The “fitness” of an infectious pathogen is defined as the ability of the pathogen to survive, reproduce, be transmitted, and cause disease. The fitness of multidrug-resistant tuberculosis (MDRTB) relative to drug-susceptible tuberculosis is cited as one of the most important determinants of MDRTB spread and epidemic size. To estimate the relative fitness of drug-resistant tuberculosis cases, we compared the incidence of tuberculosis disease among the household contacts of MDRTB index patients to that among the contacts of drug-susceptible index patients.This 3-y (2010–2013) prospective cohort household follow-up study in South Lima and Callao, Peru, measured the incidence of tuberculosis disease among 1,055 household contacts of 213 MDRTB index cases and 2,362 household contacts of 487 drug-susceptible index cases.p = 0.017). The inference of transmission in this study was limited by the lack of genotyping data for household contacts. Capturing incident disease only among household contacts may also limit the extrapolation of these findings to the community setting.A total of 35/1,055 (3.3%) household contacts of 213 MDRTB index cases developed tuberculosis disease, while 114/2,362 (4.8%) household contacts of 487 drug-susceptible index patients developed tuberculosis disease. The total follow-up time for drug-susceptible tuberculosis contacts was 2,620 person-years, while the total follow-up time for MDRTB contacts was 1,425 person-years. Using multivariate Cox regression to adjust for confounding variables including contact HIV status, contact age, socio-economic status, and index case sputum smear grade, the hazard ratio for tuberculosis disease among MDRTB household contacts was found to be half that for drug-susceptible contacts , the growth of M. tuberculosis from sputum samples, and chest X-rays.Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Every year, 8.6 million people develop active tuberculosis (tuberculosis disease), and at least 1.3 million people die as a result, mainly in resource-limited countries. M. tuberculosis strains. The fitness of a pathogen (infectious organism) is its ability to survive, reproduce, be transmitted, and cause disease in another host. Animal and laboratory studies indicate that drug-resistant M. tuberculosis strains are less fit than drug-susceptible strains, but these studies do not account for the clinical, environmental, and socio-economic variables that influence a patient’s ability to cause tuberculosis disease in a contact, and may not accurately measure the relative fitness of M. tuberculosis strains. In this prospective cohort study, the researchers estimate the fitness of drug-resistant tuberculosis relative to drug-susceptible tuberculosis by comparing the incidence of additional cases of tuberculosis disease in households with an MDRTB index case and the incidence in households with a drug-susceptible tuberculosis index case. A prospective cohort study follows a group of people over time to see whether specific baseline characteristics are associated with specific outcomes. The incidence of a disease is the number of new cases in a population over a given time period.Taking several antibiotics (including rifampicin and isoniazid) daily for six months can cure tuberculosis, but the emergence of multidrug-resistant tuberculosis (MDRTB) is making the disease increasingly hard to treat. How badly MDRTB will affect tuberculosis control efforts is likely to depend on the relative “fitness” of multi-drug resistant and drug-susceptible The researchers enrolled 1,055 household contacts of 213 MDRTB index cases  and 2,362 household contacts of 487 drug-susceptible tuberculosis index cases living in South Lima and Callao, Peru. During three years of follow-up, 35 (3.3%) of the household contacts of the MDRTB index cases and 114 (4.8%) of the household contacts of the drug-susceptible tuberculosis index cases developed tuberculosis disease. After adjusting for factors likely to affect the transmission of tuberculosis, such as HIV status, socio-economic status, and sputum smear grade of the index case (higher smear grades are associated with a higher risk of tuberculosis transmission), the hazard ratio for tuberculosis disease for household contacts of MDRTB index cases was half that of the household contacts of drug-susceptible tuberculosis index cases. That is, the household contacts of MDRTB index cases contracted tuberculosis disease half as often as those of drug-susceptible tuberculosis index cases.M. tuberculosis fitness within populations. Because the researchers did not genetically compare M. tuberculosis strains isolated from the index cases with strains isolated from the household contacts who developed tuberculosis disease, some of these contacts may have become infected outside the household. Moreover, it may not be possible to extrapolate these findings to the community setting. Nevertheless, the low relative fitness of MDRTB reported here improves our chances of controlling the spread of drug-resistant tuberculosis, with the proviso that the emergence of fitter MDRTB strains over time might yet threaten global tuberculosis control efforts.These findings indicate that, within households, MDRTB has a relatively low fitness compared to drug-susceptible tuberculosis. That is, at least during the first three years following exposure, individuals with MDRTB are less likely to transmit disease to their household contacts than individuals with drug-susceptible tuberculosis. These findings agree with those of previous animal and laboratory studies and with the findings of molecular epidemiology studies that have used genetic methods to estimate http://dx.doi.org/10.1371/journal.pmed.1001843.This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at tuberculosis and on multidrug-resistant tuberculosis; the Global Tuberculosis Report 2014 provides information about tuberculosis around the world; a supplement to the report entitled Drug-Resistant TB—Surveillance and Response is availableThe World Health Organization provides information  on Stop TB Partnership is working towards tuberculosis elimination and provides personal stories about tuberculosis (in English and Spanish); the Tuberculosis Vaccine Initiative (a not-for-profit organization) also provides personal stories about tuberculosisThe tuberculosis and about drug-resistant tuberculosis (in English and Spanish)The US Centers for Disease Control and Prevention provides information about tuberculosisThe US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis (in English and Spanish)MedlinePlus has links to further information about  Mycobacterium tuberculosis is defined as the ability of the organism to survive in the host, reproduce, be transmitted, and cause disease in another host [M. tuberculosis organisms [Natural selection of an infectious pathogen occurs as a consequence of differential reproductive success at the level of the gene or the organism during its interaction with the environment. The “fitness” of her host ,2. Matherganisms –5.M. tuberculosis bacilli, with most strains demonstrating a fitness cost and some demonstrating superior fitness [Studies by Mitchison  and Midd fitness –13. HoweVery few studies have estimated fitness in vivo by comparing the incidence of second cases of tuberculosis among contacts of patients with multidrug-resistant tuberculosis (MDRTB) to that among contacts of patients with drug-susceptible tuberculosis. Studies that have measured the incidence of second cases in households with MDRTB have lacked statistical power ,15 or haThe aim of this prospective cohort study was to estimate the fitness of drug-resistant tuberculosis cases relative to drug-susceptible tuberculosis cases by determining the incidence of second cases of tuberculosis disease in households with an MDRTB index case relative to that in households with a drug-susceptible tuberculosis index case, while considering the effect of potential confounding variables.Ethical approval was obtained from the Institutional Review Board of Universidad Peruana Cayetano Heredia (IRB00001014) before the study began . Institutional approval was also obtained from the Peruvian Ministry of Health and the regional tuberculosis control programs. Informed written consent was obtained from all study participants.p < 0.05) with a HR ≤ 0.6 . The a priori alternative hypothesis was that the incidence of second cases of tuberculosis disease among the contacts of MDRTB index cases was different from that among the contacts of drug-susceptible tuberculosis index cases, independent of potentially confounding variables. Incidence rates of tuberculosis disease from previous studies ,18 were HR ≤ 0.6 .MDRTB index patients (resistant to at least rifampicin and isoniazid) and drug-susceptible index patients (susceptible to both rifampicin and isoniazid) were identified at diagnosis from each of the regional reference laboratories. Index patients were recruited at diagnosis (MDRTB or drug-susceptible tuberculosis), and a sputum culture was obtained. All tuberculosis patients in Peru are tested for human immunodeficiency virus (HIV); these data were available from the patient record. Patients who were able to expectorate sputum had their sputum tested by serial smear microscopy on a monthly basis. For the purposes of analyzing time to sputum conversion to negative during index case follow-up, index patients were regarded as becoming smear negative on the date of the first negative sample if they remained smear negative thereafter. Index patients were also regarded as being smear negative if they could no longer produce sputum and continued as such.In order to minimize bias, for each MDRTB index patient recruited, at least two age- and sex-matched drug-susceptible controls were selected at random from the same study site as the index case for comparison. Following this, patients were visited at the health center and invited to participate in the study before providing written informed consent and completing a structured questionnaire at enrollment. The structured questionnaire recorded information on patient demographics, the household environment (crowding and house construction), and clinical data such as sputum smear result, culture results, and diagnosis dates. All variables used in the study questionnaire were field tested during a preliminary retrospective study .Index patients were followed up at the local health center by study staff every 6 mo and asked about the well-being of family members and the occurrence of second cases of tuberculosis disease in the household. Any symptomatic contacts were encouraged to attend the local health post for testing. When recruitment to the study ended in January 2013, all index patients and their families were visited at home to ensure that all incident cases of tuberculosis disease during the study period had been recorded. The final round of active household-based follow-up for all families was designed to minimize potential bias from variable follow-up between MDRTB and drug-susceptible