Protein Labeling Facilitates the Understanding of Protein Corona Formation via Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy.

Once nanoparticles enter into the biological milieu, nanoparticle-biomacromolecule complexes, especially the protein corona, swiftly form, which cause obvious effects on the physicochemical properties of both nanoparticles and proteins. Here, the thermodynamic parameters of the interactions between water-soluble GSH-CdSe/ZnS core/shell quantum dots (GSH-QDs) and human serum albumin (HSA) were investigated with the aid of labeling fluorescence of HSA. It was proved that the labeling fluorescence originating from a fluorophore (BDP-CN for instance) could be used to investigate the interactions between QDs and HSA. Gel electrophoresis displayed that the binding ratio between HSA and QDs was ∼2:1 by direct visualization. Fluorescence resonance energy transfer (FRET) results indicated that the distance between the QDs and the fluorophore BDP-CN in HSA was 7.2 nm, which indicated that the distance from the fluorophore to the surface of the QDs was ∼4.8 nm. Fluorescence correlation spectroscopy (FCS) results showed that HSA formed a monolayer of a protein corona with a thickness of 5.5 nm. According to the spatial structure of HSA, we could speculate that the binding site of QDs was located at the side edge (not the triangular plane) of HSA with an equilateral triangular prism. The elaboration of the thermodynamic parameters, binding ratio, and interaction orientation will highly improve the fundamental understanding of the formation of protein corona. This work has guiding significance for the exploration of the interactions between proteins and nanomaterials.

Stevioside attenuates bleomycin-induced pulmonary fibrosis by activating the Nrf2 pathway and inhibiting the NF-κB and TGF-ß1/Smad2/3 pathways.

Objective: This study aimed to investigate the effects of stevioside (STE) on pulmonary fibrosis (PF) and the potential mechanisms. Methods: In this study, a mouse model of PF was established by a single intratracheal injection of bleomycin (BLM, 3 mg/kg). The experiment consisted of four groups: control group, BLM group, and STE treatment groups (STE 50 and 100 mg/kg). ELISA and biochemical tests were conducted to determine the levels of TNF-α, IL-1ß, IL-6, NO, hydroxyproline (HYP), SOD, GSH, and MDA. Histopathological changes and collagen deposition in lung tissues were observed by HE and Masson staining. Immunohistochemistry was performed to determine the levels of collagen I-, collagen III-, TGF-ß1- and p-Smad2/3-positive cells. Western blot analysis was used to measure the expression of epithelial-mesenchymal transition (EMT) markers, including α-SMA, vimentin, E-cadherin, and ZO-1, as well as proteins related to the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, nuclear transcription factor-κB (NF-κB) pathway, and TGF-ß1/Smad2/3 pathway in lung tissues. Results: STE significantly alleviated BLM-induced body weight loss and lung injury in mice, decreased HYP levels, and reduced the levels of collagen I- and collagen III-positive cells, thereby decreasing extracellular matrix (ECM) deposition. Moreover, STE markedly improved oxidative stress (MDA levels were decreased, while SOD and GSH activity were enhanced), the inflammatory response (the levels of TNF-α, IL-1ß, IL-6, and NO were reduced), and EMT (the expression of α-SMA and vimentin was downregulated, and the expression of E-cadherin and ZO-1 was upregulated). Further mechanistic analysis revealed that STE could activate the Nrf2 pathway and inhibit the NF-κB and TGF-ß1/Smad2/3 pathways. Conclusion: STE may alleviate oxidative stress by activating the Nrf2 pathway, suppress the inflammatory response by downregulating the NF-κB pathway, and inhibit EMT progression by blocking the TGF-ß1/Smad2/3 pathway, thereby improving BLM-induced PF.

Genome-wide identification of the expansin gene family reveals that expansin genes are involved in fibre cell growth in cotton.

