In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.

Oxymatrine Modulation of TLR3 Signaling: A Dual-Action Mechanism for H9N2 Avian Influenza Virus Defense and Immune Regulation.

In our research, we explored a natural substance called Oxymatrine, found in a traditional Chinese medicinal plant, to fight against a common bird flu virus known as H9N2. This virus not only affects birds but can also pose a threat to human health. We focused on how this natural compound can help in stopping the virus from spreading in cells that line the lungs of birds and potentially humans. Our findings show that Oxymatrine can both directly block the virus and boost the body's immune response against it. This dual-action mechanism is particularly interesting because it indicates that Oxymatrine might be a useful tool in developing new ways to prevent and treat this type of bird flu. Understanding how Oxymatrine works against the H9N2 virus could lead to safer and more natural ways to combat viral infections in animals and humans, contributing to the health and well-being of society. The H9N2 Avian Influenza Virus (AIV) is a persistent health threat because of its rapid mutation rate and the limited efficacy of vaccines, underscoring the urgent need for innovative therapies. This study investigated the H9N2 AIV antiviral properties of Oxymatrine (OMT), a compound derived from traditional Chinese medicine, particularly focusing on its interaction with pulmonary microvascular endothelial cells (PMVECs). Employing an array of in vitro assays, including 50% tissue culture infectious dose, Cell Counting Kit-8, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot, we systematically elucidated the multifaceted effects of OMT. OMT dose-dependently inhibited critical antiviral proteins (PKR and Mx1) and modulated the expression of type I interferons and key cytokines (IFN-α, IFN-ß, IL-6, and TNF-α), thereby affecting TLR3 signaling and its downstream elements (NF-κB and IRF-3). OMT's antiviral efficacy extended beyond TLR3-mediated responses, suggesting its potential as a versatile antiviral agent. This study not only contributes to the growing body of research on the use of natural compounds as antiviral agents but also underscores the importance of further investigating the broader application of OMT for combating viral infections.

Cecropin AD reduces viral load and inflammatory response against H9N2 avian influenza virus in chickens.

Introduction: This study focuses on evaluating the therapeutic efficacy of cecropin AD, an antimicrobial peptide, against H9N2 avian influenza virus (AIV) in chickens. Given the global impact of H9N2 AIV on poultry health, identifying effective treatments is crucial. Methods: To assess the impact of cecropin AD, we conducted in vivo experiments involving 108 5-week-old chickens divided into control, infected, and various treatment groups based on cecropin AD dosage levels (high, medium, and low). The methodologies included hemagglutination (HA) tests for viral titers, histopathological examination and toluidine blue (TB) staining for lung pathology, real-time PCR for viral detection, and enzyme-linked immunosorbent assays for measuring serum levels of inflammatory markers. Results: The findings revealed that cecropin AD substantially reduced lung pathology and viral load, especially at higher dosages, comparing favorably with the effects seen from conventional treatments. Moreover, cecropin AD effectively modulated mast cell activity and the levels of inflammatory markers such as IL-6, TNF-α, IFN-γ, and 5-HT, indicating its potential to diminish inflammation and viral spread. Discussion: Cecropin AD presents a significant potential as an alternative treatment for H9N2 AIV in chickens, as evidenced by its ability to lessen lung damage, decrease viral presence, and adjust immune responses. This positions cecropin AD as a promising candidate for further exploration in the management of H9N2 AIV infections in poultry.

Heterogeneous nuclear ribonucleoprotein F deficiency in mouse podocyte promotes podocytopathy mediated by methyltransferase-like 14 nuclear translocation resulting in Sirtuin 1 gene inhibition.

