text:
The high molecular weight dipeptidyl peptidase IV Pol d 3 is a major allergen of Polistes dominula venom

Hymenoptera venom allergy can cause severe anaphylaxis in untreated patients. Polistes dominula is an important elicitor of venom allergy in Southern Europe as well as in the United States. Due to its increased spreading to more moderate climate zones, Polistes venom allergy is likely to gain importance also in these areas. So far, only few allergens of Polistes dominula venom were identified as basis for component-resolved diagnostics. Therefore, this study aimed to broaden the available panel of important Polistes venom allergens. The 100 kDa allergen Pol d 3 was identified by mass spectrometry and found to be a dipeptidyl peptidase IV. Recombinantly produced Pol d 3 exhibited sIgE-reactivity with approximately 66% of Polistes venom-sensitized patients. Moreover, its clinical relevance was supported by the potent activation of basophils from allergic patients. Cross-reactivity with the dipeptidyl peptidases IV from honeybee and yellow jacket venom suggests the presence of exclusive as well as conserved IgE epitopes. The obtained data suggest a pivotal role of Pol d 3 as sensitizing component of Polistes venom, thus supporting its status as a major allergen of clinical relevance. Therefore, Pol d 3 might become a key element for proper diagnosis of Polistes venom allergy.

between different hymenoptera species such as Vespula spp. and Polistes spp. 13 . Therefore, clinicians depend on additional diagnostic tests.
The increased knowledge of the composition of hymenoptera venoms has led to major improvements in diagnostic approaches and created the field of molecular or component-resolved diagnostics (CRD) in hymenoptera venom allergy 4,14,15 . In combination with skin testing and venom extract-based specific IgE (sIgE) diagnostics, CRD has created added clinical value for a proper allergy diagnosis. For CRD of hymenoptera venom allergy commercial allergens are available for different test platforms to determine sIgE serum titers 6 .
For the diagnosis and discrimination of honeybee venom (HBV)-and yellow jacked venom (YJV)-allergic patients, many commercial allergens are available, allowing for high diagnostic sensitivity and specificity [16][17][18][19][20][21][22] . However, for the diagnosis of PDV allergy only one allergen (antigen 5, Pol d 5) is commercially available. Furthermore, only three allergens of PDV have been identified in the past, namely phospholipase A1 (Pol d 1) 23 , protease (Pol d 4) 24 and antigen 5 (Pol d 5) 25 from which Pol d 4 is a minor allergen with restricted diagnostic importance (unpublished data). Even though, we have recently shown, that Polistes venom is free of cross-reactive carbohydrate determinant-(CCD-)based cross-reactivity 26 , available extract-based diagnostic approaches to discriminate between PDV und YJV allergy are hampered by extensive protein cross-reactivity 2,3,27,28 .
An increased knowledge of the allergen composition of PDV and the availability of further important PDV components are likely to generate added clinical benefit for proper and advanced diagnostics. Therefore, our study aimed to identify and immunologically characterise additional major allergens of PDV. The here described allergen Pol d 3 is a homologue of the prominent dipeptidyl peptidase IV (DPP IV) allergens Api m 5 and Ves v 3 from HBV and YJV 29 . Extensive sIgE reactivity and the ability to activate basophils from allergic patients clearly support the role of Pol d 3 as major allergen of PDV as well as its potential to be a key element for molecular diagnostic approaches.

Identification of Pol d 3.
Immunoblots using PDV and pooled sera of PDV-allergic patients (n = 5) revealed sIgE reactivity with several venom components (Fig. 1). Prominent bands at approximately 23 and 31 to 40 kDa were identified by mass spectrometry as the known allergens antigen 5 (Pol d 5), protease (Pol d 4) and phospholipase A1 (Pol d 1), respectively. Moreover, prominent sIgE reactivity was obtained with a high molecular weight protein of approximately 100 kDa. The protein was subjected to de novo sequencing by tandem mass spectrometry. The five identified peptides (Fig. 2) yielded hits in a database search of predicted proteins coded by the recently published genome of Polistes dominula 30 and identified the sIgE-reactive protein as venom dipeptidyl peptidase IV (GenBank accession XP_015174445). An additional matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of the protein band then led to a sequence coverage of approximately 44% with the predicted sequence.
The nucleotide sequence (GenBank accession XM 015318959) of the newly identified allergen codes a mature protein of 751 amino acids and a calculated molecular weight of 86.2 kDa. The discrepancy between the calculated and the apparent molecular mass most likely is due to posttranslational modification by glycosylation as suggested by the presence of 4 or 3 N-linked glycosylation sites based on the binary profile or on average surface accessibility, respectively 31 . The protein belongs to the dipeptidyl peptidase IV superfamily, known to cleave dipeptides from the N-terminus of peptidic substrates, including many chemokines, neuropeptides, peptide hormones and venom peptides 32,33 . Therefore, it is a homologue of the well-established allergens Api m 5 (HBV) and Ves v 3 (YJV) 29 . The identity on protein level with Api m 5 and Ves v 3 is 54.0% and 76.6%, respectively. According to its allergenic properties and the homology to YJV Ves v 3 the new PDV allergen was assigned as Pol d 3.1010 in the WHO/IUIS Allergen Nomenclature Database 34 .

Recombinant expression and characterisation of Pol d 3.
For recombinant expression, the coding region of Pol d 3 was amplified from PDV gland cDNA. Recombinant production was achieved by baculoviral infection of Sf9 insect cells and Ni 2+ -affinity chromatography yielded soluble recombinant Pol d 3 from culture supernatants with an apparent molecular weight of approximately 100 kDa as shown by Coomassie staining and reactivity with an antibody reacting with the V5-epitope tag added for recombinant expression (Fig. 3). Moreover, correct folding of the allergen was supported by enzymatic DPP IV activity ( Supplementary Fig. S1) as well as by circular dichroism (CD) spectroscopy ( Supplementary Fig. S2). For comparable analyses the homologous allergens from HBV and YJV, Api m 5 and Ves v 3, were produced as described previously 29 (Fig. 3).
Furthermore, staining with a polyclonal rabbit Api m 5 antiserum shows reactivity with Api m 5 but also with Ves v 3 and Pol d 3, suggesting cross-reactivity between the three DPP IV hymenoptera allergens. Cross-reactivity was confirmed by sIgE-reactivity of pooled sera of PDV-allergic patients with all three allergens (Fig. 3).

Activation of basophils from venom-allergic patients by Pol d 3.
To address the capability of Pol d 3 to cross-link receptor-bound IgE and, thus, to activate effector cells, basophil activation tests (BATs) were performed ( Fig. 4) as previously described 28,38 . First, 13 patients from Spain (from the area of Barcelona) with history of an allergic reaction to PDV and/or YJV were analysed for their reactivity with Pol d 3 and Ves v 3 (Fig. 4a). Three patients showed basophil activation only by Pol d 3 (patients 3,7,8), two only by Ves v 3 (patients 1, 11) and four by both allergens (patients 4, 9, 10, 13). Interestingly, for most of the patients these data nicely match the data of skin tests and/or sIgE levels to the venom extracts (i3, i77) and allergen components (i209, i210, i211)  (Table 1). Therefore, for most patients BATs with the DPPs IV would have been able to identify the insect(s), the patient most likely shows primary sensitization to. For patient 4, showing stronger basophil activation by Pol d 3 compared to Ves v 3, but higher sIgE levels to YJV compared to PDV, clinical information is too scarce to identify the allergy-eliciting venom. Moreover, patient 13 exhibits basophil activation by both allergens but much higher sIgE level to PDV compared to YJV extract and a negative skin test to YJV. However, for this patient basophil activation by Pol d 3 is much stronger compared to Ves v 3, and Ves v 3-reactivity is most likely due to cross-reactivity. For patients 9 and 10 who show comparable activation patterns by both allergens, also clinical data suggest allergy to both venoms.
In order to address cross-reactivity of Pol d 3 in BAT, 15 patients from Germany (from the area of Munich) with allergy to YJV and/or HBV were analysed for their reactivity with Pol d 3, Api m 5 and Ves v 3 (Fig. 4b). Five patients showed basophil activation by Pol d 3 (patients 15,16,18,19,28) six by Api m 5 (patients 15,17,18,21,27,28) and seven by Ves v 3 (15,18,19,20,24,26,28). Again, in most cases activation patterns by Api m 5 and Ves v 3 match sIgE and skin test data, indicating allergy to HBV, YJV or both ( Table 1). Three of the Pol d 3-reactive patients, showed basophil activation by all three allergens (patients 15,18,28) and one by Pol d 3 and Ves v 3 (patient 19). Only patient 16 showed exclusive basophil activation by Pol d 3, but the overall activation (including the positive control) was very low.  Table S1). For the remaining 6 patients (10,11,15,23,26,30) diagnostic results were less clear and, hence, allergy to PDV and YJV cannot be excluded. In the group of Spanish patients 20/30 (66.7%) exhibited pronounced sIgE-reactivity with Pol d 3 (Fig. 5a).
In order to address sIgE cross-reactivity of Pol d 3 in HBV-and YJV-allergic patients from Germany (from the area of Munich), reactivity with the recombinant allergen was assessed. The patient groups were selected either for primary sensitization to HBV or YJV by detailed clinical characterisation (Supplementary Table S1). Nevertheless, in these groups allergy to both species cannot be fully excluded. However, since PDV allergy in Germany is virtually not present, primary sensitization to PDV can be excluded with high probability. 9/28 (32.1%) of HBV-and 14/22 (63%) of YJV-allergic patients showed reactivity with Pol d 3, respectively (Fig. 5a).  Interestingly, most of the HBV-and YJV-allergic patients with sIgE to Pol d 3 also exhibited sIgE to the homologous allergens from HBV (Api m 5) (Fig. 5b) and YJV (Ves v 3) (Fig. 5c), respectively. Only for one patient with HBV allergy and for 3 YJV-allergic patients with very weak reactivity to Pol d 3, reactivity with the homologous allergens was slightly below the cut-off of the ELISA.