tuberculosis households. Chemoprophylaxis was prescribed and managed by the treating health center/physician in accordance with the national tuberculosis policy: national policy recommends chemoprophylaxis for the household contacts, <16 y of age, of patients with drug-susceptible tuberculosis and not for the household contacts of patients with MDRTB . When thDrug susceptibility testing for rifampicin and isoniazid was performed for all samples at the two regional reference laboratories  using the microscopic observation drug susceptibility assay (MODS) ,21. In aPositive sputum cultures from index cases and contacts (when available) were sub-cultured on solid Ogawa medium and transported to the laboratories of Universidad Peruana Cayetano Heredia for DNA extraction and spoligotyping by conventional methodology .p-value. Previous tuberculosis disease was defined as any disease episode in which anti-tuberculous treatment was successfully completed more than 6 mo prior to the onset of the present tuberculosis episode. Missing data were minimized by revisiting households and health centers and cross-checking interview data against medical records. Missing values were then treated as “missing at random” using Stata’s default “listwise” deletion when included in multivariate regression analysis. The study database was deposited in the Dryad Digital Repository [Household contacts were defined as any person living in the same house as the index case for more than one day a week. Follow-up time for MDRTB and drug-susceptible tuberculosis household contacts started at the time of diagnosis of index case MDRTB and drug-susceptible tuberculosis, respectively. An “event” was defined as the development of tuberculosis disease in a household contact that occurred after the diagnosis of tuberculosis in the index case. Tuberculosis disease in household contacts was defined as any patient that had evidence of tuberculosis disease from sputum smear, culture, chest X-ray, or clinical diagnosis that led to initiation of anti-tuberculous treatment. This definition was chosen in order to include children who were diagnosed with tuberculosis disease and started on anti-tuberculous treatment by the treating physician without a microbiologically confirmed diagnosis. Contacts were censured if they were lost to follow-up. Contacts were regarded as lost to follow-up if they could not be located, had left the home, or had died (not as a consequence of tuberculosis). The incidence of tuberculosis disease was calculated as the number of incident cases of tuberculosis divided by the total number of contact follow-up person-years. Odds ratios (ORs) of the differences between the two comparison groups were calculated using the STATA csi command for ORs in cohort studies with an exact pository .Details on diagnosis and treatment dates were also recorded for “co-prevalent” household contacts who had initiated treatment for tuberculosis disease before the diagnosis of the index case but who had not completed tuberculosis treatment by at least 6 mo prior to the diagnosis of the index case. Screening for co-prevalent cases was undertaken at the initial index case interview using the structured questionnaire, and this was checked again at subsequent household visits. Any co-prevalent case identified at initial screening was followed up as a household contact of the index case and was screened for signs and symptoms of tuberculosis at follow-up visits.p < 0.2 on univariate analysis, were included in the multivariate regression. The interactions between age and chemotherapy use, diabetes and index case drug resistance status, socio-economic status and index case sputum smear grade, contact employment and socio-economic status, and index case genotype and index case drug resistance status were all examined for significance in predicting second household cases of disease. A p-value of <0.05 was considered statistically significant in the multivariate regression. Analysis was undertaken using Stata . The preplanned analysis did not differ from the final analysis other than consideration of the potentially confounding association between index case genotype and second cases of tuberculosis disease, which was undertaken after reviewers’ comments.Independent predictors of second cases were determined using a multivariate Cox regression survival analysis clustered at the level of the household. Each variable was tested for potential violation of the proportional hazards assumption by minus log-log plots and examination of the Schoenfeld residuals. Correction for clustering was undertaken at the level of the household to account for the correlation of variance within households. The STATA cluster clustvar command was used as the clustering method to provide a robust estimate of the standard error according to the Huber/White/Sandwich estimate of variance . Known cA total of 306 MDRTB index patients were identified for interview from the regional reference laboratories. Ninety-three MDRTB index patients could not be recruited: 45 patients (48% of unrecruited patients) could not be located either at the health post or at home as an erroneous address had been provided or they had abandoned treatment at the health post after having left a diagnostic specimen, 20 (22%) died before an interview could be undertaken, 16 (17%) were imprisoned, and 12 (13%) chose not to consent to enter the study. This left the household contacts of 213 newly diagnosed MDRTB index cases who were followed up as part of the study.A total of 657 drug-susceptible tuberculosis index patients were identified as matched controls for the MDRTB index patients, of whom 170 could not be recruited: 147 patients (86%) could not be located either at the health post or at home as an erroneous address had been provided or they had abandoned treatment at the health post after leaving a diagnostic specimen, 20 patients (12%) chose not to consent to enter the study, and three patients (2%) died prior to interview. This left the household contacts of 487 newly diagnosed, matched drug-susceptible control index cases who were followed up as part of the study. MDRTB index cases lived with 1,055 household contacts (mean of 5.0 contacts per MDRTB index case), and drug-susceptible tuberculosis index cases lived with 2,362 household contacts (mean of 4.9 contacts per drug-susceptible tuberculosis index case) .p = 0.28, non-paired Student’s t test), nor was the likelihood of smear positivity different (90% of recruited patients and 90% of unrecruited patients). However unrecruited patients were more likely to be male  and more likely to have previously received treatment .The mean age of those recruited was not significantly different from the mean age of those not recruited .Thirty-five second cases of tuberculosis disease occurred among the household contacts of MDRTB patients . This simple proportion  was less than the proportion of household contacts of drug-susceptible tuberculosis index cases who developed tuberculosis disease , but the difference was of borderline statistical significance  also had MDRTB. Among the incident cases in drug-susceptible households that had a drug susceptibility test performed, 98%  also had drug-susceptible tuberculosis.p = 0.017; The total follow-up time of MDRTB contacts was 1,425 person-years , during which 35 second cases arose, equating to an incidence of 2,456 per 100,000 contact follow-up person-years. The total follow-up time of drug-susceptible tuberculosis contacts was 2,620 person-years , during which 114 second cases arose, equating to an incidence of 4,351 per 100,000 contact follow-up person-years , to be hospitalized with their disease , and to describe any side effects of medication . The proportion of unemployment was higher among MDRTB patients , although more MDRTB patients had completed secondary education . Sputum smear grade was not significantly different between MDRTB and drug-susceptible tuberculosis index patients.Eight percent (18/213) of MDRTB index cases were HIV positive, while only 4% (20/487) of drug-susceptible tuberculosis index cases were HIV positive , while the Latin American Mediterranean spogliotype family  and T spogliotype family  were overrepresented among MDRTB index cases.The proportion of index patients with disease caused by the Haarlem spogliotype family was greater among drug-susceptible tuberculosis index patients . Among these patients, only one drug-susceptible tuberculosis contact developed tuberculosis disease (either relapse or new infection) during the course of the study. Fewer contacts of MDRTB patients had received isoniazid chemoprophylaxis .At baseline, the contacts of MDRTB index patients were more likely to report a previous history of tuberculosis disease than the contacts of patients with drug-susceptible tuberculosis  or a T strain  were not associated with a greater incidence of second cases. However, other Euro-American strains  and the Beijing spogliotype family  gave rise to fewer second cases of tuberculosis disease among contacts , as did male household contacts compared to female contacts , contacts with a previous history of tuberculosis disease compared to those with no previous tuberculosis disease , and contacts with HIV compared to contacts without HIV . Contacts who received isoniazid chemoprophylaxis after exposure were significantly less likely to develop tuberculosis disease than those who did not receive isoniazid chemoprophylaxis . Household contacts who were employed were less likely to develop tuberculosis disease than unemployed household contacts  was included in the multivariate regression, the HR for the association of index case drug resistance status with the incidence of second cases among household contacts decreased from 0.56 to 0.52, and the p < 0.001). Continued sputum smear positivity 6 mo into tuberculosis treatment was noted for 13% (27/202) and 4% (18/467) of MDRTB and drug-susceptible tuberculosis index cases, respectively .Ninety-six percent (467/487) of drug-susceptible tuberculosis and 95% (202/213) of MDRTB index patients provided sputum samples for analysis. Of these, 80% (375/467) of drug-susceptible tuberculosis index patients had either converted to sputum smear negative or were unable to expectorate sputum within the first 8 wk of starting anti-tuberculous treatment. However, only 52% (106/202) of MDRTB index cases had converted to smear negative or were unable to expectorate sputum in the same time period , abandoned treatment , or had their treatment regime changed as a consequence of a drug susceptibility test .A greater proportion of index cases with MDRTB died as a consequence of their disease  than drug-susceptible tuberculosis, at least in households. The fitness of MDRTB relative to drug-susceptible tuberculosis is one of the most important determinants of future MDRTB spread. A low relative fitness of MDRTB improves the chances of containing and diminishing the spread of drug-resistant tuberculosis.S1 Checklist(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.


