Associative mechanism for phosphoryl transfer: a molecular dynamics simulation of Escherichia coli adenylate kinase complexed with its substrates

The ternary complex of Escherichia coli adenylate kinase (ECAK) with its substrates adenosine monophosphate (AMP) and Mg-ATP, which catalyzes the reversible transfer of a phosphoryl group between adenosine triphosphate (ATP) and AMP, was studied using molecular dynamics. The starting structure for the simulation was assembled from the crystal structures of ECAK complexed with the bisubstrate analog diadenosine pentaphosphate (AP(5)A) and of Bacillus stearothermophilus adenylate kinase complexed with AP(5)A, Mg(2+), and 4 coordinated water molecules, and by deleting 1 phosphate group from AP(5)A. The interactions of ECAK residues with the various moieties of ATP and AMP were compared to those inferred from NMR, X-ray crystallography, site-directed mutagenesis, and enzyme kinetic studies. The simulation supports the hypothesis that hydrogen bonds between AMP's adenine and the protein are at the origin of the high nucleoside monophosphate (NMP) specificity of AK. The ATP adenine and ribose moieties are only loosely bound to the protein, while the ATP phosphates are strongly bound to surrounding residues. The coordination sphere of Mg(2+), consisting of 4 waters and oxygens of the ATP beta- and gamma-phosphates, stays approximately octahedral during the simulation. The important role of the conserved Lys13 in the P loop in stabilizing the active site by bridging the ATP and AMP phosphates is evident. The influence of Mg(2+), of its coordination waters, and of surrounding charged residues in maintaining the geometry and distances of the AMP alpha-phosphate and ATP beta- and gamma-phosphates is sufficient to support an associative reaction mechanism for phosphoryl transfer.     

An in silico approach towards the identification of novel inhibitors of the TLR-4 signaling pathway

Precise functioning and fine-tuning of Toll-like receptor 4 (TLR4) signaling is a critical requirement for the smooth functioning of the innate immune system, since aberrant TLR4 activation causes excessive production of pro-inflammatory cytokines and interferons. This can result in life threatening conditions such as septic shock and other inflammatory disorders. The TRIF-related adaptor molecule (TRAM) adaptor protein is unique to the TLR4 signaling pathway and abrogation of TRAM-mediated TLR4 signaling is a promising strategy for developing therapeutics aimed at disrupting TRAM interactions with other components of the TLR4 signaling complex. The VIPER motif from the vaccinia virus-producing protein, A46 has been reported to disrupt TRAM-TLR4 interactions. We have exploited this information, in combination with homology modeling and docking approaches, to identify a potential binding site on TRAM lined by the BB loop and αC helix. Virtual screening of commercially available small molecules targeting the binding site enabled to short-list 12 small molecules to abrogate TRAM-mediated TLR4 signaling. Molecular dynamics and molecular mechanics calculations have been performed for the analysis of these receptor-ligand interactions.     

Synthesis of thiazole-based substituted piperidinone oximes: Profiling of antioxidant and antimicrobial activity

The synthesis of novel thiazole-based piperidinone oximes and screening of their antioxidant and antimicrobial activity are described. The obtained results revealed that the electronic effects of active substituents at C-4 terminals of phenyl rings on either side of piperidinone skeleton, as well as at 2-hydrazinyl thiazole, played a major role in development of antioxidant and antimicrobial activity. Antioxidant activity seems to be based also on radical dissipating ability of the thiazole ring. The nucleophilic character of sulfur in thiazole and lipophilic nature of piperidinone skeleton substantially influenced the observed antimicrobial activity of thiazole-based piperidinone oximes. Among the synthesized compounds, 2,6-bis(4-hydroxy-3-methoxyphenyl)-1-methylpiperidin-4-one O-(2-(2-(4-hydroxy-3-methoxybenzylidene)hydrazinyl)thiazol-4-yl) oxime exhibited excellent antioxidant activity whereas compound 2,6-bis(4-chloro phenyl)-1-methylpiperidin-4-one O-(2-(2-(4-nitrobenzylidene)hydrazinyl)thiazol-4-yl)oxime emerged as an outstanding antimicrobial agent.     

Root hair phenotypes influence nitrogen acquisition in maize

Background and AimsThe utility of root hairs for nitrogen (N) acquisition is poorly understood.MethodsWe explored the utility of root hairs for N acquisition in the functional–structural model SimRoot and with maize genotypes with variable root hair length (RHL) in greenhouse and field environments.Key ResultsSimulation results indicate that long, dense root hairs can improve N acquisition under varying N availability. In the greenhouse, ammonium availability had no effect on RHL and low nitrate availability increased RHL, while in the field low N reduced RHL. Longer RHL was associated with 216 % increase in biomass and 237 % increase in plant N content under low-N conditions in the greenhouse and a 250 % increase in biomass and 200 % increase in plant N content in the field compared with short-RHL phenotypes. In a low-N field environment, genotypes with long RHL had 267 % greater yield than those with short RHL. We speculate that long root hairs improve N capture by increased root surface area and expanded soil exploration beyond the N depletion zone surrounding the root surface.ConclusionsWe conclude that root hairs play an important role in N acquisition. We suggest that root hairs merit consideration as a breeding target for improved N acquisition in maize and other crops.     

