Biology subjects

Sharma, R.

Publications and source records attributed to Sharma, R..

At least 217 records · Page 12Linked to original sources

bioRxiv2020

Systematic hormone-metabolite network provides insights of high salinity tolerance in Pongamia pinnata (L.) pierre

Salinity stress results significant losses in plant productivity, and loss of cultivable lands. Although Pongamia pinnata is reported to be a salt tolerant semiarid tree crop, the adaptive mechanisms to saline environment are elusive. The present investigation describes alterations in hormonal and metabolic responses in correlation with physiological and molecular variations in leaves and roots of Pongamia at sea salinity level (3% NaCl) for 8 days. At physiological level, salinity induced adjustments in plant morphology, leaf gas exchange and ion accumulation patterns were observed. Our study also revealed that phytohormones including JAs and ABA play crucial role in promoting the salt adaptive strategies such as apoplasmic Na+ sequestration and cell wall lignification in leaves and roots of Pongamia. Correlation studies demonstrated that hormones including ABA, JAs and SA showed a positive interaction with selective compatible metabolites (sugars, polyols and organic acids) to aid in maintaining osmotic balance and conferring salt tolerance to Pongamia. At the molecular level, our data showed that differential expression of transporter genes as well as antioxidant genes regulate the ionic and ROS homeostasis in Pongamia. Collectively, these results shed new insights on an integrated physiological, structural, molecular and metabolic adaptations conferring salinity tolerance to Pongamia. High lightOur data, for the first time, provide new insights for an integrated molecular and metabolic adaptation conferring salinity tolerance in Pongamia. The present investigation describes alterations in hormonal and metabolic responses in correlation with physiological and molecular variations in Pongamia at sea salinity level (3% NaCl) for 8 days.

plant biology↗

bioRxiv2020

Microglia-targeted dendrimer-2PMPA therapy robustly inhibits GCPII and improves cognition in a mouse model of multiple sclerosis

Roughly half of all individuals with multiple sclerosis (MS) experience cognitive impairment, but there are no approved treatments that target this aspect of the disease. Recent studies link reduced brain N-acetylaspartylglutamate (NAAG) levels to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate (NAA) and glutamate. Although several GCPII inhibitors, such as 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, no GCPII inhibitors are clinically available due to poor bioavailability and limited brain penetration. Systemic hydroxyl dendrimers (~4 nm) have been successfully used to enhance brain delivery of drugs selectively to activated glia. We recently discovered that GCPII is highly upregulated in activated microglia after brain injury. To determine if dendrimer conjugation could enhance the brain delivery of GCPII inhibitors, specifically in the context of MS, we attached 2-PMPA to hydroxyl polyamidoamicne (PAMAM) dendrimers (D-2PMPA) using a highly efficient click chemistry approach. Targeted uptake of D-2PMPA into activated glia was subsequently confirmed in glial cultures where it showed robust anti-inflammatory activity, including an elevation in TGF{beta} and a reduction in TNF. Given these positive effects, D-2PMPA (20mg/kg) or vehicle dendrimer were dosed twice weekly to experimental autoimmune encephalomyelitis (EAE)-immunized mice starting at disease onset (therapeutic paradigm). D-2PMPA significantly improved cognition in EAE as assessed by Barnes maze performance, even though physical severity was not impacted. Glial target engagement was confirmed, as CD11b+ enriched cells isolated from hippocampi in D-2PMPA-treated mice exhibited almost complete loss of GCPII activity. These data demonstrate the utility of hydroxyl dendrimers to enhance brain penetration and support the development of D-2PMPA to treat cognitive impairment in MS. FundingThis work was funded by the National Multiple Sclerosis Society (RG-1507-05403 to BSS), the National Institute of Health NINDS (R01NS093416 to SK, RM and BSS), and Ashvattha Therapeutics. We would also like to acknowledge support for the statistical analysis from the National Center for Research Resources and NIH NCATS (1UL1TR001079). HighlightsO_LIThe GCPII inhibitor 2-PMPA was conjugated to hydroxyl PAMAM dendrimers (D-2PMPA) C_LIO_LID-2PMPA targeted activated glia in culture and displayed anti-inflammatory activity C_LIO_LIWhen dosed systemically to EAE mice, D-2PMPA inhibited CD11b+ cell GCPII activity C_LIO_LIWhen dosed systemically to EAE mice, D-2PMPA improved cognitive function C_LI

neuroscience↗

bioRxiv2020

Reanalysis of Genome Sequences of tomato accessions and its wild relatives: Development of tomato genomic variation (TGV) database integrating SNPs and INDELs polymorphisms

