Biology subjects

Long, M.

Publications and source records attributed to Long, M..

51 records · Page 3Linked to original sources

bioRxiv2022

Analysis of D- and L- Isomers of (Meth)amphetamine in Human K2EDTA Plasma

Methamphetamine and its metabolite amphetamine are frequently abused drugs. Whether obtained legally or from clandestine laboratories it is of relevance to determine the chiral makeup of these drugs for investigative purpose. Although urine and oral fluid matrices are commonly offered, less available to independent laboratories are techniques to verify dextro (D-) or levo (L-) (meth)amphetamine from human K2EDTA plasma. This paper outlines the development and validation of a method that includes the addition of internal standard and a two-step liquid-liquid extraction to remove the analytes from human K2EDTA plasma by triple quadrupole mass spectrometry (LC-MS/MS). The assay was validated according to the United States Food and Drug Administration and College of American Pathologists guidelines, including assessment of the following parameters in plasma validation samples: linear range, limit of detection, lower limit of quantitation, matrix effects, inter- and intra-day assay precision and accuracy, carry over, linearity of dilution, matrix effects and stability. The outcome is a validated and reliable method for the determination of D- and L-isomer concentration of meth(amphetamine) human plasma samples that can be easily adopted by independent clinical laboratories.

biochemistry↗

bioRxiv2022

Quantifying 64 drugs, illicit substances, and D- and L- isomers in human oral fluid with liquid-liquid extraction

Although human oral fluid has become more routine for quantitative drug detection in pain management, detecting a large scope of medications and substances is costly and technically challenging for laboratories. This paper presents a quantitative assay for 64 pain medications, illicit substances, and drug metabolites in human oral fluid. The novelty of this assay is that it was developed on an older model AB SCIEX 4000 instrument and renders obscure the need for more technical and expensive laboratory equipment. This method includes addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water and separation and detection was accomplished using triple quadrupole mass spectrometry (LC-MS/MS). Separation was achieved using reverse-phase liquid chromatography with detection by LC-MS/MS. A second injection was done in negative mode to determine THC-COOH concentration as an indicator of THC. An aliquot of the (already) extracted samples was analyzed for D- and L- isomers of amphetamine and methamphetamine using a chiral column. The standard curve spanned from 5 to 2000 ng/mL for most of the analytes (1 to 2000 ng/mL for fentanyl and THC-COOH) and up to 1000 ng/mL for 13 analytes. Pregabalin and gabapentin ranged from 25 to 2000 ng/mL. The result is a low-cost method for the sensitive detection of a wide-ranging oral fluid menu for pain management. This assay has a high sensitivity, and good precision and accuracy for all analytes with an older model mass spectrometer.

biochemistry↗

bioRxiv2021

Immunoglobulin-producing AT2-like cells secrete IgA into the extracellular matrix in pulmonary fibrosis

Pulmonary fibrosis is an interstitial lung disease that can be caused by various factors. Here, we first observed extensive IgA deposition in the extracellular matrix (ECM) of the lungs of mice with pulmonary fibrosis induced by silica inhalation. Consistent with this phenomenon, spatial transcriptomic sequencing of fresh mouse lung tissues from control mice and model mice showed that Igha transcripts were highly expressed in the lesion area. Single-cell RNA sequencing (scRNA-seq) and reconstruction of B cell receptor (BCR) sequences revealed a new cluster of cells with a shared BCR and high expression of genes related to immunoglobulin IgA production. Surprisingly, these clonal cells had more characteristics of AT2 (alveolar epithelial cell type 2) cells than B cells; thus, these cells were named AT2-like cells. Therefore, we propose that secretion of IgA into the ECM by AT2-like cells is an important process that occurs during lung fibrosis.

cell biology↗

bioRxiv2021

Rapid gene evolution in an ancient post-transcriptional and translationalregulatory system compensates for meiotic X chromosomal inactivation

It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4-NOT deadenylase complex involved in post-transcriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4-NOT deadenylase complex and might act in post-transcriptional mRNA regulation. The rapid gene evolution in the ancient post-transcriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.

evolutionary biology↗

bioRxiv2021

Species-specific gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new gene Exov, derived from a partial gene region duplication of its parental gene Exov-L, which is a member of an exonuclease family, into a different chromosome in Arabidopsis thaliana. We experimentally investigated the phenotypic effects of Exov and Exov-L in an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated that Exov is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded by Exov in contrast to the slowly evolving Exov-L, suggesting an important role for Exov in phenotypic evolution. We analyzed phenotypic effects of exov and exov-l single T-DNA-insertion mutants;double exov, exov-l T-DNA insertion mutants; and CRISPR/Cas9-mediated exovcrp and exov-lcrp knockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of double exov, exov-l mutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution in Arabidopsis thaliana.

