African swine fever virus protein p17 promotes mitophagy by facilitating the interaction of SQSTM1 with TOMM70.

Viruses have developed different strategies to hijack mitophagy to facilitate their replication. However, whether and how African swine fever virus (ASFV) regulates mitophagy are largely unknown. Here, we found that the ASFV-encoded p17 induced mitophagy. Coimmunoprecipitation/mass spectrometry assays identified translocase of outer mitochondrial membrane 70 (TOMM70) as the protein that interacted with p17. The binding of TOMM70 to p17 promoted the binding of the mitophagy receptor SQSTM1 to TOMM70, led to engulfment of mitochondria by autophagosomes, and consequently decreased the number of mitochondria. Consistently, the levels of TOMM70 and TOMM20 decreased substantially after p17 expression or ASFV infection. Furthermore, p17-mediated mitophagy resulted in the degradation of mitochondrial antiviral signalling proteins and inhibited the production of IFN-α, IL-6 and TNFα. Overall, our findings suggest that ASFV p17 regulates innate immunity by inducing mitophagy via the interaction of SQSTM1 with TOMM70.

Association between different MAP levels and 30-day mortality in sepsis patients: a propensity-score-matched, retrospective cohort study.

BACKGROUND: Sepsis is a life-threatening organ dysfunction caused by the infection-related host response disorder. Adequate mean arterial pressure is an important prerequisite of tissue and organ perfusion, which runs through the treatment of sepsis patients, and an appropriate mean arterial pressure titration in the early-stage correlates to the positive outcome of the treatment. Therefore, in the present study, we aimed to elucidate the relationship between early mean arterial pressure levels and short-term mortality in sepsis patients. METHODS: We included all suspected sepsis patients from MIMIC-III database with average mean arterial pressure ≥ 60 mmHg on the first day of intensive care unit stay. Those patients were then divided into a permissive low-mean arterial pressure group (60-65 mmHg) and a high-mean arterial pressure group (> 65 mmHg). Multivariate Cox regression analysis was conducted to analyze the relationship between MAP level and 30-day, 60-day, and 100-day mortality of suspected sepsis patients in the two groups. Propensity score matching, inverse probability of treatment weighing, standardized mortality ratio weighting, PA weighting, overlap weighting, and doubly robust analysis were used to verify our results. RESULTS: A total of 14,031 suspected sepsis patients were eligible for inclusion in our study, among which 1305 (9.3%) had an average first-day mean arterial pressure of 60-65 mmHg, and the remaining 12,726 patients had an average first-day mean arterial pressure of more than 65 mmHg. The risk of 30-day mortality was reduced in the high mean arterial pressure group compared with the permissive low-mean arterial pressure group (HR 0.67 (95% CI 0.60-0.75; p < 0.001)). The higher mean arterial pressure was also associated with lower 60-day and 100-day in-hospital mortality as well as with shorter duration of intensive care unit stay. Patients in the high-mean arterial pressure group also had more urine output on the first and second days of intensive care unit admission. CONCLUSIONS: After risk adjustment, the initial mean arterial pressure of above 65 mmHg was associated with reduced short-term mortality, shorter intensive care unit stay, and higher urine volume in the first two days among patients with sepsis.

ERRα contributes to HDAC6-induced chemoresistance of osteosarcoma cells.

Chemotherapy resistance is an important problem for clinical therapy of osteosarcoma (OS). The potential effects of histone deacetylases (HDACs) on OS chemoresistance are studied. The expression of HDACs in OS cells resistance to doxorubicin (Dox) and cisplatin (CDDP) is checked. Among 11 members of HDACs, levels of HDAC6 are significantly upregulated in OS cells resistance to Dox and CDDP. Inhibition of HDAC6 via its specific inhibitor ACY1215 restores chemosensitivity of OS-resistant cells. Further, HDAC6 directly binds with estrogen-related receptors alpha (ERRα) to regulate its acetylation and protein stability. Inhibition of ERRα further strengthens ACY1215-increased chemosensitivity of OS-resistant cells. Mechanistically, K129 acetylation is the key residue for HDAC6-regulated protein levels of ERRα. Collectively, we find that ERRα contributes to HDAC6-induced chemoresistance of OS cells. Inhibition of HDAC6/ERRα axis might be a potential approach to overcome chemoresistance and improve therapy efficiency for OS treatment. 1. HDAC6 was significantly upregulated in Dox and CDDP resistant OS cells; 2. Inhibition of HDAC6 can restore chemosensitivity of OS cells; 3. HDAC6 binds with ERRα at K129 to decrease its acetylation and increase protein stability; 4. ERRα contributes to HDAC6-induced chemoresistance of OS cells.

