A bimetallic peroxidase-mimicking nanozyme with antifouling property for construction of sensor array to identify phosphoproteins and diagnose cancers.

Protein phosphorylation is a significant post-translational modification that plays a decisive role in the occurrence and development of diseases. However, the rapid and accurate identification of phosphoproteins remains challenging. Herein, a high-throughput sensor array has been constructed based on a magnetic bimetallic nanozyme (Fe3O4@ZNP@UiO-66) for the identification and discrimination of phosphoproteins. Attributing to the formation of Fe-Zr bimetallic dual active centers, the as-prepared Fe3O4@ZNP@UiO-66 exhibits enhanced peroxidase-mimicking catalytic activity, which promotes the electron transfer from Zr center to Fe(II)/Fe(III). The catalytic activity of Fe3O4@ZNP@UiO-66 can be selectively inhibited by phosphoproteins due to the strong interaction between phosphate groups and Zr centers, as well as the ultra-robust antifouling capability of zwitterionic dopamine nanoparticle (ZNP). Considering the diverse binding affinities between various proteins with the nanozyme, the catalytic activity of Fe3O4@ZNP@UiO-66 can be changed to various degree, leading to the different absorption responses at 420 nm in the hydrogen peroxide (H2O2) - 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) system. By simply extracting different absorbance intensities at various time points, a sensor array based on reaction kinetics for the discrimination of phosphoproteins from other proteins is constructed through linear discriminant analysis (LDA). Besides, the quantitative determination of phosphoproteins and identification of protein mixtures have been realized. Further, based on the differential level of phosphoproteins in cells, the differentiation of cancer cells from normal cells can also be implemented by utilizing the proposed sensor array, showing great potential in disease diagnosis.

Predicting the geographical distribution and niche characteristics of Cotoneaster multiflorus based on future climate change.

Introduction: Arid and semi-arid regions are climate-sensitive areas, which account for about 40% of the world's land surface area. Future environment change will impact the environment of these area, resulting in a sharp expansion of arid and semi-arid regions. Cotoneaster multiflorus is a multi-functional tree species with extreme cold, drought and barren resistance, as well as ornamental and medicinal functions. It was found to be one of the most important tree species for ecological restoration in arid and semi-arid areas. However, bioclimatic factors play an important role in the growth, development and distribution of plants. Therefore, exploring the response pattern and ecological adaptability of C. multiflorus to future climate change is important for the long-term ecological restoration of C. multiflorus in arid and semi-arid areas. Methods: In this study, we predicted the potential distribution of C. multiflorus in China under different climate scenarios based on the MaxEnt 2.0 model, and discussed its adaptability and the major factors affecting its geographical distribution. Results: The major factors that explained the geographical distribution of C. multiflorus were Annual precipitation (Bio12), Min air temperature of the coldest month (Bio6), and Mean air temperature of the coldest quarter (Bio11). However, C. multiflorus could thrive in environments where Annual precipitation (Bio12) >150 mm, Min air temperature of the coldest month (Bio6) > -42.5°C, and Mean air temperature of the coldest quarter (Bio11) > -20°C, showcasing its characteristics of cold and drought tolerance. Under different future climate scenarios, the total suitable area for C. multiflorus ranged from 411.199×104 km² to 470.191×104 km², which was 0.8~6.14 percentage points higher than the current total suitable area. Additionally, it would further shift towards higher latitude. Discussion: The MaxEnt 2.0 model predicted the potential distribution pattern of C. multiflorus in the context of future climate change, and identified its ecological adaptability and the main climatic factors affecting its distribution. This study provides an important theoretical basis for natural vegetation restoration in arid and semi-arid areas.

Fasting alleviates metabolic alterations in mice with propionyl-CoA carboxylase deficiency due to Pcca mutation.

