Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

Conditional deletion of Zeb1 in Csf1r+ cells reduces inflammatory response of the cornea to alkali burn.

ZEB1 is an essential factor in embryonic development. In adults, it is often highly expressed in malignant tumors with low expression in normal tissues. The major biological function of ZEB1 in developing embryos and progressing cancers is to transdifferentiate cells from an epithelial to mesenchymal phenotype; but what roles ZEB1 plays in normal adult tissues are largely unknown. We previously reported that the reduction of Zeb1 in monoallelic global knockout (Zeb1+/-) mice reduced corneal inflammation-associated neovascularization following alkali burn. To uncover the cellular mechanism underlying the Zeb1 regulation of corneal inflammation, we functionally deleted Zeb1 alleles in Csf1r+ myeloid cells using a conditional knockout (cKO) strategy and found that Zeb1 cKO reduced leukocytes in the cornea after alkali burn. The reduction of immune cells was due to their increased apoptotic rate and linked to a Zeb1-downregulated apoptotic pathway. We conclude that Zeb1 facilitates corneal inflammatory response by maintaining Csf1r+ cell viability.

Dissecting Drivers of Ozone Pollution during the 2022 Multicity Lockdowns in China Sheds Light on Future Control Direction.

In 2022, many Chinese cities experienced lockdowns and heatwaves. We analyzed ground and satellite data using machine learning to elucidate chemical and meteorological drivers of changes in O3 pollution in 27 major Chinese cities during lockdowns. We found that there was an increase in O3 concentrations in 23 out of 27 cities compared with the corresponding period in 2021. Random forest modeling indicates that emission reductions in transportation and other sectors, as well as the changes in meteorology, increased the level of O3 in most cities. In cities with over 80% transportation reductions and temperature fluctuations within -2 to 2 °C, the increases in O3 concentrations were mainly attributable to reductions in nitrogen oxide (NOx) emissions. In cities that experienced heatwaves and droughts, increases in the O3 concentrations were primarily driven by increases in temperature and volatile organic compound (VOC) emissions, and reductions in NOx concentrations from ground transport were offset by increases in emissions from coal-fired power generation. Despite 3-99% reduction in passenger volume, most cities remained VOC-limited during lockdowns. These findings demonstrate that to alleviate urban O3 pollution, it will be necessary to further reduce industrial emissions along with transportation sources and to take into account the climate penalty and the impact of heatwaves on O3 pollution.

Light-degradable nanocomposite hydrogels for antibacterial wound dressing applications.

Skin injuries infected by bacteria can cause life-threatening human diseases if not treated properly. In this work, we developed a light-degradable nanocomposite hydrogel to achieve both controlled antibiotic delivery and hydrogel degradation using light as the sole stimulus. Specifically, we incorporated triclosan-loaded, poly(N-isopropylacrylamide)-based nanogels (TCS-NGs) that exhibited potent antibacterial efficacy, into a light-degradable poly (ethylene glycol) (PEG)-based hydrogel matrix via simple physical entrapment method. Upon exposure to 365 nm light, the hydrogel matrix could rapidly degrade, which subsequently released the entrapped TCS-NGs into the surrounding environment. Our results demonstrated that TCS-NGs released from light-degradable nanocomposite hydrogels still possessed remarkable antibacterial efficacy by inhibiting the growth of Staphylococcus aureus both in solution (a fivefold reduction in optical density compared to the blank control) and on bacteria-infected porcine skins (a fivefold reduction in colony-forming units compared to the blank control). Finally, using an alamarBlue assay on human dermal fibroblasts, we determined that each component of the nanocomposite hydrogel exhibited excellent biocompatibility (>90% cell viability) and would not cause significant cytotoxicity. Overall, the fabricated light-degradable nanocomposite hydrogels could serve as novel material for antibacterial wound dressing applications.

A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.

Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance.

Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.

Metabolite and protein shifts in mature erythrocyte under hypoxia.

