Biology subjects

Zhao, J.

Publications and source records attributed to Zhao, J..

At least 523 records · Page 29Linked to original sources

bioRxiv2020

Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to many countries around the world. However, where is it origin and which animals are sensitive to cross-species transmission is unclear. The interaction of virus and cell receptor is a key determinant of host range for the novel coronavirus. Angiotensin-converting enzyme 2 (ACE2) is demonstrated as the primary entry receptor for SARS-CoV-2. In this study, we evaluated the SARS-CoV-2 entry mediated by ACE2 of 11 different species of animals, and discovered that ACE2 of Rhinolophus sinicus (Chinese horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (pig), Capra hircus (goat) and especially Manis javanica (Malayan pangolin) were able to render SARS-CoV-2 entry in non-susceptible cells. This is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the presume that SARS-CoV-2 may have a pangolin origin. However, none of the ACE2 proteins from Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (chicken), Notechis scutatus (mainland tiger snake), Mus musculus (house mouse) rendered SARS-CoV-2 entry. Specifically, a natural isoform of Macaca mulatta (Rhesus monkey) ACE2 with a mutation of Y217N was resistance to infection, which rises the possible impact of this type of ACE2 during monkey studies of SARS-CoV-2. Overall, these results clarify that SARS-CoV-2 could engage receptors of multiple species of animals and it is a perplexed work to track SARS-CoV-2 origin and its intermediate hosts. IMPORTANCEIn this study, we illustrated that SARS-CoV-2 is able to engage receptors of multiple species of animals. This indicated that it may be a perplexed work to track SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is considered to potentiate its diverse cross-species transmissibility. Of note, here is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the possibility that SARS-CoV-2 may have a pangolin origin. And we also demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At last, it is also important to detect the expression ratio of the Y217N ACE2 to the prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Zinc deficiency decreases bone mineral density of rat by calmodulin-induced change in calcium metabolism

(1) BackgroundZinc deficiency decreases bone mineral density (BMD), but it is not known whether decreased BMD is a result of altered calcium absorption, excretion, and/or tissue distribution. To identified the associations between zinc deficiency, calcium metabolism and decreased BMD. (2) Methodswe performed two zinc deficiency experiments. In the first experiment, male rats (5-week-old) were fed a low zinc diet for four weeks. We measured serum zinc, alkaline phosphatase, Ca2+, osteocalcin, calcitonin, parathyroid hormone (PTH), calcium concentrations in feces and urine, BMD, and femur bone length and weight. In the second experiment, male rats (3-week-old) were fed a low zinc diet for five weeks. In addition to the aforesaid indicators, we measured the concentrations of zinc, total calcium, and calmodulin in multiple tissues. (3) ResultsIn both experiments, serum zinc, alkaline phosphatase, fecal and urine calcium, BMD, and bone weight of the low zinc diet group (LZG) were reduced compared with the normal zinc diet group (NZG)and pair-fed group (PZG); PTH increased significantly. Serum Ca2+, osteocalcin, and calcitonin concentrations were unchanged and not associated with decreased BMD. In the second experiment, zinc concentrations were reduced in serum, skeletal muscle, feces, and urine of LZG animals compared with NZG and PZG. Calmodulin in serum and skeletal muscle of the LZG group was decreased. Zinc deficiency increased total calcium concentrations in serum and skeletal muscle by promoting a decrease in calmodulin. To maintain blood Ca2+ balance, elevated PTH increased calcium reabsorption, reduced calcium excretion, stimulated bone resorption, mobilized bone calcium, and decreased BMD. (4) Conclusions: Decreased calmodulin and increased PTH induced by zinc deficiency altered calcium tissue distribution and decreased BMD of rat.

molecular biology↗

bioRxiv2020

Localization of hyphal growth associated with mycotoxin production during the malting of Fusarium head blight infected grains

