10 µg/ml) alone did not cause inflammatory responses in lung epithelial cells. The mechanisms underlying the discrepancies between these findings are not clear. Interestingly, protective role of STS on CS-induced inflammatory responses was abolished in response to CFTR inhibition. All these findings suggest that STS reduces CS-induced inflammatory responses by attenuating CFTR reduction, although there is a need to use CFTR knockout cells to further unravel its role in CS-induced inflammatory and injurious responses.
In summary, our current study demonstrates that STS inhalation effectively attenuates CS-induced clinical or pathological presentations of COPD, such as emphysema, collagen deposition in the small airway, goblet cell hyperplasia of airway epithelium and lung function decline. This was associated with protective effects of STS on CS-induced oxidative stress, NF-κB-dependent inflammatory responses. Furthermore, CS-induced down-regulation of CFTR is also prevented by STS. STS could be a potential anti-inflammatory drug for COPD.
Nevertheless, our study has some limitations, such as not figuring out the specific molecular mechanism underlying that STS inhibited CS-induced CFTR reduction in COPD. However, the study has already provided evidence to support that STS inhalation has clinical efficiency, feasibility and relatively lower cost compared with  classic COPD medication as a potential drug for COPD. STS inhalation is a valuable candidate worthy of further investigation for future CS-induced COPD therapy.

Methods
Animal model. Wide-type C57BL/6 J male mice (6-8 weeks old) were purchased from the Nanjing BioMedical Research Institute of Nanjing University (Nanjing, China). Animals were housed in the specific pathogen-free facilities and the Animal Care and Use Committee of Guangzhou Medical University approved all experimental protocols. All methods were performed in accordance with the guidelines and regulations approved by the Animal Care and Use Committee of Guangzhou Medical University. The cigarettes used in the mouse model establishment were Plum brand filtered cigarettes (Guangdong Tobacco Industry, China) and each cigarette yields 11 mg tar, 1.0 mg nicotine and 13 mg carbon monoxide. A COPD model was established using CS exposure plus intranasal inhalation of Lipopolysaccharide (LPS) (Fig. 1A). Briefly, mice received LPS (7.5 μg/ Figure 8. CFTR inhibition abolishes inhibitory effect of STS on CSE-induced release in IL-6 and IL-8, but increases CSE-induced activation in ERK/NF-κB pathway. 16HBE cells were treated with 2% CSE for 48 h with 2 h of STS (10 μg/ml) pre-incubation. In order to inhibit CFTR function, cells were treated with CFTR specific inhibitor CFTR-Inh172 (10 μM) 1 h prior to STS treatment. (A) cells were treated with CFTR-Inh172 (0-50 μM) for 48 hours and cell viability was measured by CCK8 assay. (B) IL-6 and IL-8 expression in cell culture medium was measured. (C) The levels of phosphorylated ERK1/2 in cytosol protein were determined with ERK1/2 as internal control (n = 3). The level of p65/NF-κB in nuclear extraction was determined with histone H3 as internal control (n = 6). Full-length blots are presented in supplementary Figure 3. *P < 0.05 and **P < 0.01, CFTR, cystic fibrosis transmembrane conductance regulator; CSE, cigarette smoke extract; CS, cigarette smoke; STS, Sodium tanshinone IIA sulfonate. mouse in 50 μl saline; L8643, Sigma-Aldrich) or saline (vehicle control) by intranasal inhalation at the 1 st and 14 th day. Meanwhile, mice were exposed to CS (9 cigarettes/h, 2 hours/session, twice/day and 6 days/week in a whole-body exposure chamber) from day 0 to 60 except for the days giving LPS. For STS (Jiangsu Carefree Pharmaceutical, Nanjing, China) treatment, mice were given saline or STS (5 mg/kg, 30 min per session, twice per day) by airway inhalation with a PARI Nebuliser in a whole-body exposure chamber for 30 min daily before being exposed to CS. Finally, all mice were subjected to lung function analysis, and sacrificed at day 61.

Lung function measurement. Lung function was evaluated using the Forced Pulmonary Maneuver
System (Buxco Research Systems, Wilmington, North Carolina, USA) according to the manufacturer's protocol. Briefly, mice anesthetized with pentobarbital (50 mg/kg body weight) were tracheostomized, intubated and put in the body chamber of the system. The average breathing frequency of anesthetized animals was forcibly set at 150 breaths/min. Three semiautomatic maneuvers were carried out: Boyle's law functional residual capacity (FRC), quasi-static pressure volume (PV) and fast flow volume (FV) maneuver. The FRC was determined with Boyle's law FRC. To measure total lung capacity (TLC) and chord compliance (Cchord), the quasi-static PV maneuver was performed. With the fast FV maneuver, forced expiration volume in 50 milliseconds (FEV50) and forced vital capacity (FVC) were measured.
Hematocrit measurement. Briefly, at the terminal of chronic CS exposure, whole blood was collected into capillary tubes (0.5 mm outside diameter, VWR Scientific, Radnor, PA) via right ventricle puncture with K 2 EDTA as an anticoagulant and centrifuged at 7,000 rpm for 5 min, and read on a hematocrit chart (VWR Scientific). BALF analysis. The left lung was injected with three sequential 0.6 ml of ice cold saline intra-tracheally and slowly. A total of 1 ml BALF was collected and centrifuged at 500 g for 10 minutes at 4 °C to pellet the cells. The cell pellets were re-suspended in 1 ml saline for total cell counts using haemacytomer. The cells were subjected to Giemsa staining for differential counting of neutrophils, macrophages and lymphocytes. The supernatant was stored at −80 °C for pro-inflammatory cytokine detection.
Histopathology and immunohistochemistry. The left lung specimens of mice were fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin wax, and cut into 4 mm thick slices stained with hematoxylin and eosin (H&E) to evaluate morphological changes. The Image Analysis Software Image-Pro Plus 6.0 was used to assess the average alveolar intercept, which means the ratio of total alveolar length to the number of alveoli per field under microscopy. For immunohistochemistry, the slices were immunostained with anti-CFTR antibody (1:100 ACL006AN0602, Alomone Labs, Jerusalem, Israel) and developed by diaminobenzidine (DAB) solution according to manufacturer's instructions. Periodic Acid-Schiff (PAS) stain (Shanghai Sun Biotechnology, Shanghai, China) was utilized to detect goblet cells in the airway epithelium following manufacturer's instructions. Masson Trichrome stain (KGMST-8003) was utilized to detect collagen deposition of small airway according to manufacturer's instructions. Semi-quantitative analyses of positive area in lungs were completed by two independent investigators using the Image-Pro Plus 6.0. At least three fields per mouse were taken.
Measurement of liver, spleen, and kidney indices. The liver, spleen, or kidneys were weighed and each was calibrated with whole body weight.
Preparation of nuclear protein extract. Lung tissue or 16HBE cells were extracted with NE-PER nuclear and cytoplasmic reagents (Thermo Scientific) containing protease inhibitor cocktail according to the manufacturer's instructions. Histone H3 served as the loading control in Western blotting of nuclear proteins.
Preparation of CSE. CSE was freshly prepared from Plum brand filtered cigarettes within 30 min prior to treatments according to a previously described protocol 33 . The acquired CSE suspension in yellowish color with optical density (OD) at 405 nm 0.506 ± 0.008 was adjusted to pH7.4, passed through a 0.22 μm filter to remove bacteria and particles and considered as concentration 100% in cell treatments.
Cell viability assay. The cell viability of 16HBE cells was determined by using a CCK8 assay kit (Sigma-Aldrich) according to the manufacturer's instruction. Briefly, the cells (5,000 cells/well) were seeded in 96-well plates, cultured overnight, treated with CSE (0-20% in medium containing 1% FBS) or STS (0-60 μg/ ml) or CFTR-Inh172 (0-50 μM) for 48 h. Subsequently, CCK8 solution (10 µl) was added to each well and incubated for 2 h. Finally, the absorbance at 450 nm was read in a microplate absorbance reader (Thermo Scientific, Waltham, MA). Cell viability was presented as percentage of normal control cells, i.e. cells without CSE or STS or CFTR-Inh172 treatment.
Cell culture. Human bronchial epithelial cells 16HBE were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured with DMEM medium added with 10% FBS, 100 ug/ml penicillin and 100 μg/ml streptomycin in a humidified incubator at 37 °C with 95% (v/v) air and 5% (v/v) CO 2 . The cells were treated with CSE (2%) and STS (10 μg/ml) with or without CFTR inhibitor CFTR-Inh172 (10 μM, Sigma) in serum starved medium.
Glutathione fluorometric assay. The ratio of GSH/GSSG was measured in lung using the GSH/GSSG ratio detection assay kit (BioVision, Milpitas, CA) according to manufacturer's instruction. Briefly, lung tissue was homogenized using cold glutathione assay buffer. To every 60 μl of homogenate was added 20 μl of prechilled perchloric acid (PCA, 6 N). The solution was mixed, centrifuged and the supernatant was kept at −80 °C. The PCA-preserved samples were assayed for glutathione after addition of ice cold KOH (6 N, 20 μl to 40 μl per sample) to precipitate PCA and centrifuged. For GSH measurement, 10 μl of sample was added to 90 μl of assay buffer. For GSSH measurement, 10 μl of sample was added to 70 μl of assay buffer, followed by addition of a quencher buffer to quench GSH and a reducing agent to destroy excessive GSH quencher and transform GSSG to GSH. Finally, o-phthalaldehyde (OPA) probe was added to each sample and the relative fluorescence units (RFU) were read in a fluorescence plate reader with λ ex = 340 nm and λ ex = 420 nm. A standard curve with known concentrations of GSH supplied with the kit was used to calculate GSH concentration. The results were expressed as molar ratio of GSH/GSSH. Statistical analysis. Data were analyzed with ANOVA or two-tailed Student's t-test. If significant F-ratios were acquired with ANOVA, pair-wise comparison of mean was carried out with Boferroni analysis. Data are presented as mean ± SEM. "n" means the number of repeats in cell experiments or the number of animals. P < 0.05 and P < 0.01 were considered significant.


