Protein Beta Subunit Research Articles

Murine Retina Outer Plexiform Layer Development and Transcriptome Analysis of Pre-Synapses in Photoreceptors.

Photoreceptors in the mammalian retina convert light signals into electrical and molecular signals through phototransduction and transfer the visual inputs to second-order neurons via specialized ribbon synapses. Two kinds of photoreceptors, rods and cones, possess distinct morphology and function. Currently, we have limited knowledge about rod versus (vs.) cone synapse development and the associated genes. The transcription factor neural retina leucine zipper (NRL) determines the rod vs. cone photoreceptor cell fate and is critical for rod differentiation. Nrl knockout mice fail to form rods, generating all cone or S-cone-like (SCL) photoreceptors in the retina, whereas ectopic expression of Nrl using a cone-rod homeobox (Crx) promoter (CrxpNrl) forms all rods. Here, we examined rod and cone pre-synapse development, including axonal elongation, terminal shaping, and synaptic lamination in the outer plexiform layer (OPL) in the presence or absence of Nrl. We show that NRL loss and knockdown result in delayed OPL maturation and plasticity with aberrant dendrites of bipolar neurons. The integrated analyses of the transcriptome in developing rods and SCLs with NRL CUT&RUN and synaptic gene ontology analyses identified G protein subunit beta (Gnb) 1 and p21 (RAC1) activated kinase 5 (Pak5 or Pak7) transcripts were upregulated in developing rods and down-regulated in developing SCLs. Notably, Gnb1 and Gnb5 are rod dominant, and Gnb3 is enriched in cones. NRL binds to the genes of Gnb1, Gnb3, and Gnb5. NRL also regulates pre-synapse ribbon genes, and their expression is altered in rods and SCLs. Our study of histological and gene analyses provides new insights into the morphogenesis of photoreceptor pre-synapse development and regulation of associated genes in the developing retina.

Reclassification of Aestuariicella albida as Pseudomaricurvus albidus comb. nov. and Aestuariicella hydrocarbonica as Pseudomaricurvus hydrocarbonicus comb. nov. Based on Comparative Genomics and Molecular Synapomorphies.

The genus Aestuariicella has been recently reclassified as a member of the family Cellvibrionaceae. However, the taxonomic position of the genus as a distinct member of the family has not been clarified. In the present study, we performed multilayered analyses anchored on genome sequences to clarify the relationship between the genera Aestuariicella and Pseudomaricurvus within the family Cellvibrionaceae. Phylogenetic analyses based on 16S rRNA gene, RNA polymerase beta subunit (RpoB) protein, and core gene sequences showed a well-supported tight cluster formed by the members of the two genera. Moreover, the analysis of the average amino acid identity (AAI) revealed that the members of the two genera shared 68.16-79.48% AAI, values which were within the range of observed AAI (≥ 67.23%) among the members of the same genus within the family Cellvibrionaceae. Members of the two genera also shared several common characteristics. Furthermore, molecular synapomorphies in a form of conserved signature indels were identified in six protein sequences that were exclusively shared by the members of the two genera. Based on the phylogenetic and molecular evidence presented here, we propose the reclassification of the species Aestuariicella albida and Aestuariicella hydrocarbonica as Pseudomaricurvus albidus comb. nov. and Pseudomaricurvus hydrocarbonicus comb. nov., respectively.

Gene Expression Profiling of Post Mortem Midbrain of Parkinson's Disease Patients and Healthy Controls.

Parkinson's disease (PD) stands as the most prevalent degenerative movement disorder, marked by the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. In this study, we conducted a transcriptome analysis utilizing post mortem mRNA extracted from the substantia nigra of both PD patients and healthy control (CTRL) individuals. Specifically, we acquired eight samples from individuals with PD and six samples from CTRL individuals, with no discernible pathology detected in the latter group. RNA sequencing was conducted using the TapeStation 4200 system from Agilent Technologies. A total of 16,148 transcripts were identified, with 92 mRNAs displaying differential expression between the PD and control groups. Specifically, 33 mRNAs were significantly up-regulated, while 59 mRNAs were down-regulated in PD compared to the controls. The identification of statistically significant signaling pathways, with an adjusted p-value threshold of 0.05, unveiled noteworthy insights. Specifically, the enriched categories included cardiac muscle contraction (involving genes such as ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), solute carrier family 8 member A1 (SLC8A1), and cytochrome c oxidase subunit II (COX2)), GABAergic synapse (involving GABA type A receptor-associated protein-like 1 (GABARAPL1), G protein subunit beta 5 (GNB5), and solute carrier family 38 member 2 (SLC38A2), autophagy (involving GABARAPL1 and tumor protein p53-inducible nuclear protein 2 (TP53INP2)), and Fc gamma receptor (FcγR) mediated phagocytosis (involving amphiphysin (AMPH)). These findings uncover new pathophysiological dimensions underlying PD, implicating genes associated with heart muscle contraction. This knowledge enhances diagnostic accuracy and contributes to the advancement of targeted therapies.

