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Summary: Besides traditional ubiquitin-dependent proteasome degradation, thousands of eukaryotic proteins more than previously appreciated could undergo ubiquitin-independent proteasomal degradation (UbInPD). A pathogen-encoded effector protein SAP05 secreted by phytoplasma, could hijack hostage Rpn10 subunit of proteasome derived from Arabidopsis thaliana and target the degradation of GATA BINDING FACTOR (GATA) or SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors (TFs) without ubiquitin or additional proteasome shuttle factors. To explain how could SAP05 target the degradation bypassing the ubiquitin-dependent pathway, we have determined the crystal structure of the complex between Arabidopsis thaliana Rpn10 and Aster Yellows witches'-broom phytoplasma SAP05 or onion yellow phytoplasma SAP05, which showed a previously unknown recognition interface. Sequence alignment and structural biological evidence showed that this interaction is highly conserved in various SAP05 homologs, suggesting a general mode in plant infection. After docking the complex structure to the plant proteasome, SAP05 was near to the adenosine triphosphatase (ATPase) central pore and enough to submit substrate to degradation process, which suggested a molecular glue-like role to bridge TFs close to the ATPase central pore of proteasomes for the direct degradation.

The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3′UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

The human AAA+protein p97 plays an important role in cellular protein homeostasis. Here, the authors use cryo-EM to obtain further insights into how p97 interacts with its co-factor Npl4 and they observe three distinct conformational states of Npl4 in complex with human p97, which suggests that a seesaw motion is essential for the unfolding activity of the p97 complex.

ObjectiveTo determine whether gut microbiota, fatty metabolism and cytokines were associated with immune thrombocytopenia (ITP).MethodsIn total, 29 preliminarily diagnosed ITP patients and 33 healthy volunteers were enrolled. Fecal bacterial were analyzed based on 16S rRNA sequencing. Plasma cytokines and motabolites were analyzed using flow cytometry and liquid chromatography-mass spectrometry (LC-MS), respectively.ResultsBacteroides, Phascolarctobacterium, and Lactobacillus were enriched at the genus level in ITP patients, while Ruminococcaceae UCG-002, Eubacterium coprostanoligeues, Megamonas, and Lachnospiraceae NC2004 were depleted. At the phylum level, the relative abundance of Proteobacteria and Chloroflexi increased in ITP patients, while Firmicutes, Actinobacteria, and the Firmicutes/Bacteroidetes ratio decreased. Plasma levels of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-12-epi-leukotriene B4 (6t,12e-LTB4), and resolvin D2 (RvD2) were upregulated, and stachydrine, dowicide A, dodecanoylcarnitine were downregulated in ITP patients. Furthermore, RvD2 is positively correlated with order Bacteroidetes VC2.1 Bac22, 5-HETE is positively correlated with genus Azospirillum, and 6t,12e-LTB4 is positively correlated with genus Cupriavidus. In addition, stachydrine is positively correlated with family Planococcaceae, dowicide A is positively correlated with class MVP-15, and dodecanoylcarnitine is positively correlated with order WCHB1-41. Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were upregulated in ITP patients.ConclusionOur study revealed a relationship between microbiota and fatty metabolism in ITP. Gut microbiota may participate in the pathogenesis of ITP through affecting cytokine secretion, interfering with fatty metabolism.

Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.

The thermogravimetry (TGA) and derivative thermogravimetry (DTG) methods have been used to measure the free water in low-moisture foods. In this study, the 2nd derivative thermogravimetry (2nd DTG) method distinguished the free and bound water based on the speed of moisture evaporation, which could be used for both low-moisture and high-moisture foods. First, the key factors related to moisture evaporation were optimized. Isothermal temperature of 30 ~ 50°C, dynamic temperature of 0.033 ~ 0.133°C/min, and flow rate of nitrogen of 20 ~ 40 mL/min were the optimal parameters for the 2nd DTG method. Under these conditions, the repeatability and reproducibility of the 2nd DTG method were enhanced, its applicability was expanded to high-moisture foods, and the accuracy was ± 4.0% of the nuclear magnetic resonance results. Hence, the 2nd DTG method is better suited for the measurement of free water in foods.

The leading cause of heart failure is cardiomyopathy and damage to the cardiomyocytes. Adult mammalian cardiomyocytes have the ability to regenerate, but this cannot wholly compensate for myocardial cell loss after myocardial injury. Studies have shown that exercise has a regulatory role in the activation and promotion of regeneration of healthy and injured adult cardiomyocytes. However, current research on the effects of aerobic exercise in myocardial regeneration is not comprehensive. This review discusses the relationships between aerobic exercise and the regeneration of cardiomyocytes with respect to complex molecular and cellular mechanisms, paracrine factors, transcriptional factors, signaling pathways, and microRNAs that induce cardiac regeneration. The topics discussed herein provide a knowledge base for physical activity-induced cardiomyocyte regeneration, in which exercise enhances overall heart function and improves the efficacy of cardiac rehabilitation.

Bicistronic transgene expression mediated by internal ribosome entry site (IRES) elements has been widely used. It co-expresses heterologous transgene products from a message RNA driven by a single promoter. Hematologic gene delivery is a promising treatment for both inherited and acquired diseases. A combined strategy was recently documented for potential genome editing in hematopoietic cells. A transduction efficiency exceeding ~90% can be achieved by capsid-optimized recombinant adeno-associated virus serotype 6 (rAAV6) vectors. In this study, to deliver an encephalomyocarditis virus (EMCV) IRES-containing rAAV6 genome into hematopoietic cells, we observed that EMCV IRES almost completely shut down the transgene expression during the process of mRNA−protein transition. In addition, position-dependent behavior was observed, in which only the EMCV IRES element located between a promoter and the transgenes had an inhibitory effect. Although further studies are warranted to evaluate the involvement of cellular translation machinery, our results propose the use of specific IRES elements or an alternative strategy, such as the 2A system, to achieve bicistronic transgene expression in hematopoietic cells.

Lysine 2,3-aminomutase (KAM; EC 5.4.3.2) catalyzes the interconversion of L-lysine and L-β-lysine. The transcription and regulation of the kam locus, including lysine-2,3-aminoniutase-encoding genes, in Bacillus thuringiensis were analyzed in this study. Reverse transcription-PCR (RT-PCR) analysis revealed that this locus forms two operons: yodT (yodT-yodS-yodR-yodQ-yodP-kamR) and kamA (kamA-yokU-yozE). The transcriptional start sites (TSSs) of the katnA gene were determined using 5' rapid amplification of cDNA ends (RACE). A typical --12/--24 σK binding site was identified in the promoter PkamA, which is located upstream of the kamA gene TSS. A β-galactosidase assay showed that PkamA, which directs the transcription of the katnA operon, is controlled by the σ54 factor and is activated through the σ54-dependent transcriptional regulator KamR. The kamA operon is also controlled by σK and regulated by the GerE protein in the late stage of sporulation. kamR and kamA mutants were prepared by homologous recombination to examine the role of the kam locus. The results showed that the sporulation rate in B. thuringiensis HD(ΔkamR) was slightly decreased compared to that in HD73, whereas that in HD(ΔkamA) was similar to that in HD73. This means that other genes regulated by KamR are important for sporulation. [ABSTRACT FROM AUTHOR]