MotivationFacilitated by technological advances and expeditious decrease in the sequencing costs, whole-genome sequencing (WGS) is increasingly implemented to uncover variations in cultivars/accessions of many crop plants. In tomato (Solanum lycopersicum), the availability of the genome sequence, followed by the resequencing of tomato cultivars and its wild relatives, has provided a prodigious resource for the improvement of traits. A high-quality genome resequencing of 84 tomato accessions and wild relatives generated a dataset that can be used as a resource to identify agronomically important alleles across the genome. Converting this dataset into a searchable database, including information about the influence of SNPs on protein function, provides valuable information about the genetic variations. The database will assist in searching for functional variants of a gene for introgression into tomato cultivars. ResultsA recent release of better-quality tomato genome reference assembly SL3.0, and new annotation ITAG3.2 of SL3.0, dropped 3,857 genes, added 4,900 novel genes, and updated 20,766 genes. Using the above version, we remapped the data from the tomato lines resequenced under the "100 Tomato Genome ReSequencing Project" on new tomato genome assembly SL3.0 and made an online searchable Tomato Genomic Variations (TGV) database. The TGV contains information about SNPs and InDels and expands it by functional annotation of variants with new ITAG3.2 using SIFT4G software. This database with search function assists in inferring the influence of SNPs on the function of a target gene. This database can be used for selecting SNPs, which can be potentially deployed for improving tomato traits. Availability and ImplementationTGV is freely available at http://psd.uohyd.ac.in/tgv. Contactrameshwar.sharma@gmail.com

bioinformatics↗

bioRxiv2020

A mathematical model of heat shock response to study the competition between protein folding and aggregation

Proteins, under conditions of cellular stress, typically tend to unfold and form lethal aggregates leading to neurological diseases like Parkinsons and Alzheimers. A clear understanding of the conditions that favor dis-aggregation and restore the cell to its healthy state after they have been stressed is therefore important in dealing with these diseases. The heat shock response (HSR) mechanism is a signaling network that deals with these undue protein aggregates and aids in the maintenance of homeostasis within a cell. This framework, on its own, is a mathematically well studied mechanism. However, not much is known about how the various intermediate mis-folded protein states of the aggregation process interact with some of the key components of the HSR pathway such as the Heat Shock Protein (HSP), the Heat Shock transcription Factor (HSF) and the HSP-HSF complex. In this article, using kinetic parameters from the literature, we propose and analyze two mathematical models for HSR that also include explicit reactions for the formation of protein aggregates. Deterministic analysis and stochastic simulations of these models show that the folded proteins and the misfolded aggregates exhibit bistability in a certain region of the parameter space. Further, the models also highlight the role of HSF and the HSF-HSP complex in reducing the time lag of response to stress and in re-folding all the mis-folded proteins back to their native state. These models therefore call attention to the significance of studying related pathways such as the HSR and the protein aggregation and re-folding process in conjunction with each other.

systems biology↗

bioRxiv2020

Comparative Genomic Analysis of Rapidly Evolving SARS CoV-2 Viruses Reveal Mosaic Pattern of Phylogeographical Distribution

The Coronavirus Disease-2019 (COVID-19) that started in Wuhan, China in December 2019 has spread worldwide emerging as a global pandemic. The severe respiratory pneumonia caused by the novel SARS-CoV-2 has so far claimed more than 60,000 lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 displays high transmission rates, their underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nsp13-16 (ORF1b polyprotein) and S-Protein within 10 viral isolates from the USA. These viral proteins are involved in RNA replication, indicating highly evolved viral strains circulating in the population of USA than other countries. Furthermore, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of the USA isolate (MT188341) leading to shift in amino acid frame from position 2540 onwards. Through the construction of SARS-CoV-2-human interactome, we further revealed that multiple host proteins (PHB, PPP1CA, TGF-{beta}, SOCS3, STAT3, JAK1/2, SMAD3, BCL2, CAV1 & SPECC1) are manipulated by the viral proteins (nsp2, PL-PRO, N-protein, ORF7a, M-S-ORF3a complex, nsp7-nsp8-nsp9-RdRp complex) for mediating host immune evasion. Thus, the replicative machinery of SARS-CoV-2 is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies.

