Single-cell sequencing and multiple machine learning algorithms to identify key T-cell differentiation gene for progression of NAFLD cirrhosis to hepatocellular carcinoma.

Introduction: Hepatocellular carcinoma (HCC), which is closely associated with chronicinflammation, is the most common liver cancer and primarily involves dysregulated immune responses in the precancerous microenvironment. Currently, most studies have been limited to HCC incidence. However, the immunopathogenic mechanisms underlying precancerous lesions remain unknown. Methods: We obtained single-cell sequencing data (GSE136103) from two nonalcoholic fatty liver disease (NAFLD) cirrhosis samples and five healthy samples. Using pseudo-time analysis, we systematically identified five different T-cell differentiation states. Ten machine-learning algorithms were used in 81 combinations to integrate the frameworks and establish the best T-cell differentiation-related prognostic signature in a multi-cohort bulk transcriptome analysis. Results: LDHA was considered a core gene, and the results were validated using multiple external datasets. In addition, we validated LDHA expression using immunohistochemistry and flow cytometry. Conclusion: LDHA is a crucial marker gene in T cells for the progression of NAFLD cirrhosis to HCC.

The function role of HIGD1A in nonalcoholic steatohepatitis from chronic hepatitis B.

BACKGROUND: Accompanied by the growing prevalence of nonalcoholic fatty liver disease (NAFLD), the coexistence of chronic hepatitis B (CHB) and NAFLD has increased. In the context of CHB, there is limited understanding of the factors that influence the development of NASH. METHODS: We enrolled CHB combined NAFLD patients who had liver biopsy and divided them to NASH vs. non-NASH groups. A whole transcriptome chip was used to examine the expression profiles of long noncoding RNAs (lncRNAs) and mRNA in biopsied liver tissues. The function analysis of HIGD1A were performed. We knocked down or overexpressed HIGD1A in HepG2.2.15 cells by transient transfection of siRNA-HIGD1A or pcDNA-HIGD1A. In vivo investigations were conducted using hepatitis B virus (HBV) transgenic mice. RESULTS: In 65 patients with CHB and NAFLD, 28 were patients with NASH, and 37 were those without NASH. After screening 582 differentially expressed mRNAs, GO analysis revealed differentially expressed mRNAs acting on nicotinamide adenine dinucleotide phosphate (NADPH), which influenced redox enzyme activity. KEGG analysis also shown that they were involved in the NAFLD signaling pathway. The function analysis revealed that HIGD1A was associated with the mitochondrion. Then, both in vivo and in vitro CHB model, HIGD1A was significantly higher in the NASH group than in the non-NASH group. HIGD1A knockdown impaired mitochondrial transmembrane potential and induced cell apoptosis in HepG2.2.15 cells added oleic acid and palmitate. On the contrary, hepatic HIGD1A overexpression ameliorated free fatty acids-induced apoptosis and oxidative stress. Furthermore, HIGD1A reduced reactive oxygen species (ROS) level by increasing glutathione (GSH) expression, but Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/Acetyl-CoA carboxylase (ACC) pathway was not involved. CONCLUSION: Both in vivo and in vitro CHB model, an upward trend of HIGD1A was observed in the NASH-related inflammatory response. HIGDIA played a protective role in cells against oxidative stress. Our data suggested that HIGD1A may be a positive regulator of NASH within the CHB context.

Melatonin reduced colon inflammation but had no effect on energy metabolism in ageing Mongolian gerbils (Meriones unguiculatus).