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Objectives of the current study were to localize the growth of Fusarium within FHB infected kernels and to associate it with the production of DON that occurred during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in Fusarium Tri5 DNA and trichothecene mycotoxins following malting, were screened for hyphal localization. The growth of hyphae, both on the surface of kernels and within tissues of grain and malt was, imagined by scanning electron microscopy and confocal laser scanning microscopy assisted with WGA-Alexa Fluor 488 pre-staining, respectively. In barley, hyphae were primarily present on or within husk, vascular bundle, and pericarp cavities. Following malting, large amounts of hyphal growth were observed in not only these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. Interestingly, these grains already had an extensive internal presence of hyphae in unmalted grain, occurring in the pericarp, testa, vascular bundle, nucellar projection, aleurone layer, endosperm, pericarp and endosperm cavities, and embryo. Shotgun sequencing followed by metagenomics analysis verified that Fusarium spp. accounted for above 90% of the fungal hyphae growing in the interior of grains during malting, which coincided with the significant production of mycotoxins.

plant biology↗

bioRxiv2020

Dopamine Release Neuroenergetics in Mouse Striatal Slices

Parkinsons disease (PD) is the second most common neurodegenerative disease. Dopamine (DA) neurons in the substantia nigra par compacta with axonal projections to the dorsal striatum (dSTR) degenerate in PD while in contrast, DA neurons in the ventral tegmental area with axonal projections to the ventral striatum including the nucleus accumbens (NAcc) shell, are largely spared. To understand the pathogenesis of PD, it is important to study the neuroenergetics of DA neurons. This study aims to uncover the relative contribution of glycolysis and oxidative phosphorylation (OxPhos) to evoked DA release in the striatum. We measured evoked DA release in mouse striatal brain slices by fast-scan cyclic voltammetry every 2 minutes. Blocking OxPhos caused a greater reduction in evoked DA release in the dSTR compared to the NAcc shell, and blocking glycolysis caused a greater reduction in evoked DA release in the NAcc shell than in the dSTR. Furthermore, when glycolysis was bypassed in favor of direct OxPhos, evoked DA release in the NAcc shell was decreased by [~]50% over 40 minutes whereas evoked DA release in the dSTR was largely unaffected. These results demonstrated that the dSTR relies primarily on OxPhos for energy production to maintain evoked DA release whereas the NAcc shell relies more on glycolysis. Using two-photon imaging, we consistently found that the oxidation level of the DA terminals was higher in the dSTR than in the NAcc shell. Together, these findings partially explain the specific vulnerability of DA terminals in the dSTR to degeneration in PD. Significant statementThe neuroenergetics of dopaminergic neuron is important to understand Parkinsons disease (PD), a neurodegenerative disorder associated with mitochondrial dysfunctions. However, the relative contributions of glycolysis and oxidative phosphorylation (OxPhos) to presynaptic energy demands in DA terminals are unclear. We addressed this question by measuring DA release in the dorsal striatum and nucleus accumbens (NAcc) shell of mouse brain using FSCV under reagents blocking different energy systems. We found that the NAcc shell relies on both glycolysis and OxPhos to maintain DA release while the dSTR relies heavily on OxPhos. We demonstrate the different neuroenergetics of DA terminals in these two brain areas, providing new fundamentally important insight into the specific vulnerability of DA terminals in the dSTR to degeneration in PD.

neuroscience↗

bioRxiv2020

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2-like sequences in rDNA promoter upon T cell activation in vitro. The elevated pre-rRNA level of T cells is also observed in both mouse heart or skin transplantation models, and in kidney transplanted patients. Importantly, T cell activation can be significantly suppressed by inhibiting NF45/NF90-dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off-target activity (i.e. toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90-mediated rDNA transcription as a novel signaling pathway essential for T cell activation and as a new target for the development of safe and effective immunosuppressants.

molecular biology↗

bioRxiv2020

Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

Type IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

microbiology↗

bioRxiv2020

Enantiomers of Chloroquine and Hydroxychloroquine Exhibit Different Activities Against SARS-CoV-2 in vitro, Evidencing S-Hydroxychloroquine as a Potentially Superior Drug for COVID-19

