USP7 promotes cardiometabolic disorders and mitochondrial homeostasis dysfunction in diabetic mice via stabilizing PGC1ß.

Diabetic cardiomyopathy (DCM) is a major complication of diabetes and is characterized by left ventricular dysfunction. Currently, there is a lack of effective treatments for DCM. Ubiquitin-specific protease 7 (USP7) plays a key role in various diseases. However, whether USP7 is involved in DCM has not been established. In this study, we demonstrated that USP7 was upregulated in diabetic mouse hearts and NMCMs co-treated with HG+PA or H9c2 cells treated with PA. Abnormalities in diabetic heart morphology and function were reversed by USP7 silencing through conditional gene knockout or chemical inhibition. Proteomic analysis coupled with biochemical validation confirmed that PCG1ß was one of the direct protein substrates of USP7 and aggravated myocardial damage through coactivation of the PPARα signaling pathway. USP7 silencing restored the expression of fatty acid metabolism-related proteins and restored mitochondrial homeostasis by inhibiting mitochondrial fission and promoting fusion events. Similar effects were also observed in vitro. Our data demonstrated that USP7 promoted cardiometabolic metabolism disorders and mitochondrial homeostasis dysfunction via stabilizing PCG1ß and suggested that silencing USP7 may be a therapeutic strategy for DCM.

USP7 cardiomyocyte specific knockout causes disordered mitochondrial biogenesis and dynamics and early neonatal lethality in mice.

BACKGROUND: Ubiquitination is an enzymatic modification involving ubiquitin chains, that can be reversed by deubiquitination (DUB) enzymes. Ubiquitin-specific protease 7 (USP7), which is also known as herpes virus-associated ubiquitin-specific protease (HAUSP), has been shown to play a vital role in cardiovascular diseases. However, the underlying molecular mechanism by which USP7 regulates cardiomyocyte function has not been reported. METHODS: To understand the physiological function of USP7 in the heart, we constructed cardiomyocyte-specific USP7 conditional knockout mice. RESULTS: We found that homozygous knockout mice died approximately three weeks after birth, while heterozygous knockout mice grew normally into adulthood. Severe cardiac dysfunction, hypertrophy, fibrosis, and cell apoptosis were observed in cardiomyocyte-specific USP7 knockout mice, and these effects were accompanied by disordered mitochondrial dynamics and cardiometabolic-related proteins. CONCLUSIONS: In summary, we investigated changes in the growth status and cardiac function of cardiomyocyte-specific USP7 knockout mice, and preliminarily explored the underlying mechanism.

Genome-wide identification and characterization of the sweet orange (Citrus sinensis) basic helix-loop-helix (bHLH) family reveals a role for CsbHLH085 as a regulator of citrus bacterial canker resistance.

Citrus bacterial canker (CBC) is a harmful bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), negatively impacting citrus production worldwide. The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant development and stress responses. This study aimed to identify and annotate bHLH proteins encoded in the Citrus sinensis genome and explore their involvement and functional importance in regulating CBC resistance. A total of 135 putative CsbHLHs TFs were identified and categorized into 16 subfamilies. Their chromosomal locations, collinearity, and phylogenetic relationships were comprehensively analyzed. Upon Xcc strain YN1 infection, certain CsbHLHs were differentially regulated in CBC-resistant and CBC-sensitive citrus varieties. Among these, CsbHLH085 was selected for further functional characterization. CsbHLH085 was upregulated in the CBC-resistant citrus variety, was localized in the nucleus, and had a transcriptional activation activity. CsbHLH085 overexpression in Citrus significantly enhanced CBC resistance, accompanied by increased levels of salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and decreased levels of abscisic acid (ABA) and antioxidant enzymes. Conversely, CsbHLH085 virus-induced gene silencing resulted in opposite phenotypic and biochemical responses. CsbHLH085 silencing also affected the expression of phytohormone biosynthesis and signaling genes involved in SA, JA, and ABA signaling. These findings highlight the crucial role of CsbHLH085 in regulating CBC resistance, suggesting its potential as a target for biotechnological-assisted breeding citrus varieties with improved resistance against phytopathogens.

The Chinese herbal medicine Fufang Zhenzhu Tiaozhi ameliorates diabetic cardiomyopathy by regulating cardiac abnormal lipid metabolism and mitochondrial dynamics in diabetic mice.

