Cellular ESCRT machinery plays pivotal role in HIV-1 budding and release. Extracellular stimuli that modulate HIV-1 egress are currently unknown. We found that CCL2 induced by HIV-1 clade B (HIV-1B) infection of macrophages enhanced virus production, while CCL2 immuno-depletion reversed this effect. Additionally, HIV-1 clade C (HIV-1C) was refractory to CCL2 levels. We show that CCL2-mediated increase in virus production requires Gag late motif LYPX present in HIV-1B, but absent in HIV-1C, and ALIX protein that recruits ESCRT III complex. CCL2 immuno-depletion sequestered ALIX to F-actin structures, while CCL2 addition mobilized it to cytoplasm facilitating Gag-ALIX binding. The LYPX motif improves virus replication and its absence renders the virus less fit. Interestingly, novel variants of HIV-1C with PYRE/PYKE tetrapeptide insertions in Gag-p6 conferred ALIX binding, CCL2-responsiveness and enhanced virus replication. These results, for the first time, indicate that CCL2 mediates ALIX mobilization from F-actin and enhances HIV-1 release and fitness.

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it's unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

The human leukocyte antigen (HLA) locus plays a critical role in tissue compatibility and regulates the host response to many diseases, including cancers and autoimmune di3orders. Recent improvements in the quality and accessibility of next-generation sequencing have made HLA typing from standard short-read data practical. However, this task remains challenging given the high level of polymorphism and homology between HLA genes. HLA typing from RNA sequencing is further complicated by post-transcriptional modifications and bias due to amplification.

Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs.

The activation of the mitogen-activated protein kinase (MAPK) pathway has been suggested as the major downstream target when GNAQ and GNA11 (GNAQ/11) are mutated in uveal melanoma (UM). However, clinical trials with single agent MEK inhibitor showed no clinical significance in altering the overall outcome of the disease in UM; therefore, we investigated the correlation between naturally occurring mutations in GNAQ/11 and activation of MAPK pathway in vivo in primary UM.

Cellulases play important roles in the shedding of plant organs; however, little is yet known about the functions of cellulase genes during the process of organ abscission. Abnormal fruitlet abscission is a serious problem in the production of litchi (Litchi chinensis), an economically important fruit widely grown in South Asia. In this study, two abscission-accelerating treatments (carbohydrate stress and application of ethephon) were evaluated in litchi fruitlets. Cell wall degradation and cell separation were clearly observed in the abscission zones of treated fruitlets, consistent with enhanced cellulase activities and reduced cellulose contents. The expression of two cellulase genes (LcCEL2 and LcCEL8) was strongly associated with abscission. Floral organs of transgenic Arabidopsis overexpressing LcCEL2 or LcCEL8 showed remarkably precocious abscission. Electrophoretic mobility shift assays and transient expression experiments demonstrated that a novel homeodomain-leucine zipper transcription factor, LcHB2, could directly bind to and activate HD-binding cis-elements in the LcCEL2 and LcCEL8 promoters. Our results provide new information regarding the transcriptional regulation of the cellulase genes responsible for cell wall degradation and cell separation during plant organ shedding, and raise the possibility of future manipulation of litchi fruitlet abscission by modulation of the activities of these two cellulases.

CEPs (C-TERMINALLY ENCODED PEPTIDEs) inhibit Arabidopsis primary root growth by unknown mechanisms. We investigated how CEP3 levels control primary root growth. CEP3 peptide application decreased cell division, S-phase cell number, root meristematic cell number, and meristem zone (MZ) size in a dose- and CEP RECEPTOR1-dependent manner. Grafting showed that CEP3-dependent growth inhibition requires root and shoot CEPR1. CEP3 induced mitotic quiescence in MZ cells significantly faster than that induced by nutrient limitation alone. CEP3 also inhibited the restoration of S-phase to mitotically quiescence cells by nutrient resupply without quantitatively reducing TARGET OF RAPAMYCIN (TOR) kinase activity. In contrast, cep3-1 had an increased meristem size and S-phase cell number under nitrogen (N)-limited conditions, but not under N-sufficient conditions. Furthermore, cep3-1 meristematic cells remained in S-phase longer than wild-type cells during a sustained carbon (C) and N limitation. RNA sequencing showed that CEP3 peptide down-regulated genes involved in S-phase entry, cell wall and ribosome biogenesis, DNA replication, and meristem expansion, and up-regulated genes involved in catabolic processes and proteins and peptides that negatively control meristem expansion and root growth. Many of these genes were reciprocally regulated in cep3-1. The results suggest that raising CEP3 induces starvation-related responses that curtail primary root growth under severe nutrient limitation.

