The Antimicrobial Resistance and Prevalence of Enterococcus Species in Saudi Arabia

Monitoring the distribution and resistance of antibiotics to enterococcal species is critical aspect to controlling and preventing enterococcal infection. The aim of the present study is to screen the antimicrobial resistance genes within Enterococcus species isolates that collected from Taif governorate, Saudi Arabia. Out of 134 clinical samples, nineteen enterococcal isolates were identified using 16S rRNA sequence gene. Phylogenetic tree analysis using 16S rRNA gene sequence of the 19 strains divided them into 15 strains as E. faecalis and 4 strains as E. faecium. In addition, these the species of these isolates were recognized using VITEK-2 COMPACT system. The PCR technique was used to screen the multi-drug resistant genes within enterococcal isolates. The KpsII, tetL, aac(6)-Ie-aph(2)-Ia, vanA and Erm(B) genes were found in all strains. The distribute of resistance against antibiotic drugs were differs greatly between the two species, a considerably higher prevalence of resistance to penicillin, gentamicin, cefoxitin, cefotaxime, clindamycin, erthromycin and fusidic acid was identified in E. faecalis than in E. faecium, while greater spread was detected to resist to Trim/Sulf and tetracycline in E. faecalis. Finally, rep-PCR markers investigated genomic diversity of Enterococcus strains. Results of rep-PCR markers generated 142 distinct loci; 96 were polymorphic (67.6%) and 46 were monomorphic (32.4%). Number of loci for individual rep-PCR primers ranged from 9 for rep-08 to 18 for rep-02.

INTRODUCTION
The genus Enterococcus consists of thirtyeight species. The most common identified living microorganisms usually found in the intestines of human and animal cells including E. faecalis and E. faecium 1,2 . E. faecalis is accountable for about 80% of human infections 2 . Intestinal microorganisms are opportunistic organisms that become pathogenic due to an imbalance in the immune system. Currently, Olawale et al. 3 reported that intestinal tract infections can cause infections in hospitals, especially in urinary tract, surgical sites and bloodstream. Several reports pointed out that gastroenteritis is the third organism isolated between infection in hospitals of Saudi Arabia and the most isolated microorganisms common in the bloodstream 4,5 . Approximately 90% of intestinal endocarditis occurs due to E. faecalis, with less than 5% affected by E. faecium 4 . Infrequently, additional species are documented in disease conditions, including E. avium, E. casseliflavus, E. durans, E. gallinarum, E. hirae, E. mundtii and E. malodoratus 6 . Primarily in the last twenty years, these strains caused infections in the hospitals of Saudi Arabia. Enterococcus comprises a necessary piece of the gastrointestinal microflora of people, animals and insects. Notwithstanding their reality as commensals in the gastrointestinal tract, a few strains exist as sharp pathogens. In a previous study, vancomycin resistance to intestinal microorganisms (VRE) accounts for about 43% of all intestinal catheter isolates, an amount that rises due to unavailability of vancomycin for clinical use in USA. Two types of VRE isolates are included; E. faecium and E. faecalis. Both species were isolated to resist eight antibiotics 3 . Antibiotic resistance may be help in Enterococci development and consider as a threat to public health. The high distribution of Enterococcus spp. has developed as a public health threat 7 . Enterococcus is the most common bacterial infection in hospitals 7 . Enterococcus types have many virulence factors such as gastrointestinal protein and aggregation content, which increases the colonization process of epithelial lining of host cells 8 . The gene coding for ESP was commonly identified in medical infection isolates 9 . Antibiotic resistance factors are transferred by mergers, plasmids or transposons that can act as carriers. These genes are transferred to other members of the same microbial species and transmission of horizontal genes may arise through conjugation, transduction and transformation 5,10 .
The polymerase chain response (PCR) is a less complex method that gives results inside a short timeframe and at lower costs. By this technique, diverse primer sits, homologous, aleatory or degenerated, are utilized to intensify locales of the DNA particle. Band designs are in this way delivered, allowing the gathering of comparative strains and the separation of irrelevant ones 11 . Repetitive (REP) sequences are extragenic units found in various locales of the DNA of bacterial species 12 . The amplification of the areas between these units creates a helpful unique fingerprint to separate Enterococcus strains 13 .
The purpose of the present research was to screen the distribution of the antimicrobial resistance genes within Enterococcus spp in patients from King Faisal Hospital in Taif governorate, Saudi Arabia. Moreover, the genetic diversity between the Enterococcus spp. strains were investigated using rep-PCR markers.

MATERIAL AND METHOD Molecular identification of Enterococcus strains
Out of 134 bacterial isolates, nineteen multiple-drug resistant enterococcal strains were obtained from King Faisal Hospital in Taif governorate, Saudi Arabia. Isolates were recognized by high-throughput using a VITEK-MS (bioMיrieux, France) system according to manufacturer's protocol. The DNA extraction kit (Gena Bioscience, Germany) was used to isolate genomic DNA from all 19 isolates accordance the manufacturer's directions. For each isolate and as per previously described methods, one fragment of 16S rRNA gene (about 1465 bp) was amplified from each stain as previously reported 14 . The specific band was purified from the gel using QIAGEN purification kit (QIAGEN, USA). The purified band was sequenced via DNA Analyzer 3146 (Applied Biosystems, USA). To perform the BLAST searched, NCBI service was used (http://www.ncbi.nlm.nih. gov/blast/Blast.cgi). The phylogenetic tree was constructed using neighbor joining algorithm with Kimura 2 parameter distances in MEGA 7.1 software. The bar indicates the Juke-Cantor evolutionary distance.

Antibiotic Susceptibility Testing
All nineteen Enterococcus strains were screened for antimicrobial susceptibility using an automated VITEK-2 (bioMיrieux) system. In this protocol, broth microdilution minimum inhibitory concentration (MIC) method for susceptibility testing and accomplish repetitive turbidimetric screen of bacterial growth through an abbreviated incubation period. MIC results were taken based on the Clinical and Laboratory Standards Institute guidelines 15 . The criteria of Hassan et al. were used to defined MDR isolates 16 . The susceptibility of Enterococcus isolates to 16 antibiotics including vancomycin, gentamycin, cefoxitin, efotaxime, ampicillin, daptomycin, trime/sulf, teicoplanin, vancomycin, clindamycin, erythromycin, fusidic acid, linezolid, nitrofurantoin, ciprofloxacin, moxifloxacin and tetracycline was determined.

Detection of Antimicrobial Resistance Genes
Five virulence genes [KpsII, tetL, aac(6)-Ie-aph(2)-Ia, vanA, and Erm(B)] were detected in all Enterococcus strains in multi-Drug resistant according to Hassan et al. 14 . The resistance genes against antibiotic were detected in all 19 strains as previously reported 4 . The antibiotic resistance genes were isolated from the 19 strains by PCR with primers synthesized by the macrogen Co., Ltd. (Seoul, Korea) ( Table 1). The PCR reaction for each gene was accomplished as previously described 4 .

Rep-PCR analysis
The settings of rep-PCR technique for the 19 strains of Enterococcus were standardized. Five primers (Rep-1, Rep-2, Rep-8, Rep-12 and Rep-18) were used to investigate the genetic variability between the Enterococcus strains. The primers sequence and PCR conditions were performed as previously reported 17 .

Enterococcus isolates identification
The species of the nineteen Enterococcus isolates were known by a fully automated VITEK-2 COMPACT microbiology method. Fifteen were recognized as E. faecalis and four as E. faecium. These results were confirmed by 16S rRNA sequencing. The 16S rRNA gene sequence of all 19 isolates was compared with other Enterococcus strains that placed in GenBank database. The results were somehow differed from the obtained sequence (data not shown). Ribosomal genes are greatly relevant for investigating bacterial evolution and phylogeny. When re-constructing phylogenetic relationships of microorganisms, sequencing of 16S rRNA has been generally employed. Phylogenetic tree analysis using 16S rRNA sequences of all 19 strains and other related Enterococcus species from GenBank were positioned the present Enterococcus strains into E. faecalis and E. faecium (Fig. 1).

Antibiotics sensitivity test
All Enterococcus strains showed resistance to the majority of selected antibiotics (Fig. 2). A significantly higher resistance to gentamicin, cefoxitin, cefotaxime, clindamycin, erythromycin and fusidic acid were found in E. faecalis than in E. faecium. On the contrary, a higher resistance to trim/sulf and tetracycline was observed in E. faecium. Furthermore, a low resistance to nitrofurantoin, linezolid and daptomycin was noticed in both species. In addition, these isolates   were found to be less than 50% sensitivity to ciprofloxacin, moxifloxacin and tetracycline ( Fig.  2 and 3).

