Molecular basis for RASSF10/NPM/RNF2 feedback cascade-mediated regulation of gastric cancer cell proliferation.

Ras-association domain family (RASSF) proteins are encoded by numerous tumor suppressor genes that frequently become silenced in human cancers. RASSF10 is downregulated by promoter hypermethylation in cancers and has been shown to inhibit cell proliferation; however, the molecular mechanism(s) remains poorly understood. Here, we demonstrate for the first time that RASSF10 inhibits Cdk1/cyclin-B kinase complex formation to maintain stable levels of cyclin-B for inducing mitotic arrest during cell cycle. Using LC-MS/MS, live cell imaging, and biochemical approaches, we identify Nucleophosmin (NPM) as a novel functional target of RASSF10 and revealed that RASSF10 expression promoted the nuclear accumulation of GADD45a and knockdown of either NPM or GADD45a, resulting in impairment of RASSF10-mediated G2/M phase arrest. Furthermore, we demonstrate that RASSF10 is a substrate for the E3 ligase ring finger protein 2 (RNF2) and show that an NPM-dependent downregulation of RNF2 expression is critical to maintain stable RASSF10 levels in cells for efficient mitotic arrest. Interestingly, the Kaplan-Meier plot analysis shows a positive correlation of RASSF10 and NPM expression with greater gastric cancer patient survival and the reverse with expression of RNF2, suggesting that they may have a role in cancer progression. Finally, our findings provide insights into the mode of action of the RASSF10/NPM/RNF2 signaling cascade on controlling cell proliferation and may represent a novel therapeutic avenue for the prevention of gastric cancer metastasis.

E4BP4/NFIL3 modulates the epigenetically repressed RAS effector RASSF8 function through histone methyltransferases.

RAS proteins are major human oncogenes, and most of the studies are focused on enzymatic RAS effectors. Recently, nonenzymatic RAS effectors (RASSF, RAS association domain family) have garnered special attention because of their tumor-suppressive properties in contrast to the oncogenic potential of the classical enzymatic RAS effectors. Whereas most members of RASSF family are deregulated by promoter hypermethylation, RASSF8 promoter remains unmethylated in many cancers but the mechanism(s) of its down-regulation remains unknown. Here, we unveil E4BP4 as a critical transcriptional modulator repressing RASSF8 expression through histone methyltransferases, G9a and SUV39H1. In line with these observations, we noticed a negative correlation of RASSF8 and E4BP4 expression in primary breast tumor samples. In addition, our data provide evidence that E4BP4 attenuates RASSF8-mediated anti-proliferation and apoptosis, shedding mechanistic insights into RASSF8 down-regulation in breast cancers. Collectively, our study provides a better understanding on the epigenetic regulation of RASSF8 function and implicates the development of better treatment strategies.

The non-enzymatic RAS effector RASSF7 inhibits oncogenic c-Myc function.

c-Myc is a proto-oncogene controlling expression of multiple genes involved in cell growth and differentiation. Although the functional role of c-Myc as a transcriptional regulator has been intensively studied, targeting this protein in cancer remains a challenge. Here, we report a trimodal regulation of c-Myc function by the Ras effector, Ras-association domain family member 7 (RASSF7), a nonenzymatic protein modulating protein-protein interactions to regulate cell proliferation. Using HEK293T and HeLa cell lines, we provide evidence that RASSF7 destabilizes the c-Myc protein by promoting Cullin4B-mediated polyubiquitination and degradation. Furthermore, RASSF7 competed with MYC-associated factor X (MAX) in the formation of a heterodimeric complex with c-Myc and attenuated its occupancy on target gene promoters to regulate transcription. Consequently, RASSF7 inhibited c-Myc-mediated oncogenic transformation, and an inverse correlation between the expression levels of the RASSF7 and c-Myc genes was evident in human cancers. Furthermore, we found that RASSF7 interacts with c-Myc via its RA and leucine zipper (LZ) domains and LZ domain peptide is sufficient to inhibit c-Myc function, suggesting that this peptide might be used to target oncogenic c-Myc. These results unveil that RASSF7 and c-Myc are functionally linked in the control of tumorigenesis and open up potential therapeutic avenues for targeting the "undruggable" c-Myc protein in a subset of human cancers.