Discussion
Polistes dominula is one of the main elicitors of hymenoptera venom allergy in Southern Europe as well as in parts of the United States. Moreover, Polistes dominula is a very invasive species spreading from the warmer to the more moderate climate zones and is therefore likely to gain importance also in other areas. However, compared to other species such as Apis mellifera or Vepula vulgaris, the knowledge of the composition of important PDV allergens on a molecular level is restricted. Additionally, to date the availability of diagnostic tools for the discrimination between PDV and YJV allergy is very limited. Therefore, our study aimed to identify and immunologically characterise novel important allergens of PDV to extend the available repertoire of venom allergens for analyses on a molecular level.
In this study, we were able to identify the sIgE-reactive 100 kDa allergen of PDV as dipeptidyl peptidase IV and homologue of the well-established DPP IV allergens Api m 5 and Ves v 3 of HBV and YJV, which were shown to represent relevant allergens of clinical importance 18,29,39 . Protein sequence identity with the corresponding allergens of HBV and YJV is 54% and 76.1%, respectively. Due to its allergenic properties and homology to the YJV allergen Ves v 3 the new PDV allergen was assigned as Pol d 3.1010 to the IUIS/WHO allergen nomenclature database 34 .
In order to immunologically characterise Pol d 3, the protein was recombinantly produced as soluble and properly folded protein in Sf9 insect cells. Lectin-staining confirmed that Pol d 3 is a glycoprotein, a fact that explains the difference between the calculated molecular mass of the polypeptide chain of 86.2 kDa and the apparent molecular mass of approximately 100 kDa observed in SDS-PAGE and immunoblot analyses. However, recombinant Pol d 3 was devoid of CCD-reactivity as shown previously for other allergens produced in Sf9 insect cells 29,[35][36][37] . Intriguingly, the reactivity of Pol d 3 with a polyclonal Api m 5-specific antiserum 40 hinted to a pronounced protein-based cross-reactivity of the DPP IV allergens of the different hymenoptera species. This cross-reactivity was further confirmed by the reactivity of a serum pool from Pol d 3-reactive PDV-allergic patients with the DPP IV allergens Api m 5 and Ves v 3. To access the relevance of Pol d 3 as allergen, sIgE-reactivity of PDV-allergic patients with this newly identified PDV component was addressed. Thereby, over 66% of PDV-allergic patients exhibited pronounced sIgE to Pol d 3. This is in a comparable range found for the reactivity of either HBV-or YJV-allergic patients with the corresponding DPP IV allergens Api m 5 and Ves v 3, respectively. In previous studies, it was demonstrated that 58.3% to 61.7% of HBV-allergic patients and 57% of YJV-allergic patients show sIgE to Api m 5 and Ves v 3, respectively 18,29,39 . These data clearly suggest that Pol d 3 represents a major allergen of PDV. As Polistes dominula and Vespula vulgaris coexist in Spain and are difficult to discriminate, systemic reactions due to both insects cannot be excluded in the Spanish patient population.
Additionally, 32% of HBV-and 63% of YJV-allergic patients exhibited sIgE to the new PDV allergen. The majority of these patients additionally showed reactivity with the homologous allergen of HBV (Api m 5) or YJV (Ves v 3), indicating that the sIgE to Pol d 3 is a result of extensive protein-based cross-reactivity. Moreover, the lower degree of cross-reactivity between Pol d 3 and Api m 5 compared to Pol d 3 and Ves v 3 most likely reflects the lower sequence identity between the PDV and HBV allergen and, thus, of less conserved IgE epitopes.
So far phospholipases A1 (Pol d 1 and Ves v 1) and antigens 5 (Pol d 5 and Ves v 5) are well established cross-reactive allergen pairs of PDV and YJV 2,3,28,41 . Although it is likely that also the hyaluronidases of PDV and YJV are cross-reactive, no reliable data exist. Moreover, at least YJV hyaluronidase seems to be of limited clinical relevance 37,42 . In this study DPP IV allergens were identified as novel pair of cross-reactive major allergens, responsible for the frequently observed double-positive sIgE test results with PDV and YJV. Additionally, our analyses provide for the first time a molecular basis for the observed cross-reactivity between PDV and HBV 43 . Therefore, DPP IV allergens might be pan-allergens, present in various hymenoptera venoms. Also, the presence of DPPs IV in many snake venoms 44 suggests functions of this enzyme class in the venoms of phylogenetically distinct species.
The capacity of Pol d 3 to activate effector cells and to initiate an allergic response was assessed by basophil activation testing and proved its role as potent allergen. In the Spanish patient group the basophil activation patterns by Pol d 3 and Ves v 3 matched the data of skin testing and sIgE levels to allergen extracts and components. For the German patients, this was also the case for the basophil activation data obtained with Api m 5 and Ves v 3. However, in this patient group Pol d 3 clearly demonstrated to be cross-reactive also in BAT. For these patients PDV allergy can be excluded with high probability since it is virtually not present in Germany. Therefore, Pol d 3-reactivity in BAT in these patients most likely is due to cross-reactivity. This is further supported by the fact that most Pol d 3-reactive patients additionally show basophil activation in response to Ves v 3 (and Api m 5). Only one patient showed weak basophil activation in response to Pol d 3 only but also very low general activation according to the positive control. However, if these results are of clinical relevance is difficult to determine since diagnostic sting provocation testing is ethically not justifiable. Nevertheless, the data demonstrate that BATs with recombinant cross-reactive major allergens represent a helpful tool to identify the allergy-eliciting venom as shown previously 28 .
To date, in clinical practice the discrimination between allergy to PDV and YJV is quite challenging. In contrast, for the discrimination between allergy to YJV and HBV an extended component-resolved diagnostics has evolved and has shown to provide added benefit for clinical decisions 4 . It was proposed that PDV and YJV allergy should be discriminated by measurement of the level of sIgE to phospholipases A1 (Pol d 1 and Ves v 1) and antigens 5 (Pol d 5 and Ves v 5) 3 . However, in addition to the YJV allergens, so far only Pol d 5 is available for routine diagnostics. In a former study, we were able to demonstrate that for most of the patients, for whom the allergy-relevant venom was clearly identified, the sIgE level with the appropriate antigen 5 (Pol d 5 or Ves v 5) indeed was higher 28 . However, this does not hold true for all patients. Moreover, for many patients the sIgE levels are in a very comparable range, hence, results will be difficult to interpret in many cases.
Of course, it would be of major interest to identify species-specific marker allergens that would allow a reliable and easy discrimination between PDV and YJV allergy. However, all identified major allergens of the two venoms, including phospholipases A1, antigens 5 and dipeptidyl peptidases IV, exhibit a high degree of cross-reactivity. Hence, the development of a component-resolved diagnostic approach, comparable to that for the discrimination between HBV and YJV allergy is difficult to realize, in our opinion. Nevertheless, the extension of the available panel of allergen components also for routine diagnostics of PDV (and YJV) allergy will generate added value for advanced diagnostics. Certainly, the combination of the results of sIgE measurements to more than one major allergen will help to create a clearer diagnostic picture and to facilitate diagnostic decisions also in vespid venom allergy. Therefore, the newly identified major allergen of PDV, Pol d 3, together with its counterparts of YJV and HBV, might become a key element for molecular diagnostics of hymenoptera venom allergy. Moreover, the detailed knowledge of the allergen composition of different insect venoms will help to understand the immunological mechanisms of venom allergy and therapeutic outcome.

Methods
Patients. Blood and/or sera of 108 patients with allergy either to PDV, HBV and/or YJV were analysed. 65 patients were from the area of South Bavaria (Munich, Germany), 30 patients were from the area of Córdoba, Spain and 13 patients were from Barcelona, Spain. As PDV allergy is almost not present in Germany, allergic reactions to this species can be excluded with high probability and the German patients were allergic to YJV and/ or HBV. Patients from Córdoba were primarily allergic to PDV and patients from Barcelona were allergic to PDV and/or YJV. As Polistes dominula and Vespula vulgaris coexist in Spain and are difficult to discriminate, systemic reactions due to both insects cannot be excluded.
The diagnosis of venom allergy was based on a combination of clinical history of an allergic sting reaction, a positive intradermal skin test, and/or positive sIgE levels to PDV, YJV and/or HBV (i77, i3, i1) and allergen components (i208, i209, i210, i211) (UniCAP250; Thermo Fisher Scientific, Uppsala, Sweden).
All patients had given informed written consent to draw additional blood samples. Cloning and recombinant production of venom dipeptidyl peptidases IV. The coding region of Pol d 3 was amplified from Polistes dominula venom gland cDNA and plasmids coding for Api m 5 and Ves v 3 were generated as described previously 29,45 . Cloning and recombinant production in Spodoptera frugiperda (Sf9) insect cells is described in detail in the Supplementary Methods. Immunoreactivity of patient sera with recombinant dipeptidyl peptidases IV. sIgE immunoreactivity of sera with the recombinant venom DPPs IV was assessed by ELISA. A detailed description of the ELISA is given in the Supplementary Methods. The lower end functional cut-off, indicated as dotted lines, was calculated as the mean of the negative controls summed with 3 times the standard deviation (SD) of the mean and additionally 10% of the resulting value.
Basophil activation test. Basophil activation tests were performed in 15 YJV-and/or HBV-allergic patients, and in 13 PDV-and/or YJV-allergic patients as described previously 38


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Non-invasive spinal electro-magnetic stimulation (SEMS): a tool for evaluation and modulation of lower limb spinal-muscular transmission in healthy adults

Objective Our earlier electrophysiological recordings using animal models revealed diminished transmission through spared fibers to motoneurons and leg muscles after incomplete spinal cord injury (SCI). Administration of spinal electro-magnetic stimulation (SEMS) at specific parameters induced transient improvement of transmission at neuro-muscular circuitry in SCI animals. In the current human study, we sought translate this knowledge to establish optimal parameters of SEMS for (i) neurophysiological evaluation via Compound Motor Action Potential (CMAP); and (ii) modulation at neuro-muscular circuitry via H-reflex and M-wave response in 12 healthy adults. Methods SEMS application was with a coil positioned over T12-S1 spinal levels. SEMS-evoked CMAP-responses were wirelessly measured simultaneously from biceps femoris (BF), semitendinosus (ST), vastus lateralis (VL), soleus (SOL), medial gastrocnemius (MG) and lateral gastrocnemius (LG) muscles. We also examined effects of SEMS trains on H-reflex and M-wave responses. H-reflexes and M-waves were measured simultaneously from SOL, MG and LG muscles and evoked by peripheral electrical stimulation of tibial nerves before and after each SEMS session. Results Spinal levels for SEMS application to evoke CMAP-responses in corresponding muscles and amplitude/latency of these responses have been established. SEMS applied over L4-S1 spinal levels at 0.2 Hz rate for 30 min induced facilitation of H-reflexes and M-responses. Facilitation lasted for at least 1 hour after stopping SEMS and was associated with a decrease in threshold intensity and leftward shift of recruitment curve for H-reflex and M-wave. SEMS did not alter TMS-evoked responses in hand muscles. Conclusion SEMS is a novel, non-invasive approach for sustained neuromodulation of H-reflex and M-wave responses in triceps surae muscle group. The parameters of SEMS application established in this study for evaluation and neuromodulation of neural pathways innervating leg muscles in healthy individuals may be used as a reference for neurophysiological evaluation and long-lasting plasticity of the lower limb spino-neuromuscular circuitry in individuals with SCI.

