The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Full neurological recovery from severe nonexertional heat stroke with multiple organ dysfunction: A case report.

BACKGROUND: We report a rare case of full neurological recovery from severe nonexertional heat stroke in a 67-year-old woman with an initial Glasgow Coma Scale of 3. This report raises awareness among doctors that when heatstroke is diagnosed, comprehensive treatment should be implemented as soon as possible. Moreover, targeted temperature management, combination therapy with hemodialysis and hemoperfusion, and hyperbaric oxygen therapy may alleviate multiorgan failure and prevent neurological sequelae caused by heatstroke. CASE SUMMARY: A previously healthy 67-year-old woman with an initial Glasgow Coma Scale of 3 was found lying prone on the road at noon on a summer day. Laboratory tests revealed multiorgan failure. As soon as heatstroke was diagnosed, comprehensive treatment was implemented. On hospital Day 3, the patient was extubated. Her initial Sequential Organ Failure Assessment score at hospitalization was 14 and decreased to 2 on hospital Day 4. On the seventh day following hospital admission, as the patient's general condition improved, the levels of laboratory test findings decreased rapidly. Finally, the patient gradually recovered with no other neurological symptoms (the Glasgow Coma Scale at discharge was 15, and her ability to walk independently was restored). CONCLUSION: This case demonstrated that targeted temperature management, combination therapy with hemodialysis and hemoperfusion, and hyperbaric oxygen therapy may alleviate multiorgan failure and prevent neurological sequelae caused by heatstroke.

Assembly and purification of AvrSr35-induced Sr35 resistosome and determination of its structure by cryo-EM.

Sr35, a coiled-coil nucleotide-binding leucine-rich repeat receptor (CC-NLR) from the wheat species Triticum monococcum can directly recognize the pathogen avirulence factor AvrSr35 and confers immunity against wheat stem rust caused by Puccinia graminis f.sp. tritici race Ug99. Assembly of a stable Sr35 resistosome induced by AvrSr35 in vitro is usually limited by protein expression and low assembly efficiency. Here, we describe the expression and purification of AvrSr35 and Sr35, in vitro assembly of Sr35 resistosome for structure determination by cryo-EM. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2022).

A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein.

The emerging SARS-CoV-2 variants, commonly with many mutations in S1 subunit of spike (S) protein are weakening the efficacy of the current vaccines and antibody therapeutics. This calls for the variant-proof SARS-CoV-2 vaccines targeting the more conserved regions in S protein. Here, we designed a recombinant subunit vaccine, HR121, targeting the conserved HR1 domain in S2 subunit of S protein. HR121 consisting of HR1-linker1-HR2-linker2-HR1, is conformationally and functionally analogous to the HR1 domain present in the fusion intermediate conformation of S2 subunit. Immunization with HR121 in rabbits and rhesus macaques elicited highly potent cross-neutralizing antibodies against SARS-CoV-2 and its variants, particularly Omicron sublineages. Vaccination with HR121 achieved near-full protections against prototype SARS-CoV-2 infection in hACE2 transgenic mice, Syrian golden hamsters and rhesus macaques, and effective protection against Omicron BA.2 infection in Syrian golden hamsters. This study demonstrates that HR121 is a promising candidate of variant-proof SARS-CoV-2 vaccine with a novel conserved target in the S2 subunit for application against current and future SARS-CoV-2 variants.

Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. The direct recognition mechanism of pathogen effectors by coiled-coil NLRs (CNLs) remains unclear. We demonstrate that the Triticum monococcum CNL Sr35 directly recognizes the pathogen effector AvrSr35 from Puccinia graminis f. sp. tritici and report a cryo-electron microscopy structure of Sr35 resistosome and a crystal structure of AvrSr35. We show that AvrSr35 forms homodimers that are disassociated into monomers upon direct recognition by the leucine-rich repeat domain of Sr35, which induces Sr35 resistosome assembly and the subsequent immune response. The first 20 amino-terminal residues of Sr35 are indispensable for immune signaling but not for plasma membrane association. Our findings reveal the direct recognition and activation mechanism of a plant CNL and provide insights into biochemical function of Sr35 resistosome.

Green Tea Polyphenol Epigallocatechin-3-Gallate Promotes Reendothelialization in Carotid Artery of Diabetic Rabbits by Reactivating Akt/eNOS Pathway.

