A contact-based analysis of local energetic frustration dynamics identifies key residues enabling RfaH fold-switch.

Fold-switching enables metamorphic proteins to reversibly interconvert between two highly dissimilar native states to regulate their protein functions. While about 100 proteins have been identified to undergo fold-switching, unveiling the key residues behind this mechanism for each protein remains challenging. Reasoning that fold-switching in proteins is driven by dynamic changes in local energetic frustration, we combined fold-switching simulations generated using simplified structure-based models with frustration analysis to identify key residues involved in this process based on the change in the density of minimally frustrated contacts during refolding. Using this approach to analyze the fold-switch of the bacterial transcription factor RfaH, we identified 20 residues that significantly change their frustration during its fold-switch, some of which have been experimentally and computationally reported in previous works. Our approach, which we developed as an additional module for the FrustratometeR package, highlights the role of local frustration dynamics in protein fold-switching and offers a robust tool to enhance our understanding of other proteins with significant conformational shifts.

From iPSCs to Pancreatic ß Cells: Unveiling Molecular Pathways and Enhancements with Vitamin C and Retinoic Acid in Diabetes Research.

Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.

Hepatitis B virus X protein differentially regulates the angiogenesis of Hepatocellular Carcinoma through p53-VEGF axis according to glucose levels.

INTRODUCTION AND OBJECTIVES: Blood glucose fluctuates severely in the diabetes (DM) and tumor microenvironment. Our previous works have found Hepatitis B virus X protein (HBx) differentially regulated metastasis and apoptosis of hepatoma cells depending on glucose concentration. We here aimed to explore whether HBx played dual roles in the angiogenesis of hepatocellular carcinoma varying on different glucose levels. MATERIALS AND METHODS: We collected conditioned medium from HBx-overexpressing cells cultured with two solubilities of glucose, and then applied to EA.hy926 cells. Alternatively, a co-culture cell system was established with hepatoma cells and EA.hy926 cells. We analyzed the angiogenesis of EA.hy926 cells with CCK8, wound-healing, transwell-migartion and tube formation experiment. ELISA was conducted to detect the secretion levels of angiogenesis-related factors. siRNAs were used to detect the P53-VEGF axis. RESULTS: HBx expressed in hepatoma cells suppressed VEGF secretion, and subsequently inhibited the proliferation, migration and tube formation of EA.hy926 cells in a high glucose condition, while attenuating these in the lower glucose condition. Furthermore, the p53-VEGF axis was required for the dual role of HBx in angiogenesis. Additionally, HBx mainly regulated the nuclear p53. CONCLUSIONS: These data suggest that the dual roles of HBx confer hepatoma cells to remain in a glucose-rich environment and escape from the glucose-low milieu through tumor vessels, promoting liver tumor progression overall. We exclusively revealed the dual role of HBx on the angiogenesis of liver tumors, which may shed new light on the mechanism and management strategy of HBV- and DM-related hepatocellular carcinoma.

Interactions between the AraC/XylS-like transcriptional activator InvF of Salmonella Typhimurium, the RNA polymerase alpha subunit and the chaperone SicA.

Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.

Proteomic analysis reveals the global effect of the BarA/SirA-Csr regulatory cascade in Salmonella Typhimurium grown in conditions that favor the expression of invasion genes.

In many bacteria, the BarA/SirA and Csr regulatory systems control expression of genes encoding a wide variety of cellular functions. The BarA/SirA two-component system induces the expression of CsrB and CsrC, two small non-coding RNAs that sequester CsrA, a protein that binds to target mRNAs and thus negatively or positively regulates their expression. BarA/SirA and CsrB/C induce expression of the Salmonella Pathogenicity Island 1 (SPI-1) genes required for Salmonella invasion of host cells. To further investigate the regulatory role of the BarA/SirA and Csr systems in Salmonella, we performed LC-MS/MS proteomic analysis using the WT S. Typhimurium strain and its derived ΔsirA and ΔcsrB ΔcsrC mutants grown in SPI-1-inducing conditions. The expression of 164 proteins with a wide diversity, or unknown, functions was significantly affected positively or negatively by the absence of SirA and/or CsrB/C. Interestingly, 19 proteins were identified as new targets for SirA-CsrB/C. Our results support that SirA and CsrB/C act in a cascade fashion to regulate gene expression in S. Typhimurium in the conditions tested. Notably, our results show that SirA-CsrB/C-CsrA controls expression of proteins required for the replication of Salmonella in the intestinal lumen, in an opposite way to its control exerted on the SPI-1 proteins. SIGNIFICANCE: The BarA/SirA and Csr global regulatory systems control a wide range of cellular processes, including the expression of virulence genes. For instance, in Salmonella, BarA/SirA and CsrB/C positively regulate expression of the SPI-1 genes, which are required for Salmonella invasion to host cells. In this study, by performing a proteomic analysis, we identified 164 proteins whose expression was positively or negatively controlled by SirA and CsrB/C in SPI-1-inducing conditions, including 19 new possible targets of these systems. Our results support the action of SirA and CsrB/C in a cascade fashion to control different cellular processes in Salmonella. Interestingly, our data indicate that SirA-CsrB/C-CsrA controls inversely the expression of proteins required for invasion of the intestinal epithelium and for replication in the intestinal lumen, which suggests a role for this regulatory cascade as a molecular switch for Salmonella virulence. Thus, our study further expands the insight into the regulatory mechanisms governing the virulence and physiology of an important pathogen.