Background: Anticoagulant therapy has been evaluated with respect to its potential usefulness in reducing the high mortality rates associated with severe sepsis, including sepsis-induced disseminated intravascular coagulation (DIC) after intestinal perforation. We examined the hypothesis that recombinant human soluble thrombomodulin (rhTM) is effective in the treatment of patients with septic shock with sepsis-induced DIC after laparotomy for intestinal perforation.Methods: We performed propensity-score and instrumental variable analyses of the Japanese Diagnosis Procedure Combination in-patient database, a nationwide administrative database. The main outcome was 28-day in-hospital all-cause mortality.Results: We categorized eligible patients (n = 2202) from 622 hospitals into the rhTM group (n = 726) and control group (n = 1476). Propensity-score matching created 621 matched pairs of patients with and without rhTM. There was neither significant difference in 28-day mortality between the two groups in the unmatched analysis  nor in the propensity-score-matched analysis . The logistic analysis showed no significant association between the use of rhTM and the mortality in propensity-score-matched patients . The instrumental variable analyses, using the hospital rhTM-prescribing proportion as the variable, found that receipt of rhTM was not associated with the reduction in the mortality .Conclusion: We found no association between administration of rhTM and 28-day mortality in mechanically ventilated patients with septic shock and concurrent DIC after intestinal perforation. Severe sepsis is one of the most common causes of death in critically ill patients worldwide –4. Sepsip = 0.17)  is used clinically for DIC treatment in Japan , 14. Thr = 0.17) . Based oPrevious interventional and epidemiological studies have suggested that the two major infection sites giving rise to sepsis are the lungs and abdomen –3. AlthoThe current study evaluated whether rhTM can reduce mortality in patients with sepsis-induced DIC after intestinal perforation confirmed by laparotomy.This study was approved by the institutional review board of The University of Tokyo, which waived the requirement for informed patient consent because of the anonymous nature of the data.The Japanese Association for Acute Medicine (JAAM) guidelines were published in 2006 . These gFor this study, we used the Japanese Diagnosis Procedure Combination (DPC) inpatient database, previously described in detail elsewhere , 25–27. We identified mechanically ventilated patients who required vasopressors and developed DIC after emergency open laparotomy for perforation of the lower intestinal tract as recorded in the DPC database from 1 July 2010 to 31 March 2013. The inclusion criteria were: (1) age of ≥15 years; (2) confirmed diagnosis of perforation of the lower gastrointestinal tract at admission (coded in the “primary diagnosis” or “comorbidities at admission” section of the database); (3) performance of open abdominal laparotomy excluding exploratory laparotomy on day 0 or 1; (4) presence of septic shock, defined as use of vasopressors  started on day 0 or 1; (5) performance of mechanical ventilation after surgery on day 0 and/or 1; (6) initiation of antibiotic therapy on day 0 or 1; and (7) diagnosis of DIC (coded in the “main diagnosis” or “comorbidities at admission” section of the database). The exclusion criterion was initiation of rhTM on or after day 2.In the current study, the hospital volume was defined as the number of eligible patients treated at each hospital and was subcategorized into tertiles. The hospital type was categorized as academic or non-academic.The primary endpoint used was 28-day all-cause mortality. The secondary endpoints were ventilator-free days (VFDs) , accounting for the paired nature of the matched patients . A partial F-test was performed to confirm that the patterns of hospital use of rhTM is not a weak instrument   or control (n = 1476) group and underwent propensity-score matching, from which 621 propensity-score-matched pairs were generated. The C-statistic for the goodness of fit was 0.72  in the propensity-score model. The mean length of hospital stay was 44.8 days .Of 5443 mechanically ventilated patients with septic shock after surgically confirmed perforation of the lower intestine, 3271 patients were excluded (2963 due to DIC was not shown and 308 due to delayed start of rhTM). As depicted in Figure Baseline characteristics of the unmatched and propensity-score-matched groups patients were shown in Tables F-statistic of 370 . The reduction in the 28-day mortality was not significantly associated with receipt of rhTM .The overall 28-day mortality rate was 24.1% (530/2202). No significant difference was documented in the 28-day mortality rate between the two groups in the unmatched analysis  or in the propensity-score-matched analysis . The logistic GEE analysis showed no significant association between the use of rhTM and 28-day mortality in propensity-score-matched patients . In the instrumental variable model, the null hypothesis  was rejected with an We found no significant difference in the in-hospital mortality between the two groups of unmatched patients . Moreover, no significant difference was found among the propensity-score-matched patients . Cox regression analysis showed no significant difference in in-hospital mortality between the two groups .We found no significant difference in the number of VFDs between the rhTM group and control groups among either unmatched patients  or propensity-score-matched patients .The current study suggests that there is no significant association between rhTM use and 28-day mortality in sepsis-induced DIC patients after laparotomy for intestinal perforation. Additionally, no significant difference was documented in the VFDs between the rhTM and control groups.Anticoagulant therapy agents, such as antithrombin –41 and rA novel anticoagulant therapy, rhTM, was developed for patients with DIC in Japan. Several studies have investigated the use of rhTM for sepsis-induced DIC in Japan, which is the only country where rhTM is widely used for clinical treatment of DIC –51. SevePrevious studies have suggested that patients with respiratory and/or cardiac dysfunction and evidence of DIC appear to receive the greatest rhTM-related survival benefit , 51, 52.The current results are consistent with those of two randomized trials in patients with sepsis-induced DIC , 18 as wThere are some limitations in the current study. First, our study was performed retrospectively without randomization. Although a propensity-score method was used to adjust for measured confounding factors, bias could inevitably exist in the form of confounders that were not measured. For example, although approximately 20% of patients were administered dobutamine, the necessary information for calculating cardiac index of the patients was not available in the database. More importantly, DIC scores, which may indicate the severity of DIC, were also unavailable. We therefore performed instrumental variable analyses to compensate for these potentially unmeasured confounders. Second, the definition of septic shock in the current study might be vague. Vasopressors might be used for types of shock other than septic shock. Additionally, according to the Surviving Sepsis Campaign 2012 , septic We found no association between administration of rhTM and 28-day mortality in mechanically ventilated patients with septic shock and concurrent DIC after intestinal perforation. Prospective randomized trials are required to confirm our findings.TT contributed to study design, statistical analyses, and review and drafting of the manuscript. HM contributed to study design, data acquisition, statistical analyses, and review of the manuscript. KF contributed to study design, data acquisition, and review of the manuscript. HY contributed to study design, statistical analyses, data acquisition, and review and drafting of the manuscript. All authors read and approved the final version of the 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.


Results in vitro showed successful differentiation of HMSCs into neuroglial-like cells, characterized by expression of specific neuroglial markers confirmed by immunocytochemistry and by RT-PCR and qPCR targeting specific genes expressed. In vivo testing evaluated during the healing period of 20 weeks, showed no evident positive effect of HMSCs or neuroglial-like cell enrichment at the sciatic nerve repair site on most of the functional and nerve morphometric predictors of nerve regeneration although the nociception function was almost normal. EPT on the other hand, recovered significantly better after HMSCs enriched membrane employment, to values of residual functional impairment compared to other treated groups. When the neurotmesis injury can be surgically reconstructed with an end-to-end suture or by grafting, the addition of a PLC membrane associated with HMSCs seems to bring significant advantage, especially concerning the motor function recovery.In peripheral nerves MSCs can modulate Wallerian degeneration and the overall regenerative response by acting through paracrine mechanisms directly on regenerating axons or upon the nerve-supporting Schwann cells. In the present study, the effect of human MSCs from Wharton's jelly (HMSCs), differentiated into neuroglial-like cells associated to poly (DL-lactide- Traumatic injuries affecting the central and the peripheral nervous system are often characterized by very limited recovery of lost functions and severe incapacity. In cases of no surgical treatment, spontaneous nerve regeneration is in many cases curtailed by scars, neuroma formation, mismatched nerve fascicles, or extensive splitting of the regrowing axons. Moreover, peripheral nerve damage is often associated with neuropathic pain, referred by patients as a more important reason for poor quality of life than the incomplete functional recovery . FunctioPeripheral nerve neurotmesis is a relatively common type of traumatic injury affecting the peripheral nervous system. These constitute a severe nerve damage in which both nerve fibers and the nerve sheaths suffer disruption and spontaneous recovery becomes extremely difficult in cases when the peripheral nerve is not microsurgically reconstructed . Wheneve in vivo. Stem cells from different sources are much likely the golden key for regenerative medicine i) was measured in Fura-2-loaded cells by using dual wavelength spectrofluorometry as previously described  and of HMSCs application . In particular, WRL recovered faster and better in animals treated with direct end-to-end repair  compared to those receiving a graft . Also, animals treated with differentiated (P < 0.001) and undifferentiated (P < 0.005) HMSCs showed slight worst WRL response, compared with animals without cellular