Forest protection in Central India: do differences in monitoring by state and local institutions result in diverse social and ecological impacts?

Protection of forests and wildlife outside protected areas (PAs) is necessary for the conservation of wildlife. Extension of conservation efforts outside the existing PA may result in restrictions on local forest resource use. Such situations arise due to differences in understanding of forest as a resource for communities and as a conservation space for endangered species. A clearer focus is needed on the functionality and socio-ecological outcomes of different forest management institutions to address such issues. We conducted a study in a forest landscape connecting Pench and Tadoba-Andhari Tiger Reserves (TRs) in Central India. The two main forest management institutions were the Forest Department (FD) and local communities managing forest resources. We conducted vegetation surveys and focus group discussions in 15 villages selected based on presence or absence of active protection and monitoring of forest resources by either FD or local people. We found that forests with monitoring had significantly higher tree density and vegetation species richness compared to forests without monitoring. Tree density was observed to be higher in sites monitored by villagers rather than those monitored by FD. Self-regulation and resource sharing in locally monitored forests were more acceptable to local communities. In forests monitored by the FD, local communities indicated a feeling of alienation from the forest that weakened their motivation to protect the forest and wildlife. Recognition of local community rights is essential to achieve conservation goals and reduce social conflicts outside PAs, requiring collaboration between state and local institutions.     

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.     

Functional activity limitation of leprosy cases in an endemic area in Indonesia and recommendations for integrated participation program in society

BackgroundLeprosy continues to be a health problem in Indonesia, with incidence reaching over 10,000 new cases by 2021. Leprosy-related disabilities cause limitation of patients’ activity and participation in social activities. To date, no studies have been conducted in Indonesia which investigates disability in terms of bodily function, structure impairment, limitations in performing daily activities, and restrictions in participation in social activities in leprosy patients. This study is aimed to determine the demographic and clinical characteristics that might affect functional activity limitations of leprosy patients in endemic areas in Indonesia.Methods and findingsA cross-sectional study was conducted on 267 retrospectively-diagnosed cases of leprosy. The Screening of Activity Limitation and Safety Awareness (SALSA) scale was used to measure functional activity limitation, which comprises five domains: vision, mobility, self-care, work with hands, and dexterity. Differences among variables were evaluated using Kruskal-Wallis and Mann-Whitney test. The mean age of participants was 51.89±13.66 years, the majority of which were men (62.5%), uneducated (48.3%), and classified as type 2 in the World Health Organization (WHO) disability grading for hands and feet (66.3% and 68.2%, respectively). Assessment using the SALSA Scale showed 28.5% of subjects were without limitation, 43.8% with mild limitation, 13.5% with moderate limitation, 9.4% with severe limitation, and 4.9% with extreme limitation. Significant differences in the total SALSA Scale were found between age groups (p = 0.014), educational level (p = 0.005), occupation (p<0.001), and WHO disability grades (p<0.001). Multivariate analysis showed that the most significant factor influencing the total score of SALSA was disability grading for feet (score = 0.31, p <0.001) followed by occupational status, disability grading for eyes, and age. Limitation of functional activity was significantly correlated to becoming unemployed with the odds 2.59.ConclusionPeople affected by leprosy are prone to have functional activity limitation, especially the elderly, uneducated, unemployed and those with multiple disabilities. If they can overcome their barriers in functional activities, they will have better occupational opportunities.     

Emerging perspectives on multidomain phosphatidylinositol transfer proteins

The phosphatidylinositol transfer protein domain (PITP_d) is an evolutionarily conserved protein that is able to transfer phosphatidylinositol between membranes in vitro and in vivo. However some animal genomes also include genes that encode proteins where the PITP_d is found in cis with a number of additional domains and recent large scale genome sequencing efforts indicate that this type of multidomain architecture is widespread in the animal kingdom. In Drosophila photoreceptors, the multidomain phosphatidylinositol transfer protein RDGB is required to regulate phosphoinositide turnover during G-protein activated phospholipase C signalling. Recent studies in flies and mammalian cell culture models have begun to elucidate functions for the non-PITP_d of RDGB and its vertebrate orthologs. We review emerging evidence on the genomics, functional and cell biological perspectives of these multi-domain PITP_d containing proteins.