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The efficient use of livestock and poultry manure waste has become a global challenge, with microorganisms playing an important role. To investigate the impact of novel ammonifying microorganism cultures (NAMC) on microbial community dynamics and carbon and nitrogen metabolism, five treatments [5% (v/w) sterilized distilled water, Amm-1, Amm-2, Amm-3, and Amm-4] were applied to cow manure compost. Inoculation with NAMC improved the structure of bacterial and fungal communities, enriched the populations of the functional microorganisms, enhanced the role of specific microorganisms, and promoted the formation of tight modularity within the microbial network. Further functional predictions indicated a significant increase in both carbon metabolism (CMB) and nitrogen metabolism (NMB). During the thermophilic phase, inoculated NAMC treatments boosted carbon metabolism annotation by 10.55%-33.87% and nitrogen metabolism annotation by 26.69%-63.11. Structural equation modeling supported the NAMC-mediated enhancement of NMB and CMB. In conclusion, NAMC inoculation, particularly with Amm-4, enhanced the synergistic interaction between bacteria and fungi. This collaboration promoted enzymatic catabolic and synthetic processes, resultng in positive feedback loops with the endogenous microbial community. Understanding these mechanisms not only unravels how ammonifying microorganisms influence microbial communities but also paves the way for the development of the composting industry and global waste management practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 μm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.

Excavating nitrogen-fixing bacteria with high-temperature tolerance is essential for the efficient composting of animal dung. In this study, two strains of thermophilic nitrogen-fixing bacteria, NF1 (Bacillus subtilis) and NF2 (Azotobacter chroococcum), were added to cow dung compost both individually (NF1, NF2) and mixed together (NF3; mixing NF1 and NF2 at a ratio of 1:1). The results showed that NF1, NF2, and NF3 inoculants increased the total Kjeldahl nitrogen level by 38.43%-55.35%, prolonged the thermophilic period by 1-13 d, increased the seed germination index by 17.81%, and the emissions of NH 3 and N 2 O were reduced by 25.11% and 42.75%, respectively. Microbial analysis showed that Firmicutes were the predominant bacteria at the thermophilic stage, whereas Chloroflexi, Proteobacteria, and Bacteroidetes were the predominant bacteria at the mature stage. These results confirmed that the addition of the isolated strains to cow dung composting improved the bacterial community structure and benefited nitrogen retention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The origin of T cells in the teleost's brain is unclear. While viewing the central nervous system (CNS) as immune privileged has been widely accepted, previous studies suggest that T cells residing in the thymus but not in the spleen of the teleost play an essential role in communicating with the peripheral organs. Here, we identified nine T cell subpopulations in the thymus and spleen of orange-spotted grouper (Epinephelus coioices) through single-cell RNA-sequencing analysis. After viral CNS infection with red-spotted grouper nervous necrosis virus (RGNNV), the number of slc43a2 + T cells synchronously increased in the spleen and brain. During the infection tests in asplenic zebrafish (tlx1 ▲ zebrafish model), no increase in the number of slc43a2 + T cells was observed in the brain. Single-cell transcriptomic analysis indicated that slc43a2 + T cells mature and functionally differentiate within the spleen and then migrate into the brain to trigger an immune response. This study suggests a novel route for T cell migration from the spleen to the brain during viral infection in fish., (© 2024. Science China Press.)

Background: Given the lack of research on disulfidptosis, our study aimed to dissect its role in pan-cancer and explore the crosstalk between disulfidptosis and cancer immunity., Methods: Based on TCGA, ICGC, CGGA, GSE30219, GSE31210, GSE37745, GSE50081, GSE22138, GSE41613, univariate Cox regression, LASSO regression, and multivariate Cox regression were used to construct the rough gene signature based on disulfidptosis for each type of cancer. SsGSEA and Cibersort, followed by correlation analysis, were harnessed to explore the linkage between disulfidptosis and cancer immunity. Weighted correlation network analysis (WGCNA) and Machine learning were utilized to make a refined prognosis model for pan-cancer. In particular, a customized, enhanced prognosis model was made for glioma. The siRNA transfection, FACS, ELISA, etc., were employed to validate the function of c-MET., Results: The expression comparison of the disulfidptosis-related genes (DRGs) between tumor and nontumor tissues implied a significant difference in most cancers. The correlation between disulfidptosis and immune cell infiltration, including T cell exhaustion (Tex), was evident, especially in glioma. The 7-gene signature was constructed as the rough model for the glioma prognosis. A pan-cancer suitable DSP clustering was made and validated to predict the prognosis. Furthermore, two DSP groups were defined by machine learning to predict the survival and immune therapy response in glioma, which was validated in CGGA. PD-L1 and other immune pathways were highly enriched in the core blue gene module from WGCNA. Among them, c-MET was validated as a tumor driver gene and JAK3-STAT3-PD-L1/PD1 regulator in glioma and T cells. Specifically, the down-regulation of c-MET decreased the proportion of PD1+ CD8+ T cells., Conclusion: To summarize, we dissected the roles of DRGs in the prognosis and their relationship with immunity in pan-cancer. A general prognosis model based on machine learning was constructed for pan-cancer and validated by external datasets with a consistent result. In particular, a survival-predicting model was made specifically for patients with glioma to predict its survival and immune response to ICIs. C-MET was screened and validated for its tumor driver gene and immune regulation function (inducing t-cell exhaustion) in glioma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Wang, Wan, Huang, Yin, Sun, Jin and Wang.)

For a potential application of FK506 in the treatment of acute kidney failure only the FKBP12 binding capability of the compound is required, while the immunosuppressive activity via calcineurin binding is considered as a likely risk to the patients. The methoxy groups at C13 and C15 are thought to have significant influence on the immunosuppressive activity of the molecule. Consequently, FK506 analogs with different functionalities at C13 and C15 were generated by targeted CRISPR editing of the AT domains in module 7 and 8 of the biosynthetic assembly line in Streptomyces tsukubaensis. In addition, the corresponding FK520 (C21 ethyl derivative of FK506) analogs could be obtained by media adjustments. The compounds were tested for their bioactivity in regards to FKBP12 binding, BMP potentiation and calcineurin sparing. 15-desmethoxy FK506 was superior to the other tested analogs as it did not inhibit calcineurin but retained high potency towards FKBP12 binding and BMP potentiation., (© 2023 Wiley-VCH GmbH.)

