Preparation of Pueraria lobata Root-Derived Exosome-Like Nanovesicles and Evaluation of Their Effects on Mitigating Alcoholic Intoxication and Promoting Alcohol Metabolism in Mice.

Purpose: Pueraria lobata (P. lobata), a dual-purpose food and medicine, displays limited efficacy in alcohol detoxification and liver protection, with previous research primarily focused on puerarin in its dried roots. In this study, we investigated the potential effects and mechanisms of fresh P. lobata root-derived exosome-like nanovesicles (P-ELNs) for mitigating alcoholic intoxication, promoting alcohol metabolism effects and protecting the liver in C57BL/6J mice. Methods: We isolated P-ELNs from fresh P. lobata root using differential centrifugation and characterized them via transmission electron microscopy, nanoscale particle sizing, ζ potential analysis, and biochemical assays. In Acute Alcoholism (AAI) mice pre-treated with P-ELNs, we evaluated their effects on the timing and duration of the loss of the righting reflex (LORR), liver alcohol metabolism enzymes activity, liver and serum alcohol content, and ferroptosis-related markers. Results: P-ELNs, enriched in proteins, lipids, and small RNAs, exhibited an ideal size (150.7 ± 82.8 nm) and negative surface charge (-31 mV). Pre-treatment with 10 mg/(kg.bw) P-ELNs in both male and female mice significantly prolonged ebriety time, shortened sobriety time, enhanced acetaldehyde dehydrogenase (ALDH) activity while concurrently inhibited alcohol dehydrogenase (ADH) activity, and reduced alcohol content in the liver and serum. Notably, P-ELNs demonstrated more efficacy compared to P-ELNs supernatant fluid (abundant puerarin content), suggesting alternative active components beyond puerarin. Additionally, P-ELNs prevented ferroptosis by inhibiting the reduction of glutathione peroxidase 4 (GPX4) and reduced glutathione (GSH), and suppressing acyl-CoA synthetase long-chain family member 4 (ACSL4) elevation, thereby mitigating pathological liver lipid accumulation. Conclusion: P-ELNs exhibit distinct exosomal characteristics and effectively alleviate alcoholic intoxication, improve alcohol metabolism, suppress ferroptosis, and protect the liver from alcoholic injury. Consequently, P-ELNs hold promise as a therapeutic agent for detoxification, sobriety promotion, and prevention of alcoholic liver injury.

Genomic structural variation contributes to evolved changes in gene expression in high-altitude Tibetan sheep.

Tibetan sheep were introduced to the Qinghai Tibet plateau roughly 3,000 B.P., making this species a good model for investigating genetic mechanisms of high-altitude adaptation over a relatively short timescale. Here, we characterize genomic structural variants (SVs) that distinguish Tibetan sheep from closely related, low-altitude Hu sheep, and we examine associated changes in tissue-specific gene expression. We document differentiation between the two sheep breeds in frequencies of SVs associated with genes involved in cardiac function and circulation. In Tibetan sheep, we identified high-frequency SVs in a total of 462 genes, including EPAS1, PAPSS2, and PTPRD. Single-cell RNA-Seq data and luciferase reporter assays revealed that the SVs had cis-acting effects on the expression levels of these three genes in specific tissues and cell types. In Tibetan sheep, we identified a high-frequency chromosomal inversion that exhibited modified chromatin architectures relative to the noninverted allele that predominates in Hu sheep. The inversion harbors several genes with altered expression patterns related to heart protection, brown adipocyte proliferation, angiogenesis, and DNA repair. These findings indicate that SVs represent an important source of genetic variation in gene expression and may have contributed to high-altitude adaptation in Tibetan sheep.

Serum ß-klotho is a potential biomarker for the progression of hepatitis B virus-related liver diseases.