In photoperiod-sensitive wild animals, the secretion of melatonin (MT) is modulated by external photoperiod, and MT affects inflammation and the ageing process. The beneficial effects of MT in delaying the progress of ageing have been reported in laboratory mice and rats. However, little is known about MT in wild mammals. In the current study, we investigated energy metabolism, microbial community structure and colon homeostasis in ageing Mongolian gerbils (Meriones unguiculatus) through exogenous supplementation of MT to test the hypothesis that MT has beneficial effects on gut homeostasis in ageing gerbils. Exogenous MT supplementation had no effect on energy metabolism in Mongolian gerbils but reduced the levels of circulating tumor necrosis factor-α (TNF-α), immune globulin G (IgG) and corticosterone (CORT). The increase in the level of inflammation in ageing animals was related to changes in the structure and diversity of the gut microbiota. At the genus level, the relative abundance of Prevotella, Treponema, Corynebacterium, and Sphingomonas was increased in ageing animals and decreased significantly by the treatment of MT. Christensenella and Lactobacillus were attenuated in ageing animals, and tended to be enhanced by MT treatment. Functions related to glycosphingolipid biosynthesis-ganglio series and lipopolysaccharide biosynthesis (metabolisms of cofactors, vitamins and glycan) were increased in ageing animals and decreased significantly by the treatment of MT. Our data suggest that a supplement of MT could improve colon homeostasis through changing the composition of gut microbiota and reducing inflammation in ageing gerbils.

Clinical Features of Non-Alcoholic Fatty Liver Disease in the Non-Lean Population.

INTRODUCTION: The prevalence of non-alcoholic fatty liver disease (NAFLD) in non-lean patients is significantly increased, and obesity significantly increases the risk of cirrhosis and HCC in NAFLD patients. However, whether there is a difference in clinical manifestations of NAFLD between overweight and obesity remains unclear. The objective of this study was to assess the clinical and histological features of NAFLD among a non-lean population. METHODS: Current study enrolled consecutive non-lean (body mass index [BMI] >23 kg/m2) patients with NAFLD and available liver biopsy results. Patients were stratified by BMI into two groups for the comparison of their clinical and histological variables, which included the overweight (BMI 23∼<28 kg/m2) and the obese (BMI ≥28 kg/m2). Risk factors for moderate to severe fibrosis (stage >1) were also analyzed through the logistic regression model. RESULTS: Among 184 non-lean patients with metabolic-associated fatty liver disease enrolled, 65 and 119 were overweight and obese, respectively. Patients in the obesity group had a significantly lower level of gamma-glutamyl transpeptidase, higher levels of platelet, glucose, prothrombin time, and more common of moderate to severe inflammatory activity when compared to those in the overweight group. However, a significant low frequency of moderate to severe fibrosis was found in the obesity group versus the overweight group (19.33% vs. 40.00%, p = 0.002). Binary logistics regression analysis of fibrosis found that aspartate transaminase (AST), BMI, alanine transaminase (ALT), and cholesterol (CHOL) were independent predictors for moderate to severe fibrosis in non-lean patients with NAFLD. Compared with the traditional fibrosis-4 (AUC = 0.77) and aminotransferase to platelet ratio index (AUC = 0.79) indexes, the combined index based on AST, BMI, ALT, and CHOL was more accurate in predicting moderate to severe fibrosis in non-lean patients with NAFLD (AUC = 0.87). CONCLUSIONS: Clinical and histological features differed between obesity and overweight patients with NAFLD. When compared to the traditional serum markers, the combination index including AST, BMI, ALT, and CHOL provided a better model to predict moderate to severe fibrosis in non-lean patients with NAFLD.

Recruitment of Muscle Genes as an Effect of Brown Adipose Tissue Ablation in Cold-Acclimated Brandt's Voles (Lasiopodomys brandtii).

