Scatophagus argus (S. argus) during abrupt transfer from seawater (SW) to freshwater (FW). Following the transfer, serum dopamine concentration was decreased, and dopamine activated expression of the dopamine receptor 1 (designated SaDRD1) in the kidney, triggering the osmoregulatory signaling cascade. SaDRD1 protein is expressed in the renal proximal tubule cells in vivo, and is localized to the cell membrane of renal primary cells in vitro. Knockdown of SaDRD1 mRNA by siRNA significantly increased Na+/K+-ATPase (NKA) activity in cultured renal primary cells in vitro, suggesting that expression of SaDRD1 may oppose the activity of NKA. We demonstrate that exogenous dopamine enhances the response of NKA to hyposaline stress after transferring primary renal cells from isosmotic medium to hypoosmotic medium. Our results indicate that dopamine regulation via SaDRD1 ignited the renal dopaminergic system to balance the osmotic pressure through inhibiting NKA activity, providing a new perspective on the hyposaline adaptation of fish.Dopamine is an important regulator of renal natriuresis and is critical for the adaptation of many animals to changing environmental salinity. However, the molecular mechanisms through which dopamine promotes this adaptation remain poorly understood. We studied the effects of dopamine on renal hypo-osmoregulation in the euryhaline fish  Anguilla anguilla, Sarotherodon melanotheron and Oreochromis niloticus, have a limited capability to endure salinity fluctuation after long-term acclimation34Scatophagus argus (commonly known as the spotted scat). S. argus is an estuarine inhabitant notable for its tolerance of salinity fluctuationS. argus can survive after being directly transferred from the seawater (SW) to freshwater (FW) and vice versa. Thus, its highly effective osmoregulatory mechanisms deserve further attentionSalinity is an important environmental factor that greatly affects the physiology of marine fish, influencing their survival and distribution1The chemical messenger dopamine is primarily synthesized in the brain and kidney912131-like (subtypes D1 and D5) and D2-like  receptors161-like receptors, DRD1 and DRD5, are known to play a role in dopamine-regulated maintenance of sodium homeostasis and blood pressure in the kidneyDopamine activity is mediated by its receptors, DRD1–5, which belong to the G protein-coupled receptor (GPCR) family of seven-transmembrane domain receptors161-like receptor subtype has made it challenging to distinguish the individual roles of DRD1 and DRD5 in the kidney, although some progress has been made through other methods201-like receptor-mediated regulation of the cAMP-dependent protein kinase signaling pathway in renal proximal tubule cells, as DRD1 activation increases cAMP production to a much greater extent than DRD5 activation1-like receptor subtype in renal osmotic regulationThe lack of specific agonists and antagonists for each D+/K+-ATPase (NKA) activity during periods of osmotic and ionic stresses341-like receptors on the renal response of the euryhaline fish Scatophagus argus (the spotted scat) to hyposmotic stress. We employed a targeted molecular approach to specifically knock down SaDRD1 levels in cultured primary S. argus kidney cells, and observed the resulting response to osmotic stress in the presence and absence of exogenous dopamine. These results bring us closer to understanding the mechanisms of osmotic regulation in this fish.Euryhaline fishes offer a valuable model for the study of osmoregulation in the kidney, as they frequently move between areas of high and low salinity, and must rapidly adapt to these conditions. Thus, these fish present a unique opportunity to study physiological responses in osmoregulatory organs as they occur. Most previous studies on euryhaline fish have focused on changes in NaS. argus to FW, dopamine concentrations were significantly reduced in serum from the treatment group (**p < 0.01) at 1 and 3 hours post-transfer (hpt) compared to the control. Serum dopamine in the treated fish then rose, peaking at 12 hpt before declining. Compared with the control group, an increase of about 20% (from 346.5 pg/ml ± 18.2 pg/ml to 418.2 ± 26.7 pg/ml) of dopamine (DA) concentration was observed 12 hpt (p = 0.249). Thereafter, dopamine expressions in both groups were similar (During acclimation of  similar .p < 0.05) at 0.5 h and 3 h  was decreased significantly  with 7 transmembrane domains . In SW-reared fish, SaDRD1 mRNA was broadly expressed in the eye, brain and heart, and its expression was also observed in osmoregulatory organs, including the kidney and gill , expression varied greatly within this period, suggesting that SaDRD1 was involved in acclimation to FW  at different time points after hyposaline shock. SaDRD1 mRNA expression in the kidney fluctuated highly, with significant upregulation at 3 hpt and 12 hpt (>2-fold) (*p < 0.05)  was presented in To determine effects of the hyposaline shock on the expression of D < 0.05) . In contnd 1 dpt . The oriS. argus . Renal sections were double-stained with polyclonal rabbit antibody against SaDRD1 (green) and DAPI (blue), while the red channel was set as the background. The arrows and letters in Immunohistochemical experiments were conducted to determine the localization of SaDRD1 protein in the kidney of SW-acclimated SaDRD1 were analyzed by RT-qPCR following treatment with si-SaDRD1. SaDRD1 mRNA expression was reduced in renal primary cells by ~13-fold at 40 h post-infection   but transiently, subsequently dropping to 3.62 ± 0.02 μmolPi/mgprot/h at 12 hpt. After 1 day (24hpt), NKA activity returned to levels similar to controls (* < 0.05) .SaDRD1 mRNA at 180 min after treatment with either 1 μM dopamine (18.9 fold) or 10 μM dopamine (6.7-fold) (***p < 0.001), although no increase was detected at 30 minutes (The effects of exogenous dopamine on cultured primary kidney cells were tested under isosomotic and hyposmotic conditions. The addition of exogenous dopamine to cells maintained under the isosmotic condition (300 mOsm/L) caused a significant change of  minutes .SaDRD1 mRNA in the absence of exogenous dopamine. In contrast, when the hypoosmotic shock was accompanied by exogenous dopamine (1 μM), SaDRD1 mRNA levels increased significantly at this time point (*p < 0.05). After a longer exposure to hypoosmotic medium, for 180 min, SaDRD1 mRNA expression increased significantly (***p < 0.001) in both the dopamine-treated cells and the control cells. At 180 minutes post-transfer, SaDRD1 mRNA expression levels were elevated in cells in both the presence and absence of exogenous dopamine. Notably, the increase in expression was greater in the absence of added dopamine at 180 min, showing a 26.3-fold increase, compared to a 4.4-fold increase in the medium containing exogenous dopamine  for 30 minutes did not alter the expression of dopamine . The resNKA activity was observed at 30 min and 180 min after exposure to the hyposaline medium in the presence and absence of 1 μM exogenous dopamine. While the NKA activity was not significantly altered in no-dopamine cells at 30 min, treatment with 1 μM exogenous dopamine significantly inhibited NKA activity (from 5.07 ± 0.84 μmolPi/mgprot/h to 3.45 ± 0.71 μmolPi/mgprot/h). The significantly inhibition of NKA activity was observed at 180 min post-transfer in both the dopamine-treated and no-treated cells .+ transporters, thereby favoring natriuresis through paracrine and autocrine pathways29Sarotherodon mossambicus) during SW to FW adaptationSalinity adaptation in euryhaline teleosts is a complex process involving physiological responses in several osmoregulatory organs. The endocrine system mediates many of these processes in order to maintain salt and water balance when fish are challenged with changing environmental salinity27In mammals, dopamine is synthesized in dopaminergic neurons of the brain, where it serves as a neurotransmitter. A substantial amount of dopamine circulates in the bloodstream, but its origins and functions outside of the brain are not entirely clear1134S. argus decreased significantly during the first 24-hours after transfer into FW, with the exception of the 12 hpt collection , dopamine contents in the culture medium were reduced substantially at 0.5 h and 3 h in comparison with those exposed to isosmotic culture medium  are the principal mediators of the natriuretic effects of dopamine19SaDRD1 expression  pression . Thus, DSaDRD1 gene is predicted to encode a protein of 463 amino acids with 7 transmembrane domains  stimulates the expression of SaDRD1 in renal primary cells at 3 h after exposure. Higher expression of SaDRD1 was observed in the group induced with 1 μM dopamine (18.9-fold) than with 10 μM dopamine (6.7-fold)  . There aSaDRD1  resulting in the blunted dopaminergic system activity, in accordance with a previous study in the ratS. argus.The effects of exogenous dopamine (1 μM) on NKA activity in renal primary cells were explored. The results show that dopamine enhances the NKA response early when renal cells are faced with hyposaline stress by activating dopaminergic system through SaDRD1  and D. TS. argus, intrarenal dopamine plays an important role in the regulation of osmotic balance by activating renal SaDRD1, while SaDRD5 does not appear to be involved. SaDRD1 is expressed in the proximal tubule cells, and subcellularly localized to the cell membrane of cultured renal cells. During hypoosmotic shock of cultured renal cells, exogenous dopamine enhanced the early response of NKA to hyposaline stress through SaDRD1. Based on our results, we hypothesize that the dopaminergic system responds to hypoosmotic shock via DRD1 activation, which leads to inhibition of NKA activity and maintenance of osmotic balance in the fish exposed to acute hypoosmotic shock (During the acclimation to FW shock in ic shock . FurtherScatophagus argus (32.6 ± 7.4 g) were collected from Zhuhai , Guangdong Province, China and reared for 3 weeks in 25‰ seawater  at 27 ± 2 °C. Fish for hyposaline challenge were divided into a treatment group (n = 105) and a control group (n = 105). Treated individuals were transferred from 25‰ SW to fresh water . Control individuals were transferred to another SW tank . Fifteen individuals were removed from each group at each experimental time point and anaesthetizeds 32.6 ± .4 g wereet al.Kidney tissue was collected from healthy SW-acclimated fish weighing ~40 g and used for primary cell culture as described in Zhou For hypoosmotic stress experiments, primary cells were subjected to hypoosmotic medium (100 mOsmol/L), while control cultures were exposed to fresh isosmotic medium (300 mOsmol/L) . Culture medium and cells were collected at 0.5 h and 3 h post-challenge, respectively, and used for hormone assays, RNA extraction and NKA activity measurement.4/cm2 at 28 °C for transfection and immunohistochemistry staining.Additional cultures of primary cells in isosmotic medium were maintained at a density of approximately 2 × 10et al.Dopamine in serum and culture medium was measured by indirect enzyme linked immunosorbent assay (ELISA) as described by Wu via Nanodrop-2000 spectrophotometer . RNA integrity was verified by gel electrophoresis. Only samples with A260/A280 ratios 1.8 to 2.0 were used. cDNA was synthesized by reverse transcription of 1 μg total RNA with oligo(dT) primers using the PrimeScript RT reagent kit with gDNA Eraser  and stored at −20 °C.Total RNA was isolated using Trizol Reagent  following the manufacturer’s protocol. RNA was treated with DNase I  and quantified SaDRD1, primers (http://www.ncbi.nlm.nih.gov/). SaDRD1 was amplified from S. argus brain cDNA. PCR products were purified by gel extraction  and cloned into the pMD18-T vector  for sequencing.To obtain the partial sequences of  primers  were desSaDRD1, rapid amplification of cDNA ends (RACE) was performed . Primers are listed in To obtain the full-length cDNA sequence of SaDRD1 was aligned with Clustal X 1.81 and analyzed using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to predict the amino acid sequence and open reading frames (ORFs). Transmembrane domains were predicted using TMHMM v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). A phylogenetic tree was constructed using MEGA 6.0 software with neighbor-joining method and 1000 boot-strap replicatesThe nucleotide sequence of SaDRD1 in S. argus. Primers are listed in β-actin; 55 °C, 28 cycles for SaDRD1. The PCR products were visualized by 2% gel electrophoresis and expression was quantified using densitometric analysis . SaDRD1 expression was normalized against β-actin.Semi-quantitative reverse transcriptase PCR (SqRT-PCR) was conducted to assess tissue expression patterns of Custom polyclonal antibodies were raised against SaDRD1 using a recombinant peptide (amino acids 336–463 of predicted SaDRD1 protein: NADFRKAFSILLGCHRLCPGSNAIEIVSINNNMGAPTSNPNCQYQPKSHIPKEGNHSSNYVIPHSILCQEEELQKKDACGGEIDVGMVNNALEKLSPAISGNLDSDTEVTLEKINPITQNGQHKAMSC) .S. argus and rat were homogenized on ice in normal saline (0.86% NaCl) and lysates were centrifuged at 12000 rpm 4 °C for 10 min. Supernatant was collected and protein concentration determined using a BCA Protein Assay Kit . About 75 μg total protein of S. argus or rat kidney was separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes. Following blocking in 5% skim milk in PBS , membranes were incubated with rabbit anti-SaDRD1 (1:2000 dilution) in PBS overnight at 4 °C. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody  (1:5000 dilution) for 2 h at room temperature. The signals were detected by chemiluminscence using Universal HoodII .Kidneys of S. argus were fixed for 16 h in 4% paraformaldehyde at 4 °C, dehydrated using a graded series of sucrose solutions, embedded in Tissue-Tek O.C.T , and cut into 20-μm sections at −20 °C. Sections were mounted on poly-l-lysine-coated slides , incubated in 5% skim milk for 1 h at 37 °C, incubated with primary antibody (1:400 dilution) in PBS containing 0.5% Triton X-100 (PBST) overnight at 4 °C, then incubated with FITC-conjugated goat anti-rabbit secondary antibody  (1:1000 dilution) in PBST for 3–4 h at the room temperature. Nuclei were stained with DAPI  in PBS for 5 min.Kidney tissue samples collected from Cultured primary cells were fixed for 15 min in 4% paraformaldehyde, incubated in 5% skim milk for 0.5 h at 37 °C, incubated with primary antibody (1:400 dilution) in PBS containing 0.5% Triton X-100 (PBST) for 3 h at 4 °C, then with FITC-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) in PBST for 1.5 h at room temperature. Nuclei were stained with DAPI (as above) for 3 min.Samples were visualized with a Zeiss LSM510 confocal laser scanning microscope . Negative controls were incubated with normal rabbit serum.SaDRD1 in spotted scat renal primary cells. Silencer select siRNA oligonucleotides 5′-GGUGCAUUCUGCAACGUAUtt-3′ (designated si-SaDRD1) against SaDRD1 were designed and synthesized by Genepharma . A non-specific silencer select siRNA oligonucleotides  served as negative controls. Cells were treated with siRNA in 6-well plates according to manufacturer’s instructions using Lipofectamine 3000 . Expression levels of the protein SaDRD1 and NKA activities were evaluated at 40 h post-transfection.Small interfering RNA (siRNA) was employed to knock down expression of Primary cells cultured in isosmotic medium (approximately 300 mOsmol/L) were shocked by hypotonic medium (approximately 100 mOsmol/L) containing 1 μM and 10 μM dopamine . Additional primary cells in original medium containing 1 μM and 10 μM dopamine were regarded as control groups. At 0.5 and 3 h post-challenge with dopamine, cells were washed with isosmotic/hypotonic medium and collected for RNA extraction and NKA activity measurement.SaDRD1 mRNA expression was quantified by RT-qPCR. Reactions were performed in triplicate for each sample using SYBR Premix Ex Taq II . RT-qPCR primer sequences are listed in −ΔΔCt method as described in Mu et al.SaDRD1 siRNA treatment. Subsequently, NKA activity was measured following the protocol supplied by the manufacturer of the ATPase Kit . Activity was expressed as μmolPi/mgprot/h.Immunostaining for SaDRD1 and NKA α1 of primary cells treated with siRNA was performed as above. Total protein was isolated and quantified at 40 h after t-test with SigmaStat software . * means significant (p < 0.05), and *** means significant (p < 0.001).The statistical significance of differences between groups was determined using the One-way ANOVA, followed by student’s How to cite this article: Su, M. et al. Dopamine regulates renal osmoregulation during hyposaline stress via DRD1 in the spotted scat (Scatophagus argus). Sci. Rep. 6, 37535; doi: 10.1038/srep37535 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


M = 18.59 years, SD = 1.19) and found that a two-factor model fitted the data well. However, the structure of the CFI was revised because of some modifications, which were made to the original English to match the Russian equivalents of items originally developed to assess the definite aspect of cognitive flexibility. The CFI-R showed good internal consistency and suitable 7-week test–retest reliability. The construct validity of the Russian version of the CFI was studied by computing correlations with other related measures of CF , depressive symptoms (Beck Depression Inventory), coping (Ways of Coping (Revised), and rigidity (Tomsk Rigidity Questionnaire). Furthermore, to assess whether the construct validity were affected by psychopathology we examined results for non-clinical and clinical samples, using “known-groups” method. The clinical sample reported lower CF than did the non-clinical sample on the CFI-R’s total score and its subscales’ scores. Findings in the present study suggest that the psychometric properties of the Russian CFI are comparable to the English original, making it appropriate to research assessment of the type of CF in Russian speaking population.The Cognitive Flexibility Inventory (CFI) is a brief self-report measure of the type of cognitive flexibility (CF) necessary to successfully challenge and restructure maladaptive beliefs with more balanced and adaptive thinking; it is particularly popular for use with English speakers. The CFI has recently been translated into five languages , although estimates of reliability and validity of these translated versions are scarce. This study reports on the factor structure, internal consistency, reliability, and construct validity of the CFI. We adopted the CFI for a Russian-speaking population, using student sample of 445 first and second-year undergraduates ( According to The self-report measures are attractive for psychological treatment because of “they are brief, can be completed independently, and can be quickly scored and interpreted” , p. 1382r = 0.75–0.81; p < 0.001), as well as convergent construct validity of the CFI and its two subscales via their associations with other measures of CF . The principal axes extraction method yielded a two-factor structure with 20 items loading on two factors corresponding to the following subscales: Alternatives and Control, all showing good to excellent internal consistency (αs ranging from 0.84 to 0.91). The Alternatives subscale is composed of 13 items, all of which measure the ability to perceive multiple alternative explanations for life occurrences and human behavior and the ability to generate multiple alternative solutions to difficult situations; the Control subscale is composed of 7 items, most of which were originally developed to measure the tendency to perceive difficult situations as controllable. The measure demonstrated high 7-week test–retest reliability for the CFI total score and its subscales  = 439.42, df = 169, p = 0.001, GFI = 0.88, CFI = 0.87, SRMR = 0.07, RMSEA = 0.07, 90% CI . In summary, considering the construct validity and reliability of the CFI across countries and samples, these studies demonstrated inconsistent findings of the factor structure. To date, in China with a college student sample, df = 3.52, CFI = 0.90, NNFI = 0.89, SRMR = 0.06, RMSEA = 0.06) providing support for the replicability of the original CFI.A number of subsequent studies have utilized this measure of CF in non-clinical and clinical situations and reported its good psychometric features across countries and samples. Although the CFI has been largely applied internationally, studies examining its structural validity have shown different results. The two-factor structure and internal consistency have been confirmed for the CFI in Turkish , JapanesAs there is currently no instrument in Russian that can provide a valid and reliable measure of CF within a research investigation or therapeutic intervention, the present study aims to explore the replicability of the factor structure and the psychometric properties of the CFI on university students, and to develop its Russian version (CFI-R). Our analysis focuses on the CFI-R factor structure, reliability, and construct validity. We predicted to indicate the negative associations of CF aspects measured by CFI-R with depressive symptomology, coping strategies considered to be maladaptive and personality rigidity.M = 18.59, SD = 1.19). The majority of students were Russian (96%), and in social (34%) and human sciences (66%) according to the field of study. The clinical sample consisted of 35 outpatients , ranging in age from 21 to 82 years . According to the Diagnostic and Statistical Manual of Mental Disorders , ranging in age from 16 to 25 years   .The BDI is a 21-item self-report rating inventory that measures characteristic attitudes and symptoms of depression. Participants are instructed to choose one statement from the four listed beneath each item that best describes the way they have been feeling during the past week including that day. Each item is scored from 0 to 3, with higher scores indicating more serious depressive symptoms. The scores from 0 to 9 are regarded as minimal depressive symptoms, 10–18 as mild depression, 19–29 as moderate depression, and 30–63 as severe depression. The reliability and validity of the Russian translation of the BDI (α = 0.86) and its “cognitive-affective” (α = 0.79) and somatic-performance subscales (α = 0.79) have been confirmed by The ASQ is a 48-item self-report measure of explanatory style for good and bad events. It presents 12 hypothetical events (six positive and six negative). The respondents are asked to write down one major cause of each event and then rate the cause along a seven-point Likert scale (1–7) continuum for each of the three causal dimensions: internal versus external, stable versus unstable, and global versus specific causes. The original authors  reporteddoes not apply and/or not used (0); used somewhat (1); used quite a bit (2); used a great deal (3). The WC-R is a self-report measure of coping derived from Lazarus’ transactional model of stress. It contains 50 items representing thoughts and actions which can be used to deal with the demands of a stressful encounter. Responses to the WC-R are scored on a four-point Likert scale: definitely disagree (0), probably disagree (1), probably agree (3), definitely agree (4). The TRQ was developed to capture mental rigidity that a person experiences related to situations that challenge to change individual elements of a behavior program depending on the lifestyle, stereotypes, relations, attitudes, habits, skills, life and activity rate and rhythm, means of goal attainment, etc. The TRQ (α = 0.92) comprises six main subscales, which measure the different aspects of mental rigidity, and two control scales, which measure whether the respondent has indicated the agreement to the Tomsk Rigidity Questionnaire’s statements from own experience or from some assumptions , and whether the respondent has answered truthfully in other parts of the questionnaire . The Rigidity Symptom Complex subscale  reflects a person’s predisposition to a wide range of forms of stabile behavior such as perseveration, obsession, stereotypy, obstinacy, pedantry, and rigidity proper. The latter aspect of this subscale is reflected in the Actual Rigidity subscale , which measures a person’s inability to change their opinion, relation, attitude, motives, manner of experiencing, and etc. The Sensitive Rigidity subscale  reflects a person’s emotional reaction to anything unfamiliar in situations regarding any chances (similarly to neophobia). The Attitudinal Rigidity subscale  reflects a personality level of mental rigidity manifestation expressed in a person’s set, relation or attitude to acceptance/rejection of anything new, need to change the self-concept, level of aspiration, habit complex, and etc. The Rigidity as a State subscale  reflects a person’s tendency to rigid (fixed) behavior being in a state of fear, stress (distress), impaired mood, fatigue, or sickliness. The Premorbid