Separation of two HMM-S1 species from white muscle myosin

Heavy meromyosin subfragment 1 was resolved by chromatography on DEAE-cellulose into two fractions characterized by the nature of the alkali light chains present. It was shown that even in an HMM-S1 preparation with an extensive fragmentation of the heavy chain a polyacrylamide gel electrophoresis analysis differentiates alkali light chains among the light fragmentation components. A non-fragmented HMM-S1 was obtained from a papain digest of myofibrils and the chromatographic analysis supplied further evidence of the separation of the two species of HMM-S1 present in rabbit white muscle myosin.     

Alleviation of Both Water and Nutrient Limitations is Necessary to Accelerate Ecological Restoration of Waste Rock Tips

Hard rock quarries are commonly located close to national parks and special areas of conservation and are generally regarded as visually intrusive. Consequently, restoration strategies that effectively accelerate natural plant regeneration processes are required. Slate waste tips present extreme conditions for plant establishment with multiple potential limiting factors (e.g., lack of organic matter, nutrients, and poor water retention). In this study, we investigated ecological strategies to accelerate natural regeneration at the largest slate quarry in Europe. A field experiment was conducted to assess ecosystem restoration using a contrasting set of native woody species. Treatments included amendments of waste tips with: polyacrylamide gel to increase water‐holding capacity; mineral fertilizer to increase nutrient supply; and two treatments that increased both (organic waste or boulder clay addition). Ecosystem recovery was evaluated through above‐ and below‐ground productivity (plant and microbial, respectively) and soil analyses. Neither increasing nutrient supply (with mineral fertilizer) nor water‐holding capacity (with polyacrylamide gel) was sufficient, alone, to improve plant establishment. However, both boulder clay and organic waste amendment significantly enhanced plant growth. There was a marked positive interaction in the effects on tree growth of the amendment with organic waste and boulder clay. Large interactions occurred between tree species and substrate amendments. The growth of N₂‐fixing species was strongly favored over non‐fixers where there was no addition of material increasing soil nitrogen supply, whereas the growth advantage of pioneer species over non‐pioneers was greatest with fertilizer, organic waste, or clay additions. Organic waste addition had the greatest positive impact on soil processes.     

Association of RAGE gene polymorphism with circulating AGEs level and paraoxonase activity in relation to macro-vascular complications in Indian type 2 diabetes mellitus patients

Background and aims

Sustained interaction of advanced glycation end products (AGEs) with their receptor RAGE and subsequent signaling plays an important role in the development of diabetic complications. Genetic variation of RAGE gene may be associated with the development of vascular complications in type 2 diabetes mellitus (T2DM).

Objectives

The present study aimed to explore the possible association of RAGE gene polymorphisms namely − 374T/A, − 429T/C and G82S with serum level of AGEs, paraoxonase (PON1) activity and macro-vascular complications (MVC) in Indian type 2 diabetes mellitus patients (T2DM).

Methods

A total of 265 diabetic patients, including DM without any complications (n = 135), DM-MVC (n = 130) and 171 healthy individuals were enrolled. Genotyping of RAGE variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. Serum AGEs were estimated by ELISA and fluorometrically. and PON1 activity was assessed spectrophotometrically.

Results

Of the three examined SNPs, association of − 429T/C polymorphism with MVC in T2DM was observed (OR = 3.001, p = 0.001) in the dominant model. Allele ‘A’ of − 374T/A polymorphism seems to confer better cardiac outcome in T2DM. Patients carrying C allele (− 429T/C) and S allele (G82S) had significantly higher AGEs levels. − 429T/C polymorphism was also found to be associated with low PON1 activity. Interaction analysis revealed that the risk of development of MVC was higher in T2DM patients carrying both a CC genotype of − 429T/C polymorphism and a higher level of AGEs (OR = 1.343, p = 0.040).

Conclusion

RAGE gene polymorphism has a significant effect on AGEs level and PON1 activity in diabetic subjects compared to healthy individuals. Diabetic patients with a CC genotype of − 429T/C are prone to develop MVC, more so if AGEs levels are high and PON1 activity is low.     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Distinguishing enzyme structures from non-enzymes without alignments

The ability to predict protein function from structure is becoming increasingly important as the number of structures resolved is growing more rapidly than our capacity to study function. Current methods for predicting protein function are mostly reliant on identifying a similar protein of known function. For proteins that are highly dissimilar or are only similar to proteins also lacking functional annotations, these methods fail. Here, we show that protein function can be predicted as enzymatic or not without resorting to alignments. We describe 1178 high-resolution proteins in a structurally non-redundant subset of the Protein Data Bank using simple features such as secondary-structure content, amino acid propensities, surface properties and ligands. The subset is split into two functional groupings, enzymes and non-enzymes. We use the support vector machine-learning algorithm to develop models that are capable of assigning the protein class. Validation of the method shows that the function can be predicted to an accuracy of 77% using 52 features to describe each protein. An adaptive search of possible subsets of features produces a simplified model based on 36 features that predicts at an accuracy of 80%. We compare the method to sequence-based methods that also avoid calculating alignments and predict a recently released set of unrelated proteins. The most useful features for distinguishing enzymes from non-enzymes are secondary-structure content, amino acid frequencies, number of disulphide bonds and size of the largest cleft. This method is applicable to any structure as it does not require the identification of sequence or structural similarity to a protein of known function.     

Molecular basis for substrate recognition and septum cleavage by AtlA,the major N-acetylglucosaminidase of Enterococcus faecalis

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA–substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.     

右美托咪定联合帕瑞昔布钠对老年腹腔镜胃癌手术患者应激反应、细胞免疫功能和认知功能的影响

摘要 目的：探讨帕瑞昔布钠和右美托咪定联合麻醉对老年腹腔镜胃癌手术患者细胞免疫功能、应激反应和认知功能的影响。方法：纳入2019年8月~2022年7月期间西安交通大学第二附属医院收治的150例老年腹腔镜胃癌手术患者。按照随机数字表法将患者分为帕瑞昔布钠组（n=50,帕瑞昔布钠）、右美托咪定组（n=50,右美托咪定）和联合组（n=50,右美托咪定联合帕瑞昔布钠）。对比三组临床指标、视觉模拟评分法（VAS）、简易精神状态检查表（MMSE）评分、术后认知功能障碍（POCD）发生率、应激反应指标[皮质醇（Cor）、肾上腺素（E）、促肾上腺皮质激素（ACTH）]、细胞免疫功能变化情况。结果：联合组的术后住院天数、肛门排气时间短于右美托咪定组、帕瑞昔布钠组（P<0.05）。联合组术后12 h、术后24 h、术后48 h VAS评分低于右美托咪定组、帕瑞昔布钠组（P<0.05）。联合组术后24 h、术后72 h MMSE评分高于右美托咪定组、帕瑞昔布钠组（P<0.05）。联合组的POCD发生率低于右美托咪定组、帕瑞昔布钠组（P<0.05）。三组术后1 d Cor、E、ACTH升高,但联合组低于帕瑞昔布钠组、右美托咪定组同期（P<0.05）。三组术后1 d CD8⁺升高,但联合组低于帕瑞昔布钠组、右美托咪定组同期;CD3⁺、CD4⁺、CD4⁺CD8⁺下降,但联合组高于帕瑞昔布钠组、右美托咪定组同期（P<0.05）。结论：右美托咪定联合帕瑞昔布钠应用于老年腹腔镜胃癌手术患者,镇痛效果显著,可减轻机体的应激反应、免疫抑制及对认知功能的损害。