Single-Nucleus Transcriptomic Atlas of Human Pericoronary Epicardial Adipose Tissue in Normal and Pathological Conditions.

BACKGROUND: Pericoronary epicardial adipose tissue (EAT) is a unique visceral fat depot that surrounds the adventitia of the coronary arteries without any anatomic barrier. Clinical studies have demonstrated the association between EAT volume and increased risks for coronary artery disease (CAD). However, the cellular and molecular mechanisms underlying the association remain elusive. METHODS: We performed single-nucleus RNA sequencing on pericoronary EAT samples collected from 3 groups of subjects: patients undergoing coronary bypass surgery for severe CAD (n=8), patients with CAD with concomitant type 2 diabetes (n=8), and patients with valvular diseases but without concomitant CAD and type 2 diabetes as the control group (n=8). Comparative analyses were performed among groups, including cellular compositional analysis, cell type-resolved transcriptomic changes, gene coexpression network analysis, and intercellular communication analysis. Immunofluorescence staining was performed to confirm the presence of CAD-associated subclusters. RESULTS: Unsupervised clustering of 73 386 nuclei identified 15 clusters, encompassing all known cell types in the adipose tissue. Distinct subpopulations were identified within primary cell types, including adipocytes, adipose stem and progenitor cells, and macrophages. CD83high macrophages and FOSBhigh adipocytes were significantly expanded in CAD. In comparison to normal controls, both disease groups exhibited dysregulated pathways and altered secretome in the primary cell types. Nevertheless, minimal differences were noted between the disease groups in terms of cellular composition and transcriptome. In addition, our data highlight a potential interplay between dysregulated circadian clock and altered physiological functions in adipocytes of pericoronary EAT. ANXA1 and SEMA3B were identified as important adipokines potentially involved in functional changes of pericoronary EAT and CAD pathogenesis. CONCLUSIONS: We built a complete single-nucleus transcriptomic atlas of human pericoronary EAT in normal and diseased conditions of CAD. Our study lays the foundation for developing novel therapeutic strategies for treating CAD by targeting and modifying pericoronary EAT functions.

A comparison of clinical features between neurobrucellosis and tuberculous meningitis.

BACKGROUD: This study aims to compare the clinical manifestations, imaging findings, routine tests, biochemistry indicators and cerebrospinal fluid cytology between neurobrucellosis and tuberculous meningitis. The objective is to evaluate the similarities and differences of these two diseases and improve early diagnosis. METHODS: A comprehensive evaluation was conducted by comparing clinical data, imaging results, routine tests findings, biochemistry indicators and cerebrospinal fluid cytology of patients admitted to the Department of Neurology, the Second Hospital of Hebei Medical University from 2019 to 2021. Statistical analysis was applied to identify significant differences and similarities between the two diseases. RESULTS: Preliminary analysis demonstrated both diseases commonly present with symptoms such as fever, headache. However, there were no statistical differences between neurobrucellosis and tuberculous meningitis in early clinical data, imaging results, routine tests findings, biochemistry indicators. Further analysis indicates there is a statistically significantly difference in the lymphocyte ratio and neutrophil ratio in the cerebrospinal fluid between the two groups. CONCLUSIONS: Neurobrucellosis and tuberculous meningitis share similarities in early clinical manifestations, imaging findings and initial cerebrospinal fluid parametes, making early-stage differentiation challenging. The ratio of lymphocytes and neutrophil in the cerebrospinal fluid and a detailed medical history investigation can provide clues for early clinical diagnosis. So the examination of CSF cytology might be a potential to distinguish these two diseases and become a powerful tool in the future.

Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function.

Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

Complex effects of testosterone level on ectoparasite load in a ground squirrel: an experimental test for the immunocompetence handicap hypothesis.