The role of Calmodulin Binding Transcription Activator 1 (CAMTA1) gene and its putative genetic partners in the human nervous system.

The Calmodulin Binding Transcription Activator 1 (CAMTA1) gene plays a central role in the human nervous system. Here evidence-based perspectives on its clinical value for the screening of CAMTA1 malfunction is provided and argued that in future, patients suffering from brain tumours and/or neurological disorders could benefit from this diagnostic. In neuroblastomas as well as in low-grade gliomas, the influence of reduced expression of CAMTA1 results in opposite prognosis, probably because of different carcinogenic pathways in which CAMTA1 plays different roles, but the exact genetics bases remains unsolved. Rearrangements, mutations and variants of CAMTA1 were associated with human neurodegenerative disorders, while some CAMTA1 single nucleotide polymorphisms were associated with poorer memory in clinical cases and also amyotrophic lateral sclerosis. So far, the follow-up of patients with neurological diseases with alterations in CAMTA1 indicates that defects (expression, mutations, and rearrangements) in CAMTA1 alone are not sufficient to drive carcinogenesis. It is necessary to continue studying CAMTA1 rearrangements and expression in more cases than done by now. To understand the influence of CAMTA1 variants and their role in nervous system tumours and in several psychiatric disorders is currently a challenge.

Allosteric couplings upon binding of RfaH to transcription elongation complexes.

In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.

Fmr1 exon 14 skipping in late embryonic development of the rat forebrain.

BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

HBX suppresses PTEN to promote the malignant progression of hepatocellular carcinoma through mi-R155 activation.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common and fatal tumors in the world, ranking third in cancer-related mortality. Chronic HBV infection is one of the major risk factors for hepatocellular carcinoma in China, Korea, and Sub-Saharan Africa. The HBx protein encoded by the X gene of HBV is a broadly regulated protein involved in transcriptional activation, epigenetics, apoptosis, DNA repair, and other regulatory processes. This study aimed to investigate the mechanism of HBx regulation of miR-155 and PTEN (Phosphatase and tensin homolog deleted on chromosome ten) in HBV-HCC. METHODS: Exosomal miR-155 quantity was analyzed by sampling serum exosomes of patients with hepatocellular carcinoma and normal subjects. The analysis was divided into different subgroups according to HBV positivity or negativity. At the cellular level, the biological roles of HBX, microRNA-155 and PTEN on hepatocellular carcinoma cells and their regulatory relationships with each other were verified. RESULTS: MicroRNA-155 and PTEN expression in HBV-positive HCC liver cancer tissues were negatively correlated, and HBX and miR-155 expression were positively correlated; microRNA-155 could target and inhibit PTEN expression, thereby promoting hepatocellular carcinoma cell activity, inhibiting apoptosis, and promoting invasion and migration; HBX could upregulate microRNA-155 thereby inhibit PTEN to promote malignant transformation of hepatocellular carcinoma. CONCLUSIONS: HBX could promote malignant transformation of hepatocellular carcinoma cells by upregulating microRNA-155 expression and thereby inhibiting the PTEN/PI3K-AKT pathway. Blocking miR-155 expression could attenuate the proliferation-promoting and invasive effects of HBX.

Delivery of superoxide dismutase by TAT and abalone peptides for the protection of skin cells against oxidative stress.

