Conformational plasticity of SpyCas9 induced by AcrIIA4 and AcrIIA2: Insights from molecular dynamics simulation.

CRISPR-Cas9 systems constitute bacterial adaptive immune systems that protect against phage infections. Bacteriophages encode anti-CRISPR proteins (Acrs) that mitigate the bacterial immune response. However, the structural basis for their inhibitory actions from a molecular perspective remains elusive. In this study, through microsecond atomistic molecular dynamics simulations, we demonstrated the remarkable flexibility of Streptococcus pyogenes Cas9 (SpyCas9) and its conformational adaptability during interactions with AcrIIA4 and AcrIIA2. Specifically, we demonstrated that the binding of AcrIIA4 and AcrIIA2 to SpyCas9 induces a conformational rearrangement that causes spatial separation between the nuclease and cleavage sites, thus making the endonuclease inactive. This separation disrupts the transmission of signals between the protospacer adjacent motif recognition and nuclease domains, thereby impeding the efficient processing of double-stranded DNA. The simulation also reveals that AcrIIA4 and AcrIIA2 cause different structural variations of SpyCas9. Our research illuminates the precise mechanisms underlying the suppression of SpyCas9 by AcrIIA4 and AcrIIA2, thus presenting new possibilities for controlling genome editing with higher accuracy.

Increasing dietary nutrient levels modulates colon immune adaptation and alleviates inflammation in the epithelial heterogeneous nuclear ribonucleoprotein I (Hnrnp I) knockout mice.

Heterogeneous nuclear ribonucleoprotein I (HNRNP I) is an RNA-binding protein essential for neonatal immune adaptation by downregulating interleukin-1 receptor-associated kinase (IRAK1) in toll-like receptor (TLR)-mediated NF-κB signaling pathways. TLR-mediated NF-κB is associated with chronic inflammation, including the development of inflammatory bowel diseases. Meanwhile, dietary protein intake is one of the major concerns for individuals with inflammatory bowel diseases. The present study aims to investigate the effects of a protein-enriched diet on intestinal inflammation and immune responses in a mouse model with aberrant NF-κB signaling in the colon. A transgenic mouse model with intestinal-epithelial-cell (IEC) specific Hnrnp I knocked out was used to investigate the effects of protein intake on the immune system in the colon. A control diet (CON) and a nutrient-dense modified diet (MOD) were fed to both the wild-type (WT) and the knockout (KO) male mice for 14 weeks. Inflammatory markers and colonic immune responses were examined, with gene expression and protein expression levels analyzed. IEC-specific Hnrnp I knocked out mice had significantly increased expression of the active NF-κB subunit, P65, in their colons. There was a concomitant induction of mRNA expression of Il1ß, Il6, Cxcl1, and Ccl2. The number of CD4+ T cells in the distal colon was also increased in the KO mice. The results confirmed that KO mice had proinflammatory responses with aberrant NF-κB signaling in the colon. Importantly, increased nutrient density in their diets attenuated colon inflammation by decreasing the expression of proinflammatory cytokines, reducing P65 translocation, downregulating IRAK1, and limiting the number of CD4+ T cells recruited in Hnrnp I KO mice colon. In summary, this study found that a diet with increased nutrient density relieved the inflammation induced by knockout of Hnrnp I, attributable partially to the reduced expression of inflammatory and immune-modulating cytokines in the mouse distal colon.

A comparison of the effects of resistant starch types on glycemic response in individuals with type 2 diabetes or prediabetes: A systematic review and meta-analysis.