BACKGROUND: Expansins (EXPs), a group of proteins that loosen plant cell walls and cellulosic materials, are involved in regulating cell growth and diverse developmental processes in plants. However, the biological functions of this gene family in cotton are still unknown. RESULTS: In this paper, we identified a total of 93 expansin genes in Gossypium hirsutum. These genes were classified into four subfamilies, including 67 GhEXPAs, 8 GhEXPBs, 6 GhEXLAs, and 12 GhEXLBs, and divided into 15 subgroups. The 93 expansin genes are distributed over 24 chromosomes, excluding Ghir_A02 and Ghir_D06. All GhEXP genes contain multiple exons, and each GhEXP protein has multiple conserved motifs. Transcript profiling and qPCR analysis revealed that the expansin genes have distinct expression patterns among different stages of cotton fibre development. Among them, 3 genes (GhEXPA4o, GhEXPA1A, and GhEXPA8h) were highly expressed in the initiation stage, 9 genes (GhEXPA4a, GhEXPA13a, GhEXPA4f, GhEXPA4q, GhEXPA8f, GhEXPA2, GhEXPA8g, GhEXPA8a, and GhEXPA4n) had high expression during the fast elongation stage, and GhEXLA1c and GhEXLA1f were preferentially expressed in the transition stage of fibre development. CONCLUSIONS: Our results provide a solid basis for further elucidation of the biological functions of expansin genes in relation to cotton fibre development and valuable genetic resources for future crop improvement.

[Rutaecarpine protects against bleomycin-induced pulmonary fibrosis through inhibiting Notch1/eIF3a signaling pathway in rats].

To investigate whether the protection of rutaecarpine against bleomycin-induced pulmonary fibrosis is mediated by inhibiting Notch1/eukaryotic initiation factor 3a (eIF3a) signaling pathway, and whether these effects are related to the synthesis and release of calcitonin gene-related peptide (CGRP) and inhibition of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, male Sprague-Dawley rats were randomly divided into five groups (n=12), respectively, Control group, bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group and capsaicin plus rutaecarpine (300 mg·kg⁻¹) group. Bleomycin (5 mg·kg⁻¹) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg·kg⁻¹) by subcutaneous injections 1 days before and 7, 14, 21 days after induce pulmonary fibrosis rat model to deplete endogenous CGRP. At the end of experiments, blood was collected from carotid artery to determinate the plasma levels of CGRP by ELISA. Pulmonary tissue change was observed by HE staining. Masson's trichrome stain was used to demonstration collagen deposition. The collagen I expression in pulmonary tissue was measured by immunohistochemisty. The expression of CGRP, Notch1, eIF3a, collagen I, vimentin, alpha-smooth muscle actin (α-SMA), E-cadherin and zonula occludens-1 (ZO-1) was detected by qPCR or Western blot. Compared with the control group, the pulmonary tissue of the bleomycin group showed significant fibrosis, including significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, and concomitantly with the decrease in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was decreased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was increased in bleomycin group (P<0.05 or P<0.01). Compared with the bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group significantly reduced bleomycin-induced pulmonary injury concomitantly with the increase in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was increased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was decreased by rutaecarpine treatment (P<0.05 or P<0.01). All these effects of rutaecarpine were abolished by capsaicin.These results suggest that rutaecarpine protects against bleomycin-induced pulmonary fibrosis by inhibiting Notch1/eIF3a signaling pathway, alleviating EMT process, which is related to the increased synthesis and release of CGRP.

miR-200b-containing microvesicles attenuate experimental colitis associated intestinal fibrosis by inhibiting epithelial-mesenchymal transition.