Iridoviruses are large DNA viruses that infect a wide range of invertebrates and lower vertebrates, causing serious threats to ecological security and aquaculture industry worldwide. However, the mechanisms underlying intracellular transport of iridovirus remain unknown. In this study, the transport of Singapore grouper iridovirus (SGIV) in early endosomes (EEs) and late endosomes (LEs) was explored by single-particle tracking technology. SGIV employs EEs to move rapidly from the cell membrane to the nucleus, and this long-range transport is divided into "slow-fast-slow" stages. SGIV within LEs mainly underwent oscillatory movements near the nucleus. Furthermore, SGIV entered newly formed EEs and LEs, respectively, possibly based on the interaction between the viral major capsid protein and Rab5/Rab7. Importantly, interruption of EEs and LEs by the dominant negative mutants of Rab5 and Rab7 significantly inhibited the movement of SGIV, suggesting the important roles of Rab5 and Rab7 in virus transport. In addition, it seems that SGIV needs to enter clathrin-coated vesicles to move from actin to microtubules before EEs carry the virus moving along microtubules. Together, our results for the first time provide a model whereby iridovirus transport depending on EEs and LEs, helping to clarify the mechanism underlying iridovirus infection, and provide a convenient tactic to investigate the dynamic infection of large DNA virus., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests or personal relationships regarding the publication of this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens, respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo, implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of CcCD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that CcCD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of CcCD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after CcCD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, CcCD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that CcCD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Rab32 is a member of the Rab GTPase family that is involved in membrane trafficking and immune response, which are crucial for controlling pathogen infection. However, the role of Rab32 in virus infection is not well understood. In this study, we focused on the regulation of Rab32 on virus infection and the host immunity in orange-spotted grouper, Epinephelus coioides. EcRab32 encoded a 213-amino acid polypeptide, which shared a high sequence identity with other Rab32 proteins from fishes to mammals. In healthy orange-spotted grouper, the mRNA of EcRab32 was expressed in all the detected tissues, with the more expression levels in the head kidney, liver and gill. Upon SGIV infection, the expression of EcRab32 was significantly up-regulated in vitro, indicating its potential role in viral infection. EcRab32 was observed to be distributed in the cytoplasm as punctate and vesicle-like structures. EcRab32 overexpression was found to notably inhibit SGIV infection, while the interruption of EcRab32 significantly promoted SGIV infection. In addition, using single particle imaging analysis, we found that EcRab32 overexpression prominently reduced the attachment and internalization of SGIV particles. Furthermore, the results demonstrated that EcRab32 played a positive role in regulating the interferon immune and inflammatory responses. Taken together, these findings indicated that EcRab32 influenced SGIV infection by regulating the host immune response, providing an overall understanding of the interplay between the Rab32 and innate immunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Due to a lack of research on the critical non-coding RNAs in regulating ferroptosis, our study aimed to uncover the crucial ones involved in the process. We found that LINC01133 could make pancreatic cancer cells more resistant to ferroptosis. A higher expression of LINC01133 was associated with a higher IC50 of sorafenib in clinical samples. Furthermore, we discovered that LINC01133 induced this process through enhancing the mRNA stability of FSP1. CEBPB was the transcription factor to increase the expression of LINC01133. A higher CEBPB could also indicate a higher IC50 of sorafenib in patients with cancer. Moreover, we confirmed that LINC01133 could form a triple complex with FUS and FSP1 to increase the mRNA stability of FSP1., (© 2023. The Author(s).)

Real-time and efficient driver distraction detection is of great importance for road traffic safety and assisted driving. The design of a real-time lightweight model is crucial for in-vehicle edge devices that have limited computational resources. However, most existing approaches focus on lighter and more efficient architectures, ignoring the cost of losing tiny target detection performance that comes with lightweighting. In this paper, we present MTNet, a lightweight detector for driver distraction detection scenarios. MTNet consists of a multidimensional adaptive feature extraction block, a lightweight feature fusion block and utilizes the IoU-NWD weighted loss function, all while considering the accuracy gain of tiny target detection. In the feature extraction component, a lightweight backbone network is employed in conjunction with four attention mechanisms strategically integrated across the kernel space. This approach enhances the performance limits of the lightweight network. The lightweight feature fusion module is designed to reduce computational complexity and memory access. The interaction of channel information is improved through the use of lightweight arithmetic techniques. Additionally, CFSM module and EPIEM module are employed to minimize redundant feature map computations and strike a better balance between model weights and accuracy. Finally, the IoU-NWD weighted loss function is formulated to enable more effective detection of tiny targets. We assess the performance of the proposed method on the LDDB benchmark. The experimental results demonstrate that our proposed method outperforms multiple advanced detection models.

It is essential to reduce nitrogen losses and to improve nitrogen conversion during organic waste composting because of environmental protection and sustainable development. To reveal newly domesticated ammonifying microorganisms (AM) cultures on the ammonification and nitrogen conversion during the composting, the screened microbial agents were inoculated at 5 % concentration (in weight basis) into cow manure compost under five different treatments: sterilized distilled water (Control), Amm-1 (mesophilic fungus-F1), Amm-2 (mesophilic bacterium-Z1), Amm-3 (thermotolerant bacterium-Z2), and Amm-4 (consortium: F1, Z1, and Z2), and composted for 42 days. Compared to control, AM inoculation prolonged the thermophilic phases to 9-19 days, increased the content of NH 4 + -N to 1.60-1.96 g/kg in the thermophilic phase, reduced N 2 O and NH 3 emissions by 22.85-61.13 % and 8.45-23.29 %, increased total Kjeldahl nitrogen, and improved cell count and viability by 12.09-71.33 % and 66.71-72.91 %. AM was significantly associated with different nitrogen and microbial compositions. The structural equation model (SEM) reveals NH 4 + -N is the preferable nitrogen for the majority of bacterial and fungal growth and that AM is closely associated with the conversion between NH 3 and NH 4 + -N. Among the treatments, inoculation with Amm-4 was more effective, as it significantly enhanced the driving effect of the critical microbial composition on nitrogen conversion and accelerated nitrogen ammonification and sequestration. This study provided new concepts for the dynamics of microbial in the ammonification process of new AM bacterial agents in cow manure compost, and an understanding of the ecological mechanism underlying the ammonification process and its contribution to nitrogen (N) cycling from the perspective of microbial communities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The hydroxyl group was discovered to promote gold catalyzed hydrofluorination of alkynes via hydrogen bonding interaction. Based on this strategy, propargyl alcohols could be hydrofluorinated smoothly using Et 3 N·3HF under acidic additive-free conditions, which provided a straightforward alternative protocol for the synthesis of 3-fluoroallyl alcohols.

