Expanding the DNA-encoded library toolbox: identifying small molecules targeting RNA.

DNA-encoded library (DEL) technology is a powerful tool for small molecule identification in drug discovery, yet the reported DEL selection strategies were applied primarily on protein targets in either purified form or in cellular context. To expand the application of this technology, we employed DEL selection on an RNA target HIV-1 TAR (trans-acting responsive region), but found that the majority of signals were resulted from false positive DNA-RNA binding. We thus developed an optimized selection strategy utilizing RNA patches and competitive elution to minimize unwanted DNA binding, followed by k-mer analysis and motif search to differentiate false positive signal. This optimized strategy resulted in a very clean background in a DEL selection against Escherichia coli FMN Riboswitch, and the enriched compounds were determined with double digit nanomolar binding affinity, as well as similar potency in functional FMN competition assay. These results demonstrated the feasibility of small molecule identification against RNA targets using DEL selection. The developed experimental and computational strategy provided a promising opportunity for RNA ligand screening and expanded the application of DEL selection to a much wider context in drug discovery.

A Study of Elderly Patients' Preference for "Internet + Nursing Service" Based on Discrete Choice Experiments.

Objective: To investigate the preference of elderly patients for "Internet + Nursing Service", so as to provide reference for accurately matching their service needs. Methods: Based on discrete choice experiments, the elderly patients' "Internet + Nursing Service" choice preference questionnaire was prepared, and 410 elderly patients were interviewed face-to-face. The Conditional Logit Model was used for quantitative analysis of the experimental data. Results: The six attributes included in the study had a significant impact on the elderly patients' preference for "Internet + Nursing Service" (P < .05). Among non-economic attributes, "medical insurance reimbursement ratio -70%" has the highest utility (.263, P < .001), with "hospital size - larger (.205, P < .05)" and "medical insurance reimbursement ratio -50%" (.188, P < .05) ranking second and third, respectively. The relative importance of the attribute of medical insurance reimbursement proportion is the highest (26.44%), and the relative importance of service content is the lowest (9.78%). If the proportion of medical insurance reimbursement increases from 30% to 70%, patients are willing to pay 202.9 yuan, and the probability of choosing "Internet + Nursing Service" increases by 6.1%. Conclusion: Patients prefer to choose a higher proportion of medical insurance reimbursement, a larger medical institution, a higher level of nurses, low single service costs, a basic package, and "Internet + Nursing Service" led by the government and hospitals. Improvement of the price and medical insurance payment policy is recommended, along with the enhancement of the ability of primary nursing services, strengthening of the management of Internet enterprise platforms, and scientific assessment of the needs of patients for personalized nursing services.

[Retrospective study on the diagnosis, treatment, and follow-up of 85 cases of hypophosphatemic rickets in children]. / 85ä¾‹ä½Žè¡€ç£·æ€§ä½å»ç— æ‚£å„¿è¯Šæ–­ã€æ²»ç–—ä¸Žéšè®¿çš„å›žé¡¾æ€§ç ”ç©¶.

OBJECTIVES: To study the diagnosis, treatment, and complications of hypophosphatemic rickets (HR) in children, explore effectiveness evaluation indicators for the disease, and understand the pattern in height growth among these patients. METHODS: A retrospective analysis of the initial clinical data and five-year follow-up data of 85 children with HR treated at Children's Hospital of Nanjing Medical University from January 2008 to December 2022. RESULTS: Among the 85 children with HR, there were 46 males (54%) and 39 females (46%). The age at initial diagnosis ranged from 6 months to 13 years and 9 months, with a median age of 2.75 years. The average height standard deviation score was -2.0±1.1. At initial diagnosis, children exhibited reduced blood phosphate levels and elevated alkaline phosphatase (ALP), with 99% (84/85) presenting with lower limb deformities. The positive rate for PHEX gene mutations was 93% (55/59). One year post-treatment, there was a significant reduction in ALP levels and the gap between the lower limbs (P<0.05). The fastest height growth occurred in the first year after treatment, at 8.23 cm/year, with a peak height velocity (PHV) phase lasting about two years during puberty. The height increased by 9-20 cm in male children during the PHV stage and 10-15 cm in female children. Major complications included nephrocalcinosis and hyperparathyroidism. The incidence rate of nephrocalcinosis in the first year after treatment was 55% (22/40), which increased with the duration of the disease (P<0.001); an increased urinary phosphate/creatinine ratio was positively associated with a higher risk of nephrocalcinosis (OR=1.740, P<0.001). The incidence of hyperparathyroidism in the first year after treatment was 64% (27/42). CONCLUSIONS: For children presenting with lower limb deformities, short stature, and slow growth, early testing for blood levels of phosphate, calcium, and ALP, along with imaging examinations of the lower limbs, can aid in the early diagnosis of HR. Genetic testing may be utilized for definitive confirmation when necessary. ALP combined with improvements in skeletal deformities and annual height growth can serve as indicators of therapeutic effectiveness for HR. Compared to normal children, children with HR demonstrate a lower height increase during the PHV phase, necessitating close follow-up and timely adjustment of treatment plans Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 677-682.

Well-Ordered Au Nanoarray for Sensitive and Reproducible Detection of Hepatocellular Carcinoma-Associated miRNA via CHA-Assisted SERS/Fluorescence Dual-Mode Sensing.