Skeletal muscle-based nonshivering thermogenesis (NST) plays an important role in the regulation and maintenance of body temperature in birds and large mammals, which do not contain brown adipose tissue (BAT). However, the relative contribution of muscle-based NST to thermoregulation is not clearly elucidated in wild small mammals, which have evolved an obligate thermogenic organ of BAT. In this study, we investigated whether muscle would become an important site of NST when BAT function is conditionally minimized in Brandt's voles (Lasiopodomys brandtii). We surgically removed interscapular BAT (iBAT, which constitutes 52%~56% of total BAT) and exposed the voles to prolonged cold (4 °C) for 28 days. The iBAT-ablated voles were able to maintain the same levels of NST and body temperature (~37.9 °C) during the entire period of cold acclimation as sham voles. The expression of uncoupling protein 1 (UCP1) and its transcriptional regulators at both protein and mRNA levels in the iBAT of cold-acclimated voles was higher than that in the warm group. However, no difference was observed in the protein or mRNA levels of these thermogenesis-related markers except for PGC-1α in other sites of BAT (including infrascapular region, neck, and axilla) between warm and cold groups either in sham or iBAT-ablated voles. The iBAT-ablated voles showed higher UCP1 expression in white adipose tissue (WAT) than sham voles during cold acclimation. The expression of sarcolipin (SLN) and sarcoplasmic endoplasmic reticulum Ca2+-dependent adenosine triphosphatase (SERCA) in skeletal muscles was higher in cold than in warm, but no alteration in phospholamban (PLB) and phosphorylated-PLB (P-PLB) was observed. Additionally, there was increased in iBAT-ablated voles compared to that in the sham group in cold. Moreover, these iBAT-ablated voles underwent extensive remodeling of mitochondria and genes of key components related with mitochondrial metabolism. These data collectively indicate that recruitment of skeletal muscle-based thermogenesis may compensate for BAT impairment and suggest a functional interaction between the two forms of thermogenic processes of iBAT and skeletal muscle in wild small mammals for coping cold stress.

The identify role and molecular mechanism of the MALAT1/hsa-mir-20b-5p/TXNIP axis in liver inflammation caused by CHB in patients with chronic HBV infection complicated with NAFLD.

BACKGROUND/AIMS: To identify the inflammatory damage caused by chronic hepatitis B (CHB) in patients of chronic hepatitis B virus (HBV) infection complicated with non-alcoholic fatty liver disease (NAFLD), then guiding clinicians to carry out antiviral treatment. METHODS: According to the pathological features of liver biopsy, treatment-naïve obese patients of chronic HBV infection complicated with NAFLD who had elevated alanine transaminase (ALT) were divided into CHB group and NASH group. Transcriptome chips were used to analyze the expression profiles of long non-coding RNA (lncRNA) and mRNA in liver puncture tissues from the two groups. The chip data of CHB and NASH groups were analyzed for differential expression analysis, gene function analysis, signal pathway analysis, target gene prediction and competing endogenous RNAs (ceRNA) network analysis. RESULTS: By comparing CHB group with NASH group, a total of 44 differentially expressed lncRNAs and 567 differentially expressed mRNAs were screened. GO analysis predicted that the differentially expressed mRNAs may affect monooxygenase activity and oxidoreductase activity. KEGG analysis predicted that the differentially expressed mRNAs may be related to signaling pathways involved in oxidative phosphorylation, phagosomes, and NAFLD. Differential analysis of lncRNA shown that the expression of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) in CHB group was significantly upregulated. Subsequently, through target gene prediction and ceRNA network analysis, we found thioredoxin interacting protein (TXNIP), which was significantly upregulated in the CHB group and had a ceRNA relationship with MALAT1. It is predicted that there may be a ceRNA regulation relationship of MALAT1/hsa-miR- 20b-5p/TXNIP. CONCLUSION: The MALAT1/hsa-miR-20b-5p/TXNIP axis may mediate CHB-induced inflammatory damage in chronic HBV infection complicated with NAFLD, and the mechanism may be related to the activation of NLRP3 inflammatory bodies and downstream inflammatory responses.

Pedicle complex tissue flap transfer for reconstruction of duplicated thumbs with unequal size.