Rigidity  subscale reflects whether a person has experienced difficulties in situations regarding any chances being in teen-age and preadult age. Respondents retrospectively assess how they have behaved, undergone and overcome any difficulties at school age (for patients it is a premorbid period). In the normal differential estimate of mental rigidity there are four levels of its display: “low,” “moderate,” “high,” and “very high.” The original author . All other analyses were carried out by applying confirmatory factor analysis (CFA) using EQS 6.2 for Windows , goodness-of-fit index (GFI), the standardized root mean square residual (SRMR), the root-mean-square error of approximation (RMSEA) and confidence interval (CI), to evaluate model fit , internal consistency, and intercorrelations of scales were calculated using the statistical package IBM SPSS Statistics (Version 22). Cronbach’s alpha was used to examine the internal consistency of the Russian version of the Cognitive Flexibility Inventory (CFI-R), Pearson’s correlation coefficient and intra-class correlation coefficients (ICC) were used to establish the 7-week test–retest reliability. The CFI-R’s construct validity and its two subscales were assessed through calculating correlations with related measures comprising the BDI, ASQ, WC-R, and TRQ. An EFA was performed to examine the factor structure of the CFI using a principal axes factor analysis. A Promax rotation with Kaiser normalization was utilized to test a hypothesis that two factors of the CFI are correlated, as it was anticipated by original developers of English version . Student Windows . Since todel fit .p < 0.0001). Based on the Scree plot and Kaiser’s coefficient alpha of generalizability  measure of sampling adequacy  and Bartzability , a two-f c of CF . Giving of CF  = 428.23, df = 169, p < 0.001, CFI = 0.88, GFI = 0.89, SRMR = 0.07, RMSEA = 0.06, 90% CI . The two factors were correlated 0.36 (p < 0.001) in the model. For comparison purposes, we examined the original CFI’s factorial structure. We ran CFA using the CFI’s developers scoring procedure (2(N = 445) = 956.97, df = 169, p < 0.001, CFI = 0.65, GFI = 0.77, SRMR = 0.14, RMSEA = 0.10, 90% CI, . All fit indices showed a bad fit for a two-factor model of the original CFI, although the two factors were correlated 0.51 (p < 0.001) in the model. Notwithstanding the height of the correlations, we observed that a two-factor model of the CFI-R with a specified number of items fitted the data better than a two-factor model of the original CFI. Thus, these analyses confirmed the CFI-R’s factor structure and provided further support for the modification of the original CFI.Based on the results from the EFA two subscales of the CFI-R were constructed. The Alternatives subscale with the eigenvalue of 5.10 explained 25.50% of the total variance, and the Control subscale with the eigenvalue of 2.33 explained 11.64% of the total variance. Internal consistency estimates of reliability, as indexed by Cronbach’s alpha, for the Alternatives subscale and the Control subscale were 0.82 and 0.85, respectively. The means of inter-item correlations were 0.30 for the Alternatives subscale and 0.41 for the Control subscale. Cronbach’s alpha for the CFI-R was 0.85 and it had a mean of inter-item correlations of 0.25. This two-factor CFI-R’s model further tested whether it would fit the data well. The results supported the acceptable model fit scaled χrocedure  with theM = 18.40, SD = 1.01), 231 females  and 31 males . They were invited to return for the re-testing questionnaire session 7-week later. Mean scores on the CFI-R were 106.36 (SD = 12.11) at testing and 104.86 (SD = 12.20) at re-testing. The mean scores for the Alternatives subscale were 67.02 (SD = 7.59) at testing and 66.72 (SD = 7.39). The mean scores for the Control subscale were 39.34 (SD = 7.45) at testing and 38.15 (SD = 7.58). The test–retest correlation had a moderate to strong range for the CFI-R , Alternatives subscale  and Control subscale .The test–retest reliability assessment was conducted with 262 undergraduate students who participated in a general questionnaire session (testing), ranging in age from 16 to 23 years (Table 2). The assessment was conducted with 269 undergraduate students who completed all measures comprising the BDI, ASQ, WC-R, and TRQ. They ranged in age from 17 to 24 years , 219 females  and 50 males . Given that greater cognitive rigidity on the CFI was associated with increasing depressive symptomatology  was randomly drawn from the total sample of undergraduate students. None of them met the DSM-5 depressive, anxiety and personality disorders’ criteria. Levene’s tests of homogeneity of variances revealed non-significant variance equalities among the groups, F = 1.148 (p > 0.05) for the Alternatives subscale, F = 3.205 (p > 0.05) the Control subscale, and F = 0.270 (p > 0.05) for total CFI-R. The t-statistic confirmed that the CFI-R’s total score and its subscales’ scores were significantly better for the non-clinical sample than for the clinical sample (see Table 3).Given that the CFI was developed to measure the type of CF needed to successfully challenge and replace maladaptive thoughts with more rational and balanced thinking in treatment context , we examIn the present study we examined the replicability of a two-factor structure and the psychometric properties of the CFI in Russian-speaking university students. Using exploratory and CFA we modified the original CFI. Given that the CFI-R was recommended with appropriate adaptation and further psychometric validation. EFA results indicated good internal consistency reliability for CFI-R and its subscales, and CFA results showed that a two-factor model of the CFI-R with a specified number of items fitted the data better than a two-factor model of the original CFI. The CFI-R was composed of 20 items – 12 items on the Alternatives subscale and 8 items on the Control subscale. Some modifications were made to the CFI to obtain the CFI-R included the revising items 14 and 19, originally developed to assess two aspects of cognitive flexibility: the ability to perceive multiple alternative explanations for life occurrences and human behavior and the ability to generate multiple alternative solutions to difficult situations, and items 1, 2 and 15, originally developed to assess one aspect of cognitive flexibility: the tendency to perceive difficult situations as controllable. It is important to note that A newly specified structure fitted the data better than the original CFI. All fit statistics indicated acceptable model fit for the model of the CFI-R. Results also provided evidence that the CFI-R and its two subscales had acceptable 7-week test–retest reliability.The concurrent validity of the CFI-R was confirmed by correlations between scores on the CFI-R and those on other related measures. The CFI-R was significantly and negatively related with BDI indicating that a person with a lower CF on the CFI-R was associated with increasing depressive symptoms on the BDI. Our results revealed a similar finding for associations of the original CFI and its two subscales with depressive symptomology . We alsoFurthermore, research has also shown correlations between the CFI-R and the ASQ. Greater cognitive rigidity on the ASQ was associated with greater CF on total CFI-R and the Control subscale, except with the Alternatives subscale. Although the results were consistent with the findings of the CFI developers, in this research we observed the significant associations between total CFI-R and both subscales and the rigidity subscales on the TRQ. Hence, further study is needed to better understand whether the construct of cognitive rigidity as measured by There was also evidence of convergent construct validity of the CFI-R. As expected based on prior research , the scoAdditionally preliminary support for the construct validity of the CFI-R was obtained via examining the known-groups validity. The results indicate that the CFI-R scores for participants who met the DSM-5 depressive, anxiety and personality disorders’ criteria were significantly worse than for participants who did not. This finding suggests that individuals with depressive symptoms display lower CF on the CFI-R which is indicative of greater cognitive rigidity associated with less cognitive adaptability when encountering stressful situations .Overall, the results of this study presented acceptable fit of the two-factor structure of the Russian language version of the CFI among a sample of university student, suitable reliability and validity with the exception of the associations between the CFI-R and AQS, which are unclear. This study suggests that psychometric properties of the Russian CFI are comparable to the English original, making it appropriate to research assessment of the type of CF necessary to successfully challenge and restructure maladaptive beliefs with more balanced and adaptive thinking in Russian speaking population.Several limitations should be considered in interpreting the study results. Firstly, we started from the Russian translation of the CFI and then confirmed the reliability and validity of its two-factor structure (with a specified number of items) that was consistent with the English original. This suggests that some impact of cross-cultural factors might play a role in the results of the factor analysis. Secondly, given that participants across the samples were predominantly female, a special focus should be made on the generalization of the results. It would be necessary to examine the influence of gender on the CFI-R total score and both subscales’ scores. Thirdly, the participants comprised the non-clinical sample. Future studies would benefit from assessing the reliability and validity of the CFI-R using clinical samples. A larger sample of clinical individuals would allow for a direct comparison of the factor structure in non-clinical and clinical samples. However, it is difficult to find individuals that are very similar in terms of their demographic and clinical characteristics.The findings of the study provide evidence for the satisfactory psychometric properties of the CFI by applying it to a sample of Russian university students, a population not previously studied. The factor structure of the CFI-R with a specified number of items is consistent with a two-factor model and showed an acceptable model fit. The internal consistency and test–retest reliability for the CFI-R and its two subscales were also adequate. As predicted, we indicated the negative associations of CF aspects measured by CFI-R with depressive symptomology, coping strategies considered to be maladaptive, and personality rigidity. The study succeeded in increasing the applicability of the Russian CF Inventory in research context and facilitated its utility as a measure in the treatment context.SK and EO conceived and designed the study and analyzed the data. EO carried out the implementation of the research. SK took the lead in writing the manuscript. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EditorThe following manuscript has been retracted from May-June 2015 issue on the request of theauthors who stated that “after publication, their group found that it was difficultto repeat the results. We believe that there may be some flaws or operational loopholes,hence we would like to retract this paper.”- Retraction in: Pak J Med Sci 2015;31(3):672-677. doi:http://dx.doi.org/10.12669/pjms.313.7170 Link:http://pjms.com.pk/index.php/pjms/article/view/7170


