CD11b/CD86 involved in the microenvironment of colorectal cancer by promoting Wnt signaling activation.

BACKGROUND: Colorectal cancer (CRC) is a malignancy that arises within the gastrointestinal tract. Despite ongoing research, the etiology and pathogenesis of CRC remain elusive; particularly, the distribution and characteristics of tumor-associated macrophages is currently an active area of investigation in understanding the pathological progression and prevention of CRC. METHODS: This study utilized CRC patient surgical samples, mouse models of colitis-associated cancer, colonic organoid, and co-culture cell line to examine the changes in CD11b/CD86 at different pathological region and detect the Wnt signaling pathway activity. RESULTS: Our findings revealed a sensitive and increased expression of CD11b from the early to the advanced CRC tissues and correlated with poor prognosis, while CD86 expression was reduced in advanced CRC tissues. CD133 expression was also elevated in advanced CRC tissues and mainly co-localized with CD11b, suggesting a positive regulatory effect of CD11b and CD133 expression that may contribute to CRC progression. In AOM/DSS mouse models, activation of the Wnt signaling pathway was associated with increased CD133 and CD11b expression. In vitro, THP-1 cell was induced to high expression of CD11b, and the above conditional cultural medium enhanced HCT116 cell colony number and CD133 protein expression. Furthermore, colonic crypts from AOM/DSS mouse models were isolated to culture, and the colonic organoids exhibited dilation and significant increases expression of CD133 and ß-Catenin/N-P-B-Catenin. CONCLUSIONS: CD11b might be an important factor to participate the progress of CRC. And the high CD11b of CRC microenviroment might potentially promote CD133 expression and associate with Wnt signal activation.

SLAMF1 as a novel molecule mediating the causal association between rheumatoid arthritis and interstitial lung disease: A Mendelian randomization study combined with transcriptomics and in vivo validation.

BACKGROUND: Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a common complication of rheumatoid arthritis (RA) that result in significant morbidity and mortality. Understanding the molecular mechanisms underlying RA-ILD is crucial for effective prevention. This study aims to identify the specific molecule that mediate the causal association between RA and ILD, as well as to explore its potential mechanisms in the pathogenesis of RA-ILD. METHODS: Using two-sample Mendelian randomization (MR) analyses, we investigated the causal relationship among 16,987 blood genes, RA and ILD. Subsequently, a two-step MR technique was employed to identify significant genes that mediate the association between RA and ILD, and to quantify their proportion of mediation effect. To validate the genes as mediators, the replication MR analysis was conducted and the in vivo experiment was performed using an established animal model of RA-ILD. Furthermore, integrated bioinformatic analyses were conducted to elucidate the specific biological functions of the determined mediator in pathogenesis of RA-ILD. RESULTS: Nine genes, namely MAPK8IP2, TAF11, SLAMF1, DAB2IP, GLUL, SLC4A10, PRSS35, NFX1, and PLK3, were identified as mediators. Among them, SLAMF1 was validated as the most significant mediator, accounting for 4.693% of the mediating effect on the causal relationship between RA and ILD. Upregulated mRNA expression of SLAMF1 was observed in the animal model of RA-ILD compared to controls. Bioinformatic analyses revealed that SLAMF1 was overexpressed in patients with lung fibrosis and correlated with a poor prognosis. Specifically, SLAMF1 was found to be predominantly overexpressed in T cells in lung tissues of patients with lung fibrosis. Additionally, the functional role of SLAMF1 was associated with multiple immune cell infiltrations and the biological process of extracellular matrix synthesis in pulmonary tissues from patients with lung fibrosis. CONCLUSION: SLAMF1 may play a crucial role as a molecular mediator in the causal association between RA and ILD, and participate in multiple mechanisms underlying the pathogenesis of RA-ILD. This research provides insights into how the development of RA influences the risk of ILD and offers potential interventional targets against RA-ILD.

