The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multisubunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the kinesin-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes.

MicroRNAs (miRNAs) function as key regulators of numerous types of cancers. miRNA (miR)‑421 expression is dysregulated in a variety of tumors; however, its role in non‑small cell lung cancer (NSCLC) remains unclear. In the present study, the role and molecular mechanism of miR‑421 in NSCLC was investigated. In this study, miRNA (miR)‑421 was upregulated in NSCLC tissues and cell lines used the reverse transcriptase quantitative polymerase chain reaction. Ectopic expression of miR‑421 significantly promoted cell proliferation in vitro and tumor growth in vivo by promoting cell cycle progression via CCK-8, colony formation, EdU assay, xenograft model and cell cycle assay. In addition, miR‑421 inhibited NSCLC cell apoptosis by flow cytometry apoptosis assay, as evidenced by anti‑apoptosis gene Bcl‑2 and apoptosis gene cleaved caspase‑3 and cleaved PARP using western blot assay. Furthermore, miR‑421 promoted cell migration and invasion through EMT process using Transwell and western blot assay. It was also demonstrated that miR‑421 can directly target HOPX by the EGFP reporter assay and western blot assay. MiR‑421 overexpression promoted the protein expression levels of β‑catenin, cyclin D1 and c‑myc by western blot assay, which are the downstream genes of Wnt pathway. These data indicated that miR‑421 may act as an oncogene through the effects of HOPX on the Wnt/β‑catenin signaling pathway and may provide insight into the mechanisms underlying carcinogenesis and the identification of potential biomarkers associated with NSCLC.

Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.

Influenza A virus (IAV) occasionally cross-species transmission among humans, swine and avian. The ectodomain of matrix protein 2 (M2e) is highly conserved in IAV, and multi-copy M2e from different species are usually displayed on the surface of nanoparticles to improve immunogenicity and constitute universal IAV nanovaccines. In our previous study, three M2e were inserted into the C-terminal of Cap protein of porcine circovirus type 2 (PCV2) to form a universal nanovaccine that provides protection against PCV2 and different subtypes of IAV. However, M2e adopts at least two converted conformations, and the intermolecular linker of M2e enhances the conformational instability, which limits the recognition by B cell receptors and production of high-level antibodies. Here, we report that the permutation of M2e affects effectiveness of nanovaccines. Three M2e derived from humans, swine and avian IAV were inserted into the C-terminal of Cap protein to form nanovaccines. Immunoprotective effects of different M2e arrangements were explored in mice. Results showed that the M2e closest to the surface of nanoparticle induced the most efficient protection against IAV derived from corresponding species. The results will contribute to develop more effective PCV2 and universal IAV bivalent nanovaccines for pigs, as well as species-specific universal IAV vaccines.

The aim of this study was to localize the anatomic distribution of upper motor neuron (UMN) loss through examining cortical thickness at the clinical onset of amyotrophic lateral sclerosis (ALS) and explore motor manifestation in functionally impaired body region attribute to impairment of lower motor neuron (LMN) or UMN or mixed LMN and UMN? The clinical features, cortical thickness of corresponding areas from different body regions in MRI and electromyography (EMG) data were collected from 108 classical ALS patients. The cortical thickness was thinner in ALS group than control group in bilateral head-face and upper-limb areas (p < 0.05). In head-face area, the cortical thickness of bulbar-onset group was significantly lower than that of control groups (p < 0.05). In upper-limb areas, the cortical thickness of cervical-onset group was significantly thinner than that of control group. Notably, the bulbar ALSFRS-R subscore was correlated with cortical thickness in bilateral head-face areas (p < 0.05). The bulbar ALSFRS-R subscore of the positive LMN damage group was lower compared to that of the negative LMN damage group (P < 0.001). The limb ALSFRS-R subscore correlated with compound muscle action potential (CMAP) amplitudes of median, ulnar, peroneal, and tibial nerves (P < 0.001), but was not related to cortical thickness. In conclusion, the UMN degeneration in ALS was derived from focal initiation, bulbar- and cervical-onset may date from head-face and upper-limb areas in motor homunculus cortex, respectively. The bulbar dysfunction was resulted from the mixed UMN and LMN impairment, while limb dysfunction derived mostly from LMN loss.