Synthetic macrocycles are an attractive area in drug discovery. However, their use has been hindered by a lack of versatile platforms for the generation of structurally (and thus shape) diverse macrocycle libraries. Herein, we describe a new concept in library synthesis, termed multidimensional diversity‐oriented synthesis, and its application towards macrocycles. This enabled the step‐efficient generation of a library of 45 novel, structurally diverse, and highly‐functionalized macrocycles based around a broad range of scaffolds and incorporating a wide variety of biologically relevant structural motifs. The synthesis strategy exploited the diverse reactivity of aza‐ylides and imines, and featured eight different macrocyclization methods, two of which were novel. Computational analyses reveal a broad coverage of molecular shape space by the library and provides insight into how the various diversity‐generating steps of the synthesis strategy impact on molecular shape. Macrocyclic compounds are associated with a diverse range of biological activities and they have proven therapeutic potential.Herein, we report the development a multidimensional DOS strategy towards highly functionalized macrocycles which uses fluorous‐tagged azido building blocks and exploits the pluripotent reactivity of aza‐ylides and imines Scheme .2 into 3 a,b. The aza‐ylide group served as the primary branching point and 3 a,b were reacted in situ in diverse aza‐Wittig‐type transformations to form 4–9  and X‐ray crystallography of one product .The DOS commenced with the transformation of building block 4 .In the pair stage of the DOS, the linear cyclization precursors were “folded” into diverse macrocycles with different ring architectures making use of the pluripotency of alkynes. Six known macrocyclisation methods were used initially Scheme  a–f. The4 Scheme  g. After6 into 5‐iodotriazole 24 c was achieved using an external iodide sourceIodo‐alkynes may be cyclized into their corresponding 5‐iodotriazoles, thereby generating another handle for macrocycle functionalization. Current strategies require the synthesis of the linear iodinated analog.54 a to form 61 was achieved using RuAAC . The presence of a fluorous‐tag on the common precursor 16 and which differ only in the linking motif installed in the coupling phase at the secondary branching point. The broad coverage of molecular shape space by this relatively small set of macrocycles validates the use of a secondary branching point as an efficient means to generate shape diversity. Figure Principal moments of inertia (PMI) plots are commonly used to visualize the shape diversity of compounds in “molecular shape space” spanned by the three basic extreme shape types “rod‐like”, “disk‐like” and “spherical”. Figure In summary, we have described a multidimensional DOS strategy for the step‐efficient generation of structurally complex and diverse macrocycles from simple and similar starting materials. Macrocyles of this sort are of significant interest from both a biological and synthetic perspective and their successful generation provides a validation of the multidimensional DOS concept. We report two new macrocyclization methodologies , which, we envisage will prove valuable in a wider synthetic context. PMI analysis indicated that the library has a relatively high level of shape diversity, thus demonstrating the utility of the multidimensional approach for the compound‐efficient coverage of substantial molecular shape space. Interesting insights were gained into the impact that the various diversity‐generating elements of the DOS have upon molecular shape; this new understanding may provide a basis for the deliberate synthesis of macrocycles with tailored molecular shapes.Multidimensional DOS integrates the B/C/P approach with branching diversification. The result is a modular and highly divergent concept for library synthesis that features systemic “product‐equals‐substrate” reactivity; functional groups generated in products become the new pluripotent handles for the next stage of the synthesis, which facilitates diversification at every branching point. This allows for the rapid generation of diversity and complexity from simple and similar starting materials. Thus, it is anticipated that the multidimensional concept will have broad strategic value in the DOS of other molecular libraries. Work is underway to expand this strategy to the synthesis of 12‐ to 14‐ membered rings and to study the substrate scope of the new macrocyclization methods reported herein.Dedicated to Professor Stuart L. Schreiber on the occasion of his 60th birthdayAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Thallus Sargassi Pallidi (HZ) and Radix Glycyrrhizae (GC), one of the so-called “eighteen antagonistic medicaments”, contains in HYD. To explore pharmacological mechanisms of HYD acting on iodine-deficient goiter and to provide evidence for potential roles of herb pair HZ and GC in HYD, our genome-wide microarray detection and network analysis identified a list of goiter-related genes, mainly involved into the alterations in hypothalamus-pituitary-thyroid/gonad/growth axes. Then, the disease genes-drug genes interaction network illustrated the links between HYD regulating genes and goiter-related genes, and identified the candidate targets of HYD acting on goiter. Functionally, these candidate targets were closely correlated with thyroid hormone synthesis. Moreover, the potential regulating genes of herb pair HZ and GC were revealed to be crucial components in the pathway of thyroid hormone synthesis. The prediction results were all verified by following experiments based on goiter rats. Collectively, this integrative study combining microarray gene expression profiling, network analysis and experimental validations offers the convincing evidence that HYD may alleviate iodine-deficient goiter via regulating thyroid hormone synthesis, and explains the necessity of herb pair HZ and GC in HYD. Our work provides a novel and powerful means to clarify the mechanisms of action for multi-component drugs such as herbal formulae in a holistic way, which may improve drug development and applications.Haizao Yuhu Decoction (HYD), a famous multi-component herbal formula, has been widely used to treat various thyroid-related diseases, including iodine-deficient goiter. Herb pair  These therapeutic strategies have advantages of low cost, new nodule formation prevention, marked goiter reduction and improvement in inspiratory capacity [Goiter represents thyroid pathological enlargement, which is characterized by the cellular proliferation of thyroid follicular cells with simultaneous pathological changes including the neo-angiogenesis, the accumulation and involution of colloid, and the hyperplasia of connective tissues . In clincapacity . Howevercapacity –10. TherThallus Sargassi Pallidi , Thallus Laminariae Japonicae , Rhizoma Pinelliae Preparata , Bulbus Fritillariae Thunbergii , Pericarpium Citri Reticulatae , Pericarpium Citrus reticulata , Radix Angelicae Sinensis , Rhizoma Chanxiong , Radix Angelicae Pubescentis , Fructus Forsythiae Suspensae  and Radix Glycyrrhiza , and is widely produced in China in accordance with the China Pharmacopoeia standard of quality control. HYD has prominent therapeutic effects in resolving “qi” stagnation and phlegm coagulation [Traditional Chinese Medicine (TCM), an essential component of the current medical system, has been extensively applied in clinical practice because of its valuable therapeutic efficacy with few adverse effects. There are various Chinese herbal formulae clinically prescribed to cure goiter by invigorating blood circulation, especially microcirculation, eliminating phlegm, and softening and resolving masses . Haizao gulation . Moreovegulation , 14. Espgulation , 16. NotChinese herbal formulae are complex mixtures of herbs consisting of multiple bioactive ingredients, which bind to the corresponding targets simultaneously, transiently, or weakly, and in combination to treat complex diseases in a systematic manner. Due to their complex nature and the lack of scientific methods, the molecular mechanisms of Chinese herbal formulae remain elusive. Novel and efficient approaches are urgently demanded for identifying mutual interactions and further interpreting the scientific basis of TCM formula. With the rapid advancement in bioinformatics, systems biology and polypharmacology, “network pharmacology” –18, shifTo explore the pharmacological mechanisms of HYD acting on iodine-deficient goiter and to provide evidence for the potential roles of the herb pair HZ and GC in this formula, in the current study, genome-wide microarrays based on thyroid tissues obtained from normal rats, goiter rat model, goiter rats treated with HYD or HYD deleted HZ/GC herb pair were detected to identify candidate goiter-related genes and HYD regulating genes. Then, the disease genes-drug genes interaction network was constructed, and the topological importance of nodes in the created network were evaluated to screen potential targets of HYD acting on goiter rats. After that, a series of experiments were performed to validation our prediction results Figure . This stLiquid chromatography triple quadrupole-tandem mass spectrometry (LC-QQQ-MS/MS) was performed to identify the chemical compounds of each herb containing in HYD. As shown in Compared to the normal rats, the serum levels of T3 and T4 were both significantly decreased, while the serum levels of TSH were dramatically increased in rats with oral administration of propylthiouracil for 14 days , suggestThen, we constructed the goiter imbalance network using interaction information among goiter deregulated genes. This network consists of 519 nodes and 1684 edges . A totalCompared to the goiter model rats, the serum levels of T3 and T4 were both significantly increased, while the serum levels of TSH were dramatically decreased in goiter rats treated with HYD for 28 days . The DEGBased on our previous developed target prediction system , a totalTo shed light on the pharmacological mechanisms of HYD acting on goiter, we constructed disease-related genes-drug-regulating genes network based on the interactions among 139 hub goiter deregulated genes, 10 known goiter related genes, 426 HYD regulating genes and 143 putative targets of HYD. This network consists of 422 nodes and 2039 edges .A total of 233 hub nodes, the degree values of which are more than two fold of the median degree of all nodes in the disease-related genes-drug-regulating genes network , were idAccording to the pathway enrichment analysis, the candidate targets of HYD acting on goiter were significantly associated with thyroid hormone synthesis, in which there were nine HYD candidate targets involved, including Adcy1 , Adcy2 , Creb1 , Hspa5 , Pdia4 , Plcb1 , Prkca , Prkcb  and Tpo  Figure , implyinTo verify this hypothesis, an independent experimental validation was performed. In line with findings based on samples of microarray analysis, the reduced levels of T3 and T4 in sera of goiter rats could be efficiently increased by the administration of HYD at 0.9 g/(kg·day) for 28 days ; After that, TG, carrying T4 and T3, is internalized into the follicular cell by micro- and macropinocytosis and digested in lysosomes; The thyronines T4 and T3 are released into the blood stream, while MIT and DIT are deiodinated by a dehalogenase and recycled [Thyroid hormones, one of mammalian hormones with halogen and iodide in the biochemical structure, play vital roles in several biological processes, such as metabolism of carbohydrates, lipids and proteins, and cell replication, in developing and adult mammals , 27. Durrecycled –30. Thisrecycled , 32. ConSince the normal function of the thyroid gland ultimately depends on appropriate iodide intake, it is extremely necessary to supply iodide for thyroid hormonogenesis. Several herbs containing in HYD, such as HZ and KB, have been indicated to be rich in iodine according to the previous chemical identification –35. AccuAccording to the Chinese medicinal literature, the herb pair HZ-GC is defined as one of “eighteen antagonistic medicaments”, which means that the two herbs should not be used in the same prescriptions in order to avoid mutually interference. However, this herb pair has been applied in HYD to treat thyroid-related diseases for several decades. To clarify its roles in HYD, we compared the therapeutic effects , and the gene expression profiles of goiter rats treated with HYD and HYD deleted HZ ad GC. As a result, although HYD-non-HZ and/or GC treatment had a trend to amolierate the alterations of goiter rats, the differences were not more significant than HYD treatment, suggesting that the therapeutic effects of HYD-non-HZ and/or GC treatment were weaker than HYD treatment. Notably, our network target prediction and experimental validation both confirmed the implications of the herb pair HZ-GC regulating targets into the synthesis of thyroid hormone, highlighting the critical roles of this herb pair in HYD acting on goiter.In conclusion, this integrative study combining microarray gene expression profiling, network analysis and experimental validations offers the convincing evidence that HYD may alleviate iodine-deficient goiter via regulating thyroid hormone synthesis and explains