Heterogeneous nuclear ribonucleoprotein F (HnRNP F) is a key regulator for nucleic acid metabolism; however, whether HnRNP F expression is important in maintaining podocyte integrity is unclear. Nephroseq analysis from a registry of human kidney biopsies was performed. Age- and sex-matched podocyte-specific HnRNP F knockout (HnRNP FPOD KO) mice and control (HnRNP Ffl/fl) were studied. Podocytopathy was induced in male mice (more susceptible) either by adriamycin (ADR)- or low-dose streptozotocin treatment for 2 or 8 weeks. The mouse podocyte cell line (mPODs) was used in vitro. Nephroseq data in three human cohorts were varied greatly. Both sexes of HnRNP FPOD KO mice were fertile and appeared grossly normal. However, male 20-week-old HnRNP FPOD KO than HnRNP Ffl/fl mice had increased urinary albumin/creatinine ratio, and lower expression of podocyte markers. ADR- or diabetic- HnRNP FPOD KO (vs. HnRNP Ffl/fl) mice had more severe podocytopathy. Moreover, methyltransferase-like 14 (Mettl14) gene expression was increased in podocytes from HnRNP FPOD KO mice, further enhanced in ADR- or diabetic-treated HnRNP FPOD KO mice. Consequently, this elevated Mettl14 expression led to sirtuin1 (Sirt1) inhibition, associated with podocyte loss. In mPODs, knock-down of HnRNP F promoted Mettl14 nuclear translocation, which was associated with podocyte dysmorphology and Sirt1 inhibition-mediated podocyte loss. This process was more severe in ADR- or high glucose- treated mPODs. Conclusion: HnRNP F deficiency in podocytes promotes podocytopathy through activation of Mettl14 expression and its nuclear translocation to inhibit Sirt1 expression, underscoring the protective role of HnRNP F against podocyte injury.

Simulation of thermal hazards risk in octogen based on non-isothermal DSC data.

To evaluate the possible thermal risks associated with the storage of octogen (HMX), non-isothermal differential scanning calorimetry (DSC) experiments were conducted in order to ascertain the kinetic model and parameters governing its thermal decomposition. DSC measurements indicate that HMX underwent a crystal transformation prior to thermal decomposition. A kinetic model for the autocatalytic thermal decomposition process was developed through the analysis of its primary exothermic peaks. Subsequently, numerical simulations were performed using the aforementioned kinetic model to assess the potential thermal explosion hazard of HMX under two distinct storage conditions. The comparison was made between the models of HMX autocatalytic decomposition temperature and thermal explosion critical temperature under two distinct storage conditions. The prediction of the influence of ambient temperature on the critical temperature of thermal explosion is conducted simultaneously. Finally, the thermal hazard parameters of HMX under different package quality are given.

Identification of odorant receptors of Tribolium confusum in response to limonene repellent activity.

Tribolium confusum is an important storage pest showing significant resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Present study attempts to explore the molecular mechanism about the repellent activity of limonene. When treatment concentration of limonene was 200.00 µg/cm2, the repellent level remained at grade V after 24 hours. Our study showed that limonene could be distinguished by T. confusum antenna with a maximal electroantennography test value of 0.90 mV. Simultaneously, 88 upregulated and 98 downregulated genes were sequenced in limonene-repellent T. confusum, and RT-qPCR analysis showed that four down-regulated and one up-regulated OR genes play an important role in the response to limonene. The repellent rate was decreased by 22.13% mediated with a knockdown of dsTconOR93, while the EAG value of the female and male adults was reduced to 0.26 mV (49.06%) and 0.20 mV (54.05%), respectively. In conclusion, limonene had a strong repellent activity against T. confusum and TconOR93 gene was determined to be a major effector in perception of limonene. This study provides a basis for the development of limonene as a novel botanical pesticide for the control to storage pests, which will reduce the utilization of chemical pesticides and postpone the development of resistance.

Influences on treatment-seeking and antibiotic use for common illnesses in eastern China.