Strategies to activate and engage patients and caregivers in shared decision making in the acute care setting may result in improved outcomes.

The present study aimed to investigate the potential biological functions of microRNA‑340 (miR‑340) in non‑small cell lung cancer (NSCLC) beyond its role as a critical regulator of tumorigenesis and tumor progression. The expression levels of miR‑340 and RAB27B were analyzed by reverse transcription‑quantitative polymerase chain reaction. Subsequently, the protein expression levels of RAB27A, RAB27B, RAB9A, RAB11A and BRAB21 were determined by western blot analysis. The expression levels of the aforementioned proteins in NSCLC tissues were analyzed by immunohistochemistry. RAB27B, as a potential target of miR‑340 was investigated via a dual‑luciferase reporter assay. The proliferative ability of PC9, A549 and BEAS‑2B cells was detected with a Cell Counting kit‑8 assay, while the migration and invasion of the NSCLC cells were analyzed using a Transwell assay. The results revealed that the expression levels of miR‑340 in the NSCLC cells were significantly decreased compared with those in normal cells (BEAS‑2B cells). RAB27B was proposed as a potential target gene of miR‑340, and its expression was notably increased in the NSCLC cells. miR‑340 overexpression inhibited the migration and invasion of the NSCLC cells by targeting RAB27B, while the knockdown of miR‑340 exerted opposite effects. On the whole, these findings indicate that the miR‑340/RAB27B axis may be actively involved in the occurrence of NSCLC. Thus, miR‑340 and RAB27B may be novel therapeutic targets for the treatment of NSCLC.

Glaucoma is the leading cause of irreversible blindness worldwide; the apoptosis of the retinal ganglion cells (RGCs) is a hallmark of glaucoma. Tetramethylpyrazine (TMP) is the main active component of Ligusticum wallichii Franchat, and has been demonstrated to improve a variety of injuries through its antioxidative and antiapoptotic properties. However, these effects of TMP on glaucoma have not been studied. The present study aimed to investigate the potential role of TMP in glaucoma and to elucidate its possible mechanisms responsible for these effects. An in vitro model was generated, in which primary RGCs (PRGCs) were treated with H2O2. Our study revealed that TMP protected against H2O2‑induced injury to PRGCs, as evidenced by enhanced cell viability, reduced caspase 3 activity and decreased cell apoptosis. We also reported that TMP treatment inhibited reactive oxygen species (ROS) production and malondialdehyde levels, but upregulated the antioxidative enzyme superoxide dismutase. In particular, TMP significantly increased the expression of microRNA‑182‑5p (miR‑182) in H2O2‑treated PRGCs, which was selected as the target miRNA for further research. In addition, our findings suggested that the protective effects of TMP on H2O2‑induced injury were attenuated by knockdown of miR‑182. The results of a luciferase reporter assay demonstrated that Bcl‑2 interacting protein 3 (BNIP3), an effector of mitochondria‑mediated apoptosis, was a direct target of miR‑182. In addition, TMP treatment significantly decreased the expression of BNIP3, Bax, cleaved‑caspase‑3 and cleaved‑poly(ADP‑ribose)polymerase, but increased that of Bcl‑2. Also, TMP treatment decreased the release of cytochrome c from mitochondria and improved mitochondrial membrane potential in H2O2‑treated RGCs. Of note, the inhibitory effects of TMP on the mitochondrial apoptotic pathway were suggested to be reversed by knockdown of miR‑182. Collectively, our findings provide novel evidence that TMP protects PRGCs against H2O2‑induced damage through suppressing apoptosis and oxidative stress via the miR‑182/mitochondrial apoptotic pathway.