RNA-seq reveals outcome-specific gene expression of MMP7 and PCK1 in biliary atresia.

The disease phenotype in biliary atresia (BA) is caused by a fibro-inflammatory process leading to destruction of cholangiocytes, obstruction of ductular pathways and eventual progression to liver cirrhosis. The first line of management is a Kasai portoenterostomy (KPE) followed by liver transplantation (LT) in some children. Several factors have been postulated to affect the outcome of KPE and/or the subsequent progression of liver disease. However, no biomarkers have been identified in the liver for BA. We aimed to address this deficit. Whole transcriptome mRNA sequencing was performed for 29 samples (25 BA and 4 Controls) to identify the candidate genes predicting the prognosis of KPE. These results were further confirmed with quantitative Realtime PCR (qPCR). Analysis from RNA-sequencing data identified matrix metalloproteinase7 (MMP7) and phosphoenolpyruvate carboxykinase (PCK1) as potential determinants of the outcome of KPE. MMP7 expression was significantly elevated in patients who failed to clear jaundice after KPE as well as in patients with End Stage Liver Disease (ESLD). In contrast, PCK1 level was upregulated in patients who had successful KPE, while there was a significant down regulation in patients who failed KPE. MMP7 and PCK1 expression patterns had an inverse relation to the outcome of KPE and hence could potentially be used as biomarkers to predict KPE outcome and disease progression, enabling clinicians to design new treatment strategies for BA.

Cytosine methylation by DNMT2 facilitates stability and survival of HIV-1 RNA in the host cell during infection.

The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, our results indicate that HIV-1 enhances its survivability in the host cell by utilizing this RNA methylation capability of DNMT2 to increase the stability of its own genome. Upon infection, DNMT2 re-localizes from the nucleus to the SGs and methylates HIV-1 RNA. This DNMT2-dependent methylation provided post-transcriptional stability to the HIV-1 RNA. Furthermore, DNMT2 overexpression increased the HIV-1 viral titre. This would suggest that HIV hijacks the RNA-processing machinery within the SGs to ensure its own survival in the host cell. Thus, our findings provide for a novel mechanism by which virus tries to modulate the host cell machinery to its own advantage.

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21 Cip1/Waf1.

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G1 to S phase transition of cells by enhancing CDK inhibitor p21(Cip-1/Waf1) expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB(Ser-780)) followed by the up-regulation of E2F1 target genes required to promote G1 to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB(Ser-780) levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G1 phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21(Cip-1/Waf1) pathway.

Antigenicity and immunogenicity of a trimeric envelope protein from an Indian clade C HIV-1 isolate.

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.

Characterization of protective immune response elicited by a trimeric envelope protein from an Indian clade C HIV-1 isolate in rhesus macaques.

BACKGROUND: Recent preclinical studies have demonstrated the use of properly folded trimeric HIV-1 envelope proteins as immunogen for eliciting protecting immune response in macaques. METHODS: Trimeric gp145 protein of Indian clade C HIV-1 (93IN101) was characterized for antigenicity by evaluating its binding to sCD4, and several monoclonal antibodies to HIV-1 by bio-layer interferometry. Ten macaques were immunized four times with purified gp145 in adjuplex adjuvant, and serum antibodies were characterized for binding to gp145 and neutralization. Immunized macaques were subjected to weekly low-dose vaginal challenge with SHIV1157-ipEL-p for 8 weeks. RESULTS: Env protein elicited strong antibody response in macaques. Following challenge, seven of ten immunized macaques resisted challenge, while six of eight control animals were infected. CONCLUSIONS: Env proteins from a clade C Indian isolate can elicit protective immune response and therefore may be a candidate for inclusion in a multiclade-based HIV-1 vaccine.

Signals and pathways regulating nucleolar retention of novel putative nucleolar GTPase NGP-1(GNL-2).

NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-ß and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.

Landscape of humoral immune responses against SARS-CoV-2 in patients with COVID-19 disease and the value of antibody testing.

A new pandemic is ongoing in several parts of the world. The agent responsible is the newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The symptoms associated with this virus are known as the coronavirus disease-2019 (COVID-19). In this review, we summarize the published data on virus specific antibodies in hospitalized patients with COVID-19 disease, patients recovered from the disease and the individuals who are asymptomatic with SARS-CoV-2 infections. The review highlights the following: i) an adjunct role of antibody tests in the diagnosis of COVID-19 in combination with RT-PCR; ii) status of antibodies from COVID-19 convalescent patients to select donors for plasma therapy; iii) the potential confounding effects of other coronaviruses, measles, mumps and rubella in antibody testing due to homology of certain viral genes; and iv) the role of antibody testing for conducting surveillance in populations, incidence estimation, contact tracing and epidemiologic studies.

Viral protein X unlocks the nuclear pore complex through a human Nup153-dependent pathway to promote nuclear translocation of the lentiviral genome.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus 2 (HIV-2) display unique ability to infect nondividing target cells. Viral protein X (Vpx) of HIV-2/SIV is known to be involved in the nuclear import of viral genome in nondividing cells, but the mechanism remains poorly understood. In the present investigation for the first time we provide evidence that Vpx of SIVsmPBj1.9 physically interacts with human nucleoporin 153 (Nup153), which is known to provide a docking site for protein-cargo complexes at the nuclear pore complex (NPC). Results from superresolution-structured illumination microscopy studies reveal that Vpx interaction with NPC-associated Nup153 is critical for its efficient nuclear translocation. Virion-associated MAPK/ERK-2-mediated phosphorylation of Vpx plays a critical role in its interaction with human Nup153 and this interaction was found to be evolutionarily conserved in various SIV isolates and HIV-2. Interestingly, MAPK/ERK-2 packaging defective SIV failed to promote the efficient nuclear import of viral genome and suggests that MAPK/ERK-2-mediated Vpx phosphorylation is important for its interaction with Nup153, which is critical for lentiviruses to establish infection in nondividing target cells. Together, our data elucidate the mechanism by which Vpx orchestrates the challenging task of nuclear translocation of HIV-2/SIV genome in nondividing target cells.

Guanine nucleotide binding protein like-1 (GNL1) promotes cancer cell proliferation and survival through AKT/p21 CIP1 signaling cascade.

Human guanine nucleotide binding protein like 1 (GNL1) is an evolutionary conserved putative nucleolar GTPase belonging to the HSR1_MMR1 subfamily of GTPases. GNL1 was found to be highly up-regulated in various cancers. Here, we report for the first time that GNL1 inhibits apoptosis by modulating the expression of Bcl2 family of proteins and the cleavage of caspases 7 and 8. Furthermore, GNL1 protects colon cancer cells from chemo-drug-induced apoptosis. Interestingly, GNL1 up-regulates the expression of p53 and its transcriptional target, p21 but the up-regulation of p21 was found to be p53 dependent as well as independent mechanisms. Our results further demonstrate that GNL1 promotes cell growth and survival by inducing cytoplasmic retention and stabilization of p21 through AKT-mediated phosphorylation. In addition, GNL1 failed to inhibit apoptosis under p21 knockdown conditions which suggests the critical role of p21 in GNL1-mediated cell survival. Finally, an inverse correlation of GNL1, p21, and AKT expression in primary colon and breast cancer with patient survival suggests their critical role in tumorigenesis. Collectively, our study reveals that GNL1 executes its antiapoptotic function by a novel mechanism and suggests that it may function as a regulatory component of the PI3K/AKT/p21 signaling network to promote cell proliferation and survival in cancers.

