Solvent-stable Pseudomonas aeruginosa PseA protease gene: identification, molecular characterization, phylogenetic and bioinformatic analysis to study reasons for solvent stability

We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonas aeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide. The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 alpha-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.     

Conquering the night: understanding nocturnal migration in birds

Abstract

Migration is a biologically distinct and unique phenomenon that enables the birds to migrate twice-a-year between the breeding and wintering grounds. These movements are known as spring and autumn migration, respectively. Depending on their inherent programming, the migratory birds may fly during day or night or both. Different environmental factors such as, temperature, food, predator pressure and physiological demands of energy storage and expenditure, contribute to the pattern of migrations, day or nighttime. Since, most of them are nighttime migrants they have to make dramatic changes in their physiology and behavior to transform them from being diurnal to predominantly nocturnal. These changes result in different life history stages (LHSs) such as migration, reproduction and molt, in their annual cycle, which are regulated by endogenous circadian and circannual clocks. As a result, the birds start preparing well in advance for the approaching LHS. The present review focuses on behavioral strategies of a nocturnal migrant and understanding of the possible physiological responses to ensure successful migration.     

Diversity-oriented sustainable synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives: New ligands for a metallo-β-lactamase from Klebsiella pneumonia

A simple, environmentally benign and highly proficient microwave assisted one-pot approach for the synthesis of antimicrobial spiropyrrolidine/thiapyrrolizidine oxindole derivatives is reported assembling two pharmacophoric moieties (1,3-indanedione and pyrrolidine/thiapyrrolizidine) in a single molecular framework via three-component 1,3-dipolar cycloaddition reaction of substituted isatin, sarcosine/1,3-thiazoles-4-carboxylic acid and Knoevenagel adduct (2-Cyano-3-phenyl-acrylic acid ethyl ester or 2-Benzylidene-malononitrile) in 2,2,2-trifluoroethanol as a reusable green solvent. Good functional group tolerance and broad scope of usable substrates are other prominent features of the present methodology with high degree of chemo-, regio- and stereoselectivity. The stereochemistry of synthesized compounds was confirmed by single crystal X-ray analysis. All the synthetic compounds were examined for their antimicrobial potential. The synthesized compounds having pyrrolothiazole moiety showed excellent activity against K. pneumoniae as compared to others and even more inhibitory activity than the mentioned drugs, i.e. compounds 6a (MIC = 0.09 μg/mL), 6b (MIC = 0.045 μg/mL), 6c (MIC = 0.005 μg/mL), 6d (MIC = 0.19 μg/mL). Additionally, compound 6c has shown better binding affinity against New Delhi Metallo-beta-Lactamase-1 (NDM-1) protein in computational docking studies.     

Place prioritization for biodiversity content

The prioritization of places on the basis of biodiversity content is part of any systematic biodiversity conservation planning process. The place prioritization procedure implemented in the ResNet software package is described. This procedure is primarily based on the principles of rarity and complementarity. Application of the procedure is demonstrated with two analyses, one data set consisting of the distributions of termite genera in Namibia, and the other consisting of the distributions of bird species in the Islas Malvinas/Falkland Islands. The attributes that data sets should have for the effective and reliable application of such procedures are discussed. The procedure used here is compared to some others that are also currently in use.     

Genetic transformation of sweet sorghum

Sweet sorghum has substantial potential as a biofuel feedstock, with advantages in some environments over alternatives such as sugarcane or maize. Gene technologies are likely to be important to achieve yields sufficient for food, fuel and fibre production from available global croplands, but sorghum has proven difficult to transform. Tissue culture recalcitrance and poor reproducibility of transformation protocols remain major challenges for grain sorghum, and there has been no reported success for sweet sorghum. Here we describe a repeatable transformation system for sweet sorghum, based on (1) optimized tissue culture conditions for embryogenic callus production with >90% regenerability in 12-week-old calli, and (2) an effective selection regimen for hygromycin resistance conferred by a Ubi-hpt transgene following particle bombardment. Using this method, we have produced sixteen independent transgenic lines from multiple batches at an overall efficiency of 0.09% transformants per excised immature embryo. Co-expression frequency of a non-selected luciferase reporter was 62.5%. Transgene integration and expression were confirmed in T₀ and T₁ plants by Southern analysis and luciferase assays. This success using the major international sweet sorghum cultivar Ramada provides a foundation for molecular improvement of sweet sorghum through the use of transgenes. Factors likely to be important for success with other sweet sorghum cultivars are identified.     

Survey of medical training in cytopathology carried out by the journal Cytopathology

Anshu, A. Herbert, B. Cochand‐Priollet, P. Cross, M. Desai, R. Dina, J. Duskova, A. Evered, A. Farnsworth, W. Gray, S. S. Gupta, K. Kapila, I. Kardum‐Skelin, V. Kloboves‐Prevodnik, T. K. Kobayashi, H. Koutselini, W. Olszewski, B. Onal, M. B. Pitman, ?. Marin?ek, T. Sauer, U. Schenck, F. Schmitt, I. Shabalova, J. H. F. Smith, E. Tani, L. Vass, P. Vielh and H. Wiener
Survey of medical training in cytopathology carried out by the journal Cytopathology This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3–6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6–12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides and assess adequacy of fine‐needle aspiration samples when immediate diagnosis was not required. The survey demonstrated a clear need for European and international guidelines for training in cytopathology.     

Optical band gap enhancements of chemically synthesized α-Ni(OH)2 nanoparticles by a novel technique: Precipitator molarity variation

Nickel hydroxide nanoparticles (NHNPs) are extremely important semiconducting materials for applications in energy storage and energy harvesting devices. This study uses a novel variation in molarity of the sodium hydroxide (NaOH) precipitator solution to enhance the direct optical band gap in the NHNPs chemically synthesized by using nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O) as the precursor. The simple, energy benign chemical precipitation route involved the usage of 1 M (Ni(NO₃)₂·6H₂O) solutions as the precursor and 0.4 M, 0.6 M, and 0.8 M NaOH solutions as the precipitator solutions. The simple variation in precipitator molarity induces an increase in pH from about 6.9 to 7.5 of the reactant solution. As the molarity of the precursor solution does not change, the change in pH of the reactant solution is equivalent to the change in the pH of the precipitator solution. The NHNPs characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR) and ultraviolet-visible (UV-vis) techniques confirm a reduction of the nanocrystallite size from about 6.8 to 4.5 nm with a concomitant enhancement in the direct optical band gap energy from about 2.64 to 2.74 eV. The possible mechanisms that could be operative behind obtaining microstructurally tuned (MT)-NHNPs and band gap engineering (BGE) of the MT-NHNPs are discussed from both theoretical and physical process perspectives. Further, the implications of these novel results for possible future applications are briefly touched upon. The reported results might be useful to assess the material as an active electrode to improve the performance of batteries.     

In vitro regeneration and optimization of factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation in <Emphasis Type="Italic">Artemisia Pallens</Emphasis>, an important medicinal plant

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog’s medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog’s medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD₆₀₀ of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T₀ transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.     

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.