Transcriptomics and Metabolomics Unveil the Neuroprotection Mechanism of AnGong NiuHuang (AGNH) Pill Against Ischaemic Stroke Injury.

As a famous prescription in China, AnGong NiuHuang (AGNH) pill exerts good neuroprotection for ischaemic stroke (IS), but its mechanism is still unclear. In this study, the neuroprotection of AGNH was evaluated in the rat IS model which were established with the surgery of middle cerebral artery occlusion (MCAO), and the potential mechanism was elucidated by transcriptomic analysis and metabolomic analysis. AGNH treatment obviously decreased the infarct volume and Zea-Longa 5-point neurological deficit scores, improved the survival percentage of rats, regional cerebral blood flow (rCBF), and rat activity distance and activity time. Transcriptomics showed that AGNH exerted its anti-inflammatory effects by affecting the regulatory network including Tyrobp, Syk, Tlr2, Myd88 and Ccl2 as the core. Integrating transcriptomics and metabolomics identified 8 key metabolites regulated by AGNH, including L-histidine, L-serine, L-alanine, fumaric acid, malic acid, and N-(L-arginino) succinate, 1-pyrroline-4-hydroxy-2-carboxylate and 1-methylhistamine in the rats with IS. Additionally, AGNH obviously reduced Tyrobp, Syk, Tlr2, Myd88 and Ccl2 at both the mRNA and protein levels, decreased IL-1ß, KC-GRO, IL-13, TNF-α, cleaved caspase 3 and p65 nucleus translocation, but increased IκBα expression. Network pharmacology analysis showed that quercetin, beta-sitosterol, baicalein, naringenin, acacetin, berberine and palmatine may play an important role in protecting against IS. Taken together, this study reveals that AGNH reduced neuroinflammation and protected against IS by inhibiting Tyrobp/Syk and Tlr2/Myd88, as well as NF-κB signalling pathway and regulating multiple metabolites.

COPD stage detection: leveraging the auto-metric graph neural network with inspiratory and expiratory chest CT images.

Chronic obstructive pulmonary disease (COPD) is a common lung disease that can lead to restricted airflow and respiratory problems, causing a significant health, economic, and social burden. Detecting the COPD stage can provide a timely warning for prompt intervention in COPD patients. However, existing methods based on inspiratory (IN) and expiratory (EX) chest CT images are not sufficiently accurate and efficient in COPD stage detection. The lung region images are autonomously segmented from IN and EX chest CT images to extract the 1 , 781 × 2 lung radiomics and 13 , 824 × 2 3D CNN features. Furthermore, a strategy for concatenating and selecting features was employed in COPD stage detection based on radiomics and 3D CNN features. Finally, we combine all the radiomics, 3D CNN features, and factor risks (age, gender, and smoking history) to detect the COPD stage based on the Auto-Metric Graph Neural Network (AMGNN). The AMGNN with radiomics and 3D CNN features achieves the best performance at 89.7 % of accuracy, 90.9 % of precision, 89.5 % of F1-score, and 95.8 % of AUC compared to six classic machine learning (ML) classifiers. Our proposed approach demonstrates high accuracy in detecting the stage of COPD using both IN and EX chest CT images. This method can potentially establish an efficient diagnostic tool for patients with COPD. Additionally, we have identified radiomics and 3D CNN as more appropriate biomarkers than Parametric Response Mapping (PRM). Moreover, our findings indicate that expiration yields better results than inspiration in detecting the stage of COPD.

Immunomodulatory Function of Pien Tze Huang in T Cell-Mediated Anti-tumor Activity against B16-F10, MC38 and Hep1-6 Tumor Models.

