Background. Extensive evidence, arising from models of endothelial nitric oxide synthase gene (NOS3)-knockout mice supports the role of endothelial malfunction in the pathogenesis of the metabolic syndrome (MS). Aims. The aim of this study was to evaluate the role of −786T/C polymorphism in the etiology of MS and assess previously reported interaction with cigarette smoking. Methods. Based on International Diabetes Federation 2005 criteria, we recruited randomly 152 subjects with MS and 75 subjects without MS. Results. Allelic and genotype frequencies did not differ significantly between both groups. Total cholesterol level (CHOLT) and intima-media thickness of carotid arteries were significantly higher in −786CC homozygotes, in comparison with −786TC and −786TT patients. Regarding current smoking status, −786C allele was associated with higher CHOLT than −786T allele. Conclusion. Our study indicates the putative role of −786T/C polymorphism in the development of hypercholesterolemia, in patients with MS, which might be enhanced by cigarette smoking. The metabolic syndrome (MS) is a clustering entity, characterized by obesity, hypertension, hyperglycemia, dyslipidemia, and insulin resistance. Definitions of MS were created in order to indicate the patients at increased risk of cardiovascular complications and type 2 diabetes. Currently recommended criteria for MS diagnosing were proposed by National Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP III), and International Diabetes Federation 2005 (IDF 2005). NOS3 gene, and displays anti-inflammatory, vasodilatory, and antiproliferative effects [NOS3-knockout mice develop dyslipidemia, insulin resistance, decreased insulin-dependent glucose uptake in skeletal muscles and adipose tissue, and hypertension, mimicking the phenotype of MS [NOS3 gene is located at 7q35-36 and possesses a few polymorphic variants. Several reports indicate the potential association between NOS3 gene polymorphisms and MS [NOS3 gene expression [Atherosclerotic lesions may be the complication of MS, arising from endothelial dysfunction and induced by decreased nitric oxide (NO) production. NO is synthesized by nitric oxide synthase (eNOS), encoded by  effects . Recent pe of MS –5. In hus and MS . Functiopression , 10 and pression . AlthougNOS3 genotype [Besides genetic risk factors, atherosclerotic vessel damage may occur due to modifiable, environmental insults. Moreover, a growing body of evidence suggests the role of gene × environment interactions (G × E) in the etiology of multifactorial diseases including MS, type 2 diabetes, and atherosclerosis . Followigenotype . The aim of this study was to evaluate the association between −786T/C polymorphism and MS, and to assess the putative G × E interaction with cigarette smoking. We recruited randomly 227 males without any previous treatment of dyslipidemia, diabetes, or cardiovascular diseases. To avoid the potential hormonal influences and assess the notion that gender may be the risk factor for vascular incidents, we limited our study to male individuals. All subjects were Caucasians, worked in the copper smelter, and came from the same geographic area. Written informed consent was obtained from all volunteers after the approval of local Ethics Committee. Anthropometric measurements were carried out by trained medical staff and included weight, height, waist, and hip circumference. On the basis of anthropometric measurements, waist-to-hip ratio (WHR) and body mass index (BMI) were calculated. Hypertension was diagnosed based on blood pressure measurements in a quiet room after lying down for 15 minutes, which were performed at least twice during two or three different appointments. Diabetes type 2 or impaired fasting glucose were diagnosed using WHO criteria. Triglycerides (TG), total cholesterol (CHOLT), low-density lipoproteins (LDL) and high-density lipoproteins (HDL) levels were estimated according to standard protocols. The compound panel of biochemical measurements was performed in all subjects from both groups and included coagulation parameters , hormones concentrations , C-reactive protein (CRP), uric acid (UA), and leptin and adiponectin concentrations. Subjects, who fulfilled IDF 2005 criteria for MS, were recruited into the study group  and others, without clinical and biochemical features of MS were enrolled into the control group .MspI restriction endonuclease and visualized on 2.5% agarose gel.Genetic analysis was performed according to the PCR-RFLP protocol, described by Imamura et al.  with theU test.Statistical analysis has been performed using STATISTICA v 9.0 software. Chi-square test has been used for the evaluation of genetic linkage between −786T/C polymorphism and MS. The mean values of clinical parameters with regard to −786T/C polymorphism have been analyzed in the Kruskal-Wallis test. Baseline characteristics and the influence of cigarette smoking on CHOLT and IMT have been assessed using the Mann-Whitney Characteristics of study and control group are shown in P = 0.04). Similarly, both left and right IMT were significantly lower in −786TT homozygotes, in comparison with −786TC and −786CC patients . Analysis of mean clinical parameters with regard to −786T/C polymorphism in study group revealed significant results . The difP = 0.03) in −786TC smokers (5.74 mmol/L) in comparison with −786TC nonsmokers (5.3 mmol/L). Higher CHOLT (P = 0.01) was also found in −786CC smokers (5.66 mmo/L) than in −786CC nonsmokers . The difference in CHOLT between −786TT smokers and −786TT nonsmokers was not statistically significant . Regarding allele carrier status, −786T allele was associated with significantly lower (P = 0.003) CHOLT with respect to −786C allele (5.23 mmol/L versus 5.73 mmol/L). It is noteworthy that the mean values of CHOLT did not differ significantly between subjects with distinct genotypes either in smokers or nonsmokers (data not shown).The interaction between cigarette smoking and studied polymorphism was assessed in both groups simultaneously and revealed significant results regarding only CHOLT . The preNOS3 gene and lead to reduction in NO release [NOS3 in vitro.Our study did not reveal significant association of −786T/C polymorphism with MS. The subthreshold results may arise from the study design limitations, originating from the sample size. However, interesting results were obtained concerning clinical parameters of MS. We confirmed the putative impact of −786T/C polymorphism on atherosclerotic lesions by measuring IMT in carotid arteries. These findings are consistent with previously published data, based on various imaging indices of atherosclerosis in MS patients including aortic and peripheral wave velocity  or ankle release , 17, 18. release  have fouNOS3-knockout mice display hypercholesterolemia among other clinical traits of MS [NOS3 gene on clinical features of MS including cholesterol metabolism might be less severe than the knockout of the whole gene. Moreover, the genetic etiology of MS originates from interactions between a plethora of modest effect loci, which impair different clinical parameters of this clustering entity. Hence, the only clinical outcome from this study concerns alterations in CHOLT and IMT. The novel finding, resulting from our study, concerns the influence of −786T/C on CHOLT, which was significantly higher in −786CC homozygotes, in comparison with other genotypes. Emerging data indicate that ts of MS , 20. AltNOS3 polymorphisms has been assessed in several studies with regard to coronary artery disease [NOS3 gene expression [Furthermore, we evaluated the possible G × E interactions by assessing the synergism between cigarette smoking and −786T/C polymorphism. According to our results, the influence of −786T/C polymorphism might be enhanced by tobacco smoke ingredients. Statistical analysis revealed significant correlations between CHOLT and −786T/C polymorphism in conjunction with current smoking status. The interaction between smoking and  disease , 22, per disease , and cer disease . Moreove disease , 25. Acc disease , targetipression , 28, altpression , as wellpression . The limpression .NOS3 gene expression and contribute to lipid dysmetabolism. Thus, observed impact of −786T/C polymorphism on atherosclerotic indices not only might constitute the direct effect of endothelial damage but could also appear due to elevated CHOLT. On the other hand, −786C allele may enhance deleterious effects of cigarette smoking. However, the exact mechanism of this G × E interaction requires further investigation.Our results support the influence of −786T/C polymorphism on clinical features of MS. Moreover, this study sheds new light on the role of NO in the development of hypercholesterolemia. Studied polymorphism may alter


Salmonella enterica serovar Typhi (S. Typhi) isolates causing enteric fever in and around Kolkata, India. Among the isolates resistance to ampicillin (A), chloramphenicol (C), cotrimoxazole (Co) and tetracycline (T) were plasmid mediated; the plasmid was unstable in S. Typhi, and the other enteric bacteria like Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were found to be the potential source of dissemination of such plasmids into S. Typhi. The infection with such S. Typhi strains were successfully treated with ciprofloxacin (Cp: MICs 0.0075–0.075 μg mL−1) and/or ofloxacin (Ofx: MICs 0.0125–0.075 μg mL−1), but in the later course, the S. Typhi strains, showing resistance to nalidixic acid, developed low level of resistance to Cp and Ofx, causing the treatment failure. Thus, the treatment regimen was shifted to the third generation cephalosporins like ceftriaxone (Ct) and cefotaxime (Cf). Keeping in mind the anticipation of development of resistance to Ct/Cf, we prepared the treatment regimen for MDR enteric fever, based on the double-drug synergy tests in vitro; Cp-gentamycin (FICI 0.121–0.216) and Cp-trimethoprim (FICI 0.14–0.483) combinations were found effective against S. Typhi isolates having decreased sensitivity to cp (MICs: 0.5–1.25 μg mL−1).This communication states the changing patterns of  Salmonella enterica serovar Typhi (S. Typhi), remains a serious public health problem in many developing countries, with highest incidence in parts of Africa (50 per 100.000 person-years) and Asia (274 per 100.000 person-years) [S. Typhi strains carrying resistance to multiple clinically relevant antibiotics [S. Typhi is the commonest etiological agent. Most developing countries have experienced outbreaks of multidrug-resistant (MDR) enteric fever due to S. Typhi; Kolkata  and its suburbs also faced epidemic of enteric fever caused by life threatening infection of S. Typhi [Typhoid fever, the causative agent of which is ibiotics , 4. In IS. Typhi (1991–2001) obtained at the Calcutta School of Tropical Medicine from blood cultures from enteric fever patients within and outside Kolkata, India, (ii) determine the dissemination of R-factor of S. Typhi isolates, (iii) correlate the zone diameter of inhibition (ZDI) and minimum inhibitory concentration (MIC) values for evaluation of resistance to ciprofloxacin (Cp) and to evaluate the Nalidixic acid (Nx) resistance as a marker for decreased Cp susceptibility in S. Typhi isolates, and (iv) evaluate the in vitro efficacy of various combination of antibiotics against S. Typhi in order to combat antibiotic resistance.The above background prompted us to (i) determine the antibiotic resistance pattern of outbreak causing as well as sporadic isolates of S. Typhi isolates showed flip-flop patterns of resistance to ampicillin (A), chloramphenicol (C), co-trimoxazole (Co), and tetracycline (T) with wide range of MICs, 0.1–6000, 0.1–5000, 0.5–1200, and 0.5–800 