Propionic acidemia (PA), resulting from Pcca or Pccb gene mutations, impairs propionyl-CoA metabolism and induces metabolic alterations. While speculation exists that fasting might exacerbate metabolic crises in PA patients by accelerating the breakdown of odd-chain fatty acids and amino acids into propionyl-CoA, direct evidence is lacking. Our investigation into the metabolic effects of fasting in Pcca-/-(A138T) mice, a PA model, reveals surprising outcomes. Propionylcarnitine, a PA biomarker, decreases during fasting, along with the C3/C2 (propionylcarnitine/acetylcarnitine) ratio, ammonia, and methylcitrate. Although moderate amino acid catabolism to propionyl-CoA occurs with a 23-h fasting, a significant reduction in microbiome-produced propionate and increased fatty acid oxidation mitigate metabolic alterations by decreasing propionyl-CoA synthesis and enhancing acetyl-CoA synthesis. Fasting-induced gluconeogenesis further facilitates propionyl-CoA catabolism without changing propionyl-CoA carboxylase activity. These findings suggest that fasting may alleviate metabolic alterations in Pcca-/-(A138T) mice, prompting the need for clinical evaluation of its potential impact on PA patients.

EGCG suppresses PD-1 expression of T cells via inhibiting NF-κB phosphorylation and nuclear translocation.

Epigallocatechin-3-gallate (EGCG) is an important tea polyphenol with anti-tumor potential. Our previous studies revealed that EGCG was a promising immune checkpoint inhibitor (ICI) as it could downregulate expression of programmed cell death 1 ligand 1 (PD-L1) in tumor cells, thereby resulting tumor killing effect. In particular, EGCG can effectively avoid the inflammatory storm caused by anti-tumor therapy, which is a healthy green capacity absent from many ICIs. However, the relationship between EGCG and programmed cell death 1 (PD-1) of T cells remains unclear. In this work, we explored the effect of EGCG on T cells and found that EGCG suppressed PD-1 via inhibiting NF-κB phosphorylation and nuclear translocation. Furtherly, the capability of EGCG was confirmed in tumor-bearing mice to inhibit PD-1 expression in T cells and enhance apoptosis in tumor cells. These results implied that EGCG could inhibit the expression of PD-1 in T cells, thereby promoting anti-tumor effects of T cells. EGCG will be a promising candidate in anti-tumor therapy.

BCAA-nitrogen flux in brown fat controls metabolic health independent of thermogenesis.

Brown adipose tissue (BAT) is best known for thermogenesis. Rodent studies demonstrated that enhanced BAT thermogenesis is tightly associated with increased energy expenditure, reduced body weight, and improved glucose homeostasis. However, human BAT is protective against type 2 diabetes, independent of body weight. The mechanism underlying this dissociation remains unclear. Here, we report that impaired mitochondrial catabolism of branched-chain amino acids (BCAAs) in BAT, by deleting mitochondrial BCAA carriers (MBCs), caused systemic insulin resistance without affecting energy expenditure and body weight. Brown adipocytes catabolized BCAA in the mitochondria as nitrogen donors for the biosynthesis of non-essential amino acids and glutathione. Impaired mitochondrial BCAA-nitrogen flux in BAT resulted in increased oxidative stress, decreased hepatic insulin signaling, and decreased circulating BCAA-derived metabolites. A high-fat diet attenuated BCAA-nitrogen flux and metabolite synthesis in BAT, whereas cold-activated BAT enhanced the synthesis. This work uncovers a metabolite-mediated pathway through which BAT controls metabolic health beyond thermogenesis.

Study on stabilized mechanism of high internal phase Pickering emulsions based on commercial yeast proteins: Modulating the characteristics of Pickering particle via sonication.