treatment. Untreated animals (Gap group) showed no signs of recovery of the WRL response during the healing period of 20 weeks (P < 0.001). At week 20, the mean EPT of the End-to-EndPLCCellnonDif and End-to-EndPLCCellDif group was 0.19 ± 0.17 and 0.64 ± 0.27, respectively. Concerning the graft groups where the PLC membrane associated to the cellular system the WRL mean values were 0.13 ± 0.08 and 0.51 ± 0.19, for GraftPLCCellnonDif and GraftPLCCellDif group, respectively.Immediately following sciatic nerve transection, all animals presented a severe motor deficit demonstrated by a virtual complete loss of EPT response in the affected hind limb  = 28.778; P < 0.001] but not due to grafting . Pairwise comparisons showed differences between EPT responses in animals that did not receive the cellular treatment, compared to those treated with undifferentiated (P < 0.05) and differentiated HMSCs (P < 0.001). Differences in EPT response between animals treated with undifferentiated and differentiated HMSCs were also significant (P < 0.001). An interaction effect on EPT responses could be found involving the type of nerve repair  and the application of HMSCs . Pooled EPT data suggests that undifferentiated HMSCs, but not differentiated HMSCs, enhanced EPT recovery in animals that were specifically treated with autologous sciatic nerve graft, thus helping in minimizing the negative consequences of grafting in functional outcome. Also, data suggests that undifferentiated HMSCs, but not differentiated HMSCs, enhanced EPT recovery in animals that were specifically treated with an end-to-end suture  = 10.414; P < 0.001] and TO  but not at IC  and OT . At HR, ankle velocity was significantly altered in the Gap group compared with the End-to-EndPLCCellDif, GraftPLCCellnonDif, and GraftPLCCellDif groups (P < 0.05). Also at HR, significant changes in ankle velocity could be seen for every group of sciatic nerve-injured animals compared with intact animals. Similar results were found at TO, where ankle velocity in sciatic nerve-injured animals differed from that of intact animals. However, at TO ankle velocity was similar in every group of sciatic nerve-injured animals  (GraftPLCCellnonDif and GraftPLCCellDif)   , in all Control) . Microfa Figures  in compa Figures . From a  Figures  and graf Figures  nerve re in vitro fabrication of autologous, living tissues with the potential of regeneration is a promising scientific and clinical field. Peripheral nerve regeneration should include a multidisciplinary team able to develop biomaterials, to develop cell therapies, and to elaborate in vitro analysis and preclinical trials concerning animal welfare and the most appropriate animal model before the clinical trials and clinical application approval i, using epifluorescence technique and the Fura-2AM probe, of undifferentiated and neuroglial-differentiated HMSCs. The Vivosorb membrane proved to be adequate to be used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs, so that in vivo studies could be pursued in models of severe nerve injury, such as in the present study [2+]i, measurements using epifluorescence technique with Fura-2AM probe.In a previous study, the therapeutic value of HMSCs on rat sciatic nerve after axonotmesis injury associated to the same PLC membrane was evaluated (Vivosorb). Also,nt study . The sam in vitro results demonstrated successfully the HSMCs differentiation into neuroglia-like cells, as demonstrated by changes in cell morphology and positive staining for the specific neuroglial markers GFAP, GAP-43, and NeuN. Previously, it was demonstrated by NMR, that HMSCs expansion was glycolysis-dependent but that the differentiation of these cells required the switch of the metabolic profile to oxidative metabolism. Simultaneously, in vivo studies in a sciatic nerve crush rat model showed improvement in regenerated sciatic nerve's morphology, such as increased myelin sheath thickness, in animals treated with transplanted undifferentiated and differentiated HMSCs, which was accompanied also by enhanced recovery of motor and sensory function [In this study,function . in vitro differentiated HMSCs to promote nerve regeneration and to improve functional recovery even when nerves are surgically treated by epineural end-to-end suture or autologous grafting. Nerve histology demonstrated successful nerve regeneration of the transected and repaired sciatic nerves although the extent of such regeneration was somewhat limited. Regenerated axons and nerve fibers were small in diameter and their number was clearly diminished in comparison with uninjured nerves. Not surprisingly, such changes in morphology were more severe in sciatic nerves treated with the autologous graft and the cellular system (GraftPLCCellnonDif and GraftPLCCellDif groups) compared with the direct end-to-end suture and the cellular system (End-to-EndPLCCellnonDif and End-to-EndPLCCellDif). The use of PLC membranes enwrapping the repaired sciatic nerves could not significantly alter the degree of nerve regeneration. Similarly, coating the sciatic nerves at the injury site with undifferentiated or differentiated HMSCs could not significantly modify the extent of nerve regeneration. These results should be confirmed by histomorphometric analysis, which unfortunately was not possible to be performed in this study. The results confirmed the results obtained previously by Gärtner et al., 2013 [ in vitro differentiated HMSCs in this experimental work. According to the results of RT-PCR, undifferentiated HMSCs cells in vitro secreted several factors that can aid in nerve regeneration. Consistent with the immunocytochemistry observations in which the antibodies did not recognize GFAP, NF-H, and GAP-43 proteins in the undifferentiated HMSCs in the slides, the molecular analysis showed a small expression of GFAP gene and absence of expression of the NF-H and GAP-43 genes in these same undifferentiated cells. In fact, the small detection of the GFAP gene expression may be due to the high sensitivity of the molecular tests in comparison with immunocytochemistry tests. Moreover, the expression of the remaining genes, NeuN, β-actin, GAPDH, and Nestin, was also observed in undifferentiated HMSCs. The expression of the housekeeping genes, β-actin and GAPDH, is expected to occur. As per the NeuN and Nestin gene, the observation of its expression in undifferentiated HMSCs is not new; Bertani et al. [ in vitro differentiation of HMSCs into neuron-like cells.Peripheral nerve crush injuries are appropriate to investigate the cellular and molecular mechanisms of peripheral nerve regeneration and to assess the role of different factors in the regeneration process . Nerve cl., 2013 , where ti et al.  showed ti et al.  comparedHMSCs isolated from Wharton's jelly of the umbilical cord and delivered through PLC membranes might improve clinical outcome especially after trauma to sensory nerves, particularly in the cases of nerve injuries with significant loss of nervous tissue, requiring entubulation or grafting. When the neurotmesis injury can be surgically reconstructed with an epineural end-to-end suture without tension or by grafting, the addition of a PLC membrane associated with undifferentiated HMSCs seems to bring significant advantage, especially concerning the motor function recovery, basically by the secretion of local growth factors and cytokines secretion.Table 3 represents values in seconds (s) that were obtained performing Withdrawal Reflex Latency (WRL) test to evaluate the nociceptive function. This test has been performed pre-operatively (week-0), at week 1 and 2 and after every two weeks until the end of the 20-week follow-up time. Results are presented as mean and standard deviation (SD). N corresponds to the number of rats within the experimental group. Table 4 represents values of Motor Deficit that were obtained performing Extensor Postural Thrust (EPT) test. This test has been performed pre-operatively (week-0), at week 1 and 2 and after every two weeks until the end of the 20-week follow-up time. Results are presented as mean and standard deviation (SD). N corresponds to the number of rats within the experimental group.


Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1) homogenizing, 2) effective denaturation of proteins from RNA, 3) inactivation of ribonuclease, and 4) removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols.Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results.Results: Immersing pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA) when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR.Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue. Recent studies have focused on RNA analysis as a gene expression tool in cells to detect differential gene expression between two conditions. Different methods have been presented for extracting nucleic acids such as guanidinium thiocyanate followed by phenol-chloroform extraction, chromatography by cellulose, extraction using silica matrices, magnetic bead based nucleic acid purification, and anion-exchange.3,4Accurate detection of gene expression is influenced by status of the RNA that is isolated from tissues. The quality of isolated RNA should be checked prior to its use in subsequent tests and studies. The purity and quality of the isolated RNA is a vital step in RNA dependent assays. Performing complementary molecular tests with low-quality RNA may compromise the results of downstream applications which are often labor-intensive, time consuming, and highly expensive. Researchers need high quality RNA for molecular biological tests that have various diagnostic applications such as quantitative RT-PCR, micro-arrays, ribonuclease protection assay, northern blot analysis, RNA mapping, and cDNA library construction.5,6The quality of purified RNA from tissues and cells is variable. Often, after extraction, RNA is rather unstable over a long storage time. Long mRNA fragments up to 10 kb are especially sensitive to degradation.7,8Researchers must consider various factors that affect the quality of purified RNA. Purified RNA must not be contaminated with RNases, proteins, genomic DNA, and enzymatic inhibitors. Additionally, the UV absorption ratio (260/280) of total RNA should be between 1.8-2.0 and RNA should have a minimal degree of fragmentation during electrophoresis. Recently developed laboratory techniques allow scientists to more adequately control the quality of samples used for molecular analyses.9-15Although RNA is easily and successfully isolated from most cells and tissues, intact RNA extraction from the pancreas is difficult due to the high level of its ribonucleases (RNases). Despite the improvement in several approaches, including rapid removal of pancreatic tissue from the abdominal cavity and homogenization at cold temperatures to inhibit RNases, the isolation of intact, high-quality RNA from this