ResearchPad - resources https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast]]> https://www.researchpad.co/article/elastic_article_16344 Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics’ droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.

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<![CDATA[Online Bayesian Phylodynamic Inference in BEAST with Application to Epidemic Reconstruction]]> https://www.researchpad.co/article/elastic_article_16302 Reconstructing pathogen dynamics from genetic data as they become available during an outbreak or epidemic represents an important statistical scenario in which observations arrive sequentially in time and one is interested in performing inference in an “online” fashion. Widely used Bayesian phylogenetic inference packages are not set up for this purpose, generally requiring one to recompute trees and evolutionary model parameters de novo when new data arrive. To accommodate increasing data flow in a Bayesian phylogenetic framework, we introduce a methodology to efficiently update the posterior distribution with newly available genetic data. Our procedure is implemented in the BEAST 1.10 software package, and relies on a distance-based measure to insert new taxa into the current estimate of the phylogeny and imputes plausible values for new model parameters to accommodate growing dimensionality. This augmentation creates informed starting values and re-uses optimally tuned transition kernels for posterior exploration of growing data sets, reducing the time necessary to converge to target posterior distributions. We apply our framework to data from the recent West African Ebola virus epidemic and demonstrate a considerable reduction in time required to obtain posterior estimates at different time points of the outbreak. Beyond epidemic monitoring, this framework easily finds other applications within the phylogenetics community, where changes in the data—in terms of alignment changes, sequence addition or removal—present common scenarios that can benefit from online inference.

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<![CDATA[Single-cell transcription analysis of <i>Plasmodium vivax</i> blood-stage parasites identifies stage- and species-specific profiles of expression]]> https://www.researchpad.co/article/elastic_article_14651 Analysis of individual Plasmodium vivax parasites reveals the tight control of the expression of most genes during the intra-erythrocytic cycle and the differentiation of male and female gametocytes, and highlights differences between the development of P. vivax and P. falciparum.

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<![CDATA[The improved and the unimproved: Factors influencing sanitation and diarrhoea in a peri-urban settlement of Lusaka, Zambia]]> https://www.researchpad.co/article/elastic_article_14479 Accounting for peri-urban sanitation poses a unique challenge due to its high density, unplanned stature, with limited space and funding for conventional sanitation instalment. To better understand users, needs and inform peri-urban sanitation policy, our study used multivariate stepwise logistic regression to assess the factors associated with use of improved (toilet) and unimproved (chamber) sanitation facilities among peri-urban residents. We analysed data from 205 household heads in 1 peri-urban settlement of Lusaka, Zambia on socio-demographics (economic status, education level, marital status, etc.), household sanitation characteristics (toilet facility, ownership and management) and household diarrhoea prevalence. Household water, sanitation and hygiene (WASH) facilities were assessed based on WHO-UNICEF criteria. Of particular interest was the simultaneous use of toilet facilities and chambers, an alternative form of unimproved sanitation with focus towards all-in-one suitable alternatives. Findings revealed that having a regular income, private toilet facility, improved drinking water and handwashing facility were all positively correlated to having an improved toilet facility. Interestingly, both improved toilets and chambers indicated increased odds for diarrhoea prevalence. Odds of chamber usage were also higher for females and users of unimproved toilet facilities. Moreover, when toilets were owned by residents, and hygiene was managed externally, use of chambers was more likely. Findings finally revealed higher diarrhoea prevalence for toilets with more users. Results highlight the need for a holistic, simultaneous approach to WASH for overall success in sanitation. To better access and increase peri-urban sanitation, this study recommends a separate sanitation ladder for high density areas which considers improved private and shared facilities, toilet management and all-inclusive usage (cancelling unimproved alternatives). It further calls for financial plans supporting urban poor access to basic sanitation and increased education on toilet facility models, hygiene, management and risk to help with choice and proper facility use to maximize toilet use benefit.

