Downregulated RRS1 inhibits invasion and metastasis of BT549 through RPL11­c­Myc­SNAIL axis.

Regulator of ribosome synthesis 1 (RRS1) is a key factor in ribosome biosynthesis and other cellular functions. High level of RRS1 in breast cancer cell lines is associated with increased cell proliferation, invasion and migration. RRS1 controls the assembly of the 60s subunit and maturation of 25S rRNA during ribosome biosynthesis. In this study, lentiviral transfection of sh­RNA was used to knock down the level of RRS1, to detect the effect of RRS1 on cell function and to explore the specific mechanism of RRS1 affecting cell invasion and metastasis by COIP and dual­luciferase reporter gene assays. The present study found that RRS1 knockdown reduced the accumulation of ribosome protein L11 (RPL11) in the nucleolus, which then migrated to the nucleoplasm and bound to c­Myc. This inhibited trans­activation of SNAIL by c­Myc and eventually decreased the invasion and metastasis capacity of the human breast cancer cell line BT549. Taken together, RRS1 regulates invasion and metastasis of human breast cancer cells through the RPL11­c­Myc­SNAIL axis. The findings are of great significance for exploring the mechanism of breast cancer invasion and metastasis and the corresponding regulatory factors.

circKCNN2 suppresses the recurrence of hepatocellular carcinoma at least partially via regulating miR-520c-3p/methyl-DNA-binding domain protein 2 axis.

BACKGROUND: Recurrence is the major cause of hepatocellular carcinoma (HCC) death. We aimed to identify circular RNA (circRNA) with predictive and therapeutic value for recurrent HCC. METHODS: Tissue samples from recurrent and non-recurrent HCC patients were subjected to circRNA sequencing and transcriptome sequencing. circKCNN2 was identified through multi-omics analyses. The effects of circKCNN2 on HCC were evaluated in cells, animals, database of The Cancer Genome Atlas, and a cohort with 130 HCC patients. circRNA precipitation, chromatin immunoprecipitation assay, RNA pull-down, luciferase assay, and cell experiments were applied to evaluate the interaction of circKCNN2 with miRNAs and proteins. The association between circKCNN2 and the therapeutic effect of lenvatinib was investigated in HCC cell lines and HCC tissue-derived organoids. RESULTS: The expression of circKCNN2 was downregulated in HCC tissues and predicted a favorable overall survival and recurrence-free survival. The expression of circKCNN2 was positively correlated with the parental gene, potassium calcium-activated channel subfamily N member (KCNN2). Nuclear transcription factor Y subunit alpha (NFYA) was proven to inhibit the promoter activity of KCNN2, downregulate the expression of KCNN2 and circKCNN2, and predict an unfavorable recurrence-free survival. Ectopic expression of circKCNN2 inhibited HCC cell proliferation, colony formation, migration, and tumor formation in a mouse model. miR-520c-3p sponged by circKCNN2 could reverse the inhibitory effect of circKCNN2 on HCC cells and down-regulate the expression of methyl-DNA-binding domain protein 2 (MBD2). The intratumoral expression of MBD2 predicted a favorable recurrence-free survival. circKCNN2 down-regulated the expression of fibroblast growth factor receptor 4 (FGFR4), which can be reversed by miR-520c-3p and knockdown of MBD2. Lenvatinib inhibited the expression of FGFR4 and upregulated the expression of circKCNN2 and MBD2. Ectopic expression of circKCNN2 in HCC cells enhanced the therapeutic effect of lenvatinib. However, the high inherent level of circKCNN2 in HCC cells was associated with lenvatinib resistance. CONCLUSIONS: circKCNN2, transcriptionally repressed by NFYA, suppresses HCC recurrence via the miR-520c-3p/MBD2 axis. Inherent level of circKCNN2 in HCC cells predisposes anti-tumor effect of lenvatinib possibly because both circKCNN2 and lenvatinib repress the expression of FGFR4. circKCNN2 may be a promising predictive biomarker and therapeutic agent for HCC recurrence.