In all of the clinical trials for COVID-19 conducted thus far and among those ongoing involving chloroquine or hydroxychloroquine, the drug substance used has invariably been chloroquine (CQ) diphosphate or hydroxychloroquine (HCQ) sulfate, i.e., the phosphoric or sulfuric acid salt of a racemic mixture of R- and S-enantiomer (50/50), respectively. As a result, the clinical outcome from previous CQ or HCQ trials were, in fact, the collective manifestation of both R and S- enantiomers with inherent different pharmacodynamic and pharmacokinetic properties, and toxicity liabilities. Our data for the first time demonstrated the stereoselective difference of CQ and HCQ against live SARS-CoV-2 virus in a Biosafety Level 3 laboratory. S-chloroquine (S-CQ) and S-hydroxychloroquine (S-HCQ) significantly more active against SARS-CoV-2, as compared to R-CQ and R-HCQ, respectively. In addition, Mpro, as one of the critical enzymes for viral transcription and replication, also exhibited an enantioselective binding affinity toward the S-enantiomers. The most significant finding from this study is the pronounced difference of the two enantiomers of CQ and HCQ observed in hERG inhibition assay. The IC50 value of S-HCQ was higher than 20 M against hERG channel, which was much less active over all tested CQ and HCQ compounds. Moreover, S-HCQ alone did not prolong QT interval in guinea pigs after 3 days and 6 days of administration, indicating a much lower cardiac toxicity potential. With these and previous findings on the enantio-differentiated metabolism, we recommend that future clinical studies should employ S-HCQ, substantially free of the R-enantiomer, to potentially improve the therapeutic index for the treatment of COVID-19 over the racemic CQ and HCQ.

biochemistry↗

bioRxiv2020

Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients

As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

genomics↗

bioRxiv2020

Characterization of miRNAs encoded by Autographa californica nucleopolyhedrovirus

Two Autographa californica nucleopolyhedrovirus (AcMNPV) encoded miRNAs, AcMNPV-miR-1 and -miR-3, have been reported in 2013 and 2019. Here, we present an integrated investigation of AcMNPV-encoded miRNAs. Six candidate miRNAs were predicted through small RNA deep sequencing and bioinformatics, of which, five validated by experiments. Three miRNAs perfectly matched the coding sequence of viral genes. The other two are located in coding sequences of viral genes. Targets in both virus and host were predicted and subsequently tested using dual-luciferase reporter assay. The validated targets were found mainly in AcMNPV, except for the targets of AcMNPV-miR-4, which are all host genes. Based on reporter assays, the five miRNAs predominantly function by down-regulating their targets, though individual target is slightly up-regulated. The transcription start sites of these miRNAs were analyzed. Our results suggest that AcMNPV-encoded miRNAs function as fine modulators of the interactions between host and virus by regulating viral and/or host genes. Author summaryVirus-encoded miRNAs have been widely reported as modulators participating in almost all biological processes. However, among Baculoviridae, which consists of a large family of dsDNA viruses that infect numerous beneficial insects and agricultural pests, only several have been reported encoding miRNAs. To clarify the roles of AcMNPV-encoded miRNAs in host-pathogen interactions, we employed RNA deep sequencing and series of experimental approaches identifying AcMNPV-encoded miRNAs, followed by target validation and function deduction. Among them, AcMNPV-miR-1 and AcMNPV-miR-3 have been reported in 2013 and 2019, respectively. This study reveals the sites of these miRNAs in the genome, both in coding sequences and complements, suggesting diverse functions. These miRNAs target genes in the virus itself and in the host, largely by suppressing expression, with some enhancing it. The transcription initiations of the miRNAs were analyzed. Our results provide some insight into the finely regulated process of baculovirus infection.

microbiology↗

bioRxiv2020

Skin Delivery of Modified Vaccinia Ankara Viral Vectors Generates Superior T Cell Immunity Against a Respiratory Viral Challenge