Physical inactivity, the fourth leading mortality risk factor worldwide, is associated with chronic mental illness. Identifying the mechanisms underlying different levels of baseline physical activity and the effects of these levels on the susceptibility to stress is very important. However, whether different levels of baseline physical activity influence the susceptibility and resilience to chronic social defeat stress (CSDS), and the underlying mechanisms in the brain remain unclear. The present study segregated wild-type mice into low baseline physical activity (LBPA) and high baseline physical activity (HBPA) groups based on short term voluntary wheel running (VWR). LBPA mice showed obvious susceptibility to CSDS, while HBPA mice were resilient to CSDS. In addition, the expression of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) was lower in LBPA mice than in HBPA mice. Furthermore, activation of TH neurons in the VTA of LBPA mice by chemogenetic methods increased the levels of VWR and resilience to CSDS. In contrast, inhibiting TH neurons in the VTA of HBPA mice lowered the levels of VWR and increased their susceptibility to CSDS. Thus, this study suggests that different baseline physical activities might be mediated by the dopamine system. This system also affects the susceptibility and resilience to CSDS, possibly via alteration of the baseline physical activity. This perspective on the neural control and impacts on VWR may aid the development of strategies to motivate and sustain voluntary physical activity. Furthermore, this can maximize the impacts of regular physical activity toward stress-reduction and health promotion.

Vaccination is an effective method to control the spread of classical swine fever virus (CSFV), which is a major cause of economic losses to the swine industry. Although serological detection assays are commonly used to assess immune status, current methods for monitoring of antibodies (Abs) are time-consuming, expensive, and require cell culture and virus manipulation. To address these problems, the E2 protein of CSFV was expressed in transgenic rice seeds as a labeled antigen for the development of an immunochromatographic test strip (ICTS) for rapid, precise, and cost-effective detection of Abs. The ICTS has a reasonable sensitivity of 1:128,000 for detection of serum Abs against CSFV and no cross-reactivity with Abs of other porcine viruses. The similarity of the results between the proposed ICTS and a commercial enzyme-linked immunosorbent assay was 94.1% (128/136) for detection of serum Abs from immunized animals and 92.3% (72/78) for detection of maternally derived Abs. The proposed assay was successfully used to monitor Abs against E2 of both pigs and rabbits immunized with a live attenuated vaccine or an E2 subunit vaccine. The results confirmed that the ICTS can be applied to detect Ab levels in animals with different immunological backgrounds. The ICTS based on plant-derived E2 is a relatively inexpensive, rapid, and accurate assay for detection of Abs against CSFV and avoids the risk of contamination by animal products. IMPORTANCE The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine. The assay successfully detected Ab levels in serum samples from piglets with different immunological backgrounds. In contrast to current E2 protein-based diagnostic methods using Escherichia coli or insect cells as expression systems, plant-derived E2 avoids the limitations of low immunogenicity of eukaryotic expression systems and potential contamination of fetal bovine serum with bovine viral diarrhea virus in cell culture.

To reduce water utilization, limit environmental pollution, and guarantee aquatic production and quality, the in-pond raceway recirculating culture system (IPRS) has been developed and is widely used. The effectiveness and sustainability of IPRSs rely on a good understanding of the ecological processes related to bacterial communities in the purification area. In this study, we investigated the dynamics and assembly mechanisms of benthic bacterial communities in the purification area of an industrial-scale IRPS. We found significant temporal and spatial variations in the sediment characteristics and benthic bacterial communities of the IPRS, although correlation analyses revealed a very limited relationship between them. Among the different culture stages, we identified numerous benthic bacteria with different abundances. Abundances of the phyla Bacteroidota and Desulfobacterota decreased whereas those of Myxococcota and Gemmatimonadota increased as the culture cycle progressed. Co-occurrence networks revealed that the bacterial community was less complex but more stable in the IPRS at the final stage compared with the initial stage. The neutral community model (NCM) showed that stochastic processes were the dominant ecological processes shaping the assembly of the benthic bacterial community. The null model suggested that homogenizing dispersal was more powerful than dispersal limitation and drift in regulating the assembly of the community. These findings indicate that the benthic microbial communities in purification areas of the IPRS may not be affected by the deposited wastes, and a more stable benthic microbial communities were formed and mainly driven by stochastic processes. However, the benthic microbial communities in the purification area at the end of the culturing stage was characterized by potentially inhibited organic matter degradation and carbon and sulfur cycling abilities, which was not corresponding to the purification area's function. From this point on, the IPRS, especially the purification area was needed to be further optimized and improved.