μg mL−1, respectively. A total of 421 S. Typhi isolates from blood samples of suspected enteric fever patients undergoing treatment at the Calcutta School of Tropical Medicine, Kolkata (India), were used in the present study; a single strain was isolated from a each of the cases. The control strain used in the study was Escherichia coli ATCC 25922.The S. Typhi (resistance pattern ACCoT or ACCoTNx) was established by (a) transfer of resistance from MDR S. Typhi to the Escherichia coli C600 recipients  by in vitro conjugation experiments, (b) secondary transfer of R-factor to the antibiotic-sensitive S. Typhi isolates, (c) elimination of antibiotic resistance from donors as well as transconjugants by curing experiments, and (d) isolation of plasmid DNAs from the donors, recipients, transconjugants, and cured strains. A conjugative plasmid of approximately 55 kb was associated in mediating ACCoT resistance among the MDR S. Typhi isolates, while the antibiotic sensitive isolates were plasmidless.The presence of R-factor in S. Typhi strains acquired ACCoT resistance along with the acquisition of a plasmid (comigrated with the MDR S. Typhi plasmid) from clinical isolates of MDR Escherichia coli (resistance pattern ACCoTNx or ACCoTNxCp), Klebsiella pneumoniae (resistance pattern ACCoT), and Proteus vulgaris (resistance pattern ACCoT). Resistance to Nx and Cp was not transferable. After curing experiments, the loss of ACCoT resistance from the original MDR S. Typhi strains, all types of transconjugant S. Typhi strains and MDR E. coli strains were concomitant with the loss of the plasmid.On the other hand, the antibiotic-sensitive S. Typhi isolates showed MICs 0.0075–0.075 μg mL−1 for Cp and 0.0125–0.075 μg mL−1 for ofloxacin (Ofx). The S. Typhi isolates (1995 onwards) showing Cp MICs 0.1–1.25 μg mL−1 and equivalent ZDI of ≥21 mm caused treatment failure during Cp therapy. We encountered problem in treating enteric fever with Ofx (200 mg twice daily for 5 days) too during and after 1996, although the isolates fall within the sensitive range by disc diffusion method (≥16 mm). The corresponding isolates showed high MICs of Ofx (0.5–1.5 μg mL−1). In order to determine the MICs of antibiotics and ZDIs around the antibiotic discs, the methods of the Clinical and Laboratory Standards Institute  were followed [The MDR enteric fever cases were treated successfully with Cp (500 mg) or Ofx (200 mg) twice daily for 5 days. The associated S. Typhi isolates was evaluated by comparing the MICs of Cp and that of Nx (μg Nx disc (μg mL−1 was used as a breakpoint of decreased Cp susceptibility (signifying resistance), the sensitivity of Nx disc was 100%, and the specificity was 89.20%, and when MICs of Nx were compared with that of Cp, sensitivity of the approach was 100% and specificity was 95.95%. The applicability of Nx resistance to screen for decreased susceptibility (low level of resistance) to Ofx and norfloxacin (Nfx) among the S. Typhi isolates was also explored in our study and the method emerged useful and significant.The relevance of using the resistance to Nx as a marker for Cp resistance in at of Nx , and MIC Nx disc . When CpS. Typhi isolates, we assessed the applicability of fluoroquinolone disc diffusion test by scattergram analysis between fluoroquinolone MICs and ZDI obtained around 5 μg fluoroquinolone disc. Considering Cp MIC of ≥0.1 μg mL−1 as the breakpoint for Cp resistance, Cp ZDI of ≤29 mm resulted in 100% sensitivity and 36.15% specificity in detecting Cp resistance for S. Typhi isolates. Similarly, when Ofx MIC of ≥0.5 μg/mL was used as a breakpoint for Ofx resistance, Ofx ZDI of ≤29 mm results in 100% sensitivity and 51.2% specificity in screening for Ofx resistance among the isolates. In case of Nfx, when MIC of ≥0.1 μg/mL was taken as the breakpoint, Nfx ZDI of ≤23 mm produced 100% sensitivity and 49.22% specificity in detecting reduced Nfx susceptibility among S. Typhi isolates.In order to prepare a universally observed guideline for the worldwide surveillance of the emergence and spread of fluoroquinolone-resistant S. Typhi had been checked by susceptibility testing with Ct for the isolates; the in vitro efficacy of cefotaxime (Cf) was also assessed. We observed a correlation between the results of disc diffusion and agar dilution testing for susceptibility to Ct and Cf by scattergram analysis between MICs and ZDI. For the isolates, the MICs of Ct and Cf were 0.005–0.2 μg mL−1 and 0.005–0.1 μg mL−1, respectively, and ZDIs obtained around 30 μg Ct and 30 μg Cf discs were 26–41 mm and 24–42 mm, respectively. The isolates showed excellent in vitro susceptibility to aminoglycosides .The therapeutic efficacy of ceftriaxone (Ct) against the enteric fever patients infected with Cp-resistant S. Typhi isolates constitute a major threat in modern medicine. Therefore, in order to prepare cost-effective treatment regimen of enteric fever due to the infection of Cp-resistant S. Typhi, combination effect of Cp with various other antibiotics has been evaluated in the present study by (a) agar dilution checker board, (b) double disk synergy, and (c) time kill methods. The current findings represent the first time that an antimicrobial, here Cp, has its activity significantly increased with Ax. This view is supported by very low fractional inhibitory concentration (FIC) values of Cp (0.004–0.256 μg mL−1) in combination with amoxicillin (Ax). The Cp-trimethoprim (TMP) combination exhibited synergistic effect for all the isolates tested (FIC indices 0.140–0.483). The interaction between Cp and G was found to be synergistic against S. Typhi, since, in this study, the calculated FIC indices ranged from 0.121 to 0.216. The Cp-cefazolin (Cz) combination resulted in both synergistic (for isolates with high Cz MICs) and additive (for isolates with low Cz MICs) effects. Synergy was defined as the FIC index ≤0.5, addition as an FIC index 0.5–4, and antagonism as an FIC index >4 [The acquisition and spread of multidrug resistance among S. Typhi A is endemic. A conjugative plasmid (approximately of 55 kb) was involved mediating resistance to A, C, Co, and T in outbreak causing as well sporadic MDR isolates of S. Typhi, while antibiotic-sensitive isolates were plasmidless. Previous report indicated plasmids of molecular weight 25.4 and 62.5 kb to be more frequent among strains with the resistance pattern ACoCT [S. Typhi, the R plasmid is an unstable plasmid that may appear or disappear at any time resulting in the emergence of drug-resistant or drug-sensitive isolates, and its acquisition  is mainly due to antibiotic load exposed to them during therapy.In Kolkata (India), enteric fever due to the infection of rn ACoCT . In S. TS. Typhi isolates showing sensitivity to C, A, and Co provides a strong case for reconsidering the use of the first line of antibiotics for treatment of enteric fever in this region. Another study from Kolkata has indicated similar sensitivities  [Cp should be the antibiotic of choice for MDR typhoid fever. Longer course of Ofx therapy might be instituted in the treatment of MDR cases showing nonresponsiveness to Cp. Present finding underlines the importance of Ct for treating MDR and Cp-resistant enteric fever cases, and it should be used only if the first- and second-line antibiotics fail to evoke a satisfactory response or if the isolate is resistant to Nx. However, the Ct resistance is anticipated . Emergen for Co) .S. Typhi. Nx susceptibility has been validated as a screening test for reduced susceptibility to Cp, and Nx-resistance is associated with a high MIC of Cp, which in turn is associated with treatment failure; earlier authors also reported similar phenomenon [S. Typhi and also to prevent emergence of resistance to fluoroquinolones and newer antibiotics such as third-generation cephalosporins. Thus, physicians should be aware about the overuse and misuse of fluaroquinolones and cephalosporins in the treatment of typhoid fever to overcome the burden of drug resistance, and with Ct emerging as the sole defense against Nx-resistant S. Typhi infection, it should be instituted only in the event of nonresponsiveness to Cp [S. Typhi and treatment protocol, benefiting a large number of people of this region of the world, where enteric fever is a major public health problem.Either the MIC breakpoints or the equivalent ZDI suggested by the NCCLS did not detect fluoroquinolone resistance of enomenon . The refss to Cp . The dat


Hypercholesterolemia and oxidative stress are the main stimulating factors responsible for coronary artery disease and progression of atherosclerosis. Dairy food products are rich in conjugated linoleic acid (CLA) which is considered as an important component due to its potential health benefits such as anticarcinogenic, antiatherogenic, antidiabetic and antiadipogenic properties. In the present study, the effect of CLA enriched ghee on the antioxidant enzyme system and antiatherogenic properties in Wistar rats has been studied.Female Wistar rats of 21 days were taken for the study and fed with soybean diet (Control diet), low CLA diet and high CLA ghee diet (treatments) for thirty five days for studying antioxidative enzymes and sixteen weeks in case of antiatherogenic studies.Feeding of high CLA enhanced ghee during pubescent period in rats lead to an increase in catalase (CAT) and superoxide dismutase (SOD) enzyme activities in blood and increased CAT, SOD and glutathione transferase (GST) enzymes activities in liver by 27, 130 and 168 percent, respectively. Plasma nitrate concentration and Haemoglobin levels remained the same in all the treatments. Feeding of high CLA ghee resulted in lower (P < 0.01) plasma cholesterol & triglyceride level (52.17 and 30.27%), and higher high density lipoproteins (33.26%) than feeding of soybean oil (control group) and thus manifested in decreased (P < 0.05) atherogenic index (from 0.472 to 0.244). Lesser cholesterol and triglyceride levels were observed in the liver and aorta of high CLA fed rats than in those of the other groups. Histopathological studies of liver showed normal hepatic cords with portal triad in the high CLA ghee fed rats whereas fatty degeneration of hepatocytes containing fat vacuoles was observed in the liver of the other groups.This paper is the first report of the antioxidant and antiatherogenic properties of the high CLA enriched ghee suggesting that high CLA ghee can be used as a potential food for decreasing the risk of cardiovascular diseases, particularly in India, where, ghee is widely used for culinary and medicinal purposes. Hypercholesterolemia, obesity and oxidation stress are the major risk factors that, alone or in combination, accelerate the development of cardiovascular diseases and the progression of atherosclerosis . Due to cis −9, cis −12, C18:2) with two conjugated double bonds at various carbon positions in the fatty acid chain [Butyrivibrio fibrisolvens[t-11, C18:1 (vaccenic Acid), another intermediate of linoleic or linolenic acid biohydrogenation, by Δ9-desaturase in the tissues [Conjugated linoleic acid (CLA) is an important food component that consists of a mixture of positional and geometric isomers of linoleic acid  and medium chain fatty acids (C14:0 to C16:1) contents were comparable in all the three groups. The stearic acid (C18:0) level was lower (P < 0.01) in high CLA ghee (137.05 mg/g fat) than in other groups. Significant differences exist in the level of trans vaccenic acid in all the groups. The level of cis-9, trans-11 fatty acid level in high CLA ghee was 169 and 69 percent higher than that in other fats. The level of trans-10, cis-12, the other isomer of CLA in corresponding groups was 0.68, 1.61 and 2.78 mg/g fat, and difference