Antimicrobial resistance genes prevalence
The KpsII, tetL, ermB, aac(60)-Ieaph(200)-Ia and vanA genes were recognized in all isolates. The identification of these genes in both species were showed in table 2. The ermB gene was found in the majority of ciprofloxacinresistant enterococci, indicating the occurrence of different systems related to the resistance of enterococci to fluoroquinolones. Similar gene were present in 79% among the erythromycinsensitive enterococci and 10.5% among the Fig. 3. The combination disk diffusion test of Enterococcus strain with some antibiotics linezolid-sensitive enterococci, demonstrating no expression of ermB gene in some enterococcus strains. Further, ermB gene occurred at a greater and significance role in fluoroquinolone-resistant enterococci compare to that in the sensitive species. Therefore, the existence of ermB gene was connected with the protection against fluoroquinolones in the Enterococcus species (Fig.  4).

Rep-PCR analysis
Rep-PCR markers were used to investigate the genomic diversity of Enterococcus strains. Table 3 and Figure 4 illustrates rep-PCR results. Rep-PCR markers produced 142 distinct loci; 96 bands were polymorphic (67.6%) and merely 46 bands were monomorphic (32.4%). Individual rep-PCR primers included a number of bands varying from 9 bands for rep-08 to 18 bands for rep-02. The greatest polymorphism marker was recorded for rep-08, rep-12 and rep-18. The band size of rep-18 ranged between 350 to 2100 bp (Fig. 5). The lowest polymorphism (62.5%) was detected in rep-01. Primers rep-1, rep-8, rep-12 and rep-18 produced equal mono morphism and polymorphism bands. The band size of rep-12 ranged between 100 to 1400 bp, while, the band size of rep-18 ranged between 350 to 1700 bp (Fig. 5).
The phylogenetic analysis tree using Neighbor Joint method according to the bands that produced from rep-PCR markers was shown in Fig. 6. A low genetic distance, ranged from 0.02 to 0.36, among Enterococcus isolates was determined based on Jaccard's similarity coefficients (Fig. 6). The Enterococcus strains were gathered into two groups with around 66% genetic similarity. The first group was included E. faecium samples (Entero- 4, 9, 11 and 16), with similarity 76%. While, the  second main group included E. faecalis samples, that divided into two clusters. The first cluster contained Entero-14, 15, 17, 18 and 19 isolates, and the second one contained Entero-1, 2, 3, 5, 6, 7, 8, 10, 12 and 13. These results suggested that mostly genetic distance between native Enterococcus strains was quite low among each species, while, was high between the two species (Fig. 6).

DISCUSSION
Out of 134 clinical isolates, 19 antiretroviral strains, 15 as E. faecalis and 4 as E. faecium, were obtained from King Faisal Hospital in Taif, Saudi Arabia, within four months. The antimicrobial resistance of Enterococcus spp. was recognized to be common in gastrointestinal tracts 2,14,18 . The frequency of both species in 206 strains of intestinal tract infections that obtained  from clinical specimens at Riyadh hospitals, KSA, were 69.2% for E. faecalis and 11.3% for E. faecium 19 . Also, the different microorganism that isolated from intestinal patients of the University Hospital in Taif, Saudi Arabia were documented as E. faecium with 34% and E. faecalis with 33% 4 . In agreement to these reports, the main two species, E. faecium and E. faecalis, in our clinical specimens were found to be a natural resistance to gentamicin and aminoglycosides. Although bacteria found sensitive to these drugs in laboratory trials, effectiveness was initiate to be unsatisfactory in clinical practice 20,21 . Enterococcus species has been reported as widely multipleantimicrobial resistance 4,22 . In the present study, a greater level of resistance to gentamicin, cefoxetin, cevotaxim, clindamycin, erythromycin and fucidic acid was detected in E. faecalis than E. faecium, while a greater resistance occurrence towards Trim/Sulf was detected in E. faecium. The resistance of E. faecalis to tetracycline was greater than E. faecium. Furthermore, a low predominance of protection against linezolid and nitrofurantoin was identified in both species. In this way, linezolid, vancomycin, and nitrofurantion are as of now generally utilized for viable treatment of intestinal fistula infection 23,24 . To achieve an understanding of the change in antimicrobial resistance in gastrointestinal tracts in different hospitals, a comparison of our study with other clinical intestinal strains that have resistance towards antimicrobial agents and collected from January 2014 to December 2015 was made 4 . There was a significant increase percentage of Enterococcus strains, where E. faecium was remain prevalent, but the component rate enlarged. Persistent antibiotic pressure is believed to lead to secondary resistance to gastrointestinal microbes 4,25 . Here, ermB gene was found in all intestinal cysteine resistance to ciprofloxacin. Moreover, the existence of the ermB gene was highly significant in strain of enterococci that showed resistance to fluoroquinolone than those sensitive strains. Currently, many aminoglycoside adjusting enzymes have been recognized that have tow functions of 6'-aminoglycoside acetyltransferase (AAC(6')) and 2"-aminoglycoside phosphotransferase (APH(2")). These enzymes usually encoded by aac(6')/aph (2") gene. This gene typically removes the synergistic consequence between glycopeptide or penicillin antibiotics and aminoglycosides 5 . The incidence of aac(60)-Ie-aph(200)-Ia gene was detected in all strains.
Here, the occurrence of tetracycline-resistant genes remained in E. faecium with 10.5% and in E. faecalis with 89.5%. Consequently, protection of intestinal tract species against tetracyclines is believed to be as a result of the existence of a gene that encodes the relating enzymes. One of the mechanisms that responsible for the resistance of macrolides in intestinal microorganisms is the alteration of erythromycin target site by ermB and mef genes in the genome of enterococci microorganisms 26 . In this study, vanA gene was identified in all 19 strains. Vancomycin-resistant Enterococcus species have been able to transfer vanA gene to Staphylococcus aureus, leading to the appearance of vancomycin-resistant S. aureus bacteria, leading to further difficulty in the clinical treatment of intestinal infections 4,26 . As a result, care should be taken when vancomycin is used as a drug for treatment of gastrointestinal infection and management of viomycin-resistant enterococci need to be enhanced 23 .
Molecular procedures used to identify bacterial strains vary as far as unfair power, reproducibility, standardization, cost, simplicity of advancement and interpretation 27 . REP-PCR were established and demonstrated great outcomes when utilized to type Enterococcus strains 13 . There are a few other PCR-based strategies effectively applied for enterococcal separation, for example RAPD-PCR 28 , ITS-PCR 29 , tDNA-PCR 30 , AFLP 31 , and PCR-RFLP 32 . Additionally, multilocus arrangement examination (MLSA) is likewise a procedure utilized for recognizable proof of enterococci by utilizing partial sequences of RNA polymerase alpha subunit, phenylalanyl-tRNA synthase and the alpha subunit of ATP synthase 33,34 . In any case, PCR results are progressively hard to examine, since the presence of different powerless loci in the PCR profiles makes it hard to decipher the results. Rep-PCR with oligonucleotide primers used for amplified genomic DNA from enterococci and demonstrated acceptable results when used to identify E. faecium clinical isolates 35 . Here, the Rep-PCR method approved to be a satisfied technique to discriminate Enterococcus spp., however, additional studies should be assumed to evaluate strains obtained from other hospitals.