BACKGROUND: The immunocompetence handicap hypothesis suggests that males with a higher testosterone level should be better at developing male secondary traits, but at a cost of suppressed immune performance. As a result, we should expect that males with an increased testosterone level also possess a higher parasite load. However, previous empirical studies aimed to test this prediction have generated mixed results. Meanwhile, the effect of testosterone level on parasite load in female hosts remains poorly known. METHODS: In this study, we tested this prediction by manipulating testosterone level in Daurian ground squirrels (Spermophilus dauricus), a medium-sized rodent widely distributed in northeast Asia. S. dauricus is an important host of ticks and fleas and often viewed as a considerable reservoir of plague. Live-trapped S. dauricus were injected with either tea oil (control group) or testosterone (treatment group) and then released. A total of 10 days later, the rodents were recaptured and checked for ectoparasites. Fecal samples were also collected to measure testosterone level of each individual. RESULTS: We found that testosterone manipulation and sex of hosts interacted to affect tick load. At the end of the experiment, male squirrels subjected to testosterone implantation had an averagely higher tick load than males from the control group. However, this pattern was not found in females. Moreover, testosterone manipulation did not significantly affect flea load in S. dauricus. CONCLUSIONS: Our results only lent limited support for the immunocompetence handicap hypothesis, suggesting that the role of testosterone on regulating parasite load is relatively complex, and may largely depend on parasite type and gender of hosts.

Effect of beta-cypermethrin on the reproductive capacity of female mice in advanced age.

The aim of the present study was to investigate whether exposure to pesticides beta-cypermethrin (ß-CYP) harms the reproductive capacity of advanced-age female mice. The results evidenced that peri-implantation ß-CYP exposure significantly reduced the number of fetuses per advanced-age female in the first litter, and the number and weight of implantation sites. The levels of decidualization markers were significantly reduced in ß-CYP-administered advanced-age mice. Lower expression of Pcna, Cdk6, Foxo1, Ki67, and p62 protein and mRNA was found in the decidua of ß-CYP-treated advanced-age mice. The levels of Bax, cleaved caspase-3, Lc3a/b, Atg, mTOR, and p-mTOR protein, and the ratio of p-mTOR/mTOR protein expression were clearly downregulated by peri-implantation ß-CYP exposure. These results indicated that peri-implantation ß-CYP exposure may elevate the decline in reproductive capacity of early pregnant mice in advanced age.

Pinning Asymptotic Observability of Distributed Boolean Networks.

Asymptotic observability of distributed Boolean networks (DBNs) is studied in this article. Via a parallel extension method, asymptotic observability of the original system is converted to reachability at a fixed point of the extended system. Based on the structure matrix of the extended system, a necessary and sufficient condition is presented for asymptotic observability. Further, for unobservable systems, mode-dependent pinning control is first introduced and applied to achieve asymptotic observability, including the selections of pinning nodes, the design of output feedback controls, and the adding approaches. Then, a set of matrices is defined for the construction of the desired structure matrix. Based on it, a necessary condition is given to guarantee the solvability of the corresponding output feedback controls and the adding approaches. Finally, a numerical example is presented to show the effectiveness of the obtained results.

FGF9 is required for Purkinje cell development and function in the cerebellum.

Fibroblast growth factor 9 (FGF9) is a member of the fibroblast growth factor family, which is widely expressed in the central nervous system (CNS). It has been reported that deletion of FGF9 leads to defects in cerebellum development, including Purkinje cell defect. However, it is not clear how FGF9 regulating cerebellar development remains to be determined. Our results showed that in addition to disrupt Bergmann fiber scaffold formation and granule neuron migration, deletion of neuronal FGF9 led to ataxia defects. It affected development and function of Purkinje cells, and also changed the action potential threshold and excitation frequency. Mechanistically, depletion of FGF9 significantly changed neurotransmitter contents in Purkinje cells and led to preferential increase in inflammation, even downregulation in ERK signaling. Together, the data demonstrate that neuronal FGF9 is required for the development and function of Purkinje cells in the cerebellum. Insufficient FGF9 during cerebellum development will cause ataxia defects.

Association of Magnesium Depletion Score with Congestive Heart Failure: Results from the NHANES 2007-2016.