among the groups was significant (P < 0.05). The Omega-3-fatty acids content was higher (P < 0.05) in low CLA ghee and high CLA ghee than in soybean oil whereas Omega 6 fatty acids level was similar in all the three groups and values. The ratio of Omega 6: Omega 3 fatty acids was lowest in high CLA ghee and highest in soybean oil.Fatty acid composition in all the three treatment groups has been summarized in Table Maximum CAT activity was observed in high CLA ghee group, followed by low CLA ghee group Table . Catalasth day, all the animals were sacrificed and CAT, SOD and GST activities were analyzed in liver. Liver CAT activity was the highest in high CLA ghee fed group followed by low CLA fed group and lowest in soybean oil fed group  in soybean oil fed group as compared to low and high CLA ghee fed groups. The high CLA ghee diet reduced the AI index from 0.472 to 0.244 whereas soybean oil increased AI from 0.410 to 0.971 in rats. Additional files Maximum cholesterol and triglyceride level was observed in soybean oil fed group whereas lowest amount was detected in high CLA ghee fed group in both liver and aorta Figures  and 2. SHistopathology of liver was done to study the deposition of fat in hepatocytes and sinusoids. The normal architecture of liver showed central vein surrounded by hepatocytes along with portal triad containing hepatic artery, bile duct and portal vein Figure a. In soyst day to 55th day) increased 20 & 33% CAT and 13 & 28% SOD activity, respectively, in blood as compared to the group fed on soybean oil diet. Similarly, an increased liver CAT and SOD activities were observed in high CLA group as compared to soybean oil fed group. Hence, higher CAT & SOD activities in group fed with high CLA ghee diet may offer higher protection to the cells against oxidants. Similar results were reported by Santos Zago et al., [The objective of this study was to assess the effect of CLA containing food product (ghee) on antioxidative enzyme system and to study the antiatherogenic property in the blood and liver of rats. The results indicated that feeding of low and high CLA ghee diets during pubescent period  rats by feeding CLA for 120 days. Not much significant variations in the average body weight of rats fed with different dietary treatments were observed. This proves that CLA has anti-obesity property, which may be attributed to the modulation in lipid metabolism leading to the reduction of lipid uptake by adipocytes and manifested as reduction in body fat gain [trans 18:1 and cis-9,trans-11 Conjugated Linoleic Acid has modified Plasma Lipoproteins in the Hypercholesterolemic Rabbit. Present results clearly indicated that CLA feeding, irrespective of its level in diet, narrowed the atherogenic index (0.472 to 0.244) in rats than those fed on soybean oil (0.410 to 0.971) by decreasing LDL and increasing HDL.In our study, low and high CLA feeding reduced total cholesterol by 52.17 and 26.06%, triglycerides by 23.00 and 11.00%, however, HDL increased by 17.82 and 33.26% than soybean oil feeding in rats. The supplementation of diet containing CLA has shown a reduction in the levels of serum total cholesterol and total triglyceride and increased high-density lipoprotein cholesterol level as compared to control diet groups without CLA ,22. In aOur results also indicated that the presence of CLA in the diet of rats inhibited deposition of cholesterol and triglycerides by two and three folds, respectively, in liver as well as in aorta and the response of CLA was depending upon its level in the diet. Fatty acid composition and presence of polar lipids in the diets have significant effects on lipid partitioning, gene expression, and potentially the development of liver pathology in mice . In a reAs discussed earlier, dietary CLA tends to modulate lipid metabolism in rats. In the present study, soybean oil feeding resulted in more fat deposition in hepatocytes compared to the CLA feeding (Plate 4.1B). Recent work by Kostogrys and Pisulewski  have alsThe study shows that high CLA enriched ghee increases the antioxidant enzyme activities such as CAT, SOD and GST activities; which concludes its antioxidant nature. Feeding of high CLA enriched ghee also lead to a decrease in cholesterol, triglyceride and high density lipoproteins and inturn, decreased the low density lipoproteins, which finally lead to the reduction in atherogenic index, which proves its antiatherogenic property. The present study indicates that high CLA enriched ghee (clarified butter) has antioxidant and antiatherogenic activities suggesting that ghee can be as an important food component for decreasing the risk of cardiovascular diseases.Protocols were conducted in accordance with standard ethical guidelines, it was approved by the IAEC .Low CLA 12.18 mg/g fat) and high CLA (19.54 mg/g fat) ghee was prepared by creamery method from milk of buffaloes fed on groundnut oil cake and mustard cake plus 2% mustard oil based concentrates, respectively [ mg/g fatadlibitum basis. Animals were weighed at fortnight intervals.Twenty four female rats of Wistar strain (21 days old) with body weight of 25–42 g were obtained from small animal house of National Dairy Research Institute, Karnal, Haryana, India and housed in metal cages in an air conditioned room at 24 ± 1°C with 12:12 h light/dark cycle. Rats were randomly divided into 3 groups (n = 8) namely Soybean oil, low CLA ghee and High CLA ghee diet groups  were housed in metal cages in an air conditional room at 24 ± 1°C in small animal house of National Dairy Research Institute, Karnal and housed. Animals were randomly divided into 3 groups of eight animals each and fed with the same diet as soybean oil, low CLA and high CLA diet .The washed tissue was dehydrated in the ascending grades of isopropanol and finally cleared in xylene. The tissue was then embedded in molten paraffin wax. Sections were cut at 5 mm thickness and stained with Hematoxylin and Eosin .Results are expressed as Mean ± SE unless otherwise stated. Statistical comparisons were made by Univariate General Linear Model, T-test and Mann–Whitney test using IBM SPSS software, version 20.AI: Atherogenic Index; AOE: Antioxidative Enzymes; CAT: Catalase; CD: Conjugated dienes; CDNB: 1-Chloro,2,4-DinitroBenzene; CLA: Conjugated linoleic acid; GST: Glutathione transferase; HDL: High density lipids; LDL: Low density lipids; NOS: Nitric oxide synthase; SOD: Superoxide dismutase; TBARS: Thiobarbituric acid reactive substances; VLDL: Very low density lipids.The authors report no competing interests.KC and AKT designed the research. KC performed experiments. HKK and KC analyzed data and wrote the paper. HKK, KC, AKT, CS and PR support and review the study. All authors have read and approved the final manuscript.Plasma total cholesterol (mg/dL) levels in rats fed on Soybean oil/Low CLA ghee/high CLA ghee diet.Click here for filePlasma HDL-cholesterol (mg/dL) levels in rats fed on Soybean oil/ Low CLA ghee/high CLA ghee diet.Click here for filePlasma triglycerides (mg/dL) levels in rats fed on Soybean oil/Low CLA ghee/high CLA ghee diet.Click here for filePlasma LDL (mg/dL) levels in rats fed on Soybean oil/Low CLA ghee/high CLA ghee diet.Click here for fileAtherogenic Index in rats fed on Soybean oil/Low CLA ghee/high CLA ghee diet.Click here for file


Jiao Tai Wan (JTW), a Chinese herbal formula containing Rhizoma Coptidis and Cortex Cinnamomi, has been used for diabetic treatment for many years. The aim of this study was to determine the main components in JTW and to investigate the effects of JTW on hepatic lipid accumulation in diabetic rats and humans. JTW extract was prepared and the main components were assayed by HPLC. An animal model of diabetes mellitus was established and JTW was administered intragastrically. In the clinical study, diabetic patients with poor glycemic control were treated with JTW. Blood glucose and lipid parameters, liver histology, hepatic triglyceride content and lipogenic gene expression were examined. Our data demonstrated that JTW significantly improved hyperglycemia, hyperlipidemia and hepatic lipid accumulation in diabetic rats. This was accompanied by the down-regulation of acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FAS) protein expressions, and the up-regulation of AMP-activated protein kinase (AMPK) and phosphorylated-ACC (pACC) protein expressions in the liver tissues. Diabetic patients also exhibited decreases in their hepatic triglyceride content. The results suggest that JTW attenuates hepatic lipid accumulation in diabetic rats and humans. These beneficial effects are possibly associated with the inhibition of lipogenic gene expression in the liver. Under pfunction , 5. In tfunction , 7. In tpoptosis . TherefoExcessive lipid accumulation in the liver of patients who drink little or no alcohol is called nonalcoholic fatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liver diseases ranging from simple hepatic steatosis to nonalcoholic steatohepatitis to cirrhosis. NAFLD is highly prevalent in subjects with T2DM; approximately 70% of type 2 diabetic patients are estimated to have NAFLD . This hiCoptis chinensis Franch, Ranunculaceae) and Cortex Cinnamomi. Rhizoma Coptidis (huang lian in Chinese) mainly consists of various alkaloids including berberine, coptisine, palmatine, and epiberberine, whereas Cortex Cinnamomi (rou gui in Chinese) largely contains cinnamaldehyde and cinnamic acid [Jiao Tai Wan (JTW) is a classical traditional Chinese prescription first mentioned in the book Han-Shi-Yi-Tong in the Ming Dynasty that consists of Rhizoma Coptidis  were obtained from the Hubei Province Center for Disease Control and Prevention . The rats received a standard diet with free access to tap water and were maintained on a 12:12 h light-dark cycles in a humidity- and temperature-controlled environment. All procedures were performed in accordance with the Chinese guidelines for animal experiments  and were approved by the local authorities.Seven rats were selected randomly as the control group and were injected with saline into the tail vein. The remaining rats were injected with streptozotocin  into the tail vein. An oral glucose tolerance test (OGTT) was performed two weeks later. Impaired glucose tolerance (IGT) was diagnosed when the plasma glucose levels of rats at two time points were higher than the upper limit  or when the plasma glucose levels at one time point exceeded the upper limit by more than 20% . Thereafter, twenty-one rats with IGT were randomly divided into 3 groups. Rats receiving saline served as diabetic controls. Rats receiving JTW (3 g/kg/day) were taken as the JTW group, and rats receiving metformin hydrochloride (183 mg/kg/day) were taken as the metformin (Met) group. Oral gavage was performed once a day between 8:00 and 10:00 a.m. The doses were adjusted to the body weight, which was recorded once a week. Meanwhile, the high-fat chow  was provided to the animals with IGT while the rats in the control group continued their standard diet. At the end of the 8-week period, overnight-fasted rats were weighed and then anaesthetized by pentobarbital sodium. Blood samples were obtained from the abdominal aorta. The liver was quickly excised and divided into two parts. One sample was immediately stored in liquid nitrogen, while the other was fixed with 4% paraformaldehyde. Rhizoma Coptidis and Cortex Cinnamomi used for animal experiments were obtained from the Traditional Chinese Medicine Company in Hubei Province . The voucher specimen was deposited at the herbarium of the Pharmacy Faculty at the Hubei University of Chinese Medicine and authenticated by Department of Pharmacognosy, Hubei University of Chinese Medicine . The rat doses of Rhizoma Coptidis and Cortex Cinnamomi were obtained by the conversion of the human doses  to rat equivalent doses based on body surface areas. The JTW preparation process was as follows: ninety grams Rhizoma Coptidis and forty-five grams Cortex Cinnamomi were mixed together. The mixture was decocted twice by refluxing it with water (1 : 10 w/v), 2 hours for the first time and 1 hour for the second time. The volatile oil was collected by the volatile oil extractor during the decocting procedure. The volatile oil and the solution obtained were concentrated to 450 grams. This extract represented the solution for rat administration and was stored at 4°C until use.In the clinical study, we used another form of JTW for the convenience of oral administration. JTW, containing Rhizoma Coptidisand Cortex Cinnamomi concentrated granules, was purchased from China Resources Sanjiu Medical and Pharmaceutical Co., Ltd. Rhizoma Coptidis concentrated granules largely contain berberine , coptisine , palmatine , and epiberberine . Cortex Cinnamomi is mainly composed of cinnamaldehyde  and cinnamic acid . Each pack of JTW contains two grams of Rhizoma Coptidis concentrated granules  and one gram of Cortex Cinnamomi concentrated granules . Patients mixed the granules together by boiling water and drank two packs each day.