BACKGROUND AND AIM: Epithelial-mesenchymal transition (EMT), characterized by the decrease of E-cadherin (E-Cad) and increase in vimentin and alpha-smooth muscle actin (α-SMA), was demonstrated to participate in inflammatory bowel disease-related fibrosis. miR-200b plays an anti-fibrosis role in inhibiting EMT by targeting ZEB1 and ZEB2. But the stability of exogenous miR-200b in blood limits its application. Microvesicles (MVs), which can transfer miRNAs among cells and prevent them from degradation, may provide an excellent transport system for the delivery of miR-200b in the treatment of fibrosis. METHODS: Bone marrow mesenchymal stem cells (BMSCs) were transfected with lentivirus to overexpress miR-200b. The MVs packaged with miRNA-200b were harvested for the anti-fibrotic treatment using in vitro (transforming growth factor beta 1-mediated EMT in intestinal epithelial cells: IEC-6) and in vivo (TNBS-induced intestinal fibrosis in rats) models. The pathological morphology was observed, and the fibrosis related proteins, such as E-Cad, vimentin, α-SMA, ZEB1, and ZEB2, were detected. RESULTS: MiR-200b-MVs would significantly reverse the morphology in TGF-ß1-treated IEC-6 cells and improve the TNBS-induced colon fibrosis histologically. The treatment of miR-200b-MVs increased miR-200b levels both in the IEC-6 cells and colon, resulting in a significant prevention EMT and alleviation of fibrosis. The expression of E-Cad was increased, and the expressions of vimentin and α-SMA were decreased. ZBE1 and ZEB2, the targets of miR-200b, were also decreased. CONCLUSIONS: miR-200b could be transferred from genetically modified BMSCs to the target cells or tissue by MVs. The mechanisms of miR-200b-MVs in inhibiting colonic fibrosis were related to suppressing the development of EMT by targeting ZEB1and ZEB2.

A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

Regulation of the Pacemaker Activity of Colonic Interstitial Cells of Cajal by Protease-Activated Receptors: Involvement of Hyperpolarization-Activated Cyclic Nucleotide Channels.

BACKGROUND AND PURPOSE: The exact mechanism of protease-activated receptors (PARs) on pacemaker activity of interstitial cells of Cajal (ICCs) has not been reported. We investigated the effects on pacemaker activity by the activation of PARs and its signal mechanisms in colonic ICCs. METHODS: The whole-cell patch-clamp technique, RT-PCR and Ca2+ imaging were used in cultured ICCs from mouse colon. RESULTS: PAR-1 and PAR-2 were expressed in Ano-1 positive ICCs. TFLLR-NH2 (a PAR-1 agonist) and trypsin (a PAR-2 agonist) depolarized the membrane and increased the pacemaker potential frequency. U-73122 (a phospholipase C (PLC) inhibitor) and thapsigargin (a Ca2+ ATPase inhibitor) suppressed the TFLLR-NH2- and trypsin-induced effects on pacemaker potential. TFLLR-NH2 and trypsin also increased intracellular Ca2+ ([Ca2+]i) intensity with increasing of Ca2+ oscillations. Genistein (a tyrosine kinase inhibitor), SP600125 (a JNK inhibitor), CsCl, ZD7288, clonidine (hyperpolarization-activated cyclic nucleotide (HCN) channel blockers), SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) suppressed the increased pacemaker potential frequency without effects on depolarization of the membrane induced by TFLLR-NH2 and trypsin. CONCLUSION: These results suggest that activation of PAR-1 and PAR-2 modulates the pacemaker activity of colonic ICCs through the PLC-dependent [Ca2+]i release pathway. The increased pacemaker potential frequency by PAR-1 and PAR-2 was also dependent on tyrosine kinase, JNK, and HCN activation.

Fluoxetine mitigates depressive-like behaviors in mice via anti-inflammation and enhancing neuroplasticity.

Neuroplasticity and inflammation represent a common final pathway for effective antidepressant treatment. SSRIs are the most commonly prescribed medications for depression and have demonstrated efficacy in reducing depressive symptoms. However, the precise impact of SSRIs on neuroplasticity and inflammation remains unclear. In this study, we aimed to investigate the influence of long-term treatment with SSRIs on hippocampal neuron, inflammation, synaptic function and morphology. Our findings revealed that fluoxetine treatment significantly alleviated behavioral despair, anhedonia, and anxiety in reserpine-treated mice. Moreover, fluoxetine mitigated hippocampal neuron impairment, inhibited inflammatory release, and increased the expression of synaptic proteins markers (SYP and PSD95) in mice. Notably, fluoxetine also suppressed reserpine-induced synapse loss in the hippocampus. Based on these results, fluoxetine has been demonstrated effectively to ameliorate depressive mood and cognitive dysfunction, possibly through the enhancement of synaptic plasticity. Overall, our study contributes to a further understanding of the mechanisms underlying the therapeutic effects of fluoxetine and its potential role in improving depressive symptoms and cognitive impairments.