BACKGROUND: Thumb polydactyly is one of the most common congenital hand deformities, and the Bilhaut-Cloquet procedure or a modified one is often used. However, controversy remains over the rare instances in which both thumbs are not of similar length or far apart in distance. AIM: To evaluate the clinical outcomes of pedicle complex tissue flap transfer in the treatment of duplicated thumbs with unequal size. METHODS: From January 2014 to December 2020, 15 patients underwent duplicated thumb reconstruction by pedicle complex tissue flap transfer at our hand surgery center. The technique was used when it was necessary to combine different tissues from both severed and preserved thumbs that were not of similar length or far apart in distance. Subjective parents' evaluations and functional outcomes (ALURRA and TATA criteria) were obtained. The alignment deviation, instability, range of motion (percent of opposite thumb) of the interphalangeal and metacarpophalangeal joints, and the aesthetic aspects, including circumference, length, nail size, and nail deformity, were used to assess the clinical outcomes. RESULTS: The average age of patients at the time of surgery was 13 mo, and the mean final follow-up occurred at 42 mo. An appropriate volume with a stable joint and good appearance was obtained in 14 reconstructed thumbs. An unstable interphalangeal joint occurred in one thumb. The flexion-extension arc at the metacarpophalangeal joint was good, while that at the interphalangeal joint was poor. Most of the parents were satisfied with the cosmetic and functional results of the reconstructed thumbs. The mean ALURRA score was 21.8 (range: 20-24), and the Tada score was 6.9 (range: 5-8). Compared with the non-operated side, the length of the operated thumb was approximately 95%, the girth was 89%, and the nail width was 82.9%. The mean ranges of motion were 62.1% of that of the unaffected thumb in the interphalangeal joint and 78.3% in the metacarpophalangeal joint. CONCLUSION: Harvesting a pedicle flap from a severed thumb is a safe and reliable procedure. Defects of the preserved thumb, such as the skin, nail, and bone, can be effectively restored using the complex tissue flap.

The physiological and molecular mechanisms to maintain water and salt homeostasis in response to high salt intake in Mongolian gerbils (Meriones unguiculatus).

Desert rodents are faced with many challenges such as high dietary salt in their natural habitats and they have evolved abilities to conserve water and tolerate salt. However, the physiological and molecular mechanisms involved in water and salt balances in desert rodents are unknown. We hypothesized that desert rodents regulated water and salt balances by altering the expression of AQP2 and α-ENaC in the kidney. Mongolian gerbils (Meriones unguiculatus), a desert species, were acclimated to drinking water with different salt contents: (0, control; 4% NaCl, moderate salt, MS; 8% NaCl, high salt, HS) for 4 weeks. The gerbils drinking salty water had lower body mass, food intake, water intake, metabolic water production and urine volume. The HS gerbils increased the expression of arginine vasopressin (AVP) in the hypothalamus, and also enhanced the expression of AQP2 and cAMP/PKA/CREB signaling pathway in the kidney. In addition, these gerbils reduced serum aldosterone levels and α-ENaC expression in the kidney. Creatinine clearance was lower in the HS group than that in the control group, but serum and urine creatinine levels did not change. These data indicate that desert rodents rely on AVP-dependent upregulation of AQP2 and aldosterone-dependent downregulation of α-ENaC in the kidney to promote water reabsorption and sodium excretion under high salt intake.

GnRH expression and cell proliferation are associated with seasonal breeding and food hoarding in Mongolian gerbils (Meriones unguiculatus).

Seasonal brain plasticity contributes to a variety of physiological and behavioral processes. We hypothesized that variations in GnRH expression and cell proliferation facilitated seasonal breeding and food hoarding. Here, we reported seasonal changes in sexual and social behavior, GnRH expression and brain cell proliferation, and the role of photoperiod in inducing seasonal breeding and brain plasticity in Mongolian gerbils (Meriones unguiculatus). The gerbils captured in April and July had more mature sexual development, higher exploratory behavior, and preferred novelty much more than those captured in September. Male gerbils captured in April and July had consistently higher GnRH expression than those captured in September. GnRH expression was also found to be suppressed by food-induced hoarding behavior in the breeding season. Both subadult and adult gerbils from April and July had higher cell proliferation in SVZ, hypothalamus and amygdala compared to those in September. However, adult gerbils captured in September preferred familiar objects, and no seasonal differences were found in cell proliferation in hippocampal dentate gyrus among the three seasons. The laboratory study showed that photoperiod alone did not alter reproductive traits, behavior, cell proliferation or cell survival in the detected brain regions. These findings suggest that the structural variations in GnRH expression in hypothalamus and cell proliferation in hypothalamus, amygdala and hippocampus are associated with seasonal breeding and food hoarding in gerbils. It gives a new insight into the proximate physiological and neural basis for these seasonal life-history traits of breeding and food hoarding in small mammals.