λ Absorbance Detector, and a XTerra RP18 chromatographic column  was used. The extract of JTW was dissolved in methanol. To measure berberine, all analyses were performed at a column temperature of 35°C. The mobile phase was 30 : 70 (v/v) acetonitrile-water containing potassium dihydrogen phosphate (0.1 mol/L) and sodium hydroxide (0.05 mol/L), adjusted to pH 9.5 with triethanolamine. The flow rate was 0.9 mL/min and the wavelength of detection was set at 345 nm. To measure cinnamaldehyde, the analyses were performed at a column temperature of 22°C. The mobile phase was 55 : 15 : 25 : 5 (v/v/v/v) water, acetonitrile, methanol, and tetrahydrofuran. The flow rate was 0.6 mL/min and the wavelength of detection was set at 290 nm. To measure cinnamic acid, the analyses were performed at a column temperature of 25°C. The mobile phase was 65 : 15 : 15 : 5 (v/v/v/v) water, acetonitrile, methanol, and tetrahydrofuran, adjusted to pH 5.5 with glacial acetic acid. The flow rate was 0.9 mL/min and the wavelength of detection was set at 270 nm. Berberine, cinnamaldehyde, and cinnamic acid, the main components in JTW extracts and concentrated granules, were all measured using the high-performance liquid chromatography (HPLC) method. HPLC  equipped with Waters 600 Pump, and Waters 2487 Dual The rats were fasted overnight and given a single dose of 50% glucose solution one week before being sacrificed. The glucose solution was administered by gavage at a dose of 2.0 g/kg body weight. Blood samples for glucose measurement were obtained from the tail vein at 0 hour (before glucose-loading), 1 hour, and 2 hours (after glucose-loading) by glucose-oxidase method using a glucose monitor . μIU/mL)/22.5. The plasma levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), nonesterified fatty acid (NEFA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using commercial reagents . The fasting insulin concentration (FINS) was measured by radioimmunoassay kit . The homeostasis model assessment index (HOMA-IR) was calculated using the formula of fasting glucose (mmol/L) × fasting insulin (μm thick paraffin sections of the liver were stained with hematoxylin-eosin (HE) for liver pathology. The morphology of the liver cells and inflammatory cells was observed under high-power magnification (×400). Neutral lipids were extracted from each aliquot using the Folch procedure with slight modifications [Serial 4 ications . Hepaticβ-actin . The membranes were then washed by TBST, followed by lucifugal incubation with the DyLight 800-labeled antibody to rabbit IgG (1 : 1000)  at room temperature for 1 h. The membranes were washed in a lucifugal way 3 times with TBST. Protein bands were detected by the LI-COR Odyssey Infrared Fluorescent Scanner . Band densities were determined by Bio-Rad Quantity One software v 4.62 and quantified as the ratio between the OD value of target band and the OD value of β-actin.Protein lysates extracted from liver homogenates were subjected to 10% SDS-PAGE gel  and then transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature. The blocking solution was then removed, and the membranes were washed by TBST, followed by overnight incubation at 4°C with the primary antibodies   and β-blockers and thiazides), the use of insulin, and women who were pregnant or lactating. The nature and potential risks of the study were explained to all subjects before obtaining their written informed consent. The experimental protocol was approved by the ethics committee of the Tongji Hospital affiliated with Tongji Medical College following the ethical principles outlined in the Declaration of Helsinki.Type 2 diabetic patients with poor glycemic control were enrolled in this study based on the following inclusion criteria: (1) aged 25–70 years; (2) use of stabilizing oral hypoglycemic drugs or lifestyle modification to control their blood glucose for at least 8 weeks; (3) fasting plasma glucose (FPG) 7.0–13.0 mmol or 2-hour postprandial blood glucose (2hPPG) 11.1–16.6 mmol/L and hemoglobin A1c (HbA1c) >6.5%; (4) no known acute or chronic disease based on history, physical examination, and standard laboratory tests; (5) alcohol consumption <20 g/day; and (6) no evidence of hepatitis A, B, or C as well as no evidence of autoimmune hepatitis, clinical signs or symptoms of inborn errors of metabolism, and no history of the use of toxins or drugs known to induce hepatitis. Exclusion criteria included thyroid disease, the use of antihypertensive agents that could possibly influence glucose metabolism  and the control group. Their previous diabetes management was maintained during the 12-week experimental period except for the addition of JTW in patients of the JTW-treated group. The following indicators before and after treatment were measured: body mass index (BMI), waist circumference, waist-to-hip ratio (WHR), blood pressure (BP), FPG, 2hPPG, HbA1c, plasma TC, TG, LDL-C, HDL-C, ALT, and AST. 1H-MRS) scans of their livers before and after treatment, as described previously [All of the patients received proton magnetic resonance spectroscopy  followed by Dunnett's T3 test for data with equal variances not assumed. For data with equal variances assumed, ANOVA followed by LSD test was used. Nonparametric tests were used to compare median values between the groups. Calculations were performed using SPSS 14.0 software. Statistical significance was defined as The contents of berberine, cinnamaldehyde, and cinnamic acid in the JTW extract used in the animal experiments were 23.03 mg/g, 3.12 mg/g, and 0.27 mg/g, respectively. In clinical trials, the contents of berberine, cinnamaldehyde, and cinnamic acid in the JTW concentrated granules were 79.51 mg/g, 13.39 mg/g, and 1.06 mg/g, respectively. The chromatograms of berberine, cinnamaldehyde, and cinnamic acid in JTW were presented in P < 0.05, P < 0.01). In contrast to diabetic rats, the rats in the JTW- or metformin-treated group exhibited little gain in body weight as well as a significant reduction in plasma glucose, insulin levels, and HOMA-IR .As shown in P < 0.01). However, the treatment with JTW or metformin significantly reversed these plasma lipid changes in diabetic rats .As shown in P < 0.01). These changes were accompanied by a larger increase in the severity of hepatic steatosis compared with controls as well as increased intrahepatic inflammatory cell infiltration . The degree of fatty degeneration was decreased without obvious intrahepatic inflammatory cell infiltration . However, treatment with JTW or metformin resulted in a marked increase in the hepatic expression of AMPK, pAMPK, and pACC as well as significant decreases in ACC and FAS proteins . JTW treatment did not change the ratio of pAMPK to AMPK expression but increased the ratio of pACC to ACC expression significantly (P < 0.01).As shown in P < 0.05). In addition, HTC decreased significantly after the addition of JTW to the diabetic treatment but increased in the control group (P < 0.05). However, the levels of ALT, AST, and HDL-C did not change significantly  were also monitored in the clinical study. No significant changes in plasma creatinine were observed during the 12 weeks of JTW treatment. No severe hypoglycemia was observed.Chinese medicine is an excellent alternative and complementary medicine in treating NAFLD. In traditional Chinese medicine, NAFLD falls into the category of symptoms caused by phlegm/dampness retention, qi/blood stasis, and spleen/kidney deficiency. Increasing evidence indicates that herbs that remove accumulated pathogenic phlegm and dampness are useful in treating NAFLD . In Chin1H-MRS scans of liver as an assessment tool. Previous studies have demonstrated that, although this method gives no information of liver histology, HTC obtained by spectroscopy closely coincides with biopsy or autopsy-derived triglyceride concentrations [In the animal study, the diabetic rats exhibited the characteristics of hyperglycemia, hyperinsulinemia, and hyperlipidemia. Plasma liver enzymatic activity and HTC also significantly increased. Liver histology demonstrated severe fatty degeneration and inflammation infiltration. The oral administration of JTW not only attenuated the biochemical disturbances in peripheral blood but also alleviated TG elevation and the morphological changes in liver tissues. We suggest that JTW could be an effective herbal prescription to treat nonalcoholic steatohepatitis of diabetic rats. These results were further confirmed by the clinical study, with the exception of the effects of JTW on liver histology and enzymatic activity. Invasive liver biopsy was not used in the clinical study to obtain histology data for ethical considerations. As an alternative, we applied noninvasive trations . Additiotrations . HoweverWith respect to the constituents of Rhizoma Coptidis and Cortex Cinnamomi, many substances have been identified. Among the constituents effective in treating diabetes, berberine (which is contained in Rhizoma Coptidis), cinnamaldehyde, and cinnamic acid (which are contained in Cortex Cinnamomi) have attracted significant attention , 29, 30.However, one limitation of our study must be considered. The effect of JTW on the fatty liver was evaluated using different formulations in the animal and clinical studies. In the animal experiments, the traditional Chinese herbal decoction was used, whereas concentrated herbal granules were chosen in the clinical trial. These different preparations account for the differences in the content of berberine, cinnamaldehyde, and cinnamic acid in JTW detected by HPLC. However, the daily dosage of JTW intake in diabetic rats was nearly identical to that in diabetic patients. Herbal granules, extracted from herbs with modern pharmaceutical technology, are convenient for those who cannot make an herbal decoction every day. We believe that the application of JTW granules assures quality control in the clinical trials. Nonetheless, we cannot confirm that there are any other differences in the constituents of JTW without the process of boiling. However, we have provided preliminary data that suggest that JTW is promising in the treatment of NAFLD. In summary, our study demonstrates that oral treatment with JTW attenuates hepatic lipid accumulation in diabetic rats and humans. These beneficial effects are possibly associated with the inhibition of lipogenic gene expression in the liver.