Introduction
Electro-magnetic stimulation is based on the principle of electromagnetic induction of an 70 electric field through intact tissue to underlying structures systems [16][17]. Transcranial electro-71 magnetic stimulation (TMS), since its initial introduction about three decades ago [18], has been 72 widely used for diagnostic applications and repetitive TMS was found to alter excitability at 73 cortico-motor circuitry [17,[19][20]. Responses evoked by TMS, however, are reliably recorded 74 from arm but not leg muscles, particularly in human participants with neurological impairments, 75 including spinal cord injury [21]. Consistent with these reported results of human studies, our 76 recent experiments using animal models revealed that TMS-evoked responses could be reliably 77 recorded from the hind limb muscles only in the naïve animals, but not in adult rats with SCI [22]. 78 However, responses evoked by electro-magnetic stimulation at spinal thoracic and lumbar levels 79 could be reliably recorded from hind limb muscles in rats with chronic SCI [1]. In fact, EMG    intensity were determined in their first session. To accomplish this, a total of 6-10 single pulses 205 were delivered to each spinal level from T12 to S1 and the stimulus intensity with 10% increments 206 ranged from 40% to 80% was applied until EMG recordings from the greatest number of muscles 207 were visible. After the optimal spinal level and SEMS intensity were determined, they were used 208 to deliver a 30 minutes of single pulse SEMS that was applied repetitively with 0.  Where S is the stimulus intensity, Hmax is maximum amplitude of H-reflex. S50 is stimulus 231 intensity needed to obtain 50% of Hmax, and b is the steepness of the curve. In addition, we 232 calculated the slope of the curve at S50 using following equation

278
With the same stimulation intensity, the optimal spinal level to evoke CMAP responses in 279 the triceps surae muscle group (MG, LG, SOL) was the L5-S1 spinal levels. The mean amplitude 280 for the SOL muscle at 70% of the maximum coil intensity was 0.15 ± 0.02 mV whereas the MG 281 and LG muscles showed 0.14 ± 0.3 mV and 0.10 ± 0.02 mV amplitude respectively (n=7). For 282 the VL muscle, the optimal spinal level was T12-L1. The mean amplitude for the VL muscle at 283 70% of maximum coil intensity was 0.14 ± 0.07 mV. For the BF and ST muscles, the optimal 284 spinal stimulation level was L1-L3. The mean amplitude for BF muscle at 70% of maximum coil 285 intensity was 0.03 ± 0.01 mV and ST was 0.05 ± 0.01 mV respectively.
Latencies of CMAP responses were also different depending on the muscle. Latency for 287 the VL muscle responses evoked from L1 SEMS was 11.8 ± 1.1 ms, 13.5 ± 1.3 ms for BF muscle    curve for both H-and M-responses; however, this shift was different among participants (Fig 4).       In order to understand whether SEMS induced changes in H-reflex and in M-wave are comparable, 405 we have also analyzed correlation between SEMS-induced changes in the parameters of H-reflex 406 and M-wave recruitment curves. As shown in Table 3,

Modulation of H-reflex by SEMS train is long-lasting and sustained after stop
413 of SEMS 414 We have also examined whether the effect of SEMS are long-lasting, i.e. how long the 415 observed threshold changes were sustained by measuring H-reflex before, immediately after 416 SEMS and after 1 hour post stopping of SEMS (Fig 6). The threshold intensity required for H 417 reflex was still significantly lower after 1 hour post SEMS administration;79.8 ± 3.3 % post-SEMS 418 compared to 100% pre-SEMS (n=4; p<0.05). Importantly, the decrease in the MT intensity 419 following SEMS application, however, was not associated with the changes in Hmax. Hmax   429 We have also examined whether SEMS administration may affect TMS induced responses. 430 We recorded TMS evoked responses from hand FDI muscle before and after SEMS administration.

431
Representative traces recorded from FDI muscle before and after SEMS in the same participant 432 presented in Fig 7A. We did not observe any changes in TMS evoked responses after SEMS.


extfieldsofstudy: Chemistry Medicine

text:
Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

INTRODUCTION
Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation (Wu et al., 2007) to cell-free production of recombinant proteins (Pedersen et al., 2011) and ribosome-nascent chain complexes (Rutkowska et al., 2009). The preparation of cellfree translation systems is a compromise between, on one hand, the desired properties, such as high synthetic activity and reproducibility of the system and, on the other hand, simplicity of generating robust extracts as well as economic considerations. In laboratory settings, the most convenient and readily accessible method of producing biomass is by growing cells in a batch format in flasks. In this case, large-scale production of highly translationally active exponentially growing cells can be challenging due to relatively low yields. To maximize extract yields, one can harvest cultures in late exponential/early stationary phase. While this provides more biomass, there is a drawback in that cells often reduce their translational capacity during slow growth (Dai et al., 2016). Importantly, the translational activity decreases upon exiting rapid exponential growth -and an important mechanism at play is the reduction of the concentration of active ribosomes via ribosomal sequestration into inactive complexes by dedicated regulatory protein factors.
Bacteria reduce their translational capacity either by directly inactivating 70S ribosomes (Agafonov et al., 1999) or forming inactive ribosome dimers, so-called 100S ribosomes (Ueta et al., 2010;Gohara and Yap, 2018). In Escherichia coli, formation of inactive 70S is mediated by protein Y (YfiA) that in response to cold shock associates with vacant ribosomes and precludes tRNA and mRNA binding (Vila-Sanjurjo et al., 2004). 100S ribosome formation in Gammaproteobacteria is mediated by two cooperating factors: the Hibernation Promoting Factor (HPF; homologous to YfiA (Ueta et al., 2008)) and the Ribosome Modulation Factor (RMF) (Wada et al., 1990). In E. coli the 100S is highly unstable, and therefore formation of the 100S in the stationary phase does not significantly reduce the efficiency of translational lysates (Failmezger et al., 2017). In the majority of bacterial species, 100S formation is mediated by one factor -HPF -the long version of the "short" HPF present in the Gammaproteobacteria (Akanuma et al., 2016;Beckert et al., 2017;Khusainov et al., 2017). Neither the Staphylococcus aureus nor the Bacillus subtilis genomes encode YfiA. S. aureus and B. subtilis 100S are considerably more stable, therefore 100S formation can potentially have a significant impact on translation efficiency in lysates (Ueta et al., 2010;Akanuma et al., 2016;Basu and Yap, 2016).
In budding yeast, the Stm1 protein acts as a translational repressor (Balagopal and Parker, 2011) and is recruited to 80S ribosomes upon nutrient limitation (Ben-Shem et al., 2011;van den Elzen et al., 2014). Rather than causing dimerization, Stm1 and its metazoan ortholog SERBP1 occlude the mRNA-binding channel in both the A-and P-site sites thus forming stable inactive 80S particles (Ben-Shem et al., 2011;Anger et al., 2013). As expected for a ribosome inactivation factor, when Stm1 is added to yeast translational extracts, their activity is strongly inhibited (Balagopal and Parker, 2011).
We reasoned that disrupting the genes encoding for ribosome inactivating factors would yield more reproducible and active bacterial (B. subtilis hpf ) and yeast (Saccharomyces cerevisiae stm1 ) cell-free translation systems. Addition of either HPF (Ueta et al., 2010) or Stm1 (Balagopal and Parker, 2011) purified proteins to cell-free translation systems in trans is known to inhibit the efficiency of protein production. The use of a hpf strain could be an especially promising strategy for improving the translation efficiency of B. subtilis lysates. Since this bacterium already expresses its HPF during exponential growth (although at significantly lower levels than in stationary phase) (Akanuma et al., 2016), it is expected that cell-free translation systems prepared from the hpf strain would be more active than those prepared from the wild type strain regardless of growth phase.
Another motivation for examining translational efficiency of lysates prepared from the hpf B. subtilis strain is the potential utility of hpf lysates for generating stalled ribosomal complexes. A popular strategy for preparation of stalled ribosomal complexes utilizes cell-free translation of dicistronic 2Xerm-mRNA encoding two identical Erm-stalling leader peptides (Arenz et al., 2014;Crowe-McAuliffe et al., 2018).
Stalled ribosomal dimers formed in the presence of the antibiotic erythromycin are readily separated from 70S monosomes -but not from 100S particles -on sucrose gradients. Use of the hpf strain lacking 100S ribosomes to generate extracts avoids this problem.

Elimination of HPF Improves the Efficiency of B. subtilis Coupled Transcription-Translation System
We opted for a coupled transcription-translation system utilizing the pIVEX2.3MCs FFluc plasmid (Starosta et al., 2010) that encodes the firefly luciferase ORF preceded by B. subtilis optimized ribosome binding site (RBS). Transcription of the mRNA is driven by recombinant T7 RNA polymerase added to the lysate (Antoun et al., 2004) and the efficiency of protein synthesis was quantified by measuring the luminescence of the translated luciferase protein. For preparing cell-free extracts we used the wild type 168 B. subtilis strain and an isogenic hpf mutant that displays no growth defect, except for a moderate increase in the lag phase (Akanuma et al., 2016). The cells were collected at OD 600 1.8-2.2 and the lysates prepared as described in the Materials and Methods section. The polysome profile analyses show that in the case of the wild type strain, the 100S peak is dominant and stable ( Figure 1A), while in the hpf mutant it is lacking altogether ( Figure 1B). Importantly, the situation is dramatically different in the case of E. coli: when lysates are prepared from the stationary phase E. coli cells, no 100S peak is observed (Failmezger et al., 2017).
Our protocol for preparation of a B. subtilis transcriptiontranslation system was based on that of Krinsky et al. (2016), which is specifically optimized to be rapid, cheap and relatively efficient -despite lacking a dedicated ATP regeneration system, it was able to generate 40-150 µg-protein/mL. The system is a binary mixture of cell-free extract and compound mix ( Figure 1C). The cell-free extract contains a full set of cellular components carrying out protein synthesis, i.e., ribosomes, tRNAs, aminoacyl tRNA synthetases, methionyl-tRNA formyltransferase and translational factors. The compound mix contains (i) inorganic ions, importantly Mg 2+ , the key player in ribosomal function (Nierhaus, 2014) (ii) buffering (HEPES) and reducing (DTT) agents (iii) NTPs that serve both as the energy source and as the building blocks for mRNA synthesis (iv) template DNA in a form of pIVEX2.3MCs FFluc plasmid supplemented with recombinant T7 RNAP polymerase (v) amino acids and folinic acid that serve as building blocks for protein synthesis and, finally, (vi) stabilizing agents such as PEG-8000 and glucose.
To test the robustness of the effect of HPF loss on the efficiency transcription-translation system, we performed two titrations of the key components. As a first step, we have varied the ratio between the compound mix and cell-free extract ( Figure 1D). To ensure the robustness of our results, we used at least three independently prepared cell-free extracts for each titration point. FIGURE 1 | Elimination of HPF improves the efficiency of B. subtilis coupled transcription-translation system. Polysome profile analysis of translational lysates prepared from wild type (A) and hpf (B) lysates demonstrates strictly HPF-dependent 100S ribosomes formation. (C) A cell-free translation system was assembled by combining the compound mix with the cell-free extract. The efficiency of translation was quantified by the activity of the firefly luciferase using the Steady-Glo Luciferase Assay (Promega). Titrations of the compound mix to cell-free extract ratio (D) and magnesium ion concentration (E) in the cell-free translation system. Luminescence readings were taken after incubation for 1 h at 37 • C. Error bars indicate the standard error of the geometric mean of biological replicates, i.e., independently prepared cell-free extracts (n ≥ 3).
At a 1:1 ratio the activity is optimal, and the hpf lysate is robustly approximately twofold more active.
The second titration step was aimed at identifying the optimal concentration of magnesium ions (Zaher and Green, 2014). Magnesium is important both for optimizing transcriptional and translational efficiency in the coupled system. While using in vitro transcribed mRNA it is possible to discriminate between Mg 2+ effects on transcription and translation, the specific effect of HPF elimination is unlikely to act on the transcriptional level.
Maintaining the ratio between the extract and compound mix at 1:1, we titrated the final concentration of Mg 2+ from 7 to 22 mM ( Figure 1E). While the activity of the hpf lysate peaks at 12 mM Mg 2+ , reaching an excess of 400,000 relative light units (RLU; the term "relative" is used since rather than providing absolute quantification of photons, the efficiency of detection is specific for a particular luminometer instrument), the hpf lysate is more active than the wild type at all magnesium concentrations tested. Therefore, we concluded that loss of HPF robustly improves the efficiency of B. subtilis lysates prepared from stationary phase cells.