As the only cell type responsible for oxygen delivery, erythrocytes play a crucial role in supplying oxygen to hypoxic tissues, ensuring their normal functions. Hypoxia commonly occurs under physiological or pathological conditions, and understanding how erythrocytes adapt to hypoxia is fundamental for exploring the mechanisms of hypoxic diseases. Additionally, investigating acute and chronic mountain sickness caused by plateaus, which are naturally hypoxic environments, will aid in the study of hypoxic diseases. In recent years, increasingly developed proteomics and metabolomics technologies have become powerful tools for studying mature enucleated erythrocytes, which has significantly contributed to clarifying how hypoxia affects erythrocytes. The aim of this article is to summarize the composition of the cytoskeleton and cytoplasmic proteins of hypoxia-altered erythrocytes and explore the impact of hypoxia on their essential functions. Furthermore, we discuss the role of microRNAs in the adaptation of erythrocytes to hypoxia, providing new perspectives on hypoxia-related diseases.

Prevention of distal stent graft-induced new entry after endovascular repair for type B aortic dissection: A retrospective cohort study.

OBJECTIVES: Distal stent graft-induced new entry (dSINE) can occur after thoracic endovascular aortic repair (TEVAR) for type B aortic dissection (TBAD). In this study we aimed to compare the effectiveness of restrictive bare stent (RBS), tapered stent graft (TSG), and non-TSG in TEVAR in preventing dSINE after a midterm follow-up. METHODS: This retrospective cohort study included patients with TBAD who underwent TEVAR (June 2010 to December 2018). The occurrence of dSINE during follow-up was examined. Predictors of dSINE were determined using Fine-Gray regression with death as the competing event. Survival was evaluated using Cox proportional hazards regression. RESULTS: Finally, 364 patients were included: 111 with non-TSG TEVAR, 125 with TSG TEVAR, and 128 with TEVAR with RBS. After 54.5 months, incidences of dSINE in the 3 groups were 12.61%, 4.80%, and 1.56%, respectively (P = .002). On Fine-Gray regression adjusted for clinically relevant covariates, the expansion mismatch ratio (subdistribution hazard ratio, 1.09; 95% CI, 1.07-1.12; P < .001) and complete false lumen thrombosis (subdistribution hazard ratio, 0.35; 95% CI, 0.13-0.94; P = .037) were identified as predictors of dSINE. The Cox proportional hazards regression analysis revealed that dSINE was not only a risk factor for aortic-related mortality (hazard ratio, 17.90; 95% CI, 3.27-98.12; P = .001), but also a predominant risk factor for all-cause mortality (hazard ratio, 4.91; 95% CI, 1.66-14.52; P = .004). CONCLUSIONS: dSINE can happen in TBAD patients who undergo TEVAR. Thus, long-term surveillance is crucial. TSG and RBS had lower expansion mismatch ratios, which might help prevent dSINE.

Meshless Search SR-STAP for Airborne Radar Based on Meta-Heuristic Algorithms.

The sparse recovery (SR) space-time adaptive processing (STAP) method has excellent clutter suppression performance under the condition of limited observation samples. However, when the cluttering is nonlinear in a spatial-Doppler profile, it will cause an off-grid effect and reduce the sparse recovery performance. A meshless search using a meta-heuristic algorithm (MH) can completely eliminate the off-grid effect in theory. Therefore, genetic algorithm (GA), differential evolution (DE), particle swarm optimization (PSO), and grey wolf optimization (GWO) methods are applied to SR-STAP for selecting exact clutter atoms in this paper. The simulation results show that MH-STAP can estimate the clutter subspace more accurately than the traditional algorithm; PSO-STAP and GWO-STAP showed better clutter suppression performance in four MH-STAP methods. To search for more accurate clutter atoms, PSO and GWO are combined to improve the method's capacity for global optimization. Meanwhile, the fitness function is improved by using prior knowledge of the clutter distribution. The simulation results show that the improved PSO-GWO-STAP algorithm provides excellent clutter suppression performance, which solves the off-grid problem better than does single MH-STAP.

Recombinant human adenovirus p53 combined with transcatheter arterial chemoembolization for liver cancer: A meta-analysis.

