Protein 5A Research Articles

Synergistic Antibiotic Activity of Ricini Semen Extract with Oxacillin against Methicillin-Resistant Staphylococcus aureus.

Resistant bacteria are emerging as a critical problem in the treatment of bacterial infections by neutralizing antibiotic activity. The development of new traditional mechanisms of antibiotics is not the optimal solution. A more reasonable approach may be to use relatively safe, plant-based compounds in combination with conventional antibiotics in an effort to increase their efficacy or restore their activity against resistant bacteria. We present our study of mixing Ricini Semen extract, or its constituent fatty acids, with oxacillin and testing the effects of each on the growth of methicillin-resistant Staphylococcus aureus. Changes in the cell membrane fluidity of methicillin-resistant S. aureus were found to be a major component of the mechanism of synergistic antibiotic activity of Ricini Semen extract and its constituent fatty acids. In our model, changes in cellular membrane fluidity disrupted the normal function of bacterial signaling membrane proteins BlaR1 and MecR1, which are known to detect oxacillin, and resulted in the incomplete expression of penicillin-binding proteins 2a and β-lactamase. Utilizing the mechanism presented in this study presents the possibility of developing a method for treating antibiotic-resistant bacteria using traditional antibiotics with plant-based compounds.

Functional Characterization of Lobularia maritima LmTrxh2 Gene Involved in Cold Tolerance in Tobacco through Alleviation of ROS Damage to the Plasma Membrane.

Cold stress is a key environmental factor affecting plant growth and development, crop productivity, and geographic distribution. Thioredoxins (Trxs) are small proteins that are ubiquitously expressed in all organisms and implicated in several cellular processes, including redox reactions. However, their role in the regulation of cold stress in the halophyte plant Lobularia maritima remains unknown. We recently showed that overexpression of LmTrxh2, which is the gene that encodes the h-type Trx protein previously isolated from L. maritima, led to an enhanced tolerance to salt and osmotic stress in transgenic tobacco. This study functionally characterized the LmTrxh2 gene via its overexpression in tobacco and explored its cold tolerance mechanisms. Results of the RT-qPCR and western blot analyses indicated differential temporal and spatial regulation of LmTrxh2 in L. maritima under cold stress at 4 °C. LmTrxh2 overexpression enhanced the cold tolerance of transgenic tobacco, as evidenced by increased germination rate, fresh weight and catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities; reduced malondialdehyde levels, membrane leakage, superoxide anion (O2-), and hydrogen peroxide (H2O2) levels; and higher retention of chlorophyll than in non-transgenic plants (NT). Furthermore, the transcript levels of reactive oxygen species (ROS)-related genes (NtSOD and NtCAT1), stress-responsive late embryogenis abundant protein 5 (NtLEA5), early response to dehydration 10C (NtERD10C), DRE-binding proteins 1A (NtDREB1A), and cold-responsive (COR) genes (NtCOR15A, NtCOR47, and NtKIN1) were upregulated in transgenic lines compared with those in NT plants under cold stress, indicating that LmTrxh2 conferred cold stress tolerance by enhancing the ROS scavenging ability of plants, thus enabling them to maintain membrane integrity. These results suggest that LmTrxh2 promotes cold tolerance in tobacco and provide new insight into the improvement of cold-stress resistance to cold stress in non-halophyte plants and crops.

Candidate genes affecting stomatal density in rice (Oryza sativa L.) identified by genome‐wide association

Stomata regulate photosynthesis and water loss. They have been an active subject of research for centuries, but our knowledge of the genetic components that regulate stomatal development in crops remains very limited in comparison to the model plant Arabidopsis thaliana. Leaf stomatal density was found to vary by over 2.5-fold across a panel of 235 rice accessions. Using GWAS, we successfully identified five different QTLs associated with stomatal density on chromosomes 2, 3, 9, and 12. Forty-two genes were identified within the haplotype blocks corresponding to these QTLs. Of these, nine genes contained haplotypes that were associated with different stomatal densities. These include a gene encoding a trehalose-6-phosphate synthase, an enzyme that has previously been associated with altered stomatal density in Arabidopsis, and genes encoding a B-BOX zinc finger family protein, a leucine-rich repeat family protein, and the 40 S ribosomal protein S3a, none of which have previously been linked to stomatal traits. We investigated further and show that a closely related B-BOX protein regulates stomatal development in Arabidopsis. The results of this study provide information on genetic associations with stomatal density in rice. The QTLs and candidate genes may be useful in future breeding programs for low or high stomatal density and, consequently, improved photosynthetic capacity, water use efficiency, or drought tolerance.