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma

ABSTRACT Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

G Protein Subunit Beta 3 (GNB3) Variant Is Associated with Biochemical Changes in Brazilian Patients with Hypertension.

Central Illustration : G Protein Subunit Beta 3 (GNB3) Variant Is Associated with Biochemical Changes in Brazilian Patients with Hypertension. Genes and their variants associated with environmental factors contribute to the development of the hypertensive phenotype. The G protein beta 3 subunit gene (GNB3) is involved in the intracellular signaling process, and its variants have been related to susceptibility to arterial hypertension. To determine the association of the GNB3 variant (rs5443:C>T) with arterial hypertension, biochemical parameters, age, and obesity in hypertensive and normotensive individuals from Ouro Preto, Minas Gerais, Brazil. The identification of variants was performed by real-time PCR, using the TaqMan® system, in 310 samples (155 hypertensive and 155 normotensive). Biochemical analyses (renal function, lipid profile and glycemia) were performed from the serum using UV/Vis spectrophotometry and ion-selective electrode. A multiple logistic regression model was used to identify factors associated with arterial hypertension. The analysis of continuous variables with normal distribution was performed using the unpaired Student's t test; non-normal data were analyzed using Mann-Whitney. P < 0.05 was considered significant. The rs5443:C>T variant was not associated with arterial hypertension in the evaluated population (p = 0.88). Regarding biochemical measures, the T allele was associated with high levels of triglycerides, glucose and uric acid in hypertensive individuals (p < 0.05). These results show the importance of genetic diagnosis to prevent the causes and consequences of diseases and imply that the GNB3 rs5443:C>T variant may be associated with changes in the biochemical profile in hypertensive individuals.

A secreted BPTI/Kunitz inhibitor domain-containing protein of barber’s pole worm interacts with host NLRP3 inflammasome activation-associated G protein subunit to inhibit IL-1β and IL-18 maturation in vitro

Protease inhibitors are major components of excretory/secretory products released by parasitic nematodes and have been proposed to play roles in host-parasite interactions. Haemonchus contortus (the barber’s pole worm) encodes for several serine protease inhibitors, and in a previous study we identified a trypsin inhibitor-like serine protease inhibitor of this blood-feeding nematode, SPI-I8, as necessary for anticoagulation. Here, we demonstrated that a bovine pancreatic trypsin inhibitor/Kunitz-type serine protease inhibitor (BPTI/Kunitz) domain-containing protein highly expressed in parasitic stages, HCON_00133150, is involved in suppressing proinflammatory cytokine production in mammalian cells. Fluorescent labelling of HCON_00133150 revealed a punctate localisation at the inner hypodermal membrane of H. contortus, an organ closely related to the excretory column. Yeast two-hybrid screening and immunoprecipitation-mass spectrometry identified that the recombinant HCON_00133150 physically interacted with a range of host proteins including the G protein subunit beta 1 of sheep (Ovis aries; OaGNB1), a negative regulator of NLRP3 inflammasome activation. Interestingly, heterologous expression of HCON_00133150 enhanced the inhibitory effect of OaGNB1 on NLRP3 inflammasome and the maturation of proinflammatory cytokines IL-1β and IL-18 in transfected cells. 1-to-1 orthologues (n = 33) of BPTI/Kunitz inhibitor domain-containing proteins were predicted in clades III, IV and V (but not clade I) parasitic nematodes. Structural (tandem BPTI/Kunitz inhibitor domains inverted into the globular reticulation) and functional (a GNB1 enhancer) characterisation of HCON_00133150 and its orthologues elucidated that these molecules might contribute to immune suppression by parasitic nematodes in animals and humans.

