Our recent studies showed that schisantherin A (StA) is a promising candidate for PD treatment, but the pharmacokinetic profile of StA is largely unknown. The effects of different formulations on the pharmacokinetics and bioavailability of StA were investigated by HPLC equipped with a vacuum degasser, a quaternary pump, a manual sampler, and an ultraviolet detector. The absolute bioavailability of StA in nanoemulsion formulation was significantly increased from 4.3% to 47.3%. To the best of our knowledge, this is the first report of absolute bioavailability for StA in rats and successful increase of bioavailability of StA by nanoemulsion formulation. The pharmacokinetic profiles of StA could be significantly improved by a safe nanoemulsion formulation. This study provides a successful example of advanced delivery system for improving the bioavailability of potential central nervous system (CNS) drug candidate with poor solubility. This novel approach could be an effective alternative solution to overcome the shortcomings of conventional poor drug delivery of CNS drugs. The results of present study not only indicate that StA has potential to be developed as a promising oral therapeutic agent for the management of PD but also shed light on novel way to improve bioavailability of PD drugs. Parkinson's disease (PD) is a slow-progressing neurodegenerative disease, affecting 4–6 million people worldwide, which is primarily caused by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). PD is characterized by debilitating symptoms such as resting tremor, rigidity, and bradykinesia . Current Schisandra chinensis (Turcz.) Baill, has been used as an antitussive, tonic, and sedative agent under the name of Wuweizi in Chinese traditional medicine. StA is believed to have a wide variety of therapeutic effects including antihepatotoxic effects, anticarcinogenic effects, and antioxidant effects [+ [+-induced cell death. The potential structure-activity relationship revealed a large benzoyloxy substituent group, on the cyclooctadiene ring in StA, and is probably functionally important for its neuroprotective activity. Moreover, StA conferred significant protection against 6-OHDA- or MPTP-induced damage in TH-positive dopaminergic neurons in PD zebrafish and mice model. The in-depth mechanistic assessment demonstrated that StA exhibited neuroprotection against MPP+ through the regulation of two distinct pathways including CREB-mediated Bcl-2 expression and PI3K/Akt survival signaling. All our in vitro and in vivo data provide the strong evidence of StA's promising anti-PD activity. Therefore, StA has been suggested for use as potential therapeutic candidate for treating neurodegenerative disease.The scaffold structure of the dibenzocyclooctadiene lignans exhibited protective effects against neuronal cell death in stroke and other neurodegenerative diseases and cognitive impairments in Alzheimer's disease. Schisantherin A (StA), a dibenzocyclooctadiene lignan from the fruit of effects –8. In oufects [+ , 10. AmoThose promising results prompted us to investigate the pharmacokinetics of StA and also further develop new oral formulations to improve the bioavailability of this new anti-Parkinson natural compound StA for the treatment of PD.Schisantherin A (batch number E-0134) was purchased from Shanghai Tauto Biotech Co., Ltd. . The purity of all the compounds used in this study was >98%. Soybean oil was purchased from Sigma . Solutol HS 15 was purchased from BASF . Acetonitrile and methanol (HPLC grade) were purchased from Hanbang Company . All other reagents were obtained from Sigma-Aldrich  unless stated otherwise. Ultrapure water was prepared by a Milli-Q Plus water purification system .μm) was used at 35°C with a mobile phase of water and methanol . The flow rate was 1 mL/min. The monitoring wavelength of StA was 230 nm.Quantitative analysis of StA was completed on an Agilent series 1260 HPLC apparatus  equipped with a vacuum degasser, a quaternary pump, a manual sampler, and an ultraviolet detector. RP Phenomenex C-18 column  × % and relative standard deviation (RSD%), respectively. Extraction recovery of StA in plasma sample was calculated by comparing the peak areas of the extracted QC samples to that of the unextracted standard solutions at equivalent concentrations. The calibration curve was plotted using a 1/All animal experiments were conducted according to the ethical guidelines for animal experiments of Jinan University. The experimental protocols were approved by the Ethics Committee for Animal Experiments of Jinan University (permit number 20110810). Male Sprague-Dawley rats (SD rats) with body weight between 220 and 250 g and age between 6 and 7 weeks were supplied by Guangdong Medical Experiment Animal Center . On the day before the experiment, a polyethylene catheter  was cannulated into the right jugular vein under 10% chloralic hydras (i.p. 250 mg/kg) anesthesia. After surgery, the rats were placed individually in cages and allowed to recover for at least 12 h.In order to calculate and improve the oral bioavailability of StA, StA has been formulated into different formulations including nanoemulsion for comparison. Rats were randomly divided into 3 groups (three rats per group) given dosing and formulation regimens as follows.Formulation 1 (tPAP formulation). StA was dissolved in a mixture of Tween-80, propanediol, anhydrous ethanol, and polyvinyl acetate . And then, the suspension of StA was orally administrated to rats at 300 mg/kg.Formulation 2 (dPS formulation). StA was absolutely dissolved in mixture of DMSO, PEG400, and saline (1 : 1 : 1). Before an i.v. bolus injection of StA at 30 mg/kg, the micropore filter membrane (0.22 μm) was used to remove the impurities in this solution.Formulation 3 (nanoemulsion formulation). StA was prepared as nanoemulsion for i.v. and i.g. at 30 mg/kg. The concentration of StA nanoemulsion was 4.9 mg/mL. Briefly, crystalline StA powders were completely dissolved in soybean oil containing Solutol HS 15  as a nonionic surfactant and by stirring the materials at ambient temperature to obtain 1% (w/w) StA oil phase. Oil-in-water nanoemulsion was prepared by mixing above oil phase  with aqueous phase  at ambient temperature using a sonicator with a 3 mm probe  with an output power of 75 W. The freshly prepared macroemulsion was then sealed in aluminum foil covered 50 mL test tubes and stored at ambient temperature. The mean particle diameter of nanoemulsion was measured using dynamic light scattering . The nanoemulsion was diluted 10 times with aqueous solution prior to analysis. The nanoemulsion had a relative small mean particle size of 300.7 ± 2.7 nm (n = 3) and the PDI values were less than 0.34.μL plasma which was taken from the supernatant, 200 μL of 50% acetonitrile and 50% methanol were added. After vortexing for 20 s and centrifuging at 12,000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm membrane, of which 20 μL was injected into the chromatographic system for analysis. Pharmacokinetic parameters were calculated with DAS 3.0 software .After administration of StA, blood samples were collected at 0.033, 0.167, 0.5, 1, 2, 4, 8, 12, 24, and 36 h, respectively. Plasma samples were obtained after centrifugation of the collected blood samples at 3,000 rpm for 5 min. Subsequently, in 100 μg/mL, and plasma sample obtained after administration of StA. There were no significant endogenous peaks directly interfering with the detection of analytes in plasma, respectively (data not shown). The retention time of StA was about 6.1 min. Under the established analysis method, the lower limit of detection (LOD) and lower limit of quantitation (LOQ) of StA were 0.02 and 0.025 μg/mL for plasma sample. In the range of 0.25–25 μg/mL for plasma samples, the calibration curve of StA showed good linearity (r2 ≥ 0.999) using a 1/x2 weighted linear regression. The intraday and interday precision and accuracy in rat plasma were evaluated by using the low, medium, and high concentration of QC samples (n = 6) surpassed 88.3 ± 4.53% . Compared with TPAP formulation, the absolute bioavailability of StA in nanoemulsion formulation significantly increased from 4.3% to 47.3%. To the best of our knowledge, this is the first report of absolute bioavailability for StA in rats and successful increase of bioavailability of StA by nanoemulsion formulation.The concentration-time profiles of StA in rat plasma were presented in  Schisandra chinensis (Turcz.) Baill, against two neurotoxins 6-OHDA and MPP+ was systematically investigated in our completed studies [ schisandrae extract or prescriptions [After decades of use, levodopa remains the mainstay of PD treatment. However, because PD by its nature is a progressive disease and because there are no medications proven to delay advancement of symptoms, patients are subject to a therapeutic regimen that inevitably becomes increasingly complex and burdensome. It is important that strategies are employed to minimize side-effects and that medications are monitored for both efficacy and safety. The crucial goal of PD treatments is the development of a neuroprotective treatment that slows disease progression . Chinese studies , 10. Theriptions . The resriptions . However in vitro and in vivo data indicated StA's promising anti-PD activity, but further development of StA was required to address the problem of its poor bioavailability. The high dose of StA, at 300 mg/kg, exhibited significant and comparable effectiveness to 10 mg/kg of the anti-PD drug selegiline in our previous PD mice study [Poor bioavailability is one of major hurdles in drug development, particularly in the area of central nervous system (CNS) drugs . Therefoce study , 10. In ce study . Due to ce study . Nanoemuce study , 17. Herce study . Moreovece study , and thece study . In addice study . Recent ce study . Recentlce study –25. The ce study . The pre in vivo activity of StA, where a previous study reported that StA was subjected to liver metabolism in vivo; thus far, two StA metabolites, 7,8-dihydroxy-schisandrin, and an additional metabolite yet to be confirmed (with M.W. 432 g) have been identified in rat [A previous study supported the notion that StA could pass through the blood-brain barrier (BBB) . Furtherd in rat .In conclusion, this study provides an example of advanced delivery system for poor soluble compounds like StA. This novel approach could overcome the shortcomings of conventional poor drug delivery of some CNS drugs. StA was prepared in nanoemulsion to satisfy well dispersion of the drug, to promote the dissolution and absorption, and finally to enhance the oral bioavailability. Our results confirmed that the nanoemulsion demonstrated the best performance compared with all other tested formulations. The pharmacokinetics of StA and a safe formulation of StA with high bioavailability had been determined. This data indicates that StA shall be a promising therapeutic agent for the prevention of PD by oral delivery.