Extrusion-Based 3D Printing of Photocurable Hydrogels in Presence of Flavin Mononucleotide for Tissue Engineering

A.G. Savelyev, PhD Student, Junior Researcher, Laboratory of Laser Biomedicine1; Junior Researcher, Laboratory of Photonic Bioengineering2; A.V. Sochilina, PhD Student, Laboratory “Polymers for Biology”3; Engineer-Researcher, Laboratory of Laser Biomedicine1; R.A. Akasov, PhD, Researcher, Laboratory of Photonic Bioengineering2; Researcher, Group of Biomedical Materials3; A.V. Mironov, PhD, Senior Researcher, Supercritical Fluid Technology Laboratory1; V.A. Semchishen, PhD, Leading Researcher, Laboratory for Nonlinear Optics of Surface and Laser-Plasma Processes1; A.N. Generalova, PhD, Senior Researcher, Laboratory “Polymers for Biology”3; E.V. Khaydukov, PhD, Head of the Laboratory of Laser Biomedicine1; Head of the Laboratory of Photonic Bioengineering2; V.K. Popov, DSc, Head of the Supercritical Fluid Technology Laboratory1

Introduction
Recent successes in regenerative medicine have made possible restoring of damaged tissues by assembling artificial mimicking constructs. Cartilage [1], bone [1,2], blood vessels [3], skin [4], and liver [5] are only some examples of reproduced tissues that have been reported in the past decade. Fabrication of tissue engineering constructs usually begins with producing three-dimensional scaffolds representing a structural support for living cells. Mechanical properties, morphology, adhesion, and porosity of scaffolds can be varied in a wide range, depending on the mimicking tissue requirements. However, all materials for scaffolding as well as products of their biodegradation must be nontoxic both to the cells and surrounding tissues. One of the attractive candidates for those scaffolds are various hydrogels based on biocompatible macromolecules [6], such as hyaluronic acid and its derivatives [7], as hyaluronic acid (nonsulfated glycosaminoglycan) is one of the major native component of the human extracellular matrix for connective, epithelial, and neural tissues.
In this work hyaluronic acid has been modified to produce hyaluronic acid glycidyl methacrylate (HAGM) which is capable of crosslinking initiated by light. Methacrylation of biocompatible hyaluronic acid enables wide possibilities for its successful implementing in 3D printing as a key component of photocurable composition (PCC), representing a water solution of photocrosslinkable macromolecules with a photoinitiator and/or photosensitizer. PCC can also include biofunctional molecules, catalysts, and other additional moieties. It is essential that hydrogels obtained as a result of PCC crosslinking possess properties close to those of living tissues. The precise control of mechanical and swelling properties of hydrogels can be performed by simple adjusting concentrations of precursors in the initial PCCs. In particular, the addition of small amount of nontoxic poly(ethylene glycol) diacrylate (PEGDA) [8] in PCC allows to decrease dramatically the unnecessarily high swelling ratio (SWR) of HAGM-based hydrogel (SWR>2000%) and make it more elastic [9]. SWR of the sample is calculated according to the expression: where W s and W d are the weights of the swollen and dried hydrogels, respectively. It is vitally important to provide the biocompatibility of all components of PCC for tissue engineering including photoinitiator and/or photosensitizer. Therefore, in the present work, riboflavin (vitamin B2) in its water-soluble form, flavin mononucleotide (FMN), has been proposed and used as an endogenous photosensitizer. Vitamin B2 non-toxicity and goodness as a dietary supplement have been approved by Food and Drug Administration. FMN possesses absorption in ultraviolet A and nontoxic blue ranges of the spectrum [10]. Under irradiation FMN [11] can form either singlet oxygen via energy transfer to atmospheric oxygen or hydrogen peroxide and derivatives via radicalization [12]. All these reactive oxygen species are capable to activate the crosslinking process. Moreover, riboflavin photobleaching time exceeding 8 min [13] permits its effective implementation as a photosensitizer in the 3D printing process.
Herein we have developed a HAGM-based PCC containing FMN as a photosensitizer and small amount (ca. 5 wt%) of PEGDA to optimize mechanical properties of the final hydrogel. The hydrogel scaffolds have been produced in the shape of lattices by extrusion-based 3D printing combined with simultaneous laser irradiation at 450 nm. Scaffolds have been analyzed using optical microscopy. In vitro cytotoxicity tests of printed hydrogel structures have been performed using human fibroblasts BJ-5ta.
Photocurable composition preparation. Aiming to substitute sodium atoms in hyaluronic acid sodium salt by methacrylate units the reaction with glycidyl methacrylate was applied according to the method described in [14] and hyaluronic acid glycidyl methacrylate (HAGM) capable of crosslinking has been obtained as a result.
HAGM and PEGDA were placed in PBS containing FMN and subjected to sonication for 1 h. Then this mixture was left overnight and sonicated again to produce a homogeneous solution. To avoid spontaneous crosslinking, preparation of PCCs was performed under yellow light conditions. The prepared PCCs contained 20 wt% of HAGM and 5 wt% of PEGDA. The concentration of FMN was 0.01 wt%.
Scaffold fabrication. Self-designed extrusion-based 3D printer has been used to produce experimental hydrogel structures. Initially, PCC was loaded in the syringe where the open end was fitted with 5 mm long 250 μm inner diameter capillary. In order to start the extrusion, the plunger was pushed inside along the syringe. Plunger speed and positioning of the syringe in Z-direction, as well as the movement of the substrate in X-Y coordinates, were controlled using G-code in Repetier software. Acceleration parameters of plunger and translation stages were precisely adjusted in Repetier to ensure uniform thickness of extruded PCC layer deposited onto the substrate. Photocuring of the extruded PCC was performed by laser irradiation at 450 nm wavelength with intensity of 70 mW/cm 2 . When the pattern was formed, the stage moved in Z-direction and the procedure was repeated. Inner structure and surface morphology of the scaffolds were analyzed by optical microscopy using HRM-300 microscope (Huvitz, Korea).
In vitro study. The cytotoxicity of the hydrogel samples was studied in the extract test as it was described earlier [15], and human fibroblasts BJ-5ta were used as model cells. Briefly, 3D printed hydrogel samples were incubated within RPMI-1640 cell culture medium supplemented with 10% of FBS at 37°C in 5% CO 2 humidified atmosphere, and the supernatants (extracts) were collected after 24 h. The BJ-5ta fibroblasts were seeded into 96-well plate (10 4 cells per well), and the plate was incubated overnight in CO 2 -incubator. Then the medium in each well was replaced with 100 μl of the extracts. The cells biophotonics in regenerative medicine cultivated in the medium without the extracts were used as a control. Cell viability was measured by a MTT assay, which is a colorimetric assay for assessing cell metabolic activity through the reducing of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan. For this reason, after 24 h incubation with extracts, an MTT solution (0.5 mg/ml) was added to the wells, and the cells were incubated at 37°C for 3 h. Formazan crystals formed in the living cells were dissolved in 100 μl of dimethyl sulfoxide, and an optical density was measured at 570 nm using a Multiscan plate reader (Flow Laboratories, USA). Relative cell viability (V) was calculated according to the following expression: (2) where OD test , OD control , and OD background are optical densities of sample, control, and background, respectively.
To demonstrate cell growth on the hydrogel surface, printed samples were placed into the 24-well plates and seeded with BJ-5ta fibroblasts (20,000 cells per each sample), and successive gradual cell growth was observed within 7 days.
OHAUS Starter 3100 pH-meter (OHAUS, USA) was employed to control pH of solutions.

