Variation in glucose metabolism under acidified sodium nitrite mediated nitrosative stress in Saccharomyces cerevisiae.

AIMS: The work aimed to understand the important changes during glucose metabolism in Saccharomyces cerevisiae under acidified sodium nitrite (ac.NaNO2 ) mediated nitrosative stress. METHODS AND RESULTS: Confocal microscopy and fluorescence-activated cell sorting analysis were performed to investigate the generation of reactive nitrogen and oxygen species, and redox homeostasis under nitrosative stress was also characterized. Quantitative PCR analysis revealed that the expression of ADH genes was upregulated under such condition, whereas the ACO2 gene was downregulated. Some of the enzymes of the tricarboxylic acid cycle were partially inhibited, whereas malate metabolism and alcoholic fermentation were increased under nitrosative stress. Kinetics of ethanol production was also characterized. A network analysis was conducted to validate our findings. In the presence of ac.NaNO2 , in vitro protein tyrosine nitration formation was checked by western blotting using pure alcohol dehydrogenase and aconitase. CONCLUSIONS: Alcoholic fermentation rate was increased under stress condition and this altered metabolism might be conjoined with the defence machinery to overcome the nitrosative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work of this kind where the role of metabolism under nitrosative stress has been characterized in S. cerevisiae and it will provide a base to develop an alternative method of industrial ethanol production.

Molecular profiling of microbial community structure and their CAZymes via metagenomics, from Tsomgo lake in the Eastern Himalayas.

The present study is the first of its kind which is focused on Tsomgo lake, a high-altitude lake, located in the Eastern Himalayas of Sikkim. To get a major insight into the bacterial diversity, the shotgun sequencing was carried out in Illumina platform. Our results showed that both the samples TLSS1 (soil) and TLSW1 (water), had Proteobacteria as the most abundant taxa. Cluster of Orthologous group (COG) functional category of TLSS1 has 1,46,965 predicted functions. Cluster of Orthologous Group (COG) functional category of TLSW1 has 1,34,773 predicted functions. Kyoto Encyclopedia of Gene and Genomes (KEGG) functional category of TLSS1 has 1,76,825 predicted functions, most of the sequence fall in metabolism followed by Environmental information processing function. (KEGG) functional category of TLSW1 has 1,62,696 predicted functions and it follows the same pattern as TLSS1. Our studies also provide insight into the presence of distribution of different carbohydrate-active enzymes (CAZymes) present in Tsomgo lake. We have found that in case of both the samples TLSW1 and TLSS1, GlycosylTransferases were active followed by GlycosylHydrolase. The result found, represents for the first time very important findings related to the microbial diversity and the abundance of CAZymes in Tsomgo lake one of the pristine high-altitude lakes in Sikkim.

Gedunin isolated from the mangrove plant Xylocarpus granatum exerts its anti-proliferative activity in ovarian cancer cells through G2/M-phase arrest and oxidative stress-mediated intrinsic apoptosis.

Gedunin is a natural tetranorterpenoid secondary metabolite found in plants of the Meliaceae family, which has been reported for its antiparasitic, antifungal and anticancer activities. Here, we describe the molecular mechanisms underlying the in vitro anti proliferative activity of gedunin (isolated from the mangrove plant Xylocarpus granatum) in human ovarian cancer cells. We observed that gedunin triggered severe ROS generation leading to DNA damage and cell cycle arrest in G2/M phase thus inhibiting cell proliferation. ROS upregulation also led to mitochondrial stress and membrane depolarization, which eventually resulted in mitochondria-mediated apoptosis following cytochrome C release, caspase 9, 3 activation, and PARP cleavage. Transmission electron microscopy of gedunin treated cells revealed sub-cellular features typical of apoptosis. Moreover, an upregulation in stress kinases like phospho-ERK 1/2, phospho-p38 and phospho-JNK was also observed in gedunin treated cells. Free radical scavenger N-Acetyl-L-Cysteine (NAC) reversed all these effects resulting in increased cell survival, abrogation of cell cycle arrest, rescue of mitochondrial membrane potential and suppression of apoptotic markers. Interestingly, gedunin is also an inhibitor of the evolutionarily conserved molecular chaperone Heat Shock Protein 90 (hsp90) responsible for maintaining cellular homeostasis. Targeting this chaperone could be an attractive strategy for developing cancer therapeutics since many oncogenic proteins are also client proteins of hsp90. Collectively, our findings provide insights into the molecular mechanism of action of gedunin, which may aid drug development efforts against ovarian cancer.

An optimal velocity for online limb-target regulation processes?