Contrary to our predictions, there was no evidence that related dyads of hoary bats or silver-haired bats were killed on the same night more frequently than expected by chance, or that the number of days between the fatalities of dyad members was influenced by relatedness or latitude of origin. Our data suggest that these bats do not socially transmit migration routes and behaviours among close kin.To migrate, animals rely on endogenous, genetically inherited programmes, or socially transmitted information about routes and behaviours, or a combination of the two. In long-lived animals with extended parental care, as in bats, migration tends to be socially transmitted rather than endogenous. For a young bat to learn migration via social transmission, they would need to follow an experienced individual, most likely one roosting nearby. Therefore, we predicted that bats travelling together originate from the same place. It is also likely that young bats would follow their mothers or other kin, so we predicted that bats travelling together are more closely related to each other than bats not travelling together. To test our predictions, we used microsatellite genotypes and stable isotope values of  Migratory animals rely on endogenous, genetically inherited programmes or socially transmitted information about routes and behaviours, or a combination of the two –4. The dNavigational abilities of experienced individuals are superior to those of inexperienced individuals ,7. In faGrus americana), social learning reduces suboptimal migration strategies; inexperienced birds that followed older birds were significantly better at accurately arriving at their over-wintering location compared with inexperienced birds that flew only with other inexperienced birds [Animals that learn components of migration from social transmission show increased flexibility and adaptability of migratory behaviour and a decreased use of suboptimal routes relative to those relying on genetic programmes . For exaed birds .Loxodonta africana) for example [The degree to which social transmission of migratory information occurs is correlated with life history. In long-lived birds with extended parental care, such as cranes, geese, swans, storks and bustards, migration tends to be socially transmitted, while it is under endogenous control in the majority of small songbirds ,19. Long example ,21.Social transmission of migratory information and the use of optimal migration routes may be particularly important for animals with slow life histories because they have low reproductive rates, increasing the importance of successful migrations of juveniles. Migration is a risky behaviour, with most of the annual mortality occurring during migration –24, partBats have slow life histories and long lives , and we Tadarida brasiliensis) [Eidolon helvum) [Although highly gregarious species of bat likely migrate as a group (e.g. Mexican free-tailed bats (liensis)  and stra helvum) ), it is  helvum) –47. One  helvum) . Echoloc helvum) , but it  helvum) . Regardl helvum) .Social transmission is most likely to occur from mother to offspring, but perhaps also from other conspecifics. For a young bat to learn migration via social transmission, it would need to follow an experienced individual, most likely one roosting nearby. Therefore, we predicted that bats travelling together on the same night originate from the same place. Stable isotopes can be used to determine the geographical origins of bat fur grown on the summering grounds, because fur is metabolically inert once grown and the stable isotope ratios in fur therefore reflect the environmental conditions (i.e. isoscape) where it was grown –51.Lasiurus cinereus/Aeorestes cinereus [Lasionycteris noctivagans), we hypothesized that the degree to which migration is socially transmitted differs among species, being more prevalent in colonial than in solitary bats. If so, then we predicted that the similarity in geographical origins and the degree of relatedness of individuals travelling on the same night would be greater in colonial bats than in solitary ones, if colony members are closely related, as in some species such as big brown bats (Eptesicus fuscus) [Plecotus auritus) [δ13C), nitrogen (δ15N) and hydrogen (δ2H) to analyse the temporal relatedness and geographical origins of migrating hoary bats and silver-haired bats killed at a wind energy facility in southwestern Alberta, Canada over two consecutive autumn migrations.It is likely that young bats follow their mothers or other close relatives, so we predicted that bats travelling together on the same night are more closely related to each other than bats travelling on different nights. This pattern should be particularly apparent in adult females and juveniles because they roost together in family groups, whereas males roost independently. Given that some species of migratory bats are solitary  and som fuscus) . Howeverauritus) , then weauritus)  and stab1.1.Hoary bats roost solitarily in tree foliage throughout much of North and South America . AlthougKnown wintering grounds of silver-haired bats are in the US Pacific Northwest, southwestern states and middle latitudes of the eastern United States ,63. The 2.2.1.We collected hair and skin samples from bat carcasses found under wind turbines in southwestern Alberta, Canada . We searched for bat carcasses from 15 July to 30 September 2006 and 2007. We searched 10 randomly chosen turbines every day and the remaining 29 turbines once a week (see  for detaWe collected skin tissue from the wingtips of freshly killed bat carcasses (i.e. those killed the previous night at any of the 39 turbines) using dissecting scissors. Tissue was stored in 70–90% ethanol and kept in a −20°C freezer until DNA extraction. We collected hair from between the scapulae of bats killed the previous night, placed samples into plastic microcentrifuge tubes and stored them in a freezer until analysis.2.2.−1 BSA, 0.4 µM unlabelled primer, 0.04 µM tag-labelled primer, 0.36 µM universal dye-labelled primer, 3.0 mM MgCl2, 0.8 mM dNTPs, 0.5 units AmpliTaq Gold® Polymerase (Applied Biosystems) and 20 ng DNA template) using an Applied Biosystems GeneAmp 9700. Touchdown thermal cycling programmes [enemapper v.~3.7 (Applied Biosystems).Development of primers and microsatellite analysis were conducted at the University of Georgia's Savannah River Ecology Laboratory , observed (Ho) and expected heterozygosity (He) and linkage disequilibrium (LD) using Arlequin v.~3.5.1.3 [icro-Checker v.~2.2.3 [One hundred and thirty-three hoary bats were genotyped at 19 microsatellite loci and 87 silver-haired bats were genotyped at 18 microsatellite loci. We calculated the number of alleles (~3.5.1.3 . We usedv.~2.2.3  to searc2.3.δ13C), nitrogen (δ15N) and deuterium (δ2H) analyses were conducted at the Saskatchewan Isotope Laboratory in the Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada .We examined relatedness of individual bats in multiple ways. First, we used the full-likelihood method of C~2.0.5.8  to putatoancestry v. 1.0.1.5 [oancestry calculates seven measures of relatedness simultaneously, but only the two maximum-likelihood methods (TrioML and DyadML) allow one to incorporate genotyping errors, which, if not accounted for, can affect the precision of the relatedness estimates. We used the genotyping errors calculated by Colony during the first analysis and compared the results of TrioML and DyadML to determine which model to use. For both species, the two models were highly correlated (r2 > 0.90), but the relatedness values were slightly higher in DyadML, so we used these values. However, the estimates from maximum-likelihood methods are not normally distributed, because they are bounded between zero and one, and thus result in loss of statistical power. Therefore, we used the package Related v. 1.0 in R 3.2.3 [oancestry-based analyses because it performed the best . A rela2.5.olony, we used two-tailed Fisher's exact tests and for the relatedness values of Coancestry, we used two-tailed t-tests. We restricted the analysis to within-year dyads . We used an α-value of 0.05. We present mean ± standard error where applicable.To investigate whether related individuals were killed on the same night more frequently than expected by chance, we compared related and unrelated dyads killed on the same night to related and unrelated dyads killed on different nights using two different methods. For the putative relatedness categories of Coancestry analysis as a predictor variable. As a proxy for geographical origins, we used the absolute difference in the stable isotope values of δ13C, δ15N and δ2H between members of each dyad that had stable isotope values available for both individuals  as the other predictor variables. We included the interactions between relatedness and each of the isotope variables, but removed the interactions from the model if they were not significant. For the GLM, we used the log link function for Poisson distributions, accounted for overdispersion, and used an α-value of 0.05.We also examined whether the time between individuals within a dyad (in days) was influenced by their relatedness or the similarity in their geographical origins by using generalized linear models (GLMs) in JMP 10 . We used the normally distributed Wang relatedness value from the C3.3.1.For hoary bats, the number of alleles per locus ranged from three to 66 . Average observed heterozygosity was 0.73 ± 0.23. Ten loci contained null alleles . Forty-four of 171 comparisons (25.73%) showed evidence of significant LD. In silver-haired bats, the number of alleles per locus ranged from six to 20 . Average observed heterozygosity was 0.67 ± 0.17. Eight loci contained null alleles . Thirty-seven of 153 comparisons (24.18%) showed evidence of significant LD.3.2.Details regarding overall patterns and relationships of the stable isotopes can be found in .3.3.olony identified 134 related dyads, 83 of which occurred within the same year. The majority of putatively related dyads (97%) were half-sibling dyads, but there were two full-sibling dyads, one mother–offspring dyad and one father–offspring dyad. Neither the full-sibling dyads nor the parent–offspring dyads involved individuals killed on the same night. There was no evidence that a greater number of related dyads of hoary bats were killed on the same night more frequently than expected by chance .Using the DyadML