Qualitative Shotgun Proteomics Strategy for Protein Expression Profiling of Fish Otoliths

Despite decades of research on fish otoliths and their capacity to serve as biochronological recorders, much remains unknown about their protein composition, the mechanisms by which proteins are incorporated into the otolith matrix, or the potential for using otolith proteins to provide insight into aspects of fish life history. We examined the protein composition of Atlantic cod (Gadus morhua) otoliths using a state-of-the-art shotgun proteomics approach with liquid chromatography coupled to an electrospray ionization-orbitrap tandem mass spectrometer. In addition to previously known otolith matrix proteins, we discovered over 2000 proteins not previously identified in cod otoliths and more than 1500 proteins not previously identified in any fish otoliths. These included three novel proteins (Somatolactin, F-actin-capping protein subunit beta, Annexin) primarily involved in binding calcium ions and likely mediating crystal nucleation. However, most of the otolith proteins were not necessarily related to otolith formation but rather to other aspects of fish physiology. For example, we identified sex-related biomarkers for males (SPATA6 protein) and females (Vitellogenin-2-like protein). We highlight some noteworthy classes of proteins having diverse functions; however, the primary goal here is not to discuss each protein separately. The number and diverse roles of the proteins discovered in the otoliths suggest that proteomics could reveal critical life history information from archived otolith collections that could be invaluable for understanding aspects of fish biology and population ecology. This proof-of-concept methodology paper provides a novel methodology whereby otolith proteomics can be further explored.

CEBPB upregulates P4HA2 to promote the malignant biological behavior in IDH1 wildtype glioma.

Temozolomide (TMZ), the primary drug for glioma treatment, has limited treatment efficacy. Additionally, considerable evidence shows that isocitrate dehydrogenase 1 mutation-type (IDH1 mut) gliomas have a better response to TMZ than isocitrate dehydrogenase 1 wildtype (IDH1 wt) gliomas. Here, we aimed to identify potential mechanisms underlying this phenotype. Herein, the Cancer Genome Atlas bioinformatic data and 30 clinical samples from patients were analyzed to reveal the expression level of cytosine-cytosine-adenosine-adenosine-thymidine (CCAAT) Enhancer Binding Protein Beta (CEBPB) and prolyl 4-hydroxylase subunit alpha 2 (P4HA2) in gliomas. Next, cellular and animal experiments, including cell proliferation, colony formation, transwell, CCK-8, and xenograft assays, were performed to explore the tumor-promoting effects of P4HA2 and CEBPB. Then, chromatin immunoprecipitation (ChIP) assays were used to confirm the regulatory relationships between them. Finally, a co-immunoprecipitation (Co-IP) assay was performed to confirm the effect of IDH1-132H to CEBPB proteins. We found that CEBPB and P4HA2 expression was significantly upregulated in IDH1 wt gliomas and associated with poor prognosis. CEBPB knockdown inhibited the proliferation, migration, invasion, and temozolomide resistance of glioma cells and hindered the growth of glioma xenograft tumors. CEBPE, as a transcription factor, exerted its function by transcriptionally upregulating P4HA2 expression in glioma cells. Importantly, CEBPB is prone to ubiquitin-proteasomal degradation in IDH1 R132H glioma cells. We also demonstrated that both genes are related to collagen synthesis, as confirmed by in vivo experiments. Thus, CEBPE promotes proliferation and TMZ resistance by inducing P4HA2 expression in glioma cells and offers a potential therapeutic target for glioma treatment.

Development and application of recombinase polymerase amplification for rapid detection of rice false smut pathogen (Ustilaginoidea virens)

Ustilaginoidea virens (Cke.) Tak. is the causal pathogen of rice false smut, a grain disease which affects both grain quality and yield. In this study, a recombinase polymerase amplification (RPA) assay protocol was developed to detect U. virens directly from rice spikelet. Three pairs of primer were designed from the U. virens GTP binding protein beta subunit (UvG-β1) gene to function both in RPA and in polymerase chain reaction (PCR). The developed RPA assay efficiently detected U. virens from both genomic DNA and mycelial crude extract. A comparison of sensitivity of RPA with PCR and loop-mediated isothermal amplification (LAMP) revealed that the latter two methods failed to detect U. virens from crude extract. The specificity of the RPA primers was evaluated using DNA from other rice pathogens, as well as, by sequencing the RPA amplicons. For validation, the RPA assay was evaluated on 32 different U. virens isolates from eastern and north-eastern India, as well as, on field samples. The developed RPA assay was able to detect U. virens directly from crude extract prepared from infected rice spikelet. The developed RPA assay will be effective for field monitoring of U. virens.

Branch blight of Juglans sigillata caused by Juglanconis appendiculata in China.