Responses in reproductive organs, steroid hormones and CYP450 enzymes in female Mongolian gerbil (Meriones unguiculatus) over time after quinestrol treatment.

The aim of this study was to assess the effects and reversibility of the synthetic estrogen compound, quinestrol, on the reproductive organs, steroid hormones, and drug-metabolizing enzymes CYP3A4 and CYP1A2 in liver and kidney over time after two quinestrol treatments in female Mongolian gerbils (Meriones unguiculatus). Female gerbils were treated with 4mg/kg quinestrol (9 gerbils/group, 3 treated group) (1 control group, 0mg/kg) for 3days and treated again after 25days. Animals were killed for collection of samples at 5, 10 and 15days after the second treatment ending. Two interval quinestrol treatments significantly increased uterine weight, with trend of increase over time, but no change could be detected in ovarian weights. Quinestrol treatment increased progesterone and estradiol levels, both with trend of decline over time. Quinestrol increased liver and kidney weights and total enzyme content of CYP3A4 and CYP1A2, with trend of decline over time. On the basis of reversible changes of detoxification enzymes or organs, interval quinestrol treatment effectively and reversibly influenced the reproductive hormone and organ to some extent.

A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects.

A novel neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), was found to have an important role in carbohydrate or lipid metabolism and was susceptible to dipeptidyl peptidase IV degradation. It can not only mediate glucose-dependent insulin secretion and lower blood glucose by activating VPAC2 receptor, but also raise blood glucose by promoting glucagon production by VPAC1 receptor activation. Therefore, its therapeutic application is restricted by the exceedingly short-acting half-life and the stimulatory function for glycogenolysis. Herein, we generated novel peptide-conjugated selenium nanoparticles (SeNPs; named as SCD), comprising a 32-amino acid PACAP-derived peptide DBAYL that selectively binds to VPAC2, and chitosan-modified SeNPs (SeNPs-CTS, SC) as slow-release carrier. The circulating half-life of SCD is 14.12 h in mice, which is 168.4-and 7.1-fold longer than wild PACAP (~5 min) and DBAYL (~1.98 h), respectively. SCD (10 nmol/L) significantly promotes INS-1 cell proliferation, glucose uptake, insulin secretion, insulin receptor expression and also obviously reduces intracellular reactive oxygen species levels in H2O2-injured INS-1 cells. Furthermore, the biological effects of SCD are stronger than Exendin-4 (a clinically approved drug through its insulinotropic effect), DBAYL, SeNPs or SC. A single injection of SCD (20 nmol/kg) into db/db mice with type 2 diabetes leads to enhanced insulin secretion and sustained hypoglycemic effect, and the effectiveness and duration of SCD in enhancing insulin secretion and reducing blood glucose levels are much stronger than Exendin-4, SeNPs or SC. In db/db mice, chronic administration of SCD by daily injection for 12 weeks markedly improved glucose and lipid profiles, insulin sensitivity and the structures of pancreatic and adipose tissue. The results indicate that SC can play a role as a carrier for the slow release of bioactive peptides and SCD could be a hopeful therapeutic against type 2 diabetes through the synergy effects of DBAYL and SeNPs.

Leptin regulates energy intake but fails to facilitate hibernation in fattening Daurian ground squirrels (Spermophilus dauricus).

Body fat storage before hibernation affects the timing of immergence in Daurian ground squirrels (Spermophilus dauricus). Leptin is an adipose signal and plays vital role in energy homeostasis mainly by action in brain. To test the hypothesis that leptin plays a role in facilitating the process of hibernation, squirrels were administrated with recombinant murine leptin (1µg/day) through intracerebroventricular (ICV) injection for 12 days during fattening. From day 7 to 12, animals were moved into a cold room (5±1°C) with constant darkness which functioned as hibernaculum. Energy intake, body mass and core body temperature (Tb) were continuously monitored throughout the course of experiment. Resting metabolic rate (RMR) was measured under both warm and cold conditions. At the end of leptin administration, we measured the serum concentration of hormones related to energy regulation, mRNA expression of hypothalamic neuropeptides and uncoupling protein 1 (UCP1) levels in brown adipose tissue (BAT). Our results showed that during leptin administration, the cumulative food intake and increase of body mass were suppressed while Tb and RMR were unaltered. The proportion of torpid squirrels was not different between two groups. At the end of leptin administration, the expressions of hypothalamic neuropeptide Y and agouti gene-related protein were suppressed. There were no differences in UCP1 mRNA expression or protein content in BAT between groups. Our data suggest that leptin can affect energy intake via hypothalamic neuropeptides, but is not involved in the initiation of hibernation in fattening Daurian ground squirrels.