Elimination of Stm1 Improves the Efficiency of S. cerevisiae Translation System
The S. cerevisiae stm1 strain was constructed by deleting the STM1 gene in the wild type MBS (Iizuka and Sarnow, 1997) strain. We have opted for a translation system supplemented FIGURE 2 | Elimination of Stm1 improves the efficiency of S. cerevisiae translation system. (A) A cell-free translation system was assembled by combining the compound mix with the cell-free extract. The efficiency of translation was quantified by the activity of firefly luciferase using the Steady-Glo Luciferase Assay (Promega). Titrations of the compound mix to cell-free extract ratio (B) and magnesium ion concentration (C) in the cell-free translation system. Luminescence readings were taken after incubation for 1 h at 25 • C. Error bars indicate the standard error of the geometric mean of biological replicates, i.e., independently prepared cell-free extracts (n ≥ 3).
with an in vitro transcribed, capped polyadenylated luciferase mRNA. Capping mRNA dramatically increases the efficiency of translation (Tarun and Sachs, 1995) but cannot be performed in situ in the lysate and must be added enzymatically to the mRNA after transcription. The translation protocol was based on that of Wu et al. (2007). Just as the bacterial coupled translation-transcription system, the yeast translation system is also a binary mixture of cell-free extract and a compound mix (Figure 2A). Since transcription is performed separately, the compound mix contains only the two NTP species necessary for translation, i.e., GTP and ATP, and mRNA is stabilized by RNase inhibitor (rRNasin). Creatine phosphokinase and phosphocreatine serve an energy regeneration system.
Similarly to the B. subtilis system, we have performed two titrations, using at least three independently prepared cell-free extracts for each titration point: altering the ratio between the compound mix and cellular extract ( Figure 2B) and the amount of Mg 2+ in the extract (Figure 2C). The efficiency of the system gradually increases with the percentage of the compound mix in the reaction from 33 to 66% ( Figure 2B). Notably, both at 33-50% compound mix there is no significant difference between the wild type and stm1 lysates; only in the presence of 66% compound mix does the stm1 translation reaction display higher activity. However, even in this case, the activity is two orders of magnitude lower than that seen in the B. subtilis system. A likely culprit is a sub-optimal concentration of Mg 2+ ions; as the fraction of the compound mix increases, the Mg 2+ concentration is moderately increased from 2 to 2.2 mM, following the concomitant increase in activity ( Figure 2B). Therefore, we titrated Mg 2+ from 2.5 to 5.5 mM ( Figure 2C). The activity peaked at 4.5 mM Mg 2+ , reaching an excess of 40,000 RLU with the stm1 S. cerevisiae lysate robustly out-performing the wild type lysate.

DISCUSSION
Here we demonstrated that genomic disruption of genes encoding ribosome inactivating factors -HPF in B. subtilis and Stm1 in S. cerevisiae -moderately but robustly (n ≥ 3 biological replicates -i.e., independently prepared cell-free extracts -were used for each data point) improves the activity of bacterial and yeast translation systems. Both of the proteins directly inactivate the ribosomes and are to cell-free translation lysates when added in trans (Ueta et al., 2010;Balagopal and Parker, 2011). Importantly, elimination of HPF leads to a complete loss of 100S ribosomes, thus despite only a moderate increase in the overall translational efficiency, the hpf lysate is particularly well-suited for disome-based approaches for preparation of stalled complexes for cryo-EM structural studies (Arenz et al., 2014;Crowe-McAuliffe et al., 2018).
The increase in efficiency of the delta hpf B. subtilis system is modest compared to the effects of reducing cellular proteolytic or RNase activities: a well-proven approach for genetic manipulation of strains for in vitro translation, as exemplified by the B. subtilis WB800N strain lacking eight protease-encoding genes (Wu et al., 2002) that is used for cell-free translation (Kelwick et al., 2016) and the E. coli MRE600 strain with low RNase I activity (Kurylo et al., 2016) that is used for preparation of active ribosomes (Rivera et al., 2015) and translation systems (Castro-Roa and Zenkin, 2015). In the case of B. subtilis WB800N, the cell-free translation system prepared from this strain is 72 times more active than that made from the wild type (Kelwick et al., 2016) -a significantly larger improvement compared to the twofold effect observed upon hpf deletion. A possible next step in developing strains for more efficient cell-free translation system is by combining genetic modifications targeting proteolysis, RNA degradation and ribosome hibernation. In the case of the S. cerevisiae system, optimization strategies used in recent years have been focused on cultivation conditions, preparation of lysates and approaches for replenishment of substrates (Zemella et al., 2015). Therefore, even the moderate improvement in efficiency yielded by Stm1 elimination has significance for biotechnological applications.

MATERIALS AND METHODS
Preparation 6His-Tagged Recombinant T7 RNA Polymerase (T7 RNAP) E. coli BL21 strain (NEB) lacking the λDE3 lysogen was transformed with pQE30-T7RNAP plasmid (Amp) (Antoun et al., 2004). An overnight culture in LB media supplemented with 50 µg/mL of ampicillin was used to inoculate a largescale culture in the same media grown at 37 • C with shaking. At OD 600 of 0.5-0.6 expression of 6His-tagged T7 RNAP was induced by addition of IPTG to a final concentration of 1 mM. After an additional 2 h growth, cells were harvested by centrifugation, resuspended in buffer A (150 mM NaCl, 100 mM Tris:HCl pH 7.5, 2 mM MgCl 2 , 1 mM ß-mercaptoethanol) supplemented with 0.1 mM PMSF, 35 µg/mL lysozyme and 1 u/mL DNase I and lysed by one pass via Stansted Fluid Power SFPH-10 Stansted Pressure Cell/Homogenizer (1.5 bar). After removal of the cell debris by centrifugation (35,000 rpm for 40 ), the clarified lysate was loaded onto a 1 mL HisTRAP HP column (GE Healthcare) equilibrated in buffer A. The column was washed with high salt buffer B (same as buffer A except for the addition of 2 M NaCl), and the protein was eluted by a gradient of buffer C (buffer A supplemented with 0.5 M imidazole), pure fractions were pooled, concentrated and buffer-exchanged into storage buffer (100 mM KCl, 20 mM Tris:HCl pH 7.5, 5 mM MgCl 2 , 1 mM DTT, 50% glycerol) using 50 MWCO centricons (Amicon). The purity of protein preparations was assessed by SDS PAGE and spectrophotometrically (A 280/260 ratio of approximately 1.8). The protein was stored at −20 • C.

Preparation of Firefly Luciferase mRNA for Use in Yeast Translational Lysates
Firefly luciferase mRNA containing a 30 nucleotide poly(A) tail was in vitro transcribed from the luciferase T7 control plasmid (Promega) linearized by Afel as a DNA template for the T7 RNA Polymerase (HiScribe TM T7 High Yield) RNA Synthesis Kit. A typical 20 µL reaction containing 1 pmole DNA was incubated for 2 h at 37 • C prior to mRNA isolation with MEGAclear TM Kit (Ambion), followed by capping by the Vaccinia Capping System (NEB) and re-purification of mRNA with MEGAclear TM Kit (Ambion). The quality of the final product was confirmed by denaturing agarose electrophoresis.

Preparation of B. subtilis Bacterial Cell-Free Extract
The lysate preparation protocol is based on that of Krinsky et al. (2016) with minor modifications. Fifty milliliter LB cultures of B. subtilis 168 wild type and hpf strains were inoculated with single colonies from fresh LB plates and grown at 37 • C with vigorous shaking overnight. To generate the biomass, two 2 L flasks containing 800 mL LB were inoculated to a starting OD 600 of approximately 0.05 and bacteria were grown at 37 • C with shaking. At the OD 600 of 1.8-2.2 cells were collected by centrifugation at 10,000 g for 3 min (4 • C, Beckman JLA-10.500 rotor), pellets were dissolved in 100 mL of ice-cold lysis buffer (10 mM Trisacetate pH 8.0, 60 mM potassium glutamate, 14 mM magnesium acetate, 0.5 mM PMSF, 1mM DTT, pH 8.2), pelleted (3 at 10,000 g, 4 • C), taken up in 50 mL lysis buffer, and pelleted again in 50 mL Eppendorf centrifuge tubes (30 at 3,000 g 4 • C) yielding 4-5 g of biomass that was processed directly. To lyse the cells, lysis buffer was added to 4-5 g of cells to final volume of 12 mL, and cells were passed once though Stansted Fluid Power SFPH-10 Stansted Pressure Cell/Homogenizer at 2 bar. The lysate was clarified (10 at 16,000 g, 4 • C) and the supernatant was then desalted using Zeba Spin Desalting Columns 5 mL (ThermoFisher) equilibrated with lysis buffer. After adjusting A 260 to 100 absorbance units with the lysis buffer, lysates were aliquoted in 50-100 µL fractions, snap frozen in liquid nitrogen and stored at −80 • C.

Preparation of S. cerevisiae Cell-Free Extract
The lysate preparation protocol is based on that of Wu et al. (2007) with minor modifications. The wild type MBS and MJY1079 strains were grown in YPD medium at 30 • C for 24 h. To generate the biomass, two 2L flasks containing 800 mL YPD were inoculated to a starting OD 600 of approximately 0.001 and yeast were grown overnight at 30 • C until OD 600 of 4.0-7.0. Cells were collected by centrifugation (10 at 7,000 g, 4 • C), washed (3 at 10,000 g, 4 • C) three times with 50 mL of icecold lysis buffer (30 mM HEPES, 100 mM potassium acetate, 2 mM magnesium acetate, 0.5 mM PMSF, 1 mM DTT, pH 7.6), and pelleted in 50 mL Falcon centrifuge tubes (30 at 3,000 g, 4 • C) yielding 10 g of cells. Cells were frozen as small pellets by mixing with lysis buffer and dropped into liquid nitrogen and stored at −80 • C or processed directly. To lyse the cells, 10 g of frozen cells were combined with 1 mL of frozen lysis buffer and crushed with a mortar and pestle in liquid nitrogen for 20 min. Note that more robust lysis methods have been shown to reduce the ability to translate exogenous mRNA in a yeast in vitro translation system (Leibowitz et al., 1991). The resulting lysate was transferred to a 50 mL falcon tube and incubated on ice until melted. The lysate was clarified (30 at 3,000 g followed by ultracentrifugation of the supernatant for 10 at 21,000 g, all at 4 • C). The supernatant was desalted using Zeba Spin Desalting Columns 5 mL (ThermoFisher) equilibrated with lysis buffer. After adjusting A 260 to 100 absorbance units with the lysis buffer, lysates were aliquoted in 50-100 µL fractions, snap frozen in liquid nitrogen and stored at −80 • C.