Whole-genome sequencing of foot-and-mouth disease virus serotype O/PanAsia-2/QOM-15 and comparison of its VP1-encoding region with two vaccine strains.

Despite widespread vaccination against foot-and-mouth disease, many outbreaks still occur in endemic areas. We attempted to determine the genetic and antigenic properties of the O/PanAsia-2/QOM-15 foot-and-mouth disease virus new vaccine strain. Thus, whole-genome sequencing was used to identify vulnerable pinpoint sites across the genome. The VP1 sequence (1D gene) of the O/PanAsia-2/QOM-15 viral genome was then compared to the VP1 sequences of two previously used vaccine strains, O/PanAsia (JQ321837) and O/PanAsia-2 (JN676146). The antigenic relationship of these three viruses was calculated by the two dimensional-virus neutralization test. At the nucleotide level, 47 single variants were identified, of which 19.00% were in the 5' untranslated region (UTR), 79.00% in the polyprotein region, and 2.00% in the 3' UTR region. Approximately half of the single nucleotide polymorphisms that have occurred in 1D gene resulted in amino acid (AA) substitutions in the VP1 structure. The single nucleotide polymorphisms also caused AA substitutions in other structural proteins, including VP2 and VP3, and some non-structural proteins (Lpro, 2C, and 3A). The O/PanAsia-2/QOM-15 shared higher sequence similarity with O/PanAsia-2 (91.00%) compared to O/PanAsia (87.30%). Evaluating r-value showed that the antigenic relationship of O/PanAsia-2/QOM-15 with O/PanAsia-2 (29.00%) was greater than that of the O/PanAsia (24.00%); however, all three viruses were immunologically distinct. After 10 years, the alteration of virus antigenicity and the lack of detectable adaptive pressure on VP1 sequence suggest that studying genetic dynamics beyond the VP1 region is necessary to evaluate FMDV pathogenicity and vaccine failure.

150. Low Propensity for Development of Spontaneous In Vitro Resistance to Tebipenem, Ertapenem and Meropenem Among Enterobacterales Uropathogens