BACKGROUND: Antibiotic resistance rates remain high in China where antibiotics are widely used for common illnesses. This study aimed to investigate the influences on people's decisions on treatment and antibiotic use for common illnesses in eastern China. METHODS: Semi-structured interviews were conducted with 29 patients recruited through convenience sampling between July 2020 and January 2021 in one hospital in County A in Zhejiang Province, and one hospital and one village clinic in County B in Jiangsu Province, respectively. All interviews were audio-recorded, transcribed verbatim and thematically analysed. This study is nested in a larger interdisciplinary mixed method project and we also compared our qualitative findings with quantitative results from a household survey conducted as part of this wider project. RESULTS: Participants' decisions about treatment-seeking and antibiotic use for common illnesses were found to be influenced by four interactive domains. (i) Self-evaluation of illness severity: Participants tend to self-treat minor conditions with ordinary medicines first and do not resort to antibiotics unless the condition worsens or is considered inflammation- related. Visiting healthcare facilities is seen as the final option. (ii) Access to and trust in care: These treatment-seeking practices are also associated with the perception, in contrast with retail pharmacies, hospitals provide professional and trustworthy care but are difficult to access, and hence require visiting only for severe illness. (iii) Prior experience: previous medical treatment and experiences of self-medication also influence participants' treatment decisions including the use of antibiotics. (iv) Medication characteristics: Participants view antibiotics as powerful medicines with harms and risks, requiring consumers to carefully trade off benefits and harms before use. CONCLUSIONS: People's treatment decisions in relation to antibiotic use in eastern China are influenced by an interplay of lay conceptual models of illnesses and antibiotics and broader organisational, social, and contextual factors. Interventions focusing on individual education to incorporate biomedical knowledge into lay understandings, and reducing situational and social incentives for self-medicating with antibiotics by strengthening access to quality professional care, would be helpful in promoting antibiotic stewardship.

Role of CYP6MS subfamily enzymes in detoxification of Sitophilus zeamais after exposure to terpinen-4-ol and limonene.

Food security is an important basis and guarantee to national safety, the loss caused by storage pests was a serious problem which affects the food security widely. Frequent application of chemical pesticides caused several critical crises including the development of resistance, pesticide residues, environmental pollution, and exposure risk to human or non-target organisms. The utilization of volatile components acts as a natural alternative for controlling storage pests has aroused extensive interest in recent years. It has been reported that terpinene-4-ol and limonene showed significant insecticidal activity against Sitophilus zeamais in our previous studies, which was evaluated to have strong influences to CYP450 genes. To determine the links and roles of related genes, we identified the SzCYP6MS subfamily genes which encoded a putative protein of 493 or 494 amino acids. Then, the expression of four CYP6MS subfamily genes were increased significantly under the fumigation stress by terpinen-4-ol and limonene, which was determined by the RT-qPCR analysis compared with non-fumigated colonies. In addition, we determined that RNAi-mediated CYP6MS genes knockdown significantly increased the sensitivity of S. zeamais to terpinen-4-ol and limonene, the mortality rates of insects with knocked down CYP6MS1, CYP6MS5, CYP6MS6, CYP6MS8, and CYP6MS9 genes increased by 25%, 25%, 16%, 17%, and 4% in terpinen-4-ol treatment groups and by 29%, 25%, 15%, 22%, and 3% in limonene treatment groups compared with that in the control groups, respectively. Finally, it was validated that CYP6MS5 exhibited the most stable binding with terpinen-4-ol that was similar to the result between CYP6MS8 and limonene which were verified by molecular docking analysis. In together, this study demonstrates the potential of terpinen-4-ol and limonene used as novel botanical pesticides to control storage pests, thereby reducing application of chemical pesticides and postponing resistance development.

Preparation of Spherical HMX/DMF Solvates, Spherical HMX Particles, and HMX@NTO Composites: A Way to Reduce the Sensitivity of HMX.

