An insight into molecular taxonomy of bufonids,microhylids, and dicroglossid frogs: First genetic records from Pakistan

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.     

NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR

Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.     

CD80/CD86 costimulation regulates acute vascular rejection

Xenotransplantation may provide the only solution to the shortage of human donor organs. Although hyperacute rejection associated with xenotransplantation can now be overcome, acute vascular rejection (AVR) remains a primary barrier to xenotransplantation. To date, standard immunosuppressive agents fail to block AVR or prolong xenograft survival. The present study was undertaken to determine the role of CD80/CD86 costimulatory molecules in regulating AVR. Lewis rat hearts were transplanted heterotopically into wild-type or IL-12, CD80- or CD86-deficient C57BL/6 mice. Wild-type recipients were treated with CD80 or CD86 neutralizing Ab with and without daily cyclosporin A (CsA, 15 mg/kg). Transplanted hearts in untreated wild-type recipients were rejected on postoperative days (POD) 17-21 and showed cell-mediated rejection (CMR) and AVR pathologies. In contrast, transplanted hearts in IL-12 and CD80 recipients or wild-type recipients treated with CD80 neutralizing Ab were rapidly rejected on POD 5 and 6 with AVR pathology. Interestingly, hearts transplanted into CD86 knockout recipients or wild-type recipients treated with CD86 neutralizing Ab underwent CMR on POD 17. Finally, blockade of CD86 but not CD80 rendered xenograft recipients sensitive to daily CsA therapy, leading to indefinite xenograft survival. To conclude, we demonstrate that AVR can be overcome by blocking the CD86 costimulatory pathway. Furthermore, we demonstrate that CD80 and CD86 have opposing roles in regulation of xenotransplantation rejection, where CD80 drives CMR and attenuates AVR while CD86 drives AVR. Most strikingly, indefinite xenograft survival can be achieved by suppressing AVR with CD86 neutralization in combination of CsA therapy, which inhibits CMR.     

Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link

Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H₂O₂) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H₂O₂ exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2 yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.     

Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.     

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.     

Incongruity between Affinity Patterns Based on Mandibular and Lower Dental Dimensions following the Transition to Agriculture in the Near East,Anatolia and Europe

While it has been suggested that malocclusion is linked with urbanisation, it remains unclear as to whether its high prevalence began 8,000 years earlier concomitant with the transition to agriculture. Here we investigate the extent to which patterns of affinity (i.e., among-population distances), based on mandibular form and dental dimensions, respectively, match across Epipalaeolithic, Mesolithic, and Neolithic samples from the Near East/Anatolia and Europe. Analyses were conducted using morphological distance matrices reflecting dental and mandibular form for the same 292 individuals across 21 archaeological populations. Thereafter, statistical analyses were undertaken on four sample aggregates defined on the basis of their subsistence strategy, geography, and chronology to test for potential differences in dental and mandibular form across and within groups. Results show a clear separation based on mandibular morphology between European hunter-gatherers, European farmers, and Near Eastern transitional farmers and semi-sedentary hunter-gatherers. In contrast, the dental dimensions show no such pattern and no clear association between the position of samples and their temporal or geographic attributes. Although later farming groups have, on average, smaller teeth and mandibles, shape analyses show that the mandibles of farmers are not simply size-reduced versions of earlier hunter-gatherer mandibles. Instead, it appears that mandibular form underwent a complex series of shape changes commensurate with the transition to agriculture that are not reflected in affinity patterns based on dental dimensions. In the case of hunter-gatherers there is a correlation between inter-individual mandibular and dental distances, suggesting an equilibrium between these two closely associated morphological units. However, in the case of semi-sedentary hunter-gatherers and farming groups, no such correlation was found, suggesting that the incongruity between dental and mandibular form began with the shift towards sedentism and agricultural subsistence practices in the core region of the Near East and Anatolia.