Results and Discussion
First, we performed methacrylation of hyaluronic acid to produce HAGM. The synthesized hyaluronic acid derivative combines advantages of its biocompatibility and capability to photocrosslinking. Therefore, we chose HAGM as the main component of PCC. In order to perform gelation of the entire volume of the initial multicomponent PCC after exposure process, the concentration of capable to photocrosslinking HAGM macromolecules must exceed the minimal threshold value of 16 wt% discussed in [9]. The formation of a disordered 3D polymer network threading through the entire exposed volume and triggering the appearance of elastic properties becomes possible only above this threshold concentration.
The water-soluble form of riboflavin was proposed as an endogenous photosensitizer in PCC. FMN used in our experiments is characterized by a broad UV-blue absorption band with two peaks illustrated in Figure 1 (left). Using FMN in HAGM-based PCCs makes possible photocuring at 450 nm wavelength. Additionally, in order to avoid the unnecessary high swelling of the final HAGMbased hydrogel in water solutions, we added linear polymer PEGDA to all PCCs. Thus, we prepared PCC for 3D printing containing 20 wt% of HAGM, 5 wt% of PEGDA (M n ≈575) and 0.01 wt% of FMN dissolved in PBS.
The scheme of our custom-designed extrusion-based 3D printer is presented in Figure 1 (right). The printer was equipped with 450 nm continuous wave semiconductor laser source. Owing to the focusing lens attached to the end facet of laser the divergent laser beam could expose the currently printed point or the whole sample. Depending on the irradiated area the power of the semiconductor laser was adjusted to provide 70 mW/cm 2 intensity.
To demonstrate the possibilities of our 3D printer, we fabricated scaffolds in the shape of lattices with period 1.4 mm as shown in Figure 2 (left). The lattices were formed using HAGM-based PCC by sequential printing of 25 layers. The printing speed was adjusted to 0.3 mm/s. During the extrusion process, the printing area was continuously irradiated at 450 nm wavelength to produce gelation of hydrogel layer. Finally, when the scaffold had been printed, we applied the additional exposure for Wavelength (nm) Absorption (a.u.) The surface of produced scaffolds was observed using optical microscope Huvitz with 50× objective (Huvitz, Korea). Figure 2 (right) illustrates the side wall of the scaffold. The wall represents uniform layered structure. The average height of every layer was measured as 30 µm. The total height of scaffolds was ca. 750 µm.
Prior to in vitro cytotoxicity study all printed samples were stored in PBS for 5 days to remove FMN and products of its photolysis from hydrogel structures.
The acute cytotoxicity of the hydrogel samples was evaluated using an extract test by MTT assay. It was  100% means hydrogel extract only; 75% means 75% of extract and 25% of non-conditioned RPMI-1640; 50% means 50% of extract and 50% of non-conditioned RPMI-1640; 25% means 25% of extract and 75% of non-conditioned RPMI-1640. Nonconditioned RPMI-1640 medium was used as a control. The data are the mean±SD. P-values were calculated by Mann-Whitney U test (* indicates a p-value <0.05) Extrusion-Based 3D Printing of Photocurable Hydrogels for Tissue Engineering found that hydrogel extracts slightly reduced the BJ-5ta cell growth (10-20% slower in comparison to controls) (see Figure 3). To clarify the possible reason of cell growth deceleration, the pH measurements of the extracts, both in RPMI-1640 and in de-ionized water, were conducted. Since no pH changes were observed, we suppose that cytotoxic effect could be caused by spontaneous daylight photoactivation of remaining FMN following by reactive oxygen species and free radical production [16]. Nevertheless, the fibroblasts were able to grow on the surface of hydrogels, confirming high biocompatibility of the resulted samples.

Conclusions
The printability of a new HAGM-based hydrogel photocurable composition has been studied. The elastic lattice structures have been produced using extrusion-based 3D printing combined with photocuring by laser light at 450 nm. We have demonstrated that human fibroblasts BJ-5ta were capable of attaching and growing on the surfaces of HAGM-based hydrogel scaffolds. Thus, we conclude that 3D printing of HAGMbased photocurable compositions containing flavin mononucleotide as an endogenous photosensitizer represents an attractive methodology for various tissue engineering applications.


s2fieldsofstudy: Biology Psychology Medicine

text:
Cognitive impairment in neuromuscular diseases: A systematic review

Neuromuscular diseases are multifactorial pathologies characterized by extensive muscle fiber damage that leads to the activation of satellite cells and to the exhaustion of their pool, with consequent impairment of neurobiological aspects, such as cognition and motor control. To review the knowledge and obtain a broad view of the cognitive impairment on Neuromuscular Diseases. Cognitive impairment in neuromuscular disease was explored; a literature search up to October 2017 was conducted, including experimental studies, case reports and reviews written in English. Keywords included Cognitive Impairment, Neuromuscular Diseases, Motor Neuron Diseases, Dystrophinopathies and Mitochondrial Disorders. Several cognitive evaluation scales, neuroimaging scans, genetic analysis and laboratory applications in neuromuscular diseases, especially when it comes to the Motor Neuron Diseases, Dystrophinopathies and Mitochondrial Disorders. In addition, organisms model using rats in the genetic analysis and laboratory applications to verify the cognitive and neuromuscular impacts. Several studies indicate that congenital molecular alterations in neuromuscular diseases promote cognitive dysfunctions. Understanding these mechanisms may in the future guide the proper management of the patient, evaluation, establishment of prognosis, choice of treatment and development of innovative interventions such as gene therapy.

Introduction
Neuromuscular diseases present a wide variety of clinical manifestations, but their effects on the cognitive function spectrum are still poorly understood. 1 In contrast to the studies on how altered executive functions in mental disorders such as anxiety, depression and bipolar disorder can affect motor performance, 2-4 the mechanisms by which essentially motor dysfunctions can affect cognitive performance still remain poorly understood and studied. [5][6][7][8] Although it is known that neuromuscular diseases mainly affect the motor functioning of the patient, the cognitive effects of these conditions can be significant. 5 This can occur from molecular defects that significantly affect neuromotor functioning but also participate in the functioning of neural networks involved in cognitive processes, leading to dysfunctions of executive, behavioral and psychosocial functions. [6][7][8] For example, mutations in the dystrophin protein gene, responsible for the Duchenne Muscular Dystrophy (DMD) phenotype, concurrently affect the stability and function of muscle fibers and central nervous system (CNS) function. However, the complex nature of neuromuscular diseases and the different effects on cognition make it difficult to understand the neuropathological process. 7,8 Three large, heterogeneous groups of neuromuscular diseases have been implicated in cognitive alterations. i) Motor Neuron Diseases, neurological disorders that affect the neurons that control voluntary motor activity; 9,10 ii) Muscular dystrophies, represented mainly by dystrophinopathies, diseases caused by hereditary defects in the dystrophin protein gene; 11 iii) Mitochondrial disorders, which are caused by mutations in cytoplasmic or nuclear mitochondrial DNA, which alter the structure and mitochondria function. 10,12 Often neuromuscular disease comes from an already known molecular disorder that is also associated with changes in brain function and cognition, such as mitochondrial myopathies. In other cases, neuromuscular disorders also have cognitive deficits, but without a well-defined molecular alteration such as Amyotrophic Lateral Sclerosis (ALS). 8,13 In this context, the present review aims to highlight the neurofunctional and cognitive alterations in the main congenital neuromuscular disorders.

Materials and Methods
The systematic review was conducted in line with the PRISMA statement. [Cognitive Impairment and Mitochondrial Disorders]. Abstracts were examined for references to the research question and if the study appeared relevant, then the full text was retrieved. As selection criteria: (a) Experimental studies, case reports and reviews written in English; (b) Without restriction of publication dates of the paper (c) Magnetic Resonance Imaging (MRI) scans, assessments with cognitive and functional scales applied to neuromuscular diseases, and cognitive tasks healthy individuals and/or individuals with neuromuscular diseases (e.g. children, young adults, middle-aged and elderly). In addition, experimental studies using rats were considered. Criteria for exclusion were: (a) dissertations, book reviews, conference proceedings, or editorials. Data were extracted based on study design and setting. The discussion was written after observing common points among the articles selected. This observation was conducted in order to analyze the papers results and answer the hypothesis of the present research.

Study selection and data extraction
Three reviewers (M.O, S.T and V.M) independently read the titles and/or abstracts of the identified papers and eliminated irrelevant studies. Studies considered eligible for inclusion were read in full and their suitability for inclusion was determined independently by three reviewers (M.O, S.T and V.M) Disagreements were managed by consensus. However, if this was not successful, consensus was sought by a fourth reviewer (M.R.G.F.). Data were extracted based on study design and setting. Some authors were contacted to provide supplementary information when insufficient data were provided in the study. The authors of three studies were contacted for further information having read their titles and abstracts. Two replied; since it bases the review of literature fulfilled the inclusion criteria.

Study selection
Studies were included if they met the following inclusion criteria. Initially, retrieved papers from each database were compared to remove duplicate records. Papers were then screened for eligibility based on their title and abstract and, once they were elicited, the full text publication was reviewed.
In summary: Study design: Case reports, original papers and reviews designs were included.
Population: Study population composed of healthy individuals and/or individuals with neuromuscular diseases (e.g., children, young adults, middle-aged and elderly). In addition, experimental studies with rats were included.
Intervention: Neurobiological interventions were defined as any intervention aimed at evaluating the neuromuscular diseases -Motor Neuron Diseases, Dystrophinopathies and Mitochondrial. Disorders.
Screening: Genetic analysis in vivo and post-mortem, MRI scans, mobile transcutaneous sensor for tissue blood gas tensions, ALS Functional Rating Scale, Reye Osterrieth Complex Figure Test Outcome: The primary outcome measure was the potential ability to accurately assess neuromuscular diseases and their implications for cognitive impairment through screening: Genetic analysis, MRI scans, mobile transcutaneous sensor for tissue blood gas tensions and cognitive Scales.
Papers were assessed for eligibility based on title and abstract; 80 were classified as meeting eligibility criteria. The next stage of the winnowing process involved full-text screening of the potentially relevant papers; Eventually, 63 studies were included in the review.

Results
Seventeen experimental studies and 30 case reports with cognitive scales and neuromuscular assessment using scales, as well as genetic studies and neuroimaging studies with MRI, 16 review papers about neuromuscular diseases. This study demonstrated that the neuromuscular diseases are multifactorial pathologies characterized by extensive muscle fiber damage that leads to the activation of satellite cells and to the exhaustion of their pool, with consequent impairment of the regenerative process.