Background: Type 2 diabetes (T2D) diagnoses are predicted to reach 643 million by 2030, increasing incidences of cardiovascular disease and other comorbidities. Rapidly digestible starch elevates postprandial glycemia and impinges glycemic homeostasis, elevating the risk of developing T2D. Starch can escape digestion by endogenous enzymes in the small intestine when protected by intact plant cell walls (resistant starch type 1), when there is a high concentration of amylose (resistant starch type 2) and when the molecule undergoes retrogradation (resistant starch type 3) or chemical modification (resistant starch type 4). Dietary interventions using resistant starch may improve glucose metabolism and insulin sensitivity. However, few studies have explored the differential effects of resistant starch type. This systematic review and meta-analysis aims to compare the effects of the resistant starch from intact plant cell structures (resistant starch type 1) and resistant starch from modified starch molecules (resistant starch types 2-5) on fasting and postprandial glycemia in subjects with T2D and prediabetes. Methods: Databases (PubMed, SCOPUS, Ovid MEDLINE, Cochrane, and Web of Science) were systematically searched for randomized controlled trials. Standard mean difference (SMD) with 95% confidence intervals (CI) were determined using random-effects models. Sub-group analyses were conducted between subjects with T2D versus prediabetes and types of resistant starch. Results: The search identified 36 randomized controlled trials (n = 982), 31 of which could be included in the meta-analysis. Resistant starch type 1 and type 2 lowered acute postprandial blood glucose [SMD (95% CI) = -0.54 (-1.0, -0.07)] and [-0.96 (-1.61, -0.31)]. Resistant starch type 2 improved acute postprandial insulin response [-0.71 (-1.31, -0.11)]. In chronic studies, resistant starch type 1 and 2 lowered postprandial glucose [-0.38 (-0.73, -0.02), -0.29 (-0.53, -0.04), respectively] and resistant starch type 2 intake improved fasting glucose [-0.39 (-0.66, -0.13)] and insulin [-0.40 (-0.60, -0.21)]. Conclusion: Resistant starch types 1 and 2 may influence glucose homeostasis via discrete mechanisms, as they appear to influence glycemia differently. Further research into resistant starch types 3, 4, and 5 is required to elucidate their effect on glucose metabolism. The addition of resistant starch as a dietary intervention for those with T2D or prediabetes may prevent further deterioration of glycemic control.

Multilabel Feature Selection: A Local Causal Structure Learning Approach.

Multilabel feature selection plays an essential role in high-dimensional multilabel learning tasks. Existing multilabel feature selection approaches mainly either explore the feature-label and feature-feature correlations or the label-label and feature-feature correlations. A few of them are able to deal with all three types of correlations simultaneously. To address this problem, in this article, we formulate multilabel feature selection as a local causal structure learning problem and propose a novel algorithm, M2LC. By learning the local causal structure of each class label, M2LC considers three types of feature relationships simultaneously and is scalable to high-dimensional datasets as well. To tackle false discoveries caused by the label-label correlations, M2LC consists of two novel error-correction subroutines to correct those false discoveries. Through local causal structure learning, M2LC learns the causal mechanism behind data, and thus, it can select causally informative features and visualize common features shared by class labels and specific features owned by an individual class label using the learned causal structures. Extensive experiments have been conducted to evaluate M2LC in comparison with the state-of-the-art multilabel feature selection algorithms.

A maternal low-protein diet during gestation induces hepatic autophagy-related gene expression in a sex-specific manner in Sprague-Dawley rats.

This study investigates the mechanism by which maternal protein restriction induces hepatic autophagy-related gene expression in the offspring of rats. Pregnant Sprague-Dawley rats were fed either a control diet (C, 18 % energy from protein) or a low-protein diet (LP, 8·5 % energy from protein) during gestation, followed by the control diet during lactation and post-weaning. Liver tissue was collected from the offspring at postnatal day 38 and divided into four groups according to sex and maternal diet (F-C, F-LP, M-C and M-LP) for further analysis. Autophagy-related mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. In addition, chromatin immunoprecipitation (ChIP) was performed to investigate the interactions between transcription factors and autophagy-related genes. Protein levels of p- eukaryotic translation initiation factor 2a and activating transcription factor 4 (ATF4) were increased only in the female offspring born to dams fed the LP diet. Correlatively, the mRNA expression of hepatic autophagy-related genes including Map1lc3b, P62/Sqstm1, Becn1, Atg3, Atg7 and Atg10 was significantly greater in the F-LP group than in the F-C group. Furthermore, ChIP results showed greater ATF4 and C/EBP homology protein (CHOP) binding at the regions of a set of autophagy-related genes in the F-LP group than in the F-C group. Our data demonstrated that a maternal LP diet transcriptionally programmed hepatic autophagy-related gene expression only in female rat offspring. This transcriptional programme involved the activation of the eIF2α/ATF4 pathway and intricate regulation by transcription factors ATF4 and CHOP.