Preparation of B. subtilis Transcription-Translation System: Final Optimized Protocol
The protocol was based on that of Krinsky et al. (2016), with minor modifications. The translation system was assembled by mixing the B. subtilis lysate (see above) with the compound mix (10 mM Mg(OAc) 2 , 90 mM HEPES pH 8.2, 30 mM K 2 HPO 4 pH 7.22, 135 mM potassium glutamate, 120 mM NH 4 OAc, 30 mg/mL PEG-8000, 1.8% (w/v) glucose, 3 mM DTT, 3 mM each of the 20 amino acid, 0.1 mM folinic acid, 3.5 mM ATP, 2.55 mM of GTP, CTP and UTP, 3 µg/mL plasmid pIVEX2.3MCs FFluc and 1.5 µM recombinant T7 RNAP) to a final volume of 30 µL per reaction point. The lysate and compound mix were combined at a 1:1 ratio. After gently mixing the binary system by pipetting, the reaction was incubated at 37 • C for 1 h with shaking (500 rpm), and 10 µL of the reaction were added to 50 µL of Steady-Glo Luciferase Assay (Promega) (see below).

Preparation of S. cerevisiae Translation System: Final Optimized Protocol
The protocol was based on that of Wu et al. (2007) with minor modifications. The translation system was assembled by mixing the yeast lysate (see above) with the compound mix (7 mM Mg(OAc) 2 , 20 mM HEPES pH 7.4, 200 mM KOAc, 2 mM DTT pH 7.4, 50 µM each of the 20 amino acids, 6 mM ATP, 0.5 mM GTP, 50 mM phosphocreatine, 40 µg/mL creatine phosphokinase, 10 µg/mL capped firefly mRNA with a 30 nucleotide poly(A) tail and 0.6 units/µL rRNasin) to a final volume of 30 µL per reaction. Prior to use, the lysates were treated with micrococcal nuclease (NEB) (24 units/µL) activated by 0.5 mM CaCl 2 (final concentration) at room temperature in order to degrade cellular mRNA species. After a 20 treatment with the nuclease, the reaction was stopped by addition of EGTA (pH 7.5) to a final concentration of 2 mM. Capped luciferase mRNA was refolded (7 at 70 • C and kept on ice prior to use) and added last to the compound mix immediately prior to assembling the final reaction mixture. The lysate and compound mix were combined to a 1:1 ratio. After gently mixing the binary system by pipetting, the reaction was incubated at 25 • C for 1 h on an Eppendorf Thermomixer with shaking (500 rpm), and 10 µL of the reaction were added to 50 µL of Steady-Glo Luciferase Assay (Promega) (see below).

Luciferase Assay
Steady-Glo Luciferase Assay (Promega) was used as per the manufacturer's manual. The luciferase assay reagent was aliquoted in 50 µL fractions in 1.5 mL Eppendorf tubes kept in dark at room temperature. Readings were taken after addition of 10 µL reaction mixture to prealiquoted luciferase reagent using GloMax 20/20 Luminometer (Promega).

AUTHOR CONTRIBUTIONS
VM and VH conceived and coordinated the study. TB, VM, and VH drafted the manuscript together with input from MJ, TN, and HT. VM, TB, and VH designed the experiments and analyzed the data. TB, VM, MJ, and HT performed the experiments. All authors have read and approved the manuscript as submitted.


Effect of type and concentration of cryoprotectant on post-thaw survivability of vitrified porcine follicular oocytes

A total of 950 and 510 oocytes with two or more cumulus cell layers adhered to zona pellucida obtained by aspiration from follicles (2–8 mm dia) of 315 and 135 abattoir porcine ovaries, respectively, were utilized in the study to find the effect of cryoprotectant and concentration on post-thaw survivability of porcine follicular oocytes. Vitrified post-thaw oocytes with intact zona pellucida and vitelline membrane, normal spherical shape and dark and evenly granulated cytoplasm under a stereo-zoom microscope were considered as viable. Out of the 10 cryoprotectant treatments i.e. ethylene glycol (EG), propylene glycol (PG), dimethyl sulphoxide (DMSO), glycerol (GL), EG + PG, EG + DMSO, EG + GL, PG + DMSO, PG + GL and DMSO + GL used for vitrification of oocytes at a concentration of 35%, the post-thaw survivability rate was the highest in EG +DMSO followed by EG + PG group which had significantly higher mean post-thaw survived oocytes as compared to GL, PG + DMSO, PG + GL and DMSO + GL groups. Exposing the oocytes to 30, 35 and 40% of EG + DMSO and EG + PG yielded no significant difference in post-thaw survivability rate of vitrified oocytes, although the highest value was obtained with 35%. It was concluded that 35 to 40% EG + DMSO yielded efficient vitrification of porcine oocytes.

Cryopreservation of oocytes rather than embryos provides greater flexibility in breeding programmes (Paynter and Fuller 2007), since cryopreservation of immature oocytes obtained from abattoir ovaries offers opportunity for planned in vitro maturation and in vitro fertilization in contrast to embryo cryopreservation. Vitrification of oocytes is rapid technique with radically increased cooling and warming rates that can obviate chilling injury and ice crystal formation associated with slow freezing. Although several vitrification protocols have been applied for porcine oocytes with varying survival rates, they are still associated with severely compromised embryo development. Several factors influence the survival rate of immature porcine oocytes following vitrification including type and concentration of cryoprotectant. Different cryoprotectants and their combinations with varying concentations effective for cryopreservation of mammalian oocytes were adopted for vitrification of oocytes in various species. However, the best cryoprotectant or combination of cryoprotectants and their concentrations are yet to be determined for vitrification of porcine follicular oocytes and hence the present study was taken up.

MATERIALS AND METHODS
Porcine ovaries were collected from local abattoirs immediately after slaughter of the animal and transported to the laboratory within 1-2 h in a flask containing normal saline with antibiotic at normal environmental temperature (22-28°C). Oocytes were recovered from follicles (2-8 mm dia.) of the ovaries after washing by aspiration technique and examined on a sterile petri-dish under stereo-zoom microscope and good quality oocytes were selected for vitrification. The oocytes were first exposed to equilibration solution (cryoprotectant @ 15% v/v + sucrose 0.25 M in basic solution that contained 80 ml DPBS, 20 ml FBS and 5 mg Gentamicin, pH 7.2-7.4) and then to vitrification solution (VS) (cryoprotectant @ 35% (v/v) + sucrose 0.5 M in basic solution). Cryoprotectants i.e. ethylene glycol (EG), propylene glycol (PG), glycerol (GLY) and dimethyl sulphoxide (DMSO) singly and in combinations were used for vitrification of oocytes. For combination of cryoprotectants, the total concentration of cryoprotectants was 15% and 35% in equilibration and vitrification solutions respectively. Vitrification procedure was performed at room temperature (24-25°C). Within 30 sec of exposure in VS, the oocytes with little quantity of VS were loaded in the French mini straw (0.25 ml), sealed and directly plunged into LN 2 tank. The thawing or warming of vitrified oocytes was done in a step-wise (1 min in 0.5 M sucrose followed by 2 min in each of 0.25 M and 0.125 sucrose in basic medium) manner at 37°C. The post-thaw vitrified oocytes were examined under stereo-zoom microscope to evaluate survivability based on morphology. Oocytes with intact zona pellucida and vitelline membrane, normal spherical shape, and a dark and evenly granulated cytoplasm were considered as viable. Oocytes with clear signs of membrane damage (brownish, faded cytoplasm) were considered as non-viable (Somfai et al. 2014). Two combinations of cryoprotectants that revealed higher rates of survivability of vitrified oocytes were further used separately for oocytes vitrification at total concentrations of 30, 35 and 40% in VS and survivability of vitrified oocytes was recorded as before. Statistical analysis of the data was done following SAS (version 4.04) software.