Existing evidence indicates anti-GABAB receptor encephalitis (GABABR-E) seems to occur more commonly later in life, yet the age-associated differences in clinical features and outcomes are not well determined. This study aims to explore the demographic, clinical characteristics, and prognostic differences between late-onset and early-onset GABABR-E and identify predictors of favorable long-term outcomes.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) plays an important role in viral replication. It has been reported that Vpr stimulates the nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) signaling pathways, and thereby regulates viral and host cell gene expression. However, the molecular mechanism behind this function of Vpr is not fully understood.

Whereas the mechanisms involved in autophagosome formation have been extensively studied for the past 2 decades, those responsible for autophagosome-lysosome fusion have only recently begun to garner attention. In this study, we report that the multisubunit BORC complex, previously implicated in kinesin-dependent movement of lysosomes toward the cell periphery, is required for efficient autophagosome-lysosome fusion. Knockout (KO) of BORC subunits causes not only juxtanuclear clustering of lysosomes, but also increased levels of the autophagy protein LC3B-II and the receptor SQSTM1. Increases in LC3B-II occur without changes in basal MTORC1 activity and autophagy initiation. Instead, LC3B-II accumulation largely results from decreased lysosomal degradation. Further experiments show that BORC KO impairs both the encounter and fusion of autophagosomes with lysosomes. Reduced encounters result from an inability of lysosomes to move toward the peripheral cytoplasm, where many autophagosomes are formed. However, BORC KO also reduces the recruitment of the HOPS tethering complex to lysosomes and assembly of the STX17-VAMP8-SNAP29 trans-SNARE complex involved in autophagosome-lysosome fusion. Through these dual roles, BORC integrates the kinesin-dependent movement of lysosomes toward autophagosomes with HOPS-dependent autophagosome-lysosome fusion. These findings reveal a requirement for lysosome dispersal in autophagy that is independent of changes in MTORC1 signaling, and identify BORC as a novel regulator of autophagosome-lysosome fusion.

Although hexokinase (HK) 2, pyruvate kinase muscle (PKM) isozyme 2 and lactate dehydrogenase (LDH) A predict the efficacy of medicines in various solid tumors, their ability to predict the efficacy of cetuximab in metastatic colorectal cancer (mCRC) remains unclear. mCRC patients with pathological specimens who received cetuximab and chemotherapy from 2005 to 2015 in the present institution were enrolled. Immunohistochemistry was used to detect HK2, PKM2 and LDHA expression. SPSS20 was used for statistical analysis. A total of 68 patients were included; 33 received cetuximab plus chemotherapy as first-line therapy, and the rest, as second- or later-line therapy. HK2 expression levels were increased in cancer compared with normal tissue (75.4% vs. 40%; P<0.001), however PKM2 (P=0.243) and LDHA (P=0.067) expression levels were not. For progression-free survival (PFS) with first-line cetuximab plus chemotherapy, patients with high HK2 expression exhibited longer PFS compared with those with low HK2 expression (23.9 months vs. 6.9 months; P=0.021). However, this positive association was absent in 35 cases administered first-line chemotherapy alone (13.4 months vs. 13.5 months; P=0.539). LDHA expression was associated with the PFS of patients receiving first-line chemotherapy (18.3 and 10.1 months for high and low expression, respectively; P=0.005), whereas this association was absent in cetuximab plus chemotherapy cases (19.9 months vs. 12 months; P=0.522). Furthermore, high LDHA expression correlated with high overall response rate (ORR) (72.2% vs. 15.4%, P=0.006) for chemotherapy, however not disease control rate (DCR) (P=0.074). Neither DCR nor ORR were associated with HK2 expression. PKM2 expression did not affect PFS, DCR or ORR. LDHA expression (P=0.005), pathological differentiation (P=0.019) and synchronous/metachronous metastasis (P=0.014) were independent predictive factors of PFS for all first-line patients, and tumor differentiation (P=0.002) was associated with overall survival (OS) in multivariate analysis. HK2, PKM2 and LDHA did not impact OS. It was concluded that HK2 expression was increased in colorectal cancer tissue and may predict cetuximab efficacy and LDHA for chemotherapy treatment of mCRC.