Enolase 1, a Moonlighting Protein, as a Potential Target for Cancer Treatment.

Enolase 1 (ENO1) is a moonlighting protein, function as a glycolysis enzyme, a plasminogen receptor and a DNA binding protein. ENO1 play an important role in the process of cancer development. The transcription, translation, post-translational modifying activities and the immunoregulatory role of ENO1 at the cancer development is receiving increasing attention. Some function model studies have shown that ENO1 is a potential target for cancer treatment. In this review, we provide a comprehensive overview of the characterization, function, related transduction cascades of ENO1 and its roles in the pathophysiology of cancers, which is a consequence of ENO1 signaling dysregulation. And the development of novels anticancer agents that targets ENO1 may provide a more attractive option for the treatment of cancers. The data of sarcoma and functional cancer models indicates that ENO1 may become a new potential target for anticancer therapy.

Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index.

BACKGROUND: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. RESULTS: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. CONCLUSIONS: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

Irisin attenuates lipopolysaccharide-induced acute lung injury by downregulating inflammatory cytokine expression through miR-199a-mediated Rad23b overexpression.

AIMS: Acute lung injury (ALI) is a leading cause of mortality as a result of inflammatory cytokine overexpression and increased rates of apoptosis. Therapies for ALI are yet to be thoroughly investigated. Recent evidence has shown that irisin exerts protective effects against many types of pathologies. The present study aimed to determine the function of irisin in an ALI mouse model induced by lipopolysaccharide (LPS) and the corresponding underlying mechanisms at the tissue, cellular, and molecular levels. MAIN METHODS: We assessed irisin function in A549 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. The cell apoptosis was evaluated by flow cytometry. Western blotting and RT-PCR were used to test expression level. Animal models of ALI was established. KEY FINDINGS: We found that irisin treatment maintained lung weight, significantly reduced inflammatory cytokine expression, and alleviated lung injury by downregulating miR-199a. In LPS-stimulated cells, forced miR-199a expression downregulated Rad23b expression by targeting its 3' untranslated region, indicating that Rad23b is a direct target of miR-199a. SIGNIFICANCE: These findings reveal that irisin can alleviate ALI by inhibiting miR-199a and upregulating Rad23b expression, suggesting that irisin has clinical potential for the treatment of ALI.

Acute pharmacological degradation of Helios destabilizes regulatory T cells.

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity.

Protective effect of syringic acid via restoring cells biomechanics and organelle structure in human lens epithelial cells.

We have previously reported that syringic acid (SA) extracted from D. aurantiacum var. denneanum (kerr) may be used to prevent diabetic cataract (DC). However, the underlying mechanisms through which SA prevents DC in human lens epithelial cells (HLECs) remained unclear. In the present study, we employed single-molecule optics technologies, including transmission electron microscopy (TEM), atomic force microscopy (AFM), laser scanning confocal microscopy (LSCM) and Raman spectroscopy, to monitor the effect of SA on HLECs biomechanics and organelle structure in real-time. TEM suggested that SA improved the ultrastructure of HLECs with regard to nuclear chromatin condensation and reducing mitochondrial swelling and degeneration, which may aid in the maintenance of HLECs integrity in the presence of glucose. AFM revealed a reduced surface roughness and stiffness following SA treatment, suggesting an improved viscoelasticity of HELCs. Raman spectrometry and LSCM further revealed that these changes were related to a modification of cell liquidity and cytoskeletal structure by SA. Taken together, these results provide insights into the effects of SA on the biomechanics of HLECs and further strengthen the evidence for its potential use as a novel therapeutic strategy for DC prevention.

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.

Selective targeting of the androgen receptor-DNA binding domain by the novel antiandrogen SBF-1 and inhibition of the growth of prostate cancer cells.

