LIS1 and NDEL1 Regulate Axonal Trafficking of Mitochondria in Mature Neurons.

Defective mitochondrial dynamics in axons have been linked to both developmental and late-onset neurological disorders. Axonal trafficking is in large part governed by the microtubule motors kinesin-1 and cytoplasmic dynein 1 (dynein). Dynein is the primary retrograde transport motor in axons, and mutations in dynein and many of its regulators also cause neurological diseases. Depletion of LIS1, famous for linking dynein deregulation to lissencephaly (smooth brain), in adult mice leads to severe neurological phenotypes, demonstrating post-developmental roles. LIS1 stimulates retrograde transport of acidic organelles in cultured adult rat dorsal root ganglion (DRG) axons but findings on its role in mitochondrial trafficking have been inconsistent and have not been reported for adult axons. Here we report that there is an increased number of mitochondria in cross-sections of sciatic nerve axons from adult LIS1+/- mice. This is probably related to reduced dynein activity as axons from adult rat nerves exposed to the dynein inhibitor, ciliobrevin D also had increased numbers of mitochondria. Moreover, LIS1 overexpression (OE) in cultured adult rat DRG axons stimulated retrograde mitochondrial transport while LIS1 knockdown (KD) or expression of a LIS1 dynein-binding mutant (LIS1-K147A) inhibited retrograde transport, as did KD of dynein heavy chain (DHC). These findings are consistent with our report on acidic organelles. However, KD of NDEL1, a LIS1 and dynein binding protein, or expression of a LIS1 NDEL1-binding mutant (LIS1-R212A) also dramatically impacted retrograde mitochondrial transport, which was not the case for acidic organelles. Manipulations that disrupted retrograde mitochondrial transport also increased the average length of axonal mitochondria, suggesting a role for dynein in fusion or fission events. Our data point to cargo specificity in NDEL1 function and raise the possibility that defects in the LIS1/NDEL1 dynein regulatory pathway could contribute to mitochondrial diseases with axonal pathologies.

Modeling and Optimization of Process Parameters for Nutritional Enhancement in Enzymatic Milled Rice by Multiple Linear Regression (MLR) and Artificial Neural Network (ANN).

This study involves information about the concentrations of nutrients (proteins, phenolic compounds, free amino acids, minerals (Ca, P, and Iron), hardness) in milled rice processed with enzymes; xylanase and cellulase produced by Aspergillus awamori, MTCC 9166 and Trichoderma reese, MTCC164. Brown rice was processed with 60-100% enzyme (40 mL buffer -undiluted) for 30 to 150 min at 30 °C to 50 °C followed by polishing for 20-100 s at a safe moisture level. Multiple linear regression (MLR) and artificial neural network (ANN) models were used for process optimization of enzymes. The MLR correlation coefficient (R2) varied between 0.87-0.90, and the sum of square (SSE) was placed within 0.008-8.25. While the ANN R2 (correlation coefficient) varied between 0.97 and 0.9999(1), MSE changed from 0.005 to 6.13 representing that the ANN method has better execution across MLR. The optimized cellulase process parameters (87.2% concentration, 80.1 min process time, 33.95 °C temperature and 21.8 s milling time) and xylanase process parameters (85.7% enzyme crude, 77.1 min process time, 35 °C temperature and 20 s) facilitated the increase of Ca (70%), P (64%), Iron (17%), free amino acids (34%), phenolic compounds (78%) and protein (84%) and decreased hardness (20%) in milled rice. Scanning electron micrographs showed an increased rupture attributing to enzymes action on milled rice.

Quantitative Neurotoxicology: An Assessment of the Neurotoxic Profile of Kainic Acid in Sprague Dawley Rats.

This study consisted of a qualitative and quantitative assessment of neuropathological changes in kainic acid (KA)-treated adult male rats. Rats were administered a single 10 mg/kg intraperitoneal injection of KA or the same volume of saline and sacrificed 24 or 48 hours posttreatment. Brains were collected, sectioned coronally (∼ 81 slices), and stained with amino cupric silver to reveal degenerative changes. For qualitative assessment of neural degeneration, sectioned material was evaluated by a board-certified pathologist, and the level of degeneration was graded based upon a 4-point scale. For measurement of quantitative neural degeneration in response to KA treatment, the HALO digital image analysis software tool was used. Quantitative measurements of specific regions within the brain were obtained from silver-stained tissue sections with quantitation based on stain color and optical density. This quantitative evaluation method identified degeneration primarily in the cerebral cortex, septal nuclei, amygdala, olfactory bulb, hippocampus, thalamus, and hypothalamus. The KA-produced neuronal degeneration in the cortex was primarily in the piriform, insular, rhinal, and cingulate areas. In the hippocampus, the dentate gyrus was found to be the most affected area. Our findings indicate global neurotoxicity due to KA treatment. Certain brain structures exhibited more degeneration than others, reflecting differential sensitivity or vulnerability of neurons to KA.