Proteomic profiling unveils citral modulating expression of IsaA, CodY and SaeS to inhibit biofilm and virulence in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the dangerous human pathogens and it is categorized as a high priority multi-drug resistant bacterium by WHO. Biofilm forming ability of MRSA is responsible for persistent infections and also difficult to eradicate using antibiotic therapy as biofilm is much more resistant to antibiotics. Thus, targeting biofilm and virulence has become an alternative approach to attenuate the pathogenicity of bacterium without affecting the growth. Hence, the present study was aimed at evaluation of antibiofilm potential of citral against MRSA and to decode the possible mode of action. Citral inhibited biofilm formation by MRSA without affecting growth at 100 µg/mL. Microscopic analyses evidenced that citral greatly hampered the surface adherence of MRSA. Effect of citral on cellular proteome of MRSA was studied using two-dimensional gel electrophoresis (2DGE) and differentially regulated proteins were identified using nano LC-MS/MS and MALDI-TOF/TOF analysis. Gene ontology and STRING analysis revealed that citral differentially regulated the proteins involved in pleotropic transcriptional repression (CodY), cell wall homeostasis (IsaA), regulation of exotoxin secretion (SaeS), cell adhesion, hemolysis, capsular polysaccharide biosynthesis and pathogenesis. Gene expression analysis and in vitro assays further validated the alteration in synthesis of slime, hemolysin, lipase, staphyloxanthin and oxidant susceptibility. Thus, the present study unveiled the multiple protein targeted antibiofilm potential of citral and portrays citral as a promising therapeutic agent to combat biofilm mediated MRSA infections with less possibility of resistance development.

The homologous putative GTPases Grn1p from fission yeast and the human GNL3L are required for growth and play a role in processing of nucleolar pre-rRNA.

Grn1p from fission yeast and GNL3L from human cells, two putative GTPases from the novel HSR1_MMR1 GTP-binding protein subfamily with circularly permuted G-motifs play a critical role in maintaining normal cell growth. Deletion of Grn1 resulted in a severe growth defect, a marked reduction in mature rRNA species with a concomitant accumulation of the 35S pre-rRNA transcript, and failure to export the ribosomal protein Rpl25a from the nucleolus. Deleting any of the Grn1p G-domain motifs resulted in a null phenotype and nuclear/nucleolar localization consistent with the lack of nucleolar export of preribosomes accompanied by a distortion of nucleolar structure. Heterologous expression of GNL3L in a Deltagrn1 mutant restored processing of 35S pre-rRNA, nuclear export of Rpl25a and cell growth to wild-type levels. Genetic complementation in yeast and siRNA knockdown in HeLa cells confirmed the homologous proteins Grn1p and GNL3L are required for growth. Failure of two similar HSR1_MMR1 putative nucleolar GTPases, Nucleostemin (NS), or the dose-dependent response of breast tumor autoantigen NGP-1, to rescue deltagrn1 implied the highly specific roles of Grn1p or GNL3L in nucleolar events. Our analysis uncovers an important role for Grn1p/GNL3L within this unique group of nucleolar GTPases.

A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: potential impact on CTL epitopes.

The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.

The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr.

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

Interplay between human nucleolar GNL1 and RPS20 is critical to modulate cell proliferation.

Human Guanine nucleotide binding protein like 1 (GNL1) belongs to HSR1_MMR1 subfamily of nucleolar GTPases. Here, we report for the first time that GNL1 promotes cell cycle and proliferation by inducing hyperphosphorylation of retinoblastoma protein. Using yeast two-hybrid screening, Ribosomal protein S20 (RPS20) was identified as a functional interacting partner of GNL1. Results from GST pull-down and co-immunoprecipitation assays confirmed that interaction between GNL1 and RPS20 was specific. Further, GNL1 induced cell proliferation was altered upon knockdown of RPS20 suggesting its critical role in GNL1 function. Interestingly, cell proliferation was significantly impaired upon expression of RPS20 interaction deficient GNL1 mutant suggest that GNL1 interaction with RPS20 is critical for cell growth. Finally, the inverse correlation of GNL1 and RPS20 expression in primary colon and gastric cancers with patient survival strengthen their critical importance during tumorigenesis. Collectively, our data provided evidence that cross-talk between GNL1 and RPS20 is critical to promote cell proliferation.