The primary significance of this work is that the commercial yeast proteins particles were successfully used to characterize the high internal phase Pickering emulsions (HIPPEs). The different sonication time (0,3,7,11,15 min) was used to modulate the structure and interface characteristics of yeast proteins (YPs) that as Pickering particles. Immediately afterward, the influence of YPs particles prepared at different sonication time on the rheological behavior and coalescence mechanism of HIPPEs was investigated. The results indicate that the YPs sonicated for 7 min exhibited a more relaxed molecular structures and conformation, the smallest particle size, the highest H0 and optimal amphiphilicity (the three-phase contact (θ) was 88.91°). The transition from extended to compact conformations of YPs occurred when the sonication time exceeded 7 min, resulting in an augmentation of size of YPs particles, a reduction in surface hydrophobicity (H0), and an elevation in hydrophilicity. The HIPPEs stabilized by YPs particles sonicated for 7 min exhibited the highest adsorption interface protein percentage and a more homogeneous three-dimensional (3D) protein network, resulting in the smallest droplet size and the highest storage (G'). The HIPPEs sample that stabilized by YPs particles sonicated for 15 min showed the lowest adsorption protein percentage. This caused a reduction in the thickness of its interface protein layer and an enlargement in the droplet diameter (D [3,2]). It was prone to droplet coalescence according to the equation used to evaluate the coalescence probability of droplets (Eq (2)). And the non-adsorbed YPs particles form larger aggregation structures in the continuous phase and act as "structural agents" in 3D protein network. Therefore, mechanistically, the interface protein layer formed by YPs particles sonicated 7 min contributed more to HIPPEs stability. Whereas the "structural agents" contributed more to HIPPEs stability when the sonication time exceeded 7 min. The present results shed important new light on the application of commercial YPs in the functional food fields, acting as an available and effective alternative protein.

Bovine milk ß-casein: Structure, properties, isolation, and targeted application of isolated products.

ß-Casein, an important protein found in bovine milk, has significant potential for application in the food, pharmaceutical, and other related industries. This review first introduces the composition, structure, and functional properties of ß-casein. It then reviews the techniques for isolating ß-casein. Chemical and enzymatic isolation methods result in inactivity of ß-casein and other components in the milk, and it is difficult to control the production conditions, limiting the utilization range of products. Physical technology not only achieves high product purity and activity but also effectively preserves the biological activity of the components. The isolated ß-casein needs to be utilized effectively and efficiently for various purity products in order to achieve optimal targeted application. Bovine ß-casein, which has a purity higher than or close to that of breast ß-casein, can be used in infant formulas. This is achieved by modifying its structure through dephosphorylation, resulting in a formula that closely mimics the composition of breast milk. Bovine ß-casein, which is lower in purity than breast ß-casein, can be maximized for the preparation of functional peptides and for use as natural carriers. The remaining byproducts can be utilized as food ingredients, emulsifiers, and carriers for encapsulating and delivering active substances. Thus, realizing the intensive processing and utilization of bovine ß-casein isolation. This review can promote the industrial production process of ß-casein, which is beneficial for the sustainable development of ß-casein as a food and material. It also provides valuable insights for the development of other active substances in milk.

Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding.

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

Strengthening the Combinational Immunotherapy from Modulating the Tumor Inflammatory Environment via Hypoxia-Responsive Nanogels.

Despite the success of immuno-oncology in clinical settings, the therapeutic efficacy is lower than the expectation due to the immunosuppressive inflammatory tumor microenvironment (TME) and the lack of functional lymphocytes caused by exhaustion. To enhance the efficacy of immuno-oncotherapy, a synergistic strategy should be used that can effectively improve the inflammatory TME and increase the tumor infiltration of cytotoxic T lymphocytes (CTLs). Herein, a TME hypoxia-responsive nanogel (NG) is developed to enhance the delivery and penetration of diacerein and (-)-epigallocatechin gallate (EGCG) in tumors. After systemic administration, diacerein effectively improves the tumor immunosuppressive condition through a reduction of MDSCs and Tregs in TME, and induces tumor cell apoptosis via the inhibition of IL-6/STAT3 signal pathway, realizing a strong antitumor effect. Additionally, EGCG can effectively inhibit the expression of PD-L1, restoring the tumor-killing function of CTLs. The infiltration of CTLs increases at the tumor site with activation of systemic immunity after the combination of TIM3 blockade therapy, ultimately resulting in a strong antitumor immune response. This study provides valuable insights for future research on eliciting effective antitumor immunity by suppressing adverse tumor inflammation. The feasible strategy proposed in this work may solve the urgent clinical concerns of the dissatisfactory checkpoint-based immuno-oncotherapy.