The prospective population-based study of cognitive impairment in neuromuscular diseases
The majority of neuromuscular diseases patients presented with features consistent with executive dysfunction, and have shorter survival time. This last finding, in turn, suggests that studies reliant on prevalent cases are likely to underestimate the incidence of these frontotemporal syndromes. In patients without dementia, we observed an overrepresentation of executive dysfunction. The incidence of executive impairment in this cohort was higher using the screening criteria. The cognitive evaluation and cognitive tasks demand heavily on multiple frontostriatal circuits leading to sensitivity in detecting any disturbance of neural circuits established for cognitive and motor demands.
In this present study, neuromuscular diseases patients showed generally a moderate muscular impairment, impaired cognitive performance over a broad range of functions, including frontal, visuospatial, naming and memory abilities and working memory abnormalities. The degree of cognitive impairment in patients with neuro-muscular diseases is greatly variable, and significant cognitive impairment is observed only in some patients. Symptoms such as depression and anxiety as well as deficits of visuospatial abilities, executive functions, and naming have been frequently reported. Several MRI studies have shown that neuromuscular diseases patients have generally more deficiencies of neural connections in the frontal, parietal and temporal regions and; these dysfunctions increase with the progression of the disease.

Motor neuron diseases and cognitive impairment
Motor Neuron Disease (MND) is a degenerative disease caused by motor neuron degeneration, whether inferior or superior. Amyotrophic Lateral Sclerosis (ALS), the most well-known form of MND, is a devastating severe disease that simultaneously affects the lower motor neuron (LMN) and upper motor neuron (UMN). 9,10 Descriptions in the literature have been linking, for more than a century, emotional liability with bulbar-onset ALS. After pathological and genetic discoveries, over the years, the overlap of frontotemporal dementia (FTD) and MND was strengthened. 9,10 Data from recent studies show that more than 50% of ALS patients at some point in the disease course, will develop phenotypic picture of cognitive or behavioural impairment, and about 13% will exhibit concomitant behavioural-variant frontotemporal dementia (b-FTD). [10][11][12][13][14][15][16] Patients who present cognitive or behavioural changes, that do not fulfil all the formal criteria for FTD, can be grouped into three different categories: ALS with behavioural impairment; ALS with executive dysfunction; and ALS non-executive dysfunction. 10,17,18 Frontotemporal dysfunction, when present, is strongly linked to lower survival in patients with ALS. In addition, regardless of the age of onset, delayed diagnosis, baseline severity, education and respiratory status, the presence of executive dysfunction is a factor and risk for a worse prognosis. 10,16 Unless FTD is advanced enough to cause significant frontal and temporal lobe atrophy, neuroimaging is rarely useful in diagnosing cognitive and behavioural deficits in ALS. Studies of neural networks have been increasingly used for the analysis of frontotemporal dysfunction in ALS. In studies with resting state functional MRI, it has been observed three main dysfunctional networks in ALS: the salience network, the default mode network, and the central executive network. 16,19 Several screening tools specifically created for rapid neuropsychological assessment of patients with ALS. Some of these tools, currently available, include the ALS-Brief Cognitive Assessment (ALS-BCA), the ALS-Cognitive Behavioural Screen (ALS-CBS), the ALS-FTD-Q, Edinburgh Cognitive and Behavioural ALS Screen (ECAS). Patients who are in compliance with the cut-off score of these tests have formal indication of complete neuropsychological assessment. 10,[20][21][22] In addition, the GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) is a frequent cause of familial MND, FTD and especially frontotemporal dementia-motor neuron disease. The repeat expansion of C9orf72 results in dipeptide repeat proteins (DPRs), which form inclusions in central nervous system (CNS). Finally, it induces the mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43). It is estimated that in the European population, 50 to 70% of patients with familial frontotemporal dementia-motor neuron disease and 15 to 20% with apparent sporadic familial frontotemporal dementia-motor neuron disease, exhibit the expansion of the C9orf72 gene. The penetrance of C9orf72 gene expansion has not yet been determined, but it seems unlikely to be a complete penetrance. 18,23,24 TAR DNA-binding protein of 43 kDa (TDP-43), was proved to be present in neuronal cytoplasmic inclusions and dystrophic neurites in the hippocampal region and temporal cortex of patients with frontotemporal dementiamotor neuron disease.
Although C9orf72 is the most frequent genetic cause of frontotemporal dementiamotor neuron disease, other gene mutations (VCP, SQSTM1, OPTN and UBQLN2) have already been linked to this pathology, but they are rare causes. 23 Another MND with cognitive impairment is the bulbospinal muscular atrophy or Kennedy's Disease (KD). KD is an Xlinked recessive pathology that affects men and is caused by the repetition of the CAG trinucleotide in exon 1 of the androgen receptor gene, slowly progressive, with adult-onset. It is characterized by degeneration of LMN that leads to atrophy, weakness, and fasciculation of limbs and bulbar musculature. A striking feature in KD is the presence of minipolymyoclonus. 25 It is required more than 38 repeats to consider the CAG trinucleotide expansion as pathogenic. The CAG repeat size correlates inversely with disease onset, but not with disease progression or severity. 25  symptoms are also found in KD. The most common non-neurological manifestation is gynecomastia. In addition, reduced fertility due to testicular atrophy, oligo/azoospermia, erectile dysfunction and reduced libido may be present. 25,26 Cognitive impairment may be found in KD. Guidetti et al. 26 have observed in their patients a predominant impairment in long-term memory and selective attention. Kessler et al. 27 observed clinical characteristics of frontal lobe dysfunction with disinhibition, impaired executive functions and reduced affective modulation. 27