Abstract Background Tebipenem (TBP), an oral carbapenem in development for treatment of complicated urinary tract infections and acute pyelonephritis, is active vs. uropathogens which produce extended-spectrum beta-lactamases (ESBLs) such as Escherichia coli (Ec) and Klebsiella pneumoniae (Kp). Bacteria acquire resistance to antibiotics via multiple routes, including by chromosomal mutation. In this study, spontaneous resistance to TBP among Enterobacterales, including those resistant to clinically relevant antibacterials, was assessed Methods The frequency of resistance (FOR) to TBP, meropenem (MEM) and ertapenem (ETP) was assessed for Ec (n = 8), Kp (n = 9), and other Enterobacterales (n = 7). Approximately 108 CFU were plated on agar containing 4x or 8x the MIC and incubated for 24h. Resistance was confirmed by MIC testing (CLSI M07Ed11). Serial passages were performed with two Ec and two Kp isolates, each of which included a wild type and ESBL+ isolate, using TBP, MEM and ETP over a 14-day period. Whole genome sequencing was conducted and assembled mutant sequences were aligned vs. β-lactamase-encoding genes, porins OmpF/OmpK35, OmpC/OmpK36, and OmpK37 (Kp only) and penicillin binding proteins (PBPs) 1a, 1b, 2, and 3 and compared with parent strains. Results FOR values for TBP, MEM and ETP at 4x to 8x the MIC ranged from < 6.87 × 10-9 to 5.54 × 10-5, with similar results obtained for all species and genotypes, including ESBL+ isolates and those with porin alterations. In the serial passage, MICs for each agent vs. four isolates over the 14-day period were ∼ five to seven 2x dilutions higher than baseline, resulting in similar MIC values to those observed in the FOR studies (0.12 to 2 µg/mL). Mutants isolated in the presence of TBP, MEM or ETP had similarly decreased susceptibility to each compound. In general, mutants selected during the single- and multi-step resistance studies showed alterations of OmpC in Ec and its homologue OmpK36 in Kp. Conclusion These data suggest TBP has a low propensity for spontaneous resistance at 4x to 8x MIC in vitro and was similar to MEM and ETP in both single-step and serial passage studies. Cross-resistance to TBP, MEM or ETP among mutants suggests common mechanisms contribute to elevated MICs, including through reduced permeability. Disclosures Brian D. VanScoy, B.S., Adagio Therapeutics, Inc.: Grant/Research Support|Amplyx Pharmaceuticals, Inc.: Grant/Research Support|AN2 Therapeutics,: Grant/Research Support|Antabio SAS: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc.: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceuticals: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Haley Conde, B.S., Adagio Therapeutics, Inc.: Grant/Research Support|Amplyx Pharmaceuticals, Inc: Grant/Research Support|AN2 Therapeutics,: Grant/Research Support|Antabio SAS,: Grant/Research Support|Arcutis Biotherapeutics, Inc.: Grant/Research Support|B. Braun Medical Inc.: Grant/Research Support|Basilea Pharmaceutica,: Grant/Research Support|Boston Pharmaceuticals: Grant/Research Support|Celdara Medical LLC: Grant/Research Support|Cidara Therapeutics Inc: Grant/Research Support|Cipla USA: Grant/Research Support|Crestone Inc.: Grant/Research Support|CXC,: Grant/Research Support|Debiopharm International SA: Grant/Research Support|Entasis Therapeutics: Grant/Research Support|Evopoint Biosciences Co.: Grant/Research Support|Fedora Pharmaceuticals: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Hoffmann-La Roche: Grant/Research Support|Insmed Inc.: Grant/Research Support|Iterum Therapeutics Limited,: Grant/Research Support|Kaizen Bioscience, Co.: Grant/Research Support|KBP Biosciences USA: Grant/Research Support|Lassen Therapeutics: Grant/Research Support|Matinas Biopharma,: Grant/Research Support|Meiji Seika Pharma Co., Ltd.: Grant/Research Support|Melinta Therapeutics: Grant/Research Support|Menarini Ricerche S.p.A: Grant/Research Support|Mutabilis: Grant/Research Support|Nabriva Therapeutics AG: Grant/Research Support|Novartis Pharmaceuticals Corp.: Grant/Research Support|Paratek Pharmaceuticals, Inc.: Grant/Research Support|PureTech Health: Grant/Research Support|Sfunga Therapeutics: Grant/Research Support|Spero Therapeutics: Grant/Research Support|Suzhou Sinovent Pharmaceuticals Co.: Grant/Research Support|TauRx Therapeutics: Grant/Research Support|Tetraphase Pharmaceutical: Grant/Research Support|tranScrip Partners: Grant/Research Support|Utility Therapeutics: Grant/Research Support|Valanbio Therapeutics, Inc.: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt Bio AG: Grant/Research Support Rodrigo E. Mendes, PhD, Spero Therapeutics: Grant/Research Support Nicole Cotroneo, B.S., Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds Ian A. Critchley, PhD, Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds Ian A. Critchley, PhD, Spero Therapeutics: Employee|Spero Therapeutics: Stocks/Bonds.

In silico and in vitro arboviral MHC class I-restricted-epitope signatures reveal immunodominance and poor overlapping patterns.

The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as "hot-spots". Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

Identification of immuno-infiltrating MAP1A as a prognosis-related biomarker for bladder cancer and its ceRNA network construction.