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<![CDATA[Terminal drought and heat stress alter physiological and biochemical attributes in flag leaf of bread wheat]]> https://www.researchpad.co/article/elastic_article_14475 Heat stress along with low water availability at reproductive stage (terminal growth phase of wheat crop) is major contributing factor towards less wheat production in tropics and sub-tropics. Flag leaf plays a pivotal role in assimilate partitioning and stress tolerance of wheat during terminal growth phase. However, limited is known about biochemical response of flag leaf to combined and individual heat and drought stress during terminal growth phase. Therefore, current study investigated combined and individual effect of terminal drought and heat stress on water relations, photosynthetic pigments, osmolytes accumulation and antioxidants defense mechanism in flag leaf of bread wheat. Experimental treatments comprised of control, terminal drought stress alone (50% field capacity during reproductive phase), terminal heat stress alone (wheat grown inside plastic tunnel during reproductive phase) and terminal drought stress + terminal heat stress. Individual and combined imposition of drought and heat stresses significantly (p≤0.05) altered water relations, osmolyte contents, soluble proteins and sugars along with activated antioxidant defensive system in terms of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX). Turgor potential, POD and APX activities were lowest under individual heat stress; however, these were improved when drought stress was combined with heat stress. It is concluded that combined effect of drought and heat stress was more detrimental than individual stresses. The interactive effect of both stresses was hypo-additive in nature, but for some traits (like turgor potential and APX) effect of one stress neutralized the other. To best of our knowledge, this is the first report on physiological and biochemical response of flag leaf of wheat to combine heat and drought stress. These results will help future studies dealing with improved stress tolerance in wheat. However, detailed studies are needed to fully understand the genetic mechanisms behind these physiological and biochemical changes in flag leaf in response to combined heat and drought stress.

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<![CDATA[NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in <i>Caenorhabditis elegans</i>]]> https://www.researchpad.co/article/elastic_article_14390 GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.

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<![CDATA[Investigating barriers and challenges to the integrated management of neglected tropical skin diseases in an endemic setting in Nigeria]]> https://www.researchpad.co/article/elastic_article_13828 Community perceptions of causation of neglected tropical diseases (NTDs) of the skin may play an important role in access to or utilization of health services. The World Health Organization (WHO) has recommended empowerment of populations affected by or at risk of NTDs in control interventions. Furthermore, the WHO recommends that social mobilisation needs to be maintained in order to create demand for integrated management of skin NTDs and to address specific community aspects and concerns related to the diseases. There are no studies on community knowledge, attitudes and practices (KAP) on skin NTDs co-occurring in the same community in Nigeria. We surveyed community members and health workers and also held group discussions with community members, health workers and individuals with lymphatic filariasis and Buruli ulcer in order to assess their understanding of the causes, treatment and effects of the skin NTDs (leprosy, Buruli ulcer and lymphatic filariasis) which were all occurring in the study communities. There was a shared understanding that these NTDs were caused by germ/infection or through witchcraft/curse/poison. Also, a substantial proportion of the community believed that these conditions are not amenable to treatment. The focus group discussions reinforced these findings.

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<![CDATA[Fine-scale visualizing the hierarchical structure of mouse biliary tree with fluorescence microscopy method]]> https://www.researchpad.co/article/elastic_article_13843 The liver is a vital organ and the hepatic lobule serves as the most basic structural and functional unit which is mainly assembled with parenchymal cells including hepatocytes and biliary epithelial cells. The continuous tubular arrangement of biliary cells which constitutes the biliary tracts is critical for liver function, however, the biliary tracts are often disrupted in many liver diseases such as cirrhosis and some congenital disorders. Visualization of the biliary tracts in fine-scale and three-dimension will help to understanding the structure basis of these liver diseases. In the present study, we established several biliary tract injury mouse models by diet feeding, surgery or genetic modification. The cytoplasm and nuclei of the parenchymal cells were marked by active uptake of fluorescent dyes Rhodamine B (red) and Hoechst (blue), respectively. After the removal of liver en bloc, the biliary tracts were retrogradely perfused with green fluorescent dye, fluorescein isothiocyanate (FITC). The liver was then observed under confocal microscopy. The fine-scale and three-dimensional (3D) structure of the whole biliary tree, particularly the network of the end-terminal bile canaliculi and neighboring hepatocytes were clearly visualized. The biliary tracts displayed clear distinct characteristics in normal liver and diseased liver models. Taken together, we have developed a simple and repeatable imaging method to visualize the fine-scale and hierarchical architecture of the biliary tracts spreading in the mouse liver.