RESULTS AND DISCUSSION
The mean post-thaw survivability rates following vitrification of porcine follicular oocytes in Dulbecco's phosphate buffered saline containing 35% of different cryoprotectants either alone or in combination are presented in Table 1. The post-thaw survivability rate of oocytes differed significantly (P<0.01) between cryoprotectants and the highest rate was obtained in EG+DMSO followed closely by EG+PG, the difference being non-significant. Oocytes in EG+DMSO and EG+PG vitrification groups had significantly (P<0.05) higher mean post-thaw survivability as compared to GL, PG+DMSO, PG+GL and DMSO+GL vitrification groups, the rate of survival being the least in DMSO+GL. This could indicate lower toxicity of EG than PG, GL and DMSO. The lowest survivability of vitrified oocytes in DMSO+GL could be due to higher molecular weights of DMSO (78.13g/mol) and GL (92.094 g/mol), which might had impaired their permeability into and out of oocytes thus reducing the efficiency of vitrification. Lower efficacy of GL in vitrification was reflected in the mean survivability of oocytes which was recorded to be lower although not significantly as compared to that in EG, PG and DMSO. This was in agreement with the findings of Shahat and Hamman (2014) in buffalo oocytes. Lower postthaw survivability rate obtained with GL as compared to EG was also reported by Yadav et al. (2008) in bubaline cumulus-oocytes complexes. EG+DMSO yielded significantly (P<0.05) higher post-thaw survived vitrified oocytes as compared to EG+GL, however, the difference between EG+PG and EG+GL was not significant. Significantly (P<0.05) higher post-thaw survivability of vitrified oocytes in EG+DMSO than in PG+DMSO was in conformity with the findings of Sharma et al. (2010) in buffalo oocytes. This could indicate efficacy of EG over PG in vitrification, which might be attributed to lower molecular weight of EG (62.07g/mol) which enabled it to permeate rapidly into and out of the oocytes as compared to PG (molwt: 76.09 g/mol). EG was reported to be an effective cryoprotectant for vitrification of oocytes (Somfai et al. 2006). The post-thaw survivability rates of follicular oocytes vitrified in EG+DMSO and EG+PG were apparently higher than that vitrified in EG, PG or DMSO alone. Similar observation was made by Mahmoud et al. (2008) for buffalo oocytes. It could be ascribed to reduction in the level of toxicity in oocytes exposed to cryoprotectant due to use of combinations of different permeable cryoprotectants at lower concentration thus obviating use of higher concentration of single cryoprotectant that was needed for avoiding ice crystal formation in oocytes but could be more toxic causing osmotic injury. Szurek and Eroglu (2011) also observed that combinations of different permeable cryoprotectants might be a possible means of improving oocytes cryopreservation protocol by reducing their specific toxicity. Somfai et al. (2013) reported higher post-thaw survivability of pig follicular oocytes vitrified in EG+PG as compared to EG alone. Thus maintenance of a balance between the toxicity and cryoprotective action of acryoprotectant was critical for the development of an efficient vitrification protocol. However, in the present study, some combinations of cryoprotectants had lower postthaw survivability of vitrified oocytes when compared with single cryoprotectant. This might be due to inequality or disproportion in the level of permeability and toxicity of different cryoprotectants.
The mean per cent post-thaw survivability rates of oocytes following vitrification using 30, 35 and 40% concentrations of EG+DMSO and EG+PG respectively are KALITA ET AL.
[Indian Journal of Animal Sciences 88 (11) furnished in Table 2. The survivability rate of post-thaw vitrified oocytes was higher than that reported by Somfai et al. (2013) in pig, however, it did not vary significantly among the concentrations used which was in consonance with the findings of Mohmoud et al. (2013) who reported no significant difference between 6 M and 7 M concentrations of EG+DMSO in post-thaw survivability of vitrified buffalo oocytes. In the present investigation, the percentage of mean post-thaw survived oocytes was higher in 35% EG+DMSO (86.99±3.07) and EG+PG (85.60±5.33), however, it did not differ significantly from that using 30 and 40% concentrations in both the combinations. Present findings of level of post-thaw survivability were comparable with that reported by Shi et al. (2006) and Huang et al. (2006) in pig and Attanasio et al. (2010) in buffalo. Somfai et al. (2013) also observed that a combination of equal concentration of EG and PG as permeable cryoprotectant that had a total concentration of 35% improved the efficacy of vitrification protocol of immature porcine oocytes. In the present study, concentration of 35 and 40% of EG+DMSO and 35% EG + PG yielded higher post-thaw survivability following vitrification of follicular pig oocytes than utilizing 30% of the combination of the cryoprotectants. This indicated that higher concentrations of cryoprotectants inside cells became hyaloids material under ultra-cooling conditions and thereby prevented icecrystal formation that were detrimental to cellular health. However, the toxic effects that might lead to oocyte damage emanating from using a higher concentration of cryoprotectant was evinced in the lower level of survivability of vitrified oocytes obtained by employing 40% total concentration of EG+PG in the present work. This could be due to inclusion of PG in the combination since use of 40% total concentration of EG+DMSO gave rise to much higher proportion of post-thaw survived vitrified oocytes. Somfai et al. (2015) also found that PG at high dose could be toxic. Szurek and Eroglu (2011) found that the toxicity of PG could be avoided by its combination with DMSO sharing equally for the total concentration. They further observed that toxicity of 1.5 M PG increased significantly as indicated by degeneration of vast majority of mouse oocytes with increase in exposure temperature and time while there was no significant increase in the toxic effect of 1.5 M DMSO and 1.5 EG under the same condition as revealed by viability of oocytes and fertilization and embryonic development. The mechanism of action of cryoprotectant (CPA) toxicity still remains poorly understood. Hydrophobic interactions between CPAs and proteins (Arakawa et al. 1990), the extent of hydrogen binding between CPAs and water molecules (Fahy et al. 2004) were proposed to explain cryoprotectant toxicity. Nevertheless, PG was implicated in inducing chemical toxicity (Huang et al. 2006). It could be concluded from the present study that the use of EG+DMSO as cryoprotectant at 35 to 40% concentration could bring about successful vitrification of porcine oocytes.


text:
Meta-Analysis of Association between PALB2 Polymorphisms and Breast Cancer

Background: Previous studies have assessed associations between single nucleotide polymorphisms (SNPs) of the Partner and localizer of BRCA2 (PALB2) gene and risk of breast cancer. However, the results of these studies are not consistent. Materials and Methods: We designed a meta-analysis to obtain a more reliable appraisal of the association between SNPs in the PALB2 gene and the susceptibility to breast cancer. We searched PubMed, Google scholar and Embase databases and selected six studies with sufficient data to estimate the pooled odds ratios (ORs) and 95% confidence intervals (CIs). Results: Statistical analyses showed that the rs120963 was associated with breast cancer risk in allelic (OR (95% CI) = 1.33 (1.18-1.49)), homozygous (OR (95% CI) = 1.74 (1.31-2.32)), dominant (OR (95% CI) = 1.42 (1.22, 1.65)) and recessive (OR (95% CI) = 1.54 (1.17, 2.03)) models. The rs249954 and rs16940342 were associated with breast cancer risk in allelic (OR (95% CI) = 1.13 (1.04, 1.23) and 1.12 (1.01, 1.24) respectively) and dominant (OR (95% CI) = 1.23 (1.09, 1.39) and 1.18 (1.04, 1.33) respectively) models. The rs249935 and rs447529 SNPs were associated with breast cancer in homozygous (OR (95% CI) = 0.67 (0.46, 0.97) and 0.51 (0.30, 0.89) respectively) and recessive (OR (95% CI) = 0.65 (0.45, 0.95) and 0.51 (0.30, 0.88) respectively) models. Conclusions: The current meta-analysis shows the associations between five SNPs of PALB2 and breast cancer risk and confirms the results of previous studies regarding the role of this gene in the pathogenesis of breast cancer.

Introduction
Breast cancer is a molecularly heterogeneous disorder associated with high mortality and morbidity (Seifi-Alan et al., 2013;Iranpour et al., 2016). Among the most important factors contributing in breast cancer are genes involved in maintenance of genomic integrity (Walsh and King, 2007). Partner and localizer of BRCA2 (PALB2) encodes a protein which co-localizes with BRCA2 in nuclear foci, enhances its stability in chromatin and nuclear matrix and facilitates its tumor suppression effect (Xia et al., 2006). In addition, it provides the functional link between BRCA1 and BRCA2. It coheres directly to BRCA1, and makes the molecular scaffold for establishment of the BRCA1-PALB2-BRCA2 complex. Its interaction with BRCA1 is mainly facilitated via apolar bonding between their corresponding coiled-coil domains. Noticeably, BRCA1 mutations detected in cancer patients abolish such interactions resulting in insufficiency of homologous recombination (HR) repair. In brief, PALB2 makes the molecular link between the BRCA proteins and its mutations lead to defective HR repair, genomic instability and carcinogenesis (Sy et al., 2009). As revealed by in vitro studies, many of BRCA2 functions including G2/M checkpoint, replication fork shields and repair of double strand breaks by HR depend on its interaction with PALB2 (Hartford et al., 2016). Germ line mutations in PALB2 have been shown to increase breast cancer risk in different populations (Rahman et al., 2007;Cao et al., 2009;Antoniou et al., 2014). However, breast cancer susceptibility is best explained by a "polygenic" model, in which several loci confer breast cancer risk, with each locus having only a minor influence (Pharoah et al., 2002). Consequently, in addition to high risk mutations, many low-penetrance variants such as single nucleotide polymorphisms (SNPs) in PALB2 might interactively affect the risk of breast cancer development (Cao et al., 2010). The associations between PALB2 SNPs and breast cancer risk have been evaluated in different populations (Chen et al., 2008;Cao et al., 2010;Guenard et al., 2010;Jiang et al., 2016). However, the results of these studies are inconclusive and inconsistent in some cases. Such inconsistency might be explained by the moderately small sample size of former studies or the inherent genetic heterogeneity of breast cancer in diverse populations. Therefore, we designed a meta-analysis to resolve these incompatible results and to achieve a conclusive decision regarding the role of PALB2 variants in genetic susceptibility to breast cancer. The selection of PALB2 for the meta-analysis was based on the previous reports indicating its co-localization with BRCA2 and its putative role in the pathogenesis of breast cancer.

Literature search
The studies included in the meta-analysis were chosen by searching the PubMed, Google scholar and Embase databases from their establishment to January 2018 using the following keywords: "PALB2 gene or Partner and localizer of BRCA2" AND "breast cancer or breast carcinoma". We also assessed all references in of the retrieved studies to detect extra research not included in the databases. We just assessed researches written in English. Figure 1 shows the flowchart of course of selection of articles for inclusion in the current meta-analysis.

Inclusion criteria
The following criteria was adopted to include studies in the current meta-analysis: (1) a case-control study scheme; (2) a concentration on the association of PALB2 SNPs with breast cancer risk; (3) satisfactory data to determine the odds ratio (OR), confidence interval (CI) and P value; (4) compliance of genotype distribution of all control groups with Hardy-Weinberg equilibrium (HWE).

Data extraction and quality assessment
Two researchers (AD and SF) evaluated eligible studies and extracted first author's name, publication year, sample size, country, ethnicity, source of DNA used for genotyping, and frequencies of each genotype in certain study groups. The quality of the studies was evaluated using Newcastle-Ottawa scale (NOS) (Stang, 2010). In the cases of missing data in the obtained studies, the mentioned researchers gathered the missing data through communication (via email) with the corresponding authors.

Statistical analyses
All analyses were performed in the RevMan (v.5.1) software (http://www.cochrane.org/revman). We assessed the association between PALB2 SNPs and breast cancer using Z test to appraise the significance of the pooled odds ratios (OR). ORs were computed in allelic (wild type (W) versus minor (M)), homozygote (WW versus MM), dominant (WW+WM versus MM) and recessive (WW versus WM+MM) models. The grade of heterogeneity between the studies was evaluated using Q-test and I2 parameter. The values up to 40% were considered acceptable. Based on the calculated P values, OR and 95% CI (for the pooled odds ratio) were computed using random-effect model or fixed-effect model in heterogeneous or homogenous states respectively. Publication bias was evaluated using Begg's and Egger's tests. The level of significance was set at P<0.05.

Results of literature search
After initial assessment of titles and abstracts of found manuscripts, 201 manuscripts were reviewed for inclusion ( Figure 1). A total of 187 studies were filtered due to unsuitable study design, unsuitable type of study (studies other than original research) or language. Fourteen studies were subjected to detailed evaluation. Finally, we chose six articles for inclusion in the current meta-analysis (Chen et al., 2008;Cao et al., 2010;Guenard et al., 2010;Loizidou et al., 2010;Leyton et al., 2015;Jiang et al., 2016). Table 1 shows the characteristics of studies included in the meta-analysis and the obtained NOS scores.

Discussion
PALB2 encodes a protein with fundamental role in HR and maintenance of genome stability (Leyton et al., 2015). Biallelic PALB2 mutations are associated with increased susceptibility to cancers along with hypersensitivity to DNA-damaging materials (Reid et al., 2007). Based on the functional interaction between PALB2 and the breast cancer susceptibility gene BRCA2, PALB2 SNPs have been considered as putative risk factors for breast cancer development. In the present meta-analysis, we assessed the associations between 10 SNPs within this gene and risk of breast cancer. We found that the rs120963 was associated with breast cancer risk in all assessed genetic models. This SNP resides approximately 16.5 kb from the 3'-end of PALB2 and was associated with groups (Table 2). In Asians, significant associations were found between the rs249935 breast cancer in homozygous and recessive models, rs249954 and breast cancer in dominant and allelic models, and the rs120963 and breast cancer in all inheritance models. Finally, the rs447529 was associated with breast cancer in this ethnic group only in recessive model.