The utilization of visual information for the control of ongoing voluntary limb movements has been investigated for more than a century. Recently, online sensorimotor processes for the control of upper-limb reaches were hypothesized to include a distinct process related to the comparison of limb and target positions (i.e., limb-target regulation processes: Elliott et al. in Psychol Bull 136:1023-1044. doi: 10.1037/a0020958 , 2010). In the current study, this hypothesis was tested by presenting participants with brief windows of vision (20 ms) when the real-time velocity of the reaching limb rose above selected velocity criteria. One experiment tested the perceptual judgments of endpoint bias (i.e., under- vs. over-shoot), and another experiment tested the shifts in endpoint distributions following an imperceptible target jump. Both experiments revealed that limb-target regulation processes take place at an optimal velocity or "sweet spot" between movement onset and peak limb velocity (i.e., 1.0 m/s with the employed movement amplitude and duration). In contrast with pseudo-continuous models of online control (e.g., Elliott et al. in Hum Mov Sci 10:393-418. doi: 10.1016/0167-9457(91)90013-N , 1991), humans likely optimize online limb-target regulation processes by gathering visual information at a rather limited period of time, well in advance of peak limb velocity.

Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development.

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing.

PURPOSE: Genetic testing is routinely used for second-tier confirmation of newborn sequencing results to rule out false positives and to confirm diagnoses in newborns undergoing inpatient and outpatient care. We developed a targeted next-generation sequencing panel coupled with a variant processing pipeline and demonstrated utility and performance benchmarks across multiple newborn disease presentations in a retrospective clinical study. METHODS: The test utilizes an in silico gene filter that focuses directly on 126 genes related to newborn screening diseases and is applied to the exome or a next-generation sequencing panel called NBDx. NBDx targets the 126 genes and additional newborn-specific disorders. It integrates DNA isolation from minimally invasive biological specimens, targeted next-generation screening, and rapid characterization of genetic variation. RESULTS: We report a rapid parallel processing of 8 to 20 cases within 105 hours with high coverage on our NBDx panel. Analytical sensitivity of 99.8% was observed across known mutation hotspots. Concordance calls with or without clinical summaries were 94% and 75%, respectively. CONCLUSION: Rapid, automated targeted next-generation sequencing and analysis are practical in newborns for second-tier confirmation and neonatal intensive care unit diagnoses, laying a foundation for future primary DNA-based molecular screening of additional disorders and improving existing molecular testing options for newborns.

Targeted capture and massively parallel sequencing of 12 human exomes.

Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.

Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by orally active clerodane diterpene.

This study was performed to investigate the mechanistic aspects of cell death induced by a clerodane diterpene (K-09) in Leishmania donovani promastigotes that was previously demonstrated to be safe and orally active against visceral leishmaniasis (VL). K-09 caused depolarization of the mitochondrion and the generation of reactive oxygen species, triggering an apoptotic response in L. donovani promastigotes. Mitochondrial dysfunction subsequently resulted in the release of cytochrome c into the cytosol, impairing ATP production. Oxidative stress caused the depletion of reduced glutathione, while pretreatment with antioxidant N-acetyl cysteine (NAC) was able to abrogate oxidative stress. However, NAC failed to restore the mitochondrial membrane potential or intracellular calcium homeostasis after K-09 treatment, suggesting that the generation of oxidative stress is a downstream event relative to the other events. Caspase-3/-7-like protease activity and genomic DNA fragmentation were observed. Electron microscopy studies revealed gross morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis of the nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket alterations, and the displacement of organelles. Moreover, an increased number of lipid droplets was detected after K-09 treatment, which is suggestive of altered lipid metabolism. Our results indicate that K-09 induces mitochondrial dysfunction and oxidative stress-mediated apoptotic cell death in L. donovani promastigotes, sharing many features with metazoan apoptosis. These mechanistic insights provide a basis for further investigation toward the development of K-09 as a potential drug candidate for VL.

PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.

High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing.

DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.

Temporally Local Tactile Codes Can Be Stored in Working Memory.

Tactile exploration often involves sequential touches interspersed with stimulus-free durations (e.g., the time during which the hand moves from one textured surface to the other). Whereas it is obvious that texture-related perceptual variables, irrespective of the encoding strategy, must be stored in memory for comparison, it is rather unclear which of those variables are held in memory. There are two established variables-"intensity" and "frequency", which are "temporally global" variables because of the long stimulus integration interval required to average the signal or derive spectral components, respectively; on the other hand, a recently established third contender is the "temporally local" variable that codes for kinematic profiles of very short, suprathreshold events in the vibrotactile signal. Here, we present the first psychophysical evidence that temporally local variables can be stored in memory. To that end, we asked participants to detect changes in pulsatile indentation stimuli at their fingertips with and without a gap of 1 s between stimulus presentations. The stimuli either contained global variables alone (change of pulse rate), or a mix of local and global variables (change of pulse shape). We found, first, that humans are much better at detecting a change in stimuli when local variables are available rather than global ones alone-as evident by the fact that 21 compared to only 6 participants out of 25 yielded a valid psychophysical curve, respectively. Second, this observation persists even when there is a gap between the stimuli, implying local variables must be stored in memory. Third, an extensive array of relevant intensity definitions failed to explain participants' performance in any consistent manner, which implies that perceptual decisions were less likely to be driven by intensity coding. Taken together, our results suggest that humans perform pulsatile change detection utilizing local pulse shape, and to a lesser degree global pulse rate, and that both parameters can be stored in memory.

