ABSTRACT
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.
Subject(s)
Autophagosomes , Lysosomes , Membrane Fusion , Phosphatidylinositol Phosphates , Qa-SNARE Proteins , Lysosomes/metabolism , Humans , Autophagosomes/metabolism , Membrane Fusion/drug effects , Qa-SNARE Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Autophagy/physiology , Autophagy/drug effects , Liposomes/metabolism , Molecular Dynamics Simulation , HeLa CellsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Ethanol/metabolism , Fermentation , Glucose/metabolism , Nitrosative Stress , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sodium Nitrite/metabolism , Sodium Nitrite/pharmacologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Autophagy , NF-E2-Related Factor 2 , Animals , Antioxidants/pharmacology , Autophagy/physiology , Autophagy-Related Protein 8 Family/metabolism , Carrier Proteins , Drosophila/metabolism , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Aggregates , Sequestosome-1 Protein/metabolism , Superoxides/metabolism , Ubiquitin/metabolism , Ubiquitinated Proteins/metabolismABSTRACT
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.
Subject(s)
Autophagy , Ion Channels/metabolism , Calcium/metabolism , Humans , Lysosomes/metabolism , Models, Biological , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Touch Perception , Biomechanical Phenomena , Humans , Physical Stimulation , Skin , Touch , VibrationABSTRACT
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.
Subject(s)
Bacteria , Enzymes , Lakes , Metagenomics , Microbiota , Soil Microbiology , Bacteria/classification , Bacteria/enzymology , Bacteria/genetics , Enzymes/genetics , India , Lakes/microbiology , Microbiota/genetics , PhylogenyABSTRACT
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.
Subject(s)
Autophagosomes/metabolism , Autophagy-Related Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Lipids/analysis , Animals , Autophagosomes/chemistry , Autophagosomes/genetics , Autophagy , Autophagy-Related Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Female , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Lipid Metabolism , Male , MutationABSTRACT
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 (nâ¯=â¯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.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Limonins/pharmacology , Meliaceae/chemistry , Reactive Oxygen Species/agonists , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/genetics , Caspase 3/genetics , Caspase 3/metabolism , Caspase 9/genetics , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Fruit/chemistry , G2 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Neoplastic , Histones/genetics , Histones/metabolism , Humans , Inhibitory Concentration 50 , Limonins/chemistry , Limonins/isolation & purification , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Oxidative Stress , Plant Extracts/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Pompe disease (PD) is screened by a two tier newborn screening (NBS) algorithm, the first tier of which is an enzymatic assay performed on newborn dried blood spots (DBS). As first tier enzymatic screening tests have false positive results, an immediate second tier test on the same sample is critical in resolving newborn health status. Two methodologies have been proposed for second tier testing: (a) measurement of enzymatic activities such as of Creatine/Creatinine over alpha-glucosidase ratio, and (b) DNA sequencing (a molecular genetics approach), such as targeted next generation sequencing. (tNGS). In this review, we discuss the tNGS approach, as well as the challenges in providing second tier screening and follow-up care. While tNGS can predict genotype-phenotype effects when known, these advantages may be diminished when the variants are novel, of unknown significance or not discoverable by current test methodologies. Due to the fact that criticisms of screening algorithms that utilize tNGS are based on perceived complexities, including variant detection and interpretation, we clarify the actual limitations and present the rationale that supports optimizing a molecular genetic testing approach with tNGS. Second tier tNGS can benefit clinical decision-making through the use of the initial NBS DBS punch and rapid turn-around time methodology for tNGS, that includes copy number variant analysis, variant effect prediction, and variant 'cut-off' tools for the reduction of false positive results. The availability of DNA sequence data will contribute to the improved understanding of genotype-phenotype associations and application of treatment. The ultimate goal of second tier testing should enable the earliest possible diagnosis for the earliest initiation of the most effective clinical interventions in infants with PD.