Juglans sigillata Dode, an endemic walnut species native in southwest China, is mainly used as nuts in Sichuan Province (Jin et al., 2019). In May 2021, symptoms of branches blight were observed in an orchard measuring 10 hectares located in Mianyang City, Sichuan Province (31°5' 25″N, 105°27'36″E, 365 m above sea level). About 40% of plants were diseased in the quadrat consisting of twenty walnut trees, and 20% of branches were dead on each affected tree. Initially, light brown spots appeared; then, the spots expanded to surround the whole branches; finally, the branches changed from brown to reddish-brown and died. Four symptomatic branches were sampled randomly from different trees. Next, four fungal isolates were obtained from the acervuli of each branch using the single-conidium isolation (Chomnunti et al. 2014) and cultured on potato dextrose agar (PDA). The Petri dishes were placed in an incubator and cultured at 25 °C under a 12-h photoperiod. Colonies were initially white with thin aerial mycelia and gradually turned dark grey with irregular margins. Conidiomata were acervular, black and scattered, with a diameter of 0.3 - 0.7 mm. Conidiophores were narrowly cylindrical, simple or branched at the base, 30 - 43 × 3 - 8 µm (x = 36.5 × 5.5 µm, n = 40). Conidiogenous cells were annellidic with distinct annellations. Conidia were unicellular, brown when mature, narrowly ellipsoid with gelatinous sheaths and truncate scars at the base, 17 - 32 × 7 - 12 µm (x = 27 × 9 µm, n = 40). The genomic DNA of a representative isolate SICAUCC 22-0064 was extracted, and the internal transcribed spacer (ITS) region, guanine nucleotide-binding protein subunit beta gene (ms204), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers V9G/LR5 (de Hoog & van den Ende 1998), MS-E1F1/MS-E5R1 (Walker et al. 2012), EF1-728F (Carbone & Kohn 1999)/TEF1LLErev (Jaklitsch et al. 2005), and T1/BtHV2r (Voglmayr et al. 2017), respectively. The sequences of ITS, ms204, tef1-α, and tub2 were deposited in NCBI with accession numbers ON000068, ON112376, ON112374, and ON112375, respectively. With the consideration of the sequence lack of ms204 and tub2 in the ex-type strain (D96) of Juglanconis appendiculata Voglmayr & Jaklitsch, the isolate D140 was used for nucleotide blast. The results showed 99.68%, 100%, 100%, and 100% identities of ITS, ms204, tef1-α, and tub2 with D140 (accession numbers KY427138, KY427157, KY427207, KY427226). Phylogenetic analysis based on a combined dataset showed 100% bootstrap with J. appendiculata, and the morphology was consistent with the asexual stage of J. appendiculata (Voglmayr et al., 2017). To verify Koch's postulates, five branches wounded by pin-prick were sprayed with conidial suspension (1 × 105 conidia/mL) in each plant, and three repetitions were performed on healthy 2-year-old potted plants. The same number of branches were sprayed with sterile distilled water as controls. The plants were placed in a greenhouse at 25 ℃ under 90% relative humidity and a 12-h fluorescent light/dark regime. After five weeks, all the inoculated branches showed brown necrosis similar to that observed in the field, and no symptoms occurred on the controls. The pathogens were re-isolated from the necrotic lesions and identified by morphology and phylogeny. J. appendiculata has been reported on Juglans nigra and J. regia in Austria, France, Spain and Greece (Farr & Rossman 2022). This paper is the first report of branch blight on Juglans sigillata caused by J. appendiculata in China. This result may develop the understanding of walnut diseases and lay a foundation for further management.

Changes in the Skin Proteome and Signs of Allostatic Overload Type 2, Chronic Stress, in Response to Repeated Overcrowding of Lumpfish (Cyclopterus lumpus L.)

Lumpfish (Cyclopterus lumpus L) is a North Atlantic species harvested for its roe and increasingly used as a cleanerfish in Atlantic salmon (Salmo salar L.) farming to remove salmon louse (Lepeophtheirus salmonis). In aquaculture, the health and welfare of fish depends on optimal levels of several biotic and abiotic factors. Crowding, a common abiotic stress factor in aquaculture practice, can affect the welfare and survival of fish. In this study, lumpfish was exposed to crowding stress daily at random timepoints for one month (stress group) or no crowding (control group). Blood and skin were sampled weekly for physiological parameter analysis and proteomics, respectively. Adrenocorticotropic hormone (ACTH) stimulation and dexamethasone (DEX) suppression test were conducted at the sampling timepoints. Gel-based proteomics coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS) was used to identify protein changes in skin tissues of lumpfish under crowding. The results indicated that the stress group showed signs of allostatic overload type 2 (chronic stress) due to oversensitivity to ACTH, and a reduced negative feedback system with increased baseline levels of cortisol. These chronic changes in the endocrine system promoted changes in secondary and tertiary stress responses as reduced osmoregulatory capacity and stunted growth, after 14 days of stress and onward. Calmodulin, guanine nucleotide binding protein subunit beta 2, glutathione-S-transferase Mu 3, fatty acid binding protein, heat shock cognate 70 kDa protein, keratin, histone H4 and 14-3-3 alpha/beta showed protein spot intensity changes compared with controls in lumpfish skin at one or several time points during the one month period of crowding stress. The differentially expressed proteins are related to several metabolic pathways and are involved in stress and immune responses. Overall, the study shows that lumpfish can suffer from chronic stress with possible dire consequences for the animal welfare.