estimator, C2/d.f.5 = 27.05, p < 0.001; Pearson's χ2/d.f.2061 = 12 768.07, p < 0.001; overdispersion = 6.20). The number of days between the fatalities of dyad members increased as the difference in their δ13C values increased, and as the difference in their δ15N values decreased, and was influenced by the interaction between dyad relatedness and the difference in their δ2H values  were half-sibling dyads, but there was one full-sibling dyad and one father–offspring dyad. Neither the full-sibling dyad nor the father–offspring dyad involved individuals killed on the same night. There was no evidence that a greater number of related dyads of silver-haired bats were killed on the same night more frequently than expected by chance .Using the DyadML estimator, Cχ2/d.f.4 = 19.7, p < 0.001; Pearson's χ2/d.f.842 = 7891.40, p < 0.001; overdispersion = 9.72). The number of days between the fatalities of dyad members increased as the difference in their δ15N values increased but was not influenced by any other predictor variable  or full-sibling dyads (n = 3) were killed on the same night. However, siblings from the same litter may have multiple paternity and be half-siblings rather than full-siblings, as seen in other species of bat that have twins [Contrary to our predictions, we found no conclusive evidence that either hoary bats or silver-haired bats migrate in family groups. In both species, the majority of dyads were not related and although a few related dyads were killed on the same night, 85–93% of the related dyads were killed on different nights. None of the putative parent–offspring  are relatively high for adults , butlt males .Because information obtained socially is sometimes easier to obtain, but less reliable than information obtained asocially , there is often a mixture within a group in the number of individuals acquiring information asocially and those acquiring information socially ,97. InfoThe costs and benefits of social transmission of migratory information can be divided into two categories, those associated with being in a group and those associated with the teaching or learning of behaviours. Benefits of being in a group may include increased predator detection and social thermoregulation, whereas costs may include increased conspicuousness and competition for resources ,101. BecIf the social transmission of migratory information is purposeful (i.e. migratory behaviours are taught), then it is likely that the costs are higher for the leaders/demonstrators than for the followers/observers. In fact, one of the main characteristics of teaching behaviour is that the~demonstrator incurs a cost, or at least not an immediate benefit, from altering their behaviour in the presence of a naive observer . Costs mWe also know little about the mating behaviour of hoary or silver-haired bats, but as in other temperate-zone bats, they likely mate in autumn, during migration ,62,65. MPipistrellus nathusii) [Bats may employ a ‘fly-and-forage strategy’, foraging periodically during migration flights rather than foraging solely at emergence in the evening, as seen in Nathusius' pipistrelle (athusii) . If atteathusii) ,111 and athusii) ,113, theathusii) ). Perhapathusii) .If migratory behaviours in bats are the result of a genetic programme rather than social transmission, how do individuals find their way? The literature on orientation and navigation systems of animals during migration is vast and rich, particularly for birds –118. BatPteropus poliocephalus) that follow the flowering of plants [We hypothesized that the benefits of social transmission, such as increased flexibility and decreased use of suboptimal routes , would of plants ,133? Wha


Enterococcus faecium possessing vanA gene while the rest (16%) were Enterococcus faecalis possessing vanB. The esp gene was found in all 45 VRE isolates while hyl genes were found only in E. faecium species. The E. faecium species expressed five distinct PFGE patterns. The predominant clones with similar or common patterns belonged to clones one and three, and each had 11 (29%) of the VRE isolates. Plasmid content was identified in representative isolates from each clonal group. By contrast, the E. faecalis species had one PFGE pattern suggesting the presence of an occult and limited clonal spread. The emergence of VRE in the country seems to be related to intra/interhospital dissemination of an epidemic clone carrying the vanA element. Therefore, infection control measures will be warranted to prevent any potential outbreak and spread of VRE in the country. Geographic spread of vancomycin-resistant enterococci (VRE) clones in cities, countries, or even continents has been identified by molecular techniques. This study aimed at characterizing virulent genes and determining genetic relatedness of 45 VRE isolates from Trinidad and Tobago using molecular tools, including polymerase chain reaction, pulsed-field gel electrophoresis (PFGE), and Random Amplification Polymorphic DNA (RAPD). The majority (84%) of the isolates were In addition, the presence of the variant esp gene in E. faecium was reported to be associated with in-hospital spread whereas the hyaluronidase (hyl) gene was also regarded as a potential virulence gene associated with invasive disease [Vancomycin-resistant enterococci (VRE) were first described in Great Britain in 1988 and shortly afterwards were reported in other European countries and the USA . In Lati disease , 5. Alth disease , there a esp repeat profile, and molecular relatedness among enterococci isolated from hospitals in Trinidad and Tobago. This information may provide the background level of VRE in Trinidad and Tobago and be of help in controlling nosocomial spread if a VRE outbreak occurs.Understanding the molecular epidemiology of VRE is crucial for assessing and implementing infection control measures in any healthcare institution or country . The aimThe 45 VRE bacterial isolates used for this analysis were those identified among 1,141 enterococcal isolates from previously reported study . These i Staphylococcus aureus ATCC 25923 and E. faecalis ATCC 29212 strains were used as controls. The antibiotics ampicillin, ciprofloxacin, levofloxacin, linezolid, nitrofurantoin, penicillin, quinupristin/dalfopristin, rifampicin, tetracycline, and vancomycin included in the Gram-positive panel 20 of the Microscan were tested. The MIC values were interpreted according to approved CLSI breakpoints [μg/mL; CIP ≤ 1 μg/mL; LEV ≤ 2 μg/mL; LZD ≤ 2 μg/mL; PEN ≤ 8 μg/mL; Q-D ≤ 1; RIF ≤ 1 μg/mL; TET ≤ 4 μg/mL; VAN ≤ 8 μg/mL.The antimicrobial susceptibility tests were performed by the standard disk diffusion method and minimum inhibitory concentration (MIC) determined using the Microscan WalkAway 96 SI .μL reaction mixtures containing deoxynucleoside triphosphate, two primers , Taq polymerase, MgCl2, buffer, and H2O.Determination of glycopeptide resistance genotypes and confirmation of species identification were performed by multiplex polymerase chain reaction (PCR), as previously described by Jayaratne and Rutherford . BrieflyThe samples were subjected to 30 PCR cycles, each consisting of one minute of denaturation at 94°C, one minute of annealing at 58°C, and one minute of elongation at 72°C. PCR products were analyzed by electrophoresis on 1% agarose gels and were stained with ethidium bromide. vanA strain (E. faecium ATCC 700221), a vanB strain , and a vancomycin susceptible E. faecalis (ATCC 29212), 16S rDNA Internal Amplification Control, were run with each set of reactions as quality positive and negative controls . Gel images were captured on the Gel Doc imaging system using Quality One Software version 4.4.1 . The resulting banding patterns were analyzed by visual inspection according to previously established criteria [Genomic DNA was prepared in agarose plugs as described by Murray et al. and Turabelidze et al. with some modifications , 12. Aftcriteria , 14. GelμL reaction mixture containing 20–30 g of genomic DNA, 2.5 μL 10x buffer, one-unit Taq DNA polymerase, two μmol primer, one mmol each of dCTP, dGTP, dATP, and dTT, and 2.5 mmol MgCl2. AP4 (5′ TCA CGC TGC A-3′) random primer was used for RAPD. PCR reactions were performed on Perkin Elmer 9600 under the following conditions: 30 cycles of 94°C for one minute, 36°C for one minute, and 72°C for two minutes, with a final extension of 72°C for five minutes. PCR products were run on 1.5% agarose gels and stained with ethidium bromide. DNA ladder  was used as DNA size markers. AP4 (5′ TCA CGC TGC A-3′) primers were chosen for RAPD analysis because on PCR they yielded clear patterns [PCR assays were routinely performed in a 25 patterns .μL loop of plated culture or one mL of broth culture and the Ultraclean Microbial DNA Isolation Kit  following the manufacturer's instructions. The extracted DNA was amplified using the DiversiLab Enterococcus fingerprinting  according to the manufacturer's instructions. Genomic DNA, the Rep-PCR primer (Enterococcus) AmpliTaq, and PCR buffer (Applied Biosystems) were all mixed together and subjected to thermal cycling. Amplicons were separated by 1.5% agarose gel electrophoresis  containing ethidium bromide (3 μg/mL in gel and in 1x tris-acetate-EDTA running buffer) for six hours at 120 V in a recirculating electrophoresis unit. DNA ladder  was used as DNA size markers. Gel images were captured on the Gel Doc imaging system using Quality One Software version 4.4.1 .Repetitive-sequence-based-PCR (Rep-PCR) methods are rapid typing procedures that amplify the regions between the noncoding repetitive sequences in bacterial genomes . The ERI esp and hyl genes was determined for all 45 VRE isolates by PCR as described by Vankerckhoven et al. [μm of primer hyl and 0.2 μm of esp primer including HotStar Taq Master Mixture , Taq DNA polymerase, and deoxynucleoside triphosphates were all subjected to 30 PCR cycles. The PCR products were analyzed by electrophoresis on 1.5% pronarose gel for one hour at 150 V and were stained with ethidium bromide. E. faecium strain C68 (hylEfm and espEfm) was used as the positive control. A 100 bp DNA ladder (Bio-Rad) was used as a molecular size marker.The presence ofn et al. , 19. Bac esp A and C repeats, two different primer combinations were used: espfs 7F-espfm 5R and espfs 5F-espfm 3R, respectively [μL volumes with HotStar Taq Polymerase and HotStar Master Mix buffers . Subsequently, the amplicons were subjected to agarose gel electrophoresis (1%) in order to determine their sizes. From the sizes of the amplicons the numbers of repeats were deduced. Amplicon size differences corresponded to multiples of either 252 bp (A repeats) or 246 bp (C repeats).For determining repeat number variations ofectively . Brieflyectively . PCR conA subset of isolates representing at least one isolate indicative of each major PFGE pattern was assessed for the presence of the plasmids by the SI nuclease method as described by Barton et al. . E. faecium and the rest  are E. faecalis. All enterococcal isolates had an MIC value for vancomycin ≥ 32 μg/mL; they all were 100% susceptible to linezolid. Although only 18% of the E. faecium were resistant to quinupristin-dalfopristin but 100% resistant to ciprofloxacin, erythromycin, and levofloxacin, all E. faecalis were 100% resistant to ciprofloxacin, erythromycin, levofloxacin, and quinupristin-dalfopristin.All 45 VRE isolates used for this analysis and their hospital and facility distribution were from work previously reported . More th E. faecium isolates possessed the vanA genes while all E. faecalis possessed the vanB genes. Overall, the esp gene was detected in all (100%) VRE isolates. None of the isolates had hyl genes. Analysis of the esp repeat profiles produced similar results. The numbers of A and C repeats of the esp gene seen in the E. faecium isolates varied from three to seven and from three to eight, respectively. Based on the esp A and C repeat profile, these E. faecium isolates belonged to five different groups. The most prevalent esp profile was A6-C5 , followed by A5-C6 , A6-C3 , A4-C5 , and A5-C7 .All the E. faecium isolates. The predominant clones were one and three (PFGE-1 and PFGE-3), and each clone occurred in 11 (29%) isolates, respectively. Clone one was present in two hospitals: “D” and “C” hospitals located in the southern and northern geographic areas of Trinidad. Clone three was present in four of five hospitals, “A,” “C,” “D,” and “E,” in the country. Clones two and five (PFGE-2 and PFGE-5) were represented by six (16%) and eight (21%) isolates, respectively, from the two hospitals “C” and “D.” Clone four (PFGE-4) had two isolates, one from “C” and the other from “A” hospitals and both from the urogenital tract (UGT). All the seven vancomycin-resistant E. faecalis had an identical PFGE pattern indicating they belong to the same clone (PFGE result not shown). The cluster analysis was achieved by the Bionumerics software . Percentages of similarity were determined using the Dice correlation coefficient and a dendrogram , type two , type three , type four , and type five . The vancomycin-resistant E. faecalis had one RAPD and Rep-PCR type and all had similar banding patterns. These results were concordant with PFGE types which were visually inspected according to Tenover et al. criteria [RAPD produced concordant patterns to PFGE. Five different RAPD and Rep-PCR types were obtained for the 38criteria .Seven VRE isolates representing the various clones and from different hospitals were randomly chosen for plasmid determination. All the isolates examined had at least one plasmid ranging in size from 48.5 kbp to 200 kbp. One isolate from hospital “C” had multiple plasmids. esp repeat profiles. Each method had a different ability to analyze the genotypes of VRE isolates.In the current study, the genetic characteristics of all vancomycin-resistant enterococci were investigated by PFGE, RAPD, Rep-PCR, andE. faecium remained as in many countries the most prevalent species among VRE (86% of the isolates) which is similar to reports from North America, Australia, and Italy, where prevalence ranged from 79.5% to 99% [ E. faecium from this current study were multiply resistant to antibiotics such as ciprofloxacin, erythromycin, levofloxacin, rifampicin, and tetracycline, similar to results from other places [ E. faecalis showed high (100%) resistance to erythromycin, vancomycin, and quinupristin/dalfopristin. Similar results were observed by Corso et al. [% to 99% –23. The r places , 23. In  E. faecium isolates in this study were of the vanA genotype which showed a high vancomycin MIC of ≥32 μg/mL. A similar high vancomycin MIC has been reported in the United States and Europe [ vanA E. faecium in our study is similar to findings in Northern Asia, Europe, and the United States [ vanB E. faecalis in our study is also similar to findings from Australia and Taiwan where vanB gene is more common [All vancomycin-resistantd Europe . The pred States , 25. Thee common . esp gene to be present in all the 45 VRE isolates. Although reports of esp prevalence vary according to region and population, our results are consistent with those of Shankar et al., who found the prevalence of esp to be 77% in a sample of E. faecium (predominantly vanA) from eight European countries [ esp gene is part of a putative pathogenicity island considered to be a marker for epidemicity and could putatively contribute to the spread of vancomycin-resistant E. faecium isolates in hospitals [ esp genes was not associated with the invasiveness or outbreak potential of VRE. Shankar et al. reported that esp genes can be deleted from the pathogenicity island of vancomycin-resistant E. faecalis at a high frequency [ esp gene is transferable by conjugation among enterococcal isolates [This present study found theountries . Also inountries . The espospitals –29. The requency . Oancea isolates . esp repeat profiles used to analyze the VRE isolates revealed that the E. faecium strains in the different groups had identical esp repeat profiles which were relatively stable and this was similar to other studies by Leavis et al. [ esp repeat profiles could be utilized to investigate the outbreaks of resistant clones in combination with other genotyping methods [Thes et al. . The esp methods . esp gene repeat profiles showed multiple genotypes of E. faecium isolates which were consistent with the result of RAPD and Rep-PCR. The presence of a dominant vancomycin-resistant E. faecium clone (clones 1 and 3) in several major hospitals shows that their spread has occurred not only within individual hospitals but also between hospitals of various geographic locations in the country. Other studies have documented the spread of vancomycin-resistant E. faecium and E. faecalis clones among hospitals [ esp gene encoding a surface protein associated with virulence for E. faecalis and E. faecium residing on a pathogenicity island. Studies by Harrington et al. support the hypothesis that a combination of vancomycin resistance and the esp gene could lead to dissemination of particular clones [ hyl gene in clinical VRE isolates in this current study suggests a low prevalence or nonexistence of this gene in Trinidad and Tobago. This will definitely be a sharp contrast to the prevalence of hyl gene that varies from 3% to 71% among European VRE [In this study, the PFGE and theospitals , 33. Theospitals , 28 haver clones . The finpean VRE , 18. E. faecium clones one and three in different wards of the same hospital, in different hospitals, and in different cities. The reason for this may be due to the absence of an alert system for patients infected or colonized with vancomycin-resistant enterococci in the hospitals. There are equally no consistent effective screening mechanisms or policies in place for VRE infections or colonization, and all these could contribute to this dissemination. The isolates in this study were polyclonal with two major clones, suggesting a highly diverse population of hospital acquired E. faecium strains. This picture can possibly be explained by exchange of a mobile resistance determinant between various enterococci as reported in other places [In our study, molecular typing results indicate the dominant dissemination of vancomycin-resistantr places –37. E. faecium isolates. This demonstrates that E. faecium strains could be easily differentiated by RAPD fingerprinting, thus supporting the validity of this fast and accurate technique in studying diversity of E. faecium population [ E. faecium [Five different RAPD types were obtained for the 38 vancomycin-resistant faecium . During  faecium . Therefo faecium . Acinetobacter baumannii, Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA) [The Rep-PCR has been reported to have good typeability and reproducibility  and has s (MRSA) . Rep-PCRs (MRSA) . The isoβ-lactamase producing enterococci [vanA phenotype) is due to dissemination of a gene rather than a bacterial clone or a single plasmid. Many attempts have been made to show the ability of enterococci to transfer genes encoding for antibiotic resistance with the same or different enterococci species, as well as to other members of other bacteria genera [ E. faecium identified in this present study appears to be low, limiting the usefulness of plasmid typing of these isolates.Plasmid analysis has been used for epidemiologic studies of several outbreaks involving aminoglycosides-resistant and erococci , 44. Stuerococci  of glycoa genera . The plaThe significantly higher prevalence of VRE in the two regional hospitals, “C” and “D” hospitals, suggests that these hospitals may be at a greater risk of VRE dissemination as there was evidence of high consumption and usage of antibiotics in these institutions .This analysis indicates that the prevalence of VRE is low among clinical isolates in the country. Our findings confirm the potential for interhospital spread of VRE and highlight the importance of strengthening the practice of appropriate infection control protocols or early implementation in hospitals in Trinidad and Tobago. esp gene was seen among the polyclonal VRE infection isolates and molecular analysis suggests that intra- and interhospital spread of vancomycin-resistant enterococci clone carrying vanA elements seem to be the main mechanism of vancomycin-resistance dissemination in Trinidad and Tobago.The PFGE, RAPD, and Rep-PCR proved useful in typing vancomycin-resistant enterococci isolates from Trinidad and Tobago. A high prevalence of theContinued surveillance activities for VRE are needed to detect early occurrence, dissemination, and corresponding increase in VRE prevalence locally. Further studies such as determination of sequence typing (ST) by multilocus sequence typing (MLST) or multiple-locus variable number tandem repeat analysis (MLVA) are warranted, and carriage rate of VRE among individuals in the country should be investigated.