A shotgun approach to explore the bacterial diversity and a brief insight into the glycoside hydrolases of Samiti lake located in the Eastern Himalayas.

BACKGROUND: The Himalayas have always been an enigma and, being biodiversity hotspots, are considered extremely important from an ecological point of view. Recent advances in studies regarding high-altitude lakes have garnered relevant importance as these habitats could harbor potential psychrophilic and psychrotrophic microbes with bio-prospective applications. Contemplating the above scenario, the present study has been undertaken to understand the diversity and the functional capacities of the microbes thriving in this lake. RESULTS: In our present study on Samiti Lake, the abundance of Proteobacteria as the major phylum was seen in both the soil and water samples. Incase of the ABSLW (water) and ABS1 (soil) sample, 148,066 and 239,754 predicted genes, were taken for functional analysis. The KEGG analysis showed that ABSLW and ABS1 had 122,911 and 160,268, genes assigned to KO terms respectively. Whereas in case of COG functional analysis, 104,334 and 130,191 genes were assigned to different COG classes for ABSLW and ABS1 respectively. Further, on studying the glycoside hydrolases, an abundance of GH13, GH2, GH3, GH43, and GH23 in both the soil and water samples were seen. CONCLUSION: Our study has provided a comprehensive report about the bacterial diversity and functional capacities of microbes thriving in Samiti Lake.  It has also thrown some light on the occurrence of glycoside hydrolases in this region, as they have numerous biotechnological applications in different sectors.

Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses.

SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Here, using a genetically non-redundant Drosophila model, we show that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. We find that accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response.Abbreviations: Atg8a: Autophagy-related 8a; cnc: cap-n-collar; IFM: indirect flight muscle; KEAP1: kelch like ECH associated protein 1; LIR: LC3-interacting region; NFE2L2/Nrf2: NFE2 like bZIP transcription factor 2; PB1: Phox and Bem1; ref(2)P: refractory to sigma P; SAR: selective autophagy receptor; UBA: ubiquitin-associated.

Vibrotactile masking experiments reveal accelerated somatosensory processing in congenitally blind braille readers.

Braille reading is a demanding task that requires the identification of rapidly varying tactile patterns. During proficient reading, neighboring characters impact the fingertip at ∼100 ms intervals, and adjacent raised dots within a character at 50 ms intervals. Because the brain requires time to interpret afferent sensorineural activity, among other reasons, tactile stimuli separated by such short temporal intervals pose a challenge to perception. How, then, do proficient Braille readers successfully interpret inputs arising from their fingertips at such rapid rates? We hypothesized that somatosensory perceptual consolidation occurs more rapidly in proficient Braille readers. If so, Braille readers should outperform sighted participants on masking tasks, which demand rapid perceptual processing, but would not necessarily outperform the sighted on tests of simple vibrotactile sensitivity. To investigate, we conducted two-interval forced-choice vibrotactile detection, amplitude discrimination, and masking tasks on the index fingertips of 89 sighted and 57 profoundly blind humans. Sighted and blind participants had similar unmasked detection (25 ms target tap) and amplitude discrimination (compared with 100 µm reference tap) thresholds, but congenitally blind Braille readers, the fastest readers among the blind participants, exhibited significantly less masking than the sighted (masker, 50 Hz, 50 µm; target-masker delays, ±50 and ±100 ms). Indeed, Braille reading speed correlated significantly and specifically with masking task performance, and in particular with the backward masking decay time constant. We conclude that vibrotactile sensitivity is unchanged but that perceptual processing is accelerated in congenitally blind Braille readers.

Humans Use a Temporally Local Code for Vibrotactile Perception.