INTRODUCTION: Hepatitis B virus (HBV) infection is a global epidemic that can lead to several liver diseases, seriously affecting people's health. This study aimed to investigate the clinical potential of serum ß-klotho (KLB) as a promising biomarker in HBV-related liver diseases. METHODOLOGY: This study enrolled 30 patients with chronic hepatitis B (CHB), 35 with HBV-related cirrhosis, 66 with HBV-related hepatocellular carcinoma (HCC), and 48 healthy individuals. ELISA measured the levels of serum KLB in the four groups. We then compared the differences in serum KLB levels among the groups and analyzed the relationship between serum KLB and routine clinical parameters. RESULTS: The concentrations of serum KLB levels were increased sequentially among the healthy subjects, the HBV-related CHB group, the HBV-related cirrhosis group, and the HBV-related HCC group (p < 0.05). Expression of KLB was positively correlated with alpha-fetoprotein (AFP), total bilirubin, direct bilirubin, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl-transferase, alkaline phosphatase, total bile acid, serum markers for liver fibrosis, ascites, cirrhosis, splenomegaly, and model for end-stage liver disease sodium, while negatively correlated with platelet count, albumin, and prothrombin activity (p < 0.05). In addition, serum KLB has better sensitivity in diagnosing HCC than AFP, and serum KLB combined with AFP has higher sensitivity and specificity than AFP alone in diagnosing HCC. CONCLUSIONS: Serum KLB level is associated with the severity of HBV-related liver diseases and has important diagnostic value for HCC. Therefore, it could be a predictive biomarker for monitoring disease progression.

Genomic structural variation is associated with hypoxia adaptation in high-altitude zokors.

Zokors, an Asiatic group of subterranean rodents, originated in lowlands and colonized high-elevational zones following the uplift of the Qinghai-Tibet plateau about 3.6 million years ago. Zokors live at high elevation in subterranean burrows and experience hypobaric hypoxia, including both hypoxia (low oxygen concentration) and hypercapnia (elevated partial pressure of CO2). Here we report a genomic analysis of six zokor species (genus Eospalax) with different elevational ranges to identify structural variants (deletions and inversions) that may have contributed to high-elevation adaptation. Based on an assembly of a chromosome-level genome of the high-elevation species, Eospalax baileyi, we identified 18 large inversions that distinguished this species from congeners native to lower elevations. Small-scale structural variants in the introns of EGLN1, HIF1A, HSF1 and SFTPD of E. baileyi were associated with the upregulated expression of those genes. A rearrangement on chromosome 1 was associated with altered chromatin accessibility, leading to modified gene expression profiles of key genes involved in the physiological response to hypoxia. Multigene families that underwent copy-number expansions in E. baileyi were enriched for autophagy, HIF1 signalling and immune response. E. baileyi show a significantly larger lung mass than those of other Eospalax species. These findings highlight the key role of structural variants underlying hypoxia adaptation of high-elevation species in Eospalax.

Influences of Divalent Ions in Natural Seawater/River Water on Nanofluidic Osmotic Energy Generation.

Besides the dominant NaCl, natural seawater/river water contains trace multivalent ions, which can provide effective screening of surface charges. Here, in both negatively and positively charged nanopores, influences from divalent ions as counterions and co-ions have been investigated with respect to the performance of osmotic energy conversion (OEC) under natural salt gradients. As counterions, trace Ca2+ ions can suppress the electric power and conversion efficiency significantly. The reduced OEC performance is due to the bivalence and low diffusion coefficient of Ca2+ ions instead of the uphill transport of divalent ions discovered in the previous work. Effectively screened charged surfaces by Ca2+ ions induce an enhanced diffusion of Cl- ions which simultaneously decreases the net ion penetration and ionic selectivity of the nanopore. As co-ions, Ca2+ ions have weak effects on the OEC performance. The promotion from charged exterior surfaces in OEC processes for ultrashort nanopores is also studied, with an effective region of ∼200 nm in width beyond pore boundaries independent of the presence of Ca2+ ions. Our results shed light on the physical details of the nanofluidic OEC process under natural seawater/river water conditions, which can provide a useful guide for high-performance osmotic energy harvesting.

Effective Charged Exterior Surfaces for Enhanced Ionic Diffusion through Nanopores under Salt Gradients.