OBJECTIVE: To investigate the anti-tumor effects of Pien Tze Huang (PZH) in mouse models of B16-F10 melanoma, MC38 colorectal cancer, Hep1-6 hepatocellular carcinoma and chemically induced hepatocellular carcinoma model. METHODS: Various tumor models, including B16-F10, MC38 and Hep1-6 tumor hypodermic inoculation models, B16-F10 and Hep1-6 pulmonary metastasis models, Hep1-6 orthotopic implantation model, and chemically induced hepatocellular carcinoma model, were utilized to evaluate the anti-tumor function of PZH. Tumor growth was assessed by measuring tumor size and weight of solid tumors isolated from C57BL/6 mice. For cell proliferation and death of tumor cells in vitro, as well as T cell activation markers, cytokine production and immune checkpoints analysis, single-cell suspensions were prepared from mouse spleen, lymph nodes, and tumors after PZH treatment. RESULTS: PZH demonstrated significant therapeutic efficacy in inhibiting tumor growth (P<0.01). Treatment with PZH resulted in a reduction in tumor size in subcutaneous MC38 colon adenocarcinoma and B16-F10 melanoma models, and decreased pulmonary metastasis of B16-F10 melanoma and Hep1-6 hepatoma (P<0.01). However, in vitro experiments showed that PZH only had slight impact on the cell proliferation and survival of tumor cells (P>0.05). Nevertheless, PZH exhibited a remarkable ability to enhance T cell activation and the production of interferon gamma, tumor necrosis factor alpha, and interleukin 2 in CD4+ T cells in vitro (P<0.01 or P<0.05). Importantly, PZH substantially inhibited T cell exhaustion and boosted cytokine production by tumor-infiltrating CD8+ T cells (P<0.01 or P<0.05). CONCLUSION: This study has confirmed a novel immunomodulatory function of PZH in T cell-mediated anti-tumor immunity, indicating that PZH holds promise as a potential therapeutic agent for cancer treatment.

Evaluation and mechanism study of Pien Tze Huang against EV-A71 infection.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management.

Pien Tze Huang regulates phosphorylation of metabolic enzymes in mice of hepatocellular carcinoma.

The Chinese medicine formula Pien Tze Huang (PZH) has been applied to the treatment of various diseases, the reported anti-tumor mechanisms included regulation of inflammation-associated cytokine secretion and cancer growth pathways. However, the potential influence of PZH on tumor metabolism remains unclear. This study aimed to investigate the global effect of PZH on hepatocellular carcinoma (HCC) compared with the anti-tumor agent sorafenib based on tandem mass tag (TMT) label proteomic and phosphoproteomic analysis in addition to parallel reaction monitoring (PRM) verification. It was observed that PZH could inhibit tumor weight by 59-69% in different concentrations. TMT proteomic studies indicated that fructose/mannose metabolism and glucagon signaling pathway in PZH group, and arachidonic acid metabolism and PPAR signaling pathway in sorafenib group, were significantly enriched, while glycolysis/gluconeogenesis pathway was found to be enriched remarkably both in PZH and sorafenib groups in TMT phosphoproteomic study. PRM verification further indicated that both PZH and sorafenib could down-regulate phosphorylations of the glycolytic enzymes phosphofructokinases 1, fructose-bisphosphate Aldolase A, phosphoglycerate mutase 2 and lactate dehydrogenase A chain, while phosphorylations of long chain fatty acid CoA ligase in fatty acid activation and acetyl-coenzyme A synthetase in glycolysis were significantly inhibited by PZH and sorafenib, respectively. This study proposed that PZH shared a similar anti-tumor mechanism of metabolic regulation to sorafenib, but differed in the regulation of some metabolic nodes. This is the first time to uncover the relationship between the anti-tumor effect of PZH and metabolic related enzymes, which distinguished from the known mechanisms of PZH. These data provided the potential molecular basis for PZH acting as a therapeutic drug for HCC, and offered cues of manipulation on Warburg effect under the treatment of PZH.

Partial recovery of the left DLPFC-right insula circuit with reduced craving in abstinent heroin users: a longitudinal study.

The phenomenon of brain recovery after long-term abstinence has been reported in substance use disorders. However, few longitudinal studies have been conducted to observe the potential recovery in heroin users, and little is known about the neural mechanism underlying the decreased craving after prolonged abstinence. The 8-month longitudinal study was carried out in 29 heroin users and 30 healthy controls. By choosing the L_DLPFC, which was activated by the heroin cue as the seeding region, different brain connection patterns were compared between healthy controls and heroin users by using Granger causality analysis (GCA) at baseline. Then, a paired t test was employed to detect the potential recovery of L_DLPFC circuits after prolonged abstinence. The visual analog scale (VAS) and trail-making test-A (TMT-A) were adopted to investigate craving and cognitive control impairment, respectively. The neuroimaging changes were then correlated with behavioral improvements. Similar analyses were applied for the mirrored right DLPFC to verify the lateralization hypothesis of the DLPFC in addiction. In the longitudinal study, enhanced GCA coefficients were observed in the L_DLPFC-R_insula circuit of heroin users after long-term abstinence and were associated with craving score changes. At baseline, decreased GCA coefficients from the left DLPFC to the bilateral SMA and right putamen, together with the reduced GCA strength from the bilateral OFC to the left DLPFC, were found between HUs and HCs. Our findings extended the brain recovery phenomenon into the field of heroin and suggested that the increased regulation of the L_DLPFC over the insula after prolonged abstinence was important for craving inhibition.

