Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR_n=4). Mobilities of ILPR_n=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR_n=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR_n=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR_n=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR_n=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.     

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.     

Genome-guided exploration of metabolic features of Streptomyces peucetius ATCC 27952: past,current, and prospect

Applied Microbiology and Biotechnology - Streptomyces peucetius ATCC 27952 produces two major anthracyclines, doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for...     

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)β(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.     

Long-term effects of plant hormones on K+ levels and transport in young wheat plants of different K+ status

Long-term effects of 1-naphtaleneacetic acid (NAA), benzyladenine (BA), gibberellic acid (GA₃), abscisic acid (ABA) and ethylene on K⁺ levels, K⁺ uptake and translocation to the shoot were studied in young wheat plants (Triticum aesticum L. cv. Martonvásári-8) grown at different K⁺ supplies. Na⁺ levels and K⁺/Na⁺ selectivity were also investigated. Both in shoots and roots, NAA, BA and ABA decreased K⁺ and Na⁺ levels more effectively in high-K⁺ plants than in low-K⁺ plants. GA, and ethylene did not influence K⁺ and Na⁺ levels. K⁺/Na⁺ selectivity in roots of low-K⁺ plants was increased in favour of K⁺ by BA, NAA and to a lesser extent by ABA. In high-K⁺ plants only BA increased the K⁺/Na⁺ ratio, whereas the effects of the other hormones were the opposite (NAA) or less pronounced (ABA). K⁺(⁸⁶Rb) uptake was inhibited by NAA and BA in low-K⁺ plants but not in high-K⁺ plants. K⁺(⁸⁶Rb) uptake was inhibited throughout by 10 μM ABA. K⁺(⁸⁶Rb) translocation to the shoot was influenced by the hormones similarly to the uptake patterns, with the exception of ABA, which inhibited translocation in low-K⁺ plants but not in high-K⁺ plants. The results show that hormonal effects may quantitatively and qualitatively be modified by K⁺ levels in the plant and that internal K⁺ concentration may play a role in the mechanisms regulating the effects of NAA, BA and ABA but probably not in those of GA₃ or ethylene.     

Evaluation of fungal culture filtrate containing chitinase as a biocontrol agent against Helicoverpa armigera

AIMS: To evaluate the biocontrol efficacy of culture filtrate containing chitinase from Trichoderma harzianum against Heliothis. METHODS AND RESULTS: T. harzianum was cultured by submerged fermentation using colloidal chitin as sole carbon source. The ability of the culture filtrate to hydrolyse colloidal chitin indicated the presence of chitinase as one of its components. Biocontrol assay on Heliothis showed that the culture filtrate is a potent antifeedant as it reduced the feeding rate and body weight of the larvae. It reduced the successful pupation and increased larval and pupal mortality in a dosage-dependent manner when applied topically. The highest mortalities (70%) were recorded for groups treated with 2000 U ml(-1) chitinase activity. The percentage of adult emergence was zero for the highest chitinase concentration (2000 U ml(-1)) tried. CONCLUSIONS: The studies showed that the culture filtrate containing chitinase from T. harzianum is capable of negatively affecting the growth and metamorphosis of Heliothis larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the need for safer and environmentally friendly pest management tools, the present study could help in the development of enzyme-based biopesticides against Heliothis.     

Focus: The Science of Stress: Rap1A,Rap1B,and β-Adrenergic Signaling in Autologous HCT: A Randomized Controlled Trial of Propranolol

Successful hematopoietic cell transplantation (HCT) depends on rapid engraftment of the progenitor and stem cells that will reestablish hematopoiesis. Rap1A and Rap1B are two closely related small GTPases that may affect platelet and neutrophil engraftment during HCT through their roles in cell adhesion and migration. β-adrenergic signaling may regulate the participation of Rap1A and Rap1B in engraftment through their inhibition or activation. We conducted a correlative study of a randomized controlled trial evaluating the effects of the nonselective β-antagonist propranolol on expression and prenylation of Rap1A and Rap1B during neutrophil and platelet engraftment in 25 individuals receiving an autologous HCT for multiple myeloma. Propranolol was administered for 1 week prior to and 4 weeks following HCT. Blood was collected 7 days (baseline) and 2 days (Day -2) before HCT, and 28 days after HCT (Day +28). Circulating polymorphonuclear cells (PMNC) were isolated and analyzed via immunoblotting to determine levels of prenylated and total Rap1A versus Rap1B. Twelve participants were randomized to the intervention and 13 to the control. Rap1A expression significantly correlated with Rap1B expression. Rap1B expression significantly correlated with slower platelet engraftment; however, this association was not observed in the propranolol-treated group. There were no significant associations between neutrophil engraftment and Rap1A or Rap1B expression. Post hoc exploratory analyses did not reveal an association between social health variables and Rap1A or Rap1B expression. This study identifies a greater regulatory role for Rap1B than Rap1A in platelet engraftment and suggests a possible role for β-adrenergic signaling in modulating Rap1B function during HCT.     

Saccharina japonica,a potential feedstock for pigment production using submerged fermentation

In this study, the feasibility and applicability of marine algal biomass Saccharina (Laminaria) japonica as a sole substrate for the production of pigments by Talaromyces amestolkiae GT11 in submerged fermentation was evaluated. Results indicated that the fungus T. amestolkiae GT11 produced the highest amount of extracellular yellow (444.83 ± 22) and red (200.94 ± 12), and intracellular yellow (362.28 ± 34) and red (193.87 ± 10) pigments, utilizing 1% (w/v) of S. japonica powder at an initial pH of 5 and 30°C, as compared to other physiochemical parameters tested. The pH and thermostability analysis results demonstrated that even after 5 h of incubation the pigment was found to be highly stable at pH 6 and 40 ~ 60°C with 98% and 90.56 ~ 84.69% of residual absorbance, respectively. Apart from the application of pigment as a natural colorant instead of synthetic one in biotechnology industry, the fermented substrate itself can be exploited as food and feed with enhanced nutrient content, improved protein quality and fiber digestibility, etc. However, further studies concerning the safety and functional properties of the pigment and fermented substrate are required. Furthermore, this study provides the evidences about the biological method of making easily fermentable biomass for biorefiners or other metabolite production.     

Pandemic swine influenza virus (H1N1): A threatening evolution

“Survival of the fittest” is an old axiom laid down by the great evolutionist Charles Darwin and microorganisms seem to have exploited this statement to a great extent. The ability of viruses to adapt themselves to the changing environment has made it possible to inhabit itself in this vast world for the past millions of years. Experts are well versed with the fact that influenza viruses have the capability to trade genetic components from one to the other within animal and human population. In mid April 2009, the Centers for Disease Control and Prevention and the World Health Organization had recognized a dramatic increase in number of influenza cases. These current 2009 infections were found to be caused by a new strain of influenza type A H1N1 virus which is a re-assortment of several strains of influenza viruses commonly infecting human, avian, and swine population. This evolution is quite dependent on swine population which acts as a main reservoir for the reassortment event in virus. With the current rate of progress and the efforts of heath authorities worldwide, we have still not lost the race against fighting this virus. This article gives an insight to the probable source of origin and the evolutionary progress it has gone through that makes it a potential threat in the future, the current scenario and the possible measures that may be explored to further strengthen the war against pandemic.     

Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.