Although the migration of hepatic stellate cells (HSCs) is important for hepatic fibrosis, the regulation of this migration is poorly understood. Notably, transforming growth factor (TGF)‑β1 induces monocyte migration to sites of injury or inflammation during the early phase, but inhibits cell migration during the late phase. In the present study, the role of transforming protein RhoA signaling in TGF‑β1‑induced HSC migration was investigated. TGF‑β1 was found to increase the protein and mRNA levels of smooth muscle actin and collagen type I in HSC‑T6 cells. The level of RhoA‑GTP in TGF‑β1‑stimulated cells was significantly higher than that in control cells. Furthermore, the phosphorylation of cofilin and formation of filamentous actin (F‑actin) were more marked in TGF‑β1‑stimulated cells than in control cells. Additionally, TGF‑β1 induced the activation of nuclear factor‑κB, and the expression of extracellular matrix proteins and several cytokines in HSC‑T6 cells. The active form of Rap1 (Rap1 V12) suppressed RhoA‑GTP levels, whereas the dominant‑negative form of Rap1 (Rap1 N17) augmented RhoA‑GTP levels. Therefore, the data confirmed that Rap1 regulated the activation of RhoA in TGF‑β1‑stimulated HSC‑T6 cells. These findings suggest that TGF‑β1 regulates Rap1, resulting in the suppression of RhoA, activation of and formation of F‑actin during the migration of HSCs.

IR‑783, a near‑infrared heptamethine cyanine dye, has been reported to possess cancer targeting and anticancer effects; Ηowever, the molecular mechanism by which IR‑783 exhibits anti‑breast cancer activity is unclear. In the present study, the inhibitory effects of IR‑783 on the proliferation and migration of breast cancer cells were investigated. Our results revealed that IR‑783 inhibited MDA‑MB‑231 and MCF‑7 cell proliferation in a dose‑ and time‑dependent manner by inducing cell cycle arrest at the G0/G1 phase. In addition, a Transwell assay demonstrated that IR‑783 treatment suppressed the migratory ability of MDA‑MB‑231 and MCF‑7 cells. Furthermore, IR‑783 treatment decreased the expression levels of matrix metalloproteinase (MMP)‑2 and MMP‑9 in MDA‑MB‑231 cells. Furthermore, IR‑783 induced MDA‑MB‑231 and MCF‑7 cell mitochondrial fission, and also decreased the levels of ATP. This was accompanied with a decrease in polymerized filamentous actin, which is the fundamental component of filopodia at the cell surface. Collectively, the results of the present study demonstrated that IR‑783 inhibited the proliferation and migration of MDA‑MB‑231 and MCF‑7 cells by inducing mitochondrial fission and subsequently decreasing ATP levels, resulting in cell cycle arrest and filopodia formation suppression. These findings suggest that IR‑783 may be developed into an effective novel drug for treating breast cancer.