Background: Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and has proven benefits on endothelial cells in diabetes. However, it remains unclear whether EGCG could improve function of late endothelial progenitor cells (L-EPCs) in diabetes. Methods: Thirty-six rabbits were randomized into six groups. Thirty diabetic rabbits were induced by a single dose of alloxan (100 mg/kg injection intraperitoneally). All of them were given intragastrically EGCG (50 mg/kg/day) or saline for 7 days after carotid injury. In autotransfusion experiment, L-EPCs were cultured with pre-treated EGCG (40 µM for 72 h) and then were injected into the site of injured vascular. Proliferation and migration of EGCG pre-treated L-EPCs in high glucose condition were assessed by EDU incorporation assay and modified Boyden chamber assay, respectively. The mRNA and protein expression of Akt-eNOS pathway were detected by real-time PCR and western blot. Results: Reendothelialization rate in injured carotid artery of diabetic rabbits was augmented in the EGCG group (50 mg/kg/d for 7 days) compared with the non-EGCG group (74.2 ± 4.6% vs. 25.6 ± 5.9%, P < 0.001). EGCG pre-treated L-EPCs autologous transfusion also accelerated the diabetic rabbits' carotid reendothelialization compared with the diabetic sham-operated group (65.6 ± 8.5% vs. 32.9 ± 5.0%, P = 0.011). In vitro studies showed, 40 µM EGCG treatment for 72 h recovered L-EPCs' proliferation and migration, as well as restored the phosphorylation level of Akt and eNOS blocked by high glucose condition. Conclusion: EGCG accelerated reendothelialization in diabetic rabbits after carotid injury in part by reactivating the Akt/eNOS pathway, which might contribute to recovering proliferation and migration of L-EPCs impaired by high glucose.

Mulberry granules protect against diabetic cardiomyopathy through the AMPK/Nrf2 pathway.

Mulberry granules (MLD) is a traditional Chinese medicine prescription that has been used in the treatment of diabetes for many years. Recently, we found that MLD protected the heart from diabetes-associated cardiomyopathy when it was used to treat diabetes. However, the beneficial effects and possible mechanism remain unknown. To elucidate these effects, an experimental myocardial ischemia/reperfusion (MI/R) injury model in diabetes rats was used in this study. Male C57BL/6 mice were injected with streptozotocin to induce diabetes. The mice were pretreated with MLD for one month, and then exposed to 30 min of ischemia followed by 24 h of reperfusion. Infarct size, heart function and various cytokines in the heart were assessed. Expression of AMP-activated protein kinase (AMPK) and nuclear factor erythroid 2­related factor 2 (Nrf2) were investigated by western blotting. In vitro, MLD significantly cleared oxygen-free radicals in DPPH and luminol chemiluminescence models. In vivo, fasting blood glucose, fasting blood insulin and lipids were significantly decreased by MLD. The results showed that MLD improved the cardiac function and decreased myocardial infarct size in the diabetic mice subjected to MI/R. In addition, upon pretreatment with MLD before MI/R treatment, GSH, SOD, CAT and GR were significantly increased in a dose-dependent manner. Pretreatment with MLD also significantly induced the expression of Nrf2, and the cardioprotective effects of MLD were abolished in Nrf2-knockout mice. Furthermore, we also found that AMPK increase is upstream and was required for Nrf2 activation mediated by MLD. In conclusion, MLD protects against diabetic-associated cardiomyopathy by suppressing oxidative stress induced by hyperglycemia and MI/R through the AMPK/Nrf2 signaling pathway.

[Effects of canopy density on the functional group of soil macro fauna in Pinus massoniana plantations]. / 马尾松人工林郁闭度对大型土壤动物功能群的影响.

In order to understand the effects of canopy density on the functional group characteristics of soil macrofauna in Pinus massoniana plantations, we divided the captured soil fauna into five types including xylophages, predators, saprophages, omnivores and fungal feeders. The results showed that 1) Saprozoic feeders had the highest percentage of total individuals, and the omnivores and xylophages occupied higher percentages of total taxa. 2) The individual and group number of the predators, and the group number of xylophages did not change significantly under 0.5-0.6 and then decreased significantly under 0.6-0.9 canopy density. 3) With the increasing canopy density, the individual an dgroup number of predators in litter layer decreased significantly, the saprozoic individual number in 5-10 cm soil layer represented irregular trends. The individual number of xylophage increased with the depth of soil, and the group number in litter layer, the individual and group number in 5-10 cm soil layer decreased significantly. 4) Pielou evenness of xylophage had no significant changes with the canopy density, all the other diversity index of xylophage and saprophage were various with the increasing canopy density. The predatory Simpson index was stable under 0.5-0.8, and then decreased significantly under 0.8-0.9 canopy density. 5) The CCA (canonical correlation analysis) indicated that soil bulk density and moisture content were the main environmental factors affecting functional groups of soil macro fauna. Moisture content greatly impacted on the number of saprophagous individuals. But xylophage and predators were mostly affected by soil bulk density, and the predatory Simpson index was mainly affected by soil pH value and total phosphorus. Our research indicated that the structure of soil macro faunal functional group under 0.7 canopy density was comparatively stable, which would facilitate the maintenance of soil fertility and ecological function in Pinus massoniana plantation.