microbiology↗

bioRxiv2020

Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

Clathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable transport-packets that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

neuroscience↗

bioRxiv2020

Deletion of RsmE 16S rRNA methyltransferase leads to low level increase in aminoglycoside resistance in Mycobacterium smegmatis

Owing to its central role in cellular function, ribosome is one of the most common targets of antibiotics in bacteria. Mutations in rrs gene, ribosomal protein genes, methyltransfersases or drug influx/efflux are often found to overcome the drug response. Despite modulation of methylation pattern in the ribosome through mutations in the methyltransferases as one of key modulators of drug response, rsmG (gidB) is the only conserved methyltransferase associated with low-level drug resistance in large number of mycobacterial isolates. Here, we present the first evidence of association of methylation by mycobacterial RsmE, that methylates U1498 of 16S rRNA, with low levels of drug resistance. Deletion of the RsmE-homolog of Mycobacterium smegmatis leads to at least two-fold increase in the inhibitory concentration of aminoglycosides that bind in the decoding center proximal to U1498 in the 30S subunit. The change in inhibitory concentrations was highly specific and does not show any cross-resistance to drugs of other classes. Surprisingly, Rv2372c, the RsmE-homolog of Mycobacterium tuberculosis has the largest number of mutations among conserved ribosomal methyltransfersases, after gidB, highlighting the role of mutations in the RsmE methyltransferase as a key emerging mechanism of drug resistance in clinical strains of M. tuberculosis. Our work underlies the association of methylation by the RsmE-homolog with drug resistance and lays the groundwork to tackle this emerging mechanism of drug resistane in mycobacteria.

microbiology↗

bioRxiv2019

Origin Specific Genomic Selection: a simple process to optimize the favourable contribution of parents to progeny

Modern crop breeding is in constant demand for new genetic diversity as part of the arms race with genetic gain. The elite gene pool has limited genetic variation and breeders are trying to introduce novelty from unadapted germplasm, landraces and wild relatives. For polygenic traits, currently available approaches to introgression are not ideal, as there is a demonstrable bias against exotic alleles during selection. Here, we propose a partitioned form of genomic selection, called Origin Specific Genomic Selection (OSGS), where we identify and target selection on favourable exotic alleles. Briefly, within a population derived from a bi-parental cross, we isolate alleles originating from the elite and exotic parents, which then allows us to separate out the predicted marker effects based on the allele origins. We validated the usefulness of OSGS using two nested association mapping (NAM) datasets: barley NAM (elite-exotic) and maize NAM (elite-elite), as well as by computer simulation. Our results suggest that OSGS works well in bi-parental crosses, and it is possible to extend the approach to broader multi-parental populations.

genetics↗

bioRxiv2019

Incorporating genetic selection into individual-based models (IBMs) of malaria and other infectious diseases.

Optimal control strategies for human infections are often investigated by computational approaches using individual-based models (IBMs). These typically track humans and evaluate the impact of control interventions in terms of human deaths, clinical cases averted, interruption of transmission etc. Genetic selection can be incorporated into these IBMs and used to track the spread of mutations whose origin and spread are often driven by the intervention, and which subsequently undermine the control strategy; for example, mutations which encode antimicrobial drug resistance or diagnosis- or vaccine-escape phenotypes. Basic population genetic descriptions of selection are based on infinite population sizes (so that chance fluctuations in allele frequency are absent) but IBMs track finite population sizes. We describe how the finite sizes of IBMs affect simulating the dynamics of genetic selection and how best to incorporate genetic selection into these models. We use the OpenMalaria IBM of malaria as an example, but the same principles apply to IBMs of other diseases. We identify four strategies to incorporate selection into IBMs and make the following four recommendations. Firstly, calculate and report the selection coefficients, s, of the advantageous allele as the key genetic parameter. Secondly, use these values of s to calculate the wait-time until a mutation successful establishes itself in the population. The wait time for the mutation can be added to speed of selection, s, to calculate when the mutation will reach significant, operationally important levels. Thirdly, quantify the ability of the IBM to robustly estimate small selection coefficients. Fourthly, optimise computational efficacy: when s is small it is plausible that fewer replicates of larger IBMs will be more efficient than a larger number of replicates of smaller size.

evolutionary biology↗

bioRxiv2019

Novel functional insights revealed by distinct protein-protein interactions of the residual SWI/SNF complex in SMARCA4-deficient small cell carcinoma of the ovary, hypercalcemic type