the necessity of the herb pair HZ and GC in HYD. Our work provides a novel and powerful means to clarify the mechanisms of action for multi-component drugs such as herbal formulae in a holistic way, which may improve drug development and applications.http://tcm.cmu.edu.tw/, Updated in 2012-06-28). The detailed information on the Chemical compounds containing in HYD is described in Chemical compounds containing in HYD were obtained from TCM Database@Taiwan  . We only used those drug-target interactions whose drugs are FDA approved for the treatment of goiter and whose targets are human genes/proteins. Totally, we obtained three known therapeutic targets of goiter . (2) Kyoto Encyclopedia of Genes and Genomes (KEGG) Disease Database [http://www.genome.jp/kegg/, Last updated: October 16, 2012). Totally, we obtained eight known goiter-related genes . After deleting redundancy, there were ten known goiter related genes collected in this study.Known goiter-related genes were obtained from the following two databases: (1) DrugBank database   were purchased from Charles River laboratories (production license No: SCXK 2012-0001). The rats were divided into two categories: 15 Wistar rats for microarray analysis and 32 Wistar rats for independent experimental validations. All rats were housed in a room with a constant temperature of 24±1°C (mean±SEM) and with a 12-hour light/dark cycle. Rats were allowed free access to food and water.All herbs contained in HYD were purchased from Beijing Tongrentang pharmacy. The HYD used in this study was manufactured by Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences  using 11 Chinese herbs at a composition of including HZ (5.5g), KB (9g), FBX (9g), LQ (9g), ZBM (9g), QP (9g), DH (9g), DG (9g), CX (9g), CP (9g) and GC (9g). The mixtures of crude herbs were immersed in 10 volumes of distilled water (v/w) for 30 min, followed by boiling for 1 h. Then, the residue was extracted again for 0.5 h with 5 volumes of water (v/w). Two supernates were both filtered through four layers of gauze, and finally pooled and concentrated to a final density of 0.09 g/mL. Meanwhile, HYD without HZ and/or GC (Non-HZ and/or GC) were separately prepared following the same procedures as mentioned above. In addition, the representative chemical compounds of each herb containing in HYD were determined by LC-QQQ-MS/MS.For microarray analysis, 15 male/female rats were divided into five groups with the equal number of 3: normal control group (con), goiter model group (mod), goiter rats treated with HYD formula (HYD-treatment group), goiter rats treated with HYD formula without HZ (Non-HZ group) and goiter rats treated with HYD formula without GC (Non-GC group). For independent experimental validation, 32 male/female rats were also divided into four groups with the equal number of 8: normal control group (con), goiter model group (mod), goiter rats treated with HYD formula (HYD-treatment group) and goiter rats treated with HYD formula without HZ and GC (Non-HZ-GC group).Goiter rat model caused by iodine deficiency was constructed by oral administration of propylthiouracil at a dosage of 0.01 g/(kg·day) for 14 days. Drug treatment of goiter was delivered by oral administration for 28 days. The dosage selection for each treated groups were set at 0.9 g/(kg·day) on the basis of clinical dosage for human. Rats in the control and goiter model groups were all administrated by an equal volume of saline.2 ratio ≥ 1 or log2 ratio ≤ −1 and P-value < 0.05 are tested for further analysis. The gene expression microarray data of GSE76817 were obtained from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus .Thyroid tissues were collected from rats in four groups, immersed in Trizol  and frozen in liquid nitrogen immediately for further microarray detection. Fluorescent aRNA targets were prepared from total RNA samples that were pooled from thyroid tissues using Amino Allyl MessageAmp TM II aRNA Amplification Kit  and Cy5 dyes . Then, the fluorescent aRNA targets were hybridized to the Rat OneArrayR v2 , scanned with an Agilent's High-Resolution C Scanner  and finally analyzed by GenePix 4.1 software (Molecular Devices). The spots with log2 fold change (FC)|>0.5. The hierarchical clustering analysis was performed for the identified DEGs according to the heat map package in R . Cluster analysis of the DEGs was performed by Cluster 3.0, based on Euclidean distance.Significant DEGs of the control vs. goiter rat model groups, HYD vs. goiter rat model groups, Non-HZ vs. HYD groups, Non-GC vs. HYD groups were identified using the criteria of P value <0.05 and |loghttp://string-db.org/) [The goiter-related-gene interaction network and the goiter-related-gene-HYD-regulating-gene interaction network were constructed based on the public database STRING   with theFour topological features, node's degree, betweenness, closeness and k-coreness, were calculated to evaluate the importance of a node in the interaction network. The definitions of these network topological features are described in http://www.genome.jp/kegg/, Last updated: Oct 16, 2012).Pathway enrichment analysis was performed using pathway data obtained from the FTP service of KEGG  . Blood samples were collected from the abdominal artery and immediately centrifuged at a speed of 3000 r/min for 15 min at 4°C to obtain serum, which were stored at −80°C for biochemical analysis. The serum levels of T3, T4 and TSH in different groups were determined by enzyme-linked immunosorbent assay (ELISA) using the corresponding commercially available kits according to the protocol from manufactures . All experiments were done in triple.After the tissue collection, the thyroid tissues were divided into two categories. One was fixed in 4% paraformaldehyde and embedded in paraffin. Then, 4 μm-thick tissue sections were prepared and stained with hematoxylin and eosin (H&E) for histological studies. H&E stained areas were viewed by the application of an optical microscope  to assess the pathologic alterations of thyroid tissues in different groups and to monitor the therapeutic effects of the treatment. All sections were evaluated by two observers who were blinded to the experimental conditions of the animals. The other part was immediately stored in liquid nitrogen at −80°C for real-time PCR analysis.−ΔΔCt method [Gene expression levels of candidate targets of HYD or the herb pair HZ-GC in thyroid tissues obtained from different groups were detected by quantitative real-time PCR. SYBR Green 1 kit was applied according to the manufacturer's protocol  and the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Then, 2 μg of total RNA was used for reverse transcription by using ABI High-Capacity cDNA reverse transcription Kits. The primer sequences of candidate target genes were listed in t method .Protein expression levels of candidate targets of HYD or the herb pair HZ-GC in thyroid tissues obtained from different groups were detected by Western blotting analysis as described in our previous study . AntibodP values less than 0.05 were considered as statistically significant.All statistical analyses were performed by the software of SPSS version 11.0 for Windows . Our data expressed as means ± standard deviation (SD). All parameters were analyzed by one-way analysis of ANOVA followed by LSD test.


Introduction: We evaluated and compared five currently available energy-based vessel sealing devices to assess typical surgical metrics.Methods: We tested Caiman 5 (C5), Harmonic Scalpel Ace Plus (HA), Harmonic Ace +7 (HA7), LigaSure (LS), and Enseal G2 (ES) on small (2–5 mm), medium (5.1–7 mm), and large (7.1–9 mm) vessels obtained from 15 Yorkshire pigs. Vessels were randomly sealed and transected. We recorded sealing and transection time, charring and carbonization, thermal spread, and bursting pressure (BP). Specimens were sent for histopathologic evaluation of seal quality and thermal spread.Results: A total of 246 vessels were evaluated: 125 were arteries and 121 were veins. There was no difference in BPs for small size arteries. For medium arteries, C5 provided the highest BP , followed by HA7, ES, LS, and HA . For large arteries, C5 and LS provided highest BP followed by HA7, ES, and HA . There were no bursting pressure failures for C5, HA7, and LS up to 9 mm vessels. For medium and large size arteries, HA had bursting failure of 20% and 40%, respectively. The ES was significantly less efficient with small, medium, and large arteries with bursting failure rates of 10%, 40%, and 80%, respectively.Conclusions: In this study, C5 outperformed all other devices. However, all of the devices provide a seal that was superphysiologic in that all burst pressures were >250 mm Hg. Contemporary bipolar devices utilize electrothermal energy to seal blood vessels and reduce damage to surrounding tissues by preventing electric current from spreading beyond the jaws of the device. In comparison, ultrasonic devices utilize high-frequency vibrations instead of electrical current but the energy is again limited to being delivered just between the jaws of the device with scant lateral spread.Traditional monopolar devices are associated with unpredictable and weak vessel sealing and increased lateral thermal spread.The reliability of vessel sealing using energy devices is clinically vital; in this regard, it is important for the seal quality to be consistent. Continuous research and technological advancements have significantly improved contemporary VSDs. New design improvements have been developed to provide more consistent energy delivery and a feedback loop to optimize seal quality.in vivo animal model with regard to mesenteric transection time, vessel transection time, vessel sealing time, jaw temperature, thermal spread, and seal burst pressures.We evaluated and compared five contemporary commercially available energy-based VSDs in an We obtained an Institutional Animal Care and Use Committee approval to perform the study experiments. We evaluated five VSDs: the Caiman 5 , Harmonic Scalpel Ace Plus , Harmonic Ace +7 , LigaSure , and Enseal G2 . We used each VSD according to the settings recommended by the manufacturer.in vivo dissection, ex vivo testing of bursting pressure, and finally histopathologic examination of vessel seals.There were three phases to the study methodology, including We used 15 Yorkshire pigs (30–35 kg) to evaluate the 5 devices . The surgeons performing the testing (Z.O. and J.L.) were experienced with all the energy devices. Despite surgeon familiarity, one pig was utilized to train the surgeons on tissue and vessel sealing in the porcine model to mitigate any device-specific learning curve.After adequate general endotracheal anesthesia, a midline incision was made from xiphoid process to symphysis pubis. Once exposed, we inked the colonic mesentery at 15-cm increments and performed a timed mesenteric transection. Mesentery transection time was defined as the time needed to dissect a 15 cm segment of small bowel mesentery. For each VSD, we performed a total of 10 transections 15 cm in length. Devices were cleaned between each transection to maintain device performance. The surgeon rated charring/carbonization, tissue sticking, seal quality, and transection quality on a 1–5 scale , medium (5.1–7 mm), and large (7.1–9 mm). Testing on each VSD was performed on each of these three categories. The C5 instrument has a special jaw-closing mechanism with an additional hinge at approximately half of the length of the lower jaw part. This results in even pressure distribution and tissue compression within the jaws and enables an increased jaw length in comparison with scissor-like jaw mechanisms. As such, for C5, we tested sealing and transection properties in the proximal and distal jaws separately for small and medium vessels. For large vessels, testing was performed in the middle component of C5 only as the vessels were too large to fit in the separate components. We evaluated sealing and transection of vessels with all other devices, with the vessels secured in the middle of the jaw. Once sealed and transected, each VSD and vessel was visually rated by the surgeon for charring/carbonization, tissue sticking (1 best to 5 worst), and seal quality (1 best to 5 worst) using standardized scoring criteria . In addiin vivo sealing, we performed the bursting pressure in an ex vivo setting as described previously.3 We inserted an 18-gauge angiocatheter into the lumen of