Prostate cancers are reliant on androgens for growth and survival. Clinicians and researchers are looking for potent treatments for the resistant forms of prostate cancer; however, a handful of small molecules used in the treatment of castration-resistant prostate cancer have not shown potent effects owing to the mutations in the AR (Androgen Receptor). We used SBF-1, a well-characterized antitumor agent with potent cytotoxic effects against different kinds of cancers and investigated its effect on human prostate cancer. SBF-1 substantially inhibited the proliferation, induced apoptosis, and caused cell cycle arrest in LNCaP and PC3/AR+ prostate cancer cell lines. SBF-1 inhibited the activation of the IGF-1-PNCA pathway, as demonstrated by decreased expression of IGF-1 (insulin-like growth factor 1), proliferating cell nuclear antigen (PCNA), and its downstream Bcl-2 protein. Using microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) assays, we observed a direct binding of SBF-1 to the AR. SBF-1 binds to the AR-DBD (DNA-binding domain) and blocks the transcription of its target gene. SBF-1 demonstrated a potent antitumor effect in vivo; it inhibited AR signaling and suppressed tumor growth in animals. Our study suggests that SBF-1 is an inhibitor of the AR and might be used in the treatment of prostate cancer.

Therapeutic potential of novel Cell Division Cycle Kinase 7 inhibitors on TDP-43-related pathogenesis such as Frontotemporal Lobar Degeneration (FTLD) and amyotrophic lateral sclerosis (ALS).

TDP-43 has been identified as the major component of protein aggregates found in affected neurons in FTLD-TDP and amyotrophic lateral sclerosis (ALS) patients. TDP-43 is hyperphosphorylated, ubiquitinated, and cleaved in the C-terminus. CDC-7 was reported to phosphorylate TDP-43. There are no effective treatments for either FTLD-TDP or ALS, being a pressing need for the search of new therapies. We hypothesized that modulating CDC-7 activity with small molecules that are able to interfere with TDP-43 phosphorylation could be a good therapeutic strategy for these diseases. Here, we have studied the effects of novel brain penetrant, thiopurine-based, CDC-7 inhibitors in TDP-43 homeostasis in immortalized lymphocytes from FTLD-TDP patients, carriers of a loss-of-function GRN mutation, as well as in cells derived from sporadic ALS patients. We found that selective CDC-7 inhibitors, ERP1.14a and ERP1.28a, are able to decrease the enhanced TDP-43 phosphorylation in cells derived from FTLD-TDP and ALS patients and to prevent cytosolic accumulation of TDP-43. Moreover, treatment of FTLD-TDP lymphoblasts with CDC-7 inhibitors leads to recovering the nuclear function of TDP-43-inducing CDK6 repression. We suggest that CDC-7 inhibitors, mainly the heterocyclic compounds here shown, may be considered as promising drug candidates for the ALS/FTD spectrum.

Downregulation of Xeroderma Pigmentosum Complementation Group C Expression by 17-Allylamino-17-Demethoxygeldanamycin Enhances Bevacizumab-Induced Cytotoxicity in Human Lung Cancer Cells.

INTRODUCTION: Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor involved in nucleotide excision repair and regulation of non-small-cell lung cancer (NSCLC) cell proliferation and viability. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) blocks ATP binding to heat shock protein 90 (Hsp90), resulting in destabilization of Hsp90-client protein complexes. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor expressed by many types of tumors. Bevacizumab (Avastin) is a humanized monoclonal antibody against human VEGF used as an antiangiogenesis agent in the therapy of many cancers, proving successful in increasing objective tumor response rate and prolonging overall survival in NSCLC patients. METHODS: After the bevacizumab and/or 17-AAG treatment, the expressions of XPC mRNA were determined by quantitative real-time PCR analysis. Protein levels of XPC and phospho-AKT were determined by Western blot analysis. We used specific XPC small interfering RNA and PI3K inhibitor (LY294002) to examine the role of the AKT-XPC signal in regulating the chemosensitivity of bevacizumab and 17-AAG. Cell viability was assessed by the MTS assay and trypan blue exclusion assay. RESULTS: In this study, bevacizumab decreased XPC expression in human lung squamous cell carcinoma H520 and H1703 cells via AKT inactivation. Enhancement of AKT activity by transfection with constitutively active AKT vectors increased XPC expression and cell survival after treatment with bevacizumab. In addition, 17-AAG synergistically enhanced bevacizumab-induced cytotoxicity and cell growth inhibition in H520 and H1703 cells, associated with downregulation of XPC expression and inactivation of AKT. DISCUSSION/CONCLUSION: Together, these results may provide a rationale to combine bevacizumab with Hsp90 inhibitors in future to enhance therapeutic effects for lung cancer.