GSK3ß Substrate-competitive Inhibitors Regulate the gut Homeostasis and Barrier Function to Inhibit Neuroinflammation in Scopolamine-induced Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a neurodegenerative disease mainly characterized by cognitive impairment. Glycogen synthase kinase 3 (GSK3ß) is a potential therapeutic target against AD. Isoorientin (ISO), a GSK3ß substrate competitive inhibitor, plays anti-AD effects in in vitro and in vivo AD model. TFGF-18 is an ISO synthetic analog with improved potency, but its neuroprotective effect in vivo remains to be elucidated, and the underlying mechanisms of GSK3ß inhibitor against AD need to be clarified. This study investigated the TFGF-18 and ISO effects on gut homeostasis and neuroinflammation in scopolamine (SCOP)-induced AD mice. And the protection on barrier function was observed in in vitro blood-brain barrier (BBB) model of mouse brain microvascular endothelial cells (bEnd.3). The results show that TFGF-18 and ISO improved cognitive function in SCOP-induced mice, and inhibited cholinergic system disorders and inflammation in the brain and intestine, decreased the level of lipopolysaccharides (LPS) in serum and intestine, protected the diversity and balance of intestinal microbiome, increased the expressions of tight junction protein (ZO-1, occludin), brain derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in the mouse brain and intestine. In addition, TFGF-18 and ISO protected against barrier damage in LPS-stimulated BBB model of bEnd.3 cells in vitro. TFGF-18 and ISO increased the ratio of p-GSK3ß/GSK3ß, suppressed toll-like receptors 4 (TLR-4) expression and nuclear factor kappa-B (NF-κB) activation in vivo and in vitro, and increased the expressions of ß-catenin, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in vitro. In conclusion, The GSK3ß inhibitors TFGF-18 and ISO modulate the gut homeostasis and barrier function to inhibit neuroinflammation and attenuate cognitive impairment by regulating NF-κB, ß-catenin and Nrf2/HO-1 pathways.

Metformin improves cognitive dysfunction through SIRT1/NLRP3 pathway-mediated neuroinflammation in db/db mice.

Diabetes mellitus (DM), an important public health problem, aggravates the global economic burden. Diabetic encephalopathy (DE) is a serious complication of DM in the central nervous system. Metformin has been proven to improve DE. However, the mechanism is still unclear. In this study, the db/db mice, a common model used for DE, were employed to explore and study the neuroprotective effect of metformin and related mechanisms. Behavioral tests indicated that metformin (100 or 200 mg/kg/day) could significantly improve the learning and memory abilities of db/db mice. The outcomes from the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) demonstrate that metformin effectively modulates glucose and insulin signaling pathways in db/db mice. The results of body weight and blood lipid panel (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol) show that metformin promotes the level of lipid metabolism in db/db mice. Furthermore, data from oxidative stress assays, which measured levels of malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase, suggest that metformin suppresses oxidative stress-induced brain damage in db/db mice. In addition, western blot, Nissl staining, and immunofluorescence results showed that metformin increased the expressions of nerve growth factor and postsynaptic density 95 and repaired neuronal structural damage. For the mechanism study, metformin activated SIRT1 and inhibited the expression of NLRP3 inflammasome (NLRP3, ASC, caspase-1, IL-1ß, and IL-18) and inflammatory cytokines (TNFα and IL-6). In conclusion, metformin could ameliorate cognitive dysfunction through the SIRT1/NLRP3 pathway, which might be a promising mechanism for DE treatment.