Global Quantitative Proteomics reveal Deregulation of Cytoskeletal and Apoptotic Signalling Proteins in Oral Tongue Squamous Cell Carcinoma.

Oral malignancies remain to have higher morbidity and mortality rates owing to the poor understanding of the carcinogenesis and the lack of early detection and diagnosis. The lack of established biomarkers for oral tongue squamous cell carcinoma (OTSCC) resulted in aggressive multi-modality management less effective. Here, we report for the first time that a panel of potential markers identified from tongue tumor samples using two-dimensional-differential-in-gel-electrophoresis (2D-DIGE). Our approach of combining 2D-DIGE with tandem mass spectrometry identified 24 candidate proteins including cofilins, myosin light chain family members, annexins, serpins, HSPs and tropomyosins, with significant differential expression in tongue carcinomas as compared with their matched adjacent normal tissues. The expression levels of the identified proteins were further validated in larger cohort of Indian samples using qPCR. Most of the differentially regulated proteins are involved in actin cytoskeletal dynamics, drug resistance, immune system, inflammation and apoptotic signalling pathways and are known to play critical role in oral tumorigenesis. Taken together, the results from present investigation provide a valuable base for understanding the development and progression of OTSCC. The validated panel of proteins may be used as potential biomarkers for early detection as well as in predicting therapeutic outcome of OTSCC.

Vanillic acid from Actinidia deliciosa impedes virulence in Serratia marcescens by affecting S-layer, flagellin and fatty acid biosynthesis proteins.

Serratia marcescens is one of the important nosocomial pathogens which rely on quorum sensing (QS) to regulate the production of biofilm and several virulence factors. Hence, blocking of QS has become a promising approach to quench the virulence of S. marcescens. For the first time, QS inhibitory (QSI) and antibiofilm potential of Actinidia deliciosa have been explored against S. marcescens clinical isolate (CI). A. deliciosa pulp extract significantly inhibited the virulence and biofilm production without any deleterious effect on the growth. Vanillic acid was identified as an active lead responsible for the QSI activity. Addition of vanillic acid to the growth medium significantly affected the QS regulated production of biofilm and virulence factors in a concentration dependent mode in S. marcescens CI, ATCC 14756 and MG1. Furthermore vanillic acid increased the survival of Caenorhabditis elegans upon S. marcescens infection. Proteomic analysis and mass spectrometric identification of differentially expressed proteins revealed the ability of vanillic acid to modulate the expression of proteins involved in S-layers, histidine, flagellin and fatty acid production. QSI potential of the vanillic acid observed in the current study paves the way for exploring it as a potential therapeutic candidate to treat S. marcescens infections.

Nimbolide upregulates RECK by targeting miR-21 and HIF-1α in cell lines and in a hamster oral carcinogenesis model.

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a potent inhibitor of matrix metalloproteinases (MMPs) is a common negative target of oncogenic signals and a potential therapeutic target for novel drug development. Here, we show that sequential RECKlessness stimulates angiogenesis and Notch signalling in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model, a paradigm for oral oncogenesis and chemointervention. We also report the chemotherapeutic effect of nimbolide, a limonoid from the neem tree (Azadirachta indica) based on the upregulation of RECK as well as modulation of the expression of key molecules involved in invasion and angiogenesis. We demonstrate that nimbolide upregulates RECK by targeting miR-21, and HIF-1α resulting in reduced MMP activity and blockade of VEGF and Notch signalling. Nimbolide reduced microvascular density, confirming its anti-angiogenic potential. Molecular docking analysis revealed interaction of nimbolide with HIF-1α. Additionally, we demonstrate that nimbolide upregulates RECK expression via downregulation of HIF-1α and miR-21 by overexpression and knockdown experiments in SCC4 and EAhy926 cell lines. Taken together, these findings provide compelling evidence that targeting RECK, a keystone protein that regulates mediators of invasion and angiogenesis with phytochemicals such as nimbolide may be a robust therapeutic approach to prevent oral cancer progression.