Critical overview of biomass feedstocks as sustainable substrates for the production of polyhydroxybutyrate (PHB).

Polyhydroxybutyrates (PHBs) are a class of biopolymers produced by different microbial species and are biodegradable and biocompatible in nature as opposed to petrochemically derived plastics. PHBs have advanced applications in medical sector, packaging industries, nanotechnology and agriculture, among others. PHB is produced using various feedstocks such as glycerol, dairy wastes, agro-industrial wastes, food industry waste and sugars. Current focus on PHB research has been primarily on reducing the cost of production and, on downstream processing to isolate PHB from cells. Recent advancements to improve the productivity and quality of PHB include genetic modification of producer strain and modification of PHB by blending to develop desirable properties suited to diversified applications. Selection of feedstock plays a critical role in determining the economic feasibility and sustainability of the process. This review provides a bird's eye view of the suitability of different waste resources for producing polyhydroxybutyrate; providing state-of the art information and analysis.

Dilute acid hydrolysis of spoiled wheat grains: Analysis of chemical, rheological and spectral characteristics.

In this work, hydrolysis of spoiled wheat grains using dilute acid (5, 10%; 1 N HCl) was investigated and the effect of hydrolysis conditions on reducing sugars, soluble proteins, rheology and infrared spectra of the hydrolysates was determined. Hydrolysis with 10% acid concentration released more quantities of reducing sugar (16.47 mg/mL) at shorter hydrolysis times whereas 5% acid concentration produced higher protein content (28.74 mg/mL) for similar durations. Flow characteristics demonstrated an increased apparent viscosity of the hydrolysates retrieved after 4.5 h of hydrolysis possibly due to breakdown of hemicelluloses and lignin into sugars. Infrared spectroscopy showed release of carbonates after 1.5 h and 5.5 h of hydrolysis perhaps due to oxidation of lignin or a reaction between acid and sugars. The study highlights that acid hydrolysis would be a rapid and cost effective approach to produce fermentable hydrolysates for bio-processing industry applications while generating an avenue for waste grain utilization.

Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology, US Food and Drug Administration Public Workshop-Workshop Proceedings.

PURPOSE: Next-generation sequencing (NGS) oncology panels are becoming integral in hospital and academic settings to guide patient treatment and enrollment in clinical trials. Although NGS technologies have revolutionized decision-making for cancer therapeutics, physicians may face many challenges in parsing and prioritizing NGS-based test results to determine the best course of treatment for individual patients. On January 29, 2018, the US Food and Drug Administration held a public workshop entitled, "Weighing the Evidence: Variant Classification and Interpretation in Precision Oncology." Here, we discuss the presentations and discussion highlights across the four sessions of the workshop. METHODS: The goal of the public workshop was to engage stakeholders and solicit input from experts in precision oncology to discuss the integration of complex NGS data into patient management and regulatory innovation within the precision oncology community. The US Food and Drug Administration gathered representatives from academia, industry, patient advocacy, government, and professional organizations for a series of presentations followed by panel discussions. After the workshop, the transcript and speaker presentation slides were reviewed and summarized for manuscript preparation. RESULTS: Speakers and panelists provided diverse perspectives on the integration of NGS technology into patient care for oncology and on the complexities that surround data interpretation and sharing. Discussions highlighted the challenges with standardization for variant classification while expressing the utility of consensus recommendations among stakeholders in oncology for driving innovation in the era of precision medicine. CONCLUSION: As precision medicine advances, clear communication within the field of precision oncology will be key to creating an environment that facilitates the generation and sharing of data that have value to patients.

Engineering allosteric regulation in protein kinases.

Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed "hotspots" that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.

Hierarchical Organization Endows the Kinase Domain with Regulatory Plasticity.

The functional diversity of kinases enables specificity in cellular signal transduction. Yet how more than 500 members of the human kinome specifically receive regulatory inputs and convey information to appropriate substrates-all while using the common signaling output of phosphorylation-remains enigmatic. Here, we perform statistical co-evolution analysis, mutational scanning, and quantitative live-cell assays to reveal a hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. We find that three quasi-independent "sectors"-groups of evolutionarily coupled residues-represent functional units in the kinase domain that encode for catalytic activity, substrate specificity, and regulation. Sector positions impact both disease and pharmacology: the catalytic sector is significantly enriched for somatic cancer mutations, and residues in the regulatory sector interact with allosteric kinase inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity.