CONCLUSION
Enterococci have been the major pathogenic microorganisms that make clinical contaminations because of various antimicrobial resistance, and the clinical enterococcal diseases prevalently happen in the urinary framework. Antimicrobial sensitivity changes in various Enterococcus species, and the resistance of enterococci to antimicrobial antibiotics is essentially inferable from the development of antimicrobial resistance genes. The screening of antimicrobial resistance of Enterococcus species would give a manual for the fitting determination of antimicrobial agents and avert the existence of increasingly antimicrobial-resistant enterococcal strains. The present results propose a moderately high predominance of antibiotic resistance in enterococci strains, especially to gentamicin, vancomycin, cefoxitin, and efotaxime. It is of worry that the scope of antibiotics to which resistance has been gained after some time is generally wide, to the degree that it presently incorporates new developing antibiotics utilized for the treatment of enterococcal contamination.


s2fieldsofstudy: Biology Psychology

text:
A soluble truncated tau species related to cognitive dysfunction and caspase-2 is elevated in the brain of Huntington’s disease patients

Huntington’s disease (HD) is a progressive neurodegenerative disease. Involuntary movements, cognitive impairment and psychiatric disturbance are the major clinical manifestations, and gradual atrophy and selective neuronal loss in the striatum and cerebral cortex are the pathologic hallmarks. HD is caused by expanded CAG trinucleotide repeats at the N-terminus of IT15 that encodes the huntingtin (HTT) protein, though the molecular mechanisms through which the mutant HTT (mHTT) exerts toxic effects remain obscure. Members of the caspase family, including caspase-2 (Casp2), play an important role in HD pathogenesis. Genetic ablation of Casp2 ameliorates cognitive and motor deficits of HD mice, though the molecular targets of Casp2 are still unclear. It is well established that the microtubule-associated protein tau potentiates cognitive dysfunction in a variety of neurodegenerative disorders, including HD. Our recent study indicates that Casp2-catalyzed tau cleavage at aspartate 314 (tau 2N4R isoform numbering system) mediates synaptotoxicity, cognitive deficits and neurodegeneration in cellular and mouse models of frontotemporal dementia; further, levels of Δtau314, the soluble, N-terminal cleavage product, are elevated in individuals with mild cognitive impairment and Alzheimer’s disease, compared with cognitively normal individuals. Here, we identified the presence of Δtau314 proteins in the striatum (caudate nucleus) and prefrontal cortex (Brodmann’s area 8/9) of human subjects, and showed that in both structures, levels of Casp2 and Δtau314 proteins correlate well, and both proteins are higher in HD patients than non-HD individuals. Our findings advance our understanding of the contribution of Casp2-mediated Δtau314 production to HD pathogenesis. Electronic supplementary material The online version of this article (10.1186/s40478-019-0764-9) contains supplementary material, which is available to authorized users.

Introduction
Huntington's disease (HD) is an autosomal-dominant and progressive brain disorder. The clinical manifestations of HD involve uncontrolled movements, psychiatric disturbance and cognitive decline (for review, see for example [39]); and HD is pathologically characterized by the gradual atrophy of and selective neuronal loss in the neostriatum (caudate nucleus and putamen) and cerebral cortex (for review, see for example [33]). HD is caused by an abnormal expansion of CAG trinucleotide repeats at the N-terminus of IT15 (interesting transcript 15) encoding the huntingtin (HTT) protein [15]. However, the molecular mechanisms through which the mutant HTT (mHTT) leads to the onset and development of HD are not fully understood (for review, see for example [18]).
The microtubule-associated protein tau (MAPT) is a highly soluble, unstructured molecule that is primarily present in the neurons of the central nervous system. The contribution of tau to HD pathogenesis involves, but is not limited to, the presence of aggregated tau inclusions in the brain of HD patients [7,9,11,17,23,25,26,31,38], the influence of MAPT haplotype in cognition of HD patients (patients with the H2 haplotype are more severely cognitively impaired than those with the H1 haplotype) [38], the amelioration of behavioral abnormalities of HD mice by genetic ablation of Mapt [11], and the mHTTmediated alterations in tau splicing isoform expression [11] and tau hyperphosphorylation [6,13]. Despite these findings, the exact tau species and the molecular mechanisms through which tau mediates HD pathogenesis are still largely unknown. The association of tau with cognitive dysfunction has been well established in a variety of mouse lines modeling multiple neurodegenerative diseases, and tau proteolysis and acetylation were shown to mediate cognitive impairment of animals [24,32,35,43,44].
In this study, we identified the presence of Δtau314 proteins in two HD-affected brain regions (caudate nucleus and prefrontal cortex (Brodmann's area (BA) 8/9)) of human subjects using conventional immunoprecipitation (IP)/Western blotting (WB) analysis. We showed that HD patients have higher levels of Δtau314 proteins as well as Casp2 than non-HD individuals. We also showed that levels of Casp2 and Δtau314 proteins are highly correlated. Results of this study, together with previous findings, suggest that Casp2-mediated tau cleavage to produce Δtau314 is a common pathologic mechanism that leads to synaptotoxicity in multiple neurodegenerative diseases.

Materials and methods
In vitro synthesis of human full-length tau and Δtau314 proteins Complementary DNA (cDNA) encoding the full-length tau 0N4R isoform was cloned into the pcDNA3.1(+) mammalian expression vector (Thermo Fisher Scientific, Waltham, MA). For synthesis of the truncated human tau protein Δtau314, the trinucleotides encoding amino acid residue leucine 315 were modified to a stop codon using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer's instructions. The following primers were used for the mutagenesis: forward primer, 5′ -AGTC TACAAACCAGTTGACTAGAGCAAGGTGACCTC-CAAG -3′; reverse primer, 5′ -CTTGGAGGT-CACCTTGCTCTAGTCAACTGGTTTGTAGACT -3′. Protein synthesis was performed using the TnT Coupled Reticulocyte Lysate Systems (Promega, Madison, WI) following the manufacturer's instructions.

Human brain collection
For the small cohort pilot study, frozen post-mortem specimens of dorsolateral prefrontal cortex (BA 8) from ten de-identified subjects-five HD patients and five non-HD individuals-were obtained from the Institute of Neurobiology Brain Bank (HUB-ICO-IDIBELL Biobank), Bellvitge University Hospital, Hospital del Llobregat, Spain.
For the large cohort study, frozen post-mortem specimens were obtained from the National Institutes of Health (NIH) NeuroBioBank and the New York Brain Bank (NYBB) at Columbia University, New York City, New York. Dorsolateral prefrontal cortex (BA 8) and caudate nucleus tissue from twenty-four de-identified subjects were provided by the NIH NeuroBioBank, and dorsolateral and medial prefrontal cortex (BA 9) tissue from twenty de-identified subjects were provided by NYBB. Tissue specimens from thirteen HD patients and fourteen non-HD individuals (see Additional file 1: Table  S1 for detailed demographic characteristics) were used in the study based on the following criteria: 1) the age at death, sex distribution and post-mortem interval (PMI) of brain tissue harvest of the HD patients are not significantly different from those of the non-HD individuals; 2) The ages of subjects are 50 years or older, and 3) the PMIs of brain tissue harvest are less than 22 h.
Frozen tissue was stored at − 80°C in either polypropylene bags or cryotubes prior to shipment to the University of Minnesota, Twin Cities, Minnesota.
All procedures were approved by the Institutional Review Boards (IRBs) of the HUB-ICO-IDIBELL Biobank (Spain), the Columbia University, the University of Miami, the Department of Veterans Affairs -Los Angeles and the University of Minnesota.

Brain protein extraction
Aqueous brain protein extracts were prepared based on a protocol previously published [34]. Specifically, human tissue specimens of 0.3-0.5 g were cut (or hemiforebrains of rTg4510 mice were dissected), transferred to 5 volumes (1 g per 5 mL) of ice-cold extraction buffer (25 mM tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl), pH 7.4; 140 mM NaCl and 3 mM KCl with the following protease and phosphatase inhibitors: 0.1 mM phenylmethylsulfonyl fluoride, 0.2 mM 1,10-phenanthroline monohydrate, a protease inhibitor cocktail (MilliporeSigma, Burlington, MA) and two phosphatase inhibitor cocktails (MilliporeSigma)), and homogenized using a Dounce homogenizer. The resulting material was centrifuged for 90 min (16,100 g, 4°C). The supernatant was subsequently depleted of endogenous immunoglobulin G (IgG) with Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Piscataway, NJ; 50 μL of slurry per 500 μL of sample), and stored at − 20°C. Protein concentrations of brain extracts were determined using a bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions.