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<![CDATA[Machine Learning Techniques for Classifying the Mutagenic Origins of Point Mutations]]> https://www.researchpad.co/article/elastic_article_12395 There is increasing interest in developing diagnostics that discriminate individual mutagenic mechanisms in a range of applications that include identifying population-specific mutagenesis and resolving distinct mutation signatures in cancer samples. Analyses for these applications assume that mutagenic mechanisms have a distinct relationship with neighboring bases that allows them to be distinguished. Direct support for this assumption is limited to a small number of simple cases, e.g., CpG hypermutability. We have evaluated whether the mechanistic origin of a point mutation can be resolved using only sequence context for a more complicated case. We contrasted single nucleotide variants originating from the multitude of mutagenic processes that normally operate in the mouse germline with those induced by the potent mutagen N-ethyl-N-nitrosourea (ENU). The considerable overlap in the mutation spectra of these two samples make this a challenging problem. Employing a new, robust log-linear modeling method, we demonstrate that neighboring bases contain information regarding point mutation direction that differs between the ENU-induced and spontaneous mutation variant classes. A logistic regression classifier exhibited strong performance at discriminating between the different mutation classes. Concordance between the feature set of the best classifier and information content analyses suggest our results can be generalized to other mutation classification problems. We conclude that machine learning can be used to build a practical classification tool to identify the mutation mechanism for individual genetic variants. Software implementing our approach is freely available under an open-source license.

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<![CDATA[Cell Atlas technologies and insights into tissue architecture]]> https://www.researchpad.co/article/elastic_article_9194 Since Robert Hooke first described the existence of ‘cells’ in 1665, scientists have sought to identify and further characterise these fundamental units of life. While our understanding of cell location, morphology and function has expanded greatly; our understanding of cell types and states at the molecular level, and how these function within tissue architecture, is still limited. A greater understanding of our cells could revolutionise basic biology and medicine. Atlasing initiatives like the Human Cell Atlas aim to identify all cell types at the molecular level, including their physical locations, and to make this reference data openly available to the scientific community. This is made possible by a recent technology revolution: both in single-cell molecular profiling, particularly single-cell RNA sequencing, and in spatially resolved methods for assessing gene and protein expression. Here, we review available and upcoming atlasing technologies, the biological insights gained to date and the promise of this field for the future.

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<![CDATA[5-Ethynyl-2′-deoxycytidine and 5-ethynyl-2′-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses]]> https://www.researchpad.co/article/elastic_article_7282 Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

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<![CDATA[Cre-assisted fine-mapping of neural circuits using orthogonal split inteins]]> https://www.researchpad.co/article/elastic_article_7268 In humans – as well as flies and most other animals – the brain controls how we move and behave, and regulates heartbeat, breathing and other core processes. To perform these different roles, cells known as neurons form large networks that quickly carry messages around the brain and to other parts of the body. In order to fully understand how the brain works, it is important to first understand how individual neurons connect to each other and operate within these networks.

Fruit flies and other animals with small brains are often used as models to study how the brain works. There are several methods currently available that allow researchers to manipulate small groups of fruit fly neurons for study, and in some cases it is even possible to target individual neurons. However, it remains an aspirational goal to be able to target every neuron in the fly brain individually.

The Gal4-UAS system is a way of manipulating gene activity widely used to study neurons in fruit flies. The system consists of two parts: a protein that can bind DNA and control the activity of genes (Gal4); and a genetic sequence (the UAS) that tells Gal4 where to bind and therefore which genes to activate. Fruit flies can be genetically engineered so that only specific cells make Gal4. This makes it possible, for example, to limit the activity of a gene under the control of the UAS to a specific set of neurons and therefore to identify or target these neurons. Luan et al. developed a new technique named SpaRCLIn that allows the targeting of a subset of neurons within a group already identified with the Gal4-UAS system.

During embryonic development, all neurons originate from a small pool of cells called neuroblasts, and it is possible to target the descendants of particular neuroblasts. SpaRCLIn exploits this strategy to limit the activity of Gal4 to smaller and smaller numbers of neuroblast descendants. In this way, Luan et al. found that SpaRCLIn was routinely capable of limiting patterns of Gal4 activity to one, or a few, neurons at a time. Further experiments used SpaRCLIn to identify two pairs of neurons that trigger a well-known feeding behavior in fruit flies. Luan et al. also developed a SpaRCLIn toolkit that will form the basis of a community resource other researchers can use to study neurons in fruit flies. These findings could also benefit researchers developing similar tools in mice and other animals.