Chromatin remodeling plays a critical role in tumor suppression as demonstrated by 20% of human cancers bearing inactivating mutations in SWI/SNF chromatin remodeling complex members. Mutations in different SWI/SNF subunits drive a variety of adult and pediatric tumor types, including non-small cell lung cancers, rhabdoid tumors, medulloblastomas, and ovarian cancers. Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is an aggressive subtype of ovarian cancer occurring in young women. Nearly all (>98%) SCCOHTs have inactivating mutations in SMARCA4, which encodes 1 of 2 mutually exclusive catalytic subunits of the SWI/SNF complex. Less than half of SCCOHT patients survive 5 years despite aggressive surgery and multimodal chemotherapy. Empirical support for effective SCCOHT treatments is scarce, in part because of the poor understanding of SCCOHT tumorigenesis. To gain insight into the functional consequences of SWI/SNF subunit loss, we defined SWI/SNF composition and its protein-protein interactions (PPIs) by immunoprecipitation and mass spectrometry (IP-MS) of SWI/SNF subunits in 3 SCCOHT cell lines. Comparing these results to a cell line containing a wild-type SWI/SNF complex, the interaction of most canonical core SWI/SNF subunits was observed in all SCCOHT cell lines at a lower abundance. The SCCOHT SWI/SNF also lacked ATPase module subunits and showed a drastic reduction in PBAF-specific subunit interactions. The wild-type and SCCOHT SWI/SNF subunits immunoprecipitated a shared set of 26 proteins, including core SWI/SNF subunits and RNA processing proteins. We observed 131 proteins exclusively interacting with the wild-type SWI/SNF complex including isoform-specific SWI/SNF subunits, members of the NuRD complex, and members of the MLL3/4 complex. We observed 60 PPIs exclusive to the SCCOHT residual SWI/SNF shared in at least 2 of the 3 SCCOHT cell lines, including many proteins involved in RNA processing. Differential interactions with the residual SWI/SNF complex in SCCOHT may further elucidate altered functional consequences of SMARCA4 mutations in these tumors as well as identify synthetic lethal targets that translate to other SWI/SNF-deficient tumors.

cancer biology↗

bioRxiv2019

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

ObjectivesSorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.\n\nResultsHere, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

plant biology↗

bioRxiv2019

Analytical characterization of Parylene-C degradation mechanisms on Utah arrays: evaluation of in vitro Reactive Accelerated Aging model compared to multiyear in vivo implantation

Implantable neural microelectrodes are integral components of neuroprosthetic technologies and can transform treatments for many neural-mediated disorders. However, dielectric material degradation during long-term (> 1 year) indwelling periods restricts device functional lifetimes to a few years. This comprehensive work carefully investigates in vivo material degradation and also explores the ability of in vitro Reactive Accelerated Aging (RAA) to evaluate implant stability. Parylene C-coated Utah electrode arrays (UEAs) implanted in feline peripheral nerve for 3.25 years were explanted and compared to RAA-processed devices, aged in phosphate buffered saline (PBS) + 20 mM H2O2 at either 67 or 87 {degrees}C (28 or 7 days, respectively). Electron microscopy revealed similar physical damage characteristics between explants and RAA (87{degrees} C) devices. Parylene C degradation was overwhelmingly apparent for UEAs from both RAA cohorts. Controls aged in PBS alone displayed almost no damage. Spectroscopic characterization (EDX, XPS, FTIR) found clear indications of oxidation and chlorine abstraction for parylene C aged in vivo. While in vitro aging was also accompanied by signs of oxidation, changes in the chemistry in vivo and in vitro were statistically different. Analysis of RAA- aged devices identified UEA fabrication approaches that may greatly improve device resistance to degradation. This work underscores the need for an improved understanding of in vivo damage mechanisms, to facilitate the critical need for representative in vitro accelerated testing paradigms for long-term implants.

bioengineering↗

bioRxiv2019

The genome of Peronospora belbahrii reveals high heterozygosity, a low number of canonical effectors and CT-rich promoters

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was ca. 35.4 Mbp in length, with an N50 scaffold length of ca. 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of ca. 40.1 kbp. From the repeat-masked genome 9049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16 % of the genome consists of repetitive sequences, and based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of Pe. belbahrii. Peronospora belbahrii has undergone a high degree of convergent evolution, reflecting its obligate biotrophic lifestyle. Features of its secretome, signalling networks, and promoters are presented, and some patterns are hypothesised to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

genomics↗

bioRxiv2019

Retrograde neurotensin release from dopamine neurons drives long-term depression of substantia nigra dopamine signaling