The black sea bass, Centropristis striata , is a potential candidate for commercial aquaculture. Due to inadequate removal of nitrogen in its breeding environment, C. striata exhibits increased nitrate concentration, which can cause acute toxicity, including energy metabolism damage and tissue damage. Therefore, RNA-seq technology was applied to characterize genes associated with toxicity tolerance under nitrate stress. The nitrate treatment caused significant changes in a total of 8920 genes, of which 2949 genes were up-regulated and 5971 genes were down-regulated. It was found that significantly enriched GO terms and KEGG were associated with blood microparticles, inhibitors of enzyme activity, and complement and coagulation cascade pathways. Furthermore, through bioinformatics analysis, it was found that these different pathways obtained in GO and KEGG enrichment analysis were mostly related to the immune and inflammatory response of fish. This study expands our understanding of the mechanism of nitrate stress affecting the liver function of C. striata .

Objective: To analyze the clinical features and genotype of a child with Schmid type metaphyseal chondrodysplasia., Methods: Clinical data of the child and her parents was collected. The child was subjected to high-throughput sequencing, and candidate variant was verified by Sanger sequencing of her family members., Results: Whole exome sequencing revealed that the child has harbored a heterozygous c.1772G>A (p.C591Y) variant of the COL10A1 gene, which was not found in either of her parents. The variant was not found in the HGMD and ClinVar databases, and was rated as likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG)., Conclusion: The heterozygous c.1772G>A (p.C591Y) variant of the COL10A1 gene probably underlay the Schmid type metaphyseal chondrodysplasia in this child. Genetic testing has facilitated the diagnosis and provided a basis for genetic counselling and prenatal diagnosis for this family. Above finding has also enriched the mutational spectrum of the COL10A1 gene.

Background: Immunogenic cell death (ICD) is a result of immune cell infiltration (ICI)-mediated cell death, which is also a novel acknowledgment to regulate cellular stressor-mediated cell death, including drug therapy and radiotherapy., Methods: In this study, TCGA and GEO data cohorts were put into artificial intelligence (AI) to identify ICD subtypes, and in vitro experiments were performed., Results: Gene expression, prognosis, tumor immunity, and drug sensitivity showed significance among ICD subgroups, Besides, a 14-gene-based AI model was able to represent the genome-based drug sensitivity prediction, which was further verified in clinical trials. Network analysis revealed that PTPRC was the pivotal gene in regulating drug sensitivity by regulating CD8+ T cell infiltration. Through in vitro experiments, intracellular down-regulation of PTPRC enhanced paclitaxel tolerance in triple breast cancer (TNBC) cell lines. Meanwhile, the expression level of PTPRC was positively correlated with CD8+ T cell infiltration. Furthermore, the down-regulation of PTPRC increased the level of TNBC-derived PD-L1 and IL2., Discussion: ICD-based subtype clustering of pan-cancer was helpful to evaluate chemotherapy sensitivity and immune cell infiltration, and PTPRC was a potential target to against drug resistance of breast cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Wang, Wang, Cao, Pan, Huang, Wan, Zhang, Huang, Jin and Wang.)

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acids protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Predicting and preparing for the trajectory of disease epidemics relies on a knowledge of environmental and socioeconomic factors that affect transmission rates on local and global spatial scales. This article discusses the simulation of epidemic outbreaks on human metapopulation networks with community structure, such as cities within national boundaries, for which infection rates vary both within and between communities. We demonstrate mathematically, through next-generation matrices, that the structures of these communities, setting aside all other considerations such as disease virulence and human decision-making, have a profound effect on the reproduction rate of the disease throughout the network. In high modularity networks, with high levels of separation between neighboring communities, disease epidemics tend to spread rapidly in high-risk communities and very slowly in others, whereas in low modularity networks, the epidemic spreads throughout the entire network as a steady pace, with little regard for variations in infection rate. The correlation between network modularity and effective reproduction number is stronger in population with high rates of human movement. This implies that the community structure, human diffusion rate, and disease reproduction number are all intertwined, and the relationships between them can be affected by mitigation strategies such as restricting movement between and within high-risk communities. We then test through numerical simulation the effectiveness of movement restriction and vaccination strategies in reducing the peak prevalence and spread area of outbreaks. Our results show that the effectiveness of these strategies depends on the structure of the network and the properties of the disease. For example, vaccination strategies are most effective in networks with high rates of diffusion, whereas movement restriction strategies are most effective in networks with high modularity and high infection rates. Finally, we offer guidance to epidemic modelers as to the ideal spatial resolution to balance accuracy and data collection costs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

During the COVID-19 pandemic, many patients could not receive timely healthcare services due to limited availability and access to healthcare resources and services. Previous studies found that access to intensive care unit (ICU) beds saves lives, but they overlooked the temporal dynamics in the availability of healthcare resources and COVID-19 cases. To fill this gap, our study investigated daily changes in ICU bed accessibility with an enhanced two-step floating catchment area (E2SFCA) method in the state of Texas. Along with the increased temporal granularity of measurements, we uncovered two phenomena: 1) aggravated spatial inequality of access during the pandemic, and 2) the retrospective relationship between insufficient ICU bed accessibility and the high case-fatality ratio of COVID-19 in rural areas. Our findings suggest that those locations should be supplemented with additional healthcare resources to save lives in future pandemic scenarios., (© 2023 Elsevier Ltd. All rights reserved.)