Western blotting
Western blotting (WB) was performed according to a previously published protocol [21].
To measure levels of Casp2, 100 μg of proteins from the aqueous brain extracts were electrophoretically separated by SDS-PAGE using Criterion 10% Tris-HCl Precast gels (Bio-Rad), and then transferred onto nitrocellulose membranes. Blots were probed with rabbit monoclonal anti-Casp2 (Abcam, Cambridge, MA; 1: 5,000, final concentration = 496 ng/mL). Following 5 X 5-min washes in wash buffer, membranes were incubated at room temperature in HRP-conjugated anti-rabbit IgG (Thermo Fisher Scientific; 1:200,000, final concentration = 5 ng/mL) for 1 h. The membranes were then washed again (5 X 5min in wash buffer) prior to being developed.
To measure levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 20 μg of proteins from the aqueous brain extracts were electrophoretically separated by SDS-PAGE using Criterion 10% Tris-HCl Precast gels (Bio-Rad), and then transferred onto nitrocellulose membranes. Blots were probed with rabbit monoclonal anti-GAPDH (Cell signaling Technology, Danvers, MA; 1: 4,000, final concentration = 25 ng/mL). Following 5 X 5-min washes in wash buffer, membranes were incubated at room temperature in HRP-conjugated anti-rabbit IgG (Thermo Fisher Scientific; 1:200,000, final concentration = 5 ng/mL) for 1 h. The membranes were then washed again (5 X 5-min in wash buffer) prior to being developed.
Western blots were developed using the West Pico electrochemiluminescence detection system (Thermo Fisher Scientific) except for the blots probed with biotinconjugated 4F3 and anti-Casp2, which were developed using the West Femto detection system (Thermo Fisher Scientific). Signal intensities were quantified densitometrically using Optiquant (Packard Cyclone, Perkin-Elmer Life Sciences Inc., Boston, MA).
Experimenters performing IP/WB were blind to the demographic and clinical characteristics of subjects. The levels of proteins were determined from two independent experiments, and the mean levels were used for statistical analysis.

Statistical analysis
Statistical analyses were performed using GraphPad Prism Version 7.04 (GraphPad Software, La Jolla, CA) and R Version 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). The demographic and neuropathologic characteristics were compared between HD patients and non-HD individuals using two-tailed, unpaired Mann-Whitney tests for continuous variables and two-sided Fisher's exact tests for binary variables. The associations between demographic characteristics and levels of Δtau314 proteins (normalized to levels of T-tau proteins) were estimated using Spearman's rank-order correlations for continuous variables and Mann-Whitney tests for binary variables. Finally, levels of Δtau314, T-tau, Casp2 and GAPDH were compared between HD patients and non-HD individuals using two-tailed, unpaired Mann-Whitney tests. Additionally, multiple linear regressions of the log transformed levels were fit with adjustment for the potential confounders of age at death, sex and PMI. The unadjusted version of this analysis-a two-tailed, unpaired t-test performed on the log transformed levels-is also presented. P values of less than 0.05 were considered statistically significant.

Results
Levels of Δtau314 proteins are elevated in the prefrontal cortex of HD patients in a small cohort First, using a polymerase chain reaction (PCR)-based method previously published [40], we verified that the studied HD patients harbored > 35 CAG trinucleotide repeats in at least one allele, whereas the number of CAG repeats in the non-HD individuals did not exceed 35 in either allele (data not shown).
We then asked whether Δtau314 proteins were present in the prefrontal cortex of studied subjects. For this, we used 4F3 (Table 1), a monoclonal antibody that specifically targets truncated tau proteins ending C-terminally at D314, to detect Δtau314 proteins in the prefrontal cortex (BA 8) of five HD patients and five non-HD individuals, a cohort of the HUB-ICO-IDIBELL Biobank, Spain (Additional file 1: Table S1). Age at death, sex and PMI of brain tissue harvest in the HD patients and the non-HD individuals were matched (Additional file 1: Table S2).
We identified 4F3-reactive Δtau314 proteins in all the studied subjects (Fig. 1a). The levels of Δtau314 proteins were elevated 1.6-fold in the HD patients compared to the non-HD individuals (Fig. 1b, Additional file 1: Table S3), and trended higher following adjustments for age at death, sex and PMI of brain tissue harvest (Additional file 1: Table S3).

Levels of Δtau314 proteins are elevated in the prefrontal cortex of HD patients in a large cohort
We then attempted to confirm these findings in a larger cohort of subjects. Based on the quantitative analysis of 4F3-reactive Δtau314 proteins, we determined that the lowest sample size required for each group to achieve statistical significance under type I error α = 0.05 and power (i.e., (1-β)) = 0.80 is N = 11.
We measured levels of Δtau314 proteins in the prefrontal cortex (BA 8/9) of thirteen HD patients and fourteen non-HD individuals (Additional file 1: Table S1). Age at death, sex distribution and PMI of brain tissue harvest did not significantly differ between the HD patients and the non-HD individuals ( Table 2). We found that levels of Δtau314 proteins were 3.1-fold higher in the HD patients than in the non-HD individuals ( Fig. 1c and d, Table 3).
Levels of Δtau314 proteins are elevated in the caudate nucleus of HD patients in a large cohort Next, we measured levels of 4F3-reactive Δtau314 proteins in the caudate nucleus, a severely affected region, in a subset of the large cohorteleven HD patients and twelve non-HD individuals (Additional file 1: Table S1). Age at death, but not sex distribution or PMI of brain tissue harvest, was significantly different between the HD patients and the non-HD individuals (Table 4). We found that levels of Δtau314 proteins in the caudate were 5.6-fold higher in HD patients than in non-HD individuals ( Fig. 1e and f, Table 5).  To ascertain whether the increases in Δtau314 proteins was due to an increase in soluble total tau (T-tau) proteins, we measured the levels of T-tau proteins that were immunoprecipitated by tau-13 antibody, and detected by tau-5, a monoclonal antibody directed against a midregional epitope of tau (Table 1). We quantified levels of T-tau proteins in the prefrontal cortex and caudate nucleus of subjects in the large cohort, and in the prefrontal cortex of subjects in the small cohort. Interestingly, in the large cohort, levels of T-tau proteins in the prefrontal cortex of HD patients were 53% lower than in non-HD individuals ( Fig. 2a and b, Table 3), indicating that the elevated levels of Δtau314 proteins in HD patients were not due to an overall increase in T-tau proteins. The reduction in T-tau proteins in HD patients resulted in 5.3-fold increase in the ratio of Δtau314:T-tau in HD patients versus non-HD individuals (Fig. 2c, Table 3).
Similar to the prefrontal cortex, the levels of T-tau proteins in the caudate nucleus were 69% lower in HD patients than in non-HD individuals ( Fig. 2d and e, Table 5). Normalizing Δtau314 to T-tau proteins accentuated their elevated levels, resulting in 13.8fold higher levels in HD patients compared to the non-HD individuals (Fig. 2f, Table 5). As in the prefrontal cortex, the elevation of Δtau314 proteins in the caudate nucleus of HD patients is not due to an overall increase in T-tau proteins.
In the small cohort, the levels of T-tau proteins were comparable between the HD patients and non-HD individuals ( Fig. 2g and h, Additional file 1: Table S3). Our inability to detect a significant decrease in the level of T-tau proteins in the small cohort is likely due to the small sample size (N = 5/group). Based on the quantitative analysis of tau 5-reactive proteins in the prefrontal cortex of subjects in the large cohort, we determined that the lowest sample size required to achieve statistical significance under type I error α = 0.05 and power (i.e., (1-β)) = 0.80 is N = 82.
To further investigating how levels of Δtau314 proteins associate with levels of soluble T-tau proteins in the large cohort, we measured the levels of T-tau (T-tau (WB)) proteins revealed by direct WB probed with tau-5.
(See figure on previous page.) Fig. 1 Levels of Δtau314 proteins are elevated in HD patients. a, c, e Representative IP/WB showing that the tau-13-immunoprecipitated Δtau314 proteins (arrows) were detected by the biotin-conjugated 4F3 antibody in the prefrontal cortex (Brodmann's area (BA) 8) of subjects from the HUB-ICO-IDIBELL Biobank, Spain (a), in the prefrontal cortex (BA 8/9) of subjects from the NIH NeuroBioBank and the New York Brain Bank (c), and in the caudate nucleus of subjects from the NIH NeuroBioBank (e). HD, Huntington's disease patients; non-HD, individuals without Huntington's disease. +, in vitro synthetic Δtau314 proteins (hash; 20 μL of sample used in IP, positive control). *, non-specific bands. Sample IDs and disease diagnoses (Additional file 1: Table S1) were shown for IP reactions. Ab only, IP reaction containing only 10 μg of tau-13 (negative control). MsIgG/ br. ex., IP reaction containing 10 μg of mouse IgG and 173 μg of soluble brain proteins (negative control). For the MsIgG/br. ex. lanes in figure c, brain extracts were a mixture of the nine samples studied in the same blot with each contributing 1/9th the amount (i.e., 19.    The two-tailed, unpaired t-test with Welch's correction was performed on the log transformed outcomes c Multiple linear regression was used to analyze the log transformed outcomes of protein levels with adjustment for age at death, sex and PMI of brain tissue harvest These tau-5-detected proteins are not necessarily tau-13-immunoreactive. In the prefrontal cortex, levels of T-tau (WB) proteins in the HD patients were 34% lower than in the non-HD individuals (Additional file 1: Figure S1a and S1b, Table S4). As a result, the ratio of Δtau314:T-tau (WB) in HD patients was 3.8fold higher than in non-HD individuals (Additional file 1: Figure S2a, Table S4). Similarly, in the caudate nucleus, levels of the T-tau (WB) proteins were 44% lower in HD patients than in non-HD individuals (Additional file 1: Figure S1c and S1d, Table S5). Normalizing Δtau314 to T-tau (WB) proteins resulted in 21.9-fold higher levels in HD patients than in non-HD individuals (Additional file 1: Figure S2b, Table  S5). These findings further support that the higher levels of Δtau314 proteins in HD patients are not due to an increase in T-tau proteins.
To confirm that our findings in levels of Δtau314 and T-tau proteins are not due to difference in the amount of soluble brain proteins applied to IP/WB, we analyzed levels of the housekeeping protein GAPDH. We showed that the levels of GAPDH were comparable between HD patients and non-HD individuals in both prefrontal cortex and caudate nucleus (Additional file 1: Figure S3, Tables 3 and 5).
Lastly, we examined the relationships between demographic characteristics of subjects and levels of Δtau314 proteins in both the large and small cohorts. We found no apparent correlation of Δtau314 protein levels (normalized to levels of T-tau) with either ages at death or PMIs of brain tissue harvest (Additional file 1: Figure  S4a, S4b, S4d, S4e, S4 g and S4 h). Also, normalized Δtau314 protein levels were comparable between female and male individuals (Additional file 1: Figure S4c, S4f and S4i).