In the control group (Group C) blood was filtrated with an arterial filter for 60 min; in long-term (Group L) and short-term (Group S) groups, blood was filtrated with a leukocyte filter for 60 and 5 min. We found that about 90% of leukocytes were removed after 5 min of filtration in both Groups L and S. Although leukocyte count continued to reduce, mean fluorescent intensities of CD11/CD18, free hemoglobin, and neutrophil elastase increased in Group L and were higher than those in Groups C and S at 60 min. Leukocyte rupture, cytoplasmic leakage, and circulating naked nuclei were also found in Group L. The data support our hypothesis that long-term filtration can induce inflammation and lead to leukocyte destruction.Filtration during extracorporeal circulation (ECC) not only removes but also activates leukocytes; therefore, long-term leukocyte filtration may cause adverse effects. In the present study, we tested this hypothesis by priming ECC with 300 mL of canine blood and examining filtration effects in 3 groups ( Inflammatory response induced by leukocyte activation during extracorporeal circulation (ECC) is one of the major causes of organ dysfunction –3. Blockin vivo study showed that short-term filtration effectively reduced leukocyte count, and preserved lung function better than long-term filtration. For example, neutrophil elastase (NE) and IL-8 increased during long-term filtration [in vitro.Leukocyte filtration activates leukocytes –17, whicltration . The resThe study was approved by the Animal Hospital Ethics Committee and all animals in the study received standard care in compliance with the “Guide for the Care and Use of Laboratory Animals.” Eighteen healthy adult hybrid dogs were provided by the Animal Experiment Center of Sichuan University for the study. Dogs  were anesthetized with sodium pentobarbital (25 mg/kg). After heparinization (3 mg/kg), 300 mL of blood was harvested from the left femoral artery to a blood bag. Meanwhile, 6% hydroxyethyl starch 130/0.4  was infused from the femoral vein to maintain blood pressure.A closed ECC loop was established with a blood storage device  and a regular arterial filter  or a leukocyte filter , a roller pump , and tubing. The ECC loop was primed with 300 mL of preserved blood harvested above and 300 mL hydroxyethyl starch 130/0.4. The circulating flow rate was set at 1 L/min, and ECC lasted for 60 min in all groups.Our pilot study showed that the leukocyte count decreased sharply with low levels of NE after 5 min. Therefore, 5 min was used for “short-term” test in this study. In clinical trials, however, a leukocyte filter is used throughout ECC, often longer than 60 min. Therefore, 60 min was used for “long-term” test.n = 6 in each group). Blood from the control group (Group C) was filtrated throughout ECC with a standard arterial filter. Blood from Groups L (long-term) and S (short-term) was filtrated with a leukocyte filter for 60 and 5 min (the filter was bypassed in the remaining 55 min). Blood samples were obtained at 0, 5, 30, and 60 min of ECC for indexes analyses.Established ECCs were divided randomly into 3 groups  . For meaμL of phosphate-buffered saline. Nonspecific antibody binding was blocked with FcR-blocking reagent (Miltenyi Biotec) for 20 min before staining with conjugated antibodies. Immunofluorescent cell staining was performed with Rat anti dog CD11/CD18:FITC . Isotype: IgG2c  served as a negative control. Data acquisition was performed on a FACSAria cytometer equipped with FACS Diva 5.0 software (BD) and analyzed by Flowjo software (Tree Star) [For determination of expression of CD11/CD18 on leukocyte, red blood cells were lysed with ammonium chloride solution, and acquired leukocytes were resuspended in 100 ee Star) .Circulating integral leukocytes were determined as we described previously . BrieflyAt the end of experiment, the membrane of the leukocyte filter was taken for transmission electron microscopy as reported previously , 19. SamP < 0.05 was considered statistically significant.Data were analyzed by SPSS 16.0 . Quantitative data were expressed by the mean ± SD, and one-way ANOVA with Student-Newman-Keuls test was used to compare differences among the 3 groups. Segmental data were expressed as a percentage, and the differences between the 3 groups were compared using the chi-square test. A value of Leukocyte counts were similar among the 3 groups before ECC and decreased by 90% after 5 min of filtration in groups L and S . Then, iP < 0.01). Thereafter, MFIs of CD11/CD18 in the leukocyte increased sharply in group L, while it increased gradually in groups C and S. Neutrophils and lymphocytes, and expression of CD11/CD18 had a similar pattern  of CD11/CD18 on leukocyte increased significantly in both groups L and S compared with that in Group C at 5 min ECC . Furthermore, the fHb level was lower in Group S than in Group C.To determine whether erythrocyte destruction is associated with activated neutrophils during ECC, fHb levels in plasma were measured. Hemoglobin (a) and fHb (b) levels were similar among the 3 groups before ECC . The fHbWright's stain and transmission electron microscopy analyses showed significant leukocyte rupture in Group L , indicatin vitro approach that focused on filtration induced inflammation. Using the extracorporeal circulation (ECC) model in vitro, we clearly showed that effectiveness of leukocyte filtration depends on exposure time during ECC. Short-term use of a leukocyte filter reduced circulating leukocytes; with continued usage, leukocytes that adhered to the filter membrane disrupted, leading to leakage of protease, inflammatory reaction, and tissue destruction. Our results may explain why leukocyte depletion could not improve the outcomes in some clinical studies [Our study is the first  studies –14.in vivo, because inflammatory products are processed by the immune system. For example, naked nuclei in the circulation would be engulfed. Therefore, the in vitro model may be a necessary approach. In this in vitro model, circulating leukocytes were significantly reduced with a 5 min short-term filtration (about 5–7 circles). The leukocyte filter was then bypassed, and leukocytes adhered to the membrane were no longer exposed to the circulation. Our results support a short-term usage of the filter because the concentration of fHb was lower in Group S than in Group C. This is in agreement with several clinic trials. Morioka et al. [Studying the effect of filtration on inflammation is difficult a et al.  and Chiba et al.  found lea et al. . In patia et al. .Efficiency of a filter is determined by membrane area and filtration time. The filtration would reach its maximal capacity by prolonging filtering time. Then, the number of circulating leukocytes would not change with continued use of the filter; however, damaging effects on leukocytes develop, which compromises the beneficial effect of the filter. An optimal length of time for filter use should be available.Previous studies have observed that leukocyte filtration did not inhibit, but activated leukocytes –17. ThisActivated leukocytes induce tissue injury via obstruction of microcirculation and release of proteases, such as neutrophil elastase. In the present study, neutrophil elastase and free hemoglobin increased in Group L significantly, suggesting that long-term usage of leukocyte filtration exerts deleterious side effects. This statement can be verified by the fact that aprotinin, a serine proteases inhibitor, significantly improves lung function and reduces the incidence of atrial fibrillation in patients treated with a leukocyte filter .in vivo studies.Although our results showed that short-term filtration may effectively inhibit inflammatory reactions and reduce erythrocyte destruction, whether it would improve patient outcomes requires further In conclusion, our results clearly show that long-term filtration can induce leukocyte destruction and evoke inflammatory reactions and should be avoided.


Young patients receiving chemotherapy occasionally face infertility and premature ovarian failure (POF). Numerous investigations reported that adipose-derived stem cells (ADSCs) transplantation could ameliorate the structure and function of injured tissues. The aim of this study was to explore the therapeutic efficacy of ADSC transplantation for chemotherapy-induced ovarian damage.Female mice were injected intraperitoneally with 50 mg/kg cyclophosphamide (CTX). After 15 consecutive days of injection, ADSCs were transplanted either directly into bilateral ovaries or via intravenous injection, and the ovaries were excised after either 1 week or 1 month of treatment. The follicles were counted and categorized, and ovarian histologic sections were stained for TUNEL. Ovarian function was evaluated by monitoring ovulation. ADSC tracking, microarray analyses, and real-time polymerase chain reaction (PCR) were used to assess the inner mechanism of injury and repair.The ovarian function of mice exposed to CTX injection improved after ADSC transplantation. The population of follicles at different stages and ovulation significantly increased after the treatment. Immunofluorescence revealed reduced TUNEL staining. The tracking of ADSCs revealed that these cells did not directly differentiate into the follicle component. Microarray analyses indicated that changes in different groups of genes might affect follicle formation or ovulation.ADSC transplantation improved ovarian function. Our results suggest a potential mechanism for ADSC therapy. Chemicals that are used to treat cancer are unquestionably beneficial as therapeutic agents. However, their side effect on the quality of life of female cancer survivors and their offspring cannot be ignored. Females are born with a finite number of undeveloped, primordial follicles. Chemicals that destroy primordial oocytes contained in the ovary can lead to premature ovarian failure (POF) or menopause . POF is Hormonal replacement therapy (HRT) has been used to treat common menopausal problems, but it increases the risk of cancer or recurrence in cancer survivors, forcing physicians to use alternative treatments. In recent years, interest has rapidly grown in the therapeutic potential of stem cells. Lee  concludeAdipose-derived stem cells (ADSCs) are a type of multipotent adult stem cell isolated from adipose tissue. Cultured ADSCs can differentiate into multiple cell types, including osteoblasts, cartilage cells, adipocytes, myocytes, vascular endothelial cells, and neurons ,10. The Eight-week-old female C57/BL6 mice and 8-week-old C57/BL6 female green fluorescent protein (GFP) transgenic mice were used in all of the experiments. The mice were purchased from Vital River Laboratories . All of the animal procedures were conducted in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for reporting animal studies. Ethical approval was obtained from the Ethics Committee of Military Medical Sciences  for this study. The mice were fed a standard pellet diet and allowed free access to water. Vaginal smears were obtained daily. Only mice showing at least two consecutive normal 4– to 5-day vaginal estrus cycles were included in the experiments.g for 5 minutes to obtain cellular pellets. The cellular pellets were cultured in α-MEM/10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified incubator at 37°C with 5% carbon dioxide. Third-passage ADSCs were used for all of the transplantation experiments.ADSC isolation was performed according to the protocol previously reported . In brie5 cells wase used for each sample, and cell Quest software was used for data analysis.Surface-marker expression was analyzed with flow cytometry by using the following antibodies: fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies against CD29, CD44, CD90, CD31, and CD45, and phycoerythrin (PE)-conjugated mouse monoclonal antibodies against CD34 . For flow cytometric analysis, adherent cells were detached by treatment with 0.25% trypsin-EDTA, neutralized with FBS-containing culture medium, and disaggregated into single cells by pipetting. The cells were incubated with mAbs for 30 minutes at 4°C, washed twice with PBS, resuspended in 0.5 ml PBS, and immediately analyzed by using an FACS Calibur flow cytometer . A minimum of 2 × 103 cells/cm2. After 24 hours, the medium was replaced by osteogenic differentiation medium, and the cells were induced for 3 weeks. Then cells could be fixed and stained with Alizarin