Although the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. To identify novel autophagy regulators, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroglioma cells expressing endogenous LC3B tagged with a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as autophagy regulators. We found that these enzymes cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increased autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, UBA6 or BIRC6 depletion decreased the formation of aggresome-like induced structures in H4 cells, and α-synuclein aggregates in rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B. Inhibition of UBA6/BIRC6 could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.

Bluetongue (BT) is a non-contact infectious disease caused by Bluetongue virus (BTV), which can be transmitted by vector insects such as Culicoides and Aedes mosquitoes. The BTV VP2 protein encoded by the L2 gene is located at the outermost layer of the virus particle, plays a key role on mediating the adsorption and entry of virus, and it is also a main antigenic protein widely used for vaccine development. In this study, the BTV1 VP2 gene was cloned into pFastBac™Dual vector, and expressed in insect Sf21 cells. Immunized mice with purified recombinant VP2 protein can induce higher levels of antibodies. Three anti BTV1 VP2 monoclonal antibodies (mAbs) were generated (17E9C6, 17E9C8, 17E9H12), and showed high specific reactivity with recombinant VP2 protein and inactivated BTV1 virus. Finally, a novel linear B-cell epitope 296-KEPAD-300 on recombinant VP2 protein was identified by using three mAbs react with a series of continue-truncated peptides. The results of this study may provide new information on the structure and function of BTV1 VP2 protein and lay a foundation for the development of BTV1 diagnostic and prophylactic methods.

Consolation is a complex empathic behavior that has recently been observed in some socially living rodents. Despite the growing body of literature suggesting that stress affects some simple form of empathy, the relationship between stress and consolation remains largely understudied. Using monogamous mandarin voles, we found that an acute restraint stress exposure significantly reduced consolation-like behaviors and induced anxiety-like behaviors. Along with these behavioral changes, corticotropin-releasing factor (CRF) and CRF receptor 1 (CRFR1) neurons were activated within the anterior cingulate cortex (ACC) and prelimbic cortex (PrL) but not within the infralimbic cortex (IL). Chemogenetic activation of CRF neurons in the ACC and PrL, recaptured acute stress-induced behavioral dysfunctions. We further observed that intracellular PKA and PKC signaling pathways mediate CRF-induced behavioral dysfunctions, but they work in a regional-specific, sex-biased manner. Together, these results suggest that the local CRF-CRFR1 system within the ACC and PrL is involved in the consolation deficits and anxiety induced by acute stress.

Consolation is a type of empathy-like behavior that has recently been observed in some socially living rodents. Despite the growing body of literature suggesting that stress affects empathy, the relationship between stress and consolation remains understudied at the preclinical level. Here, we examined the effects of chronic emotional stress or physical stress exposure on consolation and emotional behaviors by using the socially monogamous mandarin vole (Microtus mandarinus) in both males and females.