Lycopodium species (L. annotinum and L. clavatum) from pine, beech, oak and spruce forests across Poland. More than 400 strains were isolated but only 18 Ascomycete species were identified. Members of the Dothideomycetes dominated the fungal endophyte communities in Lycopodium. The most abundant taxa cultured were Phoma brasiliensis (from L. clavatum) and Paraconiothyrium lycopodinum (from L. annotinum). Five taxa were isolated exclusively from L. annotinum, but only two of them (Paraconiothyrium lycopodinum and Mycosphaerella sp.) were relatively abundant. Two taxa were only found in L. clavatum, namely: Stagonospora pseudovitensis and an unidentified Dothideomycete. The taxon assigned as Ascomycota 2 (SH219457.06FU) was isolated only from strobili of both host species. Direct PCR and cloning from L. annotinum shoots revealed a substantially greater endophyte richness compared with the results from culturing.Endophytes are a large and diverse group of fungi that colonize healthy plant tissues without causing any symptoms. The majority of studies have focused on angiosperm and conifer hosts and few have examined the endophytes of lycophytes. In the present study, we characterized culturable endophytic fungi in two closely related The online version of this article (doi:10.1007/s13199-014-0291-1) contains supplementary material, which is available to authorized users. Lycopodium  is a genus of flowerless, vascular, terrestrial plants that reproduce sexually via homospores, and vegetatively from rhizomes. The Lycopod lineage diversified early and was the dominant vegetation in Carboniferous period  How diverse are endophytic fungi from Lycopodium in temperate forest? 2) Is species community composition influenced by host plant, plant organ, geographic location of the host, or host habitat characteristics? 3) What is the taxonomic identity of hypothetical species isolated as endophytes?The aim of the present study was to characterize the fungal endophytic communities of two closely related L. annotinum from 36 sites, and 10 shoots, 15 strobili, 2 whole plants  of L. clavatum from 27 locations from temperate forests in Poland were collected , strobili  and whole plants  of L. annotinum and L. clavatum were washed in tap water and surface-sterilized by subsequent submersion of the plant tissue for 2 min in a 0.5 % sodium hypochlorite and for 2 min in 70 % ethanol. Finally, samples were rinse in sterile distilled water, as described by Davis et al. (o C) for 10 weeks or until fungal growth was observed. Using aseptic technique, emergent hyphae were transferred and purified on sterile PDA plates. The endophytes were grouped into morphotypes as described by Wang et al.  following the manufacturer’s instructions. The complete ITS region was amplified using the primer pair ITS1f and ITS4  morphotypes were grouped into operational taxonomic units (OTUs) according to 98.5 % similarity using BioEdit Sequence Alignment Editor v. 7.0.0 Hall . This 98L. annotinum shoot from site no. 30 (coordinates in supplement E. coli JM109 competent cells (Promega) following the manufacturer’s instructions. Colony PCRs were performed using universal primer pair M13f and M13r.In order to estimate the fraction of unculturable endophytes, total genomic DNA was extracted (as described above) from sterilized Representative vouchers specimens for each OTU were deposited at the General Herbarium, University of Warsaw. Their numbers as well as the GenBank accession numbers of their sequences are presented in Table http://unite.ut.ee; Abarenkov et al. ITS sequences representing each morphotype were queried against the UNITE database using the massBLAST algorithm . All indices were calculated using EstimateS v.9.1.0.  was calculated as the total number of plant segments colonized by fungi divided by the total number of all incubated segments, expressed as its percentage  comparison between shoots and strobili; (3) comparison between the samples coming from lowlands, highlands and mountains; (4) comparison between the samples collected from mixed pine, fresh pine, acidophilic oak, mountain spruce, pine bog, and acidophilic beech forests. To correct for inconsistent sample sizes in diversity and richness comparisons between samples, the data was reduced to the smallest sample size  as proposed by Davis and Shaw  comparison between and Shaw .In all the described variants, after the performance of the Shapiro-Wilk tests for normality for each variant separately, Kruskal-Wallis one-way analysis of variance was chosen as an appropriate method of comparison   plots. To avoid pseudoreplication, data from different tissues types of the same host individual were pooled for NMDS analyses that examine the effect of host, location, and vegetation type. The Kruskal’s stress was used to decide which grouping of data is the most accurate (commonly acceptable when lower than 0.2)  based on coordinates of collection sites (WGS84 system) were determined using own program written in C .All statistical analyses were calculated using STATISTICA v. 10 and PAST v. 2.16 software , a total of 458 isolates, representing 96 morphotypes, 18 OTUs   were relatively abundant (more than 5 isolates). There were also two taxa that were exclusive for L. clavatum, namely: Stagonospora pseudovitensis and unidentified Dothideomycetes (SH196053.06FU). The taxon assigned as Ascomycota 2 (SH219457.06FU) was isolated only from strobili of both host species.None of the isolated taxa was present in all samples. There were 5 taxa isolated exclusively from L. annotinum than L. clavatum, the number of isolated species as well as colonization factor and values of species diversity indices for random 12 samples  were very similar , total number of isolates (p = 0.1455), colonization factor (p = 0.1455), Shannon diversity (p = 0.2263), Fisher’s alpha (p = 0.0953) and evenness (p = 0.2206) between the two Lycopodium species.There were no significant differences in total number of fungal species (p = 0.1623), total number of isolates (p = 0.8355), colonization factor (p = 0.8355), Shannon diversity (p = 0.1073), Fisher’s alpha (p = 0.8020) and evenness (p = 0.1305) between different site locations  and vegetation type of sample site   also did not statistically differ.The total number of fungal species (p = 0.0294) was significantly higher in shoots than in strobili for both Lycopodium species . Variation in endophytic community structure could neither be explained by site vegetation type nor by geographic region of host plant origin Fig. . The useL. annotinum from site 30  while U’Ren et al.  were abundant (more than 5 isolates). The isolate Ascomycota 2 (SH219457.06FU) was restricted to the strobili, regardless of the host species which suggests some level of tissue-preference, for this isolate. However, this might be a result of isolate rarity.Among the isolated taxa, five were found exclusively in L. annotinum from lowland have very different endophytic community than all others. This pattern could be explained by the relatively high abundance of Mycosphaerella sp. and by the fact that both sites are relatively close together (240 m apart). As lycophytes can reproduce asexually, it is possible that the plants represent the same host individual. The abundant presence of Mycosphaerella sp. at these sites should be verified in further research.Cluster analysis of endophytic community similarity measures presented in two-dimensional NMDS plots revealed that only two samples from Hypocrea rufa and its anamorph Trichoderma viride but they have been isolated as endophytes previously  J. Pawłowska, Wilk, Śliwińska-Wyrzychowska, Mętrak & Wrzosek, comb. nov. In spite of the fact that several Paraconiothyrium species have been isolated as endophytes from asymptomatic photosynthetic tissues of plant species Below is the link to the electronic supplementary material.


Semen Persicae (Taoren) and Flos Carthami (Honghua) are known to relieve acute hepatic injury and inflammation, improve microcirculation, and reduce tissue fiber. The aim of our study is to investigate the potential mechanisms of Taoren-Honghua Herb Pair (THHP) in murine model of chronic liver disease caused by Carbon Tetrachloride (CCl4). Mice were randomly divided into seven groups: (1) blank, (2) model, (3) control , (4) THHP , and (5) Tao Hong Siwu Decoction (THSWD) (8.50 g/kg). Histological change and microvessels density were examined by microscopy. Hepatic function, serum fibrosis related factors, and hepatic vascular endothelial growth factor (VEGF) were measured with ELISA. VEGF, kinase insert domain-containing receptor (KDR), Flt-1, and Akt mRNA expression in hepatic tissue were determined with PCR. Tissues of Akt, pAkt, KDR, and Flt-1 were measured with western blotting. Data from this study showed that THHP improved hepatic function and restrained the hepatic inflammation and fibrosis. Its role in inhibiting pathological angiogenesis and hepatic fibrogenesis may be through affecting the angiogenesis-associated VEGF and its upstream and downstream signaling pathways.Chronic liver disease is one of the most common diseases that threaten human health. Effective treatment is still lacking in western medicine. Chronic liver disease describes a wide range of liver complications including fatty liver, hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. In particular, hepatic inflammation and fibrosis are hallmarks of the progression. Researches on prevention and treatment of chronic hepatitis and hepatic fibrosis are of major importance. At present, there is no effective treatment in Western medicine that produces minimum adverse effects , 2. Atte Semen Persicae (Taoren) and Flos Carthami (Honghua) with the function of promoting blood circulation. Formulas composed mainly of these two ingredients are used extensively in China for the treatments of chronic hepatitis and hepatic fibrosis and have generated satisfactory therapeutic efficacy  and Flos Carthami (Honghua) granule (product lot number 1405019) 2 g [equal to Flos Carthami (Honghua) 6 g], was purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd. . They were directly mixed with distilled water to 0.553 g/mL, 0.267 g/mL, and 0.133 g/mL, respectively. The mixture was preserved in 4°C fridge. THSWD includes Semen Persicae (Taoren) 9 g, Flos Carthami (Honghua) 6 g, Radix Rehmanniae (Shengdi) 12 g, Radix Paeoniae Rubra (Baishao) 9 g, Radix Angelicae Sinensis (Danggui) 9 g, and Rhizoma Chuanxiong (Chuanxiong) 6 g. Total 51 g of crude drug was provided by Xiamen Yanlaifu Pharmaceutical Co., Ltd. . These herbs were soaked in water for 20 min and boiled for 30 min to yield 200 mL. The decoction was performed twice to yield final volume of 400 mL. The solution was filtered with carbasus and concentrated to 0.85 g/mL. The remaining decoction was lyophilized into powder and stored at 4°C. Colchicine tablet, product lot number 131126, was purchased from the Xishuangbanna Pharmaceutical Co., Ltd. . Colchicine tablet was added to double steamed water to a concentration of 0.01 mg/mL.THHP, which is composed ofSeventy healthy, 4-week-old, Kunming (KM) mice with specific pathogen-free (SPF) degree, male and female in equal number, weighing 16.0 ± 2.0 g, were provided by the SLAC Laboratory Animal Co. Ltd. in Shanghai, China [License Number SCXK (Hu) 2012-0002]. The mice were bred in animal house (SPF degree) with barrier system assisted with apinoid laminar flow chamber in the Experimental Animal Center of Xiamen University and used for experiment after 1 week.4 and Arachis Oil solution in the left inguen to establish the chronic liver disease model. The injection was administered once every 5 days for 6 weeks with first injection containing double dosage of CCl4. The THHP-H, THHP-M, and THHP-L were treated intragastrically with 0.2 mL/10 g body weight of THHP aqueous solution at concentrations of 0.553 g/mL, 0.267 g/mL, and 0.133 g/mL, respectively, once a day, the THSWD group was treated with intragastric administration (0.2 mL/10 g) of 0.85 g/mL THSWD once a day, and the colchicine treated group underwent intragastric administration (0.2 mL/10 g) of 0.01 mg/mL colchicine solution once a day for 6 weeks. Colchicine is a traditionary drug for antagonizing hepatic fibrosis, so it was selected as a positive drug . Differences with 4, tissue sections were stained with VG for microscopy analysis. 