Modified Vaccinia Ankara (MVA) was recently approved as a Smallpox vaccine. Transmission of Variola is by respiratory droplets, and MVA delivered by skin scarification (s.s.) protected mice far more effectively against lethal respiratory challenge with VACV than any other route of delivery, and at much lower doses. Comparisons of s.s. with intradermal, subcutaneous or intramuscular routes showed that MVAOVA s.s.-generated T cells were both more abundant and transcriptionally distinct. MVAOVA s.s. produced greater numbers of lung Ova-specific CD8+ TRM and was superior in protecting mice against lethal VACVOVA respiratory challenge. Nearly as many lung TRM were generated with MVAOVA s.s. compared to direct pulmonary immunization with MVAOVA, and both routes vaccination protected mice against lethal pulmonary challenge with VACVOVA. Strikingly, MVAOVA s.s.-generated effector T cells exhibited overlapping gene transcriptional profiles to those generated via direct pulmonary immunization. Overall, our data suggest that heterologous MVA vectors delivered via s.s. are uniquely well-suited as vaccine vectors for respiratory pathogens like COVID-19. In addition, MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine.

immunology↗

bioRxiv2020

Comparison of SARS-CoV-2 spike protein binding to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 receptors

The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used recently released X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptor allows to define residues important for binding. From the 20 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S-binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites located near the S-interface. Of note, pigs and dogs which are not or not effectively infected, respectively, have only a few changes in the binding site have relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with viruses from bat and pangolin revealed that the latter contains only one substitution, whereas the bat virus exhibits five. However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-COV-2. IMPORTANCESARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the species barrier might be low. However, the lower expression of ACE2 in the upper respiratory tract of some pets and livestock means more research and monitoring should be done to explore the animal source of infection and the risk of potential cross-species transmission. Finally, the analysis also showed that SARS-CoV-2 may not specifically adapted to any of its putative intermediate hosts.

bioinformatics↗

bioRxiv2020

Transient high glucose causes delayed wound healing by the DNMT1-mediated Ang-1/NF-κB pathway

The progression of diabetic complications does not halt despite termination of hyperglycemia, suggesting a "metabolic memory" phenomenon. However, whether metabolic memory exists in and affects the healing of diabetic wounds, as well as the underlying molecular mechanisms, remain unclear. In this study, we found that wound healing was delayed and angiogenesis was decreased in diabetic mice, despite normalization of glycemic control. Thus, we hypothesized that transient hyperglycemic spikes may be a risk factor for diabetic wound healing. We showed that transient hyperglycemia caused persistent damage to the vascular endothelium. Transient hyperglycemia directly upregulated DNMT1 expression, leading to the hypermethylation of Ang-1 and reduced Ang-1 expression, which, in turn, induced long-lasting activation of nuclear factor (NF)-{kappa}B and subsequent endothelial dysfunction. An in vivo study further showed that inhibition of DNMT1 promoted angiogenesis and accelerated diabetic wound healing by regulating the Ang-1/NF-{kappa}B signaling pathway. These results highlight the dramatic and long-lasting effects of transient hyperglycemic spikes on wound healing and suggest that DNMT1 is a novel target for diabetic vascular complications.

cell biology↗

bioRxiv2020

Integrated genomics and comprehensive validation reveal novel drivers of genomic evolution in esophageal adenocarcinoma

Identification of genes driving genomic evolution can provide novel targets for cancer treatment and prevention. Here we show identification of a genomic instability gene signature, using an integrated genomics approach. Elevated expression of this signature correlated with poor survival in esophageal adenocarcinoma (EAC) as well as three other human cancers. Knockout and overexpression screens confirmed the relevance of this signature to genomic instability. Indepth evaluation of TTK (a kinase), TPX2 (spindle assembly factor) and RAD54B (recombination protein) further confirmed their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrated that DNA damage and homologous recombination were common mechanisms of genomic instability induced by these genes. Consistently, a TTK inhibitor impaired EAC cell growth in vivo, and increased chemotherapy-induced cytotoxicity while inhibiting genomic instability in surviving cells. Thus inhibitors of TTK and other genes identified in this study have potential to inhibit/delay genomic evolution and tumor growth. Such inhibitors also have potential to increase chemotherapy-induced cytotoxicity while reducing its harmful genomic impact in EAC and possibly other cancers.

cancer biology↗

bioRxiv2020

Structural and biochemical characterization of nsp12-nsp7-nsp8 core polymerase complex from COVID-19 virus

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus. The viral polymerase is a promising antiviral target. However, the structure of COVID-19 virus polymerase is yet unknown. Here, we describe the near-atomic resolution structure of its core polymerase complex, consisting of nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases, and suggests the mechanism for activation by cofactors. Biochemical studies revealed reduced activity of the core polymerase complex and lower thermostability of individual subunits of COVID-19 virus as compared to that of SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate a well adaptation of COVID-19 virus towards humans with relatively lower body temperatures than the natural bat hosts.

microbiology↗

bioRxiv2020

Genome-wide identification, characterization and expression analysis of the monovalent cation-proton antiporter superfamily, and their function analysis in maize salt tolerance.