Cognitive impairment of muscular dystrophies: dystrophinopathies
Congenital muscular dystrophies (CMDs) are clinically and genetically heterogeneous inherited disorders that compass muscle weakness typically manifested at birth or in infancy. 11 The spectrum ranges from severe floppy infant syndrome to moderate motor delay and mild or moderate limb-girdle involvement during childhood, compatible with survival into adult life and relatively good quality of life. 6 Besides muscle compromise, white matter and structural abnormalities of the brain, seizures, cognitive impairment, and eye abnormalities may occur depending on the genetic cause. Mutations occur in genes encoding structural proteins of the extracellular matrix, glycosylation enzymes, and proteins of the endoplasmic reticulum (ER) and nuclear envelope. However, just in 25-50% of CMD-affected subjects, mutations can be identified, suggesting that there are additional genes to be acknowledged. 11 On the other hand, it is frequently unfeasible to establish a one-to-one relationship between a given gene and a defined phenotype. For example, mutations in the FKRP (Fukutinrelated protein) gene may lead to manifestations extended from the severe Walker-Warburg (WW) syndrome to the late adultonset limb-girdle muscular dystrophy (LGMD). 28 The genetic defects might not only interfere with the translated sequence, but also with the posttranslational processing, as described with α-dystroglycan, a major extracellular matrix receptor on muscle. 28 The term dystroglycanopathies encompass the mutation in the dystroglycan gene (DAG1) itself or mutations in genes encoding glycosyltransferases of alpha-dystroglycan (α-DG), which are necessary to allow α-DG binding to laminin. 29,30 In brain, dystroglycan is expressed in astrocytic end feet abutting the glia limitans and the intracerebral vasculature, in photoreceptor cells of the retina, and in major neurons and glia in the developing CNS. 29 Cases of milder course of the disease seem to be related to a less severe and remaining glycosylation of α-DG. 30 In the more severe end of the clinical spectrum, there are brain and eyeabnormalities, as illustrated by: Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain (MEB) disease and WWS, examples of a continuum in increasing of severity. 30,31 The typical CNS involvement among dystroglicanopathies includes various degrees of lissencephaly type II (also known as cobblestone complex), pachygyria, neuronalheterotopias, ponto cerebellar hypoplasia, and cerebellar cysts. 28 Merosin-deficient congenital muscular dystrophy also referred to as MDC1A, is caused by mutations in the LAMA2 gene, which encodes the α2 subunit of laminin (merosin). 29 Patients manifest severe muscular weakness and atrophy, diffuse contractures, inability to walk and facial dysmorphism. 31 Ventilatory is needed within the first 10 years for most patients. 28 Since laminin-α2 chain is localized to the basal lamina of all cerebral blood vessels, the lack of α2 may lead to a disruption of the bloodbrain barrier (BBB), increased water content, and a resulting abnormal white matter signal intensity (WM). 31 The typical abnormal signal in WM seem in T2-wheited MRI (T2 MRI) spares the compacted fibre tracts such as the corpus callosum (CC). The cognitive functions are not impaired if the WM changes are isolated. 28 Nevertheless, it has been reported a child with cortical dysplasia and severe mental retardation, and a series of children who in addition to the typical findings had cerebellar hypoplasia and a lower performance IQ. 32 Duchene muscular dystrophy (DMD) is one of the most common genetic disorders, affecting about 1:3000 boys. 33,34 It is an Xlinked dystrophy caused by genetic mutations disrupting the protein dystrophin (DYS), another core protein of dystrophinglycoprotein Complex (DGC), as DG. 29,33,34 DMD is characterized by progressive muscle degeneration and non-progressive cognitive deficits. [33][34][35] DYS also plays an important role in the architectural organization of the CNS. 33 It is involved in the organization of GABAA receptors, once is normally expressed at the postsynaptic membrane in inhibitory synapses of structures related to cognition and emotional behaviour, such as hippocampus, amygdala, cerebellum and sensory cortices. 7,8 Although dystrophin expression in brain is only one-tenth of that found in muscle, brain tissue exhibits much greater variability in the protein products from the dystrophin gene. It includes the full-length protein, Dp427, and shorter proteins being Dp71 the main product expressed in the brain. Dp71 is found around perivascular astrocyte end feet, which suggests its role in BBB function and possible in the possible access of peripheral inflammatory molecules into the brain. In the CNS, Dp427 is only found in neurons, and only within specific regions of the brain such as the hippocampus, amygdala, cerebellar Purkinje cells, and neocortex. The interaction of Dp47 with DGC would be important for the formation and maintenance of new synaptic connections. 36 The lack of dystrophin in mdx mice) seems to alter neuron proliferation, survival and/or differentiation and perturb neuronal migration (The density of CA1 pyramidal neurons was reduced by ~34% in the anterodorsal hippocampus, although quantitative reductions varied along the hippocampal antero-posterior axis. 35 In their hippocampus were identified cytokines, which are also chronically elevated in peripheral tissue and plasma of patients with DMD. Key proinflammatory cytokines such as IL-1, TNF-α, and IL-6 exert neuromodulatory effects on the hippocampus and have been linked to altered capacities for learning and the formation of memories. Hence, there may be a neuroimmune contribution in the CNS disturb of dystrophindeficient patients. 36 In mice, the loss of DYS leaded to enhanced unconditioned fears, anxiety and deficits in fear conditioning and memories, suggesting the role of the protein in the functioning of the neuronal circuit of fear. 7 The absence of dystrophin resulted in a significant elevation of [Ca2+]i and [Na+]i in intact adult cortical and especially hippocampus pyramidal neurons of mice, as well as an increase in ROS production, and cell death. This ion dyshomeostasis was related to a spatial learning deficit. 34 In humans, the lack of this protein might also lead to neurobehavioral disorders, including attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorders, and obsessive-compulsive disorder. In the context, Colombo et al. 33 reported a non-progressive lower IQ in DMD boys compared to controls. Boys may present specific learning disabilities such as dyslexia and impaired executive functions, namely problem solving, inhibition and working memory. 33 In addition, Astrea et al. 37 demonstrated that 15 patients with DMD (age range = 19-44 years) demonstrated impairment in the ability to sequentially process auditory and visual information and attention. 6  um of CC and these values were positively correlated with verbal IQ. 37 A recent study assessing 47 Italian boys with DMD found that 14.89% of the boys had a diagnosis of ASD. Full Scale Intelligence Quotient (FSIQ) <70 were registered in 36.2% of them, with a greater impairment of verbal than other performance components. 33 Distal DMD mutations were implicated to more severe cognitive deficit, as reported by previous studies. 6,33 Similar results were found in a longitudinal study following 41 preschool DMD boys until school age. It was confirmed a correlation between neurodevelopmental assessments and later cognitive scales and on both evaluation the overall results were better when the mutation was upstream or in the exon 44 without affecting Dp140 expression. 38 Distal mutations are associated with loss of cerebral dystrophin isoforms, which are more expressed in the cerebellum and limbic system. It is worth to remember that literature supports the cerebellar contribution even to aspects of social emotional and regulatory behaviour. 33 Mutations in the DYS gene are also responsible for causing Becker muscular dystrophy (BMD). However, in BMD there is a production of partially functional dystrophin. Patients typically remain ambulatory at least until age 15 and commonly well into adult life. Cognitive impairment and neurobehavioral comorbidities are also not as common or severe in BMD compared with DMD. 39 Myotonic dystrophies (DM) are autosomal dominant multisystemic disorders that affect also the brain. 40 Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide repeat expansion in the non-coding region of the dystrophiamyotonica-protein kinase (DMPK) gene, resulting in alternative splicing of pre-messenger RNAs for various proteins, including the TAU and NMDA. 6,40 Cognitive deficits have been observed in 24-75% of DM1 patients and it depends on the age at onset of the disease. 6,31,40 Congenital patients tend to have mental retardation, while those with onset in the childhood have learning disabilities due to lower IQ, the ADHD, autistic behaviour, visuospatial impairment, lack of interest and inhibition. Adult onset is related to the dysexecutive syndrome and visuospatial deficit, while verbal functions and memory seem to be age dependent. Lateonset DM1 patients were reported to have predominantly age-dependent memory deficit. 40 Lower IQs seems to be correlated with longer expansions, mainly related to maternal inheritance and age of onset of symptoms although it does not represent a more severe neuromuscular impairment. 6 Neuropsychological analysis of 101 DM1 patients (childhood, juvenile, adult, and late-onset) registered normal general intellectual level. Visuospatial impairment was the main cognitive defect with prevalence of 75-86% among different phenotypes. Executive dysfunction was the second most common cognitive impairment, affecting 60-79% of the sample. Adult DM1 subjects with older age and longer duration of disease were more susceptible to memory and language defects. Late onset was associated with the affection of multiple cognitive domains including the highest prevalence of memory impairment (more than 80%). Among 46 DM2 patients, it was showed that even one-third of them had completely normal neuropsychological findings. Visuospatial and executive dysfunctions were also the most prevalent in DM2, but less common than in DM1. 40 A longitudinal neuropsychological assessment among 115 DM1 -only adult phenotypes disclosed that executive functions (Stroop color-word, fluency tests and WAIS-R for digit symbol coding and block design), language (fluency and Boston Naming Test), and visual memory (Rey Complex Figure Test) were the most impaired cognitive functions at baseline. However, they did not significantly decline through the time, in opposition to verbal memory, processing speed, and visual attention, which, actually, also imply the requirement of some specific executive functions (i.e. processing speed itself, mental flexibility, and selection and maintaining of accurate strategies). The FSIQ and the visual construction also remained stable. The adult phenotype performed more poorly than the late-onset phenotype on most of the tests, both at baseline and follow-up. Nevertheless, the rate of decline was higher in the late-onset than in the adult phenotype. The level of progression in most of the cognitive performances correlated with age and disease duration but not with the CTG repeats, educational level nor muscular impairment. These findings are consistent with the hypothesis that DM1 could be considered as a progeroid disease (an accelerated and increased ageing process). 41 A comparative study involving nine DM1 patients and age matched healthy controls using T1-weighted and DTI disclosed changes in cortical thickness and WM in the widely distributed network of the frontal, temporo-occipital, medial parietal, and sensorimotor areas. CTG repeats were negatively correlated to thickness in the right medial orbitofrontal cortex, left middle temporal, supramarginal, and the lateral occipital cortex and to the WM integrity in the left entorhinal, anterior corona radiate, orbitofrontal, and lateral occipital areas. This study revealed higher occurrence of WM than cortical abnormalities. This finding together with the correlation between WM lesion severity and disease duration suggests that DM1 represents a slow demyelinating process, which eventually progresses to axonopathy. Moreover, the decreased integrity of connectivity, as well as cortical pathology, was located mostly in the ventral side connecting the fronto-temporal cortex. These findings may be relevant to the behavioural symptoms, including dysexecutive syndrome, apathy, and depression. In that study, diffusion metrics were also affected in the external capsule, which might be related to decrease verbal fluency resulting from impairment in semantic processing. 42 Bajrami et al. 43 suggest that the WM hyperintense lesions (WMHLs) located at the temporal poles seem to be characteristics of the disease, while others, small and diffuse WMHLs would be similar to the age-related alterations. 43