Anti-oxidative stress and cognitive improvement of a semi-synthetic isoorientin-based GSK-3ß inhibitor in rat pheochromocytoma cell PC12 and scopolamine-induced AD model mice via AKT/GSK-3ß/Nrf2 pathway.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive deficits. Although the pathogenesis of AD is unclear, oxidative stress has been implicated to play a dominant role in its development. The flavonoid isoorientin (ISO) and its synthetic derivatives TFGF-18 selectively inhibit glycogen synthase kinase-3ß (GSK-3ß), a potential target of AD treatment. PURPOSE: To investigate the neuroprotective effect of TFGF-18 against oxidative stress via the GSK-3ß pathway in hydrogen peroxide (H2O2)-induced rat pheochromocytoma PC12 cells in vitro and scopolamine (SCOP)-induced AD mice in vivo. METHOD: The oxidative stress of PC12 cells was induced by H2O2 (600 µM) and the effects of TFGF-18 (2 and 8 µM) or ISO (12.5 and 50 µM) were observed. The AD mouse model was induced by SCOP (3 mg/kg), and the effects of TFGF-18 (2 and 8 mg/kg), ISO (50 mg/kg), and donepezil (DNP) (3 mg/kg) were observed. DNP, a currently accepted drug for AD was used as a positive control. The neuronal cell damages were analyzed by flow cytometry, LDH assay, JC-1 assay and Nissl staining. The oxidative stress was evaluated by the detection of MDA, SOD, GPx and ROS. The level of ACh, and the activity of AChE, ChAT were detected by the assay kit. The expressions of Bax, Bcl-2, caspase3, cleaved-caspase3, p-AKT (Thr308), AKT, p-GSK-3ß (Ser9), GSK-3ß, Nrf2, and HO-1, as well as p-CREB (Ser133), CREB, and BDNF were analyzed by western blotting. Morris water maze test was performed to analyze learning and memory ability. RESULTS: TFGF-18 inhibited neuronal damage and the expressions of Bax, caspase3 and cleaved-caspase3, and increased the expression of Bcl-2 in vitro and in vivo. The level of MDA and ROS were decreased while the activities of SOD and GPx were increased by TFGF-18. Moreover, TFGF-18 increased the p-AKT, p-GSK-3ß (Ser9), Nrf2, HO-1, p-CREB, and BDNF expression reduced by H2O2 and SCOP. Meanwhile, MK2206, an AKT inhibitor, reversed the effect of TFGF-18 on the AKT/GSK-3ß pathway. In addition, the cholinergic system (ACh, ChAT, and AChE) disorders were retrained and the learning and memory impairments were prevented by TFGF-18 in SCOP-induced AD mice. CONCLUSIONS: TFGF-18 protects against neuronal cell damage and cognitive impairment by inhibiting oxidative stress via AKT/GSK-3ß/Nrf2 pathway.

WeiNaiAn capsule attenuates intestinal mucosal injury and regulates gut microbiome in indomethacin-induced rat.

Indomethacin, as a non-steroidal anti-inflammatory drugs, is widely used in the clinic. However, it can cause severe injury to the gastrointestinal tract and the incidence is increasing. It has become an essential clinical problem in preventing intestinal damage. Teprenone has been reported to have a significant positive effect on intestinal mucosal lesions, but long-term use of teprenone can elicit adverse reactions. WeiNaiAn capsule is a traditional Chinese medicine formulation used widely in the treatment of gastric and duodenal mucosal injury. However, how WeiNaiAn protects against intestinal mucosal injury and its mechanism of action are not known. In this study, WeiNaiAn capsule or Teprenone treatment improved the intestinal mucosal pathological score and antioxidant level in indomethacin-induced rats. 16 S rRNA sequence data showed WeiNaiAn capsule reverted the structure community and replenished the beneficial bacteria. Furthermore, fingerprint analysis revealed multiple components of WeiNaiAn capsule, including calycosin glucoside, ginsenoside Rg1, ginsenoside Rb1, taurocholic acid sodium, formonetin, and calycosin glucoside. The components of WeiNaiAn capsule promoted the wound healing of the epithelial cell in vitro. Moreover, the components of WeiNaiAn capsule inhibited the protein expressions of phosphoinositide 3-kinase /protein kinase B /mammalian target of rapamycin in hydrogen peroxide or lipopolysaccharides-induced cell model. In conclusion, WeiNaiAn capsule improves intestinal mucosal injury by regulating cell migration, enhancing antioxidant activity, and promoting the structure of the bacterial community homeostasis, the multiple targets provide the parameters for the treatment in the clinic.

Sciadonic acid attenuates high-fat diet-induced bone metabolism disorders in mice.

High-fat diet (HFD) has been associated with certain negative bone-related outcomes, such as bone metabolism disruption and bone loss. Sciadonic acid (SC), one of the main nutritional and functional components of Torreya grandis seed oil, is a unique Δ5-unsaturated-polymethylene-interrupted fatty acid (Δ5-UPIFA) that has been claimed to counteract such disorders owing to some of its physiological effects. However, the role of SC in ameliorating bone metabolism disorders due to HFD remains unclear. In the present investigation, we observed that SC modulates the OPG/RANKL/RANK signaling pathway by modifying the lipid metabolic state and decreasing inflammation in mice. In turn, it could balance bone resorption and formation as well as calcium and phosphorus levels, enhance bone strength and bone mineral density (BMD), and improve its microstructure. In addition, SC could inhibit fat vacuoles in bone, reverse the phenomenon of reduced numbers and poor continuity of bone trabeculae, and promote orderly arrangement of collagen fibers and cartilage repair. This study provides some theoretical basis for SC as a dietary intervention agent to enhance bone nutrition.