AFAP1 antisense RNA 1 promotes retinoblastoma progression by sponging microRNA miR-545-3p that targets G protein subunit beta 1

ABSTRACT The oncogenic role of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) has been reported in retinoblastoma (RB). However, the underlying regulatory mechanisms remain poorly understood. In this study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were performed to analyze the expression of AFAP1-AS1, microRNA miR-545-3p, or G protein subunit beta 1 (GNB1). Cell Counting Kit-8 (CCK-8) and Transwell migration assays were used to detect cell proliferation and migration. In addition, caspase-3 activity was monitored by caspase-3 activity assay. Luciferase reporter assays combined with RNA immunoprecipitation (RIP) and pull-down assays were performed to elucidate the target relationship between miR-545-3p and AFAP1-AS1 or GNB1. Xenograft tumor experiments were performed to evaluate RB cell growth in vivo. Increased AFAP1-AS1 and GNB1 expression in RB tissues and cells was confirmed by RT-qPCR; conversely, miR-545-3p was found to be downregulated in RB tissues and cells. AFAP1-AS1 overexpression resulted in increased proliferation and migration of RB cells, whereas AFAP1-AS1 silencing resulted in decreased proliferation and migration of RB cells. Moreover, AFAP1-AS1 was found to target miR-545-3p. The anti-miR-545-3p treatment phenocopied the effect of AFAP1-AS1 overexpression and promoted RB cell growth in vivo. miR-545-3p was found to directly target GNB1. GNB1 silencing resulted in reduced proliferation and migration of RB cells and attenuated the oncogenic effect of the miR-545-3p inhibitor. Thus, in this study, a novel ceRNA regulation network of AFAP1-AS1 in RB was identified, where AFAP1-AS1 regulated GNB1 expression by targeting miR-545-3p, ultimately driving RB progression.

Disrupting circ-GNB4 mitigates high glucose-induced human mesangial cells injury by regulating the proliferation, ECM accumulation, inflammation and oxidative stress through circ-GNB4/miR-23c/EGR1 pathway [RETRACTED

Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. Although G protein subunit beta 4 (GNB4)-derived circular RNA (circ-GNB4; hsa_circ_0068087) is a promising candidate biomarker in diabetes mellitus, whether circ-GNB4 participates in DN occurrence and development remains unknown. Herein, we focused on DN-associated human renal mesangial cells (HRMCs) injury, and HRMCs were exposed in high glucose (HG) condition. Using quantitative polymerase chain reaction and western blotting, we found that circ-GNB4 and early growth response factor 1 (EGR1) were upregulated, whereas microRNA (miR)-23c was downregulated in DN patients' sera and HG-stimulated HRMCs. HG-induced injuries were measured by MTS method, western blotting, enzyme-linked immunosorbent assay and other special assay kits. Consequently, HG could inhibit superoxide dismutase activity, but induce cell proliferation and levels of malondialdehyde, Fibronectin, Collagen I, Collagen IV, interleukin-6, interleukin-1β, and tumor necrosis factor-α. However, HG-induced these injuries were overall suppressed by silencing circ-GNB4 or overexpressing miR-23c. Moreover, miR-23c knockdown could counteract the effect of circ-GNB4 deficiency, and EGR1 restoration abrogated miR-23c overexpression role in HG-stimulated HRMCs. Notably, circ-GNB4 could target miR-23c and EGR1 was targeted by miR-23c, as confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. Moreover, EGR1 expression was positively modulated by circ-GNB4 via miR-23c. Collectively, circ-GNB4 might be a novel mechanism of DN-induced HRMCs injury, and there was a circ-GNB4/miR-23c/EGR1 pathway underlying the proliferation, extracellular matrix accumulation, inflammation and oxidative stress. This study suggested circ-GNB4 as a potential target to interfere the development of DN.