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and maize protoplasts transient expression showed that ZmNHX5 and ZmCHX17 localized to cell membrane system. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analyses of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.

plant biology↗

bioRxiv2020

Comparative morphological and transcriptomic analyses reveal novel chemosensory genes in the poultry red mite, Dermanyssus gallinae and knockdown by RNA interference

Detection of chemical cues via chemosensory receptor proteins are essential for most animals, and underlies critical behaviors, including location and discrimination of food resources, identification of sexual partners and avoidance of predators. The current knowledge of how chemical cues are detected is based primarily on data acquired from studies on insects, while our understanding of the molecular basis for chemoreception in acari, mites in particular, remains limited. The poultry red mite (PRM), Dermanyssus gallinae, is one of the most important blood-feeding ectoparasites of poultry. Unlike other ectoparasites on animals, PRM feeds mainly at night. During daytime, these animals hide themselves in crevices around the poultry house. The diversity in habitat usage, as well as the demonstrated host finding and avoidance behaviors suggest that PRM relies on their sense of smell to orchestrate complex behavioral decisions. Comparative transcriptome analyses revealed the presence of candidate variant ionotropic receptors (IRs), odorant binding proteins (OBPs), niemann-pick proteins type C2 (NPC2) and sensory neuron membrane proteins (SNMPs). Some of these proteins were highly and differentially expressed in the forelegs of PRM. Rhodopsin-like G protein-coupled receptors (GPCRs) were also identified, while insect-specific odorant receptors (ORs) and odorant co-receptors (ORcos) were not detected. Furthermore, using scanning electron microscopy (SEM), the tarsomeres of all legs pairs were shown to be equipped with sensilla chaetica with or without tip pores, while wall-pored olfactory sensilla chaetica were restricted to the distal-most tarsomeres of the forelegs. Further, using the conserved odorant binding protein (OBP) as a test case, the results showed that RNA interference (RNAi) can be induced in D. gallinae chemosensory tissues. This study is the first to describe the presence of chemosensory genes in any Dermanyssidae family. It is also the first report of chemosensory gene knockdown by RNAi in any mite species and demonstrate that their diminutive size, less than 1 mm, is not a major impediment when applying gene knockdown approaches. Our findings make a significant step forward in understanding the chemosensory abilities of D. gallinae.

molecular biology↗

bioRxiv2020

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAcope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Thus, we optimized the RNAscope detection method in plants to visualize the spatial expression and semi-quantification of target RNAs which can be employed in other plants such as the widely utilized model dicot plant Arabidopsis.

plant biology↗

bioRxiv2020

Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2

The burgeoning epidemic caused by novel coronavirus 2019 (2019-nCoV) is currently a global concern. Angiotensin-converting enzyme-2 (ACE2) is a receptor of 2019-nCoV spike 1 protein (S1) and mediates viral entry into host cells. Despite the abundance of ACE2 in small intestine, few digestive symptoms are observed in patients infected by 2019-nCoV. Herein, we investigated the interactions between ACE2 and human defensins (HDs) specifically secreted by intestinal Paneth cells. The lectin-like HD5, rather than HD6, bound ACE2 with a high affinity of 39.3 nM and weakened the subsequent recruitment of 2019-nCoV S1. The cloak of HD5 on the ligand-binding domain of ACE2 was confirmed by molecular dynamic simulation. A remarkable dose-dependent preventive effect of HD5 on 2019-nCoV S1 binding to intestinal epithelial cells was further evidenced by in vitro experiments. Our findings unmasked the innate defense function of lectin-like intestinal defensin against 2019-nCoV, which may provide new insights into the prevention and treatment of 2019-nCoV infection.

microbiology↗