Trichoderma reesei superoxide dismutase (TrSOD) is a well-characterized enzyme being stable between 30 and 90°C for 1 h with activity at pH between 2.6 and 9.0. This work aimed to clone, express, purify, and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein), and Ab-TrSOD were expressed in E. coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3, and 6 µmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide and evaluated for reactive oxygen species (ROS) generation, in the presence or not of the recombinant enzymes. TAT-TrSOD-Yfp was able to decrease the generation of ROS by 15% when used in the concentrations of 3 and 6 µmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19% and 14% at the concentrations of 1 and 6 µmol/L, respectively, indicating that this recombinant form was more effective in reducing oxidative stress compared to SOD without the cell-penetrating peptide (CPP). Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development of products for antioxidant protection of the skin that may have therapeutic and cosmetic application.

IFN-γ affects pancreatic cancer properties by MACC1-AS1/MACC1 axis via AKT/mTOR signaling pathway.

BACKGROUND: Metastasis-related in colon cancer 1 (MACC1) is highly expressed in a variety of solid tumours, but its role in pancreatic cancer (PC) remains unknown. Interferon gamma (IFN-γ) affecting MACC1 expression was explored as the potential mechanism following its intervention. METHODS: Expressions of MACC1 treated with IFN-γ gradient were confirmed by quantitative real-time PCR (qRT-PCR) and western blot (WB). Proliferation, migration, and invasion abilities of PC cells treated with IFN-γ were analysed by CCK8, EDU, colony formation, Transwell (with or without matrix gel) and wound-healing assays. Expression of antisense long non-coding RNA of MACC1, MACC1-AS1, and proteins of AKT/mTOR pathway, (pho-)AKT, and (pho-)mTOR was also assessed by qRT-PCR and WB. SiRNA kit and lentiviral fluid were conducted for transient expression of MACC1 and stable expression of MACC1-AS1, respectively. Rescue assays of cells overexpressing MACC1-AS1 and of cells silencing MACC1 were performed and cellular properties and proteins were assessed by the above-mentioned assays as well. RESULTS: IFN-γ inhibited MACC1 expression in a time- and dose-dependent manner; 100 ng/mL IFN-γ generally caused downregulation of most significant (p ≤ 0.05). In vitro experiments revealed that IFN-γ decreased cellular proliferation, migration, and invasion abilities and downregulated the expression of pho-AKT and pho-mTOR (p ≤ 0.05). Conversely, overexpression of MACC1-AS1 upregulated pho-AKT and pho-mTOR proteins, and reversed cellular properties (p ≤ 0.05). Rescue assays alleviated the above changes of pho-AKT/ mTOR and cellular properties. CONCLUSION: IFN-γ affected PC properties by MACC1-AS1/MACC1 axis via AKT/mTOR signaling pathway, which provides novel insight for candidate targets for treating PC.

Balance between bacterial extracellular matrix production and intramacrophage proliferation by a Salmonella-specific SPI-2-encoded transcription factor.

Biosynthesis and secretion of a complex extracellular matrix (EM) is a hallmark of Salmonella biofilm formation, impacting on its relationship with both the environment and the host. Cellulose is a major component of Salmonella EM. It is considered an anti-virulence factor because it interferes with Salmonella proliferation inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work, we identified a SPI-2-encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage proliferation. An increased csgD expression is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase-coding gene restored intramacrophage proliferation to rates comparable to wild-type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and suggest that this repression lowers the activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.

Senescence-Associated Glycine max (Gm)NAC Genes: Integration of Natural and Stress-Induced Leaf Senescence.

Leaf senescence is a genetically regulated developmental process that can be triggered by a variety of internal and external signals, including hormones and environmental stimuli. Among the senescence-associated genes controlling leaf senescence, the transcriptional factors (TFs) comprise a functional class that is highly active at the onset and during the progression of leaf senescence. The plant-specific NAC (NAM, ATAF, and CUC) TFs are essential for controlling leaf senescence. Several members of Arabidopsis AtNAC-SAGs are well characterized as players in elucidated regulatory networks. However, only a few soybean members of this class display well-known functions; knowledge about their regulatory circuits is still rudimentary. Here, we describe the expression profile of soybean GmNAC-SAGs upregulated by natural senescence and their functional correlation with putative AtNAC-SAGs orthologs. The mechanisms and the regulatory gene networks underlying GmNAC081- and GmNAC030-positive regulation in leaf senescence are discussed. Furthermore, new insights into the role of GmNAC065 as a negative senescence regulator are presented, demonstrating extraordinary functional conservation with the Arabidopsis counterpart. Finally, we describe a regulatory circuit which integrates a stress-induced cell death program with developmental leaf senescence via the NRP-NAC-VPE signaling module.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

Salicylic acid stabilizes Staphylococcus aureus biofilm by impairing the agr quorum-sensing system.