Nutritional status and prognostic factors for mortality in patients admitted to emergency department observation units: a national multi-center study in China.

BACKGROUND: Nutritional risk is common among patients admitted to the emergency department and is associated with adverse clinical outcomes. Despite its large population, few comprehensive studies have been conducted in China concerning the nutritional status of patients admitted to emergency department observation units (EDOUs). METHODS: Patients admitted to EDOUs of 90 tertiary hospitals in China between June 2020 and December 2020 were enrolled. Demographic information, laboratory parameters, nutritional support therapies, and 28-day mortality were recorded. Risk factors for mortality were examined using multi-variate-adjusted logistic regression analysis. Receiver operating characteristic (ROC) curves for each predictor of mortality were plotted, and the area under the ROC (AUROC) curves was compared. RESULTS: A total of 2,005 eligible patients were finally enrolled. At the 28-day follow-up, 1,911 patients survived, and 94 died. The group with a Nutritional Risk Screening 2002 (NRS 2002) score of 3-4 points was the largest (52.01%). The number of patients receiving oral nutritional supplements, enteral nutrition (EN), parenteral nutrition (PN), and the combination of EN and PN was 425, 314, 853, and 413, respectively. Among the total, 77.55% of patients had nutritional risk (NRS 2002 ≥3). The proportion of patients with high nutritional risk (NRS2002≥5) in the age group >80 years was significantly higher than that in the age group 66-80 years (29.00% vs. 23.93%, P=0.032), but not significantly higher than that in the age group 18-65 years (29.00% vs. 26.54%, P=0.449). Logistic regression analysis revealed that heart failure (odds ratio [OR] 1.856, 95% confidence interval [CI] 1.087-3.167, P=0.023), consciousness (OR 2.967, 95% CI 1.894-4.648, P<0.001), Acute Physiology and Chronic Health Evaluation II (APACHE II) score (OR 1.037, 95% CI 1.017-1.058, P<0.001), NRS 2002 score (OR 1.286, 95% CI 1.115-1.483, P=0.001), and Mini Nutritional Assessment-Short Form score (OR 0.946, 95% CI 0.898-0.997, P=0.039) were all independent risk factors for 28-day mortality. APACHE II and NRS 2002 scores were superior to other predictors according to the comparison of AUROC. CONCLUSIONS: Nutritional risk is prevalent among older patients in EDOUs in China. APACHE II and NRS 2002 scores are important risk factors for mortality in patients admitted to the EDOU. Timely and appropriate nutritional screening and support measures are critical to reduce patients' length of hospital stay and mortality.

Erythronate utilization activates VdtR regulating its metabolism to promote Brucella proliferation, inducing abortion in mice.

Brucella is a facultative intracellular pathogen that preferentially colonizes reproductive organs and utilizes erythritol as a preferred carbon source for its survival and proliferation. In this study, we identified a virulence-related DeoR-family transcriptional regulator (VdtR) and an erythronate metabolic pathway responsible for four-carbon acid sugar metabolism of D-erythronate and L-threonate in Brucella. We found that VdtR plays an important role in Brucella intracellular survival and trafficking to the endoplasmic reticulum in RAW 264.7 macrophages and in virulence in a mouse model. More importantly, we found that VdtR negatively regulates the erythronate metabolic pathway to promote extracellular proliferation of Brucella, depending on utilization of D-erythronate, an oxidative product of erythritol in the host. In a pregnant mouse model, the erythronate metabolic pathway was shown to cooperate with erythritol metabolism and play a crucial role in Brucella proliferation in the placenta, inducing placentitis and finally resulting in abortion or stillbirth. Our results demonstrate that, in addition to erythritol, erythronate is a preferred carbon source for Brucella utilization to promote its extracellular proliferation. This discovery updates the information on the preferential colonization of reproductive organs by Brucella and provides a novel insight into the Brucella-associated induction of abortion in pregnant animals. IMPORTANCE Brucella is an intracellular parasitic bacterium causing zoonosis, which is distributed worldwide and mainly characterized by reproductive disorders. Erythritol is found in allantoic fluid, chorion, and placenta of aborted animals, preferentially utilized by Brucella to cause infertility and abortion. However, the erythritol metabolism-defected mutant was unable to function as a vaccine strain due to its residual virulence. Here, we found that erythronate, an oxidative product of erythritol in the host, was also preferentially utilized by Brucella relying on the function of a deoxyribonucleoside regulator-family transcriptional regulator VdtR. Erythronate utilization activates VdtR regulation of the erythronate metabolic pathway to promote Brucella extracellular proliferation, inducing placentitis/abortion in mice. Double mutations on Brucella erythritol and D-erythronate metabolisms significantly reduced bacterial virulence. This study revealed a novel mechanism of Brucella infection-induced abortion, thus providing a new clue for the study of safer Brucella attenuated vaccines.