To reduce the sensitivity of HMX (HMX = high-melting explosive-cyclotetramethylenetetranitramine), spherical HMX/DMF (DMF = dimethylformamide) solvates, spherical HMX particles, and HMX@NTO (NTO = 1,2,4-triazol-5-one) composites are prepared by crystallization. The structure and performance of spherical HMX crystals, HMX particles, and HMX@NTO composites are characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, differential scanning calorimetry, accelerating rate calorimetry, and mechanical sensitivity test. The results show that the space group of the spherical HMX/DMF solvate is R̅3c with the lattice parameters of a = 15.9159(4) Å, b = 15.9159(4) Å, and c = 30.5136(8) Å. The non-isothermal stability and adiabatic thermal stability of HMX/DMF solvates are similar to those of HMX particles. The non-isothermal stability of HMX@NTO composites is lower than that of NTO and HMX particles, while the adiabatic thermal stability of HMX@NTO composites is higher than that of NTO but lower than that of HMX particles. The mechanical sensitivities of spherical HMX/DMF cocrystals, spherical HMX particles, and HMX@NTO composites are lower than that of raw HMX. This study can provide some guidance for desensitizing HMX and other energetic materials.

Antibiotic residues of drinking-water and its human exposure risk assessment in rural Eastern China.

Trace levels of antibiotics were frequently found in drinking-water, leading a growing concern that drinking-water is an important exposure source to antibiotics in humans. In this study, we investigated antibiotics in tap water and well water in two rural residential areas in Eastern China to assess the related human health exposure risks in drinking-water. Twenty-seven antibiotics were analyzed using ultra performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS). The average daily dose (ADD) and the health risk quotient (HRQ) for exposure to antibiotics in humans were evaluated using 10000 times of Monte Carlo simulations. Ten antibiotics were detected in drinking-water samples, with the maximum concentrations of antibiotic mixture of 8.29 ng/L in tap water and 2.95 ng/L in well water, respectively. Macrolides and sulfonamides were the predominant contaminants and showed the seasonality. Azithromycin had the highest detection frequencies (79.71-100%), followed by roxithromycin (25.71-100%) and erythromycin (21.43-86.96%). The estimated ADD and HRQ for human exposure to antibiotic mixture through drinking-water was less than 0.01 µg/kg/day and 0.01, respectively, which varied over sites, water types, seasons and sex. Ingestion route was more important than dermal contact route (10-6 to 10-4 µg/kg/day magnitude vs. 10-11 to 10-8 µg/kg/day magnitude). Macrolides also contributed mainly to health exposure risks to antibiotics through drinking-water, whose HRQ accounted for 46% to 67% of the total HRQs. Although the individual antibiotic and their combined effects contributed to acceptable health risks for human, the long-term exposure patterns to low-dose antibiotics in drinking-water should not be ignored.

Hedgehog interacting protein activates sodium-glucose cotransporter 2 expression and promotes renal tubular epithelial cell senescence in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. METHODS: Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. RESULTS: Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1ß, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. CONCLUSIONS/INTERPRETATION: Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Impact of the COVID-19 Outbreak on the Antibiotic Use Patterns among a Rural Community Population in Eastern China.

There are growing concerns that the coronavirus disease of 2019 (COVID-19) pandemic may change antibiotic use patterns and accelerate antibiotic resistance, but evidence from the community level is lacking. This study aims to estimate the impact of the COVID-19 outbreak on the antibiotic use patterns among a community population in Eastern China. A self-administered medicine diary was used to collect information on antibiotic use from July 2019 to June 2021 among a rural community in Eastern China. We analyzed the changes in antibiotic use patterns over five months from August to December 2019 and the corresponding months in 2020. The risk of antibiotic use and its changes were measured with the incidence rate (IR) and relative risk (RR). In total, 1111 participants were eligible for the final analysis (440 in 2019 and 671 in 2020). After the COVID-19 outbreak, antibiotic use increased by 137% (5.43 per 100 person months in the 2019 vs. 12.89 per 100 person months in the 2020), and after the adjustment of covariates, the adjusted RR was 1.72 (95% CI: 1.10~2.34). It was higher among those who were women (RR = 2.62), aged 35−59 years old (RR = 2.72), non-farmers (RR = 2.75), had less than six years of education (RR = 2.61), had an annual household income over CNY 100,000 (USD 14,940) (RR = 2.60), and had no history of chronic diseases (RR = 2.61) (all p < 0.05). The proportion of cephalosporins consumed increased from 54.29% in 2019 to 64.92% in 2020 (p = 0.011). Among those aged 35 years and older, the proportion of antibiotics obtained from medical facilities increased, while the proportion obtained from retail pharmacies, homes, and other sources decreased (all p < 0.05). The COVID-19 outbreak changed antibiotic use patterns in this study population (Eastern China) significantly. More efforts to monitor and enhance antibiotic stewardship activities at the community level are needed in future.