Midbrain dopamine neurons play central roles in reward learning and motivated behavior, and inhibition at somatodendritic dopamine D2 receptor (D2R) synapses blunts psychostimulant reinforcement. Release of the neuropeptide neurotensin in the midbrain increases following methamphetamine exposure and induces long-term depression of D2R synaptic currents (LTDDA), however the source of neurotensin that drives LTDDA is not known. Here we show that LTDDA is driven by neurotensin released by dopamine neurons. Optogenetic stimulation of dopamine neurons was sufficient to induce LTDDA in the substantia nigra, but not the ventral tegmental area, and was dependent on neurotensin receptors, postsynaptic calcium, and vacuolar-type H+-ATPase activity in the postsynaptic cell. Further, LTDDA was enhanced in mice that had self-administered methamphetamine. These findings reveal a novel form of signaling between dopamine neurons involving release of the peptide neurotensin, which may act as a feed forward mechanism to increase dopamine neuron excitability and methamphetamine self-administration.

neuroscience↗

bioRxiv2019

Neurotensin receptor 1 deletion suppresses methamphetamine self-administration and the associated reduction in dopamine cell firing

We previously reported that pharmacological blockade of neurotensin receptors in the ventral tegmental area (VTA) decreases methamphetamine (METH) self-administration in mice. Here we explored the consequences of genetic deletion of neurotensin receptor 1 (NtsR1) in METH self-administration and VTA dopamine neuron firing activity. We implanted mice with an indwelling jugular catheter and trained them to nose-poke for intravenous infusions of METH. Mice with NtsR1 deletion (KO) acquired selfadministration similar to wildtype (WT) and heterozygous (HET) littermates. However, in NtsR1 KO and HET mice, METH intake and motivated METH seeking decreased when the response requirement was increased to a fixed ratio 3 and when mice were tested on a progressive ratio protocol. After completion of METH self-administration, single cell in vivo extracellular recordings of dopamine firing activity in the VTA were obtained in anesthetized mice. In WT METH-experienced mice, dopamine cell firing frequency dramatically decreases compared to WT drug-naive mice. NtsR1 KO and HET mice did not exhibit this decline of dopamine cell firing activity after prolonged METH selfadministration. We also observed an increase in population activity following METH selfadministration that was strongest in the WT group. Our results suggest a role for NtsR1 in METH-seeking behavior, and ablation of NtsR1 receptors prevents the detrimental effects of prolonged METH self-administration on VTA dopamine cell firing frequency.

neuroscience↗

bioRxiv2019

Enhanced polar auxin transport in tomato polycotyledon mutant seems to be related to glutathione levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analysed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in-vivo assay revealed higher glutathione level in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of short root phenotype of pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH level in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

plant biology↗

bioRxiv2019

Divergence from conserved residues underlies intracellular retention of mammalian odorant receptors

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in non-olfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here, we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing "consensus" odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations. Significance StatementOdor detection in mammals depends on the largest family of G protein-coupled receptors, the odorant receptors, which represent [~]2% of our protein-coding genes. The vast majority of odorant receptors are trapped within the cell when expressed in non-olfactory cells. The underlying causes of why odorant receptors cannot be functionally expressed in non-olfactory cells have remained enigmatic for over 20 years. Our study points to divergence from a consensus sequence as a key factor in a receptors inability to function in non-olfactory cells, which in turn, helps explain odorant receptors exceptional functional diversity and rapid evolution. We also show the success of protein engineering strategies for promoting odorant receptor cell surface expression.

cell biology↗

bioRxiv2019

A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT

Next-generation DNA sequencing is currently limited by an inability to count the number of input DNA molecules. Molecular counting is particularly needed when accurate quantification is required for diagnostic purposes, such as in single-gene non-invasive prenatal testing (sgNIPT) and liquid biopsy. We developed Quantitative Counting Template (QCT) molecular counting for reconstructing the number of input DNA molecules using sequencing data. We then used QCT molecular counting to develop sgNIPT of sickle cell disease, cystic fibrosis, spinal muscular atrophy, alpha-thalassemia, and beta-thalassemia. Incorporating molecular count information into a statistical model of disease likelihood led to analytical sensitivity and specificity of >98% and >99%, respectively. Validation of sgNIPT was further performed with maternal blood samples collected during pregnancy, and sgNIPT was 100% concordant with newborn follow-up.

genetics↗