High-performance osmotic energy conversion requires both large ionic throughput and high ionic selectivity, which can be significantly promoted by exterior surface charges simultaneously, especially for short nanopores. Here, we investigate the enhancement of ionic diffusion by charged exterior surfaces under various conditions and explore corresponding effective charged areas. From simulations, ionic diffusion is promoted more significantly by exterior surface charges through nanopores with a shorter length, wider diameter, and larger surface charge density or under higher salt gradients. Effective widths of the charged ring regions near nanopores are reversely proportional to the pore length and linearly dependent on the pore diameter, salt gradient, and surface charge density. Due to the important role of effective charged areas in the propagation of ionic diffusion through single nanopores to cases with porous membranes, our results may provide useful guidance to the design and fabrication of porous membranes for practical high-performance osmotic energy harvesting.

Genomic insights into zokors' phylogeny and speciation in China.

The phylogeny and speciation of subterranean zokors in China are unclear, as previous studies on morphology and limited molecular markers have generated conflicting results. This study unraveled the complex evolutionary history of eight zokor species in China based on de novo assembly at chromosome level and whole-genome sequencing of 23 populations. We found extensive phylogenetic discordances between nuclear and mitochondrial phylogenies, and different coalescent phylogenies, which could be explained by introgression and incomplete lineage sorting (ILS). The recent Qinghai-Tibet Plateau uplift (∼3.60 million y ago; Mya) drove Eospalax to speciate into clade A and clade B (∼3.22 Mya), and discordant phylogenies in this node were mainly attributed to introgression rather than ILS. Clade A rapidly diverged into three lineages due to geographical isolation and glaciation, while glaciation and C4 plant expansion contributed to the speciation of clade B. ILS contributed to the discordances of two rapidly radiated nodes rather than introgression. The effective population sizes (Ne's) of all the species of Eospalax were affected by three glaciations. Ancient polymorphisms and divergence hitchhiking contribute to genomic islands of all the species pairs. Positively selected genes putatively related to specific inhabitation adaptations were identified, such as heart development, neurogenesis, DNA repair, and immune response. Climate, geological tectonism, and C4 vegetation shaped the adaptation and speciation of zokors in China.

Sympatric speciation of the spiny mouse from Evolution Canyon in Israel substantiated genomically and methylomically.

SignificanceWhether sympatric speciation (SS) is rare or common is still debated. Two populations of the spiny mouse, Acomys cahirinus, from Evolution Canyon I (EC I) in Israel have been depicted earlier as speciating sympatrically by molecular markers and transcriptome. Here, we investigated SS both genomically and methylomically, demonstrating that the opposite populations of spiny mice are sister taxa and split from the common ancestor around 20,000 years ago without an allopatric history. Mate choice, olfactory receptors, and speciation genes contributed to prezygotic/postzygotic reproductive isolation. The two populations showed different methylation patterns, facilitating adaptation to their local environment. They cope with abiotic and biotic stresses, due to high solar interslope radiation differences. We conclude that our new genomic and methylomic data substantiated SS.

A new species of the genus Daplasa Moore from China based on morphological and molecular evidence (Lepidoptera: Erebidae: Lymantriinae).

A new species, Daplasa medoga sp. nov., is described from Tibet, China. The new species can be distinguished morphologically from all other known Daplasa species by its male genitalia with a sagittate uncus and distally broadly crotched valvae. The validity of new species is well supported by the molecular phylogenetic analyses of one mitochondrial gene (COI) and two nuclear genes (EF-1α RPS5), with a total length of 2,324 bp.

Hypoxia-Induced Adipose Lipolysis Requires Fibroblast Growth Factor 21.