Approximately 75% of bladder cancer occurrences are of the non-muscle-invasive type. The estimated five-year survival rate is 26%-55%. Currently, there is no reliable biomarker available for early diagnosis and prognosis of bladder cancer. The present study aims to identify a biomarker using bioinformatic approaches to provide a new insight in clinical research for early diagnosis and prognosis of bladder cancer. Clinical data and a transcriptome of bladder cancer were obtained from TCGA, GEO, GETx, and UCSC Xena. The differential expressed gene (DEG) analysis, weighted gene co-expression network analysis (WGCNA), and survival analysis using the Kaplan-Meier and Cox proportional-hazards models were used to identify the Microtubule-associated Proteins 1A (MAP1A). on overall survival (OS) and disease-free survival (DFS) was analyzed using GEPIA and GETx databases. The TIMER 2.0 database predicted the correlation between MAP1A and immunocytes and immune checkpoints. Target prediction of the regulated competing endogenous RNAs (ceRNAs) network of MAP1A was performed using starBase and TargetScan. Cystoscope v3.7.2 software was used to visualize the ceRNA coexpression network. The R programming language v4.0.2 was applied as an analytic tool. Gene expression of MAP1A verified by RT-qPCR. The low expression of MAP1A was verified in bladder cancer tissues and bladder cancer cell lines SW780 and 5637.P < 0.001 were obtained by Kaplan-Meier survival analysis and Cox proportional hazards model, with a hazard ratio (HR) of 1.4. Significant correlations between MAP1A and OS (P < 0.001, HR = 1.9) as well as DFS (P < 0.05, HR = 1.7) in bladder cancer were identified through gene expression profiling interactive analysis (GEPIA), indicating MAP1A may be a high-risk factor. Significant correlation in single copy-number variation of MAP1A gene with CD8+ T cells, and myeloid dendritic cells (MDCs) (P < 0.05) was noted. MAP1A expression was shown to be significantly correlated with the amount of CD4+ T cells and CD8+ T cells, MDCs, macrophages, and neutrophils in a statistically significant positive manner (P < 0.001). However, the MAP1A expression demonstrated a strong negative connection with B cells (P < 0.001). Except for macrophage M1 genes IRF5 and PTGS2, MAP1A expression was significantly correlated with the gene levels in immunocytes such as CD4+ T cells, CD8+ T cells, B cells, dendritic cells (DCs), macrophages, and neutrophils (Cor > 0.2, P < 0.001), as well as immune checkpoint related genes including cytotoxic t-lymphocyte-associated protein 4 (CTLA-4), programmed death 1 (PD-1), programmed death ligand 1 (PD-L1) (P < 0.001). Finally, we predicted that the MAP1A-interacting miRNA was miR-34a-5p, and the MAP1A endogenous competing RNAs were LNC00667, circ_MAP1B, and circ_MYLK, respectively. These findings support the need for further studies on the mechanism underlying the pathogenesis of this disease. MAP1A is considered as a prospective biomarker for early diagnosis, therapeutic observation, and prognosis analysis in bladder cancer.

Integrative analysis of m3C associated genes reveals METTL2A as a potential oncogene in breast Cancer

RNA methylation modifications, especially m6A mRNA modification, are known to be extensively involved in tumor development. However, the relationship between N3-methylcytidine (m3C) related genes and tumorigenesis has rarely been studied. In this research, we found that m3C-related genes were expressed at different levels and affected patients’ prognosis across multiple cancer types from The Cancer Genome Atlas and multi-omics levels. Importantly, methyltransferase-like proteins 2A (METTL2A) had a high amplification frequency (~ 7%) in patients with breast invasive carcinoma (BRCA), and its overexpression was an independent predictor of poor overall survival. Enrichment analysis of associated genes revealed that METTL2A may activate DNA synthesis and cell proliferation pathways in BRCA cells. Through drug sensitivity analysis, Trifluridine, PD407824, and Taselisib were shown to be effective drugs for METTL2A-positive BRCA patients. Overall, our research conducts a holistic view of the expression level and prognostic signature of m3C-related genes with multiple malignancies. Importantly, METTL2A has been intensely explored as a potential oncogene in BRCA, to aid the development of potential drug agents for precision therapy in breast cancer patients.

Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction induced by abdominal aorta constriction through cyclic guanosine monophosphate-dependent protein kinase Signaling Pathway.