Levels of Casp2 are higher in the prefrontal cortex and caudate nucleus of HD patients
To understand the extent to which Casp2 is involved in production of Δtau314 proteins in HD, we measured levels of Casp2 detected by a monoclonal anti-Casp2 antibody directed against a C-terminal epitope (Table 1) in the prefrontal cortex and the caudate nucleus of subjects in the large cohort. Prior to the experiments, we validated the binding specificity of the anti-Casp2 antibody to the cleaved small subunit of recombinant Casp2 (data not shown).
In the prefrontal cortex, levels of Casp2 proteins, normalized to levels of the house-keeping protein GAPDH, were 1.7-fold higher in HD patients than in non-HD individuals ( Fig. 3a and b, Table 3). In the caudate nucleus, levels of Casp2 proteins, normalized to levels of GAPDH, were 2.5-fold higher in HD patients than in non-HD individuals ( Fig. 3d and e, Table 5). The finding that levels of both Δtau314 and Casp2 proteins are higher in HD patients than in non-HD individuals, in two distinct HD-affected brain structures, supports the active involvement of Casp2 in Δtau314 production in human subjects, particularly in HD patients.
In addition, levels of Casp2 (normalized to levels of GAPDH) correlated well with levels of Δtau314 (normalized to levels of T-tau) in both the prefrontal cortex (Fig. 3c) and the caudate nucleus (Fig. 3f ), which further supports an association between Casp2 and the production of Δtau314 proteins.

Discussion
In this study, we focused on understanding the potential role of Δtau314, a soluble, cognition-related tau protein species associated with Casp2, in HD. Using biochemical analyses, we detected Δtau314 proteins in the striatum (caudate nucleus) and prefrontal cortex (BA 8/9), areas that are especially vulnerable to HD pathology. We showed that levels of Δtau314 proteins are 1.6-to 5.6-   The two-tailed, unpaired t-test with Welch's correction was performed on the log transformed outcomes c Multiple linear regression was used to analyze the log transformed outcomes of protein levels with adjustment for age at death, sex and PMI of brain tissue harvest fold higher in both regions in HD patients compared to non-HD individuals, that levels of Casp2 are 1.7-to 2.5fold higher in HD patients than in non-HD individuals, and that levels of Casp2 and Δtau314 proteins are correlated in both studied brain structures. Our results are supported by a previous finding that the immunostaining intensity of Casp2, which catalyzes Δtau314 production, is more intense in degenerating caudate neurons of HD patients than in those of age-matched controls [16]. Also of note,~35-and~40-kDa tau protein species that match the migration pattern of some of the Δtau314 proteins in SDS-PAGE show a remarkably higher level in the cortex of HD patients than control individuals [11], though their exact identities are unclear in that study. We used IP/WB, rather than WB only, for identifying Δtau314 in soluble human brain homogenates because of the enhanced sensitivity and specificity. Our choice of using monoclonal antibody tau-13 as the capture reagent for immunoprecipitation in this study are based on two considerations: 1) the sensitivity of tau-13, and 2) the relevance of measured proteins to cognitive dysfunction and dementia. Specifically, we tried several widely used, commercially available antibodies, including mouse monoclonal tau-5, rabbit polyclonal K9JA, in addition to tau-13, to immunoprecipitate tau proteins from soluble human brain homogenates. We found that tau-13 produced the most sensitive results. For example, the~45-kDa Δtau314 protein that is derived from the longest tau isoform (2N4R) can only be detected using tau-13 as the capture reagent. In addition, since Casp2-catalyzed production of the tau-13-immunoreactive Δtau314 mediates synaptotoxicity in cellular models and cognition-impairing effects in mice, and levels of the tau-13-immunoprecipitated Δtau314 proteins are higher in subjects with mild cognitive impairment and AD than cognitively normal individuals [44], we focused on tau-13-immunoprecipitated Δtau314 proteins for this study. It is possible that other Δtau314 species exist in the brain, and that N-terminally truncated Δtau314 proteins may also contribute to the pathogenesis of neurodegenerative diseases.
Δtau314 proteins are among the reported posttranslationally modified tau species whose levels are elevated in AD subjects, compared to healthy individuals, and correlate with the severity of cognitive dysfunction in a variety of mouse lines modeling frontotemporal dementia [24,43,44]. Recently, we found higher levels of Δtau314 proteins in patients with Lewy body dementia than in non-demented Parkinson's disease patients (Smith et al. Acta Neuropathol. Commun. in press). These findings, taken with the current results, suggest that dementia may depend on Casp2-cleavage of tau in several neurodegenerative conditions.
We found elevated Δtau314 protein levels not only in the prefrontal cortex but also in the caudate nucleus of HD patients, a brain structure known to be involved in motor control, as well as some forms of cognition. Thus, whether Casp2-mediated Δtau314 production plays a role in a variety of behavioral abnormalities-both related and unrelated to cognition-in HD is an important question to be addressed in future studies.
Our findings of lower total tau levels in HD are superficially inconsistent with a previous finding of increased total tau levels in the frontal cortex of HD patients [11]. This discrepancy is likely due to different experimental methods in the two studies. Specifically, detergent-containing brain extracts were used in the previous study, while detergentfree, aqueous brain extracts were used in the present study. The detergent extracts likely contained solubilized tau aggregates, which would be absent in the aqueous extracts. In addition, monoclonal 5E2 antibody-reactive,~49-kDa tau proteins were quantified in the previous study, whereas tau-13-immunoprecipitated, tau-5-reactive,~30-55-kDa tau proteins were quantified in this study.
The number of CAG repeats has profound implications in both clinical phenotype and pathologic characteristics (See figure on previous page.) Fig. 2 Levels of soluble total tau proteins are lowered in HD patients of the large, but not the small, cohort. a, d, g Representative IP/WB showing that the tau-13-immunoprecipitated soluble total tau (T-tau) proteins (vertical bars) were detected by the biotin-conjugated tau-5 antibody in the prefrontal cortex (Brodmann's area (BA) 8/9) of subjects from the NIH NeuroBioBank and the New York Brain Bank (a), in the caudate nucleus of subjects from the NIH NeuroBioBank (d), and in the prefrontal cortex (BA 8) of subjects from the HUB-ICO-IDIBELL Biobank, Spain (g). HD, Huntington's disease patients; non-HD, individuals without Huntington's disease. +, in vitro synthetic full-length tau protein (hash; 20 μL of sample used in IP, positive control). Sample IDs and disease diagnoses (Additional file 1: Table S1) were shown for IP reactions. Ab only, IP reaction containing only 10 μg of tau-13 (negative control). MsIgG/br. ex., IP reaction containing 10 μg of mouse IgG and 173 μg of soluble brain proteins (negative control). For the MsIgG/br. ex. lanes in figure a, brain extracts were a mixture of the nine samples studied in the same blot with each contributing 1/9th the amount (i.e., 19.2 μg); for the MsIgG/br. ex. lanes in figure d, 1/8th the amount (i.e., 21.6 μg). b, e, h Comparison of levels of T-tau proteins in the prefrontal cortex (BA 8/9) of HD patients and non-HD individuals from the NIH NeuroBioBank and the New York Brain Bank (b), in the caudate nucleus, from the NIH NeuroBioBank (e), and in the prefrontal cortex (BA 8), from the HUB-ICO-IDIBELL Biobank, Spain (h). c, f Comparison of levels of Δtau314 proteins, normalized to levels of T-tau proteins, in the prefrontal cortex (BA 8/9) of HD patients and non-HD individuals from the NIH NeuroBioBank and the New York Brain Bank (c), and in the caudate nucleus, from the NIH NeuroBioBank (f). The numbers of analyzed subjects are shown in parentheses. A.U. = arbitrary unit. The y-axes in figures e, f and h are in log scale. Mann-Whitney test was used for between-group comparison; medians (middle long bars), and 1st (lower short bars) and 3rd (upper short bars) quartiles are shown of HD [2,29], and its relationship to the level of soluble Δtau314 proteins warrants further investigation. Specifically, it would be interesting to extend the detection and quantitative analysis of Δtau314 proteins to juvenile-onset HD patients, a population that often has significantly larger numbers of CAG repeats than the adult-onset patients and whose cognitive abilities are significantly impaired [27,30]. In this study, however, we chose to study HD subjects of 50 years or older at the age of death to avoid potential juvenile-onset HD-associated confounding factors since adult-and juvenile-onset HD patients manifest distinct clinical, pathologic and biochemical features [4,5,37], suggesting different pathophysiological mechanisms through which mHTT exerts toxic effects. Our finding of the association of HD with elevated levels of Δtau314 supports the potential use of Casp2 inhibitors to ameliorate neurological dysfunction in HD. Broad-spectrum inhibitors of caspases were shown to reduce toxicity in cellular models of HD [1,20,42], and ameliorate behavioral abnormalities in HD mice [28], indicating the disease-modifying effects of pharmacological blockage of caspase activities. In addition, using in silico structure-based design coupled with solid-state synthesis, a group of pentapeptide aldehyde compounds were recently developed, and some showed high affinity (IC 50 in nanomolar magnitude) and selectivity (60-fold difference in IC 50 between Casp2 and caspase-3) to Casp2 [22], drawing attention to the possibility of targeting Casp2 activity as a therapeutic strategy in HD.
There are several limitations to this study. First, we did not determine the precise numbers of CAG repeats in the subjects, and thus the relationship between the degree of CAG expansion and levels of Δtau314 (and Ttau) proteins is unknown. Second, to better understand the association of Δtau314 proteins with other neurological functions, it will be necessary to perform biochemical analyses of Δtau314 proteins in other brain structures (e.g., hippocampus and cerebellum). Third, an important question concerns the extent to which levels of Δtau314 proteins correlate with the severity of cognitive dysfunction. For the subjects of the large cohort study, eight of the thirteen HD patients had cognition assessment records, and all demonstrated cognitive deficits prior to death. Meanwhile, four of the fourteen non-HD individuals had cognition assessment records, and all demonstrated normal cognitive function in their last assessment (Additional file 1: Table S1). Despite these observations, we did not perform a comprehensive evaluation of the correlation between levels of Δtau314 proteins and the severity of cognitive deficits, because cognitive function assessments were lacking or not quantitatively provided for the other subjects, and different assessment approaches were used for those subjects whose cognitive functions were quantitatively measured. This is a question that may be addressed in humans and in animal models of HD in future studies. Fourth, the Casp2 protein band that we quantified using WB does not represent the fully active form. We identified only the~48-kDa protein band, likely representing the procaspase-2 form, which, upon dimerization, shows a partial catalytic activity [3]. The fact that no cleaved small subunit of Casp2 was detected in the studied brains suggests that the fully active Casp2 may constitute a minute portion of the total pool of Casp2, and levels of the fully active Casp2 are below the limit of detection of WB. To the authors' knowledge, assays that specifically measure Casp2 activity in the brain are not yet available; development of such an assay is a priority of our future studies.