Salicylic acid (SAL) has recently been shown to induce biofilm formation in Staphylococcus aureus and to affect the expression of virulence factors. This study was aimed to investigate the effect of SAL on the regulatory agr system and its impact on S. aureus biofilm formation. The agr quorum-sensing system, which is a central regulator in S. aureus pathogenicity, plays a pivotal role in the dispersal of S. aureus mature biofilms and contributes to the creation of new colonization sites. Here, we demonstrate that SAL impairs biofilm dispersal by interfering with agr expression. As revealed by our work, protease and surfactant molecule production is diminished, and bacterial cell autolysis is also negatively affected by SAL. Furthermore, as a consequence of SAL treatment, the S. aureus biofilm matrix revealed the lack of extracellular DNA. In silico docking and simulation of molecular dynamics provided evidence for a potential interaction of AgrA and SAL, resulting in reduced activity of the agr system. In conclusion, SAL stabilized the mature S. aureus biofilms, which may prevent bacterial cell dissemination. However, it may foster the establishment of infections locally and consequently increase bacterial persistence leading to therapeutic failure.

Dual Control of Flagellar Synthesis and Exopolysaccharide Production by FlbD-FliX Class II Regulatory Proteins in Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, has two independent flagellar systems: a single subpolar flagellum and several lateral flagella. Each flagellum is a very complex organelle composed of 30 to 40 different proteins located inside and outside the cell whereby flagellar gene expression must be tightly controlled. Such control is achieved by a hierarchy of regulators that ensure the timing of synthesis and the allocation of the different flagellar substructures. Previously, we analyzed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of the response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, distal rod, and hook of the subpolar flagellum were tightly controlled by FlbD and FliX. Furthermore, we obtained evidence for the existence of cross-regulation between these gene products and the expression of LafR, the master regulator of lateral flagella. In addition, we observed that extracellular polysaccharide production and biofilm formation also responded to these flagellar regulators. In this regard, FlbD might contribute to the switch between the planktonic and sessile states.IMPORTANCE Most environmental bacteria switch between two free-living states: planktonic, in which individual cells swim propelled by flagella, and sessile, in which bacteria form biofilms. Apart from being essential for locomotion, the flagellum has accessory functions during biofilm formation. The synthesis of flagella is a highly regulated process, and coordination with accessory functions requires the interconnection of various regulatory networks. Here, we show the role of class II regulators involved in the synthesis of the B. diazoefficiens subpolar flagellum and their possible participation in cross-regulation with the lateral flagellar system and exopolysaccharide production. These findings highlight the coordination of the synthetic processes of external structures, such as subpolar and lateral flagella, with exopolysaccharides, which are the main component of the biofilm matrix.

The dynamics of epidermal stratification during post-larval development in zebrafish.

BACKGROUND: The epidermis, as a defensive barrier, is a consistent trait throughout animal evolution. During post-larval development, the zebrafish epidermis thickens by stratification or addition of new cell layers. Epidermal basal stem cells, expressing the transcription factor p63, are known to be involved in this process. Zebrafish post-larval epidermal stratification is a tractable system to study how stem cells participate in organ growth. METHODS: We used immunohistochemistry, in combination with EdU cell proliferation detection, to study zebrafish epidermal stratification. For this procedure, we selected a window of post-larval stages (5-8 mm of standard length or SL, which normalizes age by size). Simultaneously, we used markers for asymmetric cell division and the Notch signaling pathway. RESULTS: We found that epidermal stratification is the consequence of several events, including changes in cell shape, active cell proliferation and asymmetrical cell divisions. We identified a subset of highly proliferative epidermal cells with reduced levels of p63, which differed from the basal stem cells with high levels of p63. Additionally, we described different mechanisms that participate in the stratification process, including the phosphorylation of p63, asymmetric cell division regulated by the Par3 and LGN proteins, and expression of Notch genes.

LncRNA DDX11-AS1 accelerates hepatocellular carcinoma progression via the miR-195-5p/MACC1 pathway.