Presence, Subtypes, and Prognostic Significance of Tertiary Lymphoid Structures in Urothelial Carcinoma of the Bladder.

OBJECTIVE: To evaluate the presence and subtypes of tertiary lymphatic structures (TLSs) in urothelial carcinoma of the bladder (UCB) and to analyze their associated clinicopathological characteristics and prognostic significance. METHODS: The study enrolled 580 patients with surgically treated UCB, including 313 non-muscle invasive bladder cancer (NMIBC) and 267 muscle-invasive bladder cancer (MIBC). The presence and subtypes of TLSs were identified by immunohistochemistry (CD20, CD3, Bcl-6, and CD21). TLSs were classified into non-GC (nGC) TLS and GC TLS subtypes based on germinal center (GC) formation. Disease-free survival (DFS) was used as an endpoint outcome to evaluate the prognostic significance of TLS and its subtypes in UCB. RESULTS: TLSs were more common in MIBC than in NMIBC (67.8% vs 48.2%, P < .001), and the tumor-infiltrating lymphocyte (TIL) mean density was significantly higher in MIBC than in NMIBC (24.0% vs 17.5%, P < .001). Moreover, a positive correlation was found between TLS presence and GC structure formation and TIL infiltration in UCB. Endpoint events occurred in 191 patients. Compared to patients with endpoint events, patients without disease progression exhibited higher TIL density and more TLSs (P < .05). Kaplan-Meier curves showed that TLS was associated with better DFS in NMIBC (P = .041) and MIBC (P = .049). However, the Cox multivariate analysis did not demonstrate the prognostic significance of TLS. CONCLUSIONS: TLS is heterogeneous in UCB, and that TLS and GC structures are related to TIL density and prognostic events. However, TLS as a prognostic indicator remains unclear, warranting further investigation.

High Doses of Exogenous Glucagon Stimulate Insulin Secretion and Reduce Insulin Clearance in Healthy Humans.

Glucagon is generally defined as a counterregulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP) in response to hypoglycemia. However, glucagon has long been known to stimulate insulin release, and recent preclinical findings have supported a paracrine action of glucagon directly on islet ß-cells that augments their secretion. In mice, the insulinotropic effect of glucagon is glucose dependent and not present during basal euglycemia. To test the hypothesis that the relative effects of glucagon on hepatic and islet function also vary with blood glucose, a group of healthy subjects received glucagon (100 ng/kg) during fasting glycemia or experimental hyperglycemia (∼150 mg/dL) on 2 separate days. During fasting euglycemia, administration of glucagon caused blood glucose to rise due to increased EGP, with a delayed increase of insulin secretion. When given during experimental hyperglycemia, glucagon caused a rapid, threefold increase in insulin secretion, as well as a more gradual increase in EGP. Under both conditions, insulin clearance was decreased in response to glucagon infusion. The insulinotropic action of glucagon, which is proportional to the degree of blood glucose elevation, suggests distinct physiologic roles in the fasting and prandial states.

NIR-driven photoelectrochemical-fluorescent dual-mode biosensor based on bipedal DNA walker for ultrasensitive detection of microRNA.