Cold exposure inhibits hypothalamic Kiss-1 gene expression, serum leptin concentration, and delays reproductive development in male Brandt's vole (Lasiopodomys brandtii).

Cold commonly affects growth and reproductive development in small mammals. Here, we test the hypothesis that low ambient temperature will affect growth and puberty onset, associated with altered hypothalamic Kiss-1 gene expression and serum leptin concentration in wild rodents. Male Brandt's voles (Lasiopodomys brandtii) were exposed to cold (4 ± 1 °C) and warm (23 ± 1 °C) conditions from the birth and sacrificed on different developmental stages (day 26, day 40, day 60, and day 90, respectively). Brandt's voles increased the thermogenic capacity of brown adipose tissue, mobilized body fat, decreased serum leptin levels, and delayed the reproductive development especially on day 40 in the cold condition. They increased food intake to compensate for the high energy demands in the cold. The hypothalamic Kiss-1 gene expression on day 26 was decreased, associated with lower wet testis mass and testis testosterone concentration on day 40, in the cold-exposed voles compared to that in the warm. Serum leptin was positively correlated with body fat, testis mass, and testosterone concentration. These data suggested that cold exposure inhibited hypothalamic Kiss-1 gene expression during the early stage of development, decreased serum leptin concentration, and delayed reproductive development in male Brandt's voles.

[Sensitivity to chemotherapeutic drugs of polyploid tumor cells induced by a spindle poison nocodazole].

OBJECTIVE: To investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism. METHODS: Nocodazole in a dose of 100 ng/ml was used to induce polyploidization in a breast cancer cell line MDA-MB-231 cells. The polyploid cells (T-MDA-MB-231) were sorted by flow cytometry. The morphological changes and proliferation of T-MDA-MB-231 cells were compared with that of MDA-MB-231 cells. The cell growth inhibition was assessed by MTT assay. The cells were treated with paclitaxel, docetaxel, vincristine, epirubicin, 5-Fu, VP16 and oxaliplatin, respectively. Those cells were labeled with annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 was knocked down in T-MDA-MB-231 cells using SiRNA and their growth inhibition was evaluated by MTT assay to evaluate the reversing effect of Bcl-2-silencing on drug resistance. RESULTS: The polyploid T-MDA-MB-231 cells grew in vitro continuously and maintained constant DNA content. They had a larger cell size, and grew more slowly than MDA-MB-231 cells. The IC(50(s)) of T-MDA-MB-231 cells were significantly higher than that of the MDA-MB-231 cells: paclitaxel: (6.37 ± 0.07) vs. (2.05 ± 0.83) µmol/L; docetaxel: (32.98 ± 1.48) vs. (11.95 ± 0.98) µmol/L; vincristine: (35.28 ± 1.66) vs. (14.58 ± 0.94) µmol/L; oxaliplatin: (19.07 ± 0.45) vs. (9.75 ± 1.05) µmol/L; 5-Fu: (85.49 ± 3.21) vs. (31.35 ± 1.51) µmol/L; and epirubicin: (0.53 ± 0.06) vs. (0.15 ± 0.01) µmol/L, (all P < 0.05). The IC(50(s)) of VP16 in T-MDA-MB-231 cells was (2.85 ± 0.50)µmol/L, significantly lower than the (12.20 ± 1.55) µmol/L in MDA-MB-231 cells (P < 0.05), and that of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (19.59 ± 0.48) µmol/L, significantly higher than the (12.20 ± 1.55) µmol/L in the MDA-MB-231 cells (P < 0.05). The IC(50(s)) of docetaxel of T-MDA-MB-231 cells after Bcl-2-knocked down by siRNA was (21.52 ± 0.68) µmol/L, significantly decreased and lower than that before Bcl-2 silencing (32.98 ± 1.48) µmol/L. CONCLUSIONS: Our results indicate that polyploid tumor cells induced by spindle poison Nocodazole are more resistant to most of chemotherapeutic drugs. Downregulation of Bcl-2 increases the sensitivity of polyploid cells to docetaxel. The high expression of Bcl-2 may be one of the drug resistance mechanisms of polyploid tumor cells. The polyploid tumor cells are relatively sensitive to VP16, suggesting that VP16 might be an effective candidate drug for treatment of chemoresistant polyploid tumors.