4-induced hepatic inflammation to relieve hepatic injury.To examine whether THHP could ameliorate the pathological changes in chronic liver disease model mice induced by CCl4-induced chronic liver disease, we measured serum aspartate aminotransferase, alanine aminotransferase, and albumin levels at the end of study. Colchicine was used as a positive control. As shown in P > 0.05), but ALT and AST levels were significantly lower compared to the model group (P < 0.01). THHP decreased the concentrations of serum ALT in a dose-dependent manner. ALT and AST levels were also more reduced by THSWD as compared to the colchicine control though no statistical significance was observed. These results revealed that THHP has a role in inhibiting the hypohepatia of CCl4-induced chronic liver disease mice.To examine if THHP could improve hepatic function of mice with CClP < 0.01). The reduction of serum LN in THHP-H and THSWD groups was also lower than that of colchicine group (P < 0.01). These results showed that THHP is capable of postponing the formation of hepatic fibrosis to some degree.To evaluate if THHP could inhibit hepatic fibrosis, we measured serum fibrosis correlated factors: hyaluronic acid and laminin. As shown in 4-induced chronic liver disease mice, we measured the average hepatic microvessel density. As shown in Figures P < 0.01). Furthermore, the effect of THHP in reducing pathological angiogenesis was better than that of colchicine group, but both IOD were higher compared to the normal control group. These results indicated that THHP has a certain effect on restraining hepatic angiogenesis of CCl4-induced chronic liver disease mice.To check if THHP could reduce hepatic pathological angiogenesis of CClP < 0.05)  . These d4, we further measured gene transcription of VEGF, Flt-1, KDR, and Akt in hepatic tissues. As shown in P < 0.01, P < 0.05). Akt mRNA expression was reduced by THHP-M and THSWD treatment (P < 0.01). And the efficacy of THHP was superior to colchicine treatment. Our data revealed that THHP may exert its effect though lowering the transcriptions of genes involved in pathological angiogenesis.In order to examine the mechanisms behind THHP's reduction of the pathological angiogenesis caused by CCl4-induced chronic liver disease mice at the dose of 5.53 g/kg . We also found that the protein expression of Akt, pAkt, and Flt-1 in hepatic tissue could be decreased by THSWD . Combining the above results of ELISA and PCR, we considered that blockage of angiogenesis-associated VEGF signaling via inhibiting the expression of Akt and pAkt and VEGF and its receptors (KDR and Flt-1) to attenuate its signal activation is one of the possible mechanisms of THHP treating CCl4-induced chronic liver disease.As expected, in Chronic liver disease characterized by hepatic inflammation and fibrosis is caused by various factors. Its formation mechanisms are complicated. In chronic liver diseases, angiogenesis, the formation of new hepatic pathological microvessels from preexisting ones, may contribute to progressive hepatic fibrosis and the development of hepatocellular carcinoma , 15. In VEGF has high affinity for two receptors: KDR and Flt-1. Flt-1 mediates the cytoskeleton rearrangement to migrate; KDR mainly mediates the endothelial hyperplasia maintaining the cell survival. KDR exerts the major role in VEGF induced angiogenesis and vascular permeability . KDR ant4 through inhibiting HSCs activation and inducing apoptosis of HSCs [ Semen Persicae (Taoren) or Flos Carthami (Honghua) are mostly on cardiovascular pharmacology like antagonizing thrombus, inhibiting inflammation, analgesia, and ameliorating microcirculation [At present, TCMs antagonizing chronic liver diseases with hepatic fibrogenesis focus more on anti-inflammatory, inhibiting lipid peroxidation and extracellular matrix synthesis, promoting extracellular matrix degradation, restraining platelet-derived growth factor, inducing HSC apoptosis, and inhibiting collagen synthesis . For exa of HSCs . And the of HSCs . The stuculation , 28. The Radix Scutellariae Baicalensis (Huangqin) and Radix et Rhizoma Rhei Palmati (Dahuang) [Meanwhile, like effects of ameliorating dimethylnitrosamine-induced liver injury and fibrosis in rats through downregulation of antioxidant enzymes and inducing oxidative modification were testified withDahuang) , and antTHHP is composed of the two chief medicinals of THSWD and the effect is more focused on promoting blood circulation than that of the latter. Our findings revealed that specific concentration of THHP alleviated hepatic injury and inflammation and decreased hepatic pathological angiogenesis (which were slightly better than that of colchicine from the point of the pathology) and improved hepatic function to certain degree and also testified that THHP had certain effects on downregulating VEGF and its main receptors and could inhibit both mRNA and protein expression of Akt and protein expression of pAkt to inhibit the pathological angiogenesis to protect hepar. Those findings suggest that angiogenesis-associated VEGF and Akt and its upstream or downstream activating factors may be as the targets of THHP for anti-inflammation and antifibrosis. Semen Persicae (Taoren) extract has been reported to attenuate hepatic collagen fibers of experimental hepatic fibrosis rats [ Flos Carthami (Honghua) active constituent safflower yellow or hydroxy safflower yellow-A (HSYA) has also been reported to improve hepatic function and relieve hepatitis and hepatic fibrosis [ Semen Persicae (Taoren) and Flos Carthami (Honghua) are much possible to play a synergistic effect on chronic liver disease model mice derived from each herbal medicine's components.Due tosis rats ; and Flofibrosis , 32; in From our results, reducing pathological angiogenesis by inhibiting expression of VEGF and Akt is an important effect of THHP. But recent research showed that VEGF was also required for hepatic tissue repair and fibrosis resolution although in a mouse model of liver fibrosis resolution VEGF promoted fibrogenesis . Combini4-induced chronic liver disease, whether the mechanism of THHP decreasing pathological angiogenesis is responsible for the relief of hepatic inflammation and fibrosis also remains to be clarified. Third, our experimental results of THHP still need to be further confirmed in human trials. Finally, due to complex combination of components within THHP, the constituents that exert the dominant efficacy will be left for future work.There are several limitations to the present study. First, differences of therapeutic effectiveness between THHP and THSWD remain unclear. Second, although THHP showed the multiple effects of treating CCl in vivo. The mechanism of action may lie in inhibiting the VEGF and its upstream and downstream signaling pathway to reduce the pathological angiogenesis, which provides a promising therapeutic alternative for clinical patients.In summary, we demonstrated that THHP has a notable effect on decreasing hepatic pathological angiogenesis and positive therapeutic effectiveness in chronic liver disease with hepatitis and fibrosis


Mycobacterium tuberculosis (MTB) through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR.Multidrug resistance (MDR) acquired by Mycobacterium tuberculosis (MTB) continues to pose significant global health challenges that require immediate treatment regimens directed at new targets. TB is remediable; however, due to its long course of medication or mismanagement in drug regimen, it has led to the emergence of multidrug resistance tuberculosis (MDR-TB) against various frontline anti-TB drugs [Tuberculosis (TB) caused byTB drugs –3. DespiTB drugs . Iron deTB drugs , 6. Avai Candida albicans, Leishmania donovani, Staphylococcus aureus, and Streptococcus epidermidis [ Candida albicans with bathophenanthroline disulfonic acid (BPS) and ferrozine as chelators increased its sensitivity to many common antifungal drugs, including fluconazole (FLC). Many different species of Candida also showed an increase in the drug susceptibility under iron limitation. The effect of iron chelation on the growth of Leishmania (Viannia) braziliensis, expression of proteins, and ultrastructure of this parasite has also been studied. Similar study on Gram-positive bacteria Staphylococcus was done in the presence of iron chelator diamine diorthohydroxyphenyl acetic acid.The role of iron in drug susceptibility has already been established in other major human pathogens, namely,dermidis –11. It hIn mycobacteria, a link between phospholipid homeostasis, virulence, and iron acquisition has been recently explored by lipidomic approach . The antThe objective of the present study was to find out a correlation between iron availability and drug susceptibility of mycobacteria to known anti-TB drugs. In this study, for the first time, the role of iron in governing the drug susceptibility of known anti-TB drug is explored. We showed that iron deprivation leads to enhanced potency of first line anti-TB drugs  that could be reverted upon iron supplementation. We explored that iron deprivation leads to disrupted membrane homeostasis which was confirmed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent and TEM images. We also showed that iron deprivation leads to enhanced genotoxicity in the presence of ethidium bromide suggesting its possible role in DNA repair mechanisms. Together, this study revealed an intricate relationship between cellular iron and drug susceptibility of mycobacteria.2HPO4), potassium dihydrogen orthophosphate (KH2PO4), sodium dodecyl sulphate (SDS), glycerol, and D-glucose were obtained from Fischer Scientific; methanol was purchased from Merck.All Media chemicals Middlebrook 7H9 broth, Middlebrook 7H10 agar, albumin/dextrose/catalase (ADC), and oleic acid/albumin/dextrose/catalase (OADC) supplements were purchased from BD Biosciences (USA). Deferoxamine mesylate salt powder (DFO), bathophenanthroline disulfonic acid disodium salt (BPS), Tween-80, nitrocefin, ethambutol (EMB), and isoniazid (INH) were purchased from Sigma-Aldrich . 2,2, Bipyridyl , ethidium bromide (EtBr), dinitrophenol , and rifampicin (RIF) were purchased from Himedia . Dimethyl sulfoxide (DMSO), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen orthophosphate  broth supplemented with 0.05% Tween-80 (Sigma), 10% albumin/dextrose/catalase , and 0.2% glycerol (Fischer Scientific) in 100 mL flasks (Schott Duran) and the culture was incubated at 37°C and on Middlebrook 7H10 (BD Biosciences) agar media supplemented with 10% (v/v) oleic acid/albumin/dextrose/catalase  for solid agar allowing growth for 48 h at 37°C. Stock cultures of log-phase cells were maintained in 30% glycerol and stored at −80°C.μL of Middlebrook 7H9 broth was placed at each well of the 96-well plate following with the addition of the drug with the remaining media and then subsequently it was serially diluted 1 : 2. 