The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.     

COX‐2‐mediated Inflammation in Fat Is Crucial for Obesity‐linked Insulin Resistance and Fatty Liver

The aim was to examine the role of cyclooxygenase (COX)‐2‐mediated inflammation in the development of obese linked insulin resistance and fatty liver. The rats were fed separately regular diet (CONT), high‐fat diet (HFD) ad libitum, or energy restrictedly for 12 weeks. Rats fed HFD ad libitum were further divided into three subgroups co‐treated with vehicle (HFa), or a selective COX‐2 inhibitor celecoxib (HFa‐Cel) or mesulid (HFa‐Mes). Euglycemic hyperinsulinemic clamp (EHC) experiment was performed at the end of study. Another set of rats with similar grouping was further divided into those with a 4, 8, or 12‐week intervention period for hepatic sampling. Body weight was increased significantly and similarly in HFa, HFa‐Cel, and HFa‐Mes. Time‐dependent increases in plasma insulin, glucose, 8‐isoprostanes, leptin levels, homeostasis model assessment of insulin resistance (HOMA‐IR) and hepatic triglyceride contents shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. During EHC period, the reduction in stimulation of whole body glucose uptake, suppression of hepatic glucose production and metabolic clearance rate of insulin shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. The enhanced COX‐2 and tumor necrosis factor‐α (TNF‐α) but attenuated PPAR‐γ and C/EBP‐α mRNA expressions in epididymal fat shown in HFa were significantly reversed in HFa‐Cel and HFa‐Mes. The increases in average cell size of adipocytes and CD68 positive cells shown in HFa were also significantly reversed in HFa‐Cel and HFa‐Mes. Our findings suggest that COX‐2 activation in fat inflammation is important in the development of insulin resistance and fatty liver in high fat induced obese rats.     

Antagonistic activity of Lactobacillus acidophilus RY2 isolated from healthy infancy feces on the growth and adhesion characteristics of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAggEC) infection is an important cause of acute diarrhea, affecting children in developing countries and travelers visiting tropical or subtropical areas. Three probiotics can exert bacteriostatic or bactericidal effects on human and animal intestinal pathogens, the efficiency of probiotics on EAggEC infection remains unclear. In this study, the antagonistic activity of probiotic bacteria isolated from infant faeces was examined against several EAggEC stains. While three isolates, Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4 were shown to significantly inhibit the growth of EAggEC. In addition, the antagonistic activity of the Lactobacillus species was maintained despite heating (100 °C, 15 min) of cell free culture supernatant (CFCS). The antagonistic activity of the CFCS however, could be reduced following lactate dehydrogenase treatment and at pH 7.2. Furthermore, in an adhesion–inhibition assay, L. acidophilus RY2 was shown to be more effective than L. salivarius MM1 and L. paracasei EN4. This study suggests that L. acidophilus RY2 could be used as a probiotic organism against EAggEC.     

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae)

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm‐isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)‐like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria‐colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high‐affinity sucrose‐specific proton‐dependent importer. Plasma‐membrane/tonoplast localization of GeSUT4‐GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant‐microbe interactions.     

Stool DNA test targeting methylated syndecan-2 (SDC2) as a noninvasive screening method for colorectal cancer

Despite the steadily increasing worldwide incidence of colorectal cancer (CRC), an effective noninvasive approach for early detection of CRC is still under investigation. The guaiac-based fecal occult blood test (FOBT) and fecal immunochemical test (FIT) have gained popularity as noninvasive CRC screening tests owing to their convenience and relatively low costs. However, the FOBT and FIT have limited sensitivity and specificity. To develop a noninvasive tool for the detection of CRC, we investigated the sensitivity, specificity, and accuracy of a stool DNA test targeting methylated syndecan-2 (SDC2), which is frequently methylated in patients with CRC. The present study enrolled 62 patients diagnosed as having stage 0-IV CRC and 76 healthy participants between July 2018 and June 2019 from two institutions. Approximately 4.5 g of stool sample was collected from each participant for detection of human methylated SDC2 gene. In total, 48 of 62 (77.4%) patients with CRC showed positive results, whereas 67 out of 76 (88.2%) healthy participants showed negative results. The area under the curve of the receiver operating characteristic curve constructed was 0.872 for discrimination between patients with CRC and healthy individuals. The present study highlights the potential of the fecal methylated SDC2 test as a noninvasive detection method for CRC screening with a relatively favorable sensitivity of 77.4%, a specificity of 88.2% and a positive predictive value of 84.2% compared with other available fecal tests. Further multicenter clinical trials comprising subjects of varied ethnicities are required to validate this test for the mass screening of patients with CRC.     

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.     

朝鲜蓟叶中一个新的倍半萜内酯

从朝鲜蓟（Cynarascolyrnus）叶中分离得到2个倍半萜内酯,其中一个是新化合物,通过波谱学方法确定其结构为3β,8α,11α,13-四羟基-10（14）-愈创木烯-1α,4β,5α,6β氢-6α,12-内酯（1）。     

Conversion of crude chitosan to an anti-fungal protease by <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Bacillus cereus AU004, isolated from soil samples, secreted a complex of hydrolytic enzymes into the culture broth when it was grown aerobically in a medium containing crude chitosan flakes. The presence of the AU004 culture supernatant substantially influenced the growths of the plant-pathogenic fungi Fusarium oxysporum, F. solani and Pythium ultimum in terms of dry weight. AU004 excreted a protease when cultivated in a medium that contained 4% (w/v) chitosan as the major nutritional source. The protease was purified by sequential chromatography and characterized as a novel extracellularly neutral protease. The protease had an Mr of 28.8 kDa. The optimal pH and temperature for protease activity were 7 and 50°C, respectively. Antifungal activity of the protease was observed using an assay based on the inhibition of spore germination and hyphal extension of the fungal Pythium ultimum. This investigation is the first report of the production of an anti-fungal protease from Bacillus spp.