In vitro activity of a recombinant ABC transporter protein in the processing of plantaricin E pre-peptide.

Most bacteriocins of lactic acid bacteria (LAB) are initially synthesized as pre-peptides with an N-terminal extension (leader peptides). Generally, the precursor peptides containing a double-glycine-type leader are processed by a dedicated ATP-binding cassette (ABC) transporter. The ABC transporter and an accessory protein lead to the cleavage of inactive pre-peptide with the concomitant export of the mature peptide across the cytoplasmic membrane. Plantaricins E, F, J, and K belong to class IIb 2-peptide bacteriocins and are synthesized as pre-peptides containing N-terminal G-G leader peptide. In this study, the heterologous expression, purification, and characterization of PlnE pre-peptide, ABC transporter (PlnG), and accessory protein (PlnH) from Lactobacillus plantarum LR/14 in Escherichia coli BL21 (DE3) strain were reported. An in vitro assay was conducted with the inactive PlnE pre-peptide, which after cleavage by the addition of ABC transporter protein exhibited antimicrobial activity against some LAB species. The activity of cleaved pre-peptide was comparable to the activity of mature peptide. Accessory protein was also heterologously expressed and purified; however, no effect on processing activity was detected by the addition of the accessory protein, which suggests that accessory protein is not involved in cleavage, but it might help in the transport of mature plantaricins across the membrane.

Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides.

Plantaricin gene-specific primers were used to obtain plnE, -F, -J and -K structural gene amplicons from soil metagenome. These amplicons were cloned and expressed in pET32a (+) vector in Escherichia coli BL21 (DE3). PlnE, -F, -J and -K peptides were expressed as His-tagged-fusion proteins and were separated by Ni(2+) -chelating affinity chromatography. The peptides were released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. The cleaved peptides were further analysed for antimicrobial activity and found to be active against Listeria innocua NRRL B33314, Micrococcus luteus MTCC 106 and lactic acid bacteria, such as Enterococcus casseliflavus NRRL B3502, Lactococcus lactis lactis NRRL 1821, Lactobacillus curvatus NRRL B4562 and Lactobacillus plantarum NRRL B4496. E. coli has been successfully exploited as a host for heterologous expression with a significant yield of fused and cleaved peptides in the range of 8-12 and 1-1.5 mg/l of the culture, respectively. Heterologous expression, therefore, can be used to overcome the constraints of low yield often reported from a native strain.

Consequences of physical disturbance by tadpoles and snails on chironomid larvae.

Indirect interactions among community members impact on organisms. The effects of two snails, banded pond snail, Bellamya bengalensis (Lamarck), and Red-rimmed melania, Melanoides tuberculata (Müller), and tadpoles of Asian common toad, Duttaphrynus melanostictus (Schneider), on nonbiting midge larvae, Chironomus striatipennis Kieffer, were observed in experimental microcosm. Decrease in tube number and tube length of midge larvae was observed compared to control condition due to introduction of selected above mentioned organisms. The direct effects of non-predator organisms on the midge larvae are due to physical disturbance that destroys their tubes. This may result in vulnerability of midge larvae to predators in the wild. So the community structure may be altered by indirect effects, where one or more species, through their direct disturbance, indirectly change the abundance of other species.

Inhibitory effect of plantaricin peptides (Pln E/F and J/K) against Escherichia coli.

Plantaricins are small bioactive peptides produced by Lactobacillus plantarum strains that exhibit significant antimicrobial activity against closely-related Gram-positive bacteria, including food spoilage organisms. In comparison, bacteriocins including plantaricins, are usually less effective against Gram-negative organisms. In this study, we demonstrate that heterologously expressed and purified plantaricins, Pln E, -F, -J, and -K when tested against Gram negative model organism Escherichia coli K-12 were highly effective under certain conditions. The apparent tolerance of Gram-negative members to these peptides has been explained on the basis of the presence of the outer membrane (OM) that acts as a protective barrier. We have shown that agents and/or conditions that destabilize OM of E. coli K-12, make it susceptible to plantaricin peptides. In order to further strengthen this conclusion, an OM lipoprotein-defective lpp mutant strain of E. coli K-12 was also studied and compared. A significant loss of cell viability both in terms of CFU/ml as well as with live-dead dual staining combined with flow cytometry, could be demonstrated with the lpp mutant in comparison to the wild type strain. The results indicate that plantaricins can inhibit Gram-negative bacteria if the outer-membrane is weakened and it can be used in preservation of food with the help of some food-grade chelating agents.

Palladium(0)-catalysed regioselective cyclisations of 2-amino(tosyl) benzamides/sulphonamides: the stereoselective synthesis of 3-ylidene-[1,4]benzodiazepin-5-ones/benzo[f][1,2,5]thiadiazepine-1,1-dioxides.

The Pd(0) catalysed cyclisation reactions between tert-butyl propargyl carbonates and 2-aminotosyl benzamides or sulphonamides deliver 1,4-benzodiazepin-5-ones or sultam derivatives, key components of many biologically active compounds. But 2-amino benzamides/sulphonamides require propargyl carbonates substituted at acetylenic carbon to undergo the reaction resulting in the stereoselective formation of the said products.