Impacts of Antibiotic Residues in the Environment on Bacterial Resistance and Human Health in Eastern China: An Interdisciplinary Mixed-Methods Study Protocol.

Antibiotic resistance is a global health challenge that threatens human and animal lives, especially among low-income and vulnerable populations in less-developed countries. Its multi-factorial nature requires integrated studies on antibiotics and resistant bacteria in humans, animals, and the environment. To achieve a comprehensive understanding of the situation and management of antibiotic use and environmental transmission, this paper describes a study protocol to document human exposure to antibiotics from major direct and indirect sources, and its potential health outcomes. Our mixed-methods approach addresses both microbiological and pathogen genomics, and epidemiological, geospatial, anthropological, and sociological aspects. Implemented in two rural residential areas in two provinces in Eastern China, linked sub-studies assess antibiotic exposure in population cohorts through household surveys, medicine diaries, and biological sampling; identify the types and frequencies of antibiotic resistance genes in humans and food-stock animals; quantify the presence of antibiotic residues and antibiotic resistance genes in the aquatic environment, including wastewater; investigate the drivers and behaviours associated with human and livestock antibiotic use; and analyse the national and local policy context, to propose strategies and systematic measurements for optimising and monitoring antibiotic use. As a multidisciplinary collaboration between institutions in the UK and China, this study will provide an in-depth understanding of the influencing factors and allow comprehensive awareness of the complexity of AMR and antibiotic use in rural Eastern China.

Patterns and Determinants of Antibiotic Use Behaviors among Rural Community Residents in Eastern China.

Inappropriate antibiotic use may lead to antibiotic resistance, which has become a serious global crisis. Addressing suboptimal antibiotic use in the general population can play a significant role in the fight against antimicrobial resistance. This study aims to describe antibiotic use and sources of acquisition, and to identify factors influencing antibiotic access among rural community residents in Eastern China. A cross-sectional survey was conducted from July to August 2020, and 1494 participants from two villages in Eastern China were enrolled. Information was obtained using face-to-face interviews with a structured electronic questionnaire. Chi-squared and multinominal logistic regression analysis were used to explore possible determinants. In total, 1379 participants were eligible for the analysis. In the past 12 months, nearly half the respondents had taken any antibiotic (48.4%), and this proportion varied across marital status and age group. Two thirds of them (59.9%) obtained antibiotics from medical facilities with a prescription when they last took antibiotics, while 17.7% and 22.4% chose retail pharmacies and other sources, respectively. Multinominal analysis found that a higher proportion obtained antibiotics outside medical facilities among those aged 15 to 44 years, unmarried, non-white collar workers, with more years of education, lower annual household income per capita and lower levels of antibiotic knowledge. The antibiotic use behavior of rural community residents in Eastern China remains suboptimal. Antibiotic use and access behaviors need to be further addressed. Effective antibiotic stewardship in non-medical facility sources and training programs targeted for rural Chinese is warranted in future.

Angiotensin II type-2-receptor stimulation ameliorates focal and segmental glomerulosclerosis in mice.