To determine whether Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction (HFpEF) by regulating the cyclic guanosine monophosphate-dependent protein kinase (cGMP-PKG) signaling pathway. Except for control group 8 and sham surgery group 8, the remaining 32 male Sprague-Dawlay rats were developed into HFpEF rat models using the abdominal aorta constriction method. These rats in the HFpEF model were randomly divided into the model group, the Shunxin high-dose group, the Shunxin low-dose group, and the Qiliqiangxin capsule group. The three groups received high-dose Shunxin decoction, low-dose Shunxin decoction, and Qiliqiangxin capsule by gavage, respectively, for 14 d. After the intervention, the diastolic function of each rat was evaluated by testing E/A, heart index, hematoxylin-eosin staining, Masson, myocardial ultrastructure, and N-terminal pro-brain natriuretic peptide (NT-proBNP). The Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) software was used to predict targets for which Shunxin decoction acts on the cGMP-PKG pathway. Natriuretic peptide receptor A (NPRA) and guanylate cyclase (GC) were detected by immunohistochemistry, and eNOS, phosphodiesterase 5A (PDE5A), and cGMP-dependent protein kinase 1(PKG I) were determined by Western blotting. Compared to the model group, the thickness of the interventricular septum at the end of diastole (IVSd) and the thickness of the posterior wall at the end of diastole (PWd) of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group were all significantly reduced ( < 0.01). Furthermore, Shunxin decoction high-dose group E/A value was decreased ( < 0.01). Compared to the model group, the expression of NPRA and GC increased in the Shunxin decoction low-dose group and the Qiliqiangxin capsule group ( < 0.01). Compared to the model group, the expressions of eNOS and PKG I increased ( < 0.05) in the Shunxin decoction high-dose group. The expression of PDE5A expression decreased in the myocardium of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group compared to the model group ( < 0.01). Shunxin decoction can improve diastolic function in rats with HFpEF. It increases the expression of NPRA, GC, and eNOS in the myocardial cell cGMP-PKG signaling pathway, upregulates cGMP expression, decreases PDE5A expression to reduce the cGMP degradation. Thus, the cGMP continually stimulates PKG I, reversing myocardial hypertrophy and improving myocardial compliance in HFpEF rats.

Serum Level of Wingless Integration 5a Protein in Rheumatoid Arthritis

Serum Level of Wingless Integration 5a Protein in Rheumatoid Arthritis

Heat Shock Protein 60 Is Involved in Viral Replication Complex Formation and Facilitates Foot and Mouth Virus Replication by Stabilizing Viral Nonstructural Proteins 3A and 2C.

ABSTRACTThe maintenance of viral protein homeostasis depends on the machinery of the infected host cells, giving us an insight into the interplay between host and virus. Accumulating evidence suggests that heat shock protein 60 (HSP60), as one molecular chaperone, is involved in regulating virus infection. However, the role of HSP60 during foot-and-mouth disease virus (FMDV) replication and its specific mechanisms have not been reported. We demonstrate that HSP60 modulates the FMDV life cycle. HSP60 plays a role at the postentry stage of the viral life cycle, including RNA replication and mRNA translation; however, HSP60 does not affect viral replication of Seneca Valley virus (SVA) or encephalomyocarditis virus (EMCV). We found that HSP60 is involved in FMDV replication complex (RC) formation. Furthermore, our results indicate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, key elements of the viral replication complex. We also show that HSP60 regulates the stability of 3A and 2C via caspase-dependent and autophagy-lysosome-dependent degradation, thereby promoting FMDV RNA synthesis and mRNA translation mediated by the RC. Additionally, we determined that the apical domain of HSP60 is responsible for interacting with 3A and 2C. The N terminus of 3A and ATPase domain of 2C are involved in binding to HSP60. Importantly, HSP60 depletion potently reduced FMDV pathogenicity in infected mice. Altogether, this study demonstrates a specific role of HSP60 in promoting FMDV replication. Furthermore, targeting host HSP60 will help us design the FMDV-specific antiviral drugs.

CpG Oligodeoxynucleotides Inhibit RANKL-Induced Osteoclast Formation by Upregulating A20 Deubiquitinase in RAW 264.7 Cells.

Objective Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1β siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.

Transcriptomic Analysis of Human Keratinocytes Treated with Galactomyces Ferment Filtrate, a Beneficial Cosmetic Ingredient.