[Identification of host proteins interacting with African swine fever virus inner envelope protein p17].

African swine fever virus (ASFV) infection leads to a mortality rate of up to 100%, causing devastating disasters to the pig industry. Understanding the ASFV infection and replication is therefore of great importance. ASFV has more than 150 open reading frames, among which the inner coat protein p17 encoded by the D117L gene is involved in the formation of the icosahedral structure of the virus. However, little is known about the mechanism how p17 regulates host cell function. In this study, the potential host proteins interacting with ASFV p17 were screened by immunoprecipitation technique combined with protein profiling analysis. The interactions of p17 with mitochondrial membrane protein TOMM70 and heat shock protein HSPA8 were confirmed by co-immunoprecipitation technique and laser confocal experiments. This study provides important information for further exploring the function of p17 during ASFV infection.

IGF2BP1-regulated expression of ERRα is involved in metabolic reprogramming of chemotherapy resistant osteosarcoma cells.

Doxorubicin (Dox) is the standard treatment approach for osteosarcoma (OS), while acquired drug resistance seriously attenuates its treatment efficiency. The present study aimed to investigate the potential roles of metabolic reprogramming and the related regulatory mechanism in Dox-resistant OS cells. The results showed that the ATP levels, lactate generation, glucose consumption and oxygen consumption rate were significantly increased in Dox-resistant OS cells compared with parental cells. Furthermore, the results revealed that the increased expression of estrogen-related receptor alpha (ERRα) was involved in metabolic reprogramming in chemotherapy resistant OS cells, since targeted inhibition of ERRα restored the shifting of metabolic profiles. Mechanistic analysis indicated that the mRNA stability, rather than ERRα transcription was markedly increased in chemoresistant OS cells. Therefore, it was hypothesized that the 3'-untranslated region of ERRα mRNA was methylated by N6-methyladenine, which could further recruit insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to suppress mRNA decay and increase mRNA stability. IGF2BP1 knockdown downregulated ERRα and reversed the metabolic alteration of resistant OS cells. Additionally, the oncogenic effect of the IGF2BP1/ERRα axis on Dox-resistant OS cells was verified by in vitro and in vivo experiments. Clinical analysis also revealed that the expression levels of IGF2BP1 and ERRα were associated with the clinical progression of OS. Collectively, the current study suggested that the IGF2BP1/ERRα axis could regulate metabolic reprogramming to contribute to the chemoresistance of OS cells.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Oestrogen-related receptor alpha mediates chemotherapy resistance of osteosarcoma cells via regulation of ABCB1.

Chemotherapy resistance is one of the major challenges for the treatment of osteosarcoma (OS). The potential roles of oestrogenic signals in the chemoresistance of OS cells were investigated. As compare to the parental cells, the doxorubicin and cisplatin (CDDP) resistant OS cells had greater levels of oestrogen-related receptors alpha (ERRα). Targeted inhibition of ERRα by its specific siRNAs or inverse agonist XCT-790 can restore the sensitivity of OS resistant cells to chemotherapy. This might be due to that si-ERRα can decrease the expression of P-glycoprotein (P-gp, encoded by ABCB1), one important ABC membrane transporter for drug efflux. XCT-790 can decrease the transcription and mRNA stability of ABCB1, while had no effect on protein stability of P-gp. ERRα can bind to the transcription factor of SP3 to increase the transcription of ABCB1. Furthermore, XCT-790 treatment decreased the expression of miR-9, which can bind to the 3'UTR of ABCB1 and trigger its decay. Collectively, we found that ERRα can regulate the chemoresistance of OS cells via regulating the transcription and mRNA stability of ABCB1. Targeted inhibition of ERRα might be a potential approach for OS therapy.

Bioconversion of lutein by Enterobacter hormaechei to form a new compound, 8-methyl-α-ionone.

OBJECTIVES: To investigate the final product of the bioconversion of lutein by a novel lutein-degrading bacterium, Enterobacter hormaechei A20, and the kinetics of the process. RESULTS: A new product, 8-methyl-α-ionone, was resolved by GC-MS. The compound was further identified by NMR. A conversion yield of 90% was achieved by E. hormaechei in 36 h with 10 g lutein l-1. CONCLUSIONS: This is the first report of the bioconversion of lutein to form 8-methyl-α-ionone. A degradation pathway is proposed.

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation--up to 6 mM--in the medium. This yield is 21.5-fold higher than that obtained in previous studies.