tissue remains challenging because of the complexity and indefinite reproducibility of the above mentioned techniques.We aimed to design a simple, fast, and cost-effective method for complete RNA extraction that utilized the least amount of pancreatic tissue. We compared different protocols of RNA extraction and optimized the most feasible extraction method by which the highest quality RNA could be qualitatively obtained. In the current study, pancreatic tissues were taken from 30 rats and divided into several pieces (20-30 mg) for use in the following methods.In the first method, these small pieces of pancreatic tissue from 30 rats were placed into two microtubes. The first tube contained 1 ml RNX-plus solution  and the second tube contained 1 ml TriPure isolation reagent . Both solutions contained guanidinium thiocyanate which inhibits RNase. Subsequently, both tubes were snap-frozen in liquid-nitrogen for inhibition of RNase activity after which the integrity of RNA was evaluated with denaturing agarose gel electrophoresis  and 2.In the second method, pancreatic tissues were perfused with 1 ml RNA-later as the RNA stabilization reagent  by an insulin syringe. Tissues were subsequently cut into small pieces with sterile scissors. The tubes that contained pancreatic tissue and RNA-later were processed for extraction by using the RNX-plus solution, TriPure, and RNeasy Micro Kits  according to the manufacturers’ instructions after either 30 min, overnight in 4ºC, or following storage at -80ºC for one, three or seven days in order to compare the effect of preservation time on RNA integrity. In all conditions, the livers were removed from 30 rats and used as control tissue in a comparison of RNA quality between pancreatic and liver RNAs. 16The isolated RNAs were separately diluted at a ratio of 1/100 in water treated with diethylpyrocarbonate (DEPC) and the 260/280 UV absorption ratio was calculated. The integrity of total RNAs was evaluated by denaturing agarose gel (MOPS gel) electrophoresis. MOPS buffer was used as running buffer to separate several ribosomal RNA (rRNA) bands  during electrophoresis.We did not obtain acceptable bands when RNA was extracted with the RNX-plus reagent or RNA-later. However, we observed the best results when TriPure reagent was used. These results were dependent upon the tissue preservation time, temperature and perfusion method. Immersion of pancreatic tissue in RNA-later for 24 h at -80ºC yielded high quality RNA with sharp, distinct 28S/18S bands.Evaluating RNA Integrity with the RNX-Plus SolutionNo specific band was seen when we used the RNX-plus solution. According to electrophoresis results, the RNA was completely degraded .RNA Integrity with TriPure ReagentIn comparison to the liver tissue control, we noted that RNA separation was not successful when the TriPure reagent was used .RNA Integrity of Samples Immersed in RNA-Later and Extracted with RNX-Plus or TriPure ReagentThere was no band visualized when we used RNA-later along with the RNX-plus reagent . DependiRNA Integrity with RNA-Later and the Qiagen KitIn terms of purity and integrity, high-quality RNA was extracted by using RNA-later along with the Qiagen reagent .RT-PCR for β-Actin Gene Expression in Pancreatic Tissues with RNAs Extracted by Using the TriPure ReagentHigh quality RNA extracted with the TriPure procedure amplified a specific β-actin locus. The full length of actin was amplified by RT-PCR in order to evaluate the synthesized cDNA by intact RNA. The presence of 138 bp bands confirmed the cDNA quality .14,17,18Obtaining high-quality RNA is the first and most critical step in many molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis that uses next-generation sequencing, array analysis, northern analysis, and cDNA library construction. RNA extraction is complicated because of the presence of ribonuclease enzymes in cells and tissues that can rapidly degrade RNA.19RNases act without cofactors and are stable enzymes. The inactivation of RNases is difficult. Small amounts of RNase are sufficient to destroy RNA. In order to inactivate any RNases prior to surgery and in cases of removal of rat pancreatic tissue from the abdominal cavity, all surgical instruments should be cleaned with strong detergents, thoroughly rinsed, and placed in an oven for at least 4 h at 240ºC. The place of surgery should be sterilized with NaOH and mild bleach to inactive the RNases.18,19The pancreas has an extremely high level of RNase. RNA degradation occurs while the pancreas tissue is removed during dissection. In order to increase efficiency dissection should be performed as quickly as possible.20The pancreas is one of the important tissues that functions in the body’s homeostatic mechanisms. Therefore, improvement of RNA extraction procedures from the pancreas increases understanding of active pathways such as glucose regulation and redifferentiated β islets of the pancreas before and after treatment with different drugs. Knowledge of gene expression assays may potentially lead to the development of therapeutic drugs to restore β-cells and prevent apoptosis in diabetics.21-23 Therefore we have attempted to design an efficient, simple optimized method to extract RNA from the pancreas based on our laboratory facilities.To identify novel metabolic genes and pathways that may play a role in the pathogenesis or treatment of diabetes, differential expression of metabolic genes is necessary. However, RNA extraction from pancreatic tissue is difficult because of the abundance of RNase.In order to enhance and correct this method, we assessed different common RNA extraction methods from pancreatic tissue with particular focus on the effect of frozen storage and RNase inhibition strategies, both of which affect RNA quality.10,15,18 Therefore, in this study we obtained only 20-30 mg of pancreatic tissue in less than 2 min from the anesthetized rats.The duration of surgery and amount of collected pancreatic tissue are the most crucial steps for obtaining intact RNA. Recent studies have shown a positive correlation between RNA degradation and the amount of pancreatic tissue.24 RNA extraction performed with guanidinium thiocyanate followed by phenol-chloroform on different tissues has yielded good results, with the exception of pancreatic tissue (data not shown). Several studies determined that the RNA isolation procedure must include a number of important steps before, during, and after the actual RNA purification.25 Therefore we changed the RNA extraction process by using RNA-later.Several methods are used in molecular biology to isolate RNA from different samples. The most common isolation method is guanidinium thiocyanate followed by phenol-chloroform extraction that uses liquid nitrogen along with a specialized motorized grinding device which prevent RNA activity.Our results showed that RNA later rapidly permeated the pancreatic tissues, protected cellular RNA and minimized the need to immediately process the tissue samples. The best results were obtained after the samples that contained RNA-later were stored for 24 h at -80ºC. Our data confirmed that it was necessary to stabilize RNA within the pancreatic samples by using an effective reagent to delay RNA degradation, even the extraction was performed by using a Qiagen RNA extraction kit. Several kits such as TriPure and Qiagen (foreign kits) and RNX-Plus  are commercially available for RNA extraction in Iran. These kits offer the dual advantages of ease of use and effectiveness. These kits often work well and are widely used. Foreign kits are also more expensive per sample than homemade kits. 15 however we were unable to obtain good quality RNA. The only difference between these two protocols was the use of RNX-plus in our study which did not seem to be an appropriate solution for RNA extraction from pancreatic tissues compared to TRIzol. Although RNX-plus works well for extracting RNA from other tissues, we did not use RNX-plus for RNA extraction from pancreatic tissue. In this case, the possibility of the presence of active RNase during surgery possibly led to RNA degradation and could not be ruled out.We were unable to obtain high-quality RNA with Iranian reagents and snap-frozen tissues. Possibly, the low yield of RNA from the immediately frozen pancreatic samples was attributed to rapid degradation initiated by RNases in the pancreas. Although our entire experimental process was similar to a previous study that used TRIzol reagent where the researchers obtained high-quality intact RNA from the rat pancreas,In order to evaluate the quality of RNX-plus, the second RNA extraction procedure was performed using the TriPure reagent under snap-frozen conditions which led to decreased RNA degradation. However, the problem with using TriPure solution was the lack of reproducibility. In the second step, we decided to decrease autolysis during dissection and RNA extraction by using RNA-later as a pancreas RNase inhibitor. The use of RNA-later before starting the isolation method with RNX-plus did not change the quality of RNA. The RNA was degraded and 28S/18S rRNA smear bands were observed. However, when TriPure was used, RNA quality was incredibly high. Additionally, this process was reproducible. The quality of RNX-plus was questioned. We also detected DNA contamination with the use of RNX-plus that had to be reduced.°C or for one, three and seven days at -80°C. As shown in In the third step we have focused on how to perfuse RNA-later into the pancreatic tissues. Complete tissue perfusion with RNA-later after total pancreatic tissue dissection is not cost-effective. Therefore, we researched three perfusion conditions for the later use of RNA as follows. We dissected a small section of the pancreas (20-30 mg) during surgery from anesthetized rats and immersed these tissues in 1 ml RNA-later for 30 min at 4The above mentioned methods enabled a smaller amount of RNA-later to penetrate into the organ. The degradation process was halted faster because small pieces were dissected. Modifications to the basic procedures introduced by Li and Griffin et al enabled us to obtain high-quality reproducible RNA from rat pancreatic RNA which was suitable for RT-PCR of the actin gene as shown in Although isolation of intact RNA from the rat pancreas is compromised by autolysis and by the presence of endogenous RNases, our pancreas perfusion method yields excellent, high quality and integrity RNA for molecular biology studies which is comparable with Qiagen kits. In summary, the presented method is a simple, reproducible and economical procedure which does not require the use of higher amounts of RNA-later total perfusion. Using TriPure solution after RNA-later perfusion can be a good substitute for expensive and column-based RNA extraction kits. Furthermore, use of the RNX-plus kit for RNA extraction from pancreatic tissue is not recommended.