Recovery of superior frontal gyrus cortical thickness and resting-state functional connectivity in abstinent heroin users after 8 months of follow-up.

Compared with healthy controls, heroin users (HUs) show evidence of structural and functional brain alterations. However, little is known about the possibility of brain recovery after protracted heroin abstinence. The purpose of this study was to investigate whether brain recovery is possible after protracted abstinence in HUs. A total of 108 subjects with heroin addiction completed structural and functional scans, and 61 of those subjects completed 8-month follow-up scans. Resting-state data and 3D-T1 MR images were collected for all participants, first at baseline and again after 8 months. Cognitive function and craving were measured by the Trail Making Test-A (TMT-A) and Visual Analog Scale for Craving, respectively. The cortical thickness and resting-state functional connectivity (RSFC) differences were then analyzed and compared between baseline and follow-up, and correlations were obtained between neuroimaging and behavioral changes. HUs demonstrated improved cognition (shorter TMT-A time) and reduced craving at the follow-up (HU2) relative to baseline (HU1), and the cortical thickness in the bilateral superior frontal gyrus (SFG) was significantly greater at HU2 than at HU1. Additionally, the RSFC of the left SFG with the inferior frontal gyrus (IFG), insula, and nucleus accumbens and that of the right SFG with the IFG, insula and orbitofrontal cortex (OFC) were increased at HU2. The changes in TMT-A time were negatively correlated with the RSFC changes between the left SFG and the bilateral IFG, the bilateral caudate, and the right insula. The changes in craving were negatively correlated with the RSFC changes between the left OFC and the bilateral SFG. Our results demonstrated that impaired frontal-limbic neurocircuitry can be partially restored, which might enable improved cognition as well as reduced craving in substance-abusing individuals. We provided novel scientific evidence for the partial recovery of brain circuits implicated in cognition and craving after protracted abstinence.

[Effect of Pien Tze Huang against EV71].

This study aims to investigate the inhibitory effect of Pien Tze Huang(PZH) on enterovirus 71(EV71). To be speci-fic, chemiluminescence method was adopted to evaluate the toxicity of PZH to African green monkey kidney(Vero) cells and human rhabdomyosarcoma(RD) cells, and cytopathic effect(CPE) method to assess the inhibition on EV71-GFP reporter virus and EV71 C4 wild-type virus. The results showed that PZH had low cytotoxicity to Vero cells and RD cells, with the half-maximal cytotoxic concentration(CC_(50)) of about 0.691 3-0.879 2 mg·mL~(-1) for the two. In addition, PZH can effectively inhibit the replication of EV71 within the non-cytotoxic concentration range, and dose-dependently alleviate the cytopathic changes caused by virus infection, with the half-maximal effective concentration(EC_(50)) of 0.009 2-0.106 3 mg·mL~(-1). On the basis of the above results, the green fluorescent protein(GFP), indirect immunofluorescence assay(IFA), and median tissue culture infective dose(TCID_(50)) were employed to assess and verify the anti-EV71-GFP and anti-EV71 C4 activity of PZH. The results demonstrated that PZH can dose-dependently lower the expression of GFP by EV71-GFP and structural protein VP-1 by EV71 C4 and decrease the production of progeny infectious viruses. The EC_(50) of PZH for EV71-GFP and EV71 C4 was about 0.006 0-0.006 2 mg·mL~(-1) and 0.006 6-0.025 6 mg·mL~(-1), respectively. This study suggested that PZH may exert antiviral activity by acting on EV71 and interfering with the expression of VP-1. At the moment, there is still a lack of specific anti-EV71 drugs. This study proposed a new idea for the symptomatic treatment of EV71 infections such as hand-foot-mouth disease and verified an effective drug for the treatment of EV71 infections.

Traditional Chinese Medicine Pien-Tze-Huang Inhibits Colorectal Cancer Growth and Immune Evasion by Reducing ß-catenin Transcriptional Activity and PD-L1 Expression.