100 μL of cell suspension  was added to each well of the plate. Plates were incubated at 37°C for 48 hours. The MIC values were evaluated by observing the O.D600 in a microplate reader (Lisa Reader). The MIC80 was defined as the concentration at which 80% of the growth was inhibited compared with the controls.MIC was determined by broth dilution method described elsewhere  accordinμL of fivefold serial dilutions of each M. smegmatis culture  was spotted onto Middlebrook 7H10 agar plates in the absence (control) and the presence of the drugs. Growth difference was measured after incubation at 37°C for 48 hours.Spot assays for the strains were determined using a method as described elsewhere , 20. Briβ-lactamase activity for the permeabilization of M. smegmatis was determined by measuring the hydrolysis of nitrocefin by whole cells as described elsewhere [The lsewhere , 22. Bri M. smegmatis cells were observed using TEM (JEOL JEM-1011). The cells of 0.1 O.D600 were seeded to the media with and without drugs and were incubated for 24 h at 37°C. Sample preparation and analysis were done by using the method as described elsewhere [4 in 0.1 M phosphate buffer for 1 h. Cells were then dehydrated through ethanol, dried and coated with gold, and observed at magnification of 15000X.Treated and untreated cells oflsewhere . Brieflyμg/mL) in PBS to block the functionality of efflux pumps. The deenergized cells were pelleted, washed, and again resuspended as a 2% cell suspension (w/v) in PBS, to which EtBr was added at a final concentration of 4 μg/mL and incubated for 45 min at 25°C. The equilibrated cells with EtBr were then washed and resuspended as a 2% cell suspension (w/v) in PBS. Samples with a volume of 2 mL were withdrawn at the indicated time points and centrifuged at 10,000 rpm for 1 min. The supernatant was collected and absorption was measured at 285 nm.The diffusion of EtBr was determined by using protocol described elsewhere with modification , 24. Bri M. smegmatis cells and demonstrated that while 2,2, BP was sufficient to chelate iron at the concentration used in this study, it did not affect the growth of the cells. Thus, the growth of M. smegmatis cells was evaluated in the presence of 2,2, BP, a well-known iron chelator, at a concentration subsequently used in the study. To find out whether iron depletion leads to any growth defect was achieved by two different methods, namely, broth microdilution and spot assays.  M. smegmatis was completely inhibited at 40 μg/mL 2,2, BP; however, growth was not affected appreciably when cells were grown at 35 μg/mL 2,2, BP. This subinhibitory concentration was also confirmed by growth curve performed in the absence and the presence of 35 μg/mL 2,2, BP  without any iron deprivation. We found that MIC80 for above drugs alone were observed at 0.25 μg/mL, 4 μg/mL, and 2 μg/mL, respectively  were distinctly more susceptible to EMB, INH, and RIF compared to those growing under iron sufficient conditions  and BPS (368 μg/mL) at their subinhibitory concentrations. MIC80 results showed that cells grown in the presence of either DFO or BPS showed increased sensitivity as compared to that of those grown under iron sufficient conditions  tested in iron deprivation could be rescued . This implies that iron deprivation leads to enhanced membrane permeability which may be the causal reason for more drug intrusion inside the cell. This was further confirmed when the cells were spotted with the well-known membrane disrupting detergent SDS. Our results depict that, in the presence of SDS, iron deprived cells were hypersensitive in comparison to control cells , and iron deprivation  alone which showed smooth cell envelope, iron deprived drugs treated cells showed tampered morphology as visualized by decreased cell wall thickness and distortion  passive diffusion of the EtBr under iron deprivation. Next, we ascertain whether enhanced passive diffusion under iron deprivation also leads to enhanced diffusion of known anti-TB drug through membrane because of which now the same RIF can act more strongly as due to iron deprivation it can readily enter inside the cell. For this, the extracellular EtBr concentration was estimated for the cells treated with RIF in the absence and the presence of iron deprived condition. We observed that, in contrast to cells treated with RIF alone, iron deprived RIF treated cells showed increased extracellular EtBr concentration again implying enhanced passive diffusion , and reactive element species (RNI and ROS) is mounted by the host to destroy the infectious agent. The RNI and ROS have high toxicity due to their ability to impose damages to biomolecules such as DNA, proteins, and lipids [3 and observed that the sensitivity of EtBr could be rescued . Interestingly, our results as depicted by spot assays do not show any difference between the cells grown at either physiological or alkaline pH (As a known matter of fact alkaline pH mimics iron deficiency. This is particularly evident from the fact that at alkaline pH most of the available iron is present in insoluble ferric form thus representing iron deprived condition . Thus, ealine pH . This su M. smegmatis cells affects cellular membrane integrity, which in turn presumably allows faster entry of drugs leading to enhanced drug sensitivity of the cells. The possibility of coregulation of MDR, lipid biosynthesis, and iron acquisition genes through common regulators may also exist, as has already been observed in several instances, but needs further validation. In conclusion, changes in the drug susceptibility of mycobacteria due to iron represent a well-regulated new mechanism that merits a closer look.Taken together, our results demonstrate that iron deprivation of


Opioids have been traditionally used for postoperative pain control, but they have some unpleasant side effects such as respiratory depression or nausea. Some other analgesic drugs like non-steroidal anti-inflammatory drugs (NSAIDs) are also being used for pain management due to their fewer side effects.The aim of our study was to compare the analgesic effects of paracetamol, an intravenous non-opioid analgesic and morphine infusion after elective laparotomy surgeries.This randomized clinical study was performed on 157 ASA (American Society of Anesthesiology) I-II patients, who were scheduled for elective laparotomy. These patients were managed by general anesthesia with TIVA technique in both groups and 150 patients were analyzed. Paracetamol (4 g/24 hours) in group 1 and morphine (20 mg/24 hours) in group 2 were administered by infusion pump after surgery. Postoperative pain evaluation was performed by visual analog scale (VAS) during several hours postoperatively. Meperidine was administered for patients complaining of pain with VAS > 3 and repeated if essential. Total doses of infused analgesics, were recorded following the surgery and compared. Analysis was performed on the basis of VAS findings and meperidine consumption.There were no differences in demographic data between two groups. Significant difference in pain score was found between the two groups, in the first eight hours following operation , but not after 12 hours (P = 0.14) .The total dose of rescue drug (meperidine) and number of doses injected showed a meaningful difference between the two groups (P = 0.00). Also nausea, vomiting and itching showed a significant difference between the two groups and patients in morphine group, experienced higher levels of them.Paracetamol is not enough for postoperative pain relief in the first eight hour postoperatively, but it can reduce postoperative opioid need and is efficient enough for pain management as morphine after the first eight hours following surgery. Management of pain, especially postoperative pain, is a major concern for anesthetists in patients undergoing surgery. There are many ways for managing the postoperative pain. The most common way used after most surgeries is injecting analgesic drugs specially opioids. Excessive opioids administration is associated with a variety of side effects including ventilatory depression, drowsiness and sedation, nausea and vomiting, pruritus, ileus, urinary retention and constipation . UnpleasThe aim of our study was to compare the analgesic effects of paracetamol  and morphine  after elective laparotomy surgeries.The study was approved by the legal and ethics committee of Iran University of Medical Sciences in March 2011 and registered at Iranian Registry Clinical Trials site (IRCT ID: IRCT201203114969N6). This double-blinded study which in, patients and evaluators were unaware of group assignments was performed on 159 ASA (American Society of Anesthesiology) class I-II patients aged between 25-85 years. Patients scheduled for elective laparotomy  were selected by block randomization in two groups. Patients with psychiatric illnesses, addiction, allergic reactions to opioids or paracetamol (or other NSAIDs) severe renal or hepatic disease and BMI ≥ 30 were excluded from the survey. Technique of general anesthesia was similar in both groups. After establishment of IV access and monitoring, midazolam (0.02 mg/kg) and fentanyl (2 µg /kg) was injected as premedication, and propofol (2 mg/kg) and atracurium (0.5 mg/kg) for Induction of anesthesia.Propofol (100-150 µg/kg/min) as needed and remifentanil (0.4 µg/kg/min) were infused as maintenance and atracurium (0.15 mg/Kg) was injected every 30 minutes. Fifteen minutes before end of the surgery, fentanyl (1 µg /kg) was injected and at the end of the operation muscle relaxant effects was reversed by neostigmine (0.04 mg/kg) and atropine (0.02 mg/kg) .At the end of the surgery paracetamol, UniPharma, Greece (4 g/24 hours) or morphine sulphate, Daroupakhsh, Iran (20 mg/24 hours) injection would start through IV infusion pump. Visual analog scale (VAS) from 0 to 10 (with 0 representing no pain and 10 representing the worst imaginable pain) for postoperative pain was used in a time schedule  after surgery.For Any patient complaining of pain on with a score more that 3 on VAS 0.3 mg/kg of meperidine was injected and if demanded, the same dose was repeated until VAS ≤ 3. The total dose and number of doses injected was recorded. Postoperative nausea, vomiting, pruritus and respiratory rate were recorded throughout the study period. Data were registered in checklists including demographic characteristics  and nausea, vomiting, pruritus, respiratory rate and urinary retention, patient’s complaints, visual analog scale score and meperidine dose were analyzed. Numerical variables were reported as mean ± standard deviation (SD). Quantitative and qualitative variables were measured by independent t-test, and ANOVA test respectively. P value ≤ 0.05 was considered to be statistically significant. All analyses were performed using SPSS for Windows version 19 .Among all patients included in the study, 4 people in morphine group and five in paracetamol group were excluded due to different reasons and analysis was performed on the basis of VAS findings on the rest of the patients . Mean age of patients was 54 ± 15.45 years. The data analysis showed both groups were similar regarding age, sex, BMI and duration of surgery . The paiin vivo  and morphine infusion may be beneficial in the management of acute pain after major surgery in patients, prone to opioid-related complications. Although, it is acceptable that paracetamol overall is an effective postoperative sole analgesic, it is recommend on the basis of this study findings, if it is used for pain killing after laparotomy surgeries, small amounts of opioids are essential for the first eight hours after operation.Studying on various types laparotomy techniques was the limitation to this study; therefore it is better to repeat the study on specialized operations.