Prostac: A New Composite Score With Potential Predictive Value in Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed solid organ cancer in men worldwide. Current diagnosis of PCa includes use of initial prostate specific antigen assay which has a high false positive rate, low specificity, and low sensitivity. The side effects of unnecessary prostate biopsies that healthy men are subjected to, often result in unintended health complications. New PCa biomarkers are being discovered to address this unmet need. Here, we report on the creation of a composite score (Prostac) based on three recently discovered PCa biomarkers, Plasmacytoma Variant Translocation 1 (PVT1) exons 4A, 4B, and 9. Statistical analysis of copy numbers derived from a real-time quantitative polymerase chain (qPCR) reaction - based assay, showed these PCa biomarkers to be linearly separable and significantly over expressed in PCa epithelial cells. We train a supervised learning algorithm using support vector machines to generate a classification hyperplane from which a user-friendly composite score is developed. Cross validation of Prostac using data from prostate epithelial cells (RWPE1) and PCa cells (MDA PCa 2b) accurately classified 100% of PCa cells. Creation of the Prostac score lays the groundwork for clinical trial of its use in PCa diagnosis.

Revisiting Post-Sterilization Regret in India.

AIM: This study analyses the socio-demographic characteristics associated with post-sterilization regret. STUDY DESIGN: The study uses cross-sectional data from the fourth round of National Family Health Surveys (2015-2016). METHODS: Simple bivariate and binary logistic regressions analyses were used. RESULTS: Research shows that 7% of women aged 15-49 reported sterilization regret, which increased by 2% from 2005 to 2016. It was found that factors significantly associated with sterilization regret were years since sterilization, child loss experience, regions of residence, and quality of services. Women who got sterilized at the age of 30 or more were more likely to express regret, than women who were sterilised before 25 years of age, when adjusted for confounding variables (aO.R= 1.006). Women having sons were less likely to report sterilization regret than women who had only daughters (aO.R.=1.3 for each) but on the contrary women having both son and daughter are significantly less likely to express regret in comparison with women having only sons (aO.R. = 0.8 for each. Women who had experienced child loss had higher odds of reporting sterilization regret in rural (aO.R =1.2) as well as in urban (aO.R = 1.3) areas respectively, compared to those who did not experience any child loss. CONCLUSION: Women need to be counselled about the permanent nature of sterilization in order to avoid future regret as sterilization is largely dominated by socio-economic conditions. Thus, couples' decision-making towards using the contraceptive from the basket of choice would help in uplifting the social and cultural status of women in conservative societies and will have a positive effect on contraceptive use. In addition, efforts should be made to educate both the partners equally about contraceptive methods that have higher efficiency. Further, there is also a need to improve the quality of services, both in terms of counselling and service provision. Lastly, health-related policies should tackle disparities in the empowerment, and economic status of women that would result in decreased post-sterilization regret, and will improve sexual relationships following sterilization.

Oncogenic Role of PVT1 and Therapeutic Implications.

PVT1, a long non-coding RNA has been implicated in a variety of human cancers. Recent advancements have led to increasing discovery of the critical roles of PVT1 in cancer initiation and progression. Novel insight is emerging about PVT1's mechanism of action in different cancers. Identifying and understanding the variety of activities of PVT1 involved in cancers is a necessity for the development of PVT1 as a diagnostic biomarker or therapeutic target in cancers where PVT1 is dysregulated. PVT1's varied activities include overexpression, modulation of miRNA expression, protein interactions, targeting of regulatory genes, formation of fusion genes, functioning as a competing endogenous RNA (ceRNA), and interactions with MYC, among many others. Furthermore, bioinformatic analysis of PVT1 interactions in cancers has aided understanding of the numerous pathways involved in PVT1 contribution to carcinogenesis in a cancer type-specific manner. However, these recent findings show that there is much more to be learned to be able to fully exploit PVT1 for cancer prognostication and therapy. In this review, we summarize some of the latest findings on PVT1's oncogenic activities, signaling networks and how targeting these networks can be a strategy for cancer therapy.

Population Differentiation at the PVT1 Gene Locus: Implications for Prostate Cancer.

Genetic variation in susceptibility to complex diseases, such as cancer, is well-established. Enrichment of disease associated alleles in specific populations could have implications for disease incidence and prevalence. Prostate cancer (PCa) is a disease with well-established higher incidence, prevalence, and worse outcomes among men of African ancestry in comparison to other populations. PCa is a multi-factorial, complex disease, but the exact mechanisms for its development and progression are unclear. The gene desert located on chromosome 8q24 is associated with aggressiveness of PCa. Interestingly, the non-protein coding gene locus Plasmacytoma Variant Translocation (PVT1) is present at chromosome 8q24 and is overexpressed in PCa. PVT1 gives rise to multiple transcripts with potentially different molecular and cellular functions. In an analysis of the PVT1 locus using data from the 1000 Genomes Project, we found the chromosomal region spanning PVT1 exons 4A and 4B to be highly differentiated between African and non-African populations. We further investigated levels of gene expression of PVT1 exons 4A and 4B and observed significant overexpression of these exons in PCa tissues relative to benign prostatic hyperplasia and to normal prostate tissues obtained from men of African ancestry. These results indicate that PVT1 exons 4A and 4B may have clinical implications in PCa a conclusion supported by the observation that transient and stable overexpression of PVT1 exons 4A and 4B significantly induce greater prostate epithelial cell migration and proliferation. We anticipate that further exploration of the role of PVT1 exons 4A and 4B may lead to the development of diagnostic, therapeutic, and other clinical applications in PCa.