red; to achieve osteogenic differentiation, cells were seeded at a density of 1 × 104/cm2. After reaching confluence, cells were incubated in adipogenic differentiation medium for 2 weeks. Then cells were fixed and stained with Oil Red O. The differentiation medium was changed every 3 days.To confirm the multipotency of ADSCs, osteogenic and adipogenic differentiation were verified with Alizarin Red staining and Oil Red O staining, respectively . In brieTo establish the POF model of chemotherapy-induced ovarian damage, adult female C57/BL6 mice were administered CTX (50 mg/kg) for 15 consecutive days via intraperitoneal injection.6 autologous ADSCs in a volume of 0.3 ml of 0.1 M PBS (pH 7.4). The injection was repeated again the following day. In the in situ group, 1 × 105 ADSCs in 20 μl PBS were injected directly into the bilateral ovaries of POF mice with a microinjector.After establishing the POF model, we randomly divided the mice into four equal groups. The WT group consisted of normal control mice that received no treatment. In the POF group, the mice were administered CTX. In the intravenous group, POF mice were injected intravenously with 1 × 10in situ. The ovaries were collected 1 week and 1 month after treatment, and the follicles were detected and classified. Five representative sections from each ovary were selected. The ovaries were removed and fixed in 4% paraformaldehyde for at least 24 hours. After fixation, the ovaries were dehydrated, paraffin-embedded, serially sectioned at 5 μm, and mounted on glass microscope slides. Routine hematoxylin and eosin (H&E) staining was performed for histologic examination with light microscopy. Primordial, primary, secondary, and antral follicles were counted on every fifth section. Only follicles containing an oocyte were counted to avoid counting any follicle twice. Follicles were classified as follows: primordial follicle, oocyte surrounded by a single layer of squamous granulosa cells; primary follicle, intact enlarged oocyte with a visible nucleus and one layer of cuboidal granulosa cells; secondary follicle, two or three layers of cuboidal granulosa cells without antral space; early antral follicles, emerging antral spaces; and preovulatory follicles, which are largest of the follicular types and possess a defined cumulus granulosa cell layer [ADSCs were administered either intravenously or ll layer .Four groups of female C57/BL6 mice were superovulated 1 week or 1 month after transplantation via an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG), followed by intraperitoneal injection of 5 IU human chorionic gonadotropin (HCG) 48 hours later. The oocytes were collected from the ampulla portion of the oviduct 14 to 16 hours after HCG injection.in situ Cell Death Detection Kit, POD , was used to detect apoptosis in the mouse ovaries, according to manufacturer’s instruction. Next, the nucleus was dyed with Hoechst 33324, and the sections were observed with fluorescence microscopy . Apoptotic granulosa cells (GCs) in the ovary were stained green, and five random fields from each sample were counted (five samples/group).An For cell-tracking studies, C57/BL6 female GFP transgenic mice were used as donors, and C57/BL6 female mice served as receptors. GFP-positive ADSCs were acquired as described previously . The traRNA was isolated from mouse ovaries by using Trizol (Invitrogen) with standard methods. Labeling and hybridization were performed at the CapitalBio Company, according to protocols described in the 32 K mouse genome arrays user manual. The data were analyzed by using LuxScan 3.0 Image analysis software .Gapdh. For expression analysis, data from three replicates were analyzed by using the 2−ΔΔCt method.Real-time quantitative PCR reactions were set up in triplicate by using the SYBR Green Real-time PCR Master Mix  and run on a Bio-Rad CFX96 (US). The PCR primers were designed according to cDNA sequences in the NCBI database Table . All of t tests were used to determine the significance between two groups. One-way analysis of variance (ANOVA) with least significant difference (LSD) tests was used to determine significant differences between four groups. A P value < 0.05 was considered to be statistically significant.The statistical analyses were performed by using SPSS 14. The mean ± SEM of the data were calculated. Student Isolated cells were cultured in tissue-culture dishes. After 4 days in culture, the cells appeared to be spindle shaped and formed symmetric colonies Figure A. After Zuk  first rein situ routes were increased. The ovaries of mice that received ADSC transplantation after chemotherapy possessed several hundred follicles at all stages of development. In contrast, the POF mice did not recover to the normal level within 1 month. Thus, both intravenous and in situ therapy significantly reduced ovary injury. After 1 week, the number of primary (357 ± 15.38), early antral follicles (476 ± 77.73) and antral follicles (377 ± 39.45) in the in situ group were significantly greater than those of the POF group (P < 0.05). Moreover, the amounts of other types of follicles in the intravenous and in situ groups were greater than those of the POF group, but these differences did not reach statistical significance. After 1 month, the number of primordial (278 ± 24.9), secondary (470 ± 57.45), early antral (320 ± 14.14), and preovulatory follicles (321 ± 42.49) in the in situ group, and the number of primary (252 ± 33.47), secondary (456 ± 62.29), early antral (352 ± 22.8), and preovulatory follicles (316 ± 38.47) in the intravenous group were significantly increased compared with the POF group (P < 0.05)  . However, in the intravenous  and in situ groups , ovulation increased 1 week after treatment  Figure B signifi2) was significantly higher than that of the other groups (P < 0.05). The number of TUNEL-positive cells in the intravenous (42.8 ± 13.7/mm2) and in situ (50.4 ± 13.1/mm2) groups was lower than that of the POF group . Both of the therapeutic methods reduced host apoptosis.One week after ADSC transplantation, the number of cells that stained positive for TUNEL in ovary sections was quantified. The number of apoptotic GCs in the POF group 150.8 ± 48.4/mm8.4/mm2 win situ and intravenous transplantation of ADSC rescues the apoptosis of GCs in ovary sections.Previous studies conclusively demonstrated that alkylating agents induce apoptosis in GCs and oocytes, and consequently, the loss of follicles ,16. ThisCell tracking is generally used to monitor stem cell homing and engraftment. One week and 1 month after transplantation, the injected GFP (+)-ADSCs were traced to the ovary, and GFP-positive cells were located in the interstitium but not in follicles Figure . We alsoCareful observation is needed to determine whether stem cell transplantation constitutes regenerative cell therapy or cell-based cytokine therapy and to determine how long these injected cells can function in the ovary. Considering their strength and stability, GFP-positive ADSCs were used in this study. We found that ADSCs undergo a massive cell loss after intravenous injection, resulting in low signal intensity, which was consistent with previous reports. At 4 weeks, only a small portion of transplanted ADSCs could be detected in the intravenous group with immunofluorescence. We also observed that the interstitium in the therapy group exhibited fluorescent stem cells that had infused into the donor ovary, but these cells did not develop into follicles, including the GCs or the oocytes. This result indicates that transplanted ADSCs did not directly participate in follicle regeneration. Furthermore, a large number of engrafted cells died within 1 month after transplantation. Taken together, these results suggest that the direct regeneration of ADSCs played a minor role in the improved ovary structure and function.in situ group , the transforming growth factor (TGF)-β signaling pathway, natural killer cell-mediated cytotoxicity, the MAPK pathway, and the Toll-like receptor signaling pathway. Some of those pathways are important for follicle and oocyte growth.Zcchc11, Angpt 1, Onecut2, among others, were upregulated after ADSC therapy in both therapy groups, whereas Cxcr4 was downregulated, these results are in accordance with our microarray analysis. However, certain functions of these genes remain obscure , VEGF , PGF , and TGF-β. ADSCs also exhibit moderate expression of FGF (fibroblast growth factor)-2 and Ang (angiopoietin)-1, as well as low levels of Ang-2 -20. SomeSome cases of POF clearly have a genetic basis . We usedAngpt1 after therapy was in accordance with previous research. The change in Angpt1 and Angpt2 levels may be associated with follicular growth and angiogenesis during the preovulatory period [The increased expression of y period . Angpt1 y period .CXCR4, an α-chemokine receptor specific for stromal-derived-factor-1 , has potent chemotactic activity for lymphocytes and was downregulated after therapy. CXCR4/SDF1 may play an important role in retaining follicles in an inactivated state. The inactivation of CXCR4 may induce the growth of a primordial follicle into a mature follicle, and the CXCR4/SDF1 interaction can inhibit the primordial transition to a primary follicle in the neonatal mouse . The dowThe Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. Zcchc11 is also important for postnatal survival and growth. Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and insulin-like growth factor (IGF)-1 protein in the blood during the neonatal period . IGF-1, Apoptosis is likely a relevant mechanism in the genesis of POF in patients who have undergone chemotherapy or radiotherapy . After AADSC therapy may be used in clinical applications for patients who have no distant metastasis. In this scenario, we can collect the patient’s stem cells and then reinfuse them after completing chemotherapy. For aggressive tumors, stem cells can be acquired after chemotherapy, although cell viability may decline, and the risk of cancer cell reintroduction may increase. More research is needed to evaluate the efficacy and safety of ADSC acquired after chemotherapy.in vitro, and these genes can be expressed efficiently and for long periods in vivo. Kucerova [In regard to genetic defects, autologous ADSCs will presumably also carry the same genetic defect. Thus, ADSCs represent desirable target cells for gene therapy, as they are susceptible to transfection with exogenous genes. Experiments have confirmed that ADSCs can easily import exogenous genes Kucerova  used ADSOur studies revealed that ADSCs significantly improved ovarian function after chemotherapy-induced ovarian injury. Our results suggest a potential mechanism for ADSC therapy. ADSCs could increase follicle number and oocyte number through gene-expression changes and the production of paracrine cytokines. ADSC treatment also decreased granulosa cells apoptosis. Therefore, ADSCs represent an alternative strategy for POF therapy and may be useful for future regenerative medicine and clinical applications.ADSC: Adipose-derived stem cells; Ang: Angiopoietin; BMT: Bone marrow stem cells transplantation; CAMs: Cell-adhesion molecules; CTX: Cyclophosphamide; FBS: Fetal bovine serum; FGF: Fibroblast growth factor; FITC: Fluorescein isothiocyanate; FSH: Follicle-stimulating hormone; GC: Granulosa cell; GFP: Green fluorescent protein; HCG: Human chorionic gonadotropin; HGF: Hepatocyte growth factor; HRT: Hormonal replacement therapy; IGF: Insulin-like growth factor; miRNA: microRNA; PBS: Phosphate-buffered saline; PE: Phycoerythrin; PGF: Placental growth factor; PMSG: Pregnant mare serum gonadotropin; POF: Premature ovarian failure; SDF: Stromal-derived-factor; TGF: Transforming growth factor; VEGF: Vascular endothelial growth factor.The authors declare that they have no competing interests.MS conceived of and performed all the experiments, analyzed the data, and drafted the manuscript. SW conceived of and performed animal experiments and revised the manuscript. YL was responsible for the molecular biology experiments, analyzed the data, and revised the manuscript. LY performed the cell experiments and analyzed the data. FG analyzed the data and drafted the manuscript. CW contributed essential reagents and tools and revised the manuscript. YY conceived of the research, contributed essential reagents or tools, and revised the manuscript. All of the authors read and approved the final manuscript.