Conclusions
In conclusion, Δtau314 proteins are present in the prefrontal cortex and caudate nucleus of human subjects, levels of Casp2 and Δtau314 proteins are correlated, and levels of both Casp2 and Δtau314 proteins are elevated in HD patients, compared to non-HD individuals. Findings of this study extend our understanding of the contribution of Casp2-mediated tau cleavage to HD pathogenesis, and support the development and exploration of Casp2 inhibitors in the treatment of HD.
(See figure on previous page.) Fig. 3 Levels of Casp2 are higher in HD patients of the large cohort. a, d Representative WB showing that Casp2 (~48-kDa full-length form, upper panels) and GAPDH (loading control, lower panels; revealed in the same blots as Casp2) were detected in the prefrontal cortex (Brodmann's area (BA) 8/9) of subjects from the NIH NeuroBioBank and the New York Brain Bank (a), and in the caudate nucleus of subjects from the NIH NeuroBioBank (d). HD, Huntington's disease patients; non-HD, individuals without Huntington's disease. -, Casp2 knock-out FVB/129S6 mice (negative control); +, wild-type non-transgenic FVB/129S6 mice (positive control). Sample IDs and disease diagnoses (Additional file 1: Table S1) were shown. b, e Comparison of levels of Casp2 in the prefrontal cortex (BA 8/9) of HD patients and non-HD individuals from the NIH NeuroBioBank and the New York Brain Bank (b), and in the caudate nucleus, from the NIH NeuroBioBank (e). The numbers of analyzed subjects are shown in parentheses. Mann-Whitney test was used for between-group comparison; medians (middle long bars), and 1st (lower short bars) and 3rd (upper short bars) quartiles are shown. c, f Correlation of levels of Casp2 (normalized to the house-keeping protein GAPDH) and levels of Δtau314 proteins (normalized to levels of T-tau proteins) in the prefrontal cortex (BA 8/9) of HD patients and non-HD individuals from the NIH NeuroBioBank and the New York Brain Bank (c), and in the caudate nucleus, from the NIH NeuroBioBank (f). Spearman's rank-order correlation was used. The y-axes in figures c and f are in log scale. A.U. = arbitrary unit


extfieldsofstudy: Medicine Engineering

s2fieldsofstudy:
Biology
Materials Science

text:
Electrospraying: Possibilities and Challenges of Engineering Carriers for Biomedical Applications—A Mini Review

Electrospraying, a liquid atomization-based technique, has been used to produce and formulate micro/nanoparticular cargo carriers for various biomedical applications, including drug delivery, biomedical imaging, implant coatings, and tissue engineering. In this mini review, we begin with the main features of electrospraying methods to engineer carriers with various bioactive cargos, including genes, growth factors, and enzymes. In particular, this review focuses on the improvement of traditional electrospraying technology for the fabrication of carriers for living cells and providing a suitable condition for gene transformation. Subsequently, the major applications of the electrosprayed carriers in the biomedical field are highlighted. Finally, we finish with conclusions and future perspectives of electrospraying for high efficiency and safe production.