[Experimental study on the retentive force of cobalt-chromium alloy, pure titanium and vitallium cast clasps in the simulated 3-year clinical use].

PURPOSE: To investigate the changes of retentive force of cobalt-chromium alloy, pure titanium and vitallium cast clasps in the simulated 3-year clinical use. METHODS: Fifteen metal abutment crowns made of No.QT800-2 nodular cast iron were used in the test. Five clasps from each of the following alloys: cobalt-chromium alloy, pure titanium and vitallium were fabricated. The undercut depth was 0.25 mm. A masticatory simulator was used to cycle the clasp on and off the metal abutment crown 5000 times, simulating 3-year clinical use. Retentive force was measured 11 times during this process. SPSS13.0 software package was used to analyze the results. Casting defects were observed using X-ray non destructive testing (X-ray NDT) before cyclic test. Surface characteristics were qualitatively evaluated using scanning electron microscope (SEM) before and after cyclic test. RESULTS: The results indicated that there were significant differences (P=0.000) in the retentive force of the 3 groups before and after the cyclic test. The highest retentive force was recorded in the vitallium clasps, and the lowest retentive force was measured in the pure titanium clasps. The results of X-ray NDT depicted the typical casting defect seen at the end of the connector. SEM examination revealed that no evidence of pores and cracks in the inner surfaces of the 3 groups was found before cyclic test. Wear was evident in the inner surfaces of the 3 groups but none of the clasps exhibited any evidence of cracks after cyclic test through SEM examination. CONCLUSIONS: In this in vitro test, vitallium clasps show the best retentive force in the 3 groups before and after 5000 cycles at 0.25 mm undercut depth. Cobalt-chromium alloy and vitallium clasps can maintain ideal retentive force at 0.25mm undercut depth in the long-term use. Wear may be one of the reasons for the loss of retentive force of clasps in the cyclic test.

[Computer aided design and computer aided manufacture of template for crown lengthening].

PURPOSE: To introduce a method applied in computer aided design and computer aided manufacture (CAD-CAM) of template for crown lengthening. METHODS: Point cloud data of dental stone model of the patient in the plan of crown lengthening surgery was obtained by laser scanning. The following processes were carried out, constructing 3-D curve of the gingiva, drawing template outline on the triangle mesh model, shelling it for 3-D model of template, and transferring the data to rapid prototyping equipment for manufacture. RESULTS: 3-D model of the template was preliminarily accomplished. The resin template was manufactured with rapid prototyping equipment. The fitness between resin template and plaster model was good. CONCLUSIONS: This method, as an integrated procedure including data acquisition, 3-D computer modeling and fabrication by rapid prototyping, is feasible to implement CAD-CAM of template for crown lengthening.

[Effects of ketamine on proliferation and apoptosis of pheochromocytoma cell].

OBJECTIVE: To explore the effect of ketamine on adrenal pheochromocytoma (PC12) cell proliferation inhibition and induction of apoptosis and its mechanism. METHODS: PC12 cells of rats were models for dopaminergic neuron. PC12 cells were cultured with ketamine at concentrations of 0.9, 1.2, 1.5, 1.8 and 2.1 mmol/L, respectively. The cell viability was measured by MTT method after incubation at 12, 24, 48 and 72h. Hoechst stain was used to observe the morphological changes of apoptosis. PC12 cells cultured after 48 h with different concentrations of ketamine were selected to detect apoptotic rate using flow cytometry and detect the expression of bax and bcl-2 proteins using Western blotting. RESULTS: For different concentrations of ketamine, vitality of PC12 cells significantly decreased with increase of the incubation time. Apoptosis was obviously observed using Hoechst staining. Flow cytometry showed that apoptosis rates significantly increased with increasing ketamine concentrations. CONCLUSION: Ketamine can inhibit the proliferation of PC12 cell by inducing apoptosis of the PC12 cell in a concentrations-dependent manner. The underlying mechanism may be related to promoting the expression of bax and inhibiting the expression of bcl-2 in the cells.