Ascorbic Acid Promotes Plasma Cell Differentiation through Enhancing TET2/3-Mediated DNA Demethylation.

Plasma cells provide high-affinity antibodies against invading pathogens. Although transcriptional and epigenetic mechanisms have been extensively studied for plasma cell differentiation, how these mechanisms respond to environmental cues remains largely unexplored. In this study, we show that ascorbic acid (vitamin C), an essential nutrient, is able to promote plasma cell differentiation and humoral immune response by enhancing TET2/3-mediated DNA demethylation. Ascorbic acid treatment during B cell activation has persistent effects on later plasma cell differentiation by predisposing germinal center B cells toward the plasma cell lineage. Conversely, ascorbic acid deficiency in vivo blocks plasma cell differentiation and attenuates the humoral immune response following antigen immunization. We further demonstrate that such effects of ascorbic acid on plasma cell differentiation require DNA methylcytosine oxidases TET2 and TET3. Our study thus reveals a previously uncharacterized link between essential nutrients and epigenetic regulation of plasma cell differentiation and humoral immune response.

Structural and Functional Study of the Klebsiella pneumoniae VapBC Toxin-Antitoxin System, Including the Development of an Inhibitor That Activates VapC.

Klebsiella pneumoniae is one of the most critical opportunistic pathogens. TA systems are promising drug targets because they are related to the survival of bacterial pathogens. However, structural information on TA systems in K. pneumoniae remains lacking; therefore, it is necessary to explore this information for the development of antibacterial agents. Here, we present the first crystal structure of the VapBC complex from K. pneumoniae at a resolution of 2.00 Å. We determined the toxin inhibitory mechanism of the VapB antitoxin through an Mg2+ switch, in which Mg2+ is displaced by R79 of VapB. This inhibitory mechanism of the active site is a novel finding and the first to be identified in a bacterial TA system. Furthermore, inhibitors, including peptides and small molecules, that activate the VapC toxin were discovered and investigated. These inhibitors can act as antimicrobial agents by disrupting the VapBC complex and activating VapC. Our comprehensive investigation of the K. pneumoniae VapBC system will help elucidate an unsolved conundrum in VapBC systems and develop potential antimicrobial agents.

Thallium Toxicity in Caenorhabditis elegans: Involvement of the SKN-1 Pathway and Protection by S-Allylcysteine.

Monovalent thallium (Tl+) is a cation that can exert complex neurotoxic patterns in the brain by mechanisms that have yet to be completely characterized. To learn more about Tl+ toxicity, it is necessary to investigate its major effects in vivo and its ability to trigger specific signaling pathways (such as the antioxidant SKN-1 pathway) in different biological models. Caenorhabditis elegans (C. elegans) is a nematode constituting a simple in vivo biological model with a well-characterized nervous system, and high genetic homology to mammalian systems. In this study, both wild-type (N2) and skn-1 knockout (KO) mutant C. elegans strains subjected to acute and chronic exposures to Tl+ [2.5-35 µM] were evaluated for physiological stress (survival, longevity, and worm size), motor alterations (body bends), and biochemical changes (glutathione S-transferase regulation in a gst-4 fluorescence strain). While survival was affected by Tl+ in N2 and skn-1 KO (worms lacking the orthologue of mammalian Nrf2) strains in a similar manner, the longevity was more prominently decreased in the skn-1 KO strain compared with the wild-type strain. Moreover, chronic exposure led to a greater compromise in the longevity in both strains compared with acute exposure. Tl+ also induced motor alterations in both skn-1 KO and wild-type strains, as well as changes in worm size in wild-type worms. In addition, preconditioning nematodes with the well-known antioxidant S-allylcysteine (SAC) reversed the Tl+-induced decrease in survival in the N2 strain. GST fluorescent expression was also decreased by the metal in the nematode, and recovered by SAC. Our results describe and validate, for the first time, features of the toxic pattern induced by Tl+ in an in vivo biological model established with C. elegans, supporting an altered redox component in Tl+ toxicity, as previously described in mammal models. We demonstrate that the presence of the orthologous SKN-1 pathway is required for worms in evoking an efficient antioxidant defense. Therefore, the nematode represents an optimal model to reproduce mammalian Tl+ toxicity, where toxic mechanisms and novel therapeutic approaches of clinical value may be successfully pursued.