The magnesium depletion score (MDS) is considered a new valuable and reliable predictor of body magnesium status. This study aimed to explore the association between MDS and congestive heart failure (CHF) among US adults. A total of 19,227 eligible participants from the 2007-2016 National Health and Nutrition Examination Survey were enrolled in this study and then divided into three groups according to the level of MDS (none to low: MDS=0-1, middle: MDS=2, high: MDS=3-5). Sample-weighted logistic regression models were applied to calculate odds ratios (ORs) and 95% confidence intervals (CIs) exploring the independent relationship between MDS and CHF. The estimated prevalence of CHF increased with the increasing level of MDS (none to low: 0.86%, middle: 4.06%, high: 13.52%; P < 0.001). Compared to those in the none-to-low group, participants in the middle and high groups were at significantly higher risk of CHF after adjusting for various covariates (model 3: OR=1.55, 95%CI: 1.05-2.30, P < 0.001; OR=3.20, 95%CI: 2.07-4.96, P < 0.001; respectively). Subgroup analyses indicated that adequate dietary magnesium intake could reduce the risk of CHF in participants who did not meet the recommended dietary allowance (RDA) for magnesium. Besides, there was an interaction between coronary artery disease and MDS on CHF (P for interaction < 0.001). These findings indicated that MDS, a novel indicator estimating magnesium deficiency, is associated with the risk of CHF in non-institutionalized US civilians. Participants whose dietary magnesium intake reaches the RDA might be at lower risk.

Risk factors and early outcomes of prolonged mechanical ventilation following redo aortic arch surgery: A retrospective study.

BACKGROUND: Redo aortic arch surgery is complex and associated with higher risks and mortality. Prolonged mechanical ventilation (PMV) after cardiac surgery is linked to early adverse outcomes and increased costs. OBJECTIVES: Identify specific risk factors and early complications associated with PMV following redo aortic arch surgery. METHODS: Retrospective study at Fuwai Hospital involving 203 patients. Data on patient characteristics, intraoperative factors, and outcomes were analyzed. RESULTS: A total of 203 patients were included, with 42.4 % requiring PMV. PMV patients had longer ICU stays (P < 0.001), lower discharge ADL scores (P < 0.001), and higher hospitalization costs (P < 0.001). While there was no significant difference in-hospital mortality between the two groups, the long-term survival rate in the PMV group was lower than that in the non-PMV group (P = 0.029). Multivariate analysis identified longer cardiopulmonary bypass time (OR 1.008, 95% CI, 1.002 - 1.014, P = 0.006), elevated intraoperative red blood cell transfusion(OR 1.214, 95% CI, 1.057 - 1.393, P = 0.006), higher PEEP (OR 1.296, 95% CI 1.089 - 1.542, P = 0.003), and total arch replacement (OR 3.241, 95% CI 1.392 - 7.543, P = 0.006) as independent risk factors for PMV. CONCLUSION: PMV following redo aortic arch surgery is linked to early adverse outcomes, increased healthcare costs, and reduced long-term survival, with longer cardiopulmonary bypass times, elevated intraoperative red blood cell transfusion, higher PEEP, and total arch replacement as independent risk factors.

Interfering TRIB3 protects the blood brain barrier through PI3K/Akt pathway to alleviate cerebral ischemia-reperfusion injury in diabetes mellitus mice.