Galactomyces ferment filtrate (GFF, Pitera™) is a cosmetic ingredient known to have multiple skin care benefits, such as reducing redness and pore size via the topical application of its moisturizer form. Although GFF is known to act partly as an antioxidative agonist for the aryl hydrocarbon receptor (AHR), its significance in keratinocyte biology is not fully understood. In this study, we conducted a transcriptomic analysis of GFF-treated human keratinocytes. Three different lots of GFF consistently modulated 99 (22 upregulated and 77 downregulated) genes, including upregulating cytochrome P450 1A1 (CYP1A1), a specific downstream gene for AHR activation. GFF also enhanced the expression of epidermal differentiation/barrier-related genes, such as small proline-rich proteins 1A and 1B (SPRR1A and SPRR1B), as well as wound healing-related genes such as serpin B2 (SERPINB2). Genes encoding components of tight junctions claudin-1 (CLDN1) and claudin-4 (CLDN4) were also target genes upregulated in the GFF-treated keratinocytes. In contrast, the three lots of GFF consistently downregulated the expression of inflammation-related genes such as chemokine (C-X-C motif) ligand 14 (CXCL14) and interleukin-6 receptor (IL6R). These results highlight the beneficial properties of GFF in maintaining keratinocyte homeostasis.

The Effects of Cucumber Mosaic Virus and Its 2a and 2b Proteins on Interactions of Tomato Plants with the Aphid Vectors Myzus persicae and Macrosiphum euphorbiae.

Cucumber mosaic virus (CMV), a major tomato pathogen, is aphid-vectored in the non-persistent manner. We investigated if CMV-induced volatile organic compounds (VOCs) or other virus-induced cues alter aphid–tomato interactions. Y-tube olfactometry showed that VOCs emitted by plants infected with CMV (strain Fny) attracted generalist (Myzus persicae) and Solanaceae specialist (Macrosiphum euphorbiae) aphids. Myzus persicae preferred settling on infected plants (3 days post-inoculation: dpi) at 1h post-release, but at 9 and 21 dpi, aphids preferentially settled on mock-inoculated plants. Macrosiphum euphorbiae showed no strong preference for mock-inoculated versus infected plants at 3 dpi but settled preferentially on mock-inoculated plants at 9 and 21 dpi. In darkness aphids showed no settling or migration bias towards either mock-inoculated or infected plants. However, tomato VOC blends differed in light and darkness, suggesting aphids respond to a complex mix of olfactory, visual, and other cues influenced by infection. The LS-CMV strain induced no changes in aphid–plant interactions. Experiments using inter-strain recombinant and pseudorecombinant viruses showed that the Fny-CMV 2a and 2b proteins modified tomato interactions with Macrosiphum euphorbiae and Myzus persicae, respectively. The defence signal salicylic acid prevents excessive CMV-induced damage to tomato plants but is not involved in CMV-induced changes in aphid–plant interactions.

Variability among the Isolates of Broad Bean Mottle Virus and Encapsidation of Host RNAs

Broad bean mottle bromovirus infects legume plants and is transmissible by insects. Several broad bean mottle virus (BBMV) isolates have been identified, including one in England (isolate Ba) and five in the Mediterranean countries: Libya (LyV), Morocco (MV), Syria (SV), Sudan (TU) and Tunisia (TV). Previously, we analyzed the nucleotide sequence of the Ba RNA and here we report on and compare it with another five Mediterranean variants. The RNA segments in the latter ones were extensively homologous, with some SNPs, single nucleotide deletions and insertions, while the number of mutations was higher in isolate Ba. Both the 5′ and 3′ untranslated terminal regions (UTRs) among the corresponding RNAs are highly conserved, reflecting their functionality in virus replication. The AUG initiation codons are within suboptimal contexts, possibly to adjust/regulate translation. The proteins 1a, 2a, 3a and coat protein (CP) are almost identical among the five isolates, but in Ba they have more amino acid (aa) substitutions. Phylogenetic analysis revealed the isolates from Morocco and Syria clustering with the isolate from England, while the variants from Libya, Tunisia and Sudan created a different clade. The BBMV isolates encapsidate a high content of host (ribosomal and messenger) RNAs. Our studies present BBMV as a useful model for bromoviruses infecting legumes.

Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system

The enterovirus Coxsackievirus B3 (CVB3) is known to be a major source for the development of cardiac dysfunctions like viral myocarditis (VMC) and dilatative cardiomyopathy (DCM), but also results in bradycardia and fatal cardiac arrest. Besides clinical reports on bradycardia and sudden cardiac death, very little is known about the influence of CVB3 on the activity of human cardiac pacemaker cells. Here, we address this issue using the first human induced pluripotent stem cell (hiPSC)-derived pacemaker-like cells, in which the expression of a transgenic non-infectious variant of CVB3 can be controlled dose- and time-dependently. We found that CVB3 drastically changed hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) distribution and function in hiPSC-derived pacemaker-like tissue. In addition, using HCN4 cell expression systems, we found that HCN4 currents were decreased with altered voltage dependency of activation when CVB3 was expressed. Increased autophagosome formation and autophagosomal HCN4 insertion was observed in hiPSC-derived pacemaker-like cells under CVB3 expression as well. Individual effects of single, non-structural CVB3 proteins were analyzed and demonstrated that CVB3 proteins 2C and 3A had the most robust effect on HCN4 activity. Treatment of cells with the Rab7 inhibitor CID 106770 or the CVB3-3A inhibitor GW5074 led to the recovery of the cytoplasmatic HCN4 accumulation into a healthy appearing phenotype, indicating that malfunctioning Rab7-directed autophagosome transport is involved in the disturbed, cytoplasmatic HCN4 accumulation in CVB3-expressing human pacemaker-like cells. Summarizing, the enterovirus CVB3 inhibits human cardiac pacemaker function by reducing the pacemaker channel plasma membrane density, an effect that can be corrected by pharmacological intervention of endocytic vesicle trafficking.

Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua

Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.

The relative binding position of Nck and Grb2 adaptors impacts actin-based motility of Vaccinia virus.

Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to assemble complex signalling networks. The concept of phase separation has recently changed our appreciation of multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now investigated this parameter in the operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3 complex-mediated actin polymerisation. Manipulating the position of pTyr motifs in A36 and the unrelated p14 from Orthoreovirus, we find that only specific spatial arrangements of Nck and Grb2 binding sites result in robust N-WASP recruitment, Arp2/3 complex driven actin polymerisation and viral spread. This suggests that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are frequently conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those undergoing phase transitions.

Molecular profiling of kidney compartments from serial biopsies differentiate treatment responders from non-responders in lupus nephritis

The immune pathways that define treatment response and non-response in lupus nephritis (LN) are unknown. To characterize these intra-kidney pathways, transcriptomic analysis was done on protocol kidney biopsies obtained at flare (initial biopsy (Bx1)) and after treatment (second biopsy (Bx2)) in 58 patients with LN. Glomeruli and tubulointerstitial compartments were isolated using laser microdissection. RNA was extracted and analyzed by nanostring technology with transcript expression from clinically complete responders, partial responders and non-responders compared at Bx1 and Bx2 and to the healthy controls. Top transcripts that differentiate clinically complete responders from non-responders were validated at the protein level by confocal microscopy and urine ELISA. At Bx1, cluster analysis determined that glomerular integrin, neutrophil, chemokines/cytokines and tubulointerstitial chemokines, T cell and leukocyte adhesion genes were able to differentiate non-responders from clinically complete responders. At Bx2, glomerular monocyte, extracellular matrix, and interferon, and tubulointerstitial interferon, complement, and T cell transcripts differentiated non-responders from clinically complete responders. Protein analysis identified several protein products of overexpressed glomerular and tubulointerstitial transcripts at LN flare, recapitulating top transcript findings. Urine complement component 5a and fibronectin-1 protein levels reflected complement and fibronectin expression at flare and after treatment. Thus, transcript analysis of serial LN kidney biopsies demonstrated how gene expression in the kidney changes with clinically successful and unsuccessful therapy. Hence, these insights into the molecular landscape of response and non-response may help align LN management with the pathogenesis of kidney injury.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.