The impact of starchy food structure on postprandial glycemic response and appetite: a systematic review with meta-analysis of randomized crossover trials.

BACKGROUND: Starchy foods can have a profound effect on metabolism. The structural properties of starchy foods can affect their digestibility and postprandial metabolic responses, which in the long term may be associated with the risk of type 2 diabetes and obesity. OBJECTIVES: This systematic review sought to evaluate the clinical evidence regarding the impact of the microstructures within starchy foods on postprandial glucose and insulin responses alongside appetite regulation. METHODS: A systematic search was performed in the PUBMED, Ovid Medicine, EMBASE, and Google Scholar databases for data published up to 18 January 2021. Data were extracted by 3 independent reviewers from randomized crossover trials (RCTs) that investigated the effect of microstructural factors on postprandial glucose, insulin, appetite-regulating hormone responses, and subjective satiety scores in healthy participants. RESULTS: We identified 745 potential articles, and 25 RCTs (n = 369 participants) met our inclusion criteria: 6 evaluated the amylose-to-amylopectin ratio, 6 evaluated the degree of starch gelatinization, 2 evaluated the degree of starch retrogradation, 1 studied starch-protein interactions, and 12 investigated cell and tissue structures. Meta-analyses showed that significant reductions in postprandial glucose and insulin levels was caused by starch with a high amylose content [standardized mean difference (SMD) = -0.64 mmol/L*min (95% CI: -0.83 to -0.46) and SMD = -0.81 pmol/L*min (95% CI: -1.07 to -0.55), respectively], less-gelatinized starch [SMD = -0.54 mmol/L*min (95% CI: -0.75 to -0.34) and SMD = -0.48 pmol/L*min (95% CI: -0.75 to -0.21), respectively], retrograded starch (for glucose incremental AUC; SMD = -0.46 pmol/L*min; 95% CI: -0.80 to -0.12), and intact and large particles [SMD = -0.43 mmol/L*min (95% CI: -0.58 to -0.28) and SMD = -0.63 pmol/L*min (95% CI: -0.86 to -0.40), respectively]. All analyses showed minor or moderate heterogeneity (I2 < 50%). Sufficient evidence was not found to suggest how these structural factors influence appetite. CONCLUSIONS: The manipulation of microstructures in starchy food may be an effective way to improve postprandial glycemia and insulinemia in the healthy population. The protocol for this systematic review and meta-analysis was registered in the international prospective register of systematic reviews (PROSPERO) as CRD42020190873.

First Report of Green Mold Disease Caused by Trichoderma hengshanicum on Ganoderma lingzhi.

Ganoderma lingzhi is a well-known source of natural fungal medicines which has been given for the treatment of several diseases. China is one of the major commercial producers of Ganoderma mushroom worldwide. However, with the expansion of the commercial cultivation, the occurrence of the fungal diseases on G. lingzhi has also been increased. The green mold disease symptoms were observed in the cultivation base of G. lingzhi in Zuojia Town, Jilin City, Jilin Province, China, causing the basidiomes to be rotten and withered, and the green mycelium layer generated gradually. The pathogenicity tests showed the same symptoms as appeared naturally in Zuojia mushroom base. Morphology characters revealed conidia green, ellipsoid, globose, 2.56-4.83 × 2.09-4.22 µm, length-width ratio was 1.1-1.2 (n = 10). Conidiophores trichoderma-like, often asymmetry, branches solitary, paired or in whorls of 3 phialides formed solitary, paired or in whorl, variable in shape, lageniform, sometimes ampulliform or subulate. While using molecular methodology, comparing with the sequences of Trichoderma hengshanicum from GenBank, the analyzed sequence showed 97.32% homology with the RPB2 sequences, 100% with the TEF1-α sequences. A fungus isolated from the diseased tissues was identified based on morphology and molecular studies as T. hengshanicum. This is the first report of T. hengshanicum causing the green mold disease of G. lingzhi in China.