Sensory environments are commonly characterized by specific physical features, which sensory systems might exploit using dedicated processing mechanisms. In the tactile sense, one such characteristic feature is frictional movement, which gives rise to short-lasting (<10 ms), information-carrying integument vibrations. Rather than generic integrative encoding (i.e., averaging or spectral analysis capturing the "intensity" and "best frequency"), the tactile system might benefit from, what we call a "temporally local" coding scheme that instantaneously detects and analyzes shapes of these short-lasting features. Here, by employing analytic psychophysical measurements, we tested whether the prerequisite of temporally local coding exists in the human tactile system. We employed pulsatile skin indentations at the fingertip that allowed us to trade manipulation of local pulse shape against changes in global intensity and frequency, achieved by adding pulses of the same shape. We found that manipulation of local pulse shape has strong effects on psychophysical performance, arguing for the notion that humans implement a temporally local coding scheme for perceptual decisions. As we found distinct differences in performance using different kinematic layouts of pulses, we inquired whether temporally local coding is tuned to a unique kinematic variable. This was not the case, since we observed different preferred kinematic variables in different ranges of pulse shapes. Using an established encoding model for primary afferences and indentation stimuli, we were able to demonstrate that the found kinematic preferences in human performance, may well be explained by the response characteristics of Pacinian corpuscles (PCs), a class of human tactile primary afferents.

Implementation of Hospital-Based Supplemental Duchenne Muscular Dystrophy Newborn Screening (sDMDNBS): A Pathway to Broadening Adoption.

Duchenne muscular dystrophy (DMD) is not currently part of mandatory newborn screening, despite the availability of a test since 1975. In the absence of screening, a DMD diagnosis is often not established in patients until 3-6 years of age. During this time, irreversible muscle degeneration takes place, and clinicians agree that the earlier therapy is initiated, the better the long-term outcome. With recent availability of FDA-approved DMD therapies, interest has renewed for adoption by state public health programs, but such implementation is a multiyear process. To speed access to approved therapies, we implemented a unique, hospital-based program offering parents of newborns an optional, supplemental DMD newborn screen (NBS) via a two-tiered approach: utilizing a creatine kinase (CK) enzyme assay coupled with rapid targeted next-generation sequencing (tNGS) for the DMD gene (using a Whole-Exome Sequencing (WES) assay). The tNGS/WES assay integrates the ability to detect both point mutations and large deletion/duplication events. This tiered newborn screening approach allows for the opportunity to improve treatment and outcomes, avoid the diagnostic delays, and diminish healthcare disparities. To implement this screening algorithm through hospitals in a way that would ultimately be acceptable to public health laboratories, we chose an FDA-approved CK-MM immunoassay to avoid the risks of false-negative/-positive results. Because newborn CK values can be affected due to non-DMD-related causes such as birth trauma, a confirmatory repeat CK assay on a later dried blood spot (DBS) collection has been proposed. Difficulties associated with non-routine repeat DBS collection, including the tracking and recall of families, and the potential creation of parental anxiety associated with false-positive results, can be avoided with this algorithm. Whereas a DMD diagnosis is essentially ruled out by the absence of detected DMD sequence abnormalities, a subsequent CK would still be warranted to confirm resolution of the initial elevation, and thus the absence of non-DMD muscular dystrophy or other pathologies. To date, we have screened over 1500 newborns (uptake rate of ~80%) by a CK-MM assay, and reflexed DMD tNGS in 29 of those babies. We expect the experience from this screening effort will serve as a model that will allow further expansion to other hospital systems until a universal public health screening is established.

Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila.

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship.

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP3Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps-including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases-in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases.

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae.

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Deltayhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Deltayhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.

Targeted next generation sequencing for newborn screening of Menkes disease.

PURPOSE: Population-based newborn screening (NBS) allows early detection and treatment of inherited disorders. For certain medically-actionable conditions, however, NBS is limited by the absence of reliable biochemical signatures amenable to detection by current platforms. We sought to assess the analytic validity of an ATP7A targeted next generation DNA sequencing assay as a potential newborn screen for one such disorder, Menkes disease. METHODS: Dried blood spots from control or Menkes disease subjects (n = 22) were blindly analyzed for pathogenic variants in the copper transport gene, ATP7A. The analytical method was optimized to minimize cost and provide rapid turnaround time. RESULTS: The algorithm correctly identified pathogenic ATP7A variants, including missense, nonsense, small insertions/deletions, and large copy number variants, in 21/22 (95.5%) of subjects, one of whom had inconclusive diagnostic sequencing previously. For one false negative that also had not been detected by commercial molecular laboratories, we identified a deep intronic variant that impaired ATP7A mRNA splicing. CONCLUSIONS: Our results support proof-of-concept that primary DNA-based NBS would accurately detect Menkes disease, a disorder that fulfills Wilson and Jungner screening criteria and for which biochemical NBS is unavailable. Targeted next generation sequencing for NBS would enable improved Menkes disease clinical outcomes, establish a platform for early identification of other unscreened disorders, and complement current NBS by providing immediate data for molecular confirmation of numerous biochemically screened condition.