AbstractSepsidae (Diptera) for the country. Here we describe two new species in the species-poor genus Perochaeta (Perochaeta cuirassa sp. n. andPerochaeta lobo sp. n.) and one to the largest sepsid genus Sepsis (Sepsis spura sp. n.) which is also found in Sumatra and Sulawesi. Two additional Sepsis species are new records for Vietnam . We conclude with a discussion of the distribution of Perochaeta and the three Sepsis species.A recent collecting trip to Vietnam yielded three new species and two new records of  Sepsidae are a moderately large, cosmopolitan family of saprophagous flies, with over 300 extant species recorded from all zoogeographic regions (Ozerov 2005). Most species are attracted to dung, carrion, and other malodorous, decaying organic substrates (Pont and Meier 2002); i.e., by using different substrates in different microhabitats, the sepsid fauna from a specific locality can be quickly explored. Separating sepsids from the remaining saprophagous insects is also relatively straightforward because most sepsids can be easily recognized based on the constriction of the first two abdominal segments which gives the flies a wasp- or ant-like habitus.The Perochaeta cuirassa sp. n., Perochaeta lobo sp. n., Sepsis spura sp. n., Sepsis sepsi and Sepsis monostigma.Here we update an existing species list for Vietnam by adding five species: three are new to science while two others are new records. The relatively large number of additions is due to the fact that the Vietnamese sepsid fauna remains poorly studied . The current species list comprises 21 species in six genera and is based on the sepsid world catalogue (Ozerov 2005) and subsequent taxonomic research by Iwasa and Thinh (2008). We complement this list by adding five species that were collected during a brief collecting trip in July 2010: Sepsis sepsi and Sepsis spura were also collected previously in Indonesia (Sulawesi and Sumatra) in 2007 and 2009.All five species were collected between 11–16 July 2010 from Northern Vietnam . Cow dung was placed in various habitats for at least a few hours to attract sepsids, which were then caught by sweep-netting. Additional material for Perochaeta species based on the methods described in Tan et al. (2010). All type specimens and additional material are vouchered in 95% ethanol and kept in the Cryogenic Collection of the Raffles Museum of Biodiversity and Research (RMBR), National University of Singapore, Singapore. We adopt the taxonomic terminology as described by Merz and Haenni (2000) for adult morphology  and Sinclair (2000) for male genitalia.Specimens were photographed using a Leica Z16 APO-A stereomicroscope fitted with a DFC425 digital microscope camera, and then digitally traced to illustrations using a Wacom© PTZ 630 tablet. We also amplified and sequenced a 544-bp fragment of cytochrome oxidase c subunit I (COI) within the DNA barcoding region for the two new PerochaetaPageBreak, which has only three described species  and no synonyms. In addition, the descriptions and illustrations for the described species are of good quality. Furthermore, molecular data are consistent with distinct species: the two new Perochaeta species are separated by 3.3% for the barcoding gene COI (uncorrected pairwise distances); while the distances of either to Perochaeta dikowi are 11.4% and 11.8% . Of the 23 Sepsis recorded in the Oriental region (Ozerov 2005), the Sepsis species described here closely resembles the widespread Sepsis nitens Wiedemann, 1824 which has two synonyms . Duda’s (1926) description and fore leg illustration of Sepsis tuberculata are sufficiently detailed to confirm that it is indeed a synonym of Sepsis nitens. However, Brunetti’s (1910) rather vague description of Sepsis brevicosta based on one male from Calcutta and a few females from localities in other Indian localities , is more difficult to interpret. He describes Sepsis brevicosta’s fore femur as having “a small bump [on the ventromedial region] with three or four strong short spines”. This description of a “bump” is in agreement with Sepsis nitens  and Indonesia (Sumatra and Sulawesi).Describing a new Oriental s nitens  while the a spur . In addiPerochaeta) to estimate the species boundaries. The two new Perochaeta species are sympatric and the new Sepsis species is parapatric with Sepsis nitens. In both cases we have not seen any intermediate specimens so that there is no evidence for hybridization. This supports our species hypotheses. However, both Perochaeta species are so rare that this test is relatively weak. As pointed out by Lim PageBreaket al. (in press), in such cases descriptions should only be prepared if the species are unusually distinct. This is the case here, whereby the two new species can be clearly distinguished based on both morphology [cf. sternites and hypopygia of Perochaeta cuirassa . Here we apply the Hennigian species concept (Meier and Willmann 2000) and use morphology and DNA sequence data (for cuirassa  and Peroeta lobo ] and DNAeta lobo . When thAng 2010 sp. n.urn:lsid:zoobank.org:act:76BA1CDF-D467-45D0-9898-305C21045C6DHolotype. ♂ (RMBR), Vietnam, Lào Cai Province, Sa Pa Valley. Baited with cow dung at forest edge next to a small cascade alongside highway, ca. 850m along the road westward of the Thác bạc  tourist attraction . Collected 16.VII.2010 (Ang Y). Paratypes. 2 ♂ (RMBR), collected from same locality and time as holotype.th sternite, which resembles a cuirass or breastplate armor.The specific epithet refers to the shape of the main scleral plate for the 4Perochaeta cuirassa is very similar to Perochaeta lobo and can only be reliably distinguished from the latter based on the 4th sternite . Collected 16.VII.2010 (Ang Y).th sternite.The specific epithet is a phonetic translation of Greek “λοβό”, which refers to the large, distinct lobe found on each lateral half on the posterior margin of the 4Perochaeta cuirassa and can only be reliably distinguished based on the 4th sternite and hypopygium. The 4th sternite . Collected 15.VII.2010 (Ang Y).♂♂♀♀ (RMBR), Sepsis monostigma is an Oriental species that resembles Sepsis pseudomonostigma Ursu, 1969 but is geographically exclusive from Sepsis pseudomonostigma . Sepsis monostigma can be differentiated by the two long medioventral spines (one short spine in Sepsis pseudomonostigma) on the fore femur , Taiwan, India , Japan , Korea, Philippines (Luzon Is.), Sri Lanka, South Russia , Vietnam (Lào Cai).Ozerov, 2003Sepsis sepsi  Ozerov 2003. Zoologicheskiy zhurnal, 82, 10: 1276.Vietnam, Ha Tay Province, Ba Vi National Park. From ex culture established from ♀ collected from rubbish dump near temple at summit of mountain . Collected 11.VII.2010 (Ang Y). 2 ♂ (RMBR), Indonesia, West Sumatra, Bukit Tingei Regency, Tanjung Mutiara Dist., Bantar Gadang Beach, . Collected 6.VII.2007 (Lohman D).♂♂♀♀ (RMBR), Sepsis sepsi bears some resemblance to Sepsis nitens and was initially thought to be such by Hennig but later identified it as a ‘Sepsis n.sp.’ (1941), and was formally described by Ozerov (2003). Sepsis sepsi can be distinguished from Sepsis nitens based on the row of four large spines on a large rounded ventromedial bump of the fore femur , Vietnam (Ha Tay).Ang 2010 sp. n.urn:lsid:zoobank.org:act:581DEFE8-8274-4365-BCDF-7F34BB59A258Holotype. ♂ (RMBR), Vietnam, Lào Cai Province, Sa Pa Valley. Collected from dung on cow farm, 4km NE of Sa Pa town . Collected 15.VII.2010 (Ang Y). Paratypes. 2 ♂ (RMBR), Indonesia, West Sumatra, Bukit Tingei Regency, Tanjung Mutiara Dist., Bantar Gadang Beach, . Collected 6.VII.2007 (Lohman D). ♂ (RMBR), Indonesia, N. Sulawesi, Tondano Province, Kampung Jawa . Collected 12.V.2009 (Ang Y).The specific epithet old English for “spur”, and refers to the distinct spur-like medioventral tubercle found on the male fore femur.Sepsis spura closely resemble Sepsis nitens but can be distinguished by the following characters: (1) Medioventral tubercle on male fore femur of Sepsis spura is spur-like and bent at a forward angle with two smaller adjacent spines dorsally positioned at the end of the tubercle and one larger spine at the ventral end (Sepsis nitens is thicker on the base and has its three spines positioned more in a anterio-posterior fashion (Sepsis spura (Sepsis nitens (Sepsis nitens. (3) Sepsis spura  Theis spura  merges bs nitens . The shois spura  has onlys nitens  has suchs nitens  has a rais spura  has a rost flagellomere yellowish, arista brown. Proboscis whiPageBreaktish yellow. Forelegs wholly yellow. Mid and rear coxa yellow with brown base. Mid femur brown but yellow on basal and distal tips; mid tibia brown on basal half and diffuses to yellow on apical half. Rear femur yellow but brown on dorsal region, while rear tibia wholly brown. Fore tarsus with tarsomeres 3–5 brown, mid tarsus with tarsomeres 3–5 very lightly brown, rear tarsus with tarsomeres 4 and 5 brown. Wing clear except for basicostal cell and basal region of costal cell, which is light brown. Veins dark brown. Calypter clear, margin and fringe-hairs yellowish. Haltere white. Thorax mostly dark brown, but pronotopleuron is yellow. Abdominal tergites and sternites glossy dark brown.Head capsule mostly brown with a thin light brown strip on gena; fascial margin black. Vertex dark brown. Facial carina and lunule light grey-brown. Pedicel dark brown, 1ocellar, 1 divergent postocellar (ocellar longer than postocellar), 1 inner vertical, 1 outer vertical . Orbital very reduced to absent. 3–4 vibrissae. 3–4 postocular. Lower fascial margin lined with setulae.Roundish, facial carina short and shallow, facial area receding. Gena and parafacial region narrow. Largely glossy except for lightly microtomentose occipital region. Chaetotaxy: 1 PageBreakAnepimeron glossy. Katatergite, meron and metepimeron microtomentose. Chaetotaxy: 1 apical scutellar, 1 reduced basal scutellar, 1 row  dorsocentral with posterior-most two setae as bristles, 1 row acrostichial and 1 postalar, 1 supraalar, 1 notopleural, 1 postpronotal, 1 anepisternal and 2 posterior spiracular.Scutum, postpronotum and scutellum wholly microtomentose. Subscutellum microtomentose with a small glossy spot ventromedially. Mediotergite microtomentose on margins and glossy in the medial region. Scutellum twice wide as long. Pleural pruinosity pattern : Proepisanteriodorsal. Mid femur with 1 anterior. Mid tibia with 2 posterior on median and subapex, 2 dorsal on basal 2/3 and apex, 1 anterior on apex, 1 anterioventral on basal 2/3 and 1 ventral on apex. Hind femur with 1 anteriodorsal on basal 2/3 and 1 posterioventral subapically. Hind tibia with 2 dorsal at median and subapically, 1 anterior at median and 2 anterioventral medially and subapically. Rear basitarsus with 2 ventral dark spines basally.Forelegs modified; fore femur  with larWithout pterostigma. Veins bare. Covered with microtrichiae except for basal half of basal-medial cell. Anterior region of r2+3 cell with sparse microtrichiae. Radial-medial cross-vein divides discal-medial cell by ratio of slightly less than 2 : 1. Length: 2.9–3.5mm.Tergites glossy black, syntergite 1+2 – tergite 5 normal, tergite 6 missing, syntergite 7+8 present and extending ventrad as a narrow sclerite. Spiracles 1–3 on intersegmental membrane close to tergite, spiracles 4 and 5 within respective tergites near margin. Spiracles 7 and 8 adjacent on margin of syntergite 7+8. Sternite 1 a broad rectangle with posterior invaginations while sternite 2 is triangular, tapering posteriorly; sternite 3 is oblong. Sternite 4 is V-shaped with setae and one stout