Optical biosensors have become powerful tools for bioanalysis, but most of them are limited by optic damage, autofluorescence, as well as poor penetration ability of ultraviolet (UV) and visible (Vis) light. Herein, a near-infrared light (NIR)-driven photoelectrochemical (PEC)-fluorescence (FL) dual-mode biosensor has been proposed for ultrasensitive detection of microRNA (miRNA) based on bipedal DNA walker with cascade amplification. Fueled by toehold-mediated strand displacement (TMSD), the bipedal DNA walker triggered by target miRNA-21 is formed through catalytic hairpin assembly (CHA), which can efficiently move along DNA tracks on CdS nanoparticles (CdS NPs)-modified fluorine doped tin oxide (FTO) electrode, resulting in the introduction of upconversion nanoparticles (UCNPs) on electrode surface. Under 980 nm laser irradiation, the UCNPs serve as the energy donor to emit UV/Vis light and excite CdS NPs to generate photocurrent for PEC detection, while the upconversion luminescence (UCL) at 803 nm is monitored for FL detection. This PEC-FL dual-mode biosensor has achieved the ultrasensitive and accurate analysis of miRNA-21 in human serum and different gynecological cancer cells. Overall, the proposed dual-mode biosensor can not only couple the inherent features of each single-mode biosensor but also provide mutual authentication of testing results, which opens up a new avenue for early diagnosis of miRNA-related diseases in clinic.

Tibetan Plateau yak milk: A comprehensive review of nutritional values, health benefits, and processing technology.

Yak milk is a characteristic animal product of yaks in the Qinghai-Tibet Plateau. Although yak milk production is low, it is richer in nutrients such as protein, fat, and lactose, a more comprehensive range of bioactive components, and unique microbial resources than Holstein cow milk. The plateau environment makes yak milk resistant to hypoxia, anti-fatigue, antioxidant, antibacterial, and relieves chronic diseases. In this paper, based on the systematic analysis of yak milk research results in the past 20 years using CiteSpace 6.1.R2, we reviewed yak lactation performance and nutritional efficacy of yak milk. This paper summarizes the improvement of traditional yak dairy processing technology, and also focuses on the microbial diversity of yak milk sources and their beneficial effects. The purpose of this review is to provide scientific support for the development of a quality yak milk industry on the Tibetan plateau.

Isolation of endophytic fungi from Cotoneaster multiflorus and screening of drought-tolerant fungi and evaluation of their growth-promoting effects.

In the context of climate change and human factors, the drought problem is a particularly serious one, and environmental pollution caused by the abuse of chemical fertilizers and pesticides is increasingly serious. Endophytic fungi can be used as a protection option, which is ecologically friendly, to alleviate abiotic stresses on plants, promote plant growth, and promote the sustainable development of agriculture and forestry. Therefore, it is of great significance to screen and isolate endophytic fungi that are beneficial to crops from plants in special habitats. In this study, endophytic fungi were isolated from Cotoneaster multiflorus, and drought-tolerant endophytic fungi were screened by simulating drought stress with different concentrations of PEG-6000, and the growth-promoting effects of these drought-tolerant strains were evaluated. A total of 113 strains of endophytic fungi were isolated and purified from different tissues of C. multiflorus. After simulated drought stress, 25 endophytic fungi showed strong drought tolerance. After ITS sequence identification, they belonged to 7 genera and 12 species, including Aspergillus, Fusarium, Colletotrichum, Penicillium, Diaporthe, Geotrichum, and Metarhizium. According to the identification and drought stress results, 12 strains of endophytic fungi with better drought tolerance were selected to study their abilities of dissolving inorganic phosphorus and potassium feldspar powder and producing indole-3-acetic acid (IAA). It was found that the amount of dissolved phosphorus in 7 strains of endophytic fungi was significantly higher than that of CK, and the content of soluble phosphorus was 101.98-414.51 µg. ml-1; 6 endophytic fungi had significantly higher potassium solubilization than CK, and the content of water-soluble potassium ranged from 19.17 to 30.94 mg·l-1; 6 strains have the ability to produce IAA, and the yield of IAA ranged between 0.04 and 0.42 mg. ml-1. This study for the first time identified the existence of endophytic fungi with drought tolerance and growth-promoting function in C. multiflorus, which could provide new direction for plant drought tolerance and growth promotion fungi strain resources. It also provides a theoretical basis for the subsequent application of endophytic fungi of C. multiflorus in agricultural and forestry production to improve plant tolerance.