Diabetic cardiomyopathy (DCM) is an important complication leading to the death of patients with diabetes, but there is no effective strategy for clinical treatments. Fufang Zhenzhu Tiaozhi (FTZ) is a patent medicine that is a traditional Chinese medicine compound preparation with comprehensive effects for the prevention and treatment of glycolipid metabolic diseases under the guidance of "modulating liver, starting pivot and cleaning turbidity". FTZ was proposed by Professor Guo Jiao and is used for the clinical treatment of hyperlipidemia. This study was designed to explore the regulatory mechanisms of FTZ on heart lipid metabolism dysfunction and mitochondrial dynamics disorder in mice with DCM, and it provides a theoretical basis for the myocardial protective effect of FTZ in diabetes. In this study, we demonstrated that FTZ protected heart function in DCM mice and downregulated the overexpression of free fatty acids (FFAs) uptake-related proteins cluster of differentiation 36 (CD36), fatty acid binding protein 3 (FABP3) and carnitine palmitoyl transferase 1 (CPT1). Moreover, FTZ treatment showed a regulatory effect on mitochondrial dynamics by inhibiting mitochondrial fission and promoting mitochondrial fusion. We also identified in vitro that FTZ could restore lipid metabolism-related proteins, mitochondrial dynamics-related proteins and mitochondrial energy metabolism in PA-treated cardiomyocytes. Our study indicated that FTZ improves the cardiac function of diabetic mice by attenuating the increase in fasting blood glucose levels, inhibiting the decrease in body weight, alleviating disordered lipid metabolism, and restoring mitochondrial dynamics and myocardial apoptosis in diabetic mouse hearts.

CsAP2-09 confers resistance against citrus bacterial canker by regulating CsGH3.1L-mediated phytohormone biosynthesis.

Citrus bacterial canker (CBC) is a serious bacterial disease affecting citrus plantations and the citrus industry all over the world. We have previously shown that an apetala 2/ethylene response factor in Citrus sinensis, CsAP2-09, positively regulated resistance to CBC, although the regulatory mechanisms remained undetermined. Here, we demonstrated that CsAP2-09 positively and sustainably controlled resistance to CBC in three-year transgenic plants. CsAP2-09 was found to be a transcriptional activator, and qRT-PCR and dual luciferase assays showed that it controlled the expression CsGH3.1L. CsAP2-09 bound directly to the promotor of CsGH3.1L, shown by yeast one-hybrid assay, with the binding site confirmed by electrophoretic mobility shift assay. Biochemical assays showed that CsAP2-09 negatively regulated the biosynthesis of indole acetic acid (IAA) and positively regulated that of salicylic acid (SA) and ethylene, verified with transient overexpression of CsGH3.1L. The combination of these results with those of previous reports indicated that SA, ethylene, and IAA can directly regulate CBC resistance. Overall, we revealed a pathway whereby CsAP2-09 conferred CBC resistance by direct binding to the CsGH3.1L promoter, activating its expression and modulating IAA, SA, and ethylene biosynthesis. Our study indicates the potential value of manipulating CsAP2-09 and CsGH3.1L in the breeding of CBC-resistant citrus.

Taxifolin, an Inhibitor of Sortase A, Interferes With the Adhesion of Methicillin-Resistant Staphylococcal aureus.

The evolution and spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant hidden risk to human public health. The majority of antibiotics used clinically have become mostly ineffective, and so the development of novel anti-infection strategies is urgently required. Since Staphylococcus aureus (S. aureus) cysteine transpeptidase sortase A (SrtA) mediates the surface-anchoring of proteins to its surface, compounds that inhibit SrtA are considered potential antivirulence treatments. Herein, we report on the efficacy of the potent SrtA inhibitor taxifolin (Tax), a flavonoid compound isolated from Chinese herbs. It was able to reversibly block the activity of SrtA with an IC50 of 24.53 ± 0.42 µM. Tax did not display toxicity toward mammalian cells or S. aureus at a concentration of 200 µM. In addition, Tax attenuated the virulence-related phenotype of SrtA in vitro by decreasing the adherence of S. aureus, reducing the formation of a biofilm, and anchoring of S. aureus protein A on its cell wall. The mechanism of the SrtA-Tax interaction was determined using a localized surface plasmon resonance assay. Subsequent mechanistic studies confirmed that Asp-170 and Gln-172 were the principal sites on SrtA with which it binds to Tax. Importantly, in vivo experiments demonstrated that Tax protects mice against pneumonia induced by lethal doses of MRSA, significantly improving their survival rate and reducing the number of viable S. aureus in the lung tissue. The present study indicates that Tax is a useful pioneer compound for the development of novel agents against S. aureus infections.

Genome-wide identification of auxin response factor (ARF) family in kiwifruit (Actinidia chinensis) and analysis of their inducible involvements in abiotic stresses.