discal bristle on each lateral margin; sternite 5 reduced to a thin lateral crescent .Cercal plate with two very weak lobes, each with single distal setae. Hypopygium and surstylus relatively setaeless. Surstylus fused to hypopygium; bulging medially at base but thin and curved medially, slightly dentate terminally .Indonesia , Vietnam (Lào Cai).Perochaeta cuirassa and Perochaeta lobo are the first Perochaeta species from Vietnam. That they are found in the same locality is surprising, given that Perochaeta is a small Oriental genus with only three described species. As discussed in Ang et al. (2008), the species in this genus appear to be only found in mid- and high-elevation localities in Asia above 650m ASL: Perochaeta dikowi was found on Fraser’s Hill, Malaysia , Perochaeta hennigi is only known from Thawalamtenne, Sri Lanka  and Tamil Nadu, India . The type locality of Perochaeta orientalis  is the Jiji Township in Taiwan  which is within the Chung Yang mountain range that has an average elevation >1000m ASL, and can also found in regions such as Indonesia . The new species Perochaeta cuirassa and Perochaeta lobo were found at 2600m ASL. A male Perochaeta specimen from Flores Island, Indonesia, examined by Hennig (1941) was also collected from a mid elevation site (1200m ASL). Unfortunately, the latter was poorly preserved and could not be described, but based on Hennig’s illustration, it likely constitutes a new species. Given that Perochaeta species are restricted to elevated sites, we predict that the number of species will rapidly grow as more mid and high elevation localities are sampled.Perochaeta is also of interest, because this  genus is very atypical for sepsids. Males of most sepsids have modified forelegs . This has made Perochaeta a model for testing the correlation between the evolution of behavior and morphology .Adding two new species to Sepsis spura appears to be a relatively widespread species ranging between Indonesia and Vietnam, and it can be found across all elevations, ranging from highlands  through mid elevation areas  to sea level . Such widespread species of insects are relatively common in Southeast Asia and are increasingly attracting attention because they can give rise to new species . At all localities the Sepsis spura is found in low numbers on bovine dung while other Sepsis species can be very abundant. This, along with its morphological resemblance to Sepsis nitens, may explain why Sepsis spura is only described now. It is likely to belong to a clade of Sepsis species without wingspots .Sepsis sepsi was first described from Sumbawa Is., Indonesia at 450m ASL (Ozerov 2003), but has subsequently been collected at low elevation sites in Sumatra as well as now at mid-elevation sites (800m ASL) in Ba Vi, Vietnam. This suggests that Sepsis sepsi is a relatively widespread species able to live in low to mid elevation habitats. We predict that it is also likely to be found in other areas between Indonesia and Vietnam. The record of the widely distributed Sepsis monostigma in Vietnam is not unexpected given that it falls within the recorded range from India to the far east of Russia.


Thus, we carried out a case-control study and an interaction analysis to test whether ADD1 is a common candidate gene for hypertension in the Chinese population. Blood samples and information including body mass index (BMI), smoking habit, and alcohol abuse were collected. Meanwhile, total cholesterol, high density lipoprotein, triglyceride were measured by automatic biochemistry analyzer. All 6 tag single nucleotide polymorphisms (tagSNPs) within ADD1 gene were genotyped by SNPstream genotyping system. Multifactor dimensionality reduction (MDR) was used to identify the interactions among the SNPs and the non-genetic factors. Results showed that plasma triglyceride, total cholesterol, and BMI were significantly higher in the hypertensive group than in the control group. Result from genotyping indicated that rs4963 was significantly associated with essential hypertension. After stratification by gender, rs4963 was associated with essential hypertension only in males. MDR analysis indicated that interaction among BMI, rs4963, and rs16843452 were involved in susceptibility of hypertension. The present study indicated that rs4963 within ADD1 gene was associated with essential hypertension in Chinese population, which might be related to altered exonic splicing and disrupted gene regulation. The association between polymorphisms of  The α-subunit is known to increase renal sodium reabsorption and may be involved in the pathophysiology of essential hypertension [α-adducin gene (ADD1) polymorphisms with essential hypertension in the past two decades.Adducin (ADD) is a heterodimeric cytoskeleton protein consisting of an -subunit , and theADD1 gene is located on chromosome 4p16.3 and comprises 16 exons. One well-studied polymorphism in ADD1 gene is a substitution of Gly for Trp at amino acid residue 460 , which was first described by Cusi et al. [ADD1 W460W was found to be associated with a form of low renin salt-sensitive hypertension in Caucasians, with reduced response to pressure natriuretic function and with increased proximal sodium reabsorption [Human sorption . TherefoADD1 G460W polymorphism with essential hypertension in global population; but it is suggested that the W carrier might increase the risk of essential hypertension in Han Chinese population [ADD1 G460W polymorphism, and whether other single nucleotide polymorphisms (SNPs) in ADD1 gene were associated with essential hypertension is not clear. Therefore, we carried out a case-control study and an interaction analysis to verify whether ADD1 gene is associated with hypertension in the Chinese population.To convince the association of this polymorphism with essential hypertension, several meta-analyses were recently published –10. Mostpulation . HoweverThe participants were chosen from our established community-based epidemiology study of common diseases. We have collected more than 10,000 health records. Subsequently, participants who fulfilled the following criteria were put into our database: 30 to 75 years old, Han Chinese, living in Ningbo City (East Coast of China) for at least three generations and do not have a migration history. The hypertensive subjects and control subjects were matched for age and sex. In addition, only subjects who do not have other cardiovascular disease or major chronic illnesses according to their health records were included in the control group. Lastly, 905 essential hypertension cases and 905 healthy controls, including 392 males and 513 females in each group, were involved in the following studies.Blood pressure measurements were conducted in the morning after the participant had been in the sitting position for 10 minutes. Three BP measurements were obtained at 5-minute intervals using standard mercury sphygmomanometer, and the average of last two measurements was taken as the BP for that participant. Hypertension in this study is defined as a sitting systolic blood pressure (SBP) ≥ 140 mmHg and/or a diastolic blood pressure (DBP) ≥ 90 mmHg, or self-reported use of antihypertension medication . Patients with secondary hypertension were excluded. Normal blood pressure is defined with SBP ≤ 120 mmHg and DBP ≤ 80 mmHg.Blood samples were collected with informed consent. Subsequently, total cholesterol (TC), high density lipoprotein (HDL), and triglyceride (TG) were measured from these blood samples by Hitachi automatic biochemistry analyzer 7100. Clinical information including body mass index (BMI), and weekly alcohol and cigarettes consumption were also obtained. In this study, who drank ≥70 g alcohol per week for more than 1 year were defined as individuals with alcohol abuse. Moreover, who smoked ≥70 cigarettes per week for more than 1 year were defined as individuals with smoking habit. The protocol of this study was reviewed and approved by the Ethics Committees of Ningbo University.http://hapmap.ncbi.nlm.nih.gov/), with tagger pairwise method in CHB: R Square cut off = 0.8 and MAF cut off = 0.1. Genomic DNA was obtained from the whole blood using standard phenol/chloroform extraction. Genotyping was performed using the GenomeLab SNPstream Genotyping System (Beckman Coulter Inc.) according to the manufacturer's protocol [All six tagSNPs were retrieved from HapMap . Hardy-Weinberg equilibrium (HWE) was tested by the software PEDSTATS V0.6.8 (http://www.sph.umich.edu/csg/abecasis/). Linkage equilibrium (LD) blocks were defined based on the confidence intervals method [http://www.epistasis.org/mdr.html).Continuous variables are presented as the mean ± SD and analyzed by s method  and the s method . Multifas method . The sofP values less than 0.05 were considered statistically significant.All tests were two sided, and P < 0.05).The baseline characteristics of our study population are summarized in ADD1 gene were shown in P > 0.05). Only rs4963 was significantly associated with essential hypertension  = 0.85, 95% confident interval (CI) = 0.75–0.97). After stratification by gender, rs4963 was associated with essential hypertension only in males, and the other 5 SNPs were still negative (ADD1. Only one LD block and three common haplotypes within the LD block (including rs16843452 and rs12503220) were identified  activity in renal tubular cells, increased renal sodium reabsorption, and subsequently hypertension [Among the three adducin genes, ipidemia , renal dipidemia , and coripidemia . Series rtension , 19. ADD1 G460W polymorphism was able to explain only a very minute proportion of the heritability of BP traits. Therefore, it is necessary to explore whether there are any other SNPs within ADD1 gene that are also associated with essential hypertension. In order to answer this question, we carried out a case-control study in the Chinese population. The result showed that rs4963 was associated with essential hypertension, especially in males. One possible reason for this result might be that men have higher TG (1.93 versus 1.69 mM) and TC (5.39 versus 5.08 mM) level than women.Since hypertension is considered polygenic, resulting from the interaction of several genes and together with environmental factors, a single SNP has weak effects on the phenotype. A family study from van Rijin et al.  showed tADD1 gene [Out of 1,113 SNPs associated with DD1 gene , 9 are iDD1 gene . rs4963 DD1 gene . In currMoreover, environmental factors and individual's biological characteristics, including excess dietary sodium intake, alcohol abuse, smoking, obesity, stress, BMI, TC, and TG, cannot be neglected , 25. We ADD1 gene was associated with essential hypertension in Chinese population, which might be related to altered exonic splicing and disrupted gene regulation. Regulation of sodium transport is such a complex process that at least 189 genes are involved according to gene ontology (GO: 0002028). ADD1 is only a common member of this system, and polymorphisms within this gene may play a tiny role. Other genes involved in sodium transport regulation system need to be studied. In addition, in our interaction analysis, a significant interaction was found between genetic and nongenetic factors, demonstrating that genetic study alone is not a sufficient indicator for hypertension. To decipher causal factors leading to the development and the pathogenesis of hypertension, future work will require analysis of gene-environmental interaction.In conclusion, the present study indicated that rs4963 within