Stromal cell-derived factor 1alpha reduces senescence of endothelial progenitor subpopulation in lectin-binding and DiLDL-uptaking cell through telomerase activation and telomere elongation.

Recent studies have suggested that reduced endothelial progenitor subpopulation in lectin-binding and DiLDL-uptaking cell (EPC subpopulation) number and activity was associated with EPC subpopulation senescence that involved telomerase activity and telomere length. Stromal cell-derived factor-1alpha (SDF-1alpha) has been shown to augment a variety of cellular functions of EPC subpopulation and subsequently contribute to ischemic neovascularization. Therefore, we investigated whether SDF-1alpha might be able to prevent senescence of EPC subpopulation and also investigated the effects of SDF-1alpha on the telomerase activity and telomere length. EPC subpopulation were isolated from peripheral blood and characterized. After ex vivo prolonged cultivation, EPC subpopulation became senescent as determined by acidic beta-galactosidase staining. SDF-1alpha dose-dependently inhibited the onset of EPC subpopulation senescence. Moreover, SDF-1alpha increased proliferation and colony-forming activity of EPC subpopulation. SDF-1alpha also increased telomerase activity and telomere length, which was accompanied with upregulation of the catalytic subunit, telomerase reverse transcriptase (TERT). Whereas these effects of SDF-1alpha on telomerase activity and expression of hTERT mRNA were significantly attenuated by CXCR4-specific peptide antagonist (AMD3100) and phosphoinositide 3-kinase (PI3K) inhibitor (LY294002). In conclusions, SDF-1alpha delays the onset of EPC subpopulation senescence, which may be related to the activation of telomerase and elongation of telomere length. The inhibition of EPC subpopulation senescence and induction of EPC subpopulation proliferation by SDF-1alpha in vitro may importantly improve the functional activity of EPC subpopulation for potential cell therapy.

[Behavior study of ketamine-induced symptoms similar to schizophrenia in mice].

OBJECTIVE: To observe the symptoms similar to schizophrenia in mice after ketamine single or continuous injection and to evaluate the feasibility of schizophrenia model injected with different dose of ketamine. METHODS: A total of 40 male mice were randomly divided into 4 groups, which were injected intraperitoneally with physiological saline (control group), 25 mg/kg ketamine (low dose group), 50 mg/kg ketamine (middle dose group), and 100 mg/kg ketamine (high dose group) qd for 7 days continuously. The behavior changes of mice were observed. RESULTS: Hyperactivity, stereotyped behavior and ataxia (P < 0.01) were observed in high dose group after single injection. After continuous injection of ketamine for 7 days, the middle dose group showed hyperactivity, stereotyped behavior and ataxia (P < 0.05), stereotyped behavior and ataxia were more significant in high dose group (P < 0.01). CONCLUSION: Ketamine can induce the symptoms similar to schizophrenia in mice after single or continuous injection. The symptoms induced by high dose ketamine will be more prominent and stable after continuous injection.

Thymosin beta4 induces endothelial progenitor cell migration via PI3K/Akt/eNOS signal transduction pathway.

Thymosin beta4, a G-actin-sequestering peptide, has been shown to play an important role in cell migration. However, little is known about the effect of thymosin beta4 on circulating endothelial progenitor cell (EPC) directional migration, which is essential for EPC-mediated reendothelialization and neovascularization. In our study, using a transwell migration assay, we showed that thymosin beta4 induced EPC migration in a concentration-dependent manner. Western blot analysis revealed that treatment of EPCs with thymosin beta4 resulted in a time and concentration-dependent phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase (ERK)1/2. Functional analysis showed that thymosin beta4-induced EPC migration was blocked by phosphatidylinositol 3-kinase inhibitors (LY294002 or wortmannin) or eNOS inhibitor (Nomega-nitro-L-arginine methyl ester) but was not significantly attenuated by mitogen-activated protein kinase (MAPK)/ERK inhibitor (PD98059). These findings suggest that thymosin beta4 stimulates EPC directional migration via phosphatidylinositol 3-kinase/Akt/eNOS, rather than via MAPK/ERK signal transduction pathway.