ABSTRACT
Natural exploration of textures involves active sensing, i.e., voluntary movements of tactile sensors (e.g., human fingertips or rodent whiskers) across a target surface. Somatosensory input during moving tactile sensors varies according to both the movement and the surface texture. Combining motor and sensory information, the brain is capable of extracting textural features of the explored surface. Despite the ecological relevance of active sensing, psychophysical studies on active touch are largely missing. One reason for the lack of informative studies investigating active touch is the considerable challenge of assembling an appropriate experimental setup. A possible solution might be in the realm of virtual tactile reality that provides tactile finger stimulation depending on the position of the hand and the simulated texture of a target surface. In addition to rigorous behavioral studies, the investigation of the neuronal mechanisms of active tactile sensing in humans is highly warranted, requiring neurophysiological experiments using electroencephalography (EEG), magnetoencephalography (MEG) and/or functional magnetic resonance imaging (fMRI). However, current neuroimaging techniques impose specific requirements on the tactile stimulus delivery equipment in terms of compatibility with the neurophysiological methods being used. Here, we present a user-friendly, MEG compatible, tactile virtual reality simulator. The simulator consists of a piezo-electric tactile stimulator capable of independently protruding 16 plastic pistons of 1 mm diameter arranged in a 4 × 4 matrix. The stimulator delivers a spatial pattern of tactile stimuli to the tip of a finger depending on the position of the finger moving across a 2-dimensional plane. In order to demonstrate the functionality of the tactile virtual reality, we determined participants' detection thresholds in active and passive touch conditions. Thresholds in both conditions were higher than reported in the literature. It could well be that the processing of the piston-related stimulation was masked by the sensory input generated by placing the finger on the scanning probe. More so, the thresholds for both the active and passive tasks did not differ significantly. In further studies, the noise introduced by the stimulator in neuromagnetic recordings was quantified and somatosensory evoked fields for active and passive touch were recorded. Due to the compatibility of the stimulator with neuroimaging techniques such as MEG, and based on the feasibility to record somatosensory-related neuromagnetic brain activity the apparatus has immense potential for the exploration of the neural underpinnings of active tactile perception.
ABSTRACT
S.cerevisiae is an industrially important organism known for its ability to produce ethanol as the demand for ethanol is increasing day by day all over the world, the need to find better and alternative ways to increase ethanol production is also rising. In this work we have proposed such alternative but effective method for producing ethanol by S.cerevisiae. Here, we are reporting for the first time the effect of nitrosative stress on ethanol production. Under in vivo condition, nitrosative stress is marked by the modification of macromolecules in the presence of reactive nitrogen species (RNS). Our result showed that treated cells were more capable for ethanol production compared with untreated cells. Our result also showed enhanced alcohol dehydrogenase activity under stressed condition. Further ethanol production was also optimized by using Response Surface Methodology (RSM) with stressed cells. Further, production of ethanol with immobilized beads of stress affected Saccharomyces cerevisiae was also determined. Overall, the obtained data showed that under nitrosative stress, the maximum ethanol production is 34.4 g/l after 24 h and such higher production was observed even after several cycles of fermentation. This is the first report of this kind showing the relation between nitrosative stress and ethanol production in Saccharomyces cerevisiae which may have important industrial application.
Subject(s)
Ethanol/metabolism , Nitrosative Stress , Saccharomyces cerevisiae/metabolism , Alcohol Dehydrogenase/metabolism , Nitrosative Stress/drug effects , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/growth & development , Sodium Nitrite/pharmacologyABSTRACT
Isovaleric acidemia (IVA) is an inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase. IVA clinical picture includes gastroenterological and progressive neurological symptoms which can lead to permanent disability and death. Early detection by newborn screening (NBS) and treatment promotes normal development. In this study, clinical summaries, biochemical measurements and targeted next generation sequencing (tNGS) data from the IVD gene were compared in 13 Mexican patients. The main symptoms were vomiting, feeding refusal, abdominal pain, impaired alertness, lethargy, stupor, coma; hypotonia, ataxia, hallucinations, seizures; anemia, neutropenia and pancytopenia. Mean blood concentration of isovalerylcarnintine was above the reference value (0.5⯵M) in symptomatic patients (8.78⯵M), as well as in the screen positive newborns (2.23⯵M). The molecular spectrum of this cohort was heterogeneous, with 14 different variants identified, seven were previously-described, and seven were novel. The most frequent variant was c.158Gâ¯>â¯C (p.R53P). In this study, we found a long diagnostic delay (average of 44â¯months). Thus, it is essential to increase physician awareness of this treatable condition. Biochemical IVA NBS accompanied by molecular studies (e.g. tNGS) will permit identification of potentially asymptomatic forms of the disease, and improve genotype-phenotype relationship, management decisions and follow-up.
Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , DNA/genetics , High-Throughput Nucleotide Sequencing , Isovaleryl-CoA Dehydrogenase/deficiency , Sequence Analysis, DNA , Amino Acid Metabolism, Inborn Errors/blood , Biomarkers/blood , Cohort Studies , Delayed Diagnosis , Female , Humans , Infant, Newborn , Isovaleryl-CoA Dehydrogenase/blood , Isovaleryl-CoA Dehydrogenase/genetics , Male , Mexico , Neonatal Screening , Tandem Mass SpectrometryABSTRACT
Autophagy is a lysosome-dependent intracellular degradation pathway that has been implicated in the pathogenesis of various human diseases, either positively or negatively impacting disease outcomes depending on the specific context. The majority of medical conditions including cancer, neurodegenerative diseases, infections and immune system disorders and inflammatory bowel disease could probably benefit from therapeutic modulation of the autophagy machinery. Drosophila represents an excellent model animal to study disease mechanisms thanks to its sophisticated genetic toolkit, and the conservation of human disease genes and autophagic processes. Here, we provide an overview of the various autophagy pathways observed both in flies and human cells (macroautophagy, microautophagy and chaperone-mediated autophagy), and discuss Drosophila models of the above-mentioned diseases where fly research has already helped to understand how defects in autophagy genes and pathways contribute to the relevant pathomechanisms.