P < 0.05). The birth rate on regular chow was 87%. Six out of 11 DS-low rats during pregnancy produced pups while the rats fed a high-salt diet all delivered pups (P < 0.025). The pup survival rate was 67% for high-salt mothers during mating and 54% for mothers on a low-salt diet. The pup survival rate was 95% for mothers on a high-salt diet during pregnancy and 64% for mothers on a low-salt diet (P < 0.0001). Seven out of 8 DS-regular rats during mating delivered 59 neonates. However, 66% of the neonates survived. A low-salt diet during mating or pregnancy lowers birth rate and the neonates from low-salt mothers during pregnancy were more likely to die than those from high-salt mothers.We investigated the influence of maternal salt restriction during mating or gestation on birth rate and offspring growth in Dahl salt-sensitive rats (DS). DS were divided into 5 groups: DS fed a low-salt  (DS-low) or high-salt  diet (DS-high) during mating and DS-high or DS-low during gestation, and DS fed regular chow  (DS-regular) throughout mating and gestation. During the unspecified periods, the rats were given regular chow. DS-low during mating delivered fewer infants than high-salt mothers ( For the last several decades, increased salt intake has been blamed for hypertension and related diseases, and a world-wide campaign on salt reduction commenced. Since the 1980s many investigators have reported serious problems in adult physiological and psychological health that are related to excessive salt intake. Despite such trends towards salt reduction and to our surprise, a recent meta-analysis including 25 previous studies on salt intake and cardiovascular events demonstrated that excessively low-salt intake is associated with an increase in cardiovascular events rather than benefits [Some researchers reported that, particularly during pregnancy, low-salt intake is associated with adverse effects, that is, low birth weight and insulin resistance in offspring in adulthood –4. GrowtAccordingly, an increasing amount of evidence has suggested that fetal programming during gestation may play an important role in the development of metabolism-related disorders during adulthood , 6, 7. CTo our knowledge, however, few studies have investigated the influence that maternal low-salt intake has on the process of fetal growth. There is a necessity to separately examine the influence of low-salt diet on fertilization and on fetal growth. Moreover, it is of interest to disclose the influence of low-salt intake in mothers on the growth of babies after weaning.Thus, in the present study, we hypothesized that low-salt intake during mating (early pregnancy period) or pregnancy period influences fertilization and fetal growth and causes a decrease in the number of newborn pups. To test this hypothesis, we investigated the effects of a low-salt diet during mating or pregnancy on birth and growth rates of offspring in Dahl salt-sensitive (Dahl S) rats. Moreover, we examined the influence of low-salt intake on the growth of babies after weaning.We obtained 4-week-old male and female Dahl salt-sensitive (Dahl S) rats from Sankyo Laboratories, Inc., Tokyo, Japan. The rats were fed regular chow  . At 10 weeks of age, the rats were allowed to mate for 1 week (mating period (early pregnancy period)). Next, the mated female rats were placed in separate cages for the pregnancy period. Three weeks after mating, the mothers began delivering pups. We monitored the number of mothers that delivered pups, the number of pups delivered, and the growth of pups until weaning.Dahl S rats were divided into 5 groups : (1) Dah ad libitum.Dahl S rats were fed the regular  chow during the periods unspecified. Water was availableχ2 and one-way factorial analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) test, Mann-Whitney U test, or Kaplan-Meier survival curve. P values less than 0.05 were considered statistically significant.All statistical analyses were performed using STATISTICA software . Values were expressed as means ± SD. Differences were assessed by We followed the guidelines for experimental animal handling, and our study was approved by the Animal Care Committee of the Kyoritsu Women's University. The experiment was conducted in accordance with the National Institutes of Health (NIH) guidelines.Dahl S rats fed a low-salt diet during the mating (early pregnancy) period delivered less pups compared with those fed a high-salt diet . In DahlSimilarly, Dahl S rats fed the low-salt diet during the pregnancy period delivered less pups compared with those fed the high-salt diet. There were no differences in the birth rates between Dahl S rats that were fed the high-salt rats and those on regular chow.The number of babies delivered from one mother was equal among the 5 mother groups .The pups that were born in each group and the offspring that survived during the lactation period are shown in χ2 = 19.88; P < 0.0001).In contrast, only 64% pups from mothers that were fed the low-salt diet during the pregnancy period survived the lactation period, while the survival rate was 95% for the pups whose mothers were fed the high-salt diet  diet during the mating pregnancy period produced lower number of pups than those on a high-salt (4% NaCl) diet. Since 7 out of 8 mothers that were fed regular chow throughout the mating and pregnancy periods produced babies, the low-birth rate of pups from mothers that were fed the low-salt diet during the mating period may be due to the failure to achieve fertilization. However, the mating season usually lasts 6-7 days in rats, and this means that time point of fertilization varied in mothers tested. The separation on the influence on fertilization and pregnancy process is technically difficult in the present experimental setting.Similarly, the mothers that were fed the low-salt diet during the pregnancy period produced less pups than those that were fed the high-salt diet. As shown in mothers that were fed 0.75% NaCl throughout the mating and pregnancy periods, 7 out of 8 mothers produced pups, suggesting that the low-birth rate of pups from mothers that were fed the low-salt diet during the pregnancy period was due to a failure to maintain the pregnancy. In the present study, we were not able to conclude that fertilization is affected by the low-salt diet; however, it may be concluded that low-salt diet during the mating period and following pregnancy decreases birth rate.In addition, we demonstrated that there were no differences in the number of babies born per pregnant mother among the experimental groups. These findings suggested that ovulation may not be affected by a low-salt diet.The mechanism underlying why fewer pregnant mothers on a low-salt diet delivered pups is unclear. We speculate that low-salt intake enhanced the activity of the renin angiotensin system (RAS), and this may affect the intrauterine growth of pups. Moreover, some studies suggest that the overexpression of RAS transgenic mice results in a placenta-fetus imbalance and leads to preeclampsia –10. In tWe demonstrated in the present study that rat pups born to mothers that were on a low-salt diet during a pregnancy period were more likely to die before weaning than those born to mothers that were fed a high-salt diet. However, it was also demonstrated that there were no differences in survival rate among the pups from mothers fed 0.75% NaCl or a low-salt diets during a pregnancy period. The 0.75% NaCl chow did not influence the birth rate of pups, but it lowered the survival rate of pups from mothers in comparison with pups from mothers fed a high-salt diet during a pregnancy period. This was demonstrated in Kaplan-Meier survival analysis. Since 95% of the pups from mothers on a high-salt diet during a pregnancy period survived until weaning, it was suggested that the regular (0.75% NaCl) diet was less likely to prevent the death of pups during a lactation period. The sensitivity of salt intake to benefits for mothers and pups differed.In the present study we investigated Dahl S rats for the implications of mating or gestational salt intake in the birth and growth of the offspring. It is unclear whether salt sensitivity contributes to the relationship of mothers that were on a low-salt diet and their offspring. The future direction of research regarding salt intake will entail evaluating whether these findings are also true in other genetic rat strains of spontaneous hypertension.We investigated the influence of salt intake on gestation and baby growth using Dahl S rats. Dahl S rats are a genetic model for salt-induced hypertension in humans , 12. ThiThe role of salt intake in the development of hypertension in humans has been the main theme over several decades, and this led to a world-wide campaign of salt reduction. However, the extent of salt reduction is controversial. Recently, a meta-analysis involving 25 studies reported that excessive salt intake is associated with an increased incidence of cardiovascular diseases . MoreoveIn conclusion, low-salt intake during mating and pregnancy is associated with low-birth rates and high death rates among the surviving offspring. We urge additional investigations into the effects of paragestational salt reduction in humans as the level of salt intake that is required for optimal health varies depending on the subjects' age or stage of growth. Furthermore, the current finding provides evidence that supports the recommendation that the amount of salt consumed during pregnancy should be considered carefully based on data regarding the relationship between mothers and their offspring.


The roles of inflammatory macrophages in pancreatic tissue and the development of pancreatic cancer have not been well characterized. Recently it was shown that inflammatory macrophages, besides their function in clearing dead cells, also initiate pancreatic acinar cell metaplasia to duct-like progenitor cells. While in pancreatitis this is a reversible process, in context of an oncogenic stimulus this process is irreversible and can lead to the formation of precancerous lesions. Recent work now indicates that acquisition of an activating Kras mutation in acinar cells initiates signaling that leads to chemoattraction of M1-poliarized macrophages. This oncogene-caused chronic microinflammation can accelerate the pathogenesis of pancreatic cancers. Infiltration of macrophages into the pancreas occurs during acute or chronic pancreatitis; and chronic pancreatitis is tightly-linked to development and progression of pancreatic ductal adenocarcinoma (PDA). While the roles of macrophages in pancreatitis are somewhat well established, only little is known on their role in the development of PDA. Differently polarized macrophages M1 or M2) can exhibit pro- and anti-inflammatory properties. M1-polarized macrophages not only remove dead or dying cells via phagocytosis, but are also involved in inflammation-mediated tissue remodeling, while M2-polarized macrophages restrain the inflammatory response and also inhibit the T-cell response  or M2 ca.Acute pancreatitis is a sudden inflammation of the pancreas that can have multiple causes including alcohol abuse, smoking or unhealthy diet. It is driven by damage of pancreatic acinar cells and release of digestive enzymes and pro-inflammatory messengers that lead to macrophage infiltration and activation . During While rapid production of anti-inflammatory cytokines self-limits acute pancreatitis, the failure to terminate pro-inflammatory signaling prevents regeneration and leads to chronic pancreatitis . In humaIn order to drive the development of pancreatic cancer, oncogenic Kras needs additional inflammatory stimuli to reach pathological activity levels , 14. EveUnderstanding the crosstalk between the pancreatic microenvironment and acinar cells expressing oncogenic mutations is important for several reasons. First, it may lead to discovery of early markers predicting cancer development; and second it could reveal new strategies for intervention.The finding that M1-polarized macrophages can contribute to tumor initiation is somewhat surprising, since tumor associated macrophages (TAM) often are of a M2-polarized phenotype. M1-polarized macrophages so far mainly have been seen as anti-tumorigenic. An immediate question is why M1-polarized macrophages, once attracted to regions of ADM, do not kill precancerous cells? This may be explained by the high antioxidant capacity of pancreatic cells that express an oncogenic version of Kras . MacrophIt is also unclear if at some point during initiation or progression of pancreatic lesions signaling is initiated that leads to a switch from M1- to M2-polarized phenotypes . M2-polain vivo [Eventually, the understanding of the crosstalk between macrophages, pancreatic cells and the pancreatic microenvironment can be important to develop new treatment strategies. Different possibilities arise to target macrophage functions in pancreatitis or developing pancreatic cancer. First, chemoattraction could be blocked using neutralizing antibodies targeting soluble ICAM-1. We have demonstrated that such a strategy is effective in mice to inhibit formation of KrasG12D-caused PanINs . Similarin vivo , 22.Second, macrophages could be directly targeted. In animal studies, macrophages can be inhibited experimentally with gadolinium chloride , liposomin vitro after treatment with IL-4, IL-10 or IL-13 [in vivo in a pancreas [Macrophages show high plasticity and can change their phenotype and physiology dependent on the microenvironment. Therefore, a third possibility for intervention is to modulate macrophage phenotypes. For example preventing a M1 to M2 subtype switch in pancreata with KrasG12D-caused lesions, although it may increase ADM events, may also prevent progression to PanINs and pancreatic cancer. A conversion from M1 to M2 can be achieved or IL-13 , but it pancreas .In summary, to target macrophages or macrophage populations in the developing pancreatic cancer for therapeutic applications requires a full understanding of the phenotypes involved, how they crosstalk with other pancreatic or infiltrated cells, and how this crosstalk contributes to the formation and progression of pre-neoplastic lesions.