SKN-1 is involved in combination of apple peels and blueberry extracts synergistically protecting against oxidative stress in Caenorhabditis elegans.

Increased consumption of fruits and vegetables is associated with reduced risk of age-related functional declines and chronic diseases, primarily attributed to their bioactive phytochemicals. Apples and blueberries are rich in phytochemicals with a wide range of biological activities and health benefits. Our previous research has shown the combination of apple peel extracts (APE) and blueberry extracts (BE) can synergistically promote the lifespan of Caenorhabditis elegans (C. elegans). The objectives of this study were to determine whether the extension of lifespan was involved in regulation of oxidative stress, and to explore the underlying mechanisms of action. The results showed that the combination of APE and BE could synergistically ameliorate oxidative stress by improving antioxidant enzyme activities and enhancing resistance to paraquat. Meanwhile, treatment with APE plus BE could down-regulate the overexpression of reactive oxygen species (ROS) and affect the expression of antioxidant related genes, including sod-3, cat-1, ctl-1, skn-1, mev-1 and isp-1. However, administration with APE plus BE abolished the extension of the lifespan of skn-1(zu135) mutants, and inhibited the expression of skn-1 downstream genes, including gcs-1, gst-4 and gst-7. In addition, supplementation with APE plus BE could promote the migration of SKN-1 into the nucleus, which eliminated improvement to ROS and paraquat. In conclusion, the combination of APE and BE could synergistically protect against oxidative stress in C. elegans via the SKN-1/Nrf2 pathway. This study provided the theoretical basis to explore the combination of phytochemicals in the prevention of aging regulated by oxidative stress.

The structural basis of African swine fever virus pA104R binding to DNA and its inhibition by stilbene derivatives.

African swine fever virus (ASFV) is a highly contagious nucleocytoplasmic large DNA virus (NCLDV) that causes nearly 100% mortality in swine. The development of effective vaccines and drugs against this virus is urgently needed. pA104R, an ASFV-derived histone-like protein, shares sequence and functional similarity with bacterial HU/IHF family members and is essential for viral replication. Herein, we solved the crystal structures of pA104R in its apo state as well as in complex with DNA. Apo-pA104R forms a homodimer and folds into an architecture conserved in bacterial heat-unstable nucleoid proteins/integration host factors (HUs/IHFs). The pA104R-DNA complex structure, however, uncovers that pA104R has a DNA binding pattern distinct from its bacterial homologs, that is, the ß-ribbon arms of pA104R stabilize DNA binding by contacting the major groove instead of the minor groove. Mutations of the basic residues at the base region of the ß-strand DNA binding region (BDR), rather than those in the ß-ribbon arms, completely abolished DNA binding, highlighting the major role of the BDR base in DNA binding. An overall DNA bending angle of 93.8° is observed in crystal packing of the pA104R-DNA complex structure, which is close to the DNA bending angle in the HU-DNA complex. Stilbene derivatives SD1 and SD4 were shown to disrupt the binding between pA104R and DNA and inhibit the replication of ASFV in primary porcine alveolar macrophages. Collectively, these results reveal the structural basis of pA104R binding to DNA highlighting the importance of the pA104R-DNA interaction in the ASFV replication cycle and provide inhibitor leads for ASFV chemotherapy.