Advancements of Macrophages Involvement in Pathological Progression of Colitis-Associated Colorectal Cancer and Associated Pharmacological Interventions.

Intestinal macrophages play crucial roles in both intestinal inflammation and immune homeostasis. They can adopt two distinct phenotypes, primarily determined by environmental cues. These phenotypes encompass the classically activated pro-inflammatory M1 phenotype, as well as the alternatively activated anti-inflammatory M2 phenotype. In regular conditions, intestinal macrophages serve to shield the gut from inflammatory harm. However, when a combination of genetic and environmental elements influences the polarization of these macrophages, it can result in an M1/M2 macrophage activation imbalance, subsequently leading to a loss of control over intestinal inflammation. This shift transforms normal inflammatory responses into pathological damage within the intestines. In patients with ulcerative colitis-associated colorectal cancer (UC-CRC), disorders related to intestinal inflammation are closely correlated with an imbalance in the polarization of intestinal M1/M2 macrophages. Therefore, reinstating the equilibrium in M1/M2 macrophage polarization could potentially serve as an effective approach to the prevention and treatment of UC-CRC. This paper aims to scrutinize the clinical evidence regarding Chinese medicine (CM) in the treatment of UC-CRC, the pivotal role of macrophage polarization in UC-CRC pathogenesis, and the potential mechanisms through which CM regulates macrophage polarization to address UC-CRC. Our objective is to offer fresh perspectives for clinical application, fundamental research, and pharmaceutical advancement in UC-CRC.

Effects of topography and stand spatial structure on the diameter at breast height growth of major pioneer tree species of natural broad-leaved mixed forests in Zhejiang Province, China.

Based on the continuous inventory data of forest resources in Zhejiang Province in 2019 and 2021, we used statistical methods such as polynomial regression to analyze the impacts of topography and forest spatial structure on average annual diameter at breast height (DBH) growth of main pioneer tree species in natural broad-leaved mixed forests. The results showed that DBH of Schima superba, Quercus glauca, Quercus fabri, Lithocarpus glaber, Castanopsis eyrei, and Castanopsis sclerophylla were between 5-50.8, 5-41.5, 5-50.8, 5-43.9, 5-55.5, and 5-46.1 cm, respectively. We classified all the trees into three classes based on DBH: small (6-12 cm), medium (12-14 cm), and large (>26 cm). The average annual DBH growth of S. superba and Q. glauca was the highest on semi-shady slope and shady slope, with increases of 2.9%-15.7% and 1.1%-41.2%, respectively. The average annual DBH growth of large-diameter S. superba, L. glaber, C. eyrei and C. sclerophylla decreased with the increase of slope, with a maximum decrease of 27.0% for S. superba, with no significant difference among small- and medium-diameter trees as a whole. The slope position did not affect the annual DBH growth of small-diameter trees, while that of medium- and large-diameter S. superba, Q. glauca, and large-diameter Q. fabri, L. glaber decreased with the change of slope position from downhill, mesoslope, uphill to ridge, with a maximum decrease of 28.1% for L. glabe, and the major-diameter C. eyrei was on the contrary. Appropriate increase in the mingling was beneficial to the average annual DBH growth of medium- and large-diameter trees. Moderate mixing was suitable for S. superba, while low degree mixing and moderate mixing was suitable for Q. glauca, Q. fabri and L. glaber, and intensive mixing was suitable for C. eyrei and C. sclerophylla. No significant difference was observed for minor-diameter trees under the mingling. The neighborhood comparison only had a significant effect on the average annual DBH growth of large-diameter Q. glauca, Q. fabri, and L. glaber, which was significantly higher under subdominance-moderation than moderation-inferiority. The average annual DBH growth in the study area was mainly affected by aspect and mixing degree.

Sinomenine regulates the cholinergic anti-inflammatory pathway to inhibit TLR4/NF-κB pathway and protect the homeostasis in brain and gut in scopolamine-induced Alzheimer's disease mice.

