Network of epistatic interactions in an enzyme active site revealed by large-scale deep mutational scanning.

Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M ß-lactamase as a model system. CTX-M hydrolyzes ß-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the ß-lactamase enzyme and quantitated the function of variants against two ß-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.

Estimation on the hourly near-surface temperature lapse rate and its time-varying characteristics.

Since much of the current researches have focused on daily, monthly or annual near-surface (2 m) temperature lapse rate (NSTLR), there is little guidance on best estimation practices and analyses of time-varying characteristics for the hourly NSTLR. To estimate hourly NSTLR and identify its time-varying characteristics accurately and objectively, this study proposed a robust estimation strategy based on IGGIII equivalent weight using multiple linear regression models. The accuracy and reliability of the proposed method was verified. The results show that the robust estimation strategy can further improve the hourly NSTLR solution accuracy relative to the least square (LSQ) method, especially in the time period of relatively high temperature. The hourly NSTLR was positively correlated with temperature, with a 24-h average maximum of 0.604 °C/100 m at universal time coordinated (UTC) 7.2 h and minimum of 0.284 °C/100 m at UTC 20.5 h, respectively. Throughout the year, the NSTLR was the largest from June to August, with an average median of around 0.492 °C/100 m. However, from November to the following January, the NSTLR value was the smallest, with a mean median of about 0.323 °C/100 m. In addition, the hourly NSTLR values were essentially less than the constant value of 0.65 °C/100 m. When the hourly NSTLR estimated based on the proposed method was applied to the temperature interpolation, the interpolation accuracies at the highest altitude (1545 m) and other meteorological stations (below 310 m) can increase by 22.4 % and 8.1 %, respectively, relative to the hourly NSTLR calculated by the LSQ method, and increased by 55.6 % and 13.0 %, respectively, relative to the no-NSTLR correction. The results are important for the fine establishment of high spatiotemporal resolution temperature fields and for the study of climatic phenomena characterized with rapid spatiotemporal variation.

Treatment with palbociclib and tislelizumab for CDKN2A-mutated and PD-L1-positive advanced intrahepatic cholangiocarcinoma: a case report and literature review.

Background: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignancy with a steadily increasing incidence worldwide. ICC has insidious onset, rapid progression, and poor prognosis. More multidisciplinary clinical studies are needed to continuously explore safer and more efficient diagnosis and treatment modes for ICC. Methods and results: A 66-year-old female patient with ICC rapidly developed systemic multiple metastases after surgery, and the first-line two-drug combination chemotherapy was not effective. Due to cyclin-dependent kinase inhibitor 2A mutation and programmed cell death-ligand 1-positive, a partial response and progression-free survival of 9.5 months were achieved after a second-line treatment with cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) combined with immunotherapy. The patient developed thromboembolism 7 months after treatment and died due to disseminated intravascular coagulation. Conclusion: The combination of targeted and immune therapy has revealed a potentially effective regimen for the effective treatment of patients with ICC, which needs to be observed in larger clinical studies. The thromboembolism rates in real-world patients treated with CDK4/6 inhibitors are higher than those reported in clinical trials, and the application of prophylactic anticoagulation in this patient population may be questionable.

Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

Exploiting the Carboxylate-Binding Pocket of ß-Lactamase Enzymes Using a Focused DNA-Encoded Chemical Library.

ß-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important ß-lactam class of antibiotics. The OXA-48 and NDM-1 ß-lactamases cause resistance to the last-resort ß-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel ß-lactamase inhibitors. We exploited the ß-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all ß-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-ß-lactam pharmacophores for the development of ß-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Association between monocyte lymphocyte ratio and abdominal aortic calcification in US adults: A cross-sectional study

Abstract Background This study aimed to evaluate the association between Monocyte Lymphocyte Ratio (MLR) and Abdominal Aortic Calcification (AAC) in adults over 40 years of age in the United States. Methods Data were collected from the 2013-2014 National Health and Nutrition Examination Survey (NHANES). AAC was quantified by the Kauppila score system based on dual-energy X-Ray absorptiometry. Severe AAC was defined as a total AAC score > 6. The lymphocyte count and monocyte count can be directly obtained from laboratory data files. Multivariable logistic regression models were used to determine the association between MLR and the AAC score and severe AAC. Results A total of 3,045 participants were included in the present study. After adjusting for multiple covariates, MLR was positively associated with higher AAC score (β = 0.21, 95% CI 0.07, 0.34, p = 0.0032) and the odds of severe AAC increased by 14% per 0.1 unit increase in the MLR (OR = 1.14, 95% CI 1.00, 1.31, p = 0.0541). The Odds Ratio (OR) (95% CI) of severe AAC for participants in MLR tertile 3 was 1.88 (1.02, 3.47) compared with those in tertile 1 (p for trend = 0.0341). Subgroup analyses showed that a stronger association was detected in the elderly compared with non-elderly (p for interaction = 0.0346) and diabetes compared with non-diabetes (borderline significant p for interaction = 0.0578). Conclusion In adults in the United States, MLR was associated with higher AAC scores and a higher probability of severe AAC. MLR may become a promising tool to predict the risk of AAC.

MiR-216a-5p alleviates LPS-induced inflammation in the human bronchial epithelial cell by inhibition of TGF-b1 signaling via down-regulating TGFBR2

Objective: Bronchopneumonia is a common respiratory infection disease and is the leading cause of hospitalization in children under 5 years of age. Inflammation is the primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation induced by bronchopneumonia and investigate the potential mechanism underlying it. Methods: Human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccha-rides (LPS) to trigger bronchopneumonia in vitro. The production of interleukin (IL)-1β, IL-6, and Tumor necrosis factor (TNF)-α was measured using the enzyme-linked immunosorbent assay. The luciferase assay was conducted to explore the relationship between miR-216a-5p and TGFBR2. Quantitative real-time polymerase chain reaction and western blot were used to detect the gene expression. Results: miR-216a-5p gene expression decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of IL-1β, IL-6, and TNF-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor (TGF)-β1, and phosphorylation of SMAD family member 2 (smad2),. This ectopic expression of miR-216a-5p was restored by overexpressed TGFBR2. Conclusion: miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-β1 signaling via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia (AU)