AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei—Crystal structure,mode of action,and biological activity

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC₅₀ of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.     

Novel pH-dependent regulation of human cytosolic sialidase 2 (NEU2) activities by siastatin B and structural prediction of NEU2/siastatin B complex

Human cytosolic sialidase (Neuraminidase 2, NEU2) catalyzes the removal of terminal sialic acid residues from glycoconjugates. The effect of siastatin B, known as a sialidase inhibitor, has not been evaluated toward human NEU2 yet. We studied the regulation of NEU2 activity by siastatin B in vitro and predicted the interaction in silico. Inhibitory and stabilizing effects of siastatin B were analyzed in comparison with DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) toward 4-umbelliferyl N-acetylneuraminic acid (4-MU-NANA)- and α2,3-sialyllactose-degrading activities of recombinant NEU2 produced by E. coli GST-fusion gene expression. Siastatin B exhibited to have higher competitive inhibitory activity toward NEU2 than DANA at pH 4.0. We also revealed the stabilizing effect of siastatin B toward NEU2 activity at acidic pH. Docking model was constructed on the basis of the crystal structure of NEU2/DANA complex (PDB code: 1VCU). Molecular docking predicted that electrostatic neutralization of E111 and E218 residues of the active pocket should not prevent siastatin B from binding at pH 4.0. The imino group (¹NH) of siastatin B can also interact with D46, neutralized at pH 4.0. Siastatin B was suggested to have higher affinity to the active pocket of NEU2 than DANA, although it has no C7–9 fragment corresponding to that of DANA. We demonstrated here the pH-dependent affinity of siastatin B toward NEU2 to exhibit potent inhibitory and stabilizing activities. Molecular interaction between siastatin B and NEU2 will be utilized to develop specific inhibitors and stabilizers (chemical chaperones) not only for NEU2 but also the other human sialidases, including NEU1, NEU3 and NEU4, based on homology modeling.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

Identification of novel telomeric G-quadruplex-targeting chemical scaffolds through screening of three NCI libraries

Thirteen compounds with diverse chemical structures have been identified as selective telomeric G-quadruplex-binding ligands through screening the NCI Diversity Set II, the NCI Natural Products Set II and the NCI Mechanistic Diversity Set libraries containing a total of 2307 members against a human telomeric G-quadruplex using a FRET-based DNA melting assay. These compounds show significant selectivity towards a telomeric G-quadruplex compared to duplex DNA, fall within a molecular weight range of 327-533, and are generally consistent with the Lipinski Rule of Five for drug-likeness. Thus they provide new chemical scaffolds for the development of novel classes of G-quadruplex-targeting agents.     

Assessing Essential Trace Elements in Cave Nectar Bat (Eonycteris spelaea): A Study in Barak Valley of Assam,India

Biological Trace Element Research - This study investigated trace elements in the different organs of Eonycteris spelaea, a hill cave from the Bhuban Hills of Sonai Reserve Forest, Cachar, Assam...     

Substantial genetic divergence and lack of recent gene flow support cryptic speciation in a colour polymorphic bumble bee (Bombus bifarius) species complex

Phenotypic polymorphism can constitute an inherent challenge for species delimitation. This issue is exemplified in bumble bees (Bombus), where species can exhibit high colour variation across their range, but otherwise exhibit little morphological variation to distinguish them from close relatives. We examine the species status of one of the most abundant North American bumble bees, Bombus bifarius Cresson, which historically comprised two major taxa, bifarius s.s. and nearcticus. These lineages are recognized primarily by red and black variation in their mid-abdominal coloration; however, a continuum from black (nearcticus) to red (bifarius s.s.) variation has led to their historic synonymization. Integrating mitochondrial and nuclear data and whole-genome sequencing, we reveal a high level of both mitochondrial and nuclear divergence delimiting two morphologically cryptic species – the red bifarius s.s. and the colour-variable (black to red) nearcticus. Population genomic analysis supports an absence of recent genomic admixture and a strong population structure between the two clades, even in sympatry. Species distribution models predict partially differentiated niches between the genetically inferred clades with annual precipitation being a leading differentiating variable. The bifarius s.s. lineage also occupies significantly higher elevations, with regions of sympatry being among the highest elevations in nearcticus. Our data also support a subspecies-level divergence between the broadly distributed nearcticus and the island population vancouverensis. In this paper, we formally recognize the two species, Bombus bifarius Cresson and Bombus vancouverensis Cresson, the latter including the subspecies B. vancouverensis vancouverensis comb.n. and B. vancouverensis nearcticus comb.n ., with vancouverensis the name bearer due to year priority.     

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The objective was to evaluate the effect of the interval between ovarian hyperstimulation and laparoscopic ovum pick-up (LOPU) on quality and developmental competence of goat oocytes before and after in vitro maturation (IVM) and intracytoplasmic sperm injection (ICSI). Estrus was synchronized with an intravaginal insert containing 0.3g progesterone (CIDR) for 10d, combined with a luteolytic treatment of 125 microg cloprostenol 36 h prior to CIDR removal. Ovaries were hyperstimulated with 70 mg FSH and 500 IU hCG given im 36, 60, or 72 h prior to LOPU (n=15, 16, and 7 does, respectively). For these groups, oocyte retrieval rates (mean+/-S.E.M.) were 24.7+/-2.9, 54.5+/-4.7, and 82.8+/-4.6% (P<0.001), and the proportions of cumulus-oocyte complexes (COC) with more than five layers of cumulus cells were 29.7+/-8.3, 37.6+/-6.9, and 37.3+/-7.0% (P<0.001). The proportion of IVM oocytes was highest at 72 h (82.1+/-2.8%; P<0.05), with no significant difference between 36 and 60 h (57.3+/-8.9% and 69.0+/-8.4%). Cleavage rates of ICSI embryos were 4.2+/-4.2, 70.9+/-8.4, and 78.9+/-8.2% with LOPU 36, 60, and 72 h post FSH/hCG (P<0.01), with a lower proportion of Grade-A embryos (P<0.05) following LOPU at 36 h compared to 60 and 72 h (29.7+/-8.3%, 37.6+/-6.9%, and 37.3+/-7.0%). In summary, a prolonged interval from FSH/hCG to LOPU improved oocyte retrieval rate and oocyte quality. Therefore, under the present conditions, LOPU 60 or 72 h after FSH/hCG optimized yields of good-quality oocytes for IVM and embryo production in goats.     

Identification of conformational B-cell Epitopes in an antigen from its primary sequence

Background

One of the major challenges in the field of vaccine design is to predict conformational B-cell epitopes in an antigen. In the past, several methods have been developed for predicting conformational B-cell epitopes in an antigen from its tertiary structure. This is the first attempt in this area to predict conformational B-cell epitope in an antigen from its amino acid sequence.

Results

All Support vector machine (SVM) models were trained and tested on 187 non-redundant protein chains consisting of 2261 antibody interacting residues of B-cell epitopes. Models have been developed using binary profile of pattern (BPP) and physiochemical profile of patterns (PPP) and achieved a maximum MCC of 0.22 and 0.17 respectively. In this study, for the first time SVM model has been developed using composition profile of patterns (CPP) and achieved a maximum MCC of 0.73 with accuracy 86.59%. We compare our CPP based model with existing structure based methods and observed that our sequence based model is as good as structure based methods.

Conclusion

This study demonstrates that prediction of conformational B-cell epitope in an antigen is possible from is primary sequence. This study will be very useful in predicting conformational B-cell epitopes in antigens whose tertiary structures are not available. A web server CBTOPE has been developed for predicting B-cell epitope http://www.imtech.res.in/raghava/cbtope/.