Dairy goats are the goats bred with the ability to produce large quantities of milk, and the increase of the female kid rate of breeding dairy goats is beneficial for milk production and economic benefits of dairy goat farms. Our previous study revealed that regulating the pH of dairy goat semen diluent to 6.2 or 7.4 respectively, the proportion of X chromosome bearing sperm (X-sperm) in the up and down layers of the tube after incubation was significantly higher than that of Y chromosome bearing sperm (Y-sperm) i.e. enriched X-sperm. In this study, fresh dairy goat semen collected in different seasons was diluted in different pH solutions to calculate the number and rate of X-sperm and to measure the functional parameters of enriched sperm. The artificial insemination experiments were performed with enriched X-sperm. The mechanisms of regulating the pH of diluent affecting sperm enrichment were further studied. The results showed that the proportion of enriched X-sperm in pH 6.2 and 7.4 diluents of sperm collected in different seasons showed no significantly different, but were significantly higher than that of the control group (pH 6.8). The in vitro functional parameters of X-sperm enriched in pH 6.2 and 7.4 diluent solution were not significantly different from those of the control group (P > 0.05). After artificial insemination with X-sperm enriched in pH7.4 diluent, the proportion of female offspring was significantly higher than that of the control group. It was found that the regulating pH of the diluent affected sperm mitochondrial activity and glucose uptake capacity via phosphorylating NF-κB and GSK3α/β proteins. The motility activity of X-sperm was enhanced under acidic conditions and weakened under alkaline conditions, which was conducive to the effective enrichment of X-sperm. This study demonstrated that the number and proportion of X-sperm enriched using pH 7.4 diluent were elevated, and the proportion of female kids was increased. This technology can be used for the reproduction and production of dairy goats in farms at large scales., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have identified that single-nucleotide polymorphisms (SNPs) rs1258267 in CHAT and rs3753841 in COL11A1 are associated with primary angle-closure glaucoma (PACG). The purpose of the study was to evaluate the association of CHAT rs1258267 and COL11A1 rs3753841 with PACG. A comprehensive electronic database search was performed to include eligible studies, published from October 2010 to March 2022. By calculating summary odds ratios (ORs) and 95% confidence intervals (CI) under five genetic models, the risk of PACG related to these two SNPs could be estimated. Heterogeneity was measured with a Chi-square-based Q statistic test and the I 2 statistic. By the Z test, we analyzed the overall effect of OR. We used funnel plots and Begg's funnel plots to evaluate the publication bias of included studies. The meta-analysis was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 checklist. There were eighteen studies associating CHAT rs1258267 with PACG indicating evidently decreased PACG risk in five genetic models. Thirty studies were included to demonstrate a notable increase in the risk of PACG-carrying COL11A1 rs3753841 genotypes. Subgroup analyses showed that the association of CHAT rs1258267 and COL11A1 rs3753841 with PACG was obvious in Asians, while no evidence was found to confirm this connection in Caucasians. This meta-analysis suggests that CHAT rs1258267 G/A polymorphisms could bring about a decreased risk of PACG susceptibility and COL11A1 rs3753841 G/A polymorphisms could cause an increased risk. These effects mainly manifest in Asians., Competing Interests: None

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

In the present study, a homemade probe-based nanometric morphology measurement system is proposed, which can be easily integrated with other probes, such as a diamond probe and an electrochemical electrode. In this system, an intermittent-contact mode is adopted, which is based on a set of micro-force servo modules. The micro-force serve module is mainly composed of a piezoelectric ceramic transducer, a capacitive displacement sensor, an excitation piezoelectric ceramic ring, and a four-beam spring. The four-beam spring integrated with a diamond probe is driven by the excitation piezoelectric ceramic ring. The mechanical structure and the control system of the measurement system are also designed. The vibration amplitude and the resolution of a normal load are calibrated during the engagement process under open-loop control. Moreover, the optimal values for parameters P, I, and D are obtained for the closed-loop measurement. The performance of the developed system is verified by measuring a standard sample. The measured depths agree well with the results obtained by commercial atomic force microscopy. The developed system can be used to measure nanostructures with high precision.

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP&#95;CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

G protein-coupled receptors can signal through both G proteins and ß -arrestin2. For the µ -opioid receptor (MOR), early experimental evidence from a single study suggested that G protein signaling mediates analgesia and sedation, whereas ß -arrestin signaling mediates respiratory depression and constipation. Then, receptor mutations were used to clarify which residues interact with ligands to selectively regulate signals in a ligand-specific manner. However, there is no systematic study on how to determine these residues and clarify the molecular mechanism of their influence on signal pathways. We have therefore used molecular docking to predict the amino acid sites that affect the binding of ligands and MOR. Then, the corresponding sites were mutated to determine the effect of the structural determinant of MOR on G i/o protein and ß -arrestin pathways. The pharmacological and animal behavioral experiments in combination with molecular dynamics simulations were used to elucidate the molecular mechanism of key residues governing the signaling. Without affecting ligand binding to MOR, MOR Y7.43A attenuated the activation of both G i/o protein and ß -arrestin signaling pathways stimulated by fentanyl, whereas it did not change these two pathways stimulated by morphine. Likewise, the activation peak time of extracellular regulated protein kinases was significantly prolonged at MOR Y7.43A compared with that at MOR wildtype stimulated by fentanyl, but there was no difference stimulated by morphine. In addition, MOR Y7.43A significantly enhanced analgesia by fentanyl but not by morphine in the mice behavioral experiment. Furthermore, the molecular dynamics simulations showed that H6 moves toward the cellular membrane. H6 of the fentanyl-Y7.43A system moved outward more than that in the morphine-Y7.43A system. Y7.43 mutation disrupted hydrophobic interactions between W6.48 and Y7.43 in the fentanyl-Y7.43A system but not in the morphine-Y7.43A system. Our results have disclosed novel mechanisms of Y7.43 mutation affecting MOR signaling pathways. Y7.43 mutation reduced the activation of the G i/o protein pathway and blocked the ß -arrestin2 recruitment, increased the H6 outward movement of MOR, and disrupted hydrophobic interactions. This may be responsible for the enhanced fentanyl analgesia. These findings are conducive to designing new drugs from the perspective of ligand and receptor binding, and Y7.43 is also expected to be a key site to structure optimization of synthesized compounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tian, Zhang, Wang, Gao, Sun, Liu, Fu, Tan and Su.)