OBJECTIVES: To compare the clinical curative effects, survival and complications of recombinant human adenovirus-p53 (rAd-p53) combined with transcatheter arterial chemoembolization (TACE) versus TACE for the treatment of liver cancer. METHODS: We searched all the eligible studies of rAd-p53 plus TACE versus control group had only TACE in the treatment of liver cancer, which were retrieved from CNKI, Wanfang database, CBM, VIP, PubMed, EMBase, The Chrance of Library, Web of Science from its inception to august 2022. RESULTS: A total of 17 studies were included, which involved 1045 patients. The results of the meta analysis indicated that the the rAd-p53combined with TACE markedly improved the patients' complete remission(OR = 2.19, 95% CI:1.13-4.22, P = 0.02), partial remission (OR = 2.22, 95% CI:1.67-2.94, P<0.00001), objective tumor response rate (OR = 2.58, 95% CI:1.95-3.41, P<0.00001) and disease control rate(OR = 2.39, 95% CI:1.65-3.47, P<0.00001) compared with TACE alone. And our results showed that rAd-p53combined with TACE had better survival benefit [6-month OS (OR = 3.41, 95% CI: 1.62-7.14, p = 0.001); 1-year OS (OR = 1.95, 95% CI: 1.28-2.96, p = 0.002)] and better quality of life(MD = 5.84, 95% CI:2.09-9.60, P = 0.002). In addition, the immunity of the patients was enhanced by the combination therapy, as demonstrated by the increase in the ratio of CD4+ to CD4+/CD8+. In adverse effects, except for fever in the TACE combined with rAd-p53 group, which was higher than that in the TACE group(OR = 2.62, 95% CI:2.02-3.49, P<0.00001), all other adverse effects were lower in the TACE combined with rAd-p53 group than in the TACE group. CONCLUSION: RAd-p53 combined with TACE for liver cancer showed significant advantages in terms of clinical efficacy, survival rate, and safety compared to the TACE alone, and effectively improved patient quality of life and immune function. SYSTEMATIC REVIEW REGISTRATION: https://inplasy.com/inplasy-2022-9-0127/.

Mouse Corneal Epithelial and Stromal Cell Isolation and Culture.

Corneal epithelium and stroma are the major cellular structures for ocular protection and vision accuracy; they play important roles in corneal wound healing and inflammation under pathological conditions. Unlike human, murine corneal and stromal fibroblast cells are difficult to isolate for cell culture. In our laboratory, we successfully used an ex vivo culture procedure and an enzymatic procedure to isolate, purify, and culture mouse corneal epithelial and stromal fibroblast cells. Key features • Primary cell culture models of a disease are critical for cellular and molecular mechanism studies. • Corneal tissues with the limbus contain stem cells to generate both epithelial and stromal cells. • An ex vivo corneal culture provides a constant generation of primary corneal cells for multiple passages. • The isolated cells are validated by the corneal epithelial cell markers Krt12 and Cdh1 and the stromal fibroblast marker Vim.

Double-Edged Role of VOCs Reduction in Nitrate Formation: Insights from Observations during the China International Import Expo 2018.

Aerosol nitrate (NO3-) constitutes a significant component of fine particles in China. Prioritizing the control of volatile organic compounds (VOCs) is a crucial step toward achieving clean air, yet its impact on NO3- pollution remains inadequately understood. Here, we examined the role of VOCs in NO3- formation by combining comprehensive field measurements conducted during the China International Import Expo (CIIE) in Shanghai (from 10 October to 22 November 2018) and multiphase chemical modeling. Despite a decline in primary pollutants during the CIIE, NO3- levels increased compared to pre-CIIE and post-CIIE─NO3- concentrations decreased in the daytime (by -10 and -26%) while increasing in the nighttime (by 8 and 30%). Analysis of the observations and backward trajectory indicates that the diurnal variation in NO3- was mainly attributed to local chemistry rather than meteorological conditions. Decreasing VOCs lowered the daytime NO3- production by reducing the hydroxyl radical level, whereas the greater VOCs reduction at night than that in the daytime increased the nitrate radical level, thereby promoting the nocturnal NO3- production. These results reveal the double-edged role of VOCs in NO3- formation, underscoring the need for transferring large VOC-emitting enterprises from the daytime to the nighttime, which should be considered in formulating corresponding policies.