Action of lipopolysaccharide on interstitial cells of cajal from mouse small intestine.

BACKGROUND AND PURPOSE: Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs. METHODS: We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine. RESULTS: LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4(+/+) mice was not present in TLR4(-/-) mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K(+) channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E(2) (PGE(2))-EP(2) receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs. CONCLUSIONS: LPS can activate ICCs to release NO and PGE(2) through TLR4 activation. The released NO and PGE(2) inhibit pacemaker currents by activating ATP-sensitive K(+) channels. The LPS actions are mediated by tyrosine kinase signaling pathways.

Brucella induces heme oxygenase-1 expression to promote its infection.

Brucellosis is a zoonotic and contagious infectious disease caused by Brucella spp, which causes substantial economic losses to animal husbandry and leads to severe public health problems. Brucella have evolved multiple strategies to escape host immunity and survive within host cells. Elucidating the immune evasion strategies during Brucella infection will facilitate the control of brucellosis. The host enzyme, heme oxygenase-1 (HO-1), is a multifunctional protein that functions during inflammatory diseases and microbial infections. However, how HO-1 functions during Brucella infection is rarely studied. In this study, we evaluated the role of HO-1 during Brucella infection. We found that Brucella infection induced HO-1 expression in macrophages. We further showed that HO-1 was regulated by PI3K, AMPK kinase, and nuclear erythroid-related factor 2 (Nrf2) in macrophages. Interestingly, knocking out HO-1 or inhibiting the activity of HO-1 significantly decreased Brucella intracellular growth. Inducing the expression of HO-1 by treatment with CoPP promoted Brucella intracellular growth. Mechanistic analyses indicated that the effect of HO-1 was not meditated by HO-1 metabolites, but by decreasing the production of reactive oxygen species (ROS), TNF-α, and IL-1ß. Moreover, Brucella induced HO-1 expression in bone marrow-derived macrophages (BMDMs) and mice. When the expression of HO-1 was knocked down in BMDMs, the intracellular survival of Brucella was reduced. Furthermore, the induction of HO-1 by CoPP significantly increased bacterial loads in vivo. Thus, we demonstrated that Brucella induced HO-1 expression to promote its survival and growth in vitro and in vivo. This study also identified HO-1 as a novel innate immune evasion factor during Brucella infection.

A homolog of low molecular weight protein tyrosine phosphatase isolated from Brucella melitensis displays an acidic dual specific phosphatase activity, nonessential for bacterial resistance to bactericidal factors and virulence.

Brucellosis is a bacterial infectious zoonosis which is spread worldwide, caused by Brucella, with infertility and abortion in domestic animals. Protein-tyrosine phosphatase (PTPs) have been discovered in many kinds of bacterial species, which play crucial roles in many aspects, such as bacterial physiology and virulence. However, no PTPs have been identified in Brucella to date. Here, we identified a novel gene BM28_RS15985 in Brucella melitensis that encodes a homolog of a low weight molecular PTP. Enzyme activity analysis showed that this PTP is a dual specific phosphatase, removing phosphate group from phosphotyrosine and phosphoserine/phosphothreonine peptides, which was designated as Dsp1. The optimal pH of the Dsp1 enzyme activity were 5.5, suggesting that the Dsp1 is an acidic phosphatase, and the optimal reaction temperature of the Dsp1 was 35.0 °C. Besides, the Michaelis constant and maximum reaction velocity of the Dsp1 were 40.17 mM and 24.33 nM/min/mg, respectively. In further study, we investigated the role of Dsp1 in B. melitensis phenotype and virulence. Growth curve and resistance test exhibited that the dsp1 had no role in Brucella growth and resisting bactericidal factors. Cell and animal infection experiment showed that the dsp1 deletion did not affect the intracellular survival and virulence of B. melitensis. In summary, we identified a novel acidic dual specific phosphatase in B. melitensis and evaluated its characteristics of the enzyme activity, this study will expand the understanding of Brucella phosphatase.