Auxin response factor (ARF) acts as a vital component of auxin signaling and participates in growth, development, and stress responses in plants. In the present study, we comprehensively analyzed kiwifruit's (Actinidia chinensis) ARF genes (AcARFs) and their involvement in abiotic stress response. We identified a total of 41 AcARFs encoding ARFs in the A. chinensis genome. AcARF genes were characterized by the classic ARF_resp and a B3 domain and primarily localized on the cytoplasm and nucleus. AcARFs were categorized into eight subgroups as per the phylogenetic analysis. Synteny analysis showed that 35 gene pairs in AcARF family underwent segmental and whole genome duplication events. Promoter cis-element prediction revealed that AcARFs might be involved in abiotic factors related to stress response, which was later assessed and validated by qRT-PCR based expression analysis. Additionally, AcARFs showed tissue-specific expression. These findings extend our understanding of the functional roles of AcARFs in stress responses. Taken together, the systematic annotation of the AcARF family genes provides a platform for the functional and evolutionary study, which might help in elucidating the precise roles of the AcARFs in stress responses. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01011-4.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos.

SummaryTRIM28/KAP1/TIF1ß was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

Carotenoid accumulation during tomato fruit ripening is modulated by the auxin-ethylene balance.

BACKGROUND: Tomato fruit ripening is controlled by ethylene and is characterized by a shift in color from green to red, a strong accumulation of lycopene, and a decrease in ß-xanthophylls and chlorophylls. The role of other hormones, such as auxin, has been less studied. Auxin is retarding the fruit ripening. In tomato, there is no study of the carotenoid content and related transcript after treatment with auxin. RESULTS: We followed the effects of application of various hormone-like substances to "Mature-Green" fruits. Application of an ethylene precursor (ACC) or of an auxin antagonist (PCIB) to tomato fruits accelerated the color shift, the accumulation of lycopene, α-, ß-, and δ-carotenes and the disappearance of ß-xanthophylls and chlorophyll b. By contrast, application of auxin (IAA) delayed the color shift, the lycopene accumulation and the decrease of chlorophyll a. Combined application of IAA + ACC led to an intermediate phenotype. The levels of transcripts coding for carotenoid biosynthesis enzymes, for the ripening regulator Rin, for chlorophyllase, and the levels of ethylene and abscisic acid (ABA) were monitored in the treated fruits. Correlation network analyses suggest that ABA, may also be a key regulator of several responses to auxin and ethylene treatments. CONCLUSIONS: The results suggest that IAA retards tomato ripening by affecting a set of (i) key regulators, such as Rin, ethylene and ABA, and (ii) key effectors, such as genes for lycopene and ß-xanthophyll biosynthesis and for chlorophyll degradation.

The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

[Hemorrheological changes in irreversible hemorrhagic shock].

OBJECTIVE: To observe the characteristics of changes in hemorheology at the early stage of irreversible hemorrhagic shock in a rodent model. METHODS: Rodent model of irreversible hemorrhagic shock was reproduced. Animals were randomized into 4 groups. In the first group, survival rate and mean arterial pressure (MAP) in 180 minutes were observed after hemorrhagic shock (S group). In the second group, animals were sacrificed soon after hemorrhagic shock (S0 group). In the third group, animals were sacrificed 60 minutes after hemorrhagic shock (S1 group). In the fourth group, animals were sacrificed 120 minutes after hemorrhagic shock (S2 group). Blood samples of animals of S0, S1 and S2 were all obtained before hemorrhagic shock. Blood lactate, hemorheological parameters, red blood cell (RBC) deformability and RBC aggregation index were determined. RESULTS: Mean blood loss of S group was (22.9+/-3.8) ml/kg, constituting about (38.1+/-6.3)% of total blood volume. At 60, 120 and 180 minutes after hemorrhagic shock, survival rates were 100%, 72% and 64%, respectively. Compared with baseline, 0, 60 and 120 minutes after hemorrhagic shock, blood lactate increased significantly (all P<0.01), but 120 minutes after hemorrhagic shock, it decreased significantly compared with 0 minute after hemorrhagic shock (P<0.05). Compared with baseline, 0 minute and 60 minutes after hemorrhagic shock, blood viscosity was found to be decreased at shear rate of 10 s(-1), 60 s(-1) and 100 s(-1) (all P<0.01); 120 minutes after hemorrhagic shock, at shear rate of 10 s-1 and 60 s(-1), blood viscosity decreased significantly (both P<0.01); 0, 60 and 120 minutes after hemorrhagic shock, plasma viscosity, RBC deformability and RBC aggregation index at shear rates of 600 s(-1), 800 s(-1) and 1 000 s(-1) decreased significantly (all P<0.01). CONCLUSION: At the early stage of irreversible hemorrhagic shock, blood lactate increased significantly, and decreased afterwards. These indicate reversal of deterioration of metabolism. At different time after the early stage of irreversible hemorrhagic shock, blood and plasma viscosity, RBC deformability and aggregation index lowered significantly and did not improve. Changes in viscosity and RBC aggregation are different from the changes in late stage, and this indicates that hemorheological disorders should be corrected in the treatment at the early stage after hemorrhagic shock.