Mitochondrial disorders and cognitive impairment
Mitochondrial diseases (mtD) are those caused by mutations in mitochondrial DNA (mtDNA) and/or nuclear DNA (nDNA) genes due the dual genetic control (nDNA and mtDNA) of the respiratory chain (RC), the complexity of intergenomic signalling and its functional consequences. 44,45 The mutation rate for mtDNA is much higher than that for nuclear DNA, especially for deletions. 46 The diagnosis of mtD is challenging since the clinical picture is varied and can occur at almost any stage of life, often with involvement of unusual combination of organs. 45,47 Systems/organs most frequently clinically or subclinically affected in mtD are the peripheral nervous system (PNS), the central nervous system (CNS), endocrine glands, heart, ears, eyes, gastrointestinal tract, liver, kidneys, bone marrow, and dermi. The second most frequently affected system is the CNS. 48 Common CNS findings are fluctuating encephalopathy, seizures, migraine, stroke-like episodes, ataxia, spasticity and cognitive decline through dementia. 49 The cognitive decline among manifestations of CNS is increasingly recognized and diagnosed, it is essential to guide the optimal management of these patients.
Mitochondrial syndromes associated with cognitive impairment due to mtDNA mutations include mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome, myoclonus epilepsy with ragged-red fibres (MERRF) syndrome, maternally inherited diabetes and deafness (MIDD), mitochondrial chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS) and neuropathy, ataxia and retinitis pigments (NARP) syndrome. 50 The assessment of cognitive functions in these patients is made through a neuropsychological test, and the domains impaired were abstract reasoning, verbal memory, visual memory, language, executive or constructive functions, calculation, attention or visuospacial functions. Cognitive functions and intellectual abilities may decline from initially focal cognitive impairment to lastly dementia. 51 In addition, the investigation proposed to detect mitochondrial dementia is based on imaging studies, including MRI, PET, SPECT or MR-spectroscopy, cerebrospinal fluid (CSF) investigations or electroencephalography. 52 The mitochondrial disorders are a genetically heterogeneous group and variable phenotype, therefore we select the most frequent mitochondrial diseases with cognitive impairment to approach: MELAS -mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome is typified by characteristic neurological manifestations including seizures, encephalopathy, and stroke-like episodes, and other secondary manifestations just like short stature, cognitive impairment, migraines, depression, cardiomyopathy, cardiac conduction defects and diabetes mellitus. 53 The patients suffer from cognitive dysfunction of nuclear pathogenesis. Emmanuele et al. 54 present a study exploring a possible link between cognitive dysfunction and hippocampal expression of calbindin D 28KD (CB), a high-affinity calcium-binding protein, in four MELAS patients, using post mortem hippocampal tissues. Reduction in CB expression is associated with aging and with neurodegenerative disorders. They found a reduction in CB expression in these patients and postulated that the reduced CB expression may play a role in the cognitive abnormalities in MELAS. 54 MERRF -Myoclonus Epilepsy associated with Ragged-Red Fibers had as diagnostic criteria typical manifestations of the disease such as myoclonus, generalized epilepsy, cerebellar ataxia and ragged-red fibres on muscle biopsy. Other multisystemic alterations can be found as: deafness, exercise intolerance, peripheral neuropathy, abnormal cardiac conduction, dementia, among others. 55 Kaufmann et al. 56 studied 91 individuals from 34 families with MELAS and the A3242G point mutation and 15 individuals from two families with MEERF and the A8344G mutation. The patients were divided into groups (asymptomatic, oligosymptomatic and symptomatic), and submitted to neurologic examination, neuropsychological testing and spectral resonances. The study showed that the MELAS probands had higher ventricular lactate estimates on spectral resonances than the MEERF probands, and the MELAS probands had lower scores on formal neuropsychological testing than MEERF probands. In this way the authors correlated high levels of ventricular lactate, the brain spectroscopic signature of MELAS are associated with more severe neurologic impairment. The study also evidenced that the 15 patients with MEERF had score for degree of impairment on neurologic examination lower than MELAS patients and that worsening of neuropsychological functions was independent of the severity of clinical manifestations. 56 Kearns-Sayre syndrome (KSS) presents that classic triad: pigmentary retinopathy, chronic progressive external ophthalmoplegia and heart block, beyond features like cerebellar ataxia. 57 Chronic Progressive External Ophthalmoplegia (CPEO) is a mitochondrial myopathy with slowly progressive, often symmetric blepharoptosis and limitation of ocular motility in all directions of gaze. 58 Bosbach et al. 59 performed a study with 22 patients with CPEO or KSS included screening for mitochondrial DNA (mtDNA) point mutations (3243/8344) and mtDNA deletions. All patients underwent a neuropsychological test battery. The neuropsychological testing did not reveal signs that would suggest general intellectual decline or dementia, but provided evidence of specific focal neuropsychological deficits, suggesting particular impairment of visuospatial perception associated to parieto-occipital lobes and executive deficits associated to the prefrontal cortex. 59 Autosomal CPEO presents with similar manifestations as mitochondrial CPEO but is due to mutations in the twinkle, POLG1 or ANT1 genes. 50 Simoncini et al. 60 reported a case of a woman with ANT1-related adPEO and dementia. The patient started to report disorders of short-term memory, and the neuropsychological evaluation showed a mild impairment of verbal memory and the attentive functions, magnetic resonance of brain revealed widespread moderate cortical atrophy, and magnetic resonance spectroscopy of brain disclosed mild elevation of lactate in the posterior interhemispheric region and in ventricular region. However, studies are needed to assess the prevalence of central neurological manifestations in ANT1 mitochondrial disease. 60 Leigh syndrome also referred to as subacute necrotizing encephalomyelopathy is a  Executive impairment was associated with higher rates of disability or death and faster ALS Functional Rating Scale (ALSFRS-R), Boston Naming Test, Rey-Osterrieth Complex Figure, rates of motor functional decline, particularly decline in bulbar function. and a mobile transcutaneous sensor for tissue blood gas tensions.
Decline in cognitive function was faster in patients who were cognitively impaired at baseline. Normal cognition at baseline was associated with tendency to remain cognitively intact, and with slower motor and cognitive progression. The results showed an increase in the number of CAG repeats in 6 affected males (including 2 presymptomatic patients), and in 10 females, of whom 5 were obligate carriers.
All symptomatic patients showed, besides the typical manifestation of X-BSMA, neurophysiological signs of sensory nerve involvement, and abnormal findings in neuropsychological tests.
disease that results in the regression of both mental and motor skills, leading to disability and rapid progression to death. 61 The patients also present refractory seizures, optic atrophy, ophthalmoparesis. Mental development is typically delayed at early age. Imaging studies show focal, bilaterally symmetric lesions of the basal ganglia, the thalamus and/or the brainstem. Moreover, there is raised lactate levels in blood and/or cerebrospinal fluid. 50,61 Mitochondrial neurogastroinstestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder in which the patients suffer from gastrointestinal dysmotility, cachexia, ptosis, external ophthalmoparesis, myopathy, and polyneuropathy. MRI shows leukoencephalopathy. 62 Carod-Artal et al. 63 reported a case of a Brazilian man with MNGIE who had poor weight gain, muscular atrophy, and chronic diarrhoea since childhood. He presents episodic gastric pain, recurrent nausea and vomiting, muscle aching and cramps, and had signs of memory impairment. On neuropsychological examination, patient scored 28/30 on MMSE. Working memory scores were 14/25 and 7/25 on immediate recall, and 6/25 and 1/25 on long-term recall. The patient could repeat seven digits in direct order and only four digits in reverse sequence. Verbal learning was involved (7/15 words). 63 The treatment of mitochondrial dementia includes general measures and specific therapy, which encompass: physiotherapy and physical exercise below the maximal individual limit; dietary measures; avoidance of mental and physical stress, alcohol, nicotine and infections; supplementation of respiratory chain components such as coenzyme Q., Various studies have reported a positive effect of coenzyme Q in mitochondrial dementia; administration of artificial electron acceptors, such as vitamin C or K; administration of metabolites or cofactors like carnitine. Overall, cognitive decline in mitochondrial dementia needs to be included in the differentials of dementia. 52

Limitations of existing research
There are limitations to our study. In terms of research strategy and data collection, we opted to evaluate only studies that we found through the ISI Web of Science and PubMed database, which may have limited the number of included studies. Furthermore, we only looked at studies written in English. Concerns about the reliability and validity of the applicability conditions of the questionnaires, as well as their potential subjectivity of neurofunctional and cognitive evaluation, to biased estimates of intervention effectiveness. Another potential limitation is the possible confounding variables in case reports and the low sample size.
The majority of the findings are based cross-sectional methods; further longitudinal studies are needed in order to model within-person change and avoid cohort effects, have been conducted. There are also other sources of variability within the literature that may have limited the conclusions drawn from this review, such as the lack of standardization in the cognitive and neuromuscular evaluation. This plethora of effects poses a serious challenge to researchers seeking neuromuscular diseases unifying principles. Limitations were also secondary to population size and phenotypes in relation to sample size in genetic research. In addition, the studies differ in terms of neuroimaging analysis.

Conclusions
The literature about cognition in neuro muscular diseases is still scarce, but studies converge on the influence of these disorders on mental processes (see Tables 1-3 for a summary on previous studies). Congenital disorders are common to neuromuscular function and brain function impaired. Considering that severe neuromuscular conditions such as DMD and ALS have shown a marked improvement in prognosis in the last decades, the search for the improvement of patients' quality of life has become increasingly important, which can be compromised by the primary and secondary cognitive effects the evolution of the disease. More work should be done to better evaluate cognitive deficits in patients with neuromuscular disorders, in order to improve the patient's relationship with the medical staff, the family, and society and improve the patient's quality of life. In addition, knowing the evolution of the cognitive symptoms of each disease is important for proper management of the patient, evaluation of survival, establishment of prognosis and choice of appropriate therapeutic intervention.


extfieldsofstudy:
Computer Science
Medicine

text:
rClone Red facilitates bacterial gene expression research by undergraduates in the teaching laboratory

Abstract rClone Red is a low-cost and student-friendly research tool that has been used successfully in undergraduate teaching laboratories. It enables students to perform original research within the financial and time constraints of a typical undergraduate environment. Students can strengthen their understanding of the initiation of bacterial translation by cloning ribosomal binding sites of their own design and using a red fluorescent protein reporter to measure translation efficiency. Online microbial genome sequences and the mFold website enable students to explore homologous rRNA gene sequences and RNA folding, respectively. In this report, we described how students in a genetics course who were given the opportunity to use rClone Red demonstrated significant learning gains on 16 of 20 concepts, and made original discoveries about the function of ribosome binding sites. By combining the highly successful cloning method of golden gate assembly with the dual reporter proteins of green fluorescent protein and red fluorescent protein, rClone Red enables novice undergraduates to make new discoveries about the mechanisms of translational initiation, while learning the core concepts of genetic information flow in bacteria.