INTRODUCTION
Electrospraying, also known as electrodynamic spraying, is capable of producing diminutive droplets with submicron sizes by means of an electric field (Khan et al., 2017). Electrospraying can also be used to produce fine polymeric particles, which are widely used for biomedical applications, particularly drug encapsulation. For the purpose of polymeric particle production, the common setup of electrospraying consists of a high-voltage power supply, a plastic/glass syringe capped by a metallic capillary (e.g., a 16-to 26-gauge needle) to hold a polymer solution, a syringe pump to control the flow of the solutions, and a grounded collector. When a high electric field is applied at the needle, a charged liquid jet will break up into droplets, which form small particles with generally narrow size distribution on the collector; the general setup can be found in Figure 1. The size and morphology of electrosprayed particles can be controlled to a certain extent by the factors related to polymer solutions [e.g., concentration, shear viscosity , polymer molecular weight, solvent (Bohr et al., 2015;Felice et al., 2015;Lu et al., 2015)] and electrospraying process [e.g., flow rate, electric potential difference, distance between the tip of the needle and the collector (Smeets et al., 2017)]. More details about the setup of electrospraying and its physical operating conditions can be found in other reviews (Bock et al., 2011;Ganan-Calvo et al., 2018;Jaworek et al., 2018).
Compared to the other conventional methods to produce particles, electrospraying has a few advantages, making it attractive to produce cargo carriers for biomedical applications. Firstly, the process can be performed at ambient conditions (temperature and pressure), which is beneficial for sensitive biomolecules and even living cells; secondly, due to the possible absence of an external medium that allows the dissolution or migration of water-soluble cargos, the encapsulation efficiency using electrospraying can be maximized (Sosnik, 2014). Thirdly, it can reproducibly provide drug-loaded nano/microparticles (5 nm−100 µm) with a narrow size distribution (Chen and Pui, 1997); Coulombic repulsion between the highly charged electrosprayed droplets results in selfdispersion particles without coalescence (Xu and Hanna, 2006). Last, but not least, through adjustment of the above-mentioned solution factors and processing parameters, carriers in different structures can be obtained, such as hollow microspheres (Jafari-Nodoushan et al., 2015;Zhou et al., 2017), nanocups (Kiran et al., 2016), porous microcarriers (Gao et al., 2015;Karimian et al., 2016;Huang et al., 2017), cell-shaped microparticles (Khanum et al., 2015;Ju et al., 2017), core-shell/multilayered microspheres (Rasekh et al., 2017;Zhang et al., 2017a), and Janus particles (Sanchez-Vazquez et al., 2017;Li et al., 2018). The schematic diagram of the structures can be found in Figure 1, and the real images can be found in the referred publications.
In the past decades, there are several comprehensive reviews published about electrospraying from different perspectives, including electrohydrodynamic technique, nature material-based systems, type of encapsulated drugs, and its applications (food, drug delivery, bone tissue engineering, etc.; Bock et al., 2012;Jayaraman et al., 2015;Arzi and Sosnik, 2018;Jacob et al., 2018;Jacobsen et al., 2018;Pawar et al., 2018;Rosell-Llompart et al., 2018). Recently, this technique continued to evolve in the field of bioengineering. Bioactive compounds, as well as cells, have been electrosprayed apart from conventional drugs. This mini review focuses on the novel bioactive cargo carriers prepared by electrospraying technique and the conditions used to maintain the bioactivity during the process. Then, we summarize the most recent development of this technology in biomedical applications regarding drug delivery, diagnostic and therapeutic biomedical imaging, implant coating, and tissue engineering. Figure 1 gives an overview of this mini review.

BIOACTIVE CARGOS ELECTROSPRAYED INSIDE CARRIERS
Due to the special character of bioactive cargos which is sensitive to the ambient environment, it has been a challenge to fabricate the bioactive cargo-loaded carrier. Here, we highlight the technical aspect of loading different bioactive cargos in terms of cells, genes, growth factors, and enzymes via the electrospraying method. Small-molecule drugs are not included in this section, which have been reviewed before (Nguyen et al., 2016).

Cells
Electrospraying has been explored to handle living cells and whole organisms (Jayasinghe, 2011), which is now known as "bioelectrospraying" (BES). BES has become an appealing tool for cell delivery in scaffolds for tissue engineering applications. BES is considered safe for cells due to the low current (usually in the nanoampere range) albeit the high voltage up to several kilovolts. Recently, a variety of cells have been electrosprayed, including fibroblasts, adipose-derived stem cells (ADSCs), periodontal ligament cells, retinal pigment epithelial cells, umbilical vascular endothelial cells, gastric epithelial cells (Xin et al., 2016), and bone marrow-derived mesenchymal stem cells (McCrea et al., 2018). The overall results demonstrated no distinct negative effect of the electrospraying process on cell vitality, morphology, and proliferation. This confirms the safety and efficiency of BES to encapsulate cells into different carriers and extracellular matrix for the study of diverse diseases.
Many polymers have been proven to be compatible with BES, including natural polymers subject to ionotropic or physical gelation (e.g., alginate, chitosan) (Qayyum et al., 2017) and some synthetic hydrogels [e.g., polyethylene glycol (PEG)]. The achieved cell encapsulation efficiency in PEG can be up to 90% (Qayyum et al., 2017). Cell aggregation was observed within the PEG hydrogel microspheres due to the lack of cell attachment site of PEG. Arg-Gly-Asp-Ser peptide-tethered PEG showed improved cell attachment, and the microencapsulated cells remained viable in the tethered PEG hydrogel microspheres for up to 7 days. Esfahani et al. (2017) encapsulated cells into semipermeable poly(lactide-co-glycolide) (PLGA) microspheres to release biologicals produced by the encapsulated cells. A coaxial system was adopted to avoid the toxicity of the solvent (chloroform/dimethylformamide) used to dissolve PLGA. Consequently, the cells and PLGA solution were only in contact at the tip of the needle before electrospraying.
BES has also been combined with electrospinning to obtain cellular tissue constructs. Yunmin et al. (2015) developed a micro integration scaffold by simultaneously electrospraying ADSCs and electrospin polyvinyl alcohol (PVA). Compared to scaffolds prepared by electrospinning a solution of ADSCs suspended in PVA, the integrated electrospraying/electrospinning one showed a larger number of surviving cells.
For further development, the BES parameters, such as cell concentration, nozzle size, polymer flow rate, voltage, and distance between the needle and the collector, need to be optimized. For example, voltage is very critical as it affects the cell vitality. Therefore, a low voltage is preferred (3-6 kV; McCrea et al., 2018). Ye et al. (2015) found that ADSCs were still viable when an electrospraying voltage of 10 kV was used, whereas cell viability became reduced at 20 kV and the spraying process became unstable.
In summary, BES extends the available toolkit for cell microencapsulation, and integration with other techniques, such as electrospinning, provides the possibility to form simultaneously complex living three-dimensional (3D) architectures for potential applications in regenerative medicine (Jayasinghe et al., 2015).

Genes
Gene therapy is developing rapidly as a new treatment strategy for cellular, tissue, and organ disorders, ranging from diabetes to cancer (Ward et al., 2010). To transform bacterial cells with exogenous DNA, chemo-transformation and electroporation are commonly used. However, these methods are complex and expensive, and the step of preparing competent cells is timeconsuming (Abyadeh et al., 2017). Electrospraying of DNA plasmid (pDNA) in a suitable buffer (such as high-conductivity culture medium) had been suggested as an alternative for bacterial/cell transformation with high efficiency (Boda et al., 2018). Abyadeh et al. pioneered a rapid transformation method to electrospray chitosan/pDNA into a bacterial culture to evoke a mild damage to the cell surface to increase the penetration of plasmid into bacteria. The transformation efficiency of this method was 1.7 × 10 8 CFU/µg plasmid, which is comparable to the traditional methods (Abyadeh et al., 2017).

Growth Factors
Growth factor delivery with good efficiency and efficacy is still a challenge for tissue regeneration therapies. As mentioned above, electrospraying has great benefit of reducing growth factor denaturation due to the limited exposure to organic solvents and the possibility of the dry encapsulation method (Bock et al., 2016). Zhang et al. (2017b) prepared rhBMP-2loaded PLGA microspheres with a core-shell structure via the coaxial electrospraying method and the microspheres promoted cell proliferation of bone marrow stromal cells. In order to assist growth factor bioactivity, stabilizer PEG and trehalose were added to PLGA to prepare vascular endothelial growth factor (VEGF)-or bone morphogenetic protein 7 (BMP-7)loaded carriers via electrospraying (Bock et al., 2016). Growth factor bioactivity was verified, when tested with cells, at all stages of microparticle preparation (including protein aggregation and contact with an organic solvent) and in the presence of a stabilizer in the formulation. Moreover, pre-osteoblasts (MC3T3-E1) directly co-cultured with the BMP-7-loaded carriers showed significant cell differentiation into osteoblasts. In a dual growth factor release system, double-layered microspheres were prepared by a two-step electrospraying . The inner layer of microspheres was first fabricated by electrospraying BMP-2/alginic acid sodium salt solution to a dish filled with CaCl 2 solution to form alginate microspheres. After being coated with chitosan, the microspheres were mixed with stromal cellderived factor-1 (SDF-1) in alginic acid sodium salt solution to go through a second electrospraying process to form the outer layer of the microspheres. The retention of bioactivities of both growth factors after the electrospraying process was confirmed.