Fibroblast growth factor 21 (FGF21) is a recently discovered hepatokine that regulates lipid and glucose metabolism and is upregulated in response to numerous physiological and pathological stimuli. Herein, we demonstrate that both physical and chemical hypoxia increase the systemic and hepatic expression of FGF21 in mice; by contrast, hypoxia induces a reduction of FGF21 expression in hepatocytes, indicating that hypoxia-induced FGF21 expression is differentially regulated in intact animals and in hepatocytes. Furthermore, we demonstrate that hypoxia treatment increases hormone-sensitive lipase-mediated adipose tissue lipolysis in mice, which is reduced in Fgf21 knockout mice, thereby implying that FGF21 plays a critical role in hypoxia-related adipose lipolysis. Adipose tissue lipolysis causes an increase in the amount of circulating free fatty acids, which leads to the activation of peroxisome proliferators-activated receptor alpha and an increased expression of FGF21 in hepatocytes. We further show that hypoxia-induced elevation of reactive oxygen species, but not the hypoxia-inducible factor, is responsible for the lipolysis and FGF21 expression. In conclusion, our data clearly demonstrate that FGF21 plays a critical role in hypoxia-induced adipose lipolysis, which induces hepatic expression of FGF21. Clarification of hypoxia-regulated FGF21 regulation will enhance our understanding of the pathophysiology of hypoxia-related diseases, such as sleep disorders and metabolic diseases.

A single-pair method to screen Rickettsia-infected and uninfected whitefly Bemisia tabaci populations.

Bacterial endosymbionts such as Rickettsia and Wolbachia play prominent roles in the development and behaviour of their insect hosts, such as whiteflies, aphids, psyllids and mealybugs. Accumulating studies have emphasized the importance of establishing experimental insect populations that are either lacking or bearing certain species of endosymbionts, because they are the basis in which to reveal the biological role of individual symbionts. In this study, using Rickettsia as an example, we explored a "single-pair screening" method to establish Rickettsia infected and uninfected populations of whitefly Bemisia tabaci MEAM1 for further experimental use. The original host population had a relatively low infection rate of Rickettsia (< 35%). When B. tabaci adults newly emerged, unmated males and females were randomly selected, and released into a leaf cage that covered a healthy plant leaf in order to oviposit F1 generation eggs. Following 6 days of oviposition, the parents were recaptured and used for PCR detection. The F1 progeny, for which parents were either Rickettsia positive or negative, were used to produce the F2 generation, and similarly in turn for the F3, F4 and F5 generations respectively; if the infection status of Rickettsia was consistent in the F1 to F5 generations, then the populations can be used as Rickettsia positive or negative lines for further experiments. In addition, our phylogenetic analyses revealed that Rickettsia has high fidelity during the maternal transmission in different generations.

Morphological and molecular evidence for a new species of the genus Panolis Hübner, [1821] (Lepidoptera: Noctuidae).

A new species of the genus Panolis Hübner, [1821], P. xundian sp. nov., is described and illustrated from southwestern China. It is well-defined morphologically by the male genitalia, with a well-developed pollex at the distal terminal of the sacculus and a broad, ventrally concave cucullus, the female corpus bursae with four long signum-stripes. Based on a 658 bp segment of the mitochondrial cytochrome c oxidase subunit I gene, we report the pairwise genetic distance of 2.5% from its allied species P. exquisita Draudt, 1950. Molecular phylogenetic analyses using three genes (2189 bp in total length) indicate that the new species belongs to the P. exquisita species group.

Fast, sensitive, selective and reversible fluorescence monitoring of TATP in a vapor phase.

The development of sensors for the detection of triacetone triperoxide (TATP) has attracted great attention. Here, we constructed a low-cost, portable, reusable, visible paper-based fluorescent sensor for the sensitive detection of TATP via vapor sampling. Under optimized conditions, the fluorescent film showed a high sensitivity to TATP with a detection limit of lower than 0.5 µg mL-1 in air. The linear range of the response is from 0.5 to 8.0 µg mL-1. In addition, the paper-based sensor exhibited high selectivity to TATP. The presence of potential interferents showed little effect on sensing. Moreover, sensing is fully reversible. Fortunately, the test can also be conducted in a visualized way.

Infection dynamics of endosymbionts reveal three novel localization patterns of Rickettsia during the development of whitefly Bemisia tabaci.