MicroRNAs (miRs) have been proposed to be involved in the pathological processes of cerebral ischemia/reperfusion (CIR) injury. The present study aimed to investigate the potential role and molecular mechanisms of miR‑217 in the regulation of neuronal survival in CIR injury. To perform the investigation, an in vitro cellular model of CIR injury was established by treating neurons with oxygen‑glucose deprivation and reoxygenation (OGD/R). miR‑217 levels in neurons were detected using reverse transcription‑quantitative PCR. The association between miR‑217 and sirtuin 1 (SIRT1) was identified using TargetScan and validated in a dual‑luciferase reporter assay. Cell viability and apoptosis were measured using a Cell Counting Kit‑8 assay and flow cytometry, respectively. The release of lactate dehydrogenase, and the production of proinflammatory factors and oxidative stress biomarkers were analyzed by ELISAs and using specific assay kits. It was revealed that miR‑217 was significantly upregulated in OGD/R‑treated neurons. SIRT1 was a direct target of miR‑217, and was downregulated in neurons following OGD/R treatment. Downregulation of miR‑217 significantly ameliorated OGD/R‑induced neuronal injury, inflammatory responses and oxidative stress. The effects of miR‑217 inhibitor on OGD/R treated neurons were attenuated by SIRT1 knockdown. Additionally, western blotting revealed that the SIRT1/AMP‑activated protein kinase‑α/NF‑κB pathway was partially involved in the regulation of OGD/R‑induced neuronal injury by miR‑217. In conclusion, the data of the present study indicated that the downregulation of miR‑217 protected neurons against OGD/R‑induced injury by targeting SIRT1.

Hepatocellular carcinoma (HCC) accounts for ~85% of primary liver cancer cases and is a leading cause of mortality worldwide. Effective early diagnosis is difficult for HCC; however, effective biomarkers may be beneficial for diagnosis. In the current study, serum samples, and HCC and adjacent tissue samples were obtained from patients with HCC for the detection of biomarkers using 2‑D gel electrophoresis (2‑DE) and matrix‑assisted laser desorption/ionization‑time of flight (TOF)/TOF mass spectrometry. The crude serum samples did not need to be prepared for removal of high abundance proteins. The mRNA expression levels of HCC‑associated proteins were detected in tissues using reverse transcription‑quantitative PCR. Statistical analysis and database matching were used to identify the differentially expressed proteins detected in the serum and tissue groups. Immunohistochemistry (IHC) was performed to detect the expression of significant proteins in HCC and adjacent tissues. The results revealed ~800 protein spots on a 2‑DE gel that were detected in serum samples, and 1,200 spots were identified in the tissue samples. The protein and mRNA expression levels of oxysterol binding protein‑like 11 (OSBPL11) in HCC serum and tissue samples were consistent. Pathway analysis demonstrated that members of the apolipoprotein family, particularly apolipoprotein E (APOE), and RAS family members were closely associated in HCC, either directly or via ferratin heavy polypeptide 1. IHC results demonstrated that the APOE protein serves an important role in liver cancer development. The lysis buffer used in the current study was effective for serum protein separation in 2‑DE sample preparation. In addition, the present study revealed that downregulated OSBPL11 may be a potential indicator for HCC, and the apolipoprotein family, particularly APOE, and the RAS family may cooperatively serve an important role.

Electroacupuncture (EA), a traditional Chinese therapeutic technique, is considered an effective method for treating certain painful neuropathies induced by various neuropathological damage. The current study investigated the effect of EA on intrathecal (IT) morphine‑induced hyperalgesia (MIH) and examined the hypothesis that activation of cannabinoid receptor 1 (CB1) could enhance the antinociceptive effect of EA on MIH via regulation of the extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling pathway. Using a rat model of IT MIH, mechanical and thermal hyperalgesia were evaluated by an electronic von Frey filament and hotplate at baseline (1 day before IT administration) and at days 1, 3, 5 and 7 after IT administration. Rats received IT normal saline, IT morphine or IT morphine + EA at ST36‑GB34. The protein levels of ERK1/2, phosphorylated (p)‑ERK1/2 and CB1 in the spinal cord were assayed by western blotting. Furthermore, the effect of IT injection of the CB1 agonist WIN 55,212‑2 and the CB1 antagonist SR141716 on the antinociceptive effect of EA in rats with MIH was investigated. Nociceptive behavior and ERK1/2, phosphorylated (p)‑ERK1/2 and CB1 protein levels were evaluated as mentioned above. The results revealed that chronic IT injections of morphine induced a significant decrease in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) accompanied with remarkable upregulation of p‑ERK1/2 in the spinal cord, which could be attenuated by EA at the ST36‑GB34 acupoints. In the rat model of MIH, IT injection of WIN 55,212‑2 combined with EA induced a significant increase in MWT and TWL accompanied with a significant decrease in p‑ERK1/2 and a significant increase in CB1 protein level compared with EA alone, while SR141716 induced the opposite results. The present study suggests that EA alleviates hyperalgesia induced by IT injection of morphine partially through the inhibition of ERK1/2 activation. Activation of the CB1 receptor enhances the antinociceptive effect of EA in rats with MIH partly through the regulation of the spinal CB1‑ERK1/2 signaling pathway.