INTRODUCTION AND AIM: Long non-coding RNA (lncRNA) has been shown to be a vital regulator of cancer progression, including hepatocellular carcinoma (HCC). However, the role of DEAD/H box protein 11 antisense RNA 1 (DDX11-AS1) in HCC remains to be further studied. MATERIAL AND METHODS: The expression levels of DDX11-AS1, miR-195-5p and metastasis-associated in colon cancer-1 (MACC1) were determined by quantitative real-time PCR (qRT-PCR). Cell counting kit-8 (CCK-8), transwell and apoptosis determination assays were used to evaluate cell proliferation, migration, invasion and apoptosis, respectively. Mice xenograft models were constructed to verify the effect of DDX11-AS1 on HCC tumor growth in vivo. Furthermore, lactate production, glucose consumption, ATP level and glucose uptake were detected to assess cell glucose metabolism. The interactions among DDX11-AS1, miR-195-5p and MACC1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Moreover, western blot (WB) analysis was performed to evaluate the protein levels. RESULTS: DDX11-AS1 was upregulated in HCC tissues and cells, and its silencing could inhibit HCC cell proliferation, migration, invasion and glucose metabolism, and promote apoptosis in vitro. Also, DDX11-AS1 knockdown reduced HCC tumor growth in vivo. Besides, DDX11-AS1 could interact with miR-195-5p, and miR-195-5p inhibitor reversed the inhibitory effect of silenced DDX11-AS1 on HCC cell progression. In addition, MACC1 was a target of miR-195-5p, and its overexpression reversed the suppression effect of miR-195-5p on HCC cell progression. CONCLUSION: Our data revealed that DDX11-AS1 could act as an oncogenic regulator in HCC, providing a potential therapeutic target for HCC treatment.

Vancomycin MIC and agr dysfunction in invasive MRSA infections in southern Brazil.

In methicillin-resistant Staphylococcus aureus (MRSA) treatment, the vancomycin minimum inhibitory concentration (MIC) increase, vancomycin heteroresistance (hVISA) presence, and accessory gene regulator (agr) dysfunction are predictors of vancomycin therapy failure. This study evaluated the association between vancomycin MIC (≥ 1.0 µg/mL) and agr dysfunction in invasive MRSA isolates. Vancomycin MIC, hVISA phenotype, agr group, and function were determined in 171 MRSA isolates obtained between 2014 and 2019 from hospitals in Porto Alegre, Brazil. All MRSA were susceptible to vancomycin; 16.4% of these had MIC ≥ 1.0 µg/mL. Seventeen MRSA isolates expressed the hVISA phenotype; 35.3% of them had MIC of 1.5 µg/mL. agr groups I (40.9%) and II (47.1%) were the most found groups for MRSA and hVISA isolates, respectively. The proportion of MRSA with vancomycin MIC ≥ 1.0 µg/mL in agr group II was significantly higher than in agr groups I and III (p = 0.002). agr dysfunction was observed in 4.7% (8/171) of MRSA, especially those with vancomycin MIC ≥ 1.0 µg/mL (p < 0.001). In addition, six isolates (35.3%; 6/17) with hVISA phenotype presented agr dysfunction, which was significantly higher than that in non-hVISA phenotype (p < 0.001). In conclusion, agr dysfunction in MRSA is associated with vancomycin MIC ≥ 1.0 µg/mL and hVISA phenotype, which suggests that agr dysfunction might confer potential advantages on MRSA to survive in invasive infections.

Functional evaluation of human papillomavirus type 31 long control region variants.

Persistent infections by high-risk human papillomavirus (HR-HPV) are a necessary condition, but not sufficient for cervical cancer development. Genetic variants of HR-HPV appear to be related to the risk of persistent infections. The study performed a functional evaluation of variants of the HPV-31 promoter region (LCR). For this, cloning and subcloning of variants HPV-31/UFPE-21 HPV-31/UFPE-89, HPV-31/UFPE-66, E2 gene and prototype HPV-31 were performed. Transfection with different concentrations of E2 was done and the concentration of 25 ng was determined to be ideal for LCR activation. HPV-31/UFPE-21 and HPV-31/UFPE-89 have a greater ability to alter Nluc reporter gene expression levels and HPV-31/UFPE-66 showed decreased levels of gene expression of Nluc reporter gene compared to control. Statistical analysis showed a significant difference between the polymorphic LCR regions and the control (p < 0.0001). A more refined profile of variants of HPV-31 and its importance for the prognosis of cervical lesions begins to be drawn.