This study aimed to treat diabetic cerebral ischemia-reperfusion injury (CI/RI) by affecting blood brain barrier (BBB) permeability and integrity. The CI/RI model in DM mice and a high glucose (HG) treated oxygen and glucose deprivation/reoxygenation (OGD/R) brain endothelial cell model were established for the study. Evans blue (EB) staining was used to evaluate the permeability of BBB in vivo. TTC staining was used to analyze cerebral infarction. The location and expression of tribbles homolog 3 (TRIB3) in endothelial cells were detected by immunofluorescence. Western blotting was used to detect the protein expressions of TRIB3, tight junction molecules, adhesion molecules, phosphorylated protein kinase B (p-AKT) and AKT. The levels of pro-inflammatory cytokines were detected by qRT-PCR. Trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran were used to measure vascular permeability in vitro. TRIB3 ubiquitination and acetylation levels were detected. Acetyltransferase bound to TRIB3 were identified by immunoprecipitation. TRIB3 was localized in cerebral endothelial cells and was highly expressed in diabetic CI/R mice. The BBB permeability in diabetic CI/R mice and HG-treated OGD/R cells was increased, while the junction integrity was decreased. Interference with TRIB3 in vitro reduces BBB permeability and increases junction integrity. In vivo interfering with TRIB3 reduced cerebral infarction volume, BBB permeability and inflammation levels, and upregulated p-AKT levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin reversed the effects of TRIB3-interfering plasmid. In vitro HG treatment induced TRIB3 acetylation through acetyltransferase p300, which in turn reduced ubiquitination and stabilized TRIB3. Interfering TRIB3 protects BBB by activating PI3K/AKT pathway and alleviates brain injury, which provides a new target for diabetic CI/RI.

Investigating chimeric antigen receptor T cell therapy and the potential for cancer immunotherapy (Review).

Immunotherapy has emerged as a crucial treatment option, particularly for types of cancer that display resistance to conventional therapies. A remarkable breakthrough in this field is the development of chimeric antigen receptor (CAR) T cell therapy. CAR T cells are generated by engineering the T cells of a patient to express receptors that can recognize specific tumor antigens. This groundbreaking approach has demonstrated impressive outcomes in hematologic malignancies, including diffuse large B cell lymphoma, B cell acute lymphoblastic leukemia and multiple myeloma. Despite these significant successes, CAR T cell therapy has encountered challenges in its application against solid tumors, leading to limited success in these cases. Consequently, researchers are actively exploring novel strategies to enhance the efficacy of CAR T cells. The focus lies on augmenting CAR T cell trafficking to tumors while preventing the development of CAR T cell exhaustion and dysfunction. The present review aimed to provide a comprehensive analysis of the achievements and limitations of CAR T cell therapy in the context of cancer treatment. By understanding both the successes and hurdles, further advancements in this promising area of research can be developed. Overall, immunotherapy, particularly CAR T cell therapy, has opened up novel possibilities for cancer treatment, offering hope to patients with previously untreatable malignancies. However, to fully realize its potential, ongoing research and innovative strategies are essential in overcoming the challenges posed by solid tumors and maximizing CAR T cell efficacy in clinical settings.

Role of T Cells in Microbial Pathogenesis.

The immune system functions as a sophisticated defense mechanism, shielding the body from harmful pathogenic invaders [...].

Control of CD4+ T cells to restrain inflammatory diseases via eukaryotic elongation factor 2 kinase.

CD4+ T cells, particularly IL-17-secreting helper CD4+ T cells, play a central role in the inflammatory processes underlying autoimmune disorders. Eukaryotic Elongation Factor 2 Kinase (eEF2K) is pivotal in CD8+ T cells and has important implications in vascular dysfunction and inflammation-related diseases such as hypertension. However, its specific immunological role in CD4+ T cell activities and related inflammatory diseases remains elusive. Our investigation has uncovered that the deficiency of eEF2K disrupts the survival and proliferation of CD4+ T cells, impairs their ability to secrete cytokines. Notably, this dysregulation leads to heightened production of pro-inflammatory cytokine IL-17, fosters a pro-inflammatory microenvironment in the absence of eEF2K in CD4+ T cells. Furthermore, the absence of eEF2K in CD4+ T cells is linked to increased metabolic activity and mitochondrial bioenergetics. We have shown that eEF2K regulates mitochondrial function and CD4+ T cell activity through the upregulation of the transcription factor, signal transducer and activator of transcription 3 (STAT3). Crucially, the deficiency of eEF2K exacerbates the severity of inflammation-related diseases, including rheumatoid arthritis, multiple sclerosis, and ulcerative colitis. Strikingly, the use of C188-9, a small molecule targeting STAT3, mitigates colitis in a murine immunodeficiency model receiving eEF2K knockout (KO) CD4+ T cells. These findings emphasize the pivotal role of eEF2K in controlling the function and metabolism of CD4+ T cells and its indispensable involvement in inflammation-related diseases. Manipulating eEF2K represents a promising avenue for novel therapeutic approaches in the treatment of inflammation-related disorders.