COVID-19 infection with keratitis as the first clinical manifestation.

AIM: To report a case which keratitis is the first clinical manifestation of COVID-19 that occurred 3d earlier than the common COVID-19 symptoms. METHODS: Regular slit lamp examination, corneal scraping test, and chest computed tomography (CT) were performed for patients with COVID-19 infection. The ophthalmologic treatment included ganciclovir eye drop (50 mg/mL, 6 times/d). The treatment for diarrhea included Guifu Lizhong pills (TID). The antiviral therapy consisted of oseltamivir (75 mg capsule Q12H); therapy preventing bacterial infection consisted of azithromycin (250 mg tablet QD) and moxifloxacin (0.4 g tablet Q12H); and therapy for cough relief and fever prevention consisted of Chinese herbal decoction. RESULTS: A 35-year-old male suddenly suffered pain, photophobia, and tears in his right eye for one day without systemic COVID-19 symptoms. Patient was diagnosed with keratitis, which was seemingly different from common keratitis. Ganciclovir eye drop was initiated. The corneal scraping test for COVID-19 was positive. The chest CT images were abnormal confirming the diagnosis of COVID-19 infection. The antiviral and antibacterial therapies were initiated. Chinese herbal therapy was used for cough relief and fever prevention. After roughly two weeks, patient recovered from COVID-19. CONCLUSION: A new type of keratitis, atypical keratitis, is a clinical manifestation of COVID-19, and this clinical manifestation could appear 3d earlier than fever and cough. The earlier a COVID-19 clinical manifestation is identified, the earlier can a patient be directed to stay at home, and significantly fewer people would be infected.

The novel LysR-family transcriptional regulator BvtR is involved in the resistance of Brucella abortus to nitrosative stress, detergents and virulence through the genetic regulation of diverse pathways.

Brucella is a facultative intracellular bacterium lacking classical virulence factors; its virulence instead depends on its ability to invade and proliferate within host cells. After entering cells, Brucella rapidly modulates the expression of a series of genes involved in metabolism and immune evasion. Here, a novel LysR-family transcriptional regulator, designated Brucellavirulence-related transcriptional regulator (BvtR), was found to be associated with Brucella abortus virulence. We first successfully constructed a BvtR mutant, ΔbvtR, and a complemented strain, ΔbvtR-Com. Subsequently, we performed cell infection experiments, which indicated that the ΔbvtR strain exhibited similar adhesion, invasion and survival within HeLa cells or RAW264.7 macrophages to those of the wild-type strain. In stress resistance tests, the ΔbvtR strain showed enhanced sensitivity to sodium nitroprusside and sodium dodecyl sulfate, but not to hydrogen peroxide, cumene hydroperoxide, polymyxin B and natural serum. Mouse infection experiments indicated that the virulence of the ΔbvtR strain significantly decreased at 4 weeks post-infection. Finally, we analyzed differentially expressed genes regulated by BvtR with RNA-seq, COG classification and KEGG pathway analysis. Nitrogen metabolism, siderophore biosynthesis and oligopeptide transport were found to be the predominantly altered functions, and key metabolic and regulatory networks were delineated in the ΔbvtR mutant. Thus, we identified a novel Brucella virulence-related regulator, BvtR, and demonstrated that BvtR regulation affects Brucella resistance to killing by sodium nitroprusside and sodium dodecyl sulfate. The differentially expressed genes responding to BvtR are involved in diverse functions or pathways in Brucella, thus, suggesting the breadth of BvtR's regulatory functions. This study provides novel clues regarding Brucella pathogenesis.

