Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase,Reveals Novel Properties

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.     

The EAR Motif Controls the Early Flowering and Senescence Phenotype Mediated by Over-Expression of SlERF36 and Is Partly Responsible for Changes in Stomatal Density and Photosynthesis

The EAR motif is a small seven amino acid motif associated with active repression of several target genes. We had previously identified SlERF36 as an EAR motif containing gene from tomato and shown that its over-expression results in early flowering and senescence and a 25–35% reduction of stomatal density, photosynthesis and stomatal conductance in transgenic tobacco. In order to understand the role of the EAR motif in governing the phenotypes, we have expressed the full-length SlERF36 and a truncated form, lacking the EAR motif under the CaMV35S promoter, in transgenic Arabidopsis. Plants over-expressing the full-length SlERF36 show prominent early flowering under long day as well as short day conditions. The early flowering leads to an earlier onset of senescence in these transgenic plants which in turn reduces vegetative growth, affecting rosette, flower and silique sizes. Stomatal number is reduced by 38–39% while photosynthesis and stomatal conductance decrease by about 30–40%. Transgenic plants over-expressing the truncated version of SlERF36 (lacking the C-terminal EAR motif), show phenotypes largely matching the control with normal flowering and senescence indicating that the early flowering and senescence is governed by the EAR motif. On the other hand, photosynthetic rates and stomatal number were also reduced in plants expressing SlERF36ΔEAR although to a lesser degree compared to the full- length version indicating that these are partly controlled by the EAR motif. These studies show that the major phenotypic changes in plant growth caused by over-expression of SlERF36 are actually mediated by the EAR motif.     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.     

Novel genetic tools for Hansenula polymorpha

Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H.?polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H.?polymorpha. For this, we generated a yku80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H.?polymorpha.     

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

Background  

Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.     

Compensating Human–Wildlife Conflict in Protected Area Communities: Ground-Level Perspectives from Uttarakhand, India

This paper examines people’s experiences with economic compensation for losses due to human–wildlife conflict (HWC) in Uttarakhand, India. Employing a combination of qualitative and quantitative approaches, we used a case study approach to investigate (1) socio-economic characteristics of applicant versus non-applicant households, (2) explanations for why only some households chose to apply, and (3) perceptions of program effectiveness. We found that despite widespread complaints, the participation rate was only 37%. Our results broadly support the findings of other studies which have identified inadequate remuneration, processing delays, and corruption as key problems. However, we also found that non-participation was itself a critical problem. Our study indicates that participation in the scheme was shaped by factors including wealth, gender, social networks, and pre-existing expectations. We highlight the need for improved communication about what “compensation” can and should be, advocate for reconceptualizations of compensation that are more closely based on ground-level realities, and point to the potential for alternative forms of payment to be more sustainable and socially just.

Effect of steric molecular field settings on CoMFA predictivity

Steric molecular field can be represented in a number of ways in comparative molecular field analysis (CoMFA). This study aimed to investigate whether the choice of steric molecular field settings significantly influences the predictive performance of CoMFA and, if so, which is the best. The three-dimensional quantitative structure activity relationship (3D-QSAR) models based on Lennard–Jones, indicator, parabolic and Gaussian steric fields were compared using 28 datasets taken from the literature. The analysis of the predictive ability of these models (cross validated R²) indicates that steric fields in which the value drops off quickly with distance (i.e. Lennard–Jones and indicator fields) tend to perform better than the Gaussian version, which has a slower and smoother decrease. Furthermore, depending on the steric field type used, the field sampling density (i.e. grid spacing) has a variable influence on the predictive ability of the models generated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of the Chemotypes of Ocimum forskolei and Ocimum basilicum by NMR Spectroscopy

The chemotypes of Ocimum forskolei Benth and Ocimum basilicum L. growing wild in Oman have been established by ¹³C‐NMR analyses of the vegetative and floral oils of the plants. The chemotypes, estragole for O. forskolei and linalool for O. basilicum, suggested by ¹³C‐NMR fingerprinting were also confirmed by GC‐FID and GC/MS analyses. The oil of O. forskolei demonstrated better activities against bacteria and dermatophytes. The significance of the presence of estragole and linalool in the volatile oils of plants whose fragrances are traditionally inhaled, added to food, or rubbed on the skin are discussed.