Accomplishing the goals outlined in "Ending the HIV (Human Immunodeficiency Virus) Epidemic: A Plan for America Initiative" will require properly estimating and increasing access to HIV testing, treatment, and prevention services. In this research, a computational spatial method for estimating access was applied to measure distance to services from all points of a city or state while considering the size of the population in need for services as well as both driving and public transportation. Specifically, this study employed the enhanced two-step floating catchment area (E2SFCA) method to measure spatial accessibility to HIV testing, treatment (i.e., Ryan White HIV/AIDS program), and prevention (i.e., Pre-Exposure Prophylaxis [PrEP]) services. The method considered the spatial location of MSM (Men Who have Sex with Men), PLWH (People Living with HIV), and the general adult population 15-64 depending on what HIV services the U.S. Centers for Disease Control (CDC) recommends for each group. The study delineated service- and population-specific accessibility maps, demonstrating the method's utility by analyzing data corresponding to the city of Chicago and the state of Illinois. Findings indicated health disparities in the south and the northwest of Chicago and particular areas in Illinois, as well as unique health disparities for public transportation compared to driving. The methodology details and computer code are shared for use in research and public policy., Competing Interests: The authors have declared that no competing interests exist.

Largemouth bass virus (LMBV) is a major viral pathogen in largemouth bass culture, usually causing high mortality and heavy economic losses. Accurate and early detection of LMBV is crucial for diagnosis and control of the diseases caused by LMBV. Previously, we selected the specific aptamers, LA38 and LA13, targeting LMBV by systematic evolution of ligands by exponential enrichment (SELEX). In this study, we further generated truncated LA38 and LA13 (named as LA38s and LA13s) with high specificity and affinities and developed an aptamer-based sandwich enzyme-linked apta-sorbent assay (ELASA) for LMBV diagnosis. The sandwich ELASA showed high specificity and sensitivity for the LMBV detection, without cross reaction with other viruses. The detection limit of the ELASA was as low as 1.25 × 10 2 LMBV-infected cells, and the incubation time of the lysate and biotin labeled aptamer was as short as 10 min. The ELASA could still detect LMBV infection in spleen lysates at dilutions of 1/25, with good consistency of qRT-PCR. For the fish samples collected from the field, the sensitivity of ELASA was 13.3% less than PCR, but the ELASA was much more convenient and less time consuming. The procedure of ELASA mainly requires washing and incubation, with completion in approximately 4 h. The sandwich ELASA offers a useful tool to rapidly detect LMBV rapidly, contributing to control and prevention of LMBV infection.

The key to promoting the EPC (Engineering, Procurement, Construction) model in China's public construction projects is to alter the path dependence of a project owner's choice of project delivery model (PDM). This study uses evolutionary game theory to discuss the mechanism of government incentives as an external motivation to alter path dependence in the PDM. In addition, a cellular automata simulation to examine the influence of various government incentives on the project owner's choice. The results show that the combination of subsidies and penalties can produce the best incentive. Subsidies are more effective at promoting PDM institutional change, whereas penalties are more effective at preventing PDM institutional change from anti-recession effects. Based on our results, we propose that the Chinese government should take active subsidy measures at the initial stage of EPC promotion, and adopt a dynamic incentive strategy of continuously reducing subsidies and increasing penalties according to the improvement of the development degree of EPC model., Competing Interests: I declare that I have no competing interests

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mammalian studies have shown that the nuclear transcription factor Y (NFYC) regulates the expression of major histocompatibility complex (MHC) by binding to CCAAT-box on promoters. However, few studies have focused on the regulatory mechanisms of NFYC in MHC pathway in fish. To explore the transcriptional regulatory mechanism of MHCIa in fish, we characterized NFYC and MHCIa of red-spotted grouper (Epinephelus akaara) (named EaNFYC and EaMHCIa, respectively). The EaNFYC genome sequence is 13,796 bp and contains 1,065 bp open reading frame. It is composed of ten exons and nine introns and encode a 354 amino acid sequence. The putative EaNFYC protein sequence shared 67.2-99.4% identity to vertebrate NFYC and possesses a typically conserved domain (histone- or haem-associated protein 5 domain (HAP5)) at the N-terminus. Transcripts of both EaNFYC and EaMHCIa were ubiquitously expressed in all detect tissues, and higher mRNA levels were detected in immune-relevant tissues (middle-kidney). EaNFYC expression increased after treatment with polyinosinic: polycytidylic acid, lipopolysaccharide, nervous necrosis virus, zymosan A, and Singapore grouper iridovirus. Analysis of subcellular localization indicated that EaNFYC was localized at the cell nucleus only. Furthermore, overexpression of EaNFYC significantly stimulated the expression of EaMHCIa, interferon signalling molecules and inflammatory cytokine. The region -878 bp to +82 bp of EaMHCIa promoter was identified to be the core promoter which EaNFYC take effect on. Additionally, point mutations and electrophoretic mobility shift assays verified that NFYC activate MHCIa expression by binding at the M1 and M2 binding sites that do not contain CCAAT-box. These results contribute to elucidating the function of fish NFYC on MHC transcriptional mechanisms, and provide the first evidence of positive regulation of MHCIa expression by NFYC in fish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Regulatory factor X 5 (RFX 5) is a member of the RFX family, and it forms the transcription factor complex RFX with RFXANK/B and RFXAP. The RFX complex can activate MHC expression by binding to the MHC promoter. However, the regulate mechanism of RFX in fish species is not been fully elucidated. In this study, we investigated the transcriptional regulation of Epinephelus akaara RFX5 (EaRFX5) on EaMHCI, and its effect on immune pathways. The genomic sequence of EaRFX5 was 35,774 bp and consisted of ten exons and nine introns. The length of EaRFX5 ORF sequence is 2,160 bp, encoding 719 amino acids. By qRT-PCR, EaRFX5 was detected constitutively expressed in twelve selected tissues, showing a wide range of expression. EaRFX5 expression parttern in response to poly (I:C), LPS, Zymosan A, SGIV, and NNV challenges showed that EaRFX5 plays a differentiated immunomodulatory role in response to various stimuli in different tissues, and EaRFX5 was most significantly upregulated in the kidney after challenge with SGIV. Subcellular localization assays showed that EaRFX5 is a typical nuclear protein. Based on the in vitro overexpression experiments, EaRFX5 appeared to promote the expression of EaMHCIa gene, interferon signalling pathway and inflammatory cytokine. Luciferase reporter assay showed that the -267 bp to +82 bp region of EaMHCIa promoter was the core region where EaRFX5 modulated. Additionally, point mutations and electrophoretic mobility shift assays indicating M3 is the EaRFX5 binding sites in the EaMHCIa promoter. These results contribute to elucidating the function of EaRFX5 in fish immune response, and provide the first evidence of positive regulation of MHCIa expression by RFX5 in fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Midwestern "Corn-Belt" in the United States is the most productive agricultural region on the planet despite being predominantly rainfed. In this region, global climate change is driving precipitation patterns toward wetter springs and drier mid- to late-summers, a trend that is likely to intensify in the future. The lack of precipitation can lead to crop water limitations that ultimately impact growth and yields. Young plants exposed to water stress will often invest more resources into their root systems, possibly priming the crop for any subsequent mid- or late-season drought. The trend toward wetter springs, however, suggests that opportunities for crop priming may lessen in the future. Here, we test the hypothesis that early season dry conditions lead to drought priming in field-grown crops and this response will protect crops against growth and yield losses from late-season droughts. This hypothesis was tested for the two major Midwestern crop, maize and soybean, using high-resolution daily weather data, satellite-derived phenological metrics, field yield data, and ecosystem-scale model (Agricultural Production System Simulator) simulations. The results from this study showed that priming mitigated yield losses from a late season drought of up to 4.0% and 7.0% for maize and soybean compared with unprimed crops experiencing a late season drought. These results suggest that if the trend toward wet springs with drier summers continues, the relative impact of droughts on crop productivity is likely to worsen. Alternatively, identifying opportunities to breed or genetically modify pre-primed crop species may provide improved resilience to future climate change., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Food and Energy Security published by John Wiley & Sons Ltd.)