Predicting ventricular tachycardia circuits in patients with arrhythmogenic right ventricular cardiomyopathy using genotype-specific heart digital twins.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that leads to ventricular tachycardia (VT), a life-threatening heart rhythm disorder. Treating ARVC remains challenging due to the complex underlying arrhythmogenic mechanisms, which involve structural and electrophysiological (EP) remodeling. Here, we developed a novel genotype-specific heart digital twin (Geno-DT) approach to investigate the role of pathophysiological remodeling in sustaining VT reentrant circuits and to predict the VT circuits in ARVC patients of different genotypes. This approach integrates the patient's disease-induced structural remodeling reconstructed from contrast-enhanced magnetic-resonance imaging and genotype-specific cellular EP properties. In our retrospective study of 16 ARVC patients with two genotypes: plakophilin-2 (PKP2, n = 8) and gene-elusive (GE, n = 8), we found that Geno-DT accurately and non-invasively predicted the VT circuit locations for both genotypes (with 100%, 94%, 96% sensitivity, specificity, and accuracy for GE patient group, and 86%, 90%, 89% sensitivity, specificity, and accuracy for PKP2 patient group), when compared to VT circuit locations identified during clinical EP studies. Moreover, our results revealed that the underlying VT mechanisms differ among ARVC genotypes. We determined that in GE patients, fibrotic remodeling is the primary contributor to VT circuits, while in PKP2 patients, slowed conduction velocity and altered restitution properties of cardiac tissue, in addition to the structural substrate, are directly responsible for the formation of VT circuits. Our novel Geno-DT approach has the potential to augment therapeutic precision in the clinical setting and lead to more personalized treatment strategies in ARVC.

Predicting Ventricular Tachycardia Circuits in Patients with Arrhythmogenic Right Ventricular Cardiomyopathy using Genotype-specific Heart Digital Twins.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that leads to ventricular tachycardia (VT), a life-threatening heart rhythm disorder. Treating ARVC remains challenging due to the complex underlying arrhythmogenic mechanisms, which involve structural and electrophysiological (EP) remodeling. Here, we developed a novel genotype-specific heart digital twin (Geno-DT) approach to investigate the role of pathophysiological remodeling in sustaining VT reentrant circuits and to predict the VT circuits in ARVC patients of different genotypes. This approach integrates the patient's disease-induced structural remodeling reconstructed from contrast-enhanced magnetic-resonance imaging and genotype-specific cellular EP properties. In our retrospective study of 16 ARVC patients with two genotypes: plakophilin-2 (PKP2, n = 8) and gene-elusive (GE, n = 8), we found that Geno-DT accurately and non-invasively predicted the VT circuit locations for both genotypes (with 100%, 94%, 96% sensitivity, specificity, and accuracy for GE patient group, and 86%, 90%, 89% sensitivity, specificity, and accuracy for PKP2 patient group), when compared to VT circuit locations identified during clinical EP studies. Moreover, our results revealed that the underlying VT mechanisms differ among ARVC genotypes. We determined that in GE patients, fibrotic remodeling is the primary contributor to VT circuits, while in PKP2 patients, slowed conduction velocity and altered restitution properties of cardiac tissue, in addition to the structural substrate, are directly responsible for the formation of VT circuits. Our novel Geno-DT approach has the potential to augment therapeutic precision in the clinical setting and lead to more personalized treatment strategies in ARVC.

Multiplex-GAM: genome-wide identification of chromatin contacts yields insights overlooked by Hi-C.

Technology for measuring 3D genome topology is increasingly important for studying gene regulation, for genome assembly and for mapping of genome rearrangements. Hi-C and other ligation-based methods have become routine but have specific biases. Here, we develop multiplex-GAM, a faster and more affordable version of genome architecture mapping (GAM), a ligation-free technique that maps chromatin contacts genome-wide. We perform a detailed comparison of multiplex-GAM and Hi-C using mouse embryonic stem cells. When examining the strongest contacts detected by either method, we find that only one-third of these are shared. The strongest contacts specifically found in GAM often involve 'active' regions, including many transcribed genes and super-enhancers, whereas in Hi-C they more often contain 'inactive' regions. Our work shows that active genomic regions are involved in extensive complex contacts that are currently underestimated in ligation-based approaches, and highlights the need for orthogonal advances in genome-wide contact mapping technologies.

Zeb1 facilitates corneal epithelial wound healing by maintaining corneal epithelial cell viability and mobility.