Comparative proteomic study of pig muscle proteins during growth and development of an animal

The production of high-quality pork is closely related to the growth and development of muscle tissue. The present article provides a comparative proteomic research of l. dorsi, b. femoris, m. brachiocephalicus during the pigs’ growth and development (at age of 60 days and 180 days). This work was supported by data of electrophoretic methods: one-dimensional electrophoresis according to Laemmli with densitometric assessment in the ImageJ software and two-dimensional electrophoresis according to O’Farrell method with its further processing on the software ImageMaster. The mass spectrometric identification was conducted with the help of the high-performance liquid chromatography (HPLC) system connected to a mass spectrometer; further the data were interpreted by search algorithm Andromeda. When comparing frequency diagrams of one-dimensional electrophoregrams of all three muscle tissues of weaned pigs, the greatest difference was observed for the muscle sample l. dorsi. Comparison of diagrams of muscle tissue samples taken for mature pigs showed a great similarity of all three studied muscles samples. Within the framework of the research, the Fold indicator was calculated. The exceeding its value by more than 2 units is generally considered to be a statistically significant difference. When analyzing two-dimensional electrophoretograms of weaned pigs’ muscles, 18 protein fractions were revealed with Fold &gt; 2. When examining the muscle tissue of mature pigs, 15 of those proteins were found; the differences were mostly detected in the minor protein fractions. The mass spectrometric analysis of the cut bands with well-pronounced differences from the onedimensional electrophoretogram revealed 214 proteins involved to a greater extent in cellular and metabolic processes, physical activity and localization. Growth and development protein — semaphorin‑6B (96.78 kDa) — was revealed in muscle tissue of l. dorsi, a. Also in l. dorsi and b. femoris the growth and development proteins were found: cadherin‑13 (78.23 kDa), cadherin‑7 (87.01 kDa), the F‑actin-cap protein beta subunit (30.66 kDa), and two uncharacterized proteins at 65.60 kDa and 63.88 kDa.

Analysis of cancer-promoting genes related to chemotherapy resistance in esophageal squamous cell carcinoma

BackgroundAccording to histopathology, esophageal cancer can be divided into squamous cell carcinoma (SCC) and esophageal adenocarcinoma (adeno arcinoma). In China, 90% of esophageal cancer patients are squamous cell carcinoma. Cisplatin and fluaziridine are the main chemotherapy before and after surgery. Long-term drug treatment is often accompanied by the emergence of drug resistance of tumor cells. There are many mechanisms for the emergence of drug resistance of tumor cells, including the increase of drug efflux, the decrease of drug intake, the inhibition of cell apoptosis, and so on. This study aimed to investigate the key cancer-promoting genes related to chemotherapy resistance in esophageal squamous cell carcinoma (ESCC).MethodsTwo datasets from the Gene Expression Omnibus (GEO) database (GSE86099 and GSE50224) were retrieved. We performed microRNA (miRNA) and messenger RNA (mRNA) expression analysis to identify differentially expressed genes (DEGs). The intersection of the downregulated miRNA targets and the upregulated mRNAs were used for Gene Ontology (GO) enrichment analysis, and survival risk was assessed using data from The Cancer Genome Atlas (TCGA).ResultsThere were 35 common genes, of which, based on GO enrichment, most were related to the cardiac muscle cell action. Four genes showed significant association with the estimated half-maximal inhibitory concentration (IC50) of paclitaxel: bone morphogenetic protein 1 (BMP1), dumbbell former 4 protein (DBF4), angiogenin (ANG), and MAP7 domain containing 2 (MAP7D2). Four risk factors (MP1, HIP1, ANG, and MAP7D2) were selected to generate a signature using least absolute shrinkage and selection operator (LASSO) regression. Protein-protein interaction (PPI) analysis showed guanine nucleotide-binding protein subunit beta 4 (GNB4), calcium voltage-gated channel auxiliary subunit beta 2 (CACNB2), and sodium voltage-gated channel alpha subunit 1 (SCN1A) were located at key positions of the network. Among potential risk genes, only the high expression of dedicator of cytokinesis 8 (DOCK8) was associated with poorer survival.ConclusionsThe 35 oncogenes may be involved in mechanisms of chemotherapy resistance in ESCC, as well as the corresponding enrichment and regulatory network. The signature containing 4 key risk genes merits further investigation and may provide a deeper understanding of the molecular mechanisms in ESCC treatment failure.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

First report of brown leaf spot on Juglans sigillata caused by Ophiognomonia leptostyla in Sichuan, China.