Effects of corticosterone within the hypothalamic arcuate nucleus on food intake and body weight in male rats.

OBJECTIVE: Obesity is a major cause of morbidity and mortality. Few weight-reducing medications are available, and these have limited efficacy. Cushing's Syndrome (caused by elevated glucocorticoid levels) and obesity have similar metabolic features. Though circulating glucocorticoid levels are not elevated in obesity, tissue-specific glucocorticoid levels have been implicated in the development of the metabolic phenotype of obesity. Tissue glucocorticoid levels are regulated by 11ß-hydroxysteroid dehydrogenase type1 (11ßHSD1), which increases the local concentration of active glucocorticoids by the production of corticosterone from 11-dehydrocorticosterone. 11ßHSD1 is expressed in the hypothalamic arcuate nucleus (ARC), a major weight and appetite-regulating centre, and therefore represents a target for novel anti-obesity therapeutic agents. Thus, we sought to investigate the effect of chronic alterations of ARC corticosterone levels (mediated by 11ßHSD1) on food intake and body weight in adult male rats. METHODS: Recombinant adeno-associated virus particles bearing sense 11ßHSD1 (rAAV-S11ßHSD1) and small interfering 11ßHSD1 (rAAV-si11ßHSD1), respectively, were stereotactically injected into the ARC (bilaterally) of adult male Wistar rats. rAAV-GFP was injected into control groups of male Wistar rats. Food intake and body weight were measured three times a week for 70 days. Terminal brain, plasma and intrascapular brown adipose tissue (iBAT) samples were taken for measurement of mRNA expression and hormone levels. RESULTS: Compared to controls, rAAV-S11ßHSD1 injection resulted in higher ARC corticosterone levels, hyperphagia and increased weight gain. Conversely, rAAV-si11ßHSD1 injection (compared to controls) resulted in lower ARC corticosterone levels, higher iBAT uncoupling protein-1 mRNA expression and less weight gain despite similar food intake. CONCLUSIONS: Therefore ARC corticosterone, regulated by 11ßHSD1, may play a role in food intake and body weight regulation. These data have important implications for the development of centrally-acting 11ßHSD1 inhibitors, which are currently being developed for the treatment of obesity, metabolic disorders, and other conditions.

Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis.

Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake (3)H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis.

Functional definition of BirA suggests a biotin utilization pathway in the zoonotic pathogen Streptococcus suis.

Biotin protein ligase is universal in three domains of life. The paradigm version of BPL is the Escherichia coli BirA that is also a repressor for the biotin biosynthesis pathway. Streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. Unlike the scenario in E. coli, S. suis lacks the de novo biotin biosynthesis pathway. In contrast, it retains a bioY, a biotin transporter-encoding gene, indicating an alternative survival strategy for S. suis to scavenge biotin from its inhabiting niche. Here we report functional definition of S. suis birA homologue. The in vivo functions of the birA paralogue with only 23.6% identity to the counterpart of E. coli, was judged by its ability to complement the conditional lethal mutants of E. coli birA. The recombinant BirA protein of S. suis was overexpressed in E. coli, purified to homogeneity and verified with MS. Both cellulose TLC and MALDI-TOFF-MS assays demonstrated that the S. suis BirA protein catalyzed the biotinylation reaction of its acceptor biotin carboxyl carrier protein. EMSA assays confirmed binding of the bioY gene to the S. suis BirA. The data defined the first example of the bifunctional BirA ligase/repressor in Streptococcus.

Interaction of Cy3 dye with CCG and its application for BSA detection.

Chemically converted graphene (CCG) was found to effectively quench the fluorescence emission of Cy3 dye 1 (the intensity is down to 1/38 of 1 alone) in aqueous solution, due to the formation of a CCG-1 hybrid. After the addition of a certain amount of bovine serum albumin (BSA) into the above-mentioned system, about 60 times fluorescence enhancement was observed for the hybrid CCG-1. This was employed to discriminate BSA: the fluorescence intensity was found to be proportional to the BSA added in the concentration range from 0 to 8 × 10-6 M, and the BSA detection limit was down to 5 × 10-8 M.