[The CD133 polyclonal antibody generation and cancer stem cells identification].

OBJECTIVE: To generate the cancer stem cells (CSCs) specific protein CD133 polyclonal antibody for the study of the biological characteristics of CSCs in tumor tissues and CSCs screening for the mouse model. METHODS: The extracellular peptide of the human CD133 was injected into rabbits to generate polyclonal antibody which was used for glioblastoma(GBM) Western blot and immunohistochemistry. RESULTS: The CD133 antiserum we made could detect both overexpressed myc-CD133 and endogenous CD133 efficiently by Western blot. Immunohistochemistry indicated that the CD133 polyclonal antibody can label CSCs in GBM sections. CONCLUSION: High efficient and specific CD133 antibody was generated successfully and could be used to label CSCs in tumor sections and screen CSCs for the mouse model.

Hypothalamic neuropeptides, not leptin sensitivity, contributes to the hyperphagia in lactating Brandt's voles, Lasiopodomys brandtii.

Both pregnancy and lactation are associated with hyperphagia, and circulating leptin levels are elevated during pregnancy but decreased during lactation in Brandt's voles, Lasiopodomys brandtii. Previous findings suggest that impaired leptin sensitivity contributes to hyperphagia during pregnancy. The present study aimed to examine whether the decreased circulating leptin level and/or hypothalamic leptin sensitivity contributed to the hyperphagia during lactation in Brandt's voles. The serum leptin level and mRNA expression of the long form of the leptin receptor (Ob-Rb), suppressor-of-cytokine-signalling-3 (SOCS-3), neuropeptide Y (NPY), agouti-related protein (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus were examined on dioestrous, day 5, day 17 of lactation and day 27 (1 week after weaning) in Brandt's voles. Compared with controls, hypothalamic Ob-Rb and SOCS-3 mRNA expression was not significantly changed during lactation. The serum leptin level was significantly lower in lactating females than in the non-reproductive group. Hypothalamic NPY and AgRP mRNA expression significantly increased whereas POMC mRNA expression was significantly decreased during lactation compared with controls. However, there were no significant changes in hypothalamic CART mRNA expression. Food intake was positively correlated with NPY and AgRP mRNA expression but negatively correlated with POMC mRNA expression during lactation. These data suggest that hyperphagia during lactation was associated with low leptin levels, but not impaired leptin sensitivity, and that the hypothalamic neuropeptides NPY, AgRP and POMC are involved in mediating the role of leptin in food intake regulation in lactating Brandt's voles.

Food hoarding and associated neuronal activation in brain reward circuitry in Mongolian gerbils.