Juglans sigillata Dode (Iron walnut) is mostly distributed in southwestern China, and valued for wood and nuts (Feng et al. 2018). In April 2020, we surveyed a walnut garden located in Baisha Town, Wanyuan City, (Sichuan, China), where brown spot symptoms were observed on leaves of ten trees among of 100 plants, and this disease can result in a reduced growth potential when trees are severely infected. Necrotic and subcircular lesions with conidiamata were observed on diseased leaves. Symptomatic leaves were collected and taken back to the laboratory forfurther analysis. Using the single spore isolation technique developed by Chomnunti et al. (2014), five isolates were grown from the infected leaves on Potato Dextrose Agar medium (PDA). The five isolates had similar colony morphology, which was initially white, suborbicular, gradually turning yellowish with black spots, developing fluffy aerial mycelium. Morphological characteristics were examined using light microscopy on the PDA. Conidiogenous cells were subcylindrical to cylindrical, or ampulliform, hyaline, rarely branched. Macroconidia were lunate, reniform, hyaline, 1-3-septate, mostly 1-septate, distinctly constricted at the septum, the basal cell was bluntly rounded, the apical cell had an acute end, and the basal cell was equal to or larger than the apical cell, measuring 22 to 40.5 × 2.5 to 8.3 μm (mean = 32 × 6.2 μm, n = 50). Microconidia were botuliform, or subfusiform, hyaline, both ends were rounded, straight or curved, aseptate, and measured 10 to 28.5 × 1.9 to 3.7 μm (mean= 17.2 × 2.7 μm, n = 20). A multilocus approach was conducted for precise identification of a representative isolate SICAUCC 20-0012. The internal transcribed spacer regions (ITS), guanine nucleotide-binding protein subunit beta gene (MS204), and translation elongation factor 1-alpha (tef1-α) of isolate SICAUCC 20-0012 were amplified and sequenced as described by Sogonov et al. (2008) and Walker et al. (2012a). GenBank Accession Nos. for ITS, MS204, and tef1-α are MW250303, MW246773, and MW246775, respectively. Phylogenetic analyses showed 100% support with Ophiognomonia leptostyla (Fr.) Sogonov, and the morphology was consistent with the asexual stage of O. leptostyla documented by Walker et al. (2012b). To test Koch's postulates, five healthy plants of J. sigillata (2- to 3-year-old) with 5-8 leaves per plant were inoculated with conidial suspensions (104 conidia/mL) after wounded with a small pin as described by Desai et al. (2019), and the same number of healthy plants were wounded and sprayed with sterile distilled water as controls. Plants were sprayed regularly with distilled water every day and placed in a growth chamber at 25℃ with a 12-h fluorescent light/dark regimen. After 15 days, typical brown spot symptoms developed on inoculated leaves, but not on the controls. The fungus O. leptostyla was reisolated from the lesion as described above but not from non-inoculated leaves. O. leptostyla has been reported on some walnut trees; for example: J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. nigra, and J. regia (Farr & Rossman 2020). However, to our knowledge, this is the first report of O. leptostyla causing brown leaf spot on J. sigillata. J. sigillata is an economically important tree in southwest China, and fungicide treatments should be considered to prevent the spread of this fungus before it becomes more widespread. Chunlin Yang, Yu Deng, and Feihu Wang contributed equally to this work. This research was supported by the Key Research and Development Project of Sichuan Province (2021YFYZ0032).

First Report of Brown Leaf Spot of Juglans hybrid Caused by Ophiognomonia leptostyla in China.