evolutionary biology↗

bioRxiv2021

Dinophyceae use exudates as weapons against the parasite Amoebophrya sp. (Syndiniales)

Parasites of the genus Amoebophrya sp. are important contributors to marine ecosystems and can be determining factors in the demise of blooms of Dinophyceae, including microalgae commonly responsible for toxic red tides. Yet they rarely lead to the total collapse of Dinophyceae blooms. The addition of resistant Dinophyceae (Alexandrium minutum or Scrippsiella donghaienis) or their exudate into a well-established host-parasite culture (Scrippsiella acuminata-Amoebophrya sp.) mitigated the success of the parasite and increased the survival of the sensitive host. Effect were mediated via water-borne molecules without the need of a physical contact. Severity of the anti-parasitic defenses fluctuated depending on the species, the strain and its concentration, but never totally prevented the parasite transmission. The survival time of Amoebophrya sp. free-living stages (dinospores) decreased in presence of A. minutum but not of S. donghaienis. The progeny drastically decreased with both species. Integrity of the membrane of dinospores was altered by A. minutum which provided a first indication on the mode of action of these anti-parasitic molecules. These results demonstrate that extracellular defenses are an effective strategy against parasites that does not only protect the resistant cells but also have the potential to affect the whole surrounding community.

microbiology↗

bioRxiv2020

New Genes in Drosophila Quickly Evolved Essential Functions in Viability During Development

It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the analyses of gene effects and improvements in the computational identification of new genes, which has led to large sample sizes of new genes, open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues related to phenotypic effect analyses of genes, particularly of those that recently originated. Here we reported our analyses of these issues, including the dating of gene ages, the interpretation of RNAi data that may confuse false positive/false negative rates, and the potential confounding impact of compensation and developmental effects that were not considered during previous CRISPR knockout experiments. We further analyzed new data from knockdowns of 702 new genes (~66% of total 1,070 Drosophila melanogaster new genes), revealing a similarly high proportion of essential genes from recent evolution, compared to those found in distant ancestors of D. melanogaster. Knockout of a few young genes detected analogous essentiality. Furthermore, our experimentally determined distribution and comparison of knockdown efficiency in different RNAi libraries provided valuable data for general functional analyses of genes. Taken together, these data, along with an improved understanding of the phenotypic effect analyses of new genes, provide further evidence to the conclusion that new genes in Drosophila quickly evolved essential functions in viability during development.

developmental biology↗

bioRxiv2020

A CRISPR-Cas9-engineered mouse model for GPI anchor deficiency mirrors human phenotype and shows hippocampal synaptic dysfunctions.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI) anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C>A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This new mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development. Significance statementInherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated the first viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using the new model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.

neuroscience↗

bioRxiv2019

Evolutionary dynamics of abundant 7 bp satellites in the genome of Drosophila virilis

The factors that drive the rapid changes in satellite DNA genomic composition we see in eukaryotes are not well understood. Drosophila virilis has one of the highest relative amounts of simple satellites of any organism that has been studied, with an estimated >40% of its genome composed of a few related 7 bp satellites. Here we use D. virilis as a model to understand technical biases affecting satellite sequencing and the evolutionary processes that drive satellite composition. By analyzing sequencing data from Illumina, PacBio, and Nanopore platforms, we identify platform-specific biases and suggest best practices for accurate characterization of satellites by sequencing. We use comparative genomics and cytogenetics to demonstrate that the highly abundant satellite family arose from a related satellite in the branch leading to the virilis phylad 4.5 - 11 million years ago before exploding in abundance in some species of the clade. The most abundant satellite is conserved in sequence and location in the pericentromeric region but has diverged widely in abundance among species, whereas the satellites nearest the centromere are rapidly turning over in sequence composition. By analyzing multiple strains of D. virilis, we saw that one centromere-proximal satellite is increasing in abundance along a geographical gradient while the other is contracting in an anti-correlated manner, suggesting ongoing conflicts at the centromere. In conclusion, we illuminate several key attributes of satellite evolutionary dynamics that we hypothesize to be driven by processes like selection, meiotic drive, and constraints on satellite sequence and abundance.

genomics↗

bioRxiv2019

A microtubule-mediated mechanical feedback controls leaf blade development in three dimensions