Prevalence of propionic acidemia in China.

Propionic acidemia (PA) is a rare autosomal recessive congenital disease caused by mutations in the PCCA or PCCB genes. Elevated propionylcarnitine, 2-methylcitric acid (2MCA), propionylglycine, glycine and 3-hydroxypropionate can be used to diagnose PA. Early-onset PA can lead to acute deterioration, metabolic acidosis, and hyperammonemia shortly after birth, which can result in high mortality and disability. Late-onset cases of PA have a more heterogeneous clinical spectra, including growth retardation, intellectual disability, seizures, basal ganglia lesions, pancreatitis, cardiomyopathy, arrhythmias, adaptive immune defects, rhabdomyolysis, optic atrophy, hearing loss, premature ovarian failure, and chronic kidney disease. Timely and accurate diagnosis and appropriate treatment are crucial to saving patients' lives and improving their prognosis. Recently, the number of reported PA cases in China has increased due to advanced diagnostic techniques and increased research attention. However, an overview of PA prevalence in China is lacking. Therefore, this review provides an overview of recent advances in the pathogenesis, diagnostic strategies, and treatment of PA, including epidemiological data on PA in China. The most frequent variants among Chinese PA patients are c.2002G > A in PCCA and c.1301C > T in PCCB, which are often associated with severe clinical symptoms. At present, liver transplantation from a living (heterozygous parental) donor is a better option for treating PA in China, especially for those exhibiting a severe metabolic phenotype and/or end-organ dysfunction. However, a comprehensive risk-benefit analysis should be conducted as an integral part of the decision-making process. This review will provide valuable information for the medical care of Chinese patients with PA.

Dairy products and constituents: a review of their effects on obesity and related metabolic diseases.

Obesity has become a global public health problem that seriously affects the quality of life. As an important part of human diet, dairy products contain a large number of nutrients that are essential for maintaining human health, such as proteins, peptides, lipids, vitamins, and minerals. A growing number of epidemiological investigations provide strong evidence on dairy interventions for weight loss in overweight/obese populations. Therefore, this paper outlines the relationship between the consumption of different dairy products and obesity and related metabolic diseases. In addition, we dive into the mechanisms related to the regulation of glucose and lipid metabolism by functional components in dairy products and the interaction with gut microbes. Lastly, the role of dairy products on obesity of children and adolescents is revisited. We conclude that whole dairy products exert more beneficial effect than single milk constituent on alleviating obesity and that dairy matrix has important implications for metabolic health.

First Report of Leaf Spot Caused by Botrytis cinerea on Strawberry in China.