SMARCE1 is required for the invasive progression of in situ cancers.

Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis.

Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

Biomarkers: A potential prognostic tool for silicosis.

Long-term exposure to respirable dust containing silica leads to pneumoconiosis/silicosis. The disease is irreversible and incurable, and only preventive steps such as job rotation, use of personal protective equipment, etc., remain solutions to the problem. Under such a situation, early diagnosis or prediction may become very useful to control the disease. Biomarkers are biological parameters in blood serum that change their values with deposition of dust in the lung and onset of lung fibrosis. Since these biomarkers can help us to diagnose and in the prognosis of the disease before it is actually diagnosed by the conventional X-ray technique and lung function test used for diagnosis of silicosis. The present paper describes the various types of available biomarkers, their application and usefulness. The paper also suggests that further study of the behavior/level of these biomarkers on a specific subject with time may provide more useful information of a confirmatory nature for prevention of dust-linked diseases.

A Cdk5-dependent switch regulates Lis1/Ndel1/dynein-driven organelle transport in adult axons.

Lissencephaly is a human developmental brain abnormality caused by LIS1 haploinsufficiency. This disorder is in large part attributed to altered mitosis and migration in the developing brain. LIS1 and an interacting protein, NDEL1, bind to cytoplasmic dynein, a microtubule motor protein. While the tripartite complex is clearly important for developmental events, we are intrigued by the fact that Lis1 and Ndel1 expression remain high in the adult mouse nervous system. Dynein plays a crucial role in retrograde axonal transport, a process that is used by mature neurons. Here, we monitored acidic organelles moving in axons of adult rat sensory neurons to determine whether Lis1 and Ndel1 contribute to axonal transport. Lis1 RNAi significantly reduced axon transport of these organelles. Ndel1 RNAi had little impact, but combined Lis1 and Ndel1 RNAi caused a more severe phenotype than Lis1 RNAi alone, essentially shutting down transport. Lis1 overexpression stimulated retrograde transport, while a Lis1 dynein-binding mutant severely disrupted transport. Overexpression of Ndel1 or a Lis1 Ndel1-binding mutant only mildly perturbed transport. However, expressing a mutant Ndel1 lacking key phosphorylation sites shut down transport completely, as did a dominant-negative Cdk5 construct. We propose that, in axons, unphosphorylated Ndel1 inhibits the capacity of dynein to transport acidic organelles. Phosphorylation of Ndel1 by Cdk5 not only reduces this inhibition but also allows Lis1 to further stimulate the cargo transport capacity of dynein. Our data raise the possibility that defects in a Lis1/Ndel1 regulatory switch could contribute to neurodegenerative diseases linked to axonal pathology in adults.

Carbon footprint: current methods of estimation.

Increasing greenhouse gaseous concentration in the atmosphere is perturbing the environment to cause grievous global warming and associated consequences. Following the rule that only measurable is manageable, mensuration of greenhouse gas intensiveness of different products, bodies, and processes is going on worldwide, expressed as their carbon footprints. The methodologies for carbon footprint calculations are still evolving and it is emerging as an important tool for greenhouse gas management. The concept of carbon footprinting has permeated and is being commercialized in all the areas of life and economy, but there is little coherence in definitions and calculations of carbon footprints among the studies. There are disagreements in the selection of gases, and the order of emissions to be covered in footprint calculations. Standards of greenhouse gas accounting are the common resources used in footprint calculations, although there is no mandatory provision of footprint verification. Carbon footprinting is intended to be a tool to guide the relevant emission cuts and verifications, its standardization at international level are therefore necessary. Present review describes the prevailing carbon footprinting methods and raises the related issues.

Assessment of environmental water demands (EWD) of forests for two distinct Indian ecosystems.

Sustainable use of water and land resources requires that these scarce resources be appropriately allocated among various competing human activities. Worldwide, there is a realization now that sustainable river basin management should be accorded the highest priority, because it deals not only with technical, but also with ecological and socioeconomic aspects, and thus calls for a multidisciplinary and integrated approach. However, most of the policy and planning documents have either remained silent, or have made only implicit reference to the importance of environmental water demand (EWD) and its quantification. Therefore, in the light of its importance, a methodology has been evolved in this article for quantifying EWD for various forested areas in two distinctly different Indian river basins: Brahmani (humid zone) and Sabarmati (dry zone). The article analyzes and discusses EWD estimates at three different spatial levels: river basins, states, and districts within them, and finally presents a comparative analysis of all these results. Findings of the present study will be immensely useful in understanding various ecological issues connected with water resource projects and proposals in these river basins.