Enzymes
A few studies explored the possibility of using electrospraying to encapsulate enzymes. Fung et al. (2016) electrosprayed coenzyme Q10 in copovidone (Kollidon R VA64) using acetone as the solvent. Both in vitro and in vivo evaluation showed enhanced bioavailability of the electrosprayed microparticles. Compared with the physical mixture of raw materials, electrosprayed microparticles revealed also enhanced dissolution properties. Yaghoobi et al. (2017) electrosprayed streptokinase-loaded PLGA nanoparticles, but found the activity of streptokinase decreased to 19.2% after electrospraying. Therefore, more research has to be done to maintain the biological activity of the enzyme during the electrospray process.

APPLICATION OF THE ELECTROSPRAYED CARRIERS IN BIOMEDICINE
Electrosprayed cargo carriers have been widely explored for various biomedical applications. In this part, the most recent applications related to electrosprayed carriers were summarized to follow the newest trend of this technique.

Drug Delivery
Electrospraying has been widely studied to prepare and encapsulate drugs in particles that can function as a drug delivery system. This topic has been covered by the other reviews (Nguyen et al., 2016;Boda et al., 2018;Pawar et al., 2018). In Table 1, we summarize the most recent, published from year 2015 onward, drug delivery systems developed by electrospraying to achieve particular therapeutic effects.

Diagnostic and Therapeutic Biomedical Imaging
Electrospraying has been combined with the sol-gel synthesis technique to prepare ternary phosphate-based glass nanospheres, which can be used as contrast agent for ultrasound imaging (Foroutan et al., 2015). These nanospheres are not cytotoxic at the used doses, and their function has been confirmed in vivo. The nanospheres also degrade in aqueous media, and the degradation products are easily metabolized in the body. Taking a step ahead, Rasekh et al. (2017) applied coaxial electrospraying to encapsulate genistein (model drug), superparamagnetic iron oxide nanoparticles (10-15 nm), PEG, and a fluorescent dye in triglyceride tristearin, and demonstrated a drug release time of over 30 h. The stable process and high drug encapsulation efficiency (around 92%) make coaxial electrospraying a promising choice to encapsulate nanoparticles together with sensitive drugs for combined imaging and therapeutic application.
In another study, Zhang et al. (2017a) engineered particles with combined diagnostic and therapeutic functions using a trineedle coaxial electrospraying. The particles were constructed with a core-shell structure separated by an oil layer. Magnetic Fe 3 O 4 nanoparticles were embedded in the polymeric shell to enable both ultrasound and magnetic resonance imaging capacity. Meanwhile, therapeutic drugs could be incorporated in both the core and the shell compartments and their release could be regulated by an external auxiliary magnetic field. A similar study was performed by electrospraying Janus particles composed of [PLGA/TbLa 3 (Bim) 12 ]/[PLGA/Fe 3 O 4 ] . These Janus particles showed good magnetic properties, thermal stability, biocompatibility, and enhanced fluorescent properties, displaying their potential use for biological probing and biomedical imaging.

Implant Coating
As mentioned in the Introduction, electrospraying is a versatile technique that can produce an aerosol of charged droplets with precise control of size and shape. This feature can be used to produce different morphologies of polymeric coatings on medical implants, which can guide cellular functions at the cell-implant interface. Biodegradable polyurethane with tailored microtopography was electrosprayed onto commercial coronary stents (Guo et al., 2015). The authors found the topography of coating could be manipulated by tuning the processing conditions, which influences Coulombic fission of the electrosprayed droplets. Li et al. (2017a) prepared heparin (an effective natural anticoagulant)-loaded polycaprolactone/PEG microspheres by coaxial electrospraying to coat blood vessel substitutes. Heparin could maintain its activity and sustained release for 15 days under the protection of the shell layer. This coating was able to prevent platelet adhesion and blood coagulation (Li et al., 2017a).
Montelukast is a selective cysLT 1 receptor antagonist and able to preserve the proliferation and migration of coronary artery endothelial cells (Zamani et al., 2016). Drug-eluting stents coated by electrosprayed montelukast-loaded PLGA was developed to inhibit the formation of neointimal hyperplasia (Zamani et al., 2016).
As the conventional polymer coatings may lose mechanical properties after implantation, novel poly (polyol sebacate)derived polymers have been explored to be used as the electrospraying coating material for metallic stents (Navarro et al., 2016). The coating can be tuned from flexible to rigid and shows no cytotoxicity on ADSCs.

Tissue Engineering
Electrosprayed particles have been widely used as biomolecule carriers for tissue regeneration, particularly bone tissue engineering. In most studies, the electrosprayed carriers were implanted into the defects or combined with a 3D scaffold. Modified coaxial electrospraying was used to prepare carriers consisting of a shell (PLGA with VEGF for angiogenesis) and core (PLGA with BMP-2 for osteogenesis; Wang et al., 2015). The obtained carriers showed an initial burst release of VEGF and a sustained release of BMP-2 with maintained bioactivity. An in vivo experiment in a rat cranial bone defect model demonstrated that growth factor-loaded spheres enhanced significantly new bone formation. In another study, nanoparticles of cartilage-specific proteins, e.g., collagen type II, hyaluronic acid, and chondroitin sulfate, were developed by electrospraying for articular cartilage repair (Yang et al., 2018). Those nanoparticles could be taken up by chondrocytes via nonspecific pinocytosis, and the gene expression of collagen type II, aggrecan, and transforming growth factor beta 1 was up-regulated, suggesting enhanced chondrocyte functionality. In another study, hydroxyapatite and calcium-deficient hydroxyapatite particles were synthesized via a sol-gel-assisted electrospraying process (Chakrapani Venkatesan et al., 2018). The calcium-deficient hydroxyapatite particles tend to display the capability to differentiate rBMSCs into the osteogenic lineage. They showed a better drug loading and release compared to the microwave-synthesized particles, with the advantages in both osteogenic differentiation and drug release for bone tissue engineering.
To enhance bone regeneration, electrospraying was combined with electrospinning to prepare a nanofibrous structured bone graft substitute. Zhu et al. (2015) used this combined fabrication method for neural tissue regeneration and engineered a highly aligned polycaprolactone microfibrous framework with embedded PLGA core-shell nanospheres for bioactive factor encapsulation. The released bioactive factor promoted rat pheochromocytoma cell proliferation and the highly aligned scaffold directed neurite extension along the fibers.

CHALLENGES AND POSSIBILITIES FOR ELECTROSPRAYED CARRIERS
Electrospraying is capable to produce fine carriers with controlled shapes/sizes and high encapsulation efficiency. It is not a complex biological or chemical modification but involves delicate engineering and material manipulation (Naqvi et al., 2016). From a technical perspective, it is a rapid, single-step approach to prepare carriers for biomedical use. However, the upscaling of this process needs a lot of investigation. To increase the preparation efficiency, Jordahl et al. (2017) invented a novel preparation process using a needleless apparatus with two parallel glass plates with narrow spaces in between (0.35 mm) as microchannels for electrospraying fluid. The plate edge at the outlet of the microchannels was sharpened and grooved to aid in the fluid flow. Multiple spraying jets were formed after application of electric potential, resulting in a very high production rate. In the other report, Zhang et al. (2015) described a flute-like multipore emitter device to replace conventional electrospraying capillaries for large-scale production. In BES, Zhang et al. (2016) designed a customized multihole spinneret that could produce continuous, stable jets with a five to seven times increased efficiency without affecting morphology, viability, and proliferation of human umbilical vascular endothelial cells.
Another limitation is the organic solvents used during electrospraying that may damage the bioactivity of genes, enzymes, and cell vitality. "Green electrospraying, " which makes use of benign or aqueous solvents, would be the alternative to reduce toxicity (Agarwal and Greiner, 2011). Also, BES gives us an opportunity to encapsulate cells directly in scaffolds. This technique also has great potential to be used for organs-onchip, which are used to mimic the real organ to replace part of animal experiments.
In conclusion, electrospraying is a versatile technique to prepare polymeric carriers for genes, drugs, proteins, enzymes, growth factor, and cells. The application of multiple needles to improve the spraying process brings the technology closer to commercial production. Without, the use of organic solvents, green electrospraying and BES both allow the application for tissue engineering.

AUTHOR CONTRIBUTIONS
JW wrote the manuscript. JJ and FY helped to revise the manuscript.