Introduction
Education in STEM (science, technology, engineering and math) has reached an intersection where opportunities can meet challenges. We want our students to learn cutting edge methods but do so with meaningful learning outcomes. Students, teaching faculty, professional societies and the government have all called for better methods of hands-on learning that can improve student performance in core competencies (1,2). Increasingly, STEM educators are turning to course-based undergraduate research experiences (CUREs) as a way to reach a larger number of students with diverse demographics, while containing expenditures (3)(4)(5)(6). The redesigned Advanced Placement (AP) Biology course has urged teachers to provide laboratory experiences for high school students despite limitations associated with cost, facilities and teacher training (7). The Next Generation Science Standards (NGSS) also calls on high school biology teachers to provide students with hands-on learning opportunities with open-end laboratory experiences (8). The field of synthetic biology has grown rapidly to provide inexpensive, student-friendly lab-based options, some of which were developed by undergraduate students in the International Genetically Engineered Machines (iGEM) competition (9).
Students who use a traditional Genetics textbook to learn about protein-encoding gene expression initially focus on the flow of genetic information from DNA to RNA to proteins, according to the 'central dogma'. Thereafter, they learn that transcription is regulated largely by promoters and nearby regulatory DNA elements located upstream of the protein-encoding coding DNA sequence (10). When students study translation, they usually focus on the protein coding sequence that begins with the start codon and ends with a stop codon. However, teachers and students alike often skip over a very important control element that regulates the initiation of translation. The ribosomal binding site (RBS) is a short segment of bacterial mRNA located upstream of the start codon ( Figure 1). The mRNA sequence of the RBS is a key factor in determining the overall efficiency of translation because the 16S rRNA of the 30S ribosomal subunit must base pair with the mRNA in order for translation to begin. It would be especially helpful if students could conduct research on the RBS using cost-effective synthetic biology methods that are easy to master.
We developed pClone Red to allow students to conduct research on promoters (11). Even novice students are able to clone new promoters into pClone Red because it takes advantage of golden gate assembly (GGA) cloning (12,13). GGA uses type IIs restriction enzymes that cut outside their asymmetric recognition sequences and produce sticky ends that can be selected by users. We have found that first year college students and high school students are remarkably successful when using GGA to clone novel promoters and carry out mutational analysis of existing promoters from synthetic oligonucleotides. In previous education publications, we described our use of pClone Red to help undergraduates better understand the regulation of the initiation of transcription by investigating promoter sequences (14,15). In this education research, we describe how rClone Red can be used by students to design and test RBSs. Previously, we described rClone Red in a basic research publication (16). Independent research students working outside laboratory courses conducted research to examine different RBS designs and 'anti-RBS' sequences within the mRNA that reduced reporter protein production. The current education study explains how faculty can foster student understanding, while adhering to typical laboratory course budgets and schedule constraints. We explain how the design of rClone Red makes it easy for early career STEM students to identify clones that contain the RBS they designed, and to measure RBS strengths with a reporter gene. We used pre-/post-assessment to measure, which concepts students learned from a laboratory-based curriculum that included rClone Red.

Cloning methods
For use in cloning with rClone Red, students design two oligonucleotides between 15 and 60 bases long that are ordered from Integrated DNA Technologies, Inc. as 'lab ready' solutions of 100 mM each. Students have access to a free online tool we developed called Oligator (http://gcat.davidson.edu/iGem10/index. html) that reduces errors in sequence design. In the final design, the top strand sequence must begin with the left sticky end of 5 0 CGAC 3 0 , and the bottom strand must begin with the right sticky end of 5 0 CCGC 3 0 . The Oligator ensures that neither strand contains a Bsa I recognition site. Oligos are annealed to form double-stranded DNA using a simple overnight protocol. At least one day before lab, one student from each group prepares an annealing solution that contains 100 mM NaCl, 10 mM Tris-HCl, pH 7.4 and both oligonucleotides at 5 mM in a final volume of 20 mL. Tubes (500 ml) with the mixed oligonucleotides are floated for 10 min in a beaker containing 600 ml of boiling water, after which time the heat is turned off and the mixed oligonucleotides are allowed to cool slowly and anneal in the water bath overnight. Annealed oligonucleotides are used the next day or stored frozen at À20 C and used later.
GGA combines BsaI-HF v2 type IIs restriction enzyme (New England Biolabs) and T4 DNA ligase (Promega) as described (12,13). The plasmid rClone Red (J100272; Genbank accession number MH549194) produces GFP when unaltered because it has a 'left-facing' promoter, RBS and GFP ORF, all of which is flanked by two BsaI restriction sites. To the left of this backwards GFP expression cassette is a 'right facing' promoter. To the right of the GFP cassette is a RFP ORF. When GGA is performed, the GFP cassette is removed and a new RBS encoded by students' annealed oligonucleotides is cloned between the right-facing promoter and the RFP ORF, producing a new RFP expression cassette. Each GGA reaction contains 50 ng of rClone Red plasmid, 4 nM annealed oligonucleotides, 0.5 ml BsaI HFv2, 0.5 ml ligase and 1X ligase buffer in a final volume of 10 ml. A thermal cycler is used to perform 30 cycles of 1 min at 37 C followed by 1 min at 16 C. A final 5 min incubation at 37 C reduces the frequency of background green colonies. Competent JM109 Escherichia coli (Zymo Research) are transformed following the manufacturers recommendations, and transformed cells are plated on LB ampicillin (50 mg/ml) agar plates. Carolina Biological sells an rClone Red kit that contains all reagents, including a positive control RBS, competent cells and antibiotic-containing agar plates. Student-designed RBSs must be purchased separately.
For each RBS tested, three non-green candidate colonies are picked with a sterile toothpick and grown in shaking incubator overnight at 37 C in 4 ml of LB ampicillin (50 mg/ml) liquid media. The next day, 200 ml are placed in 96 well plates for quantification of RFP fluorescence intensity with a BioTek Synergy H1 microplate reader. RFP was measured in arbitrary fluorescence units using 585 nm for excitation and 615 nm for emission, and the concentration of bacteria was measured using optical density at 590 nM. Students calculate the ratio of fluorescence divided by absorbance to normalize for cell culture density. Students convert their raw fluorescence values into normalized values because not all cultures will have the same density of cells.
For two-dimensional RNA folding analysis, students use Mfold, a free online tool (17). Students can submit their RBS designs to Mfold to predict the stability of any secondary RNA structures that might prevent the initiation of translation. Mfold reports the stability of several possible RNA folding structures with DG, where larger negative DG values indicate more stable structures.
Photographs of colonies from rClone experiments are taken by normal digital cameras and not fluorescence microscopes or dissecting scopes. Our students routinely use their cell phones to capture images similar to the ones in this report for use in their oral presentations and laboratory reports.

Educational methods
The genetics course for this educational research project was taught at Missouri Western State University (MWSU), a primarily undergraduate institution with open admissions. Most MWSU students live within a 50 mile radius and many are first generation college students, which means they are the first in their families to attend college. The genetics course is required for Biology majors and most students enrolled in the course are sophomores. The course included three 50 min lecture/discussion periods and one 3 h laboratory session per week. The prerequisites for the course are one semester of introductory cellular biology and one semester of general chemistry. There were 50 students enrolled in the course, which was divided into three laboratory sections. One of us (T.T.E.) was the sole instructor for all lecture and laboratory sections of the course. Four weeks of laboratory sessions were dedicated to an rClone Red laboratory module that complemented lecture material from the course. Prior to the laboratory module, students had already learned to pipet small volumes of liquid and work sterilely with bacteria, and they had learned about DNA base pairing, transcription and translation.
To assess learning gains among undergraduates using rClone Red, we developed a 20 question multiple choice assessment instrument (available to instructors upon request). In the MWSU genetics course, the instrument was administered to 50 MWSU genetics students at the beginning of the fall 2015 semester and again at the end of the semester to 48 students (one week after the rClone Red module had been completed). IRB exemption #2416 was granted prior to the study. Significant differences between pre-and post-survey scores in Figure 4 were determined using an unpaired z test for difference in proportion. The assessment data in this study were collected in the fall of 2015, but rClone Red has been used repeatedly in subsequent semesters at MWSU and at Davidson College. There is only one Genetics class each semester at MWSU, so it was not possible to have a control group of students taking Genetics that was not using rClone Red.

Biological context
Our goal in developing rClone Red was to offer students a CURE without having to reconfigure our normal laboratory schedules or exceeding our existing budgets for laboratory supplies. rClone Red empowers undergraduates to conduct original research on the control of translation initiation without the allocation of additional time or funding. The simplicity and efficiency of GGA makes rClone Red appropriate for teaching laboratory courses with novice students. The design of rClone Red enables students to easily determine, which colonies in a cloning experiment have successfully incorporated a new RBS (Figure 2). Bacteria that have been transformed with the original rClone Red plasmid express GFP using the left-facing expression cassette, and are easily visible on a UV or blue light box as bright green colonies. Because GGA replaces the backwards GFP expression cassette with a new RBS, colonies resulting from successful GGA are not green. Our students who are color blind learned to detect the difference between fluorescing green and non-fluorescent colonies. The extent to which successful colonies glow red from the resulting RFP reporter gene using the right-facing promoter and the new RBS depends on the ability of the new RBS to initiate translation. The colonies in Figure 2 are from a strong RBS, so the colonies appear bright red. Once they are shown the differences in control GFP and RFP plates, color blind students learned to detect the difference between GFP and RFP because GFP is so much brighter than RFP (see Figure 2).
Genetics students at MWSU learned in lecture about the basic mechanisms by which RBSs initiate translation and were given the opportunity to enhance their learning by conducting original research by participating in an rClone Red CURE. They began their laboratory investigation by examining the sequence of a strong RBS used as a positive control that produced red colonies ( Figure 3A, top). The genetics students proposed new mutations that would enable them to explore the sequence requirements for RBS function. The strong RBS (B21) is composed of eight bases, but the oligonucleotide students used also contained flanking bases and sticky ends to produce directional ligation upstream of the RFP start codon in rClone Red. One group of students designed mutant #6 which differs from B21 by one base upstream of the RBS and one base in the RBS itself (see Figure 3A, middle). Another group designed mutant #2, which has five mutations upstream of the RBS and two mutations in the RBS (see Figure 3A, bottom). The photograph of 2 ml drops of cultures on a plate shows that mutant #6 does not produce as much red fluorescence as B21, and mutant #2 does not produce any visible RFP. Because color perception is influenced by genetics (18), students used a fluorometer to quantify fluorescence intensity as an indirect measure of translation efficiency ( Figure 3B). When the students tried to understand their results, they were confused initially because mutants #6 and #2 base pair with the 16S rRNA as well as the strong RBS B21 ( Figure 3C). For all three RBSs, seven of the eight bases form hydrogen bonds with the 16S rRNA of the small ribosomal subunit. The students expanded their understanding of RBS secondary structure when they used the online RNA folding tool Mfold ( Figure 3D) (17,19,20). Both B21 and mutant #6 are predicted to form loop structures that are anchored by a four base pair stem, including two bases of the RBS. In contrast, the predicted structure for mutant #2 contains a six base pair stem including four bases of the RBS. These RNA folding predictions allowed the students to propose a structural explanation why the more energetically stable mutant #2 (DG ¼ À2.40 kcal/mol) produced a weaker RBS than B21 or mutant #6, which are energetically less stable and have more bases available for pairing to the 16S rRNA.

