Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish

Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.     

Profiles of Serum Cytokines in Acute Drug-Induced Liver Injury and Their Prognostic Significance

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)].

Conclusions

Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance.     

Different population histories of the Mundari- and Mon-Khmer-speaking Austro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertion polymorphism in Indian populations

Length variation in the human mtDNA intergenic region between the cytochrome oxidase II (COII) and tRNA lysine (tRNA^lys) genes has been widely studied in world populations. Specifically, Austronesian populations of the Pacific and Austro-Asiatic populations of southeast Asia most frequently carry the 9-bp deletion in that region implying their shared common ancestry in haplogroup B. Furthermore, multiple independent origins of the 9-bp deletion at the background of other mtDNA haplogroups has been shown in populations of Africa, Europe, Australia, and India. We have analyzed 3293 Indian individuals belonging to 58 populations, representing different caste, tribal, and religious groups, for the length variation in the 9-bp motif. The 9-bp deletion (one copy) and insertion (three copies) alleles were observed in 2.51% (2.15% deletion and 0.36% insertion) of the individuals. The maximum frequency of the deletion (45.8%) was observed in the Nicobarese in association with the haplogroup B5a D-loop motif that is common throughout southeast Asia. The low polymorphism in the D-loop sequence of the Nicobarese B5a samples suggests their recent origin and a founder effect, probably involving migration from southeast Asia. Interestingly, none of the 302 (except one Munda sample, which has 9-bp insertion) from Mundari-speaking Austro-Asiatic populations from the Indian mainland showed the length polymorphism of the 9-bp motif, pointing either to their independent origin from the Mon-Khmeric-speaking Nicobarese or to an extensive admixture with neighboring Indo-European-speaking populations. Consistent with previous reports, the Indo-European and Dravidic populations of India showed low frequency of the 9-bp deletion/insertion. More than 18 independent origins of the deletion or insertion mutation could be inferred in the phylogenetic analysis of the D-loop sequences.     

QTL-seq for the identification of candidate genes for days to flowering and leaf shape in pigeonpea

To identify genomic segments associated with days to flowering (DF) and leaf shape in pigeonpea, QTL-seq approach has been used in the present study. Genome-wide SNP profiling of extreme phenotypic bulks was conducted for both the traits from the segregating population (F₂) derived from the cross combination- ICP 5529 × ICP 11605. A total of 126.63 million paired-end (PE) whole-genome resequencing data were generated for five samples, including one parent ICP 5529 (obcordate leaf and late-flowering plant), early and late flowering pools (EF and LF) and obcordate and lanceolate leaf shape pools (OLF and LLS). The QTL-seq identified two significant genomic regions, one on CcLG03 (1.58 Mb region spanned from 19.22 to 20.80 Mb interval) for days to flowering (LF and EF pools) and another on CcLG08 (2.19 Mb region spanned from 6.69 to 8.88 Mb interval) for OLF and LLF pools, respectively. Analysis of genomic regions associated SNPs with days to flowering and leaf shape revealed 5 genic SNPs present in the unique regions. The identified genomic regions for days to flowering were also validated with the genotyping-by-sequencing based classical QTL mapping method. A comparative analysis of the identified seven genes associated with days to flowering on 12 Fabaceae genomes, showed synteny with 9 genomes. A total of 153 genes were identified through the synteny analysis ranging from 13 to 36. This study demonstrates the usefulness of QTL-seq approach in precise identification of candidate gene(s) for days to flowering and leaf shape which can be deployed for pigeonpea improvement.Subject terms: Genetic association study, Plant hybridization

QTL-seq approach was utilized for mapping of genomic regions/genes associated with days to flowering and leaf shape in pigeonpea. Analysis of genomic regions and associated SNPs with days to flowering and leaf shape revealed 1 and 4 non-synonymous SNPs, respectively. The study demonstrated sequencing-based trait mapping approach can accelerate trait mapping of the targeted traits.     

Biomethanation of poultry litter leachate in UASB reactor coupled with ammonia stripper for enhancement of overall performance

In the present study possibility of coupling stripper to remove ammonia to the UASB reactor treating poultry litter leachate was studied to enhance the overall performance of the reactor. UASB reactor with stripper as ammonia inhibition control mechanism exhibited better performance in terms of COD reduction (96%), methane yield (0.26m(3)CH(4)/kg COD reduced), organic loading rate (OLR) (18.5kg COD m(-3)day(-1)) and Hydraulic residence time (HRT) (12h) compared to the UASB reactor without stripper (COD reduction: 92%; methane yield: 0.21m(3)CH(4)/kg COD reduced; OLR: 13.6kg CODm(-3)day(-1); HRT: 16h). The improved performance was due to the reduction of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) in the range of 75-95% and 80-95%, respectively by the use of stripper. G/L (air flow rate/poultry leachate flow rate) in the range of 60-70 and HRT in the range of 7-9min are found to be optimum parameters for the operation of the stripper.     

Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection

HIV immunity is likely CD4 T cell dependent. HIV-specific CD4 T cell proliferative responses are reported to correlate inversely with virus load and directly with specific CD8 responses. However, the phenotype and cytokine profile of specific CD4 T cells that correlate with disease is unknown. We compared the number/function of Gag p24-specific CD4 T cells in 17 HIV-infected long-term nonprogressors (LTNPs) infected for a median of 14.6 years with those of 16 slow progressors (SPs), also HIV infected for a median of 14 years but whose CD4 count had declined to <500 cells/ micro l. Compared with SPs, LTNPs had higher numbers of specific CD4s that were double positive for IFN-gamma and IL-2 as well as CD28 and IL-2. However, CD4 T cells that produced IL-2 alone (IL-2(+)IFN-gamma(-)) or IFN-gamma alone (IFN-gamma(+)IL-2(-)) did not differ between LTNPs and SPs. The decrease in p24-specific CD28(+)IL-2(+) cells with a concomitant increase of p24-specific CD28(-)IL-2(+) cells occurred before those specific for a non-HIV Ag, CMV. p24-specific CD28(-)IL-2(+) cells were evident in LTNPs and SPs, whereas the CMV-specific CD28(-)IL-2(+) response was confined to SPs. The difference between LTNPs and SPs in the Gag p24 IFN-gamma(+)IL-2(+) response was maintained when responses to total Gag (p17 plus p24) were measured. The percentage and absolute number of Gag-specific IFN-gamma(+)IL-2(+) but not of IFN-gamma(+)IL-2(-) CD4s correlated inversely with virus load. The Gag-specific IFN-gamma(+)IL-2(+) CD4 response also correlated positively with the percentage of Gag-specific IFN-gamma(+) CD8 T cells in these subjects. Accumulation of specific CD28(-)IL-2(+) helpers and loss of IFN-gamma(+)IL-2(+) CD4 T cells may compromise specific CD8 responses and, in turn, immunity to HIV.     

Mutations in QARS,Encoding Glutaminyl-tRNA Synthetase,Cause Progressive Microcephaly,Cerebral-Cerebellar Atrophy,and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.