Effectiveness of VA-ECMO plus intra-aortic balloon pump for cardiac shock in patients with type A aortic dissection: a case series.

Limited reports exist on the utilization of venoarterial extracorporeal membrane oxygenation (VA-ECMO) following aortic dissection surgery, possibly due to concerns regarding complications. This case series aimed to evaluate the effectiveness and safety of using VA-ECMO in combination with intra-aortic balloon pump (IABP) for managing postoperative cardiogenic shock in patients with type A aortic dissection (AAD). The study included nine patients with an average age of 57.0 ± 9.5 years. The patients underwent various surgical procedures, including coronary artery bypass grafting (CABG) and aortic root reconstruction. The results showed that the combined use of VA-ECMO and IABP was feasible and effective in managing postoperative cardiogenic shock in AAD patients. However, the in-hospital mortality rate was high, with six out of nine patients succumbing to the condition. Among the patients who received VA-ECMO plus IABP in the operating room, four were successfully weaned from VA-ECMO, and three survived with a mean follow-up of 20 months. The study also highlighted the potential risks of renal complications associated with VA-ECMO and IABP. The findings suggest that the combined therapy of VA-ECMO and IABP may be beneficial for patients who have difficulty weaning from cardiopulmonary bypass (CPB) after AAD surgery.

[Multicenter Prospective Study of Different Induction Regimens of Azacytidine in Treatment of Elderly Patients with Acute Myeloid Leukemia].

OBJECTIVE: To observe the efficacy and safety of different induction regimens of same total dosage of azacitidine (Aza), including standard dose (standard dose group) and low-dose long-term (adjusted dose group), in the treatment of elderly acute myeloid leukemia (AML). METHODS: A total of 103 elderly patients with AML (non-acute promyelocytic leukemia) from January 2020 to June 2021 were enrolled. Aza was administered at the standard dose of 75 mg/(m2·d) for 7 days in the standard dose group (50 cases), while at 100 mg/d for 7-12 days in the adjusted dose group (53 cases). The administration days in adjusted dose group was calculated based on the total standard dose of the patient's single course of treatment. The efficacy and safety between standard dose group and adjusted dose group were compared. Subgroup analysis were performed in the two groups for Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy for efficacy and safety. RESULTS: There were no significant differences in overall response rate (ORR), incidence of adverse reaction, and 1-year overall survival (OS) rate between standard dose group and adjusted dose group (P >0.05). The ORR of combination was higher than that of Aza alone (P < 0.05), while there was no significant difference in ORR between Aza combined with BCL-2 inhibitor and Aza combined with low-dose chemotherapy (P >0.05). The combination of BCL-2 inhibitor did not increase the incidence of adverse reactions compared wtih Aza alone. There was a higher risk of myelosuppression and pulmonary infection with a combination of low-dose chemotherapy than with a combination of BCL-2 inhibitor and Aza alone (P <0.05). No significant difference was observed in 1-year OS between Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy (P >0.05). CONCLUSIONS: Both two induction regimens can be used in elderly AML patients who cannot tolerate intensive chemotherapy with similar overall effectiveness and safety. Aza combined with low-dose chemotherapy may result in increased ORR and an increased incidence of serious adverse reactions, and may not result in longer survival compared with Aza alone. Aza combined with BCL-2 inhibitor not only has similar effect in complete remission, objective response rate, and OS compared with Aza combined with low-dose chemotherapy, but also has higher safety.