Due to climate change and rapid urbanization, Urban Heat Island (UHI), featuring significantly higher temperature in metropolitan areas than surrounding areas, has caused negative impacts on urban communities. Temporal granularity is often limited in UHI studies based on satellite remote sensing data that typically has multi-day frequency coverage of a particular urban area. This low temporal frequency has restricted the development of models for predicting UHI. To resolve this limitation, this study has developed a cyber-based geographic information science and systems (cyberGIS) framework encompassing multiple machine learning models for predicting UHI with high-frequency urban sensor network data combined with remote sensing data focused on Chicago, Illinois, from 2018 to 2020. Enabled by rapid advances in urban sensor network technologies and high-performance computing, this framework is designed to predict UHI in Chicago with fine spatiotemporal granularity based on environmental data collected with the Array of Things (AoT) urban sensor network and Landsat-8 remote sensing imagery. Our computational experiments revealed that a random forest regression (RFR) model outperforms other models with the prediction accuracy of 0.45 degree Celsius in 2020 and 0.8 degree Celsius in 2018 and 2019 with mean absolute error as the evaluation metric. Humidity, distance to geographic center, and PM 2.5 concentration are identified as important factors contributing to the model performance. Furthermore, we estimate UHI in Chicago with 10-min temporal frequency and 1-km spatial resolution on the hottest day in 2018. It is demonstrated that the RFR model can accurately predict UHI at fine spatiotemporal scales with high-frequency urban sensor network data integrated with satellite remote sensing data., Competing Interests: Competing interestsProf. Shaowen Wang is an EBM of Urban Informatics. He was not involved in the peer-review or handling of the manuscript. The authors have no other competing interests to disclose., (© The Author(s) 2022.)

The relevance of Tregs in the induction of tolerance against corneal allografts has been well established. Although it is well known that the conversion of Tregs into effector-like cells contributes to the loss of corneal immune privilege, the underlying mechanism is still not fully understood. Using heterologous penetrating keratoplasty model, we found that Tregs from corneal allograft rejected mice (inflam-Tregs) exhibit impaired function and characteristics of effector T cells. Further study showed that the expression of NF-κB c-Rel, a key mediator of effector T cell function, was significantly increased in inflam-Tregs. Mechanistic study revealed that elevated NF-κB c-Rel level in inflam-Tregs impaired Treg function through the promotion of inflammatory cytokine production and glycolysis. More importantly, we demonstrated that targeting NF-κB c-Rel was able to improve the immune suppressive function of inflam-Tregs in vitro and enhance the potential of them to suppress corneal transplantation rejection. Therefore, our current study identified NF-κB c-Rel as a key mediator of the conversion of Tregs into effector-like cells when under inflammatory environment., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling., Competing Interests: Declaration of interests The authors are either employees and/or shareholders of Novartis. J.D., M-H.L., D.M.R., N.S., and S.W. are inventors on patents US10259827B2 and WO2018087677A1 for BMP potentiators., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The emergence of the CRISPR/Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR/Cas13 system has been used to target RNA. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the red-spotted grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo . These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. IMPORTANCE RNA viruses are important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find widely effective ways to inhibit RNA viruses, and we urgently need to develop effective antiviral methods. CasRx is a small type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a nonenveloped positive-strand RNA virus, is one of the most serious viral pathogens, infecting more than 40 cultured fish species and resulting in huge economic losses worldwide. Here, we establish a novel effective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioides) as a model. Our data showed that CasRx was most robust for RNA virus interference applications in fish, and we demonstrate its suitability for studying key questions related to virus biology.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS., Competing Interests: The authors have declared that no competing interests exist.