The lower mortality rate of obese patients with heart failure (HF) has been partly attributed to reverse causation bias due to weight loss caused by disease. Using data about weight both before and after HF, this study aimed to adjust for reverse causation and examine the association of obesity both before and after HF with mortality.Using the Atherosclerosis Risk in Communities (ARIC) study, 308 patients with data available from before and after the incidence of HF were included. Pre-morbid and post-morbid obesity were defined based on body mass index measurements at least three months before and after incident HF. The associations of pre-morbid and post-morbid obesity and weight change with survival after HF were evaluated using a Cox proportional hazard model.Pre-morbid obesity was associated with higher mortality  but post-morbid obesity was associated with increased survival . Adjusting for weight change due to disease as a confounder of the obesity-mortality relationship resulted in the absence of any significant associations between post-morbid obesity and mortality.This study demonstrated that controlling for reverse causality by adjusting for the confounder of weight change may remove or reverse the protective effect of obesity on mortality among patients with incident HF. Despite the fact that obesity is positively related to all-cause mortality in the general population ,2, the “In light of the discordant opinions regarding the obesity-mortality relationship, we conducted an analysis using the Atherosclerosis Risk in Communities (ARIC) study to investigate the association between obesity and future mortality in patients with incident HF. One aim of this study was to examine the effect of reverse causation by controlling for weight change as confounder. The hypothesis was that if weight loss distorts the true association between body weight and mortality, the obesity-mortality relationship will disappear as a result of adjusting for weight change. The other aim of this study was to estimate the association of obesity both before and after the development of HF with mortality. If greater metabolic reserve contributes to lower mortality, then obesity before the development of HF would be associated with better survival.The ARIC study was a community-based prospective cohort study designed to investigate the causes of atherosclerosis. The study recruited 15,792 adults aged 45 years to 64 years at baseline who resided in four US communities. The first examinations of participants (visit 1) took place during 1987 to 1989, followed by three visits at 3-year intervals. The follow-up rates for the first, second, and third intervals were 91%, 82%, and 74%, respectively. At each visit, participants responded to an interviewer-administered questionnaire and underwent an extensive physical examination . The curIncident HF was defined as death or hospitalization with an International Classification of Diseases, 9th revision (ICD-9) code for HF  given the absence of prevalent HF at visit 1. HF cases were determined through annual telephone interviews regarding their experiences of hospitalization, reviewing hospital discharge lists, and a survey of the national death index and death certificate files. The latter two methods were performed through community-wide surveillance of the communities involved in the ARIC study. The response rate for telephone interviews was 93% to 96%. Mortality outcomes were evaluated in the same way.2) and obese (BMI ≥30 kg/m2). Weight change was defined as percentage change in BMI and calculated as the change in BMI value divided by the original (pre-HF) values of BMI multiplied by 100. Weight change was then categorized based on a priori cutpoint [For this study, demographic variables including sex, race (black or white), and education level  were determined at visit 1. The data before and after the development of HF were considered to define pre-morbid and post-morbid variables. Premorbid variables were based the measurement of covariates ≥3 months before incident HF and post-morbid variables were defined as measurements of covariates ≥3 months after incident HF. On this basis, pre-morbid and post-morbid values for comorbidities including a history of cancer, coronary heart disease, stroke and chronic lung disease, as well as covariates including smoking and drinking status, diabetes mellitus, hypertension, and total serum cholesterol were obtained. Similarly, pre-morbid and post-morbid obesity were defined using BMI measurements from study visits occurring ≥3 months prior and ≥3 months after the date of incident HF, respectively. Due to the small sample of available patients, we categorized BMI into non-obese  or frequency (percentage) and compared using the t-test or the chi-square test. The pre-morbid and post-morbid measurements of risk factors were compared using the paired t-test or the McNemar test. Correlations between pre-morbid and post-morbid covariates were evaluated using the Spearman test. In the multivariate adjusted model, pre-morbid covariates were dropped if they were highly correlated with post-morbid covariate values (r≥0.80). The endpoint of interest was time to all-cause mortality after incident HF. A Cox proportional hazard model with age as the underlying time scale was used to examine the effect of pre-morbid and post-morbid obesity and other covariates on survival. The proportionality assumption was assessed using a graphical approach and testing the slope of Schoenfeld residuals. The Cox model used the date of birth as the origin of the time scale. In order to calculate weight change as a confounder, we used each participant’s weight before the potential onset of disease as their original weight. Weight change naturally occurs before post-morbid obesity, so it was not an intermediate variable between the exposure of interest and outcome. All statistical analyses were performed using Stata version 13.0 .The median and interquartile range of the duration from the previous visit to incident HF was 24.5 (14 to 31) months and from incident HF to the post-visit follow-up was 17 (9 to 27) months. Among 308 patients with incident HF and data available for the pre-morbid and post-morbid covariates, 47% were obese pre-morbidity, 61% were male, 33% were black, and 44% had a basic education. The mean age at the incidence of HF was 61 years old . A total of 3,027 person-years were incorporated into this study, during which 231 deaths occurred. The median follow-up was 9.8 years and the death rate was 76 per 1,000 person-years. A multivariate adjusted model was used to determine the associations of pre-morbid and post-morbid obesity with mortality. Based on subject matter knowledge and the graphical representation of variables as a directed acyclic graph in According to the reverse causation hypothesis, as presented in In this study, reverse causation was revisited and evaluated using a new approach that treated weight change due to disease as a confounder. Our results demonstrate that reverse causation may result in the protective effect of post-morbid obesity on all-cause mortality in patients with incident HF. This conclusion is supported by the strong association we found between weight change due to disease and post-morbid obesity and the association of weight loss with all-cause mortality. Therefore, mortality in obese patients may be reduced due to their lower likelihood of having experienced weight loss. We used a relative measure for weight change in which all patients who lost more than 6% of their original BMI were categorized as experiencing weight loss, irrespective of their original weight.The issue of reverse causation as a form of bias occurring in studies of obesity and mortality has generally been accounted for in the general population ,13,17,18In this study, pre-morbid obese patients who eventually developed HF had a higher risk of mortality than pre-morbid non-obese patients. This finding is in contrast with a previous report that found better survival rates among pre-morbid obese patients . The maiAlthough the term “obesity paradox” is accepted by many scientific communities and listed in some guidelines for cardiovascular disease, it seems that some methodological flaws still persist in estimating the effect of obesity among patients suffering from chronic diseases . This stThis study suffers from some limitations. The identification of incident HF cases was only based on ICD-9 codes for hospitalizations and deaths due to HF, which were not validated, while outpatients with HF were included through responses from physicians using a form. However, the validation of ICD-9 codes by a subsequent medical record review following the ARIC criteria in the ARIC community surveillance study showed a sensitivity of 95% and a positive predictive value of 85%, which are both higher than the Framingham criteria . AdditioIn this study, obese patients who developed HF had a higher risk of mortality than non-obese patients. Thus, greater metabolic reserve did not serve as a protective factor against mortality among obese patients. Post-morbid obesity was not a significant prognostic factor for mortality when the relationship was adjusted for weight change due to HF. Reverse causation may be a plausible explanation for the lower mortality risk of post-morbid obese patients.