Anti-tumoral Effect of a Cell Penetrating and Interfering Peptide Targeting PP2A/SET Interaction.

OBJECTIVE: To test cell penetrating and interfering peptide Mut3DPT-PP2A/SET in interaction between serine threonine phosphatase PP2A and its physiological inhibitor, the oncoprotein SET. MATERIALS AND METHODS: Adult male C3H/S-strain mice, 60 days old, were given a graft of breast adenocarcinoma cells (TN60) into subcutaneous tissue. Mut3DPT-PP2A/SET peptide was used to block PP2A and SET oncoprotein interaction. The graft-bearing animals were divided into a control group (injected with saline buffer), and an intervention group injected intraperitoneally with Mut3DPT-PP2A/SET peptide (5 mg/kg) every day from day 5 to day 37. The variables we used to compare the outcome in both groups were tumor size in mm (length×width) and histological changes. In the statistical analysis we used ANOVA and Student-Keuls multiple comparisons test and Tuckey for the post-test analysis. RESULTS: 48 mice were grafted at day 0 with breast UNLP-C3H/S tumor cells, and after randomization, they were assigned to one of the two study groups. At day 5 all mice were injected either with placebo or with the peptide. The treated group showed significant tumor reduction (p<0.07). Histological changes showed presence of apoptosis and necrosis of tumor in treated group. CONCLUSION: The peptide Mut3DPT-PP2A/SET has demonstrated anti-tumor activity by reduction in vivo of tumor growth becoming a promising future in anticancer therapy.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

Valproic Acid Promotes Early Neural Differentiation in Adult Mesenchymal Stem Cells Through Protein Signalling Pathways.

Regenerative medicine is a rapidly expanding area in research and clinical applications. Therapies involving the use of small molecule chemicals aim to simplify the creation of specific drugs for clinical applications. Adult mesenchymal stem cells have recently shown the capacity to differentiate into several cell types applicable for regenerative medicine (specifically neural cells, using chemicals). Valproic acid was an ideal candidate due to its clinical stability. It has been implicated in the induction of neural differentiation; however, the mechanism and the downstream events were not known. In this study, we showed that using valproic acid on adult mesenchymal stem cells induced neural differentiation within 24 h by upregulating the expression of suppressor of cytokine signaling 5 (SOCS5) and Fibroblast growth factor 21 (FGF21), without increasing the potential death rate of the cells. Through this, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is downregulated, and the mitogen-activated protein kinase (MAPK) cascade is activated. The bioinformatics analyses revealed the expression of several neuro-specific proteins as well as a range of functional and structural proteins involved in the formation and development of the neural cells.

Exposure to sub-inhibitory ciprofloxacin and nitrofurantoin concentrations increases recA gene expression in uropathogenic Escherichia coli: The role of RecA protein as a drug target.

Sub-inhibitory concentrations (sub-MIC) of antimicrobial agents can lead to genetic changes in bacteria, modulating the expression of genes related to bacterial stress and leading to drug resistance. Herein we describe the impact of sub-MIC of ciprofloxacin and nitrofurantoin on three uropathogenic Escherichia coli strains. Disk-diffusion assays with different antimicrobial agents were tested to detect phenotype alterations, and quantitative real-time PCR (qRT-PCR) was performed to analyze the expression of ompF and recA genes. Significant reduction on the susceptibility to ciprofloxacin and nitrofurantoin was detected on disk diffusion test. The qRT-PCR results revealed a 1.2-4.7 increase in recA expression in all E. coli studied, while the ompF expression varied. Because RecA was pointed as an important component to the development of drug resistance, molecular docking studies were performed with three experimentally known inhibitors of this enzyme. These studies aimed to understand the inhibitory binding mode of such compounds. The results confirmed the ADP/ATP binding site as a potential site of inhibitor recognition and a binding mode based on π-stacking interactions with Tyr103 and hydrogen bonds with Tyr264. These findings can be useful for guiding the search and design of new antimicrobial agents, mainly concerning the treatment of infections with resistant bacterial strains.