each vessel tested. We allowed at least 1 cm vessel length to provide enough space between the needle tip and the vessel seal. Once inserted, we secured the angiocatheter to the vessel with a silk suture and a hemostat clamp. The angiocatheter was then attached to both a digital pressure manometer  and a saline pump with tubing. Physiologic saline was instilled at a steady rate until leakage or vessel bursting was observed on the sealed section of the vessel. Bursting pressure was recorded in mm Hg.After 5–7We defined the arterial bursting failure as vessel bursting less than a pressure of 300 mm Hg. We defined the venous bursting pressure failure as vessel seal bursting at a pressure <30 mm Hg.For histopathologic evaluation of thermal spread and seal quality, we collected treated vessels that had not been burst tested after sealing. These vessels were placed into 10% neutral buffered formalin and subsequently were embedded in paraffin wax and sectioned. These segments were then evaluated by an expert pathologist for thermal damage and seal quality (hematoxylin and eosin staining). The pathologist was blinded as to which VSD was used. The extent of thermal energy damage was defined by measuring the length of coagulation necrosis from the seal.F-test, Kruskal–Wallis test) to test for the differences among the groups; each device served as an independent grouping factor. Differences between distal and proximal vessel sealing were explored by adding an additional grouping factor. We used SYSTAT statistical software, version 13.0, to analyze the data . Statistical significance was defined as p-values <0.05.We used analysis of variance ; ES>HA(<0.001); ES>HA7(<0.001); LS>C5(0.001); LS>HA(<0.001); LS>HA7(<0.001)]. There were no differences in the median charring/carbonization scores . The median tissue sticking scores were 1.0, 1.2, 1.6, 1.0, and 4.2 for C5, HA, HA7, ES, and LS, respectively . Transection quality was equal among all VSDs: 1 for C5, 1 for HA, 1.2 for HA7, 1.2 for LS, and 1.0 for ES (no pairwise differences were significant). Maximum jaw temperature recorded during the mesenteric transections was highest for HA7 followed by HA, ES, C5, and LS ; HA7>ES(<0.001); HA7>HA(<0.001); HA7>LS(<0.001); HA>C5(<0.001); HA>ES(<0.001); HA>LS(<0.001); C5>LS(0.04); ES>LS(0.002)].in vivo failure, mean vessel bursting pressures, and mean maximum jaw temperature for arteries and veins comparing all VSDs are given in Mean vessel sizes, percentage of p = 0.046; no pairwise differences were significant).For small arteries, seal quality scores were equal for distal and proximal C5, HA, HA7, and LS, which were superior to ES . For medium size arteries, seal quality scores for distal and proximal C5, HA, HA7, LS, and ES were 1.0, 1.0, 1.0, 2.8, 1.6, and 1.4, respectively, . For large size arteries, the mean seal quality scores were 1.3, 1.0, 3.7, 1.0, and 1.0 for C5, HA, HA7, LS, and ES, respectively ; HA<LS(0.002); HA7<LS(0.006); HA<ES(0.006); HA7<ES(0.017)]. Tissue sticking scores for medium size arteries were better for distal and proximal C5 followed by HA, ES, HA7, and LS . Tissue sticking for large arteries was superior for HA7 and ES followed by C5, LS, and HA .Tissue sticking scores for small arteries were significantly better for distal and proximal C5, HA, and HA7 than for LS and ES . Charring and carbonization for medium size arteries were 1.2, 1.2, 1.3, 1.0, 4.5, and 2.3 for distal and proximal C5, HA, HA7, LS, and ES, respectively . Charring and carbonization for large arteries were 2.3, 2.3, 1.0, 2.3, and 1.0 for distal and proximal C5, HA, HA7, LS, and ES, respectively .The LS resulted in significantly more charring and carbonization than the other devices. Specifically, charring and carbonization for small arteries were 1.4, 1.6, 1.1, 1.4, 2.8, and 1.3 for distal and proximal C5, HA, HA7, LS, and ES, respectively . For medium size veins, seal quality scores were better for distal and proximal C5, HA, and LS and inferior for HA7 and ES . For large size veins, C5 and LS demonstrated significantly better seal quality followed by HA, ES, and HA7 .For small size veins, seal quality scores were better for distal and proximal C5, LS and inferior for HA, HA7, and ES . Tissue sticking scores for medium size veins were better for distal and proximal C5 and ES followed by HA, HA7, and LS . Tissue sticking for large veins was superior for C5, HA7, and ES followed by HA and LS .Tissue sticking scores for small veins were significantly better for distal and proximal C5, HA, and HA7 and inferior for LS and ES . Charring and carbonization for medium size veins for distal and proximal C5, HA, HA7, LS, and ES were 1.8, 1.4, 1.3, 1.0, 4.2, and 2.2 . Charring and carbonization for large size veins for distal and proximal C5, HA, HA7, LS, and ES were 1.7, 2.3, 1.0, 2.3, and 1.3 .Charring and carbonization for small veins were significantly better for distal and proximal C5, HA, HA7, and ES and inferior for LS [1.2, 1.6, 2.1, 1.3, 2.7, and 1.3, respectively, p = 0.721; no pairwise differences were significant). For veins, C5, HA, HA7, LS, and ES had a mean thermal spread of 2.1, 2.9, 2.3, 3.4, and 3.9, respectively . Example slides of arterial and vein seal quality for each VSD are shown in Thermal spread for each VSD is provided in 9Contemporary VSDs differ in instrument design and the type of energy they deploy in the act of sealing and transecting arteries and veins during laparoscopic procedures. Although early generations of ultrasonic VSDs were limited in use to 3 mm vessels, subsequent advances, including tissue feedback algorithms, have allowed contemporary technology to be relevant in addressing vessels up to 7 mm.8 Specifically, we aimed to test the VSDs on small (2–5 mm), medium (5.1–7 mm), and large (>7 mm) arteries and veins. For the bursting pressure trials, C5, at both distal and proximal jaws, provided the highest bursting pressure measurements across all vessel sizes. For small and medium size vessels HA7, LS, and ES also demonstrated equivalent bursting pressures. However, all three VSDs demonstrated significantly lower bursting pressures when they were used for large size vessels. HA demonstrated the lowest bursting pressures for all three vessel sizes.The primary outcome of this study was to test vessel sealing quality of five contemporary VSDs measured by the maximum bursting pressure, which defines the efficacy of VSD.10 Again, C5, HA7, and LS were all able to effectively seal arteries and veins up to 9 mm with no failures. HA and ES performed slightly less favorably. For medium and large size arteries, HA had bursting failure of 20% and 40%, respectively. ES was significantly less efficient with small, medium, and large arteries with bursting failure rates of 10%, 40%, and 80%, respectively.Bursting pressure failure was defined by threshold of 300 mm Hg for arteries and 30 mm Hg for veins.12 In addition, adequate and even compression force throughout the entire length of the jaw is crucial for the creation of a stable and reliable seal.14 Uniform compression is very much related to the jaw configuration of the VSD. Most contemporary VSDs utilize either a scissor-like or pivoting jaw design. It has been shown that the scissor-like design is associated with a less uniform distribution of compressive forces with decreasing pressure force from the proximal to the distal end of the jaws, resulting in a weaker sealing quality at the tip of the instrument.13 C5 is designed with a novel pivoting jaw to address the limitations of the scissor-like jaw configuration. Specifically, the C5 device evaluated in this study consists of a novel pivoting jaw design and closing mechanism with the sealing electrodes distributed in both the upper and lower jaws.14 This mechanism allows for a more homogeneous pressure distribution in the entire jaws. Our data with the C5 support these earlier findings.The quality of vessel sealing and amount of thermal spread are dependent on several factors, including vessel type, vessel size, temperature, time, and the type of energy used.5 For both mesenteric and vessel tissues, ES and LS demonstrated the lowest jaw temperatures, whereas HA and HA7 consistently demonstrated the highest jaw temperatures across all vessel sizes. These data are consistent with previously reported studies.16 ES and LS employ a pulsed bipolar energy and a feedback energy control output during tissue sealing and transection, which maintain the jaw temperature levels below 100°C.17 However, of even greater importance than the recorded jaw temperature is the degree of coagulation necrosis on either side of the jaws. In this regard, HA and HA7 had the least thermal spread for both veins and arteries compared with the other three VSDs. It may seem counterintuitive that the devices with the highest jaw temperature resulted in the least energy damage on histopathology. However, it is clear that many factors affect tissue response. The advantage of high energy is because of a feedback loop that delivers fast and highly efficient energy with the least amount of transection time. Reyes and colleagues evaluated optimal temperature and duration of clamp time to achieve a complete vessel seal. Vessel seals tested at 40°C, 60°C, and 80°C demonstrated 25%, 17%, and 2.8% failure rates, respectively. There were no failures at 90°C applied for 10 seconds. These investigators concluded that average of 2.4–3.8 MPa (348–551 psi) pressure force with 90°C with at least 10 seconds of clamp time is required for optimal and reliable vessel sealing.10 Current prices of energy devices are HA7—$507, HA—$48, C5—$450, LS—$470, and ES—$487. These prices were taken from the Emergency Care Research Institute (ECRI) national supplies database that captures reported purchase price from hospitals across the country .All VSDs should be used with great care adjacent to vital organs, and a margin of at least 5 mm is recommended to avoid any thermal damage.16 in which they demonstrated that LS had poor tissue sticking results compared with ES and HA.Finally, we evaluated tissue sticking, charring, and carbonization for all VSDs. Tissue sticking on the instrument's jaws can create increased resistance to energy transmission, thereby prolonging the sealing time and subsequent coagulation necrosis on either side of the jaws. C5, in both proximal and distal jaw positions, had the lowest tissue sticking scores followed by HA, ES, LS, and HA7 when used on small and medium vessels. However, for large arteries, ES and HA7 demonstrated best results followed by C5, LS, and HA. Our data are similar to results reported by Milsom and colleagues,Overall, all VSDs created a reliable seal for small and medium size arteries. For large arteries, C5, HA7, and LS were the most reliable VSDs with the highest bursting pressures and no bursting failures <300 mm Hg. HA and HA7 are associated with highest jaw temperatures but both provide the fastest sealing and transection time with less thermal damage.This study has several limitations. The main limitation is that the findings for VSDs in an animal model may not be similar in humans. However, previous comparison studies have shown that swine is the most optimal model for studying vessel anatomy and physiology. Another limitation is that all procedures were performed through an open rather than laparoscopic approach. The two environments are markedly different with respect to air flow and other parameters that might affect jaw temperature. In addition, the list of energy devices evaluated in this study is not exhaustive. There are multiple other devices that are routinely used in clinical practice in the United States. However, our goal was to test the most commonly used devices and compare them with a novel Caiman device. In addition, our sample size was limited to only 10 trials with each instrument. Finally, all vessels were evaluated by two surgeons who were not blinded and this may have introduced some bias to the study results.All tested VSDs provided excellent seal quality as corroborated by histopathology and supraphysiologic bursting pressures. C5 provided the highest bursting pressure measurements at both proximal and distal jaws and consistently scored better than the other devices with regard to tissue sticking, charring, and carbonization. However, HA and HA7 provided the least thermal damage, whereas LS and ES provided the lowest jaw temperatures, although with a broader area of thermal damage.