The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a severe agricultural pest that harbors at least seven endosymbionts. Many important aspects of the symbiosis mechanism between these bacterial endosymbionts and their hosts are poorly understood, such as endosymbiont proliferation dynamics, spatial distribution and titer regulation during host development. In this study, infection by bacterial endosymbionts in the whitefly B. tabaci Middle East-Asia Minor-1 (MEAM1, formerly B biotype) South China population, their infection titers in various stages of whitefly host development and their spatial localization were investigated. Results revealed that the MEAM1 B. tabaci harbors the primary symbiont Portiera and secondary symbionts Rickettsia and Hamiltonella. The titers of these three endosymbionts increased with the development of their B. tabaci host. Significant proliferation of Portiera and Hamiltonella mainly occurred during the second to fourth instar nymphal stages, while Rickettsia proliferated mainly during adult eclosion. Fluorescence in situ hybridization analysis of B. tabaci adults revealed three novel infection patterns of Rickettsia: assemblage in the bacteriocytes that scattered through the entire abdomen of the female host, localization in wax glands and localization in the colleterial gland. These novel infection patterns may help to uncover the function of Rickettsia in its insect hosts.

Wolbachia Has Two Different Localization Patterns in Whitefly Bemisia tabaci AsiaII7 Species.

The whitefly Bemisia tabaci is a cosmopolitan insect species complex that harbors the obligate primary symbiont Portiera aleyrodidarum and several facultative secondary symbionts including Wolbachia, which have diverse influences on the host biology. Here, for the first time, we revealed two different localization patterns of Wolbachia present in the immature and adult stages of B. tabaci AsiaII7 cryptic species. In the confined pattern, Wolbachia was restricted to the bacteriocytes, while in the scattered pattern Wolbachia localized in the bacteriocytes, haemolymph and other organs simultaneously. Our results further indicated that, the proportion of B. tabaci AsiaII7 individuals with scattered Wolbachia were significantly lower than that of confined Wolbachia, and the distribution patterns of Wolbachia were not associated with the developmental stage or sex of whitefly host. This study will provide a new insight into the various transmission routes of Wolbachia in different whitefly species.

Changes in glucose-6-phosphate dehydrogenase expression results in altered behavior of HBV-associated liver cancer cells.

Hepatocellular carcinoma (HCC) is regarded as a major global health care issue, and chronic hepatitis B virus (HBV) infection is considered to be involved in pathogenesis of HCC. To increase knowledge of HCC pathogenesis, as well as discover potential novel molecules for anti-cancer therapy, mass spectrometry and isobaric tag for relative and absolute quantitation (iTARQ) were employed. The differences between nine HBV-related HCC and adjacent non-HCC tissue specimens were studied. In total, 222 proteins were analyzed for differential expression in the two types of samples. Among these proteins, several were further confirmed by immunohistochemical, immunoblotting, and real-time RT-PCR analysis. RNA interference induced downregulation of glucose-6-phosphate dehydrogenase (G6PD) and decreased HBV replication by fivefold by the IFN pathway. Decreased G6PD expression resulted in decreased hepatoma cell migration and invasion in cell culture. In summary, the investigation provides new information on pathogenesis of HBV infection and suggests G6PD as a novel anti-HCC target. G6PD suppression may contribute to treatment strategies for inhibiting tumor progression.

Keratin 8 is involved in hepatitis B virus replication.

Hepatitis B virus (HBV) infection can result in fatal liver diseases, including cirrhosis or liver failure, and its replication and pathogenesis depend on the critical interplay between viral and host factors. This study investigated HBV replication-related host proteins and the effect of candidate proteins on HBV replication. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to measure HBV replication-related proteins in HepG2 cells and HepG2.2.15 cells. KRT8 was up-regulated in HepG2.2.15 cells but not in HepG2 cells, and KRT8 was overexpressed in an HBV-infected patient's liver tissue. This result suggested that KRT8 is involved in HBV replication. To further clarify the relationship between KRT8 and HBV replication, KRT8 gene expression was inhibited by siRNA. The silencing of KRT8 mildly suppressed HBV replication. Moreover, overexpressed KRT8 significantly increased HBV replication, and the inhibition of HBV DNA did not suppress KRT8 expression. Thus, the host protein KRT8 is involved in the replication of HBV DNA, and it dramatically enhances HBV replication.