Interleukin (IL)‑12 modulates the generation and function of various immune cells and plays a vital role in the pathogenesis of Sjögren's syndrome (SS). Myeloid‑derived suppressor cells (MDSCs) are involved in autoimmune diseases by regulating various immune responses. However, it has not been confirmed whether inflammatory IL‑12 participates in the progression of SS via regulating MSDCs. In the present study, the plasma levels of IL‑12 were detected by ELISA in SS‑like non‑obese diabetic (NOD) mice. The mice were treated by intraperitoneal injection of IL‑12 and anti‑IL‑12 antibody, respectively, and then the salivary flow rate was detected. The pathology of submandibular glands was evaluated in tissue sections stained with hematoxylin and eosin. The proportion of MDSCs was assessed by flow cytometry. The results showed that plasma IL‑12 was significantly increased in the SS‑like NOD mice comparing with that noted in the control mice. The exogenous IL‑12 exacerbated SS‑like symptoms of NOD mice and promoted the generation of both bone marrow (BM) and splenic MDSCs in the SS‑like NOD mice. Of note, anti‑IL‑12 alleviated SS‑like symptoms of NOD mice and inhibited the generation of BM and splenic MDSCs. Moreover, the generation of MDSCs was crippled in the IL‑12‑deficient C57BL/6 (Il‑12‑/‑ B6) mice. Our findings suggest that aggravation of SS‑like symptoms by IL‑12 in NOD mice may be attributed to its promotion of MDSC development.

The progression of disease caused by fungal infection is closely associated with the human immune system. Macrophages and natural killer cells (NK cells) are two important types of innate immune cells that serve an important role in anti‑infection immunity. There has been limited research into the interactions between fungi and macrophages. In the present in vitro study, reverse transcription‑quantitative PCR, ELISA and flow cytometry were performed to reveal that the interaction between macrophages and NK cells, regulated by Aspergillus fumigatus conidia, induced macrophages to polarize into M1 macrophages by secreting large quantities of tumor necrosis factor‑α, interleukin‑18 and Galectin‑9. In addition, when NK cells were co‑cultured with the conidia of A. fumigatus‑stimulated M1 macrophages, they exhibited increased activation levels and secretion of interferon‑γ (IFN‑γ). It was further demonstrated via antibody neutralization and gene silencing experiments that galectin‑9 served an important role in the interaction between macrophages and NK cells regulated by A. fumigatus. In conclusion, it was demonstrated that A. fumigatus induced the polarization of macrophages into M1 macrophages by secreting Galectin‑9, which then promoted NK cell activity and IFN‑γ secretion. The results provided improved understanding of the role of innate immune cells in invasive fungal infections. The present study also provided novel insight into the study of macrophages and NK cells in inflammatory infections caused by A. fumigatus and potential strategies to control the progression of inflammation.