Pien Tze Huang (PZH) is a valuable traditional Chinese medicine, which has a variety of biological activities such as clearing heat-toxin, resolving blood stasis, detoxifying, relieving pain, and anti-inflammation. PZH has a partial role in suppressing the progression of CRC, while the underlying mechanism is a pending mystery; especially whether PZH mediates the immune escape of CRC remains unclear. Our study reported that PZH suppressed the proliferative activity of CRC by inhibiting Wnt/ß-catenin signaling to down-regulate the expression of PCNA and Cyclin D1. In addition, PZH suppressed the immune escape of CRC and elevated the infiltration of CD8+ T cells in tumor tissues, which depends on the suppression of PD-L1 levels via inhibiting IFNGR1-JAK1-STAT3-IRF1 signaling. More importantly, PZH pharmacologically elevated the antitumor efficacy of anti-PD-1/PD-L1 immunotherapy as demonstrated by slower tumor growth, higher infiltration and function of CD8+ T cells in the combination of PZH and PD-1/PD-L1 antibody compared with monotherapy with either agent. These results demonstrate that PZH has the potential role in inhibiting CRC proliferation and immune evasion, especially the synergistic enhancement effect of PZH on immunotherapy.

Reduced midbrain functional connectivity and recovery in abstinent heroin users.

Dopaminergic pathways from the midbrain to striatum as well as cortex are involved in addiction. However, the alternations of these pathways and whether the recoveries of aberrant circuits would be detected after prolonged abstinence in heroin users are rarely known. The resting-state functional connectivity (RSFC) patterns of midbrain (i.e., the ventral tegmental area (VTA) and substantia nigra (SN)) were compared between 40 abstinent heroin users with opioid use disorder (HUs) and 35 healthy controls (HCs). Then, we tested the functional recovery hypothesis by both cross-sectional and longitudinal design. For cross-sectional design, HUs were separated into short-term abstainers (STs) (3-15 days) and long-term abstainers (LTs) (>15 days). With regard to longitudinal design, 22 subjects among HUs were followed up for 10 months. A sandwich estimator method was used to analyze the differences between baseline HUs and follow-up HUs. HUs showed lower RSFC between midbrain and several cortical areas (medial orbitofrontal cortex (mOFC) and anterior cingulate cortex) compared with HCs. Besides, lower RSFC of VTA-right nucleus accumbens circuit as well as right SN- caudate circuit was also found in HUs. The enhanced RSFC value of VTA-left mOFC circuit was observed in LTs, compared with STs. Additionally, longitudinal design also revealed the increased RSFC values of the midbrain with frontal cortex after 10 months prolonged abstinence. We revealed abnormal functional organizations of midbrain-striato and midbrain-cortical circuits in HUs. More importantly, partially recovery of these dysfunctions can be found after long-term abstinence.

Preclinical Characterization of GLS-010 (Zimberelimab), a Novel Fully Human Anti-PD-1 Therapeutic Monoclonal Antibody for Cancer.

BACKGROUND: Zimberelimab (GLS-010) is a novel fully human monoclonal immunoglobulin G4 (IgG4) against the programmed cell death-1 (PD-1) receptor. AIM: To evaluate the affinity, competitive blocking capability, T cell activation effect, cytotoxic effector functions by Fc, preliminary anti-tumor activity, and pharmacokinetics of GLS-010. METHODS: The affinity of GLS-010 to PD-1 and the ability of GLS-010 to block the PD-L1/2 to PD-1 interaction on the cell surface were measured. An allogeneic mixed lymphocyte reaction was conducted to evaluate the inhibitory effect of GLS-010 on Tregs and stimulatory effect on T cell proliferation and activation. Pharmacodynamics and pharmacokinetics were evaluated in tumor-bearing mice and cynomolgus monkeys, respectively. RESULTS: The equilibrium dissociation constant (KD) for the association between GLS-010 and PD-1 was 1.75×10-10 M. GLS-010 could effectively block the binding of PD-L1/2 to PD-1. GLS-010 showed statistically significant anti-tumor effects in the MC38 model in human PD-1 knock-in mice. The RO rate on in the low-, moderate-, and high-dose groups were 64.50%-48.53% in CD3+T, 58.87%-40.12% in CD8+T, and 66.26%-49.07% in CD4+T, respectively. With the increasing dose from 2 mg/kg to 18 mg/kg, the systemic exposure level of GLS-010 (AUC0-last) and C0 increased proportionally, while the proportion of AUC0-last was higher than the proportion of the increase in the dose. CONCLUSIONS: As a fully human anti-PD-1 monoclonal antibody, GLS-010 has a high affinity to PD-1 and shows potent anti-tumor effects in vivo and in vitro. The results support that GLS-010 could be investigated in clinical trials in tumor patients.