The preparation process did not involve chemical treatments, such as removal of protein and calcium carbonate with sodium hydroxide and hydrochloric acid, respectively. Thus, it was economically and environmentally friendly. The nanofibers obtained had uniform width and dispersed homogeneously in water. Nanofibers were characterized in morphology, transparency, and viscosity. Results indicated that the shell was mostly disintegrated into nanofibers at above five cycles of the HPWJ system. The chemical structure of the nanofiber was maintained even after extensive mechanical treatments. Subsequently, the nanofiber complex was found to improve the growth of tomatoes in a hydroponics system, suggesting the mechanical treatments efficiently released minerals into the system. The homogeneous dispersion of the nanofiber complex enabled easier application as a fertilizer compared to the crab shell flakes.A protein/CaCO Chitin is a highly abundant carbohydrate polymer occurring primarily in crab shells. Crab shells have a hierarchically-ordered organization . The rig3/chitin nanofiber complex in detail, and discuss its potential application as a plant fertilizer since it is well known that crab shells show enhancement of plant growth.The chitin in crab shells is partially preserved in the fishing industry as an intermediate of chitosan and glucosamine, even though the other parts of the shell are disposed of as industrial waste. The chitin is prepared from crab shells by treatment with NaOH and HCl aqueous solutions to remove proteins and calcium carbonate . A majorw/w, respectively, indicating the largest proportion of the red crab shells was calcium carbonate.Red snow crab shells were used as a starting material in this study. The contents of the main components in the crab shells  were estimated by the ninhydrin-hydrindantin protein test and gravimetric analysis; they were approximately 30%, 16%, and 55% as 3/chitin nanofiber complex was successfully obtained from crab shells by HPWJ mechanical treatments. To further characterize the morphology of the nanofiber, calcium carbonate was removed from samples treated with 0, 1, and 5 passes. Calcium carbonate was neutralized by HCl and dissolved in water as CaCl2. SEM images revealed that nanofibrillation of the crab shells proceeded by repeated HPWJ treatments and appeared complete at 5 passes  from those of protein/CaCO3/chitin nanofibers with respect to the number of leaves and stem diameter of the plants. However, the effects of the two supplements on the height of the tomatoes after five treatments were significantly different (p < 0.05); with the protein/CaCO3/chitin nanofibers exhibiting the better effect on tomato growth. The difference was attributed to the effect of mechanical treatment in producing the fast release of minerals deposited in the nanofibers network to the hydroponics system. In addition, mechanically-disintegrated protein/CaCO3/chitin nanofibers were easy to apply due to their liquid state, compared to the insoluble, roughly-crushed crab shell powder.Tomato plants grown in a hydroponic system were treated with nanofibers once a week. After the fifth nanofiber treatment, the tomato plants treated with protein/CaCOcontrols , Table 13/chitin nanofibers or crab shells showed healthy growth. These results indicated that the protein/CaCO3/chitin nanofibers might act as a fertilizer. Plants require not only essential elements—such as nitrogen, phosphorus, and potassium—but also calcium, sulfur, magnesium, and trace minerals for their growth. Although chitin nanofibers and protein/chitin nanofibers contain essential nitrogen, these nanofibers might not contain sufficient amounts of minerals to promote plant growth. Even though the effects of protein/CaCO3/chitin nanofibers were not as dramatic as those of the commercial nutrient HYPONeX; after further improvements, nanofibers might show good performance as a fertilizer. The results in 3/chitin nanofibers, which was roughly crushed by grinder treatment with two passes, might be a cost-effective material for use as a fertilizer for plant growth.On the other hand, neither protein/chitin nanofibers nor chitin nanofibers had any effect on tomato growth. Tomato plants treated with protein/chitin nanofiber, chitin nanofiber and distilled water (DW) died at the ninth nanofibers treatment; whereas tomato plants treated with protein/CaCOChionoecetes opilio (red snow crab) and α-chitin powder with a 6.4% deacetylation degree were obtained from Koyo Chemicals. Other chemicals were purchased from Aldrich or Kanto Chemicals and were used as received. Tomato seeds were purchased from the Marutane Seed Co., Ltd., Kyoto, Japan.The raw shells of Raw crab shells were roughly crushed with a domestic blender under wet conditions. The crushed sample was then disintegrated with a grinder  at 1500 rpm for two cycles. The air bubbles were removed by a super stirrer . The concentration of the total solid was adjusted to 3.6 wt % for a chitin weight percentage of around 1 wt %. The samples were then passed through a high-pressure water-jet system  equipped with a ball collision chamber. The slurry was ejected from a small nozzle with a diameter of 100 µm under high pressure of 200 MPa. This mechanical treatment was repeated for 1, 5, 10, 30, and 50 cycles.3/chitin nanofiber complex. The protein/chitin nanofiber complex was prepared using the previously reported procedure, with slight modifications [Protein/chitin nanofiber complex and pure chitin nanofibers were prepared in order to compare their effects on tomato growth with those of the protein/CaCOications . BrieflyFor comparison, pure chitin nanofiber was also prepared as described in our previous study, and then modified as follows . Dry pur−1. The light transmittances of the protein/mineral/chitin nanofiber complex dispersion were measured using a UV-Vis spectrophotometer . The viscosities of the complex slurries were measured with a Brookfield digital viscometer DV-E using the LV-4 spindle  at 30 °C. The protein, calcium carbonate, and chitin contents of the original crab shells were estimated by means of a ninhydrin-hyrindantin protein test [For field emission scanning electron microscopic (FE-SEM) observation, the disintegrated samples were diluted with ethanol and dried in an oven. The dried samples were coated with an approximately 2 nm layers of platinum, using an ion sputter coater, and were observed by an FE-SEM  operating at 2.0 kV. Infrared spectra of the samples was recorded with an FT-IR spectrophotometer  equipped with an ATR attachment  with four scans at a resolution of 4 cmein test  and gravSolanum lycopersicum) cv. Chibikko (Marutane Seed) was grown hydroponically with rockwool grow cubes . Three weeks after seeding, the tomato plants were treated with a series of nanofibers, which contained chitin at a final concentration of 0.01% to each rockwool cube. The number of true leaves, the stem diameter (at cotyledon node), and the plant height (stems length above the cotyledon) were measured after the fifth and ninth nanofiber treatment. The plants were grown under environmentally-controlled conditions with 14 h of light/10 h of dark cycles at 25 °C, and treated with nanofibers once a week. Distilled water (DW) was used as a negative control, and commercial nutrition HYPONeX  at a concentration of 0.001% was used as a positive control. Roughly-crushed crab shell powder was treated as a control.Tomato (3/chitin nanofiber complex was prepared from crab shells using a simple mechanical treatment. Because this nanofiber preparation process did not include the conventional protein and calcium carbonate removal processes, it could bring down the production cost and environmental load. The resulting protein/CaCO3/chitin nanofiber would be available as a fertilizer to improve plant growth in hydroponic systems. Indeed, the mineral part of the nanofiber complex was found to be vital for plant growth and maturation. Finally, the novel nanofiber complex could be prepared at a low cost through an eco-friendly process. Thus, the results of this close analysis of the unique nanofiber-complex characteristics could lead to more effective utilization of crab shell waste.A protein/CaCO


It is well-known that high levels of Synaptic vesicle 2C proteins (SV2C) were found primarily in old brain regions like pallidum, substantia nigra, midbrain, brainstem, and olfactory bulb. They have also show that SV2C level was relatively low in the cerebellar cortex and untraceable in the hippocampus and cerebral cortex. Synaptic vesicle protein family members are closely related and they are distributed in general (SV2A) or in a very restricted manner (SV2C) depending on their functional regulation of synaptic vesicle proteins . Dardou et al. have suggested that SV2C may contribute significantly in the regulation of neurotransmitter release and synaptic transmission in the basal ganglia, including cholinergic striatal interneurons and nigrostriatal/mesolimbic dopamine neurons  mediated protection of Parkinson's disease (PD) is due to genetic modulation of the SV2c gene  mice and proved that SV2C plays a significant role in dopamine function inside the basal ganglia. They have also performed HPLC quantification of dopamine content and its metabolites in SV2C-KO mice model. Authors also investigated the alternation in motor behavior and dopamine content in PD due to loss of α-Synuclein (α-Syn) and subsequent interaction with SV2C. In the presence of nicotine, SV2C-KO mice show a significant decrease in dopamine release in the dorsal striatum compared to wild-type mice. Lastly, they link the altered expression of SV2C with a mutation in α-Syn in the basal ganglia of postmortem neurodegenerative cases including PD (Dunn et al., Dunn et al. proposed that alteration in SV2C significantly affect the dopamine release and neurotransmission leads to PD pathogenesis. SV2C is expressed in the vesicles of dopaminergic neurons and there is a significant reduction of synaptic release of dopamine following genetic deletion of SV2C, this cause reduced motor activity in PD. They have suggested the isolated neurochemical response to nicotine in SV2C-KO mice and the neuroprotective effect of nicotine is mediated through SV2C. Finally, they have demonstrated that in humans with PD and in mice that express mutated α-Syn, there is a specific and significant disruption of SV2C expression. Collectively, this paper has suggested that SV2C plays a significant role as a mediator of dopamine homeostasis and acts as a possible and potent player in the pathogenesis of PD (Dunn et al., PD is characterized by death of neurons in the mesencephalon that utilize dopamine as a neurotransmitter for synaptic communication. If dopamine is not properly stored and metabolized it causes severe cytotoxicity to dopaminergic neurons. Thus, the researchers have tried to target any of the pathways that tightly control this neurotransmitter and holds greater therapeutic expectations (Bisaglia et al., The paper published by Dunn et al. does explain the potential role of SV2C in the pathogenesis of human neurodegenerative diseases. Although this article does not show any potential relationship between VMAT2 and SV2C since both are involved in dopamine homeostasis. Authors have gone through the idea of making SV2C-KO mice, and then they should also have thought of making VMAT2-KO mice too. This is suggested because both VMAT2 and SV2C are equally responsible for taking up the free dopamine from the cytosol, store it into synaptic vesicles and release at the time of requirement, hence playing a synergistic role in maintaining dopamine homeostasis. Moreover, authors have also not observed the relationship between SV2C and MAO-B. MAO-B converts free dopamine present in the cytosol to toxic dopamine metabolite, thereby causing trouble in L-Dopa therapy given to PD patients. Additionally, the level of SV2C, VMAT2, and MAO-B should have been studied first at the transcriptional level in healthy and control mice, and then SV2C-KO mice should have been made to justify their results more appropriately. This would have resulted in a correct understanding of dopamine homeostasis. Authors should also have seen the effect of the α-Syn mutation on VMAT 2 expression too, as the mutation might result in decreased VMAT2 expression along with SV2C expression.Overall, this interesting study opens a window to search for therapeutic modifiers which can modulate expression of SV2C in dopaminergic neurons to inhibit PD pathogenesis which is subject to further investigations.