Sinomenine (SIN), an alkaloid extracted from the Chinese herbal medicine Sinomenium acutum, has great potential in anti-inflammatory, immune regulation, analgesic and sedative, and is already a clinical drug for the treatment of rheumatoid arthritis in China. Our previous studies show SIN inhibits inflammation by regulating ɑ7nAChR, a key receptor of cholinergic anti-inflammatory pathway (CAP), which plays an important role in regulating peripheral and central nervous system inflammation. Growing evidence supports the cholinergic dysregulation and inflammatory responses play the key role in the pathogenesis of AD. The intervention effects of SIN on AD by regulating CAP and homeostasis in brain and gut were analyzed for the first time in the present study using scopolamine-induced AD model mice. Behavioral tests were used to assess the cognitive performance. The neurons loss, cholinergic function, inflammation responses, biological barrier function in the mouse brain and intestinal tissues were evaluated through a variety of techniques, and the gut microbiota was detected using 16SrRNA sequencing. The results showed that SIN significantly inhibited the cognitive decline, dysregulation of cholinergic system, peripheral and central inflammation, biological barrier damage as well as intestinal flora disturbance caused by SCOP in mice. More importantly, SIN effectively regulated CAP to suppress the activation of TLR4/NF-κB and protect the homeostasis in brain and gut to alleviate cognitive impairment.

Bushen-Yizhi formula exerts neuroprotective effect via inhibiting excessive mitophagy in rats with chronic cerebral hypoperfusion.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen-Yizhi formula (BSYZ), a traditional Chinese medicine (TCM) prescription widely used in treating mental retardation and neurodegenerative diseases with kidney deficiency, has been reported to attenuate oxidative stress-related neuronal apoptosis. Chronic cerebral hypoperfusion (CCH) is considered to be related to cognitive and emotional disorders. However, it remains to be clarified that the effect of BSYZ on CCH and its underlying mechanism. AIM OF THE STUDY: In the present study, we aimed to investigate the therapeutic effects and underlying mechanisms of BSYZ on CCH- injured rats based on the domination of oxidative stress balance and mitochondrial homeostasis through inhibiting abnormal excessive mitophagy. MATERIALS AND METHODS: The in vivo rat model of CCH was established by bilateral common carotid artery occlusion (BCCAo), while the in vitro PC12 cell model was exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) condition, and a mitophagy inhibitor (chloroquine) by decreasing autophagosome-lysosome fusion was used as reverse validation in vitro. The protective role of BSYZ on CCH-injured rats was measured by open field test, morris water maze test, analysis of amyloid fibrils and apoptosis, and oxidative stress kit. The expression of mitochondria-related and mitophagy-related proteins was evaluated by Western blot, immunofluorescence, JC-1 staining assay and Mito-Tracker Red CMXRos assay. The components of BSYZ extracts were identified by HPLC-MS. The molecular docking studies were used to investigate the potential interactions of characteristic compounds in BSYZ with lysosomal membrane protein 1 (LAMP1). RESULTS: Our result indicated that BSYZ improved the cognition and memory abilities of the BCCAo rats by diminishing the occurrence of apoptosis and abnormal amyloid deposition accumulation, suppressing oxidative stress damage for abnormal excessive mitophagy activation in the hippocampus. Moreover, in OGD/R-damaged PC12 cells, BSYZ drug serum treatment substantially enhanced the PC12 cell viability and suppressed intracellular reactive oxygen species (ROS) accumulation for protecting against oxidative stress, along with the improvement of mitochondrial membrane activity and lysosomal proteins. Our studies also showed that inhibiting of autophagosome-lysosome fusion to generate autolysosomes by using chloroquine abrogated the neuroprotective effects of BSYZ on PC12 cells regarding the modulation of antioxidant defence and mitochondrial membrane activity. Furthermore, the molecular docking studies supported the direct bindings between lysosomal associated membrane protein 1 (LAMP1) and compounds in BSYZ extract to inhibit excessive mitophagy. CONCLUSION: Our study demonstrated that BSYZ played a neuroprotective role in rats with CCH and reduced neuronal oxidative stress via promoting the formation of autolysosomes to inhibit abnormal excessive mitophagy.

Developability profiling of a panel of Fc engineered SARS-CoV-2 neutralizing antibodies.

To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography.