Brain responses to drug cues predict craving changes in abstinent heroin users: A preliminary study.

BACKGROUND: Loss of control over drug intake occurring in drug addiction is believed to result from disruption of reward circuits, including reduced responsiveness to natural rewards (e.g., monetary, sex) and heightened responsiveness to drug reward. Yet few studies have assessed reward deficiency and related brain responses in abstinent heroin users with opioid use disorder, and less is known whether the brain responses can predict cue-induced craving changes following by prolonged abstinence. METHOD: 31 heroin users (age: 44.13±7.68 years, male: 18 (58%), duration of abstinence: 85.2 ± 52.5 days) were enrolled at a mandatory detoxification center. By employing a cue-reactivity paradigm including three types of cues (drug, sexual, neutral), brain regional activations and circuit-level functional coupling were extracted. Among the 31 heroin users, 15 were followed up longitudinally to assess cue induced craving changes in the ensuing 6 months. RESULTS: One way analysis of variance results showed that heroin users have differential brain activations to the three cues (neutral, drug and sexual) in the left dorsolateral prefrontal cortex (DLPFC), insula, orbiotofrontal cortex (OFC) and the bilateral thalamus. Drug cue induced greater activations in left DLPFC, insula and OFC compared to sexual cue. The psychophysiological interactions (PPI) analysis revealed negative couplings of the left DLPFC and the left OFC, bilateral thalamus, putamen in heroin users during drug cue exposure. In the 6-month follow-up study, both drug cue induced activation of the left DLPFC and the functional coupling of the left DLPFC-bilateral thalamus at baseline was correlated with craving reductions, which were not found for sexual cues. CONCLUSION: Our preliminary study provided novel evidence for the reward deficiency theory of opioid use disorder. Our findings also have clinical implications, as drug cue induced activation of the left DLPFC and functional coupling of left DLPFC-bilateral thalamus may be potential neuroimaging markers for craving changes during prolonged abstinence. Evidently, the findings in the current preliminary study should be confirmed by large sample size in the future.

Reduced thalamic resting-state functional connectivity and impaired cognition in acute abstinent heroin users.

As a critical component of cortico-striato-thalamo-cortical loop in addiction, our understanding of the thalamus in impaired cognition of heroin users (HU) has been limited. Due to the complex thalamic connection with cortical and subcortical regions, thalamus was divided into prefrontal (PFC), occipital (OC), premotor, primary motor, sensory, temporal, and posterior parietal association subregions according to white matter tractography. We adopted seven subregions of bilateral thalamus as regions of interest to systematically study the implications of distinct thalamic nuclei in acute abstinent HU. The volume and resting-state functional connectivity (RSFC) differences of the thalamus were investigated between age-, gender-, and alcohol-matched 37 HU and 33 healthy controls (HCs). Trail making test-A (TMT-A) was adopted to assess cognitive function deficits, which were then correlated with neuroimaging findings. Although no significant different volumes were found, HU group showed decreased RSFC between left PFC_thalamus and middle temporal gyrus as well as between left OC_thalamus and inferior frontal gyrus and supplementary motor area relative to HCs. Meanwhile, the higher TMT-A scores in HU were negatively correlated with PFC_thalamic RSFC with inferior temporal gyrus, fusiform, and precuneus. Craving scores were negatively correlated with OC_thalamic RSFC with accumbens, hippocampus, and insula. Opiate Withdrawal Scale scores were negatively correlated with left PFC/OC_thalamic RSFC with orbitofrontal cortex and medial PFC. We indicated two thalamus subregions separately involvement in cognitive control and craving to reveal the implications of thalamic subnucleus in pathology of acute abstinent HU.

Novel circuit biomarker of impulsivity and craving in male heroin-dependent individuals.