In the winter of 2022, circular or irregular leaf spots were observed on strawberry (Fragaria × ananassa) planted in commercial fields (cultivar 'xuetu', 'mengzhifu') in Yinzhou, Ningbo, Zhejiang, China (N29°48'48″, E121°39'47″), with disease incidence ranging from 10 to 15% in a field approximately 0.67 ha in size. The estimated crop loss associated with this disease was ~10%. Symptoms included circular or irregular lesions with brown halos and wheel marks, which eventually developed into leaf blight and petiole decay, but spore masses were seldom found on the leaf surface. In severe cases, leaves withered and abscissed. To isolate the causal agent, ten diseased leaves from ten different plants were collected, surface-sterilized with 75% ethanol for 50 s, rinsed twice with sterile distilled water, cut into small pieces (0.5 cm × 0.5 cm), and plated on potato dextrose agar (PDA), then incubated at 25°C in darkness for 5 days. Isolates , which displayed one kind of colony morphology were consistently obtained from each of the ten samples, and 58 single-conidium isolates with the same colony morphology were obtained. The isolation frequency was 58 of 60 samples. The colonies that grew on PDA produced white mycelia, which sporulated after 1 week, producing typical Botrytis-like gray spores. Three isolates (NBCM-1, NBCM-2, NBCM-3) were selected for identification and pathogenicity assays. Conidia were round to ellipsoid, 9.2 to 14.3 µm long (n=50), and 6.4 to 9.2 µm wide (n=50). Sclerotia were not observed on PDA. Based on these characteristics, the pathogen was tentatively identified as Botrytis cinerea (Zhang 2001). PCR was conducted for each of the three isolates to amplify the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, which are typically used for molecular identification of Botrytis species (Staats et al. 2005; Liu et al. 2016). The resulting amplicons were sequenced, and the sequences were processed using BLAST in the National Center for Biotechnology Information. Sequences of the three isolates were deposited in GenBank (accession nos. OR052082 to OR052086, OR493405 to OR493414). BLASTn analyses showed that isolates were 99 to 100% identical to B.cinerea reported causing leaf spot on strawberry in California; accession numbers MK919496 (G3PDH, 883/883 bp), MK919494 (HSP60, 992/992 bp), and MK919495 (RPB2, 1081/1081 bp). The resulting concatenated data set of G3PDH-HSP60-RPB2-NEP1-NEP2 was used to conduct a multilocus phylogenetic analysis (MLSA) using the maximum likelihood method. The MLSA tree indicated that the three isolates belonged to Botrytis cinerea. To test for pathogenicity, three 1-month-old strawberry (cultivar 'xuetu') plants were inoculated with each isolate (NBCM-1, NBCM-2, NBCM-3). A noninoculated control (sterile water only) was also included. The strawberry plants were inoculated by spraying with conidia suspension (1.0 × 105/ml) until run-off. Inoculations with sterile water served as controls. All plants were kept at 28/25°C (day/night), under a 12:12-h light/dark photoperiod. All plants were covered with transparent plastic bags to maintain humidity for the first 48 h, after which the bags were removed. After 4 to 7 days, leaf spot symptoms similar to those observed in the field were observed in all inoculated plants, while the controls remained healthy. The experiment was repeated three times. The pathogen was reisolated from the inoculated leaves and again identified as B. cinerea, with the same methodology used for the initial identification. Leaf spot caused by B. cinerea on strawberry was recently reported in California (Mansouripour and Holmes 2020) and Florida (Marin and Peres 2022). To our knowledge, this is the first report of B. cinerea causing leaf spot on strawberry in China. The pathogen is also the causal agent of Botrytis fruit rot on strawberry. Given the high variability of this pathogen (Marin and Peres 2022), further studies on its occurrence, spread, management, and control are required. The identification of this pathogen provides a basis for further research on its management and control.

[Clinical features and risk factors for secondary hemophagocytic lymphohistiocytosis in elderly patients with severe SARS-CoV-2 infection: a multicenter retrospective cohort study].