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

Effects of precipitation seasonal distribution on net ecosystem CO2 exchange over an alpine meadow in the southeastern Tibetan Plateau.

Ecosystem carbon balance might be affected by the variability of seasonal distribution of precipitation under global climate change. Using the eddy covariance (EC) technique, long-term observations of ecosystem net CO2 exchange (NEE) were acquired over Lijiang alpine meadow in the southeastern Tibetan Plateau from January 2014 to August 2019. During the wet season (from June to October), Lijiang meadow functioned as a carbon sink (- 37.6 ± 22.5 g C m-2 month-1), while in dry season, the meadow varied between a weak carbon source and sink with an average monthly NEE of - 3.9 ± 11.9 g C m-2 month-1. Monthly CO2 fluxes were mainly controlled by air temperature and soil water content. A large annual variation of CO2 uptake was observed. The annual NEE was - 140.3 g C m-2 year-1 in 2014 while - 247.0 g C m-2 year-1 in 2016. Correspondingly, the precipitation in wet season accounted 90% of annual precipitation in 2014 and 74% of that in 2016 despite the annual precipitation was larger than 1200 mm in both years. More precipitation in dry season can lead to longer period of net CO2 uptake, while more precipitation concentrated in wet season depressed the meadow's light response through the decrease of the magnitude of light-saturated net CO2 exchange (NEEsat) at the onset and the end of growing season.

Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1.

BACKGROUND: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of α7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on α7nAChR remain unclear. PURPOSE: To analyze the role of α7nAChR in inflammation and the effect and mechanism of SIN regulation of α7nAChR. METHODS: The effects of SIN on α7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting α7nAChR. The antagonists of α7nAChR, α-bungarotoxin (BTX) and mecamylamine (Me), were used to block α7nAChR. In RAW264.7 macrophage cells in vitro, α7nAChR short hairpin RNA (shRNA) was used to knock down α7nAChR. Macrophage polarization was analyzed by the detection of TNF-α, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines α7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. RESULTS: SIN decreased the levels of TNF-α, IL-6 and the expression of α7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the α7nAChR shRNA transfected RAW264.7 cells, compared with the control, α7nAChR was knocked down, and M1 phenotype markers (including TNF-α, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of α7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. CONCLUSIONS: SIN acts on α7nAChR to inhibit inflammatory responses and downregulates high expression of α7nAChR in vivo and in vitro. The increase of α7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Electrostatically Driven Protein-Protein Interactions: Quantitative Prediction of Second Osmotic Virial Coefficients to Aid Antibody Design.

Electrostatically driven attractions between proteins can result in issues for therapeutic protein formulations such as solubility limits, aggregation, and high solution viscosity. Previous work showed that a model monoclonal antibody displayed large and potentially problematic electrostatically driven attractions at typical pH (5-8) and ionic strength conditions (∼10-100 mM). Molecular simulations of a hybrid coarse-grained model (1bC/D, one bead per charged site and per domain) were used to predict potential point mutations to identify key charge changes (charge-to-neutral or charge-swap) that could greatly reduce the net attractive protein-protein self-interactions. A series of variants were tested experimentally with static and dynamic light scattering to quantify interactions and compared to model predictions at low and intermediate ionic strength. Differential scanning calorimetry and circular dichroism confirmed minimal impact on structural or thermal stability of the variants. The model provided quantitative/semiquantitative predictions of protein self-interactions compared to experimental results as well as showed which amino acid pairings or groups had the most impact.

Explore the Anti-Acne Mechanism of Licorice Flavonoids Based on Metabonomics and Microbiome.

Acne vulgaris is one of the most common inflammatory dermatoses in dermatological practice and can affect any gender or ethnic group. Although in previous studies, we had found that licorice flavonoids (LCF) play an anti-acne role by inhibiting PI3K-Akt signaling pathways and mitochondrial activity, the mechanism of LCF regulating skin metabolism, serum metabolism and skin microbes is still unclear. Here, we performed a full spectrum analysis of metabolites in the skin and serum using UHPLC-Triple TOF-MS. The results showed that LCF could treat acne by regulating the metabolic balance of amino acids, lipids and fatty acids in serum and skin. Similarly, we performed Illumina Hiseq sequencing of DNA from the skin microbes using 16S ribosomal DNA identification techniques. The results showed that LCF could treat acne by regulating the skin microbes to interfere with acne and make the microecology close to the normal skin state of rats. In summary, this study confirmed the anti-acne mechanism of LCF, namely by regulating metabolic balance and microbial balance. Therefore, this discovery will provide theoretical guidance for the preparation development and clinical application of the drug.