INTRODUCTION: The striatum mediates reward processing in addiction, and previous fMRI (functional Magnetic Resonance Imaging) studies have revealed abnormal striatofrontal functional connectivity in heroin addiction. However, little is known about whether there is abnormal structural connectivity of the striatal circuit in heroin addiction. This study investigated the structural connectivity of striatal circuits in abstinent heroin-dependent individuals (HDIs) without methadone treatment. METHODS: Forty-three (age: 38.8 ± 7.1) male HDIs and twenty-one (age: 42.4 ± 7.9) matched healthy controls underwent high-resolution T1 and whole-brain diffusion tensor imaging (64 directions) magnetic resonance imaging. Connectivity-based seed classification probabilistic tractography was used to detect the tract strengths of striatal circuits with 10 a priori target masks. Tract strengths were compared between groups and correlated with impulsivity behavior, evaluated using the Barratt Impulsivity Scale (BIS), and craving, measured on visual analogue scale (VAS). RESULTS: HDIs showed significantly weaker tract strength of the left striatum-medial orbitofrontal cortex (mOFC) (Bonferroni corrected, p < 0.05/20 = 0.0025) and significantly higher BIS total, attention, motor, and non-planning scores (Bonferroni corrected, p < 0.05/4 = 0.0125) than controls. In HDIs, negative correlations were observed between the left striatum- mOFC tract strengths and the BIS total, attention and non-planning scores (r1=-0.410, p1 = 0.005; r2=-0.432, p2 = 0.003; r3=-0.506, p3<0.001) and between the right striatum-posterior cingulate cortex (PCC) tract strengths and craving scores (r=-0.433, p = 0.009) in HDIs. CONCLUSION: HDIs displayed decreased structural connectivity of the striatum-mOFC circuit and higher impulsivity. Higher impulsive behavior was associated with decreased left striatal circuit connectivity. These findings suggest that the striatal circuit tract strengths might be a novel potential biomarker in heroin and, potentially, general opioid addiction.

Reaction of the Liver upon Long-Term Treatment of Fluoxetine and Atorvastatin Compared with Alcohol in a Mouse Model.

BACKGROUND: Alcoholism is known to cause liver toxicity and is extensively researched. On the other hand, stress, depression, and obesity are interrelated conditions with alcoholism, and their medications would affect the liver itself. In this study, we investigated the effects of the drugs fluoxetine and atorvastatin on the liver and compared with those of alcohol in a mouse model. METHODS: Comparisons of animals treated with the three drugs were carried out: serum aspartate transaminase (AST), alanine transaminase (ALT), and albumin were measured; liver tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta-1) levels were evaluated; proliferative cells were detected via immunohistochemistry (IHC) targeting on proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2); for apoptosis, IHC targeting on activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were employed; and histopathology was also documented in all groups. RESULTS: For ALT, AST, albumin, and liver TNF alpha, only the ethanol group surged to significantly higher levels. For TGF beta-1, both ethanol and atorvastatin groups reached a significantly higher level. PCNA and MCM2 showed increased proliferation in the livers of all three groups, with the ethanol group having the highest number of positive cells followed by atorvastatin and then the fluoxetine group. As for cell death, both ethanol and fluoxetine groups showed significantly more apoptosis than control in TUNEL and activated caspase-3, while in the atorvastatin group, activated caspase-3 positive cells increased significantly, but the increase in TUNEL-positive cells did not reach statistical significance.

RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking.

The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR.

A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

Small GTPase Rab40c associates with lipid droplets and modulates the biogenesis of lipid droplets.

The subcellular location and cell biological function of small GTPase Rab40c in mammalian cells have not been investigated in detail. In this study, we demonstrated that the exogenously expressed GFP-Rab40c associates with lipid droplets marked by neutral lipid specific dye Oil red or Nile red, but not with the Golgi or endosomal markers. Further examination demonstrated that Rab40c is also associated with ERGIC-53 containing structures, especially under the serum starvation condition. Rab40c is increasingly recruited to the surface of lipid droplets during lipid droplets formation and maturation in HepG2 cells. Rab40c knockdown moderately decreases the size of lipid droplets, suggesting that Rab40c is involved in the biogenesis of lipid droplets. Stimulation for adipocyte differentiation increases the expression of Rab40c in 3T3-L1 cells. Rab40c interacts with TIP47, and is appositionally associated with TIP47-labeled lipid droplets. In addition, over-expression of Rab40c causes the clustering of lipid droplets independent of its GTPase activity, but completely dependent of the intact SOCS box domain of Rab40c. In addition, Rab40c displayed self-interaction as well as interaction with TIP47 and the SOCS box is essential for its ability to induce clustering of lipid droplets. Our results suggest that Rab40c is a novel Rab protein associated with lipid droplets, and is likely involved in modulating the biogenesis of lipid droplets.

Rab7: roles in membrane trafficking and disease.

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.