Copy number-based quantification assay for non-invasive detection of PVT1-derived transcripts.

BACKGROUND: One of the most important susceptibility loci for cancer is the 8q24 human chromosomal region. The non-protein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in prostate cancer. PVT1 gives rise to multiple transcripts which may have different functions. Here, we describe a real-time quantitative polymerase chain reaction (qPCR)-based assay for copy number-based quantitation of PVT1 exons 4A, 4B, and 9 to enable accurate, reproducible, and quantifiable detection. METHODS: PVT1 exons 4A, 4B, and 9 were cloned into a plasmid vector to create standards for subsequent creation of linear standard curves representing a broad range of concentrations. PCR was carried out using SYBR-Green signal detection to quantify PVT1 exons 4A, 4B, and 9. The efficacy of this assay was evaluated by using it to detect these transcripts in prostate epithelial and prostate cancer cell lines, normal and cancerous human prostate tissues, human serum, mouse plasma, and urine samples. RESULTS: The results indicate that the assay can be used to quantify both low and high copy numbers of PVT1-derived transcripts. This is the first report of a copy number-based quantification assay for non-invasive detection of PVT1 derived transcripts. CONCLUSIONS: This novel assay holds promise for routine non-invasive testing in diseases where PVT1 is dysregulated.

Long Noncoding RNA from PVT1 Exon 9 Is Overexpressed in Prostate Cancer and Induces Malignant Transformation and Castration Resistance in Prostate Epithelial Cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer and second leading cause of cancer-related death for men in the United States. The nonprotein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in different cancers. PVT1 gives rise to several alternatively spliced transcripts and microRNAs. There are at least twelve exons of PVT1, which make separate transcripts, and likely have different functions. Here, we demonstrate that PVT1 exon 9 is significantly overexpressed in PCa tissues in comparison to normal prostate tissues. Both transient and stable overexpression of PVT1 exon 9 significantly induced greater prostate epithelial cell migration, as well as increased proliferation and corresponding proliferating cell nuclear antigen (PCNA) expression. Notably, implantation into mice of a non-tumorigenic prostate epithelial cell line stably overexpressing PVT1 exon 9 resulted in the formation of malignant tumors. Furthermore, PVT1 exon 9 overexpression significantly induced castration resistance. Consequently, PVT1 exon 9 expression is important for PCa initiation and progression, and holds promise as a therapeutic target in PCa.

Mutagenesis Reveals an Unusual Combination of Guanines in RNA G-Quadruplex Formation.

The classic G-quadruplex motif consists of a continuous array of 3-4 guanine residues with an intermittent loop size of 1-7 nucleotides (G3-4N1-7G3-4N1-7G3-4N1-7G3-4). An RNA G-quadruplex is able to attain only one parallel G-quadruplex topology owing to steric constraints. Investigating the possibilities of the formation of RNA G-quadruplexes with a stretch of sequences deviating from this classic motif will add to the overall conformations of RNA G-quadruplexes, bestowing diversity to this structure. Here, we report unusual combinations of guanine residues involved in RNA G-quadruplex formation in the 5' untranslated region (UTR) of the von Willebrand factor (VWF) mRNA using the mutagenesis approach. Different permutations and combinations of guanine residues form G-quadruplexes. Upon investigation, G-quadruplexes in 5' UTR of VWF mRNA are shown to exhibit an inhibitory function.

Scaling Up the Production of Recombinant Antimicrobial Plantaricin E from a Heterologous Host, Escherichia coli.

Enhanced production of heterologously expressed plantaricin (plnE) from Escherichia coli BL21 (DE3) was achieved from a small- to large-scale batch culture. Starting from a 15-ml shake-flask culture grown in Luria-Bertani (LB) broth, the protein expression could be scaled up using 50 ml, 100 ml, 1 l, and 2 l batch culture. Using similar condition, plantaricin E (PlnE) was successfully expressed in a 30-l stirred fermenter. The protein was expressed as TRX-(His)6-fusion protein and separated by Ni(2+) affinity chromatography. Growth in two complex media, LB and Terrific broth (TB), was optimized and compared for the production of PlnE, which was higher in LB in comparison with that of TB. In the fermenter, 140 and 180 mg of PlnE could be produced from 12 l of culture volume at 30 and 25 °C, respectively. The yield of heterologously purified PlnE was found to be 1.2-1.5%, which was much higher in comparison with the plantaricins produced from the native strain of Lactobacillus plantarum (0.3-0.7%). Overproduction of PlnE with the help of heterologous expression can overcome the constraint of the low yield from producer strain and provides an easy and low-cost strategy for large-scale production.