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<![CDATA[Top-down machine learning approach for high-throughput single-molecule analysis]]> https://www.researchpad.co/article/N957aad02-2c00-4587-a7f5-2b73aea07b8d During a chemical or biological process, a molecule may transition through a series of states, many of which are rare or short-lived. Advances in technology have made it easier to detect these states by gathering large amounts of data on individual molecules. However, the increasing size of these datasets has put a strain on the algorithms and software used to identify different molecular states.

Now, White et al. have developed a new algorithm called DISC which overcomes this technical limitation. Unlike most other algorithms, DISC requires minimal input from the user and uses a new method to group the data into categories that represent distinct molecular states. Although this new approach produces a similar end-result, it reaches this conclusion much faster than more commonly used algorithms.

To test the effectiveness of the algorithm, White et al. studied how individual molecules of a chemical known as cAMP bind to parts of proteins called cyclic nucleotide binding domains (or CNDBs for short). A fluorescent tag was attached to single molecules of cAMP and data were collected on the behavior of each molecule. Previous evidence suggested that when four CNDBs join together to form a so-called tetramer complex, this affects the binding of cAMP. Using the DISC system, White et al. showed that individual cAMP molecules interact with all four domains in a similar way, suggesting that the binding of cAMP is not impacted by the formation of a tetramer complex.

Analyzing this data took DISC less than 20 minutes compared to existing algorithms which took anywhere between four hours and two weeks to complete. The enhanced speed of the DISC algorithm could make it easier to analyze much larger datasets from other techniques in addition to fluorescence. This means that a greater number of states can be sampled, providing a deeper insight into the inner workings of biological and chemical processes.

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<![CDATA[The UCSF Mouse Inventory Database Application, an Open Source Web App for Sharing Mutant Mice Within a Research Community]]> https://www.researchpad.co/article/Nbb3b2ed7-43fd-4a80-9469-797d6b2ba821 The UCSF Mouse Inventory Database Application is an open-source Web App that provides information about the mutant alleles, transgenes, and inbred strains maintained by investigators at the university and facilitates sharing of these resources within the university community. The Application is designed to promote collaboration, decrease the costs associated with obtaining genetically-modified mice, and increase access to mouse lines that are difficult to obtain. An inventory of the genetically-modified mice on campus and the investigators who maintain them is compiled from records of purchases from external sources, transfers from researchers within and outside the university, and from data provided by users. These data are verified and augmented with relevant information harvested from public databases, and stored in a succinct, searchable database secured on the university network. Here we describe this resource and provide information about how to implement and maintain such a mouse inventory database application at other institutions.

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<![CDATA[A GoldenBraid cloning system for synthetic biology in social amoebae]]> https://www.researchpad.co/article/Ncce59dea-a6d8-4706-b66d-8586f5d28a63 GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria–host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.

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<![CDATA[Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes]]> https://www.researchpad.co/article/N75efa8e7-95dd-4c39-b2cd-36b25226ef1c Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

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<![CDATA[A biochemical comparison of the lung, colonic, brain, renal, and ovarian cancer cell lines using <sup>1</sup>H-NMR spectroscopy]]> https://www.researchpad.co/article/Nf9e958e6-47b5-498d-afb8-80cfab8461fe Cancer cell lines are often used for cancer research. However, continuous genetic instability-induced heterogeneity of cell lines can hinder the reproducibility of cancer research. Molecular profiling approaches including transcriptomics, chromatin modification profiling, and proteomics are used to evaluate the phenotypic characteristics of cell lines. However, these do not reflect the metabolic function at the molecular level. Metabolic phenotyping is a powerful tool to profile the biochemical composition of cell lines. In the present study, 1H-NMR spectroscopy-based metabolic phenotyping was used to detect metabolic differences among five cancer cell lines, namely, lung (A549), colonic (Caco2), brain (H4), renal (RCC), and ovarian (SKOV3) cancer cells. The concentrations of choline, creatine, lactate, alanine, fumarate and succinate varied remarkably among different cell types. The significantly higher intracellular concentrations of glutathione, myo-inositol, and phosphocholine were found in the SKOV3 cell line relative to other cell lines. The concentration of glutamate was higher in both SKOV3 and RCC cells compared with other cell lines. For cell culture media analysis, isopropanol was found to be the highest in RCC media, followed by A549 and SKOV3 media, while acetone was the highest in A549, followed by RCC and SKOV3. These results demonstrated that 1H-NMR-based metabolic phenotyping approach allows us to characterize specific metabolic signatures of cancer cell lines and provides phenotypical information of cellular metabolism.