Population dynamics of Phaius flavus in southeast China: Reproductive strategies and plants conservation.

Because of species diversity and troubling conservation status in the wild, Orchidaceae has been one of the taxa with most concern in population ecological research for a long time. Although Orchidaceae is a group with high adaptability, they have become endangered for complex and various reasons such as the germination? difficulty and habitat loss, which makes it difficult to develop an accurate protection strategy. Phaius flavus is a terrestrial orchid which used to be widely distributed in central and southern Asia; however, large populations are difficult to find in the wild. Thus, the aim of this study was to provide a new perspective for conserving endangered P. flavus by investigating the mechanisms of its population decline; we established time-specific life and fertility tables, age pyramids, survival curves, and mortality curves for this plant and then conducted Leslie matrix model. We found that both of the populations from Wuhu Mount (WM) and Luohan Mount (LM) showed declining trends and exhibited pot-shaped age pyramids, low net reproductive rates, and negative intrinsic growth rates. The population from the Beikengding Mount (BM) showed a stable status with a bell-shaped age pyramid. However, it has a significant risk of decline because of the low net reproductive rate and intrinsic growth rate. This study use time-specific life and fertility tables, age pyramids, survival curves, and mortality curves, showed that the population decline of P. flavus could be attributed to 1) the shortage of seedlings caused by the low germination rate in the wild and 2) the loss of adult individuals caused by anthropogenic disturbances. To protect this species from extinction in these areas, we suggest that human activities in these habitats should be strictly forbidden and ex situ conservation of this plant in botanical gardens is also necessary.

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium-wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium-wheat interactions.

Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

[Study on the extraction of total flavonoids from Mentha spicata by ultrasonic method].

The optimal conditions of total flavonoids extraction from Mentha spicata were identified by orthogonal test. The results showed that the ethanol concentration and the ultrasonic time had significant effect on the extraction of total flavonoids and the optimum conditions of extraction were A3B3C1D1, which were as follows: the ethanol concentration was 50%, the ultrasonic time was 40 min, the solid-liquid ratio was 1 g: 50 mL and the temperature of extraction was 60 degrees C, On the basis of the extracting method above, the content of total flavonoids in Mentha spicata was 0.0951 mg/mg. The linear equation was A = 11.51143C - 0.00029, r = 0.9999. This method is simple, quick and accurate with good prosperity.

[Effect of calcium on proliferation, migration and osteogenic differentiation of human dental follicle cells].

PURPOSE: To determine the role of Ca2+ in proliferation,migration and osteogenic differentiation of human dental follicle cells(hDFCs). METHODS: hDFCs were isolated and cultured. The source of hDFCs was detected by immunofluorescence staining. Osteogenesis and adipogenic differentiation of hDFCs was detected by alizarin red staining and oil red O staining, to identify its multi-directional differentiation ability. A series of Ca2+ solutions with different concentrations was prepared, CCK8 assay was used to detect the proliferative abilities at 1, 3, 5, and 7 d；migratory ability of 24 h was detected by Transwell assay. Calcium nodules were detected by semiquantitative analysis of alizarin red staining. mRNA expression of osteogenic differentiation related genes was examined by real-time quantitative polymerase chain reaction (RT-qPCR).Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Compared with the control group, 3,4 and 5 mmol/L Ca2+ significantly promoted proliferation of hDFCs at 3, 5 and 7 d (P<0.05). 3, 4, 5 and 6 mmol/L Ca2+ significantly promoted the migration of hDFCs at 24 h(P<0.01). High concentration of Ca2+ had no significant effect on its proliferation and migration. The results of alizarin red staining showed that when Ca2+ concentration reached 4 mmol/L, formation of mineralized nodules were increased(P<0.01), and Ca2+ concentration-dependent. RT-qPCR results showed that Ca2+ up-regulated the expression of RUNX2 and OCN in osteogenic differentiation genes (P<0.01). CONCLUSIONS: Low Ca2+ concentration is beneficial to proliferation and migration, and high Ca2+ concentration is beneficial to osteogenic differentiation of human dental follicle cells.