"Chuanzao 2" is a walnut variety derived from the hybridization of Juglans regia L. and J. sigillata Dode distributed in southwest China, where it is an economically important tree species in rural regions (Xiao et al. 2012). In April 2020, the variety in a walnut garden showed symptoms of brown leaf spot in Beishan Town (107°21'43.93″E, 31°28'12.34″N), Dazhou City in Sichuan, China, with 5% to 10% of leaves per plant affected (5 plants). Symptomatic leaves showed brown to dark brown spots (2 to 5 mm) with a dark brown to black halo and grayish-tan center. The spots were subcircular to irregular in shape, and gradually expanded and formed necrotic spots. A single conidium isolation was performed (Senanayake et al. 2020) and transferred to Potato Dextrose Agar (PDA). Five isolates were obtained from five different infected leaves. Colonies of five isolates were subcircular, erose or dentate, flat or effuse, white initially, gradually becoming yellowish with white margins, developed and fluffy aerial mycelia, and conidiogenensis was produced underneath mycelia after 25-days-incubation. Conidiogenous cells were subcylindrical to cylindrical, or irregular in shape, and hyaline. Macroconidia were lunate, reniform, hyaline, basal cell bluntly rounded, apical cell with acute end, 1-septate, rarely aseptate, sometimes slightly constricted at septum, basal cell equal or larger than apical cell, and measured 16.5 to 30.5 × 5 to 8.5 μm (mean = 23.2 × 6.3 μm, n = 50). Microconidia were not observed. These morphological characteristics resembled those of Ophiognomonia leptostyla (Fr.) Sogonov (Walker et al. 2012a). For molecular identification, genomic DNA (isolates SICAUCC 21-0008 and SICAUCC 21-0010) was extracted, and the internal transcribed spacers (ITS) region, guanine nucleotide-binding protein subunit beta (MS204) gene, and translation elongation factor 1-alpha (tef1-α) were amplified and sequenced by using the primers ITS5/ITS4 (White et al. 1990), E1F1/E5R1a (Walker et al. 2012a), and EF1-728F/EF1-1567R (Walker et al. 2012b), respectively. Phylogenetic analyses (maximum likelihood) based on a combined dataset showed 100% bootstrap support values in a clade with O. leptostyla. The sequences of ITS, MS204, and tef1-α genes were deposited in GenBank with accession numbers MW493111/MZ026300, MW495270/MZ031975, and MW495271/MZ031974, respectively. To fulfill Koch's postulates, five healthy hybrid plants (2 to 3 years old) with 5 to 8 leaves per plant were spray inoculated with conidium suspensions (104 conidia/mL; isolate SICAUCC 21-0008) prepared from 40-days-old cultures onto the wounded sites via pin-prick inoculation. Similarly, five noninoculated plants sprayed with sterile water served as controls. Plants were placed in a growth chamber at 25℃ on a 12-h fluorescent light/dark regime and daily sprayed with sterile distilled water. After two weeks, observed symptoms were similar to those from natural infections. No disease symptoms were found on control plants. The fungus O. leptostyla was reisolated from the diseased leaves and characterized morphologically. O. leptostyla is a global pathogen and has been reported to cause the leaf spot in many walnut trees, viz. J. ailantifolia, J. californica, J. cinerea, and J. major, etc. To our knowledge, this is the first report of O. leptostyla causing brown leaf spot on Juglans hybrid (J. regia × J. sigillata) in China. The increasing risk of this pathogen in the walnut-growing areas of Sichuan Province of China needs a further exploration and outreach effort to develop effective control measures. Chunlin Yang, Feng Liu, and Qian Zeng contributed equally to this paper.

High G protein subunit beta 4 protein level is correlated to poor prognosis of urothelial carcinoma.

Data mining on a public domain detected eight potential transcripts which were upregulated in advanced UBUCs, suggesting that they may take part in UC development or/and progression. Retrospectively, immunohistochemistry along with H-score recording was carried out to evaluate the GNB4 protein levels on tissues from UC patients. Correlations between GNB4 H-score and imperative clinicopathological factors, as well as the implication of GNB4 protein level on disease-specific and metastasis-free survivals were assessed. In UTUCs (n = 340) and UBUCs (n = 295), 170 (50.0%) and 148 (50.0%) cases, respectively, were identified to be of high GNB4 expression. The GNB4 protein levels were correlated to numerous clinicopathological features and patients' survivals. Upregulation of the GNB4 protein was significantly associated with primary tumor, nodal metastasis, histological grade, vascular invasion and mitotic rate. High GNB4 protein levels independently and significantly predicted poor disease-specific and metastasis-free in UTUC and UBUC, respectively. Ingenuity pathway analysis furthermore showed that multiple signaling pathways were enriched including 'Communication between Innate and Adaptive Immune Cells' and 'NFκB Signaling'. Our findings demonstrated that the upregulation of the GNB4 protein is an independent unfavorable prognosticator in UC. High GNB4 gene expression plays an important role in UC progression.

A Zinc Metalloprotease nas-33 Is Required for Molting and Survival in Parasitic Nematode Haemonchus contortus.

Molting is of great importance for the survival and development of nematodes. Nematode astacins (NAS), a large family of zinc metalloproteases, have been proposed as novel anthelmintic targets due to their multiple roles in biological processes of parasitic nematodes. In this study, we report a well conserved nas-33 gene in nematodes of clade V and elucidate how this gene is involved in the molting process of the free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. A predominant transcription of nas-33 is detected in the larval stages of these worms, particularly in the molting process. Knockdown of this gene results in marked molecular changes of genes involved in cuticle synthesis and ecdysis, compromised shedding of the old cuticle, and reduced worm viability in H. contortus. The crucial role of nas-33 in molting is closely associated with a G protein beta subunit (GPB-1). Suppression of both nas-33 and gpb-1 blocks shedding of the old cuticle, compromises the connection between the cuticle and hypodermis, and leads to an increased number of sick and dead worms, indicating essentiality of this module in nematode development and survival. These findings reveal the functional role of nas-33 in nematode molting process and identify astacins as novel anthelmintic targets for parasitic nematodes of socioeconomic significance.