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<![CDATA[Future trends of water resources and influences on agriculture in China]]> https://www.researchpad.co/article/N87c2566a-6970-4c5f-9ecf-ecac024e386a

Water resources are indispensable for all social-economic activities and ecosystem functions. In addition, changes in water resources have great significance for agricultural production. This paper uses five global climate models from CMIP5 to evaluate the future spatiotemporal variation in water resources in China under four RCP scenarios. The results show that the available precipitation significantly decreases due to evapotranspiration. Comparing the four RCP scenarios, the national average of the available precipitation is the highest under the RCP 2.6 and 4.5 scenarios, followed by that under the RCP 8.5 scenario. In terms of spatial distribution, the amount of available precipitation shows a decreasing trend from southeast to northwest. Regarding temporal changes, the available precipitation under RCP 8.5 exhibits a trend of first increasing and then decreasing, while the available precipitation under the RCP 6.0 scenario exhibits a trend of first decreasing and then increasing. Under the RCP 2.6 and 4.5 scenarios, the available precipitation increases, and the RCP 4.5 scenario has a higher rate of increase than that of RCP 2.6. In the context of climate change, changes in water resources and temperature cause widespread increases in potential agricultural productivity around Hu’s line, especially in southwestern China. However, the potential agricultural productivity decreases in a large area of southeastern China. Hu’s line has a partial breakthrough in the locking of agriculture, mainly in eastern Tibet, western Sichuan, northern Yunnan and northwestern Inner Mongolia. The results provide a reference for the management and deployment of future water resources and can aid in agricultural production in China.

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<![CDATA[MEHP interferes with mitochondrial functions and homeostasis in skeletal muscle cells]]> https://www.researchpad.co/article/Na411c472-0723-403f-89af-3381276a1e18

Abstract

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer frequently leached out from polyvinyl chloride (PVC) products and is quickly metabolized to its monoester equivalent mono(2-ethylhexyl) phthalate (MEHP) once enters organisms. Exposure to DEHP/MEHP through food chain intake has been shown to modified metabolism but its effect on the development of metabolic myopathy of skeletal muscle (SKM) has not been revealed so far. Here, we found that MEHP repressed myogenic terminal differentiation of proliferating myoblasts (PMB) and confluent myoblasts (CMB) but had weak effect on this process once it had been initiated. The transition of mitochondria (MITO) morphology from high efficient filamentary network to low efficient vesicles was triggered by MEHP, implying its negative effects on MITO functions. The impaired MITO functions was further demonstrated by reduced MITO DNA (mtDNA) level and SDH enzyme activity as well as highly increased reactive oxygen species (ROS) in cells after MEHP treatment. The expression of metabolic genes, including PDK4, CPT1b, UCP2, and HO1, was highly increased by MEHP and the promoters of PDK4 and CPT1b were also activated by MEHP. Additionally, the stability of some subunits in the oxidative phosphorylation system (OXPHOS) complexes was found to be reduced by MEHP, implying defective oxidative metabolism in MITO and which was confirmed by repressed palmitic acid oxidation in MEHP-treated cells. Besides, MEHP also blocked insulin-induced glucose uptake. Taken together, our results suggest that MEHP is inhibitory to myogenesis and is harmful to MITO functions in SKM, so its exposure should be avoided or limited.

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<![CDATA[A calibrated optogenetic toolbox of stable zebrafish opsin lines]]> https://www.researchpad.co/article/N1ccb8a53-68b9-48c2-94c3-754ddf9cf911

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

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