Keystone microalgae species determine the removal efficiency of sulfamethoxazole: a case study of Chlorella pyrenoidosa and microalgae consortia.

In recent years, antibiotics pollution has caused serious harm to the aquatic environment, and microalgae mediated degradation of antibiotics has attracted increasing attention. However, the potential toxicity of antibiotics to keystone microalgae species or their microalgae consortia, and the impact of microalgal diversity on antibiotic removal need to be further studied. In this study, we investigated the removal efficiency and tolerance of five freshwater microalgae (Chlorella pyrenoidosa, Scenedesmus quadricauda, Dictyosphaerium sp., Haematoccocus pluvialis, and Botryococcus braunii) and their microalgae consortia to sulfamethoxazole (SMX). We found that the removal efficiency of SMX by C. pyrenoidosa reached 49%, while the other four microalgae ranged between 9% and 16%. In addition, C. pyrenoidosa, S. quadricauda, and Dictyosphaerium sp. had better tolerance to SMX than H. pluvialis, and their growth and photosynthesis were less affected. At 10 and 50 mg/L SMX, the removal capacity of SMX by mixed microalgae consortia was lower than that of C. pyrenoidos except for the consortium with C. pyrenoidos and S. quadricauda. The consortia generally showed higher sensitivity towards SMX than the individual species, and the biochemical characteristics (photosynthetic pigment, chlorophyll fluorescence parameters, superoxide anion (O2 -), superoxide dismutase activity (SOD), malondialdehyde (MDA) and extracellular enzymes) were significantly influenced by SMX stress. Therefore, the removal of antibiotics by microalgae consortia did not increase with the number of microalgae species. Our study provides a new perspective for the selection of microalgal consortia to degrade antibiotics.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

NAC1 confines virus-specific memory formation of CD4+ T cells through the ROCK1-mediated pathway.

Nucleus accumbens-associated protein 1 (NAC1), a transcriptional cofactor, has been found to play important roles in regulating regulatory T cells, CD8+ T cells, and antitumor immunity, but little is known about its effects on T-cell memory. In this study, we found that NAC1 expression restricts memory formation of CD4+ T cells during viral infection. Analysis of CD4+ T cells from wild-type (WT) and NAC1-deficient (-/- ) mice showed that NAC1 is essential for T-cell metabolism, including glycolysis and oxidative phosphorylation, and supports CD4+ T-cell survival in vitro. We further demonstrated that a deficiency of NAC1 downregulates glycolysis and correlates with the AMPK-mTOR pathway and causes autophagy defective in CD4+ T cells. Loss of NAC1 reduced the expression of ROCK1 and the phosphorylation and stabilization of BECLIN1. However, a forced expression of ROCK1 in NAC1-/- CD4+ T cells restored autophagy and the activity of the AMPK-mTOR pathway. In animal experiments, adoptively transferred NAC1-/- CD4+ T cells or NAC1-/- mice challenged with VACV showed enhanced formation of VACV-specific CD4+ memory T cells compared to adoptively transferred WT CD4+ T cells or WT mice. This memory T-cell formation enhancement was abrogated by forcing expression of ROCK1. Our study reveals a novel role for NAC1 as a suppressor of CD4+ T-cell memory formation and suggests that targeting NAC1 could be a new approach to promoting memory CD4+ T-cell development, which is critical for an effective immune response against pathogens.

CRISPR/Cas9-induced asap1a and asap1b co-knockout mutant zebrafish displayed abnormal embryonic development and impaired neutrophil migration.

ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.

Regulatory considerations for paediatric drug evaluation in China.

The regulatory guidelines for the research and development of paediatric drugs are still evolving in China. The formulation of the guidelines started from learning and borrowing existing experience, and gradually changed to the exploration and improvement of local guidelines, which was not only in line with international standards but also had breakthroughs, innovations and Chinese characteristics. In this paper, the current setting of paediatric drug research and development in China and corresponding technical guidelines have been introduced from regulatory perspectives, and the accessibility of further improvement in regulatory strategies has also been discussed.