HSPB1 is an intracellular antiviral factor against hepatitis B virus.

Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans and it is considered a major global health problem. However, the mechanisms of HBV replication are complex and not yet fully understood. In this study, the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass-spectrophotometry (2D LC-MS/MS), a successfully exploited high-throughput proteomic technology. In total, 2,028 unique proteins were identified and 170 proteins were differentially expressed in HepG2.2.15 cells as compared with that in HepG2. Several differentially expressed proteins were further validated by Western blot and real-time quantitative reverse transcription-PCR. Furthermore, the association of HBV replication with heat shock protein B1, one of the highly expressed proteins in HepG2.2.15 cells, was verified. HSPB1 functions as a anti-viral protein during HBV infection by specifically inducing type interferon and some downstream antiviral effectors. This study is the first to report the application of iTRAQ technology to analyze the underlying mechanisms of HBV replication. Many of the differentially expressed proteins identified have not been linked to HBV replication before, and may provide valuable novel insights into HBV replication.

Proteomic investigation of 5-fluorouracil resistance in a human hepatocellular carcinoma cell line.

Multi-drug resistance (MDR) is a major obstacle towards a successful treatment of hepatocellular carcinoma (HCC). The mechanisms of MDR are intricate and have not been fully understood. Therefore, we employed a cell-line model consisting of the 5-fluorouracil (5-FU) resistant BEL7402/5-FU cell line and its parental BEL7402 cell line. Using relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, in total, 660 unique proteins were identified and 52 proteins showed to be differentially expressed in BEL7402/5-FU compared with BEL7402. Several differentially expressed proteins were further validated by Western blot and real-time quantitative RT-PCR analysis. Furthermore, the association of MDR with ANXA3, one of the highly expressed proteins in BEL7402/5-FU, was verified. Our study represents the first successful application of iTRAQ technology for MDR mechanisms analysis in HCC. Many of the differentially expressed proteins identified had not been linked to MDR in HCC before, which provide valuable information for further understanding of MDR.

Long-term nucleos(t)ide analogues therapy for adults with chronic hepatitis B reduces the risk of long-term complications: a meta-analysis.

BACKGROUND: The effect of antiviral therapy in chronic hepatitis B (CHB) on reducing the risk of long-term complications (LTCs) remains unclear so far. To study whether long-term nucleos(t)ide analogues therapy can reduce the risk of long-term complications. METHODS: We searched MEDLINE, EMBASE, OVID, the Cochrane Central Register of Controlled Trials. Relative risks (RRs) of long-term complications with or without treatment were studied. Also subgroup analyses including the status of drug-resistance, HBeAg and pre-existing compensated cirrhosis were done using relative risks of long-term complications either with or without treatment or among nucleos(t)ide analogues treatment groups. RESULTS: Six eligible studies (3644 patients in all) were included. Data showed the incidence of long-term complications in treatment groups was induced by 74%(RR:0.26, 95% CI: 0.15-0.47) compared with no treatment. Whether drug-resistant happened or not during the long-term therapy, the incidence of long-term complications was still significantly induced respectively by 45%(RR: 0.55,95%CI:0.40-0.76) and 78% (RR:0.22, 95%CI: 0.13-0.36). For both different status of HBeAg and pre-existing compensated cirrhosis, there was significant lower incidence of long-term complications in treatment groups compared with no treatment, too. Moreover, among the NA treatment groups, patients with drug-resistance had 2.64 times (RR:2.64, 95%CI: 1.58-4.41) higher chance of developing to long-term complications, and patients with pre-existing compensated cirrhosis also had 3.07 times (RR:3.07, 95%CI: 1.04-9.11) higher chance of developing to long-term complications. CONCLUSIONS: Long-term nucleos(t)ide analogue therapy for adults with CHB prevents or delays the development of long-term complications including decompensated cirrhosis, CHB-related death or CHB-related HCC in patients with CHB. The patients who need take antiviral drugs should receive the antiviral therapy as soon as possible.