Assessment of publication bias
The funnel plots were illustrated to evaluate the existence of publication bias in the meta-analysis of the mentioned SNPs in allelic, homozygous, dominant and recessive models (Supplementary Figure 1A-D respectively). The shape of the funnel plot was symmetrical implying lack of publication bias.  (Chen et al., 2008;Jiang et al., 2016). Moreover, the rs249954 and rs16940342 were associated with breast cancer risk in allelic and dominant models. The rs249935 and rs447529 SNPs were associated with breast cancer in homozygous and recessive models. Other SNPs were not associated with breast cancer risk in any of the assessed genetic models. The rs152451 and rs45551636 are located in exons 4 and 9 of this gene respectively and have been previously identified through sequence analysis of the complete coding region of PALB2 in 100 probands from South-American breast cancer families negative for BRCA1 and BRCA2 point mutations (Leyton et al., 2015). However, our metaanalysis revealed no association between these variants and breast cancer risk. For the rs45532440, rs45478192  and rs8053188 the results of the meta-analysis were in line with the results of individual studies regarding lack of association with breast cancer risk. In brief, our study provides further evidences for participation of PALB2 in breast cancer risk and warrants future studies to explore the biological functions of these variants. As certain genetic variations in PALB2 alter its functional interactions with BRCA2 in DNA repair mechanisms and cell cycle control (Teo et al., 2013), it is possible that functional polymorphisms within this gene also affect such interactions.
Based on the detected association between five SNPs and breast cancer, we suggest the possibility of combined effects of certain SNPs in conferring breast cancer risk which should be assessed through haplotype analysis in future studies.
Our study has some limitations. First, due to the relative small number of eligible studies which assessed association of each PALB2 SNP with breast cancer risk, we could not perform subgroup analysis. Subgroup  analysis in patients with/ without family history of breast cancer, early/ late onset of breast cancer, distinct tumor subgroups or menopausal status in addition to the ethnic based analysis would explore definite role of PALB2 in distinct types of breast cancer. As revealed recently, the association of certain SNPs with breast cancer risk may be altered by the tumor pathological features or menopausal status (Jiang et al., 2016). Second, we did not assess linkage disequilibrium (LD) between these common variants. The detected associations between these SNPs and breast cancer risk might be due to high level of LD with another causal variant which should be evaluated in future studies. Consequently, evaluation of associations between PALB2 SNPs and breast cancer risk in large sample sizes with focus on haplotypes would clarify the complicated mechanism of maintenance of genome integrity by BRCA1 and BRCA2 proteins in the context of breast cancer.
In conclusion, the current meta-analysis shows the associations between five SNPs of PALB2 and breast  Table 2. Meta-analyses of PALB2 Polymorphisms and Risk of Breast Cancer in Ethnicity-based Subgroups (NA: not assessed) cancer risk and confirms the results of previous studies regarding the role of this gene in the pathogenesis of breast cancer.


extfieldsofstudy: Medicine Biology

text:
MicroRNA-665 suppresses the growth and migration of ovarian cancer cells by targeting HOXA10

Ovarian cancer is the most lethal gynecological cancer and its metastasis leads to a poor prognosis. The present study was designed to elucidate how microRNA (miR)-665 regulates the proliferation and migration of ovarian tumor cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that miR-665 expression was decreased in ovarian cancer tissues. Increased expression of miR-665 suppressed the growth and migration of ovarian cancer cells, whereas the downregulated expression of miR-665 led to the opposite results. Bioinformatics tools identified homeobox A10 (HOXA10) as a target of miR-665. Following miR-665 overexpression, HOXA10 protein expression was significantly reduced. A dual luciferase assay revealed that miR-665 bound to the 3′-untranslated region of HOXA10. Immunohistochemistry and RT-PCR revealed that the expression of HOXA10 was negatively correlated with the expression of miR-665. It was concluded that miR-665 targets HOXA10 and may act as a tumor-suppressing gene in ovarian cancer. This pathway may be involved in the development and metastasis of ovarian cancer.

Introduction
Epithelial ovarian cancer (EOC) is a major cause for malignancy-associated female mortality (1). The mortality rate is frequently increased by a delay in diagnosis and drug resistance (2). Investigating the mechanisms behind EOC initiation and progression can help us to find a therapeutic target.
MicroRNAs (miRs), non-coding RNAs with a length of 20-24 nucleotides, regulate gene expression by inhibiting translation or degrading messenger RNA (3). Increasing evidence reveals that miRs regulate cellular processes, including proliferation, differentiation and apoptosis (3)(4)(5). A number of miRs are associated with ovarian cancer, and may regulate tumor progression, function as potential prognostic markers and contribute to drug resistance (6)(7)(8)(9). The functions of miRs in carcinogenesis have been illustrated in multiple studies. For example, miR-224, as an oncogene in EOC, improves cancer cell proliferation by downregulating KILLIN expression (10). By targeting pyruvate dehydrogenase E1 β subunit, miR-203 promotes the proliferation and migration of EOC cells (11). Other miRs function as tumor suppressors expressed (12,13). For example, by targeting cyclin dependent kinase (CDK1), miR-490-3p inhibits the proliferation, migration and invasion of EOC cells (12). miR-101 suppresses the expression of the suppressor of cytokine signaling 2 gene and inhibits the proliferation and invasion of EOC cells (13). Among them, miR-655 was the focus of the present study as it has been demonstrated to play a role in certain malignancies, including esophageal squamous cell carcinoma (14), bladder urothelial carcinoma (15) and gastric signet ring cell carcinoma (16). Currently, to the best of our knowledge, no studies have been conducted to investigate the association between miR-665 and EOC.
Homeobox A10 (HOXA10), one member of the homeobox gene family, acts as a transcription factor in embryonic development (17). The aberrant expression of HOXA10 was first observed in leukemia (18). The association between HOXA10 and EOC can be demonstrated by the downregulation and upregulation of HOXA10 in cancer cellular processes, including cell proliferation, epithelial-mesenchymal transition, apoptosis and drug resistance (6,(18)(19)(20)(21)(22). HOXA10 was demonstrated to participate in G1 phase arrest of endometrial cancer that may be caused by P 21 expression (23). It was hypothesized that miR-665 can regulate HOXA10 expression, based on the data from PicTar, TargetScan and miRBase. However, the role of miR-665 in the development of EOC and its association with HOXA10 remains uninvestigated.
In the present study, the expression of miR-665 in human EOC and normal ovary tissues were compared, and the impact of miR-665 expression on cell proliferation and migration in vitro was investigated. The results of the present study suggested that miR-665 serves a suppressive role in human EOC pathogenesis.

Materials and methods
Tissue samples and cell lines. Informed consent was obtained from each patient. The present study was approved by the Institutional Ethic Committee of Nanjing Medical University (Nanjing, China). Tissues from EOC patients were frozen immediately and stored at -80˚C. EOC tissue specimens (n=28) and normal ovarian tissue specimens (n=15) were collected from patients (age range, 24-73) who underwent surgery at the Department of Gynecology at the First Affiliated Hospital of Nanjing Medical University (Nanjing, China) between December 2014 and December 2015. EOC cell lines (HO8910 and OVCAR-3) and 293T cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 mg/ml (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) at 37˚C with 5% CO 2 .
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from ovarian tissues and ovarian cancer cell lines using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Following isolation, the integrity of RNA was determined using Agilent Bioanalyzer 2100 and RNA 6000 Nano kit (Agilent Technologies, Inc., Santa Clara, CA, USA). According to manufacturer's protocol, single-stranded complementary DNA (cDNA) was synthesized from 1 µg RNA in a 20 µl reaction volume with the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.), at 25˚C for 10 min, 37˚C for 120 min and 85˚C for 5 sec, followed by a 4˚C hold. Quantification of miR and mRNA was carried out using a SYBR Green PCR kit (Thermo Fisher Scientific, Inc.), and the cycle quantification (Cq) of each gene was recorded. The relative expression of miR-665 mRNA and HOXA10 was normalized to U6, and calculated using the 2 -∆∆Cq method (ΔCq=Cq target gene -Cq internal control ) (24). The qPCR was performed using the following parameters: 95˚C, 10 min; 40 cycles, 95˚C, 15 sec; 67˚C, 30 sec; 72˚C, 30 sec; and 72˚C, 5 min. The primers used are illustrated in Table I.
Protein extraction and western blotting. At 48 h following transfection, the cells were harvested, washed twice with phosphate-buffered saline, lysed using M-PER™ Mammalian Protein Extraction reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 0.01% protease and phosphatase inhibitor, and incubated on ice for 30 min. The cell lysate was centrifuged at 4˚C and 12,000 x g for 15 min. Protein concentrations were determined by a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology, Haimen, China). The supernatant (50 µg) of total protein was run on 10% SDS-PAGE and transferred electrophoretically to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking in Western Blocking Reagent (10%; Hoffman; Roche Diagnostics, Basel, Switzerland) for 15 min at room temperature, the membrane was incubated overnight at 4˚C with polyclonal rabbit anti-human HOXA10 (1:1,000; Abcam, Cambridge, MA, USA; cat. no. ab90641) and mouse anti-human β-actin (1:10,000; cat. no. ab49900; Abcam), respectively. The membranes were washed with 1X Tris-buffered saline containing 0.1% Tween-20 (TBST), incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h. Membranes were washed again with TBST three times for 10 min each, prior to visualization and analysis using the Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE, USA).
Transwell migration assay. Following being placed into a 24-well plate, the migration assays of OVCAR-3 and HO8910 cells were carried out using Transwell chambers (EMD Millipore). For the migration assay, a total of 1x10 5 cells were resuspended in 200 µl serum-free medium and placed in the top chambers. RPMI-1640 medium (600 µl) containing 10% FBS was added into the bottom chambers. Cells were incubated for another 20 h at 37˚C with 5% CO 2 . The cells were fixed with 4% polyoxymethylene following the incubation for 20 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature and observed using a light microscope (magnification, x100; Olympus Corporation, Tokyo, Japan). The numbers of migrated cells were calculated from five randomly selected fields.
Colony formation assay. Cells were transfected with NC, miR-665, as described above. Then 24 h later, transfected cells were trypsinized, counted and replated at a concentration of 500 cells/well. Following another 10 days, colonies formed by the surviving cells were fixed with 3.7% methanol for 20 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature and counted. Colonies containing ≥50 cells were scored. Each assay was performed in triplicate.
CCK-8 assay. The cell proliferation reagent WST-8 (Roche Diagnostics GmbH, Mannheim, Germany) was used to measure cell growth. Cells were seeded into 96-well microtiter plates (Corning Inc., Corning, NY, USA) at a density of 1.0x10 3 cells/well. CCK-8 was added to each well according to the manufacturer's protocol. Following incubation at 37˚C with 5% CO 2 for 2 h, the absorbance of the converted dye was detected at 450 nm to determine the cellular viability.
Dual-luciferase reporter assay. The 3'-untranslated regions (3'-UTRs) of human HOXA10 cDNA with the potential target sites for miR-665 were synthesized and inserted at the XbaI site downstream of the luciferase gene in the pGL3-control (Promega Corporation, Madison, WI, USA) vector by Integrated Biotech Solutions Co., Ltd., (Shanghai, China).
At 24 h prior to transfection, cells were seeded in 24-well plates (1.5x10 5 wells/well). Then, 200 ng of pGL3-HOXA10-3'-UTR and 80 ng of pRL-TK (Promega Corporation) were co-transfected with 60 pmol of miR-665 mimic or NC using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h following the transfection, the Dual-luciferase assay system (Promega Corporation) was used to determine luciferase activity as previously described (6). The firefly luciferase activity in each well was normalized to that of the Renilla luciferase. Three independent experiments were performed in duplicate. The sequence of 3' UTR of human HOXA10 cDNA containing the putative target site for the miR-665; the underlined, italicized area indicates the putative target site for miR-665: 5 '-TGA ATC TCC AGG CGA CGC GGT TTT TTC  ACT TCC CGA GCG CTG GTC CCC TCC CTC TGT CTT CAG  GCT CTG CCC AGG AAC TCG CAC CTG TGC TGG AGC CCT  GTT CCT CCC TCC CAC ACT CGC CAT CTC CTG GGC CGT  TAC ATC TGT GCA GGG CTG GTT TGT TCT GAC TTT TTG  TTT CTT TGT GTT TGC TTG GTG CTG GTT TAT TTG TTG  TTT TCT GGG GGA AAA AGC CAT ATC ATG CTA AAA TTC  TAT AGA GAT A-3'. Immunohistochemistry. The tissues were fixed in 10% formalin for 24 h at room temperature, embedded in paraffin and 4 µm thick sections were prepared. Microwave irradiation in 10 mol/l citrate buffer (pH 6.0) was used for antigen retrieval for tissue slides incubated with a HOXA10 antibody (1:500; cat. no. ab90641; Abcam) at 4˚C overnight, followed by incubation with a HRP-conjugated secondary antibody (1:1,000; cat. no. ab6721; Abcam) for 1 h at room temperature. The staining was repeated if the result was uncertain. Immunostaining of the slides was objectively evaluated by two pathologists under a light microscope (magnification, x200; Olympus Corporation, Tokyo, Japan). Discordant scores were reevaluated until consensus was reached. The level of positive HOXA10 expression in cancer cells was analyzed by HMIAS-2000 automatic medical color image analysis system (Qianping Image Technology Co., Ltd., Wuhan, China). HOXA10 staining was determined semi-quantitatively according to the staining intensity observed (0, no staining; 1,weak staining; 2, moderate staining; and 3, strong staining) and the percentage of positive cells (0, none or <10; 1, 11-25; 2, 26-50; and 3, 51-75%; 4, >75%). Scores of 0-3 were considered to indicate negative expression, and scores of 3-12 were considered to indicate positive expression. Cells were counted in at least three randomly selected fields (magnification, x200) in the tumor areas.
Statistical analysis. All experiments were repeated three times independently. The results were summarized as the mean ± standard error. Independent sample t-tests were performed to compare differences between two groups with SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Spearman's correlation test was used to analyze the correlation between miR-655 and HOXA10 expression. P<0.05 was considered to indicate a statistically significant difference.