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The extracellular matrix (ECM) is a fibrillar protein-based network, the physical and chemical properties of which can influence a multitude of cellular processes. Despite having an important role in cell and tissue signaling, a complete chemo-mechanical characterization of ECM proteins such as fibronectin (FN) is lacking. In this study, we engineered monodisperse FN nanofibers using a surface-initiated assembly technique in order to provide new insight into the elastic behavior of this material over large deformations. FN nanofibers were patterned on surfaces in a pre-stressed state and when released from the surface underwent rapid contraction. We found that the FN nanofibers underwent 3.3-fold and 9-fold changes in length and width, respectively, and that the nanofiber volume was conserved. Volume was also conserved following uniaxial extension of the FN nanofibers of ~2-fold relative to the patterned state. This data suggests that the FN networks we engineered formed an incompressible material with a Poisson’s ratio of ~0.5. While the Poisson’s ratio of cells and other biological materials are widely estimated as 0.5, our experimental results demonstrate that for FN networks this is a reasonable approximation. Fibronectin (FN) is a ubiquitous ECM glycoprotein of particular interest because it is the initial component of the ECM during embryonic development and wound healing, serving as a provisional scaffold for the assembly of other ECM fibrils such as collagen5. Structurally, FN is a disulfide-linked, 440 kDa homodimer with multiple binding sites including those for integrins6, collagen5, heparin7, growth factors9, and self-assembly (polymerization)10. Some of these sites are cryptic and require conformational changes of the secondary and/or tertiary structure from a compact, folded state to an extended, unfolded state in order to become exposed and active. In many cases these cryptic sites are exposed under cell-generated forces, forming an insoluble signalling network critical to normal cell communication. Thus, understanding how FN fibrils are mechanically manipulated by cells to expose cryptic domains is critical in developing a mechanistic model of ECM mechanobiology.The extracellular matrix (ECM) is a heterogeneous network of protein-based fibrils that constitutes the physical and chemical properties of the tissue microenvironment and mediates cell processes such as adhesion, differentiation, migration and signallingin vitro or in vivo is challenging because there is a large variability in the length and diameter of cell-derived FN fibers, and these fibers are typically integrated into a more complex ECM containing many other molecules. This limits our ability to study how strain-induced changes in molecular conformation relate to their mechanics and biological function. To address this, researchers have developed several FN fiber fabrication methods that use denaturants12, surface tension at the air-liquid-solid interface of a FN solution14, or protein-surface interactions16 to polymerize FN dimers into insoluble fibers. These studies have demonstrated that through various methods, exposing cryptic self-assembly sites via an induced, conformational change of FN is sufficient to drive fibrillogenesis in a cell-free system.Studying the properties of FN fibrils 17, key mechanical properties such as Poisson’s ratio remain unknown. Specifically, whether FN can behave as an ideal elastomer with Poisson’s ratio of 0.5 has been difficult to determine because this requires precise measurement of fiber cross-sectional area during tensile strain. To address this, we first used SIA to create an array of FN nanofibers initially patterned as 50 μm long, 20 μm wide rectangles that each easily fit within the AFM scan area. This enabled us to establish that the nanofibers were indeed monodisperse in their dimensions and to then track changes in morphology from the as patterned, pre-release state to the contracted, post-release state. Further, as a second approach we used a modified SIA process to fabricate 1 cm long, 20 μm wide FN nanofibers with regularly spaced fiducial marks, uniaxially strained these nanofibers, and then immobilized these in the strained state and performed AFM to determine morphology. While not identical to cell-generated FN fibers, our results provide key insight into the elastic behavior of insoluble FN networks and how strain-dependent changes in molecular conformation may underlie the mechanobiology of these materials in the native ECM.In this study, we used a surface-initiated assembly (SIA) process in combination with atomic force microscopy (AFM) to engineer FN nanofibers and study their change in morphology over large strains. We hypothesized that FN nanofibers would exhibit elastomeric behavior due to reversible conformational changes of the constituent FN molecules. While previous studies have used tensile testing of FN fibers to demonstrate that they can behave elastically over specific strain regimes19. Briefly, FN in solution was adsorbed onto a PDMS stamp used for microcontact printing with a surface pattern consisting of 50 μm × 20 μm rectangles  and subsequent cooling below the lower critical solution temperature (LCST) of ~32 °C caused the PIPAAm to swell and dissolve and release the assembled FN nanofibers off of the surface into solution  deoxycholate (DOC) or 2% (w/v) sodium dodecylsulfate (SDS) solutions ,o, A1, and A2 represent the final contracted length, the decay amplitude, and the fitting parameter, respectively. The FN nanofibers were found to have a fast contraction regime, τ1, of 2.88 seconds and a slow contraction regime, τ2, of 157.41 seconds. These two rates suggest that FN nanofibers may contract via multiple mechanisms, a rapid entropic-driven contraction and a slower, molecular folding driven contraction. Determining the exact cause is beyond the scope of this manuscript, but will be important to understand in future studies. Comparing the FN nanofiber before and after assembly, we found that the length pre-release was 50.12 ± 0.41 μm, confirming monodispersity . The photoresist was then exposed to UV light through a photomask and developed using MF-319 developer (Microchem). A negative of the patterned photoresist wafer was formed by casting PDMS prepolymer  over it and curing it at 65 °C for 4 hours. The cured PDMS was then peeled off the glass wafer and 1 cm2 stamps were cut out.FN nanofibers were prepared via an SIA technique based on previously described2O water for 30 minutes, washed to remove excess FN, and then dried under stream of nitrogen. FN coated PDMS stamps were then brought into conformal contact with PIPAAm coated coverslips for 10 minutes to create arrays of 50 × 20 μm FN rectangles on the PIPAAm surface. The PIPAAm coated coverslips were prepared by spincoating a 10% PIPAAm (Polysciences Inc.) in 1-butanol (w/v) solution. Upon removal of the stamps, the quality of the FN rectangles on the PIPAAm was inspected using phase contrast microscopy. The FN patterns were released from the PIPAAm surface by adding warm, ~40 °C, ddH2O and allowing the temperature to gradually drop below the LCST of PIPAAm, resulting in the dissolution of the PIPAAm layer and the non-destructive release of the nanofibers. To test detergent solubility, the nanofibers were released in either a solution containing 2% (w/v) sodium dodecyl sulphate dissolved in ddH2O or a solution containing 2% (w/v) sodium deoxycholate dissolved in ddH2O.Prior to use, the PDMS stamps were sonicated in a 50% ethanol solution for 30 minutes and then dried under a stream of nitrogen. The stamps were then incubated with FN (BD Biosciences) at a concentration of 50 μg/ml in ddH33.The FN nanofiber release was monitored optically using phase contrast microscopy and time-lapse images were captured using a Photometrics CoolSnap ES digital camera (1392 × 1040 pixels) at a frame rate of 1 fps. The contour length of 15 nanofibers was measured pre-release (−77 and −23 sec), at the initiation of release (0 sec), and post-release from the PIPAAm surface  using ImageJ 2O to remove the dissolved PIPAAm, and then dried in a 65 °C oven. FN nanofibers pre-release were scanned with a scan size of 512 × 512 lines over a scan area of ~52 μm × 52 μm and FN nanofibers post-release were scanned with a scan size of 512 × 512 lines over a scan area of ~15 μm × 15 μm. High-resolution AFM images were obtained using a scan size of 1024 × 1024 lines over a scan area of 3 μm × 3 μm for the nanofibers pre-release (zoomed in to a 500 × 500 nm area after acquisition) and 2 × 2 μm for nanofibers post-release.AFM  was used to analyze the nanostructure and quantify the morphology of FN nanofibers both pre-release and post-release. The FN nanofibers were scanned in air using AC mode with AC160TS cantilevers (Olympus Corporation). Nanofibers pre-release were scanned on the PIPAAm surface whereas post-release FN nanofibers were first allowed to adsorb back onto the glass coverslip, washed three times with ddH31. We first microcontact printed 1 cm long and 67 μm wide lines of FN that have been tagged with fiducial marks (Supplementary Figure To strain FN nanofibers we used our previously published techniquest order planefit and a 2nd order flattening. The width at five transverse locations and contour length for ten nanofibers were then measured using the IGOR Pro software environment. FN nanofiber volume and thickness were also measured using the IGOR Pro software environment. The FN nanofiber volume was calculated by first applying a mask to the FN nanofibers. The volume of each pixel within the confines of the mask was calculated by multiplying the AFM height value of a given pixel by its area. The total FN nanofiber volume was measured as the summation of the volume of each pixel within the masked area. For FN nanofibers that were uniaxially stretched, only the regions of the nanofibers between fiducial marks was masked and the volume was measured as previously described. Since thickness was heterogeneous along the length and width of the nanofibers (Fig. To accurately quantify FN nanofiber morphology, the AFM height signal of each nanofiber was first processed to ensure the background surface was flat and centred at 0 nm. To accomplish this, the FN nanofibers in the image were masked and the non-masked area was subject to a 1ers Fig. , the aveSupplementary InformationSupplementary Video 1