Podocyte damage and loss are the early event in the development of focal segmental glomerulosclerosis (FSGS). Podocytes express angiotensin II type-2-receptor (AT2R), which may play a key role in maintaining kidney integrity and function. Here, we examined the effects of AT2R deletion and AT2R agonist compound 21 (C21) on the evolution of FSGS. FSGS was induced by adriamycin (ADR) injection in both male wild-type (WT) and AT2R knockout (KO) mice. C21 was administered to WT-FSGS mice either one day before or 7 days after ADR (Pre-C21 or Post-C21), using two doses of C21 at either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day. ADR-induced FSGS was more severe in AT2RKO mice compared with WT-FSGS mice, and included profound podocyte loss, glomerular fibrosis, and albuminuria. Glomerular cathepsin L expression increased more in AT2RKO-FSGS than in WT-FSGS mice. C21 treatment ameliorated podocyte injury, most significantly in the Pre C21-HD group, and inhibited glomerular cathepsin L expression. In vitro, Agtr2 knock-down in mouse podocyte cell line given ADR confirmed the in vivo data. Mechanistically, C21 inhibited cathepsin L expression, which protected synaptopodin from destruction and stabilized actin cytoskeleton. C21 also prevented podocyte apoptosis. In conclusion, AT2R activation by C21 ameliorated ADR-induced podocyte injury in mice by the inhibition of glomerular cathepsin L leading to the maintenance of podocyte integrity and prevention of podocyte apoptosis.

Genome-Wide Identification of the Long Noncoding RNAs of Tribolium castaneum in Response to Terpinen-4-ol Fumigation.

Long noncoding RNAs (lncRNAs) are important regulatory factors in multiple biological processes, and several lncRNAs are known to respond to insecticides. However, the lncRNA functions that are associated with terpinen-4-ol resistance in the red flour beetle (Tribolium castaneum) have not yet been identified. In this study, we determined the differentially transcribed lncRNAs between fumigated and control experimental groups. In the six libraries that underwent RNA sequencing, 34,546 transcripts were identified, including 8267 novel lncRNAs, 4155 novel mRNAs, 1151 known lncRNAs, and 20,973 known mRNAs. Among these, we found that the expression of 1858 mRNAs and 1663 lncRNAs was significantly different in the fumigated group compared with the control group. Among the differentially transcribed lncRNAs, 453 were up-regulated and 1210 were down-regulated lncRNAs. In addition, we identified the regulatory function targets of the lncRNAs. Functionally, all lncRNAs and target genes associated with terpinen-4-ol metabolism were enriched in several metabolic pathways, like the ATP-binding cassette transporter, pentose interconversion, and glucuronate interconversion. To the best of our knowledge, this study represents the first global identification of lncRNAs and their potential association with terpinen-4-ol metabolism in the red flour beetle. These results will provide reference information for studies on the resistance to terpinen-4-ol and other essential oil compounds and chemical pesticides, as well as an understanding of other biological processes in T. castaneum.

Estimates of Dietary Exposure to Antibiotics among a Community Population in East China.

BACKGROUND: Antibiotics are widely used in clinics, livestock farms and the aquaculture industry. A variety of antibiotics in foods and drinking water may lead to important and inadvertent dietary exposure However, the profile of dietary exposure to antibiotics in humans is not well-explored. East China is an economically developed area with a high usage of antibiotics and a high rate of antibiotic resistance (ABR). This study aimed to evaluate the total intake level of antibiotics in humans via foods and drinking water based on a community population in East China. METHODS: A total of 600 local residents from 194 households were recruited into this study in Deqing County of Zhejiang Province since June 2019. Each subject was asked to fill a food frequency questionnaire to report their daily consumption of foods and drinking water. Tap water samples were collected from ten households and twenty-one antibiotics of five categories were selected to detect in drinking water. Data of antibiotic residues in animal-derived foods were obtained from the notification of unqualified edible agricultural products after special supervision sampling inspection in Deqing County. The human dietary exposure to antibiotics was estimated by combining the data of antibiotic contamination in foods and drinking water, and the information of dietary consumption. RESULTS: Of twenty-one antibiotics selected, subjects were exposed to a total of sixteen antibiotics, ranging from 15.12 to 1128 µg/day via two main dietary routes (animal-derived foods and drinking water). The overall dietary exposure level varied greatly in the antibiotics detected and their sources. Compared with other antibiotics, enrofloxacin made the most contributions in terms of dietary exposure, with a median exposure level of 120.19 µg/day (IQR: 8.39-698.78 µg/day), followed by sulfamethazine (median: 32.95 µg/day, IQR: 2.77-162.55 µg/day) and oxytetracycline (median: 28.50 µg/day, IQR: 2.22-146.58 µg/day). The estimated exposure level via drinking water (at the ng/day level, median: 26.74 ng/day, IQR: 16.05-37.44 ng/day) was significantly and substantially lower than those via animal-derived foods (at the µg/day level, median: 216.38 µg/day, IQR: 87.52-323.00 µg/day). The overall dietary exposure level also showed differences in sex and age. Males and youths were more likely to be exposed to antibiotics via dietary routes than others. CONCLUSIONS: The community population investigated in East China was extensively exposed to multiple antibiotics via dietary routes. Long-term exposure to low-dose antibiotics in animal-derived foods was the primary dietary exposure route, compared with drinking water. Enrofloxacin contributed to the major body burden of dietary exposure, based on the combination of consumption of aquatic products and considerable enrofloxacin residues in them. Although the human dietary exposure level to antibiotics via drinking water and animal-derived foods ranged from ng/day to µg/L, their chronic toxicity and the accumulation and spread of ABR may be potential hazards to humans. Therefore, long-term monitoring of antibiotic contaminations in foods and drinking water, and human dietary antibiotic exposure is warranted.