ATXN3 gene.The number of studies highlighting the role of microRNAs (miRNAs) in human physiology and diseases is growing, but many miRNA-driven regulatory mechanisms remain elusive. A proper understanding of the exact functions of individual miRNAs and their interaction with specific targets is vitally important because such knowledge might help cure diseases for which no effective treatment currently exists. Herein, we present current views on the role of the miRNA-mediated regulation of gene expression in the case of select spinocerebellar ataxias (SCAs) and their potential involvement in the pathogenesis of these diseases. Specifically, we summarize published data showing the known links between miRNAs and CAG repeat-dependent SCAs. Moreover, using the example of SCA type 3 (SCA3), we refer to the issue of prediction and validation of miRNA targets, and we demonstrate that miR-181a-1 may regulate the 3′-UTR of the  Several spinocerebellar ataxias (SCAs) belong to trinucleotide repeat expansion disorders (TREDs), which are a relatively large group of incurable dominantly inherited neurological diseases. More specifically, six SCAs belong to polyglutamine (PolyQ) diseases and are triggered by an expansion of the glutamine-coding CAG repeat, which is responsible for polyQ tract formation. A long polyQ stretch can elicit numerous aberrant cellular processes that lead to the degeneration and death of neuronal cells.MicroRNAs (miRNAs) are potent regulators of gene expression. They act at the post-transcriptional level by inhibiting protein synthesis that occurs with or without transcript degradation. It appears that these small RNAs are engaged in a wide range of physiological and pathological activities in cells; therefore, their function and contribution to the pathogenesis of SCAs is of special consideration.This review summarizes the current knowledge regarding the putative involvement of miRNAs in the pathogenesis of CAG repeat-dependent SCAs and the experimentally proven associations between specific miRNAs and these diseases that have been reported. Moreover, it addresses some aspects of miRNA target prediction and validation using the example of our own research on the regulation of the ataxin-3 transcript.ATXN) genes. The pathogenic glutamine repeat threshold in most of these SCAs is approximately 35, except for SCA3 and SCA6, in which cases the pathogenic repeat range starts from 50 and 21, respectively. The ataxias that fall under the category of polyglutamine diseases have similar clinical presentations; however, molecular studies are necessary to distinguish particular SCA types. Current views on the identified molecular processes causing or modulating the neurodegenerative phenotype in spinocerebellar ataxias have been discussed recently and reviewed thoroughly [There are six SCAs  among the polyQ diseases [oroughly .+ signaling/homeostasis [The normal function of the polyglutamine proteins implicated in SCAs seems to be quite disparate and may be succinctly defined as follows: ataxin-1 and ataxin-7 play a role in transcription regulation –7, ataxieostasis , and ataeostasis  (revieweeostasis ).The expanded CAGs may trigger pathogenic effects based on three types of mechanisms: (1) toxic RNA gain-of-function, (2) toxic protein gain-of-function, or 3) both transcript and protein loss-of-function  both tra, 14. TheTo date, more than 2,500 mature human miRNAs have been deposited in the miRNA registry  ). RegardGenerally, the level of the polyQ-expanded protein is among the factors that contribute to disease severity –30; howeSpecific links between miRNA regulation and CAG repeat-dependent SCAs have been described in several studies (reviewed in ) Table . HoweverATXN1 has a long 3′-UTR region (approximately 7 kb), which implies an important role in the post-transcriptional regulation governed by miRNAs. Moreover, cerebellar degeneration and ataxia in mice was reported to be interconnected with the impairment of miRNA biogenesis in Purkinje cells [, these findings show that miRNA-mediated posttranscriptional regulation of the ATXN1 gene may modulate SCA1-related neuropathology by affecting protein levels.A study on SCA1 was the first to reveal that some miRNAs can regulate the expression of target transcript mRNA containing a CAG repeat expansion. More specifically, that study showed that miR-19a, miR-101, and miR-130a co-regulate the 3′-UTR of ATXN1 through the inhibition of ATXN1 translation . The genje cells . In a reje cells . InteresAnother key example of the potential involvement of miRNAs, especially miR-144, in brain aging and SCA1 pathogenesis has been provided by a genome-wide microarray analysis . This stRgs8 and Vegfa, were reduced. The latter target was further validated in Neuro2A cells using miR-150 mimics and reporter assays; analyses of both types confirmed the functionality of binding sites for miR-150 that are present in Vegfa mRNA. Collectively, these data demonstrated that the expression levels of mutAtxn1 and miR-150 together with its target mRNA are highly interconnected. It was proposed that the mutAtxn1 de-represses activity at miRNA promoters, leading to an increase in miRNA levels, or acts through yet unknown a gain-of-toxic function mechanism. Therefore, evidence that miRNA misregulation during the critical post-natal period may underlay SCA1 pathogenicity was provided. Moreover, in this work, a promising therapeutic approach based on the RNAi-mediated suppression of mutant Atxn1 was presented. This therapeutic intervention for SCA1 has been developed further by the same group and described elsewhere [Global profiling of miRNA expression in a mouse model of SCA1 was performed to study alterations in miRNA expression in the cerebellum at both pre-symptomatic and symptomatic stages of pathogenesis . More splsewhere .Drosophila transgenic model of SCA1 with the use of deep sequencing [The miRNA deregulation at the beginning of the pathological process has been also investigated in quencing . Specifibantam (ban) was identified as a downstream modulator/suppressor of polyQ toxicity in a Drosophila model of SCA3 that prevents degeneration [ban miRNA suppressed degeneration and diminished the toxicity of ataxin-3, which is the disease-causing protein for SCA3. Suppressing ban activity was connected with neither a change in the level of the pathogenic protein nor its accumulation and was shown to occur downstream of the polyQ protein accumulation. Moreover, reduced miRNA processing due to the knockout of Dicer1 enhanced the neurodegeneration induced by ataxin-3 in both flies and human cells by modulating pathways that normally contribute to polyQ toxicity [The miRNA neration . Upregultoxicity . The proDrosophila[A protective role of miRNAs in age-associated processes and polyQ disorders was further supported by demonstrating that miR-34b mitigates the toxicity of ataxin-3 in osophila. SpecifiDrosophila transgenic model of SCA3 has been also used to investigate deregulation of miRNA expression by deep sequencing, as described for SCA1 with regard to the same analysis [analysis . The stuATXN3 gene. All miRNAs proposed by Shi et al. as potential biomarkers were earlier reported to be connected with neuronal physiology and pathology in terms of neurodegeneration [Most recently, it has been demonstrated that miRNAs are useful biomarkers for SCA3, as specific changes in regulatory miRNA levels were identified in the serum obtained from patients suffering from this disease . The micneration –63. Moreneration . These fDrosophila transgenic model of this disease, along with studying their role in SCA1 and SCA3 [The involvement of miRNAs in the pathogenesis of SCA7 has been investigated in and SCA3 . As aforWith regard to other SCA types, the link between neurodegeneration and miRNAs was demonstrated by showing that the gene encoding TATA-binding protein (TBP), which is mutated in SCA type 17 (SCA17), is downregulated by miR-146a . FinallyAll of the above-mentioned findings suggest that miRNA pathways can modulate polyQ-induced neurodegeneration but that the pathogenesis of polyQ diseases can also influence miRNA expression. It has been proven that miRNAs are implicated in the pathogenicity associated with SCAs at many levels of different pathological processes; however, miRNA specific functions remain largely unknown. Moreover, the question persists, how exactly miRNAs affect CAG repeat-mediated neurodegeneration and whether such miRNA activity is similar in all SCA types. A concept relating miRNAs and ataxias could be built on the known miRNA mode of action Figure . The miRIdentifying biologically relevant miRNA targets is of paramount importance and involves the reliable prediction of miRNA-mRNA interactions and the positive validation of functionality of these interactions. Many different algorithms have been developed to predict miRNA-mRNA binding; computational tools such as miRANDA , PicTar luc2) as the primary reporter for monitoring mRNA regulation and Renilla luciferase (hRluc-neo) as a control reporter for normalization. Typically, in a reporter system, three types of constructs are prepared; a wild-type (WT) construct bearing an intact binding site(s) for the studied miRNA, a construct with mutations in its binding site(s) (MUT), and a perfect match (PM) construct  is among the most common dominantly inherited ataxias –89. The To validate the interactions between our candidate miRNAs, miR-9-2 and miR-181a-1, and the ATXN3 transcript, we transfected HEK293T cells with either reporter carrying potential miRNA binding sites. Four constructs were transfected into cells and tested in parallel. These constructs were co-transfected with appropriate miRNA-coding plasmid vectors (System Biosciences) because our experimental system required miRNA overexpression. The transient transfection of cells with reporter constructs was followed by measuring the reporter activity, which enabled the validation of predicted miRNA-mRNA interactions. Specifically, we obtained considerable repression of the luciferase expression after the transfection of reporter constructs for miR-181a-1, whereas no repression was measured after the transfection of reporters for miR-9-2  with the substrates and procedures from the Dual Luciferase Assay Kit (Promega). The values for firefly luciferase activity for every reporter construct were normalized to the corresponding values of Renilla luciferase activity to account for varying transfection efficiency. Relative expression values for all constructs were obtained by comparing their normalized luciferase activities with those of the control plasmid. Transfection was repeated 6 and 8 times for miR-9-2 and miR-181a-1, respectively.The reduction in luciferase activity in the case of the WT construct carrying b.s. for miR-181a-1 was reproducible and statistically significant (suppression to 64%). The PM construct repressed luciferase to a very low level, while the luciferase activity for the MUT construct showed efficient de-repression. In contrast, we did not observe any decrease in luciferase activity when the WT construct carrying b.s. for miR-9-2 was transfected to cells. As for the experimental procedures, enzymatic activities of firefly and ATXN3 gene.Overall, using the luciferase reporter system, we have validated the interaction between miR-181a-1 and the 3′-UTR of ATXN3 mRNA positively and the interaction between miR-9-2 and the 3′-UTR of ATXN3 mRNA negatively. This is only a preliminary validation of miRNA-mRNA interactions with respect to SCA3 and needs to be elaborated farther, and it is an example of the utility of the reporter system in the search for miRNA targets. Nevertheless, this is the first evidence for the direct binding of miR-181a-1 to the 3′-UTR of the human Many studies have shown that miRNAs play a crucial role in the development and functional regulation of the nervous system, and the deregulation of miRNAs was postulated and proved in numerous neurological disorders of different etiology, including TREDs and even strictly polyQ diseases (reviewed in ). Modest