Expression level of HOXA10 increases in EOC tissues.
Immunohistochemical staining was used to determine the level of HOXA10 expression in both normal and ovarian cancer tissues. HOXA10 expression of 15 EOC specimens and 8 normal specimens were analyzed in the present study. Immunohistochemical staining identified that the HOXA10 protein levels were significantly increased in the ovarian cancer tissues compared with the normal tissues (P<0.01; Fig. 1).

miR-665 targets the 3'-UTR of HOXA10.
TargetScan7.1 (http://www.targetscan.org) was used to predict whether HOXA10 is targeted by miR-665 ( Fig. 2A). In the 293T cell line, relative activity of luciferase was significantly decreased (P<0.01) following the co-transfection of the pGL3-HOXA10-3'-UTR vector with the miR-665 mimic, but not following the co-transfection of pGL3-HOXA10-3'-UTR vector with mimic NC, suggesting that HOXA10 is the target gene of miR-665 (Fig. 2B). Western blotting was performed to confirm the downregulation of the HOXA10 protein following the miR-665 transfection in HO8910 and OVCAR-3 EOC cells. The protein expression level of HOXA10 significantly decreased in miR-665-transfected cells, compared with cells transfected with miR-NC (P<0.01; Fig. 2C and D). Then   miR-665 mimic and mimic NC was then transfected into 293T cells.
miR-665 is negatively associated with HOXA10. qPCR was used to determine the expression of miR-665 in 28 EOC specimens and 15 normal specimens. Compared with normal tissues, expression level of miR-665 significantly dropped in ovarian cancer tissues, suggesting that miR-665 is downregulated in ovarian cancer tissues and may be involved in the development of ovarian cancer (P<0.01; Fig. 3A). A significant inverse correlation (R²=0.7496; P<0.01) was observed between miR-665 and HOXA10 using Spearman's correlation analysis (Fig. 3B).
miR-665 suppresses cell growth in vitro. HO8910 and OVCAR-3 cells were transfected with the miR-665 mimic. Subsequently, CCK-8 assays were performed to determine the impact of miR-665 on the proliferation of ovarian cancer cells.
The results identified that increased expression of miR-665 significantly suppressed the proliferation of ovarian cancer cells in both cell lines (P<0.05; Fig. 4A and B). The colony formation capacity of HO8910 and OVCAR-3 cells transfected with miR-665 mimic was significantly inhibited compared with the miR-NC group (P<0.05; Fig. 4C and D). These results demonstrated that miR-665 inhibits the proliferative ability of HO8910 and OVCAR-3 cells.

miR-665 suppresses the migration of ovarian cancer cells.
Transwell migration assay demonstrated that miR-665-overexpressed HO8910 and OVCAR-3 cells exhibited a significantly decreased ability to migrate compared with the control cells (P<0.05; Fig. 5).
Inhibition of miR-665 promotes the growth and migration of ovarian cancer cell lines. The colony formation rate of HO8910 and OVCAR-3 cells transfected with miR-665 inhibitor, was significantly increased, compared to cells transfected with the miR-NC inhibitor (P<0.05; Fig. 6A and B). The migratory abilities of miR-665-downregulated HO8910 and OVCAR-3 cells were significantly enhanced compared with the miR-NC inhibitor (P<0.05; Fig. 6C and D).

Discussion
Ovarian cancer is the most lethal cancer for women, with an overall survival rate of ~35% (25). Although modified chemotherapy can improve the prognosis, its effectiveness has reached its limit. Consequently, novel therapies, such as targeted therapy combined with standard treatment, are being investigated in clinical trials (26). In these trials, predictive markers can be used to personalize and optimize the therapeutic strategy for ovarian cancer (27). miRs modulate gene expression in a post-transcriptional manner either by inhibiting translation or destroying the target mRNA (28). miRs are aberrantly expressed in ovarian cancer. For example, the expression of miR-145 is significantly reduced in human ovarian cancer tissues, leading to relapse and poorer outcomes of ovarian cancer (29). miR-490-3p overexpression inhibits the proliferation, migration and invasion of tumor cells by directly targeting CDK1 (12). In addition, miR-497 downregulation triggers chemotherapy resistance in ovarian cancer cells (30). The level of miR-125b expression in ovarian cancer tissue is significantly lower compared with normal ovarian tissues; the increased expression of miR-125b induces cell cycle arrest and inhibits the proliferation and clonal formation of ovarian cancer cells by targeting BCL-3 (31).
In carcinogenesis, miR-665 demonstrates diverse functions (14)(15)(16) that are co-regulated by its targets (32). miR-665, located at 14q32.2, with a length of 20 amino acids, can inhibit B7-H3 expression. However, the underlying mechanism remains uninvestigated. In the present study, the analysis of RNA expression revealed that the expression of hsa-miR-665 was decreased in EOC, which is consistent with the results of a previous study on breast cancer (33). Therefore, it is hypothesized that miR-665 may function as a tumor-suppressor gene in ovarian cancer. Certain studies demonstrated that miR-665 expression was dysregulated in a number of types of cancer, including esophageal squamous cell carcinoma (14), bladder urothelial carcinoma (15) and gastric signet ring cell carcinoma (16).
HOXA10, from the homeobox gene family, functions as a transcription factor in embryonic development (17). It has been suggested that HOXA10 acts as a key factor in endometrial receptivity and embryo implantation, and is expressed in endometrial glandular epithelium and mesenchymal cells in normal humans (34). The HOXA10 expression level in the middle and late stages of secretion is increased compared with the period of endometrial proliferation and the early stage of secretion (35). As the progesterone concentration rises during implantation and embryonic circulation, the level of HOXA10 gradually reaches a peak, indicating that HOX genes regulate endometrial development and embryonic planting (35,36). In addition, HOXA10 is highly expressed in endometroid, clear or mucinous cells, but not in serous epithelial ovary cancer cells (37,38). Aberrant HOX gene expression has been reported in several types of cancer, including glioblastoma (39), oral cancer and gastric cancer (21,22,40). Increased expression of HOXA10 promotes the proliferation, migration and invasion of clear cell adenocarcinoma of the ovary, reducing the survival of patients (41). The present study identified that miR-665 was downregulated and negatively correlated with the expression of HOXA10 in ovarian tumor tissues, indicating that miR-665 may be a tumor suppressor gene in the development of ovarian cancer and a potential therapeutic target for ovarian cancer.
In the present study, the RT-qPCR results demonstrated that miR-665 was downregulated in ovarian cancer tissues compared with normal tissues. Immunohistochemistry revealed that HOXA10 was overexpressed in ovarian cancer tissues and this expression was negatively correlated with the expression of miR-665. It was also demonstrated that miR-665 suppressed the proliferation and migration of cell lines, whereas the downregulation of miR-665 led to the opposite effect, as it bound to the 3'-UTR of HOXA10, and downregulated HOX10 by reducing HOXA10 protein levels. Further studies are needed to elucidate the mechanism of HOXA10 silencing.
The present study investigated the expression and biological function of miR-665 in ovarian cancer. miR-665 downregulated the expression of HOXA10 and weakened the ability of ovarian cancer cells to proliferate and migrate. miR-665, through targeting HOXA10, serves as a suppressor gene in ovarian cancer. Therefore, therapeutic miR that mimics miR-665 could possibly be developed to treat ovarian cancer. and 81272871) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (grant no. JX22013366)

Availability of data and materials
Not applicable.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Nanjing Medical University and samples were obtained with informed consent from all patients.