Deletion of heterogeneous nuclear ribonucleoprotein F in renal tubules downregulates SGLT2 expression and attenuates hyperfiltration and kidney injury in a mouse model of diabetes.

AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in HnrnpfRT knockout (KO) mice. Non-diabetic HnrnpfRT KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita HnrnpfRT KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita HnrnpfRT KO mice were studied up to the age of 24 weeks (n = 8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita HnrnpfRT KO mice than Akita mice. Compared with Akita mice, Akita HnrnpfRT KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita HnrnpfRT KO mice. Treatment of Akita HnrnpfRT KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita HnrnpfRT KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita HnrnpfRT KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

Generation and validation of APOE knockout human iPSC-derived cerebral organoids.

Apolipoprotein E (apoE) is a major lipid carrier in the brain and closely associated with the pathogenesis of Alzheimer's disease (AD). Here, we describe a protocol for efficient knockout of APOE in human induced pluripotent stem cells (iPSCs) using the CRISPR-Cas9 system. We obtain homozygous APOE knockout (APOE-/- ) iPSCs and further validate the deficiency of apoE in iPSC-derived cerebral organoids. APOE-/- cerebral organoids can serve as a useful tool to study apoE functions and apoE-related pathogenic mechanisms in AD. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

AT2R deficiency in mice accelerates podocyte dysfunction in diabetic progeny in a sex-dependent manner.

AIMS/HYPOTHESIS: The angiotensin II receptor type 2 (AT2R) may be a potential therapeutic target for the treatment of hypertension and chronic kidney disease (CKD). The expression and function of AT2R in the vasculature and kidney appear sexually dimorphic. We hypothesised that Agtr2 knockout dams (AT2RKO) with gestational diabetes would program their offspring for subsequent hypertension and CKD in a sex-dependent manner. METHODS: Age- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and AT2RKO mice were followed from 4 to 20 weeks of age and were monitored for development of hypertension and nephropathy; a mouse podocyte cell line (mPOD) was also studied. RESULTS: Body weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic AT2RKO dams developed insulin resistance. Compared with the offspring of non-diabetic dams, the progeny of diabetic dams had developed more hypertension and nephropathy (apparent glomerulosclerosis with podocyte loss) at 20 weeks of age; this programming was more pronounced in the offspring of AT2RKO diabetic dams, particularly female AT2RKO progeny. Female AT2RKO offspring had lower basal ACE2 glomerular expression, resulting in podocyte loss. The aberrant ACE2/ACE ratio was far more diminished in glomeruli of female progeny of diabetic AT2RKO dams than in male progeny. Knock-down of Agtr2 in mPODs confirmed the in vivo data. CONCLUSIONS/INTERPRETATION: AT2R deficiency accelerated kidney programming in female progeny of diabetic dams, possibly due to loss of protective effects of ACE2 expression in the kidney.