Educational outcomes
Before we implemented rClone Red in the Genetics teaching laboratory, we developed a list of 20 concepts that we anticipated students might learn as a result of their laboratory research ( Table 1). All 20 concepts are addressed by the instructor to varying degrees, while students are working in the lab, but we wanted to know which ones would stick with the students and which ones would not. We developed a 20 question multiple choice assessment instrument to determine what students knew coming into the class (pre-test) and what they knew 1 week after the rClone Red CURE was completed (post-test; Figure 4A). On average, students correctly answered 5.28 questions on the 20 question pre-test (26.4%). After completing their RBS research projects, students correctly answered an average of 11.6 out of 20 questions (55.6%).
Because average scores for the overall 20 question assessment obscured which topics students learned, we examined each of the 20 concepts individually and determined which ones showed significant learning gains and which ones did not ( Figure 4B). Students showed significant improvements for 16 of the 20 concepts. The four concepts that students did not learn were: #8, the GGA method; #9, characteristics of type IIs restriction enzymes; #17, altering RBS strength to regulate protein production level; and #18, selective advantage for bacteria to have different strength RBSs. We had hypothesized that students would acquire all 20 concepts equally well, but the assessment demonstrated that either the students did not learn these four, or our ability to measure these four was imperfect. Regardless of the reason, we can revise our teaching and assessment approach for these four concepts if we feel they are important enough to do so. Only through assessment can we know which portions of our curriculum works, and what needs improvement.

Discussion
Since 2015, we have been using rClone Red for teaching Genetics at MWSU and Introductory Biology for majors at Davidson College. rClone Red is a powerful tool that enables any student to have a classroom-based research experience (CURE) (3)(4)(5)(6). Every biology student is taught the core concept of central dogma and our impression from other faculty is that transcriptional regulation is often emphasized more than translational regulation. Some students are given opportunities to explore experimentally the mechanisms by which transcription is initiated and regulated, but it seems that very few get the chance to study the initiation and regulation of translation. The initiation of bacterial translation involves base pairing between the mRNA and the 16S rRNA of the small subunit of the ribosome (Figure 1). However, scientists have a limited understanding of the roles of bases flanking the RBS and RNA secondary structure, which creates opportunities for students to make new discoveries. Before modification, rClone Red expresses GFP so that students can easily detect which colonies contain the original plasmid. Cloning by GGA works nearly 100% of the time even with novice students. Beginners quickly realize that non-green colonies contain GGA products in which the GFP expression cassette was successfully replaced with their RBS (see Figure 2). The intensity of red fluorescence is determined by the strength of the RBS, which is influenced by rRNA base pairing and the three-dimensional folding of the mRNA (Figure 3) (19,20). Students conducted RBS mutational analyses, collected original data on the strengths of mutant RBSs, and provided explanations for their results using Mfold (see Figure 3) (17). RNA folding can be explored further by using the online RBS calculator tool, which predicts RBS strength (20).
Many teaching laboratories do not have access to a fluorescence microscope to photograph colonies, or a fluorometer to quantify fluorescence intensity. However, cell phones are capable of producing the images shown in this report, as long as a UV or blue light source is available. We reported previously that cell phone images can be used in conjunction with the free software ImageJ to compare relative RFP intensities (11,21). rClone Red can be used anywhere there is a UV or blue light source, cell phones and computers with ImageJ installed. Instructors who do not feel comfortable teaching ImageJ could rely upon visual perception by students to rank color intensity, though colorblind students would require assistance from peers.
In this education research project with genetics students at MWSU, we found that students learned most of the 20 concepts that we had identified prior to using rClone Red (Table 1 and Figure 4). It would have been nice to compare students in the rClone Red lab to an equivalent population not using rClone Red, but there is only one Genetics class each semester at MWSU, and it is taught by the same instructor each time. It would be interesting to have faculty at larger campuses conduct similar rClone Red educational research along with a control population. One of us (A.M.C.) is conducting additional education research using rClone Red as well as pClone Red to measure self-efficacy, self-identity and project ownership by students in a CURE setting.  Numbers match learning objectives in Table 1, and asterisk (*) denotes significant differences pre-test versus post-test, P < 0.05.
When pClone was used with genetics and introductory biology students on both campuses, there were some learning outcome differences between the populations (11). For example, the differences between type IIs and type II restriction enzymes was better understood by the Genetics students. Conversely, GGA was better understood by the introductory students. These learning outcome differences are intriguing and may indicate that beginning students are more amenable to learning a new way of cloning if they have not learned a traditional cloning method in a previous course. Genetic students might be better prepared to distinguish the two types of restriction enzymes due to prior experience with restriction enzymes in general. In this educational study using rClone Red, the MWSU genetics students failed to improve their understanding of GGA similar to previous MWSU genetics students had when using pClone Red. Instruction was altered during the genetics laboratory sessions to review GGA each lab and yet this concept did not stick well with the students. Further investigation is required before we can determine the best way to help genetics students learn GGA as a new and alternative way to clone DNA.
Many education scholars have studied the value of multiple choice questions (22)(23)(24)(25). From the literature, it is clear that multiple choice questions can reveal student learning outcomes. Our results show that students learned some concepts but not others because students improved their scores on several concepts but not all of them. Higher order thinking skills are difficult to assess with multiple choice questions, which is why MWSU students also must submit laboratory reports and give oral presentations for their genetics course grades.
In addition to its utility for CUREs, the simple elegance of the rClone Red design also makes it amendable for undergraduates or high schoolers who want to have summer undergraduate research experiences (SUREs) (16,26). There are several interesting areas of investigation that students could explore with rClone Red. Because E. coli has more than one 16S rRNA gene, students could design RBS sequences that target one or more of the seven rRNA paralogous operons (27,28). Students could generate libraries of potential RBS sequences by inserting 1-8 N bases during oligonucleotide synthesis and selecting RBS strengths for specific desired applications (16). Another application of rClone Red is using it for testing the specificity of a riboswitch and its potential for translational actuation (29). Riboswitches are RNA biosensors incorporated into the 5 0 end of mRNAs that use ligand-dependent RNA folding to control gene expression at the levels of transcription or translation (30).
Students could use rClone Red to investigate the functions of natural and synthetic riboswitches or engineer new ones.
rClone Red is one member of a family of plasmids we have developed to blur the lines between teaching and research laboratories. In addition to pClone Red (11), we have designed and built tClone Red, actClone Red and repClone Red. Each of these plasmids uses GGA, a GFP reporter gene that is silenced when students successfully clone their new DNA part, and a RFP reporter to measure gene expression. tClone Red (http://parts. igem.org/Part: BBa_J119361) supports the investigation of rhoindependent transcriptional terminators and riboswitches that use alternative RNA folding states to control gene expression. tClone Red has been used successfully in the genetics teaching laboratory at MWSU and in independent undergraduate research projects on both of our campuses. actClone Red (http:// parts.igem.org/wiki/index.php/Part:BBa_J100435) enables students to reengineer the E. coli ompC promoter, which requires three endogenous ompR proteins for activation. Student can change the order and/or sequence of the ompR binding sites to test how malleable the promoter is to sequence perturbations. repClone Red (http://parts.igem.org/wiki/index.php/Part:BBa_ J100342) is built for the pTet promoter and the tetR repressor protein. Students can alter the sequence and order of two tetR binding sites to explore this interesting promoter. actClone Red and repClone Red have been used in teaching laboratories at Davidson College, but they are unstable constructs and suffer from unwanted recombination events. We are working to improve their designs so that they are more student-friendly.
rClone Red is a cost-effective educational tool for use in teaching laboratory courses that allow students to conduct original research within typical laboratory course time constraints and budget limitations. It has been used with sophomore genetics students as well as first year introductory biology students. rClone Red is appropriate for any laboratory course where the central dogma or bioengineering are included in the learning goals. We have shown that students learn many of the concepts we hoped they would, but there is room for improvement for a subset of these concepts. Additional education research by others, with a control population, would enhance our understanding of how rClone Red improves student understanding while simultaneously providing them with a CURE. testing of rClone Red, and the participation of the fall 2015 and spring 2018 Missouri Western genetics students using rClone Red in class.

Funding
This work was supported from NSF RUI grants MCB-1120578 to Davidson College and MCB-1329350 to Missouri Western State University, and from the James G. Martin Genomics Program at Davidson College are gratefully acknowledged.