Subject(s)
Autophagy , Disease Models, Animal , Disease , Drosophila melanogaster , Animals , Disease Progression , Humans , Neurons/pathologyABSTRACT
Host-guest interaction of two significant drugs, phenylephrine hydrochloride and synephrine with α and ß-cyclodextrins were studied systematically. Initially two simple but reliable physicochemical techniques namely conductance and surface tension were employed to find out saturation concentration for the inclusion and its stoichiometry. The obtained 1:1 stoichiometry was further confirmed by two spectrometric methods, UV-Vis study and spectrofluorimetry. Significant shifts in IR stretching frequency also support the inclusion process. Relative stabilities of the inclusion complexes were established by the association constants obtained from UV-Vis spectroscopic measurements, program based mathematical calculation of conductivity data. Calculations of the thermodynamic parameters dictates thermodynamic feasibility of the inclusion process. Spectrofluorometric measurement scaffolds the UV-Vis spectroscopic measurement validating stability of the ICs once again. Mass spectroscopic measurement gives the molecular ion peaks corresponding to the inclusion complex of 1:1 molar ratio of host and guest molecules. The mechanism of inclusion was drawn by 1H-NMR and 2D ROESY spectroscopic analysis. Surface texture of the inclusion complexes was studied by SEM. Finally, the cytotoxic activities of the inclusion complexes were analyzed and found, Cell viability also balances for non-toxic behavior of the ICs. Moreover, all the studies reveal the formation of inclusion complexes of two ephedra free, alternatively emerging drugs (after their banned product having ephedra) SNP, PEH with α and ß-CD which enriches the drug delivery system with their regulatory release without any chemical modification.
Subject(s)
Anti-Obesity Agents/pharmacology , Cyclodextrins/pharmacology , Phenylephrine/pharmacology , Synephrine/pharmacology , alpha-Cyclodextrins/pharmacology , beta-Cyclodextrins/pharmacology , Anti-Obesity Agents/chemical synthesis , Anti-Obesity Agents/chemistry , Anti-Obesity Agents/toxicity , Cyclodextrins/chemical synthesis , Cyclodextrins/chemistry , Cyclodextrins/toxicity , Drug Stability , Microbial Viability/drug effects , Phenylephrine/chemical synthesis , Phenylephrine/chemistry , Phenylephrine/toxicity , Spectrum Analysis , Synephrine/chemical synthesis , Synephrine/chemistry , Synephrine/toxicity , alpha-Cyclodextrins/chemical synthesis , alpha-Cyclodextrins/chemistry , alpha-Cyclodextrins/toxicity , beta-Cyclodextrins/chemical synthesis , beta-Cyclodextrins/chemistry , beta-Cyclodextrins/toxicityABSTRACT
Autophagy, a regulated nutrient recycling program can affect both cell survival and cell death. Here, we show that Ormeloxifene (ORM), a selective estrogen receptor modulator approved for oral contraceptive use induces autophagic flux in ovarian cancer cells, which is activated by an ER stress response upstream of autophagy. The ER stress response is characterized by activation of IRE1α, PERK and ATF6 and is under regulation of JNK. Pharmacological inhibition of either autophagy or ER stress increased cell survival, as did silencing of autophagy proteins LC3 and Beclin 1, implying that ORM-induced autophagy is pro-death in nature. Ultrastructural observations of treated cells confirmed stages of autophagic maturation. Caspase-dependent apoptosis succeeded these events and was characterized by generation of reactive oxygen species and disruption of mitochondrial membrane potential. A concomitant inhibition of the Akt/mTOR axis was also observed with possible regulation of Akt by ORM. ORM inhibited tumor growth in ovarian xenograft model and displayed autophagic activity. In summary, in vitro and in vivo results reveal that ORM induces autophagy-associated cell death to attenuate proliferation of ovarian cancer cells. Our results demonstrate that using ORM in combination with ER stress and autophagy modulators could offer better therapeutic outcome in ovarian cancer.
Subject(s)
Antineoplastic Agents/metabolism , Apoptosis , Autophagy , Benzopyrans/metabolism , Signal Transduction , Unfolded Protein Response , Animals , Antineoplastic Agents/administration & dosage , Benzopyrans/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , Female , Heterografts , Humans , Mice , Neoplasm Transplantation , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Treatment OutcomeABSTRACT
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.