Many plant species have thin leaf blades, which is an important adaptation that optimizes the exchanges with the environment. Here, we provide evidence that their three-dimensional geometry is governed by microtubule alignment along mechanical stress patterns in internal walls. Depending on the primary shape of the primordium, this process has the potential to amplify an initial degree of flatness, or promote the formation of nearly axisymmetric, mostly elongating organs, such as stems and roots. This mechanism may explain leaf evolution from branches, which is alternative to Zimmermanns influential, but widely questioned, telome theory.\n\nOne Sentence SummaryMechanical feedback controls leaf development in three dimensions

developmental biology↗

bioRxiv2019

Evolutionary patterns of the chimerical retrogenes in Oryza

Chimerical retroposition delineate a process by which RNA reverse transcribed integration into genome accompanied with recruiting flanking sequence, which is asserted to play essential roles and drive genome evolution. Although chimerical retrogenes hold high origination rate in plant genome, the evolutionary pattern of retrogenes and their parental genes are not well understood in rice genome. In this study, using maximum likelihood method, we evaluated the substitution ratio along lineages of 24 retrogenes and parental gene pairs to retrospect the evolutionary patterns. The results indicate that some specific lineages in 7 pairs underwent positive selection. Besides the rapid evolution in the initial stage of new chimerical retrogene evolution, an unexpected pattern was revealed: soon or some uncertain period after the origination of new chimerical retrogenes, their parental genes evolved rapidly under positive selection, rather than the rapid evolution of the new chimerical retrogenes themselves. This result lend support to the hypothesis that the new copy assistant the function evolution among parental gene and retrogene. Transcriptionally, we also found that one retrogene (RCG3) have a high expression at the period of calli infection which supported by chip data while its parental gene doesnt have. Finally, by calibration to Ka/Ks analysis results in other species including Apis mellifera, we concluded that chimerical retrogenes are higher proportionally positive selected than the regular genes in the rice genome.

evolutionary biology↗

bioRxiv2018

A novel histone H4 variant regulates rDNA transcription in breast cancer

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. H4G expression was found in a variety of cell lines and was particularly overexpressed in the tissues of breast cancer patients. H4G was found to localize primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha helix 3 of the histone fold motif with the histone chaperone, nucleophosphomin 1. In addition, we found that H4G nucleolar localization increased rRNA levels, protein synthesis rates, and cell cycle progression. Furthermore, micrococcal nuclease digestion of H4G-containing nucleosomes reconstituted in vitro indicated that H4G destabilizes the nucleosome, which may serve to alter nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

molecular biology↗

bioRxiv2018

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-{kappa}B activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-{kappa}B at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

cell biology↗

bioRxiv2018

Baculovirus utilizes cholesterol transporter Niemann-Pick C1 for host cell entry

The dual roles of baculovirus for the control of natural insect populations as an insecticide, and for foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for baculovirus infection in insect cells. Both pretreatment of Bombyx mori embryonic cells (BmE) with NPC1 antagonists (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV (Bombyx mori nuclear polyhedrosis virus) infectivity. Furthermore, we show that the major glycoprotein gp64 of BmNPV, responsible for both receptor binding and fusion, is able to interact predominantly with the BmNPC1 C domain, with an enhanced binding capacity at low pH conditions, indicating that NPC1 most likely plays a role during viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of hNPC1 in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of exploiting host NPC1 proteins during virus intracellular entry events.\n\nIMPORTANCEBmNPV is one of the most important members of the Baculoviridae; many viruses in this family have been frequently employed as viral vectors for foreign gene delivery or expression and as biopesticides, but their host receptors still remain unclear. Here, we describe that the intracellular cholesterol transporter BmNPC1 is indispensable for BmNPV infection in insect cells, and it interacts with the major viral glycoprotein gp64. Our study on the role of BmNPC1 in baculovirus infection has further expanded the list of the enveloped viruses that require host NPC1 proteins for entry, and will ultimately help us to uncover the molecular mechanism of the involvement of NPC1 proteins in the entry process of many enveloped viruses.

microbiology↗

bioRxiv2017

Rapid evolution of gained essential developmental functions of a young gene via interactions with other essential genes

New genes originated relatively recently and are only present in a subset of species in a phylogeny. Accumulated evidence suggests that new genes, like old genes that are conserved across species, can also take on important functions and be essential for the survival and reproductive success of organisms. While there are detailed analyses of the mechanisms underlying gained fertility functions by new genes, how new genes rapidly became essential for viability remains unclear. We focused on a young retro-duplicated gene (CG7804, which we named Cocoon) in Drosophila that originated three million years ago. We found that, unlike its evolutionarily conserved and broadly expressed parental gene, Cocoon has evolved rapidly under positive selection since its birth and accumulates many amino acid divergences at functional sites from the parental gene. Despite its young age, Cocoon is essential for the survival of D. melanogaster at multiple developmental stages, including the critical embryonic stage, and its expression is essential in different tissues from its parental gene. Functional genomic analyses found that Cocoon gained multiple DNA binding targets, which regulates the expression of genes that have other essential functions and/or have multiple gene-gene interactions. Our observations suggest that Cocoon acquired essential function to survival through forming interactions that have large impacts on the gene interaction network. Our study is an important step towards deciphering the evolutionary trajectory by which new genes functionally diverge from the parental gene and become essential.

evolutionary biology↗