Mongolian gerbils (Meriones unguiculatus) display food hoarding and thus provide an opportunity to study the neuromechanisms underlying this behavior. In the present study, male gerbils exhibited a bimodal expression of food hoarding behavior-some displayed high levels of food hoarding whereas others virtually lacked this behavior under normal laboratory conditions with free access to food. Food hoarding was found to be associated with an increase in neuronal activation, indicated by Fos immunoreactive (ir) staining, in several brain areas including the nucleus accumbens, ventral tegmental area (VTA), and lateral hypothalamus. Food hoarding was also associated with increases in the number of cells labeled for tyrosine hydroxylase (TH-ir), the rate limiting enzyme for dopamine conversion, and the number of cells co-labeled for TH-ir/Fos-ir in the VTA, suggesting that dopamine in the brain reward circuitry may be involved in food hoarding. Further, we found that 22 h of food deprivation induced food hoarding in some, but not all, males that naturally did not display food hoarding. In these males, however, food hoarding did not increase TH-ir or TH-ir/Fos-ir expression in the VTA. Together, these data indicate that male Mongolian gerbils display diverse phenotypes of food hoarding behavior and that dopamine in the brain reward circuitry may be involved in the control of naturally occurring, but not food deprivation-induced, food hoarding.

Thermal physiology and energetics in male desert hamsters (Phodopus roborovskii) during cold acclimation.

The adjustments in thermal physiology and energetics were investigated in male desert hamsters (Phodopus roborovskii) which were acclimated to 5°C for 4 weeks. Mean core body temperature in cold acclimated animals decreased by 0.21°C compared with controls. Further analysis revealed that the decrease mainly occurred in the scotophase, while in the photophase core body temperature remained constant during the whole cold acclimation. Thermogenic capacity, represented by resting metabolic rate and nonshivering thermogenesis increased in cold acclimated hamsters from initial values of 1.38 ± 0.05 and 5.32 ± 0.30 to 1.77 ± 0.08 and 8.79 ± 0.31 mlO(2) g(-1) h(-1), respectively. After cold acclimation, desert hamsters maintained a relative stable body mass of 21.7 ± 0.1 g very similar to the controls kept at 23°C (21.8 ± 0.1 g). The mean values of food intake and digestible energy (metabolisable energy) in cold acclimated hamsters were 5.3 ± 0.1 g day(-1) and 76.3 ± 0.9 kJ day(-1) (74.8 ± 0.9), respectively, which were significantly elevated by 76.7 and 80.4% compared to that in control group. The apparent digestibility was 81.0 ± 0.3% in cold acclimated animals which was also higher than the 79.7 ± 0.2% observed in controls. This increase corresponded with adaptive adjustments in morphology of digestive tracts with 20.2 and 36.8% increases in total length and wet mass, respectively. Body fat mass and serum leptin levels in cold acclimated hamsters decreased by 40.7 and 67.1%, respectively. The wheel running turns and the onset of wheel running remained unchanged. Our study indicated that desert hamsters remained very active during cold acclimation and displayed adaptive changes in thermal physiology and energy metabolism, such as enhanced thermogenic and energy processing capacities.

Different physiological roles of serum leptin in the regulation of energy intake and thermogenesis between pregnancy and lactation in primiparous Brandt's voles (Lasiopodomys brandtii).

Reproduction, especially lactation, is associated with major metabolic adaptive changes. In this study, we investigated the metabolic changes and the roles of leptin during different periods of reproduction in primiparous Brandt's voles (Lasiopodomys brandtii). Energy intake, thermogenic capacity and serum leptin levels were examined in non-reproductive, mid pregnant, late pregnant, early lactating and peak lactating voles. Voles increased body mass by nearly 70% during late pregnancy compared to the non-breeding controls. The increase in body mass was mainly due to the increase in body fat mass which increased by 56%, and the growth of the reproductive tissues and digestive organs. Lactating voles decreased body fat by nearly 27% at peak lactation compared to the controls, and 53% compared to late pregnant voles. At the same time they increased food intake significantly. Uncoupling protein 1 (UCP1) content in brown adipose tissue (BAT) decreased significantly at peak lactation. Serum leptin increased significantly in the mid pregnancy, at a time when there was no increase in body fat, and remained at this high level in late pregnancy. Leptin levels decreased after parturition and reached a nadir at peak lactation. Serum leptin was negatively correlated with energy intake during lactation, but not during pregnancy. These data suggest that Brandt's voles adjust energy intake, thermogenic capacity and body reserves to match the high energy demands for reproduction. Hyperleptinemia, without decreased energy intake suggests a state of leptin resistance during pregnancy, and hypoleptinemia during lactation might act as a signal to stimulate energy intake.