The cornea is the outmost ocular tissue and plays an important role in protecting the eye from environmental insults. Corneal epithelial wounding provokes pain and fear and contributes to the most ocular trauma emergency assessments worldwide. ZEB1 is an essential transcription factor in development; but its roles in adult tissues are not clear. We identify Zeb1 is an intrinsic factor that facilitates corneal epithelial wound healing. In this study, we demonstrate that monoallelic deletion of Zeb1 significantly expedites corneal cell death and inhibits corneal epithelial EMT-related cell migration upon an epithelial debridement. We provide evidence that Zeb1-regulation of corneal epithelial wound healing is through the repression of genes required for Tnfa-induced epithelial cell death and the induction of genes beneficial for epithelial cell migration. We suggest utilizing TNF-α antagonists would reduce TNF/TNFR1-induced cell death in the corneal epithelium and inflammation in the corneal stroma to help corneal wound healing.

Immunological effect of Lactic acid bacteria adjuvant on in ovo injection of Newcastle disease vaccine.

In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against various viral pathogens. The immunogenic efficacies of in ovo administration of lactic acid bacteria (LAB) in combination with live Newcastle disease (ND) vaccine were investigated in this study. Four hundred healthy 1-day-old fertilized specific pathogen-free (SPF) eggs of similar weights were randomly assigned to one of four treatments, with five replicates of each treatment and a total of 20 for each replicate. On day 18.5 of incubation, in ovo injections were given. The treatment groups are as follows: (I) no injection, (II) 0.9% physiological saline injection, (III) ND vaccine injection, and (IV) LAB as an adjuvant for ND vaccine injection. The ND vaccine adjuvanted with LAB significantly increased the daily weight gain, immune organ index, and small intestine histomorphological development in layer chicks while decreasing the feed conversion ratio (FCR). The results suggested that the LAB-adjuvant group significantly affected the relative expression of mucosal mucin protein (mucin-1) and zoccluding small circle protein-1 (ZO-1) (P < 0.05), whereas the relative expression of occludin mRNA was not significantly affected (P > 0.05) compared with the non-injected group. Meanwhile, we indicated that intra-amniotic synbiotic injection significantly maintained the balance of flora (P < 0.05). Compared with the non-injected group, the ND vaccine adjuvanted with the LAB group exhibited significant promotion of the HI and SIgA antibody titers in serum on day 21 (P < 0.05), induction of higher production of cytokines (IL-2, IL-4, IL-6, IFN-γ) in serum. In summary, in ovo injection of ND vaccine adjuvanted with LAB has a positive impact on the growth performance, immune function, and microbiome of growing chicks.

Novel Detection of Atmospheric Turbulence Profile Using Mie-Scattering Lidar Based on Non-Kolmogorov Turbulence Theory.

Turbulence can cause effects such as light intensity fluctuations and phase fluctuations when a laser is transmitted in the atmosphere, which has serious impacts on a number of optical engineering application effects and on climate improvement. Therefore, accurately obtaining real-time turbulence intensity information using lidar-active remote sensing technology is of great significance. In this paper, based on residual turbulent scintillation theory, a Mie-scattering lidar method was developed to detect atmospheric turbulence intensity. By extracting light intensity fluctuation information from a Mie-scattering lidar return signal, the atmospheric refractive index structure constant, Cn2, representing the atmospheric turbulence intensity, could be obtained. Specifically, the scintillation effect on the detection path was analyzed, and the probability density distribution of the light intensity of the Mie-scattering lidar return signal was studied. It was verified that the probability density of logarithmic light intensity basically follows a normal distribution under weak fluctuation conditions. The Cn2 profile based on Kolmogorov turbulence theory was retrieved using a layered, iterative method through the scintillation index. The method for detecting Kolmogorov turbulence intensity was applied to the detection of the non-Kolmogorov turbulence intensity. Through detection using the scintillation index, the corresponding C˜n2 profile could be calculated. The detection of the C˜n2 and Cn2 profiles were compared with the Hufnagel-Valley (HV) night model in the Yinchuan area. The results show that the detection results are consistent with the overall change trend of the model. In general, it is feasible to detect a non-Kolmogorov turbulence profile using Mie-scattering lidar.