The value of positron emission tomography computed tomography in predicting invasiveness of ground glass nodules: A protocol for systematic review and meta-analysis.

BACKGROUND: The study was conducted to investigate the value of Positron emission tomography computed tomography (PET/CT) in predicting invasiveness of ground glass nodule (GGN) by the method of meta-analysis. METHODS: Two researchers independently searched for published literature on PET/CT diagnosis of GGN as of November 30, 2020. After extracting the data, RevMan5.3 was used to evaluate the quality of the included literature. The Stata14 software was used to test the heterogeneity of the original study that met the inclusion criteria, to calculate the combined sensitivity, specificity, positive likelihood ratio and negative likelihood ratio, the prior probability and posttest probability. The summary receiver operator characteristic curve was drawn and the area under the curve was calculated. Using Deeks funnel plot to evaluate publication bias. RESULTS: Five studies were finally included, including 298 GGN cases. The included studies had no obvious heterogeneity and publication bias. The combined sensitivity and specificity of PET/CT for predicting invasive adenocarcinoma presenting as GGN were 0.74 (95% confidence interval [CI]: 0.68-0.79), 0.82 (95% CI: 0.71-0.90), positive likelihood ratio and negative likelihood ratio were 4.1 (95% CI: 2.5-6.9), 0.32 (95% CI: 0.25-0.40), and the diagnostic odds ratio was 13 (95% CI: 7-26). The prior probability is 20%, the probability of GGN being invasive adenocarcinoma when PET/CT was negative was reduced to 7%, and the probability of GGN being invasive adenocarcinoma when PET/CT was positive was increased to 51%. The area under the curve of the summary receiver operator characteristic curve was 0.85. CONCLUSION: PET/CT has high diagnostic accuracy for invasive adenocarcinoma presenting as GGN.

Sinomenine increases adenosine A2A receptor and inhibits NF-κB to inhibit arthritis in adjuvant-induced-arthritis rats and fibroblast-like synoviocytes through α7nAChR.

Sinomenine (SIN) is a clinical drug for treating rheumatoid arthritis (RA) in China. Our previous study found SIN inhibited inflammation via alpha7 nicotinic acetylcholine receptor (α7nAChR) in macrophages in vitro. Adenosine receptor A2A has anti-inflammatory and immunosuppressive function. However, the mechanisms of SIN acting on α7nAChR and the effect on adenosine A2A receptor (A2A R) in RA are not clear. In the present study, the effects of SIN on adjuvant-induced-arthritis (AIA) rats in vivo and on fibroblast-like synoviocytes (FLSs) in vitro were investigated. Indomethacin (Indo) and methotrexate (MTX), the clinical anti-arthritis drugs, were used as controls. Nicotine (Nic), a specific agonist of α7nAChR, was used as a control for targeting α7nAChR. Alpha-bungarotoxin (α-BTX), the antagonist of α7nAChR or small interference RNA (siRNA) was used to block or knock down α7nAChR. Results showed that SIN decreased arthritis index, hind paw volume, erythrocyte sedimentation (ESR) and serum TNF-α in AIA rats, and α-BTX attenuated the earlier-mentioned effects of SIN and Nic, but not Indo and MTX. The expressions of A2A R in synovium declined in AIA rats, but remarkably increased after the intervention of SIN. The expression of A2A R decreased by LPS or TNF-α, but increased by SIN; cAMP also increased by SIN in FLSs in vitro. SIN inhibited the expression of MCP-1, IL-6, and vascular endothelial growth factor in LPS-induced FLSs. SIN inhibited the activation of NF-κB. Meanwhile, α-BTX or α7nAChR siRNA blocked the earlier-mentioned effects of SIN in FLSs. Results suggested the expressions of A2A R in synovium and FLSs are negatively correlated with the arthritis progression of AIA rats and the activation of FLSs. SIN increases A2A R and inhibits the activation of NF-κB pathway via α7nAChR in AIA rats and FLSs.