OBJECTIVE: To explore the incidence of secondary hemophagocytic lymphohistiocytosis (sHLH) in elderly patients with severe SARS-CoV-2 infection, and to analyze and summarize its clinical features and risk factors for early identification of high-risk groups. METHODS: A retrospective cohort study was conducted. From January to May 2020, No. 960 Hospital of People's Liberation Army, the Second Hospital Affiliated to Cheeloo College of Medicine of Shandong Province, the First Rehabilitation Hospital of Shandong Province, the Public Health Clinical Center Affiliated to Shandong University, and Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine received 248 patients over 60 years old who were diagnosed with severe SARS-CoV-2 infection during their assistance to Hubei or support for diagnosis and treatment of SARS-CoV-2 infection in Shandong Province. The clinical data of patients were collected. According to the hemophagocytic lymphohistiocytosis diagnosis scoring (HScore) criteria, the patients were divided into sHLH group (HScore > 169) and non-sHLH group (HScore < 98). The demographic data, clinical features, laboratory results, the proportion of organ failure and 60-day mortality of patients were collected and compared between the two groups. The risk factors of sHLH and 60-day death were evaluated through binary multivariate Logistic regression analysis in elderly patients with severe SARS-CoV-2 infection. The receiver operator characteristic curve (ROC curve) was plotted to analyze the diagnostic value of indicators only or combined for sHLH. RESULTS: Among 248 elderly patients with severe SARS-CoV-2 infection, 82 patients with incomplete data and untraceable clinical outcomes, and 35 patients with HScore of 98-169 were excluded. Finally, 131 patients were enrolled in the final follow-up and statistics, including 25 patients in the sHLH group and 106 patients in the non-sHLH group. Compared with the non-sHLH group, plasma albumin (ALB), hemoglobin (Hb), lymphocyte count (LYM), platelet count (PLT), fibrinogen (Fib) and prealbumin (PAB) in the sHLH group were significantly reduced, while alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), MB isoenzyme of creatine kinase (CK-MB), serum creatinine (SCr), C-reactive protein (CRP), D-dimer, ferritin (Fer), lactate dehydrogenase (LDH), procalcitonin (PCT), cardiac troponin I (cTnI), triglycerides (TG), interleukin-6 (IL-6), total bilirubin (TBil) were significantly higher. The fever and fatigue in the sHLH group were more severe than those in the non-sHLH group, and the patients in the sHLH group had higher rates of shock, acute kidney injury, liver dysfunction, and cardiac injury than the non-sHLH group. The 60-day mortality of patient in the sHLH group was significantly higher than that in the non-sHLH group [84.0% (21/25) vs. 40.6% (43/106), P < 0.01]. Binary multivariate Logistic regression analysis showed that high Fer [odds ratio (OR) = 0.997, 95% confidence interval (95%CI) was 0.996-0.998], D-dimer (OR = 0.960, 95%CI was 0.944-0.977), LDH (OR = 0.998, 95%CI was 0.997-0.999) and TG (OR = 0.706, 95%CI was 0.579-0.860) were independent risk factors for sHLH in elderly patients with severe SARS-CoV-2 infection (all P < 0.01), while elevated Fer (OR = 1.001, 95%CI was 1.001-1.002), LDH (OR = 1.004, 95%CI was 1.002-1.005) and D-dimer (OR = 1.036, 95%CI was 1.018-1.055) were independent risk factors for 60-day death of patients (all P < 0.01). The death risk of the sHLH patients was 7.692 times higher than that of the non-sHLH patients (OR = 7.692, 95%CI was 2.466-23.987, P = 0.000). ROC curve analysis showed that a three-composite-index composed of LDH, D-dimer and TG had good diagnostic value for sHLH in elderly patients with severe SARS-CoV-2 infection [area under the ROC curve (AUC) = 0.920, 95%CI was 0.866-0.973, P = 0.000]. CONCLUSIONS: Elderly patients with severe SARS-CoV-2 infection complicated by sHLH tend to be critically ill and have refractory status and worse prognosis. High Fer, LDH, D-dimer and TG are independent risk factors for sHLH, and are highly suggestive of poor outcome. The comprehensive index composed of LDH, D-dimer and TG has good diagnostic value, and can be used as an early screening tool for sHLH in elderly patients with severe SARS-CoV-2 infection.

Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.

Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled, since elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underlie many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential in neutralizing ROS and establish a proosteogenic reduction and oxidation reaction (REDOX) environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis led to acute degradation of RUNX2, impaired osteoblast differentiation, and reduced bone formation. Conversely, reducing ROS using catalase enhanced RUNX2 stability and promoted osteoblast differentiation and bone formation when GSH biosynthesis was limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilized RUNX2 and improved bone development in the Runx2+/- haplo-insufficient mouse model of human cleidocranial dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarify how ROS negatively impacts osteoblast differentiation and bone formation.