Synaptic failure and neurofibrillary degeneration are two major neuropathological substrates of cognitive dysfunction in Alzheimer’s disease (AD). Only a few studies have demonstrated a direct relationship between these two AD hallmarks. To investigate tau mediated synaptic injury we used rat model of tauopathy that develops extensive neurofibrillary pathology in the cortex. Using fractionation of cortical synapses, we identified an increase in endogenous rat tau isoforms in presynaptic compartment, and their mis-sorting to the postsynaptic density (PSD). Truncated transgenic tau was distributed in both compartments exhibiting specific phospho-pattern that was characteristic for each synaptic compartment. In the presynaptic compartment, truncated tau was associated with impairment of dynamic stability of microtubules which could be responsible for reduction of synaptic vesicles. In the PSD, truncated tau lowered the levels of neurofilaments. Truncated tau also significantly decreased the synaptic levels of Aβ40 but not Aβ42. These data show that truncated tau differentially deregulates synaptic proteome in pre- and postsynaptic compartments. Importantly, we show that alteration of Aβ can arise downstream of truncated tau pathology. Neurofibrillary tangles, amyloid plaques, neuronal loss and synaptic failure represent the major hallmarks of Alzheimer’s disease  on Image Reader LAS-3000 . Modified tubulin blots were stripped and re-probed using total α-tubulin antibody for quantitation. Actin was used as loading control and all relevant data were normalized to actin. The western blots (WB) were quantified using Advanced Image Data Analyzer software .Total synaptosomes were extracted as described earlier and used to quantify amyloid-β40 (Aβ40) and β42. Protein concentration was determined using BCA protein estimation and 400 μg of total synaptosomes were used for assay. Eight samples per group were analyzed. Amyloid β assay kits  were used for assay and experiments in duplicates were performed according to manufacturer’s instructions.Rats were perfused with phosphate saline buffer  followed by 4% paraformaldehyde , and the brains were removed. Tissues were postfixed in 4% PFA overnight, followed by treatment with 25% sucrose for 48 h to provide cryoprotection. Brains were then frozen in isopentane at −40°C. Coronal sections (40 μm) were cut in a cryostat at −18°C. Free-floating sections were incubated with primary antibodies  overnight at 4°C. Sections were subsequently incubated with secondary antibodies conjugated with ALEXA 488 or ALEXA 546 fluorescent dyes  for 1 h at room temperature. After washing, the sections were mounted onto slides using Vectashield mounting medium (Vector Laboratories), and examined with an Olympus IX 71 Fluorview laser scanning confocal microscope.n = 3) were perfused with 300 mmol/l glutaraldehyde in 100 mmol/l cacodylate buffer, their brains were extracted and fixed in the same buffer overnight. Brains were cut to pieces (1 mm3), and postfixed in 40 mmol/l osmium tetroxide in 100 mmol/l cacodylate buffer (1 h). After rinsing in cacodylate buffer and dehydration in ethanol, samples were embedded in araldite resin . Ultrathin sections (60 nm thick) were cut using Leica EM UC6 ultramicrotome and stained with uranylacetate and lead citrate. Sections were examined under FEI Morgagni 268D electron microscope  at 70 kV. Comparative images were captured using the same resolution as indicated in the EM micrographs.Rats  on the significance level α = 0.05. Bootstrap two-sample t-statistics were used as the test criterion. Additionally, we calculated the 95% bootstrap-t empirical confidence intervals  estimates of the mean differences, and MC estimates of upper and lower bounds of 95% CI. The statistical results are expressed by p-values, mean differences, and CI in the form “”. If the p-value is greater or equal than 0.05, the CI includes zero; if the p-value is smaller than 0.05, the CI does not include zero. Graphs representing data points and mean for individual protein per group were generated using PRISM , where *p ∈ 〈0.01, 0.05); **p ∈ 〈0.001, 0.01); ***p ∈  are used to indicate statistical significance.All experiments were repeated three times for consistency. Only one randomly selected value per experiment, however, was used for statistical analysis. Representative blots are shown. Statistical analyses were performed with R software , Figure p = 0.002; 150.79; , Figure To investigate synaptic damage induced and driven by truncated tau we assessed the distribution of rat endogenous tau and human truncated tau in the synaptic membrane fraction (SMF) and postsynaptic density fraction (PSD). In SMF, pan tau antibody DC25 recognized endogenous rat tau in both wild-type and transgenic animals Figure . PostsynWe evaluated the purity of synaptic fractions using either synaptophysin—a specific marker for presynaptic compartment, or PSD95—a specific marker for the PSD Figure . Blots wPhosphorylation regulates microtubule binding activity of the tau protein. In AD brains, site specific phosphorylation of tau correlates with neuronal pathology ; pS214, p = 0.003; –0.28; ; pS262, p = 0.009; –0.27; ; pS356, p = 0.006; –0.39; ), while antibody specific for phospho-T212 showed increased immunoreactivity in the PSD when compared to SMF )  Figure .p = 0.002; 154.97; ) and a sizeable increase in levels of total β-tubulin ) in the SMF fractions of transgenic animals when compared with wild-type rats ) and acetylated tubulin ) in the presynaptic compartments of the transgenic rats compared to wild-type rats. However, we observed no significant change in the levels of tyrosinated tubulin when compared to wild-type rats ).We next investigated whether the specific increase of tubulin might affect the dynamic instability of microtubules in the presynaptic compartment in transgenic rats. Microtubule polymerization is associated with several posttranslational modifications of tubulin and specific modifications influence the microtubule stability p = 0.224; 144.46; ; total β-tubulin p = 0.902; –12.71; ). In addition, we did not observe any change in acetylated tubulin levels in the PSD (data not shown). Furthermore, the levels of MAP2—a dendrite specific microtubule binding protein also remained unaltered. However, we found significant changes in neurofilament proteins ) and neurofilament M ). Additionally, we also observed a slight nonsignificant decrease in drebrin E/A ), a dendrite-specific actin-binding protein, that regulates spine morphology and size levels ). In contrast to synaptophysin, we observed only nonsignificant increase in bassoon—a synaptic vesicle clustering protein ), and no change in the levels of GAP 43 protein, a neurite growth cone protein ). In the PSD, we did not detect any significant changes in the levels of PSD95 and neuroligin, a ligand for presynaptic β-neurexins , Neuroligin: p = 0.197; 111.42; ) . EM micrographs revealed a decrease in synaptic vesicles in the synapses of transgenic animals  and transgenic animals (n = 3) by a blind observer. The number of vesicles per synapse decreased significantly in transgenic rats compared to wild type rats ), corresponding to the decrease in synaptophysin levels ). Furthermore, we quantified the levels of amyloid peptides Aβ40 and Aβ42 in total synaptosomes by quantitative ELISA. The quantification did not reveal any changes in the levels of toxic Aβ42 in transgenic animals when compared to wild-type rats ). The levels of Aβ40 were, however, significantly reduced ) in synapses of transgenic animals.To identify the position of amyloid cascade in truncated tau-mediated synaptic deficits, we compared the levels of APP, amyloid-β 42 (Aβ42) and Aβ40 in synapses of transgenic and wild-type animals. Staining using anti APP antibody (clone 22C11) revealed no change in the levels of total APP in the transgenic animals ), α- tubulin ) and β-tubulin ) suggesting that the pathological changes were present exclusively in the synaptic terminals of the transgenic animals.To verify that the observed differences were specific for synapses, we also quantified the levels of selected proteins in total brain extracts Figures . Immuno-Synaptic failure and neurofibrillary tangles strongly correlate with cognitive decline in AD  is sufficient to induce synaptic and dendritic loss and reduce the field excitatory post-synaptic potential leading to the cognitive decline (Zhang et al., Finally, we cannot completely exclude the effect of tau overexpression on the synaptic damage, however we can see specific pattern of synaptic damage in the cortex of transgenic rats. First of all, not all proteins are affected. Furthermore microtubular network is mainly damaged in the presynaptic compartment while neurofilaments are altered in postsynaptic compartment. It is important to mention that overexpression of truncated tau is similar in both compartments. Therefore we suggest that dysregulation of synaptic markers are caused by tau truncation rather than tau overexpression.In conclusion, truncated tau protein displayed different phospho-patterns in the pre- and postsynaptic compartments, which was associated with selective damage of microtubular network in the presynaptic compartment and deregulation of neurofilaments in the post-synaptic density. Importantly, although the truncated tau protein orchestrated the synaptic deficits independently from Aβ42, it significantly regulated Aβ40. The study opens new avenues for better understanding of synaptic pathology in AD and related neurodegenerative diseases.Santosh Jadhav performed isolation of synaptic fractions, sarkosyl fractionation, western blotting experiments, and analysis of EM images. Norbert Zilka designed the protocol of the study. Santosh Jadhav, Norbert Zilka and Andrej Kovac performed EM experiments. Stanislav Katina did the statistical analysis. Santosh Jadhav and Zuzana Kazmerova performed ELISA experiments. Santosh Jadhav, Norbert Zilka and Michal Novak wrote the paper. All authors read and accepted 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.