African swine fever virus (ASFV) gives rise to a grievous transboundary and infectious disease, African swine fever (ASF), which has caused a great economic loss in the swine industry. To prevent and control ASF, once suspicious symptoms have presented, the movement of animal and pork products should be stopped, and then, laboratory testing should be adopted to diagnose ASF. A method for ASFV DNA quantification is presented in this research, which utilizes the next-generation PCR platform, nanofluidic chip digital PCR (cdPCR). The cdPCR detection showed good linearity and repeatability. The limit of detection for cdPCR is 30.1995 copies per reaction, whereas no non-specific amplification curve was found with other swine viruses. In the detection of 69 clinical samples, the cdPCR showed significant consistency [91.30% (63/69)] to the Office International des Epizooties-approved quantitative PCR. Compared with the commercial quantitative PCR kit, the sensitivity of the cdPCR assay was 86.27% (44/50), and the specificity was 94.44% (17/18). The positive coincidence rate of the cdPCR assay was 88% (44/50). The total coincidence rate of the cdPCR and kit was 89.86% (62/69), and the kappa value reached 0.800 (P < 0.0001). This is the first time that cdPCR has been applied to detecting ASFV successfully.

In the regulation of emotional and social behaviors, both oxytocin (OT) and vasopressin (AVP) are sex specific. Although significant sex differences have been reported in the context of behavioral and hormonal responses to social stress, such differences in response to chronic social defeat stress (CSDS) and the underlying neural mechanisms remain largely unknown. By investigating monogamous mandarin voles (Microtus mandarinus), CSDS was found to decrease the percentages of time spent in the central area of the open field, in the open arms of the elevated plus maze, as well as in the light area of the light and dark boxes in both male and female voles. CSDS also increased the observed level of social withdrawal in both sex groups. However, CSDS exposure increased the percentages of immobile time in both the tail suspension test and the forced swim test and reduced the locomotor activity in the open field (in females only). Along with these behavioral changes, the oxytocin receptor (OTR) levels in the nucleus accumbens (NAc) were significantly lower in CSDS-exposed voles of both sexes; however, in males, the levels of OTR in the paraventricular nucleus (PVN) were reduced. CSDS-exposed males showed lower levels of V1aR in the NAc than CSDS-exposed females. Furthermore, induced by a single social defeat event, CSDS reduced c-Fos and OT double labeling in the PVN of females but increased c-Fos and AVP double-labeled neurons in the PVN of males exposed to a single social defeat event. Collectively, the present study indicates that OT and AVP systems may play important regulatory roles in the sex differences of behavioral performances in response to CSDS. These findings suggest mandarin voles as a useful animal model for studying sex-specific behavioral performance and the underlying neurobiological mechanisms of stress-related mental disorders in preclinical studies.

Baicalin, a main bioactive compound of Scutellaria baicalensis, has a variety of pharmacological activities including antioxidation, anti-inflammation and hepatoprotection. However, there are few reports on these biological activities in fish. Therefore, the aim of this study was to assess the effects of baicalin on growth performance, antioxidative status and hepatoprotection in tilapia. The fish were fed on different doses of baicalin (0, 0.4, 0.8 and 1.6 g/kg diet). After feeding 60 days, parts of fishes were netted, and the blood, liver, gills and muscle tissues were collected to analyze the antioxidative effect. The remaining fishes were injected with saline or hydrogen peroxide (H2O2) for challenge test. The results showed that the specific growth rate of fish was slightly increased in three baicalin treatments, and the feed efficiency was clearly improved in 0.4 g/kg baicalin treatment. Meanwhile, the antioxidative capacity in blood, liver and/or gill was enhanced in treatments with 0.4, 0.8 and/or 1.6 g/kg baicalin. After challenge test, the pre-treatments with baicalin effectively alleviated H2O2-induced liver injury. In serum and liver, pre-treatments with 0.8 and/or 1.6 g/kg baicalin suppressed the oxidative damage induced by H2O2, as evidenced by improvement of the levels of SOD, T-AOC and GSH and the decline of MDA level. More important, pre-treatments with 0.4, 0.8 and/or 1.6 g/kg baicalin blocked the upregulation of mRNA levels of tlr1, myd88, irak4, rela, tnf-α and il-1β in H2O2-induced liver injury. In summary, dietary baicalin supplementation could improve feed efficiency, enhance antioxidative ability and alleviate oxidative stress-induced hepatotoxicity in tilapia.

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants. In naïve animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccine's targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection against Delta and Omicron BA.1variants. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine-primed populations.