Angelman syndrome (AS) is a congenital neuro-developmental disorder typically occurring due to functional defects of the UBE3A gene caused by uniparental disomy (UPD), translocation or single gene mutation. UBE3A gene exhibits imprinting expression, and only maternal inherited alleles express functional UBE3A protein in the brain. The common method to diagnose AS is single nucleotide polymorphism array or methylation‑specific multiplex ligation‑dependent probe amplification (MS‑MLPA). In recent years, whole exome sequencing (WES) has been increasingly used in the genetic diagnosis of a variety of indications, exhibiting great advantages as a comprehensive and unbiased testing method. In the present study, the cases of two unrelated patients with Robertsonian‑like translocation in chromosome 15, namely 45,XX,der(15;15)(q10;q10) and 45,XY,der(15;15)(q10;q10), are reported. The first case was diagnosed with AS by WES and validated by Sanger sequencing. In contrast to 42.84%  homozygous variants on all chromosomes, 92.69%  homozygosity variants were observed on chromosome 15. A homozygous stretch identifier was applied and identified a homozygous region across the entire chromosome 15. Sanger sequencing was used to further determine the subtype and confirm that two homozygous variants on chromosome 15 with low allele frequency (<0.01) were derived only from the father and not from the mother, thereby indicating a paternal UPD case, classified as isodisomy. MS‑MLPA results of the other AS patient with the same karyotype indicated that he had a high possibility of paternal UPD at chromosome 15. Taken together, the current study suggested the potential application of WES in detecting and facilitating the diagnosis of UPD.

The expression of microRNA‑802 (miR‑802) is known to be associated with insulin resistance (IR); however, the mechanism remains unclear. The present study investigated how miR‑802 contributes to the development of IR using C57BL/6J mice fed a high‑fat diet (HFD) to establish a model of IR. Adeno‑associated virus overexpressing miR‑802 was administered to the mice via tail vein injection. The effects of miR‑802 on reactive oxygen species (ROS), lipid peroxidation (LPO) and the activities of multiple ROS‑related enzymes were investigated. Western blot analysis was used to estimate the protein levels of extracellular signal regulated kinase (ERK), p38mitogen‑activated protein kinases (p38MAPK), c‑Jun N‑terminal kinase (JNK), insulin receptor substrate 1 (IRS‑1) and protein kinase B (AKT1). The results demonstrated that the levels of ROS and LPO production were increased in the livers of the miR‑802‑treated group compared with the control group. The activities of the ROS‑related enzymes were reduced. Furthermore, the expression of phosphorylated (phosphor)‑p38MAPK and phosphor‑JNK were upregulated in the miR‑802 overexpression group, whereas there was no difference in the expression levels of phosphor‑ERK. The expression levels of phosphor‑AKT1 were reduced in the miR‑802‑treated group and these effects were reversed by miR‑802 knockdown. In conclusion, the results demonstrate that miR‑802 may cause IR by activating the JNK and p38MAPK pathways to increase hepatic oxidative stress.

Stanniocalcin 2 (STC2), a secretory glycoprotein hormone, regulates many biological processes including cell proliferation, apoptosis, tumorigenesis and atherosclerosis. However, the effect of STC2 on proliferation, migration and epithelial‑mesenchymal transition (EMT) progression in human colorectal cancer (CRC) cells remains poorly understood. The expression level of STC2 was determined by quantitative real‑time polymerase chain reaction (qPCR) and western blot analysis. Cell Counting Kit‑8 (CCK‑8) was used to detect the viability of SW480 cells. The invasion and migration of cells were identified by wound healing and Transwell assays. The mRNA and protein expression levels of β‑catenin, matrix metalloproteinase (MMP)‑2, MMP‑9, E‑cadherin and vimentin were assessed by qPCR and western blot analysis. In the present study, it was demonstrated that STC2 was highly expressed in the CRC cell lines. After silencing of STC2, the cell viability, migration and invasion were significantly reduced. Silencing of STC2 in the CRC Sw480 cells increased the expression of E‑cadherin and decreased the expression of vimentin, MMP‑2 and MMP‑9, compared to those in the normal and empty vector group. Furthermore, the expression of β‑catenin in the STC2 gene silenced group was suppressed, and the expression of β‑catenin was reversed by Wnt activator, SB216763. These results demonstrated that STC2 participates in the development and progression of CRC by promoting CRC cell proliferation, survival and migration and activating the Wnt/β‑catenin signaling pathway.