Brazilian grain production increased more than fourfold from 1980 to 2016. The grain boom was achieved primarily by soybean-corn double cropping and cropland expansion-both show changing spatiotemporal patterns since the 1980s. Here, we quantified the contributions of these two strategies to corn and soybean production in Brazil using municipality-level data from 1980 to 2016. We found the contribution of double cropping to the grain boom steadily increased to 35% and the largest driving force was the increasing demand for grain export. While double cropping dominated the conventional agricultural regions, cropland expansion was still the major strategy in agricultural frontiers such as the Centre-West and Matopiba. The implementation of double cropping offset the equivalent of 76.7 million ha of Brazilian arable land for grain production from 2003 to 2016. Double cropping in Brazil has the potential to help alleviate land burdens in other pantropical countries with increasing global food demand., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper ( Epinephelus coioides ) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens , respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1-45), IMD (aa 46-67), CIL (aa 68-93), TMD (aa 94-119), and CTD (aa 120-131). In vitro , the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro . All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Wang, Zheng, Huang, Wang, Huang, Qin and Huang.)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19) pandemic, has infected millions of people and caused hundreds of thousands of deaths. While COVID-19 has overwhelmed healthcare resources (e.g., healthcare personnel, testing resources, hospital beds, and ventilators) in a number of countries, limited research has been conducted to understand spatial accessibility of such resources. This study fills this gap by rapidly measuring the spatial accessibility of COVID-19 healthcare resources with a particular focus on Illinois, USA., Method: The rapid measurement is achieved by resolving computational intensity of an enhanced two-step floating catchment area (E2SFCA) method through a parallel computing strategy based on cyberGIS (cyber geographic information science and systems). The E2SFCA has two major steps. First, it calculates a bed-to-population ratio for each hospital location. Second, it sums these ratios for residential locations where hospital locations overlap., Results: The comparison of the spatial accessibility measures for COVID-19 patients to those of population at risk identifies which geographic areas need additional healthcare resources to improve access. The results also help delineate the areas that may face a COVID-19-induced shortage of healthcare resources. The Chicagoland, particularly the southern Chicago, shows an additional need for resources. This study also identified vulnerable population residing in the areas with low spatial accessibility in Chicago., Conclusion: Rapidly measuring spatial accessibility of healthcare resources provides an improved understanding of how well the healthcare infrastructure is equipped to save people's lives during the COVID-19 pandemic. The findings are relevant for policymakers and public health practitioners to allocate existing healthcare resources or distribute new resources for maximum access to health services.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides , and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus , and Homo sapiens , respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions., (Copyright © 2020 Wang, Li, Zhang, Yang, Wei, Huang, Qin and Wang.)

Peroxisome proliferative-activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α, IFN-γ, ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rain-fed corn system has varied optimal environmental requirements by growth phases and regions. Understanding spatiotemporal characteristics of such requirements are important to ensure food security. To capture the stage-variant growing requirements, we develop and compare statistical models with various spatial and temporal resolutions to quantify the relationships between corn yield and meteorological factors. Multilinear regression models are trained using cross-sectional datasets pooled at three magnitudes (state, district, county) with temperature and precipitation related predictors according to three temporal resolutions (growing season, fixed month, growing phase). The models are applied to the U.S. Corn Belt for the time period of 1981-2016. The results show that average corn yield variation explained by meteorological factors can be improved to 50.2% at the agricultural district scale with growth phase resolution from ~30% at the state-level with growing season resolution. The results reveal that corn yield is most sensitive to extreme heat stress during the grain filling phase. From a spatial perspective, the northern counties in the U.S. Corn Belt are less limited by precipitation resources but are more vulnerable to extreme heat. The spatiotemporal explicit statistic modeling approach quantifies the impact and adaptation potential of changing the planting date for production. Appropriate adaptions by changing plant dates can increase the potential of corn production by 0.87 million Mg year -1 in the Corn Belt., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The transcription factor nuclear factor kappa B (NF-κB) is a critical regulator of immune and inflammatory responses with crucial roles in various pathophysiologic conditions involving cell survival and death. Recent studies in mammals showed that NF-κB was also involved in peroxisome proliferator-activated receptors (PPARs)-mediated immune responses However, the mechanism by which NF-κB regulates PPARδ in teleosts remains unclear. In the present study, we analyzed the potential role of NF-κB/p65 (Ecp65) in the immune response stimulated by various pathogens in the grouper Epinephelus coioides. Ecp65 expression was significantly induced soon after infection with lipopolysaccharide, nervous necrosis virus, poly(I:C), and zymosan A. We also analyzed the promoter to determine the regulatory effect of Ecp65 on PPARδ expression, using progressive EcPPARδ promoter deletion mutations. Among the five truncated mutants, the luciferase reporter activity of the PPARδ-5 promoter region was highest in response to Ecp65, indicating that the core p65-binding region was located in the PPARδ-5 promoter region (+122 bp to +383 bp). Mutation analyses indicated that the luciferase reporter activity of the EcPPARδ promoter was dramatically decreased by mutation of the M3 (+305 bp to +324 bp) and M4 (+346 bp to +365 bp) binding sites, respectively. We further confirmed that Ecp65 bound to the M3 and M4 binding sites in the 5'-untranslated region of EcPPARδ by electrophoretic mobility shift assay. Finally, overexpression of Ecp65 in vitro notably promoted the transcription of EcPPARδ, interferon-related genes, and several inflammatory cytokines. This study demonstrated that Ecp65 plays an important role in modulating the innate immune responses in groupers. These results also further our understanding of the mechanisms involved in the transcriptional regulation of PPARs by p65 in bony fish., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biomarkers have important roles in various physiological functions and disease pathogenesis. As a nucleocytoplasmic DNA virus, Singapore grouper iridovirus (SGIV) causes high economic losses in the mariculture industry. Aptamer-Q5-complexed major capsid protein (MCP) in the membrane of SGIV-infected cells can be used as a specific molecular probe to investigate the crucial events of MCP endocytosis into SGIV-infected host cells during viral infection. Chlorpromazine blocks clathrin-mediated endocytosis, and MCP endocytosis into SGIV-infected cells decreased significantly when the cells were pretreated with chlorpromazine. The disruption of cellular cholesterol by methyl-β-cyclodextrin also significantly reduced MCP endocytosis. In contrast, inhibitors of key regulators of caveolae/raft-dependent endocytosis and macropinocytosis, including genistein, Na + /H + exchanger, p21-activated kinase 1 (PAK1), myosin II, Rac1 GTPase, and protein kinase C (PKC), had no effect on MCP endocytosis. The endocytosis of the biomarker MCP is dependent on low pH and cytoskeletal actin filaments, as shown with various inhibitors (chloroquine, ammonia chloride, cytochalasin D). Therefore, MCP enters SGIV-infected host cells via clathrin-mediated endocytosis, which is dependent on dynamin, cholesterol, low pH, and cytoskeletal actin filaments. This is the first report of a specific aptamer-based probe used to analyze MCP endocytosis into SGIV-infected host cells during viral infection. This method provides a convenient strategy for exploring viral pathogenesis and facilitates the development of diagnostic tools for and therapeutic approaches to viral infection., (Copyright © 2020 Yu, Liu, Wu, Wei, Xiao, Yi, Cheng, Wang, Zhang, Qin and Li.)

Nervous necrosis virus (NNV) has infected more than 50 fish species worldwide, and has caused serious economic losses in the aquaculture industries. However, there is no effective antiviral therapy. The development of a rapid and accurate point-of-care diagnostic method for the prevention and control of NNV infection is urgently required. Commonly used methods for NNV detection include the cell culture-based assay, antibody-based assay and polymerase chain reaction (PCR)-based assay. However, these methods have disadvantages as they are time-consuming and complex. In the present study, we developed a simple and sensitive aptamer-based lateral flow biosensor (LFB) method for the rapid detection of red-spotted grouper nervous necrosis virus (RGNNV). An aptamer is a single-stranded nucleotide, which can specifically bind to the target and has many advantages. Based on a previously selected aptamer, which specifically bound to the coat protein of RGNNV (RGNNV-CP), two modified aptamers were used in this study. One aptamer was used for magnetic bead enrichment and the other was used for isothermal strand displacement amplification (SDA). After amplification, the product was further tested by the LFB, and the detection results were observed by the naked eye within 5 min with high specificity and sensitivity. The LFB method could detect RGNNV-CP protein as low as 5 ng/mL or 5 × 10 3 RGNNV-infected GB (grouper brain) cells. Overall, it is the first application of a LFB combined with aptamer in the rapid diagnosis of virus from aquatic animals, which provides a new option for virus detection in aquaculture., (Copyright © 2020 Liu, Qin, Zhang, Li, Yu, Huang, Mukama, Zeng and Wang.)

Nervous necrosis virus (NNV) causes viral nervous necrosis, the most devastating disease in more than 50 fish species worldwide, with massive mortality rates up to 100%, resulting in great economic losses to mariculture. However, few methods are available for the efficient diagnosis and treatment of viral nervous necrosis. Aptamers are molecular recognition ligands characterized by their remarkably high specificity and affinity, great stability, and ease of synthesis, and have been widely studied in application of disease diagnosis and therapies. In this study, we generated three aptamers against red-spotted grouper nervous necrosis virus (RGNNV)-infected grouper brain (GB) cells using the Cell-SELEX (cell based-systematic evolution of ligands by exponential enrichment) technology. The selected aptamers formed stable stem-loop structures, and could specifically recognize RGNNV-infected GB cells, with calculated dissociation constants ( K d ) of 27.96, 29.3, and 59.5 nM for aptamers GBN2, GBN10, and GBN34, respectively. They also recognized RGNNV-infected brain tissues. The three aptamers were non-toxic and showed antiviral activities both in vitro and in vivo . Fluorescence microscopy and flow cytometry also demonstrated that aptamer GBN34 could be efficiently and specifically internalized into RGNNV-infected GB cells. The targeted cellular delivery of aptamer-small interfering RNA (siRNA) conjugates remarkably inhibited RGNNV infection in GB cells. The efficiency of the aptamer-based targeted delivery system was about 75% reduction in infection after 48 h, which was similar to that of transfection. These aptamers have great potential utility in the rapid diagnosis and inhibition of RGNNV infection in mariculture., (Copyright © 2020 Zhou, Wang, Yu, Wei, Liu, Wei, Huang, Huang, Li and Qin.)

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of EcKLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys 2 -His 2 ) domain in the C-terminal region. Basing on the detection of the mRNA levels of viral genes, western blotting of MCP protein, and morphological CPEs, we found that the overexpression of EcKLF6 suppressed the replication of Singapore grouper iridovirus (SGIV), exerting its antiviral activity against fish virus. Moreover, promoter analysis was performed to investigate whether EcKLF6 was a regulator of EcPPAR-δ. The luciferase reporter assay and real time PCR results indicated a negative regulatory role of EcKLF6 on EcPPAR-δ transcription in grouper. Further experimental analysis shows that the potential EcKLF6 binding sites may locate in the EcPPAR-δ-4-M3 (+133 to +154) and EcPPAR-δ-4-M4 (+354 to +368) region of the EcPPAR-δ promoter. Electrophoretic mobile shift assays (EMSAs) verified that EcKLF6 interacted with the binding site of the EcPPAR-δ-4-M4 promoter region. In addition, we also found that KLF6 promotes inflammatory responses in GS cells. Considering that KLF6 and PPAR-δ play opposite roles in regulating inflammatory responses, we speculated the promoting effect of KLF6 on inflammatory response may be related to its negative regulation on EcPPAR-δ. In conclusion, the present study provides the first evidence of the negative regulation of EcPPAR-δ transcription by EcKLF6 and contributes to a better understanding of the transcriptional mechanisms of EcKLF6 in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Understanding large-scale crop growth and its responses to climate change are critical for yield estimation and prediction, especially under the increased frequency of extreme climate and weather events. County-level corn phenology varies spatially and interannually across the Corn Belt in the United States, where precipitation and heat stress presents a temporal pattern among growth phases (GPs) and vary interannually. In this study, we developed a long short-term memory (LSTM) model that integrates heterogeneous crop phenology, meteorology, and remote sensing data to estimate county-level corn yields. By conflating heterogeneous phenology-based remote sensing and meteorological indices, the LSTM model accounted for 76% of yield variations across the Corn Belt, improved from 39% of yield variations explained by phenology-based meteorological indices alone. The LSTM model outperformed least absolute shrinkage and selection operator (LASSO) regression and random forest (RF) approaches for end-of-the-season yield estimation, as a result of its recurrent neural network structure that can incorporate cumulative and nonlinear relationships between corn yield and environmental factors. The results showed that the period from silking to dough was most critical for crop yield estimation. The LSTM model presented a robust yield estimation under extreme weather events in 2012, which reduced the root-mean-square error to 1.47 Mg/ha from 1.93 Mg/ha for LASSO and 2.43 Mg/ha for RF. The LSTM model has the capability to learn general patterns from high-dimensional (spectral, spatial, and temporal) input features to achieve a robust county-level crop yield estimation. This deep learning approach holds great promise for better understanding the global condition of crop growth based on publicly available remote sensing and meteorological data., (© 2019 John Wiley & Sons Ltd.)