Ultra-sensitive detection of cancer-related biomarkers in serum is of great significance for early diagnosis, treatment, prognosis, and staging of cancer. In this work, we proposed a surface-enhanced Raman scattering and fluorescence (SERS/FL) dual-mode biosensor for hepatocellular carcinoma (HCC)-related miRNA (miR-224) detection using the composition of well-arranged Au nanoarrays (Au NAs) substrate coupled with the target-catalyzed hairpin assembly (CHA) strategy. The hot spots densely and uniformly distributed on the Au array offers considerably enhanced and reproducible SERS signals, along with their wide and open surface to facilitate miR-224 adsorption. By this sensing strategy, the target miR-224 can be detected in a wide linear range (1 fM to 1 nM) with a limit of detection of 0.34 fM in the SERS mode and 0.39 fM in the FL mode. Meanwhile, this biosensor with exceptional specificity and anti-interference ability can discriminate target miR-224 from other interference miRNAs. Practical analysis of human blood samples also demonstrated considerable reliability and repeatability of our developed strategy. Furthermore, this biosensor can distinguish HCC cancer subjects from normal ones and monitor HCC patients before and after hepatectomy as well as guide the distinct Barcelona clinic liver cancer (BCLC) stages. Overall, benefiting from a well-arranged Au nanoarray, CHA amplification strategy, and SERS/metal enhanced fluorescence effect, this established biosensor opens new avenues for the early prediction, warning, monitoring, and staging of HCC.

Growth hormone treatment in pre-pubertal short Chinese children with chronic kidney disease prior to transplantation.

BACKGROUND: The effect of recombinant human GH (rhGH) in Chinese children with chronic kidney disease (CKD) is unclear. METHODS: This was a 52-week, multicenter, randomized, open-label, negative-controlled phase 3 study. Prepubertal subjects were randomized 1:1 to either daily subcutaneous injections of rhGH 0.05 mg/kg/day or no treatment for 52 weeks. RESULTS: A total of 68 subjects with a mean age of 7.8 ± 3.27 years were enrolled. At week 52, the height standard deviation score (HT-SDS) in the treated group increased by 0.75 ± 0.58, which was significantly higher compared with 0.17 ± 0.47 in the untreated group (least squares mean 0.58, 95% confidence interval, 0.32-0.84; P < 0.001). At week 52, significant improvements were observed in other growth parameters (height velocity [P < 0.001]), insulin-like growth factor 1 (IGF-1) SDS [P < 0.001], IFG-1/insulin-like growth factor binding protein-3 molar ratio [P < 0.001], and height [P < 0.001]) compared with the untreated control. Seven patients reported treatment-related adverse events (TRAEs) and most TRAEs were mild in severity. Most subjects recovered without further intervention. CONCLUSIONS: Daily rhGH for 52 weeks in children with CKD-induced growth retardation significantly improved HT-SDS and other growth parameters without compromising safety. IMPACT: The efficacy and safety of growth hormone (GH) therapy in Chinese children with chronic kidney disease (CKD) are unclear. This study found that giving short stature Chinese children with CKD daily recombinant human growth hormone (rhGH) for 52 weeks improved growth parameters without compromising safety. This study's information can give physicians the confidence to treat these patients in their clinical practice.

[Clinical characteristics and prognosis of 12 cases of lupus nephritis complicated with thrombotic microangiopathy]. / 12ä¾‹ç‹¼ç–®æ€§è‚¾ç‚Žåˆå¹¶è¡€æ “æ€§å¾®è¡€ç®¡ç— å˜ä¸´åºŠç‰¹ç‚¹åŠé¢„åŽåˆ†æž.

OBJECTIVES: To investigate the clinical characteristics, pathological features, treatment regimen, and prognosis of children with lupus nephritis (LN) and thrombotic microangiopathy (TMA), as well as the treatment outcome of these children and the clinical and pathological differences between LN children with TMA and those without TMA. METHODS: A retrospective analysis was conducted on 12 children with LN and TMA (TMA group) who were admitted to the Department of Nephrology, Children's Hospital of Nanjing Medical University, from December 2010 to December 2021. Twenty-four LN children without TMA who underwent renal biopsy during the same period were included as the non-TMA group. The two groups were compared in terms of clinical manifestations, laboratory examination results, and pathological results. RESULTS: Among the 12 children with TMA, 8 (67%) had hypertension and 3 (25%) progressed to stage 5 chronic kidney disease. Compared with the non-TMA group, the TMA group had more severe tubulointerstitial damage, a higher Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score at onset, and higher cholesterol levels (P<0.05). There were no significant differences between the two groups in the percentage of crescent bodies and the levels of hemoglobin and platelets (P>0.05). CONCLUSIONS: There is a higher proportion of individuals with hypertension among the children with LN and TMA, as well as more severe tubulointerstitial damage. These children have a higher SLEDAI score and a higher cholesterol level.

Role of exogenous abscisic acid in freezing tolerance of mangrove Kandelia obovata under natural frost condition at near 32°N.

BACKGROUND: Mangroves possess substantial ecological, social, and economic functions in tropical and subtropical coastal wetlands. Kandelia obovata is the most cold-resistance species among mangrove plants, with a widespread distribution in China that ranges from Sanya (18° 12' N) to Wenzhou (28° 20' N). Here, we explored the temporal variations in physiological status and transcriptome profiling of K. obovata under natural frost conditions at ~ 32oN, as well as the positive role of exogenous abscisic acid (ABA) in cold resistance. RESULTS: The soluble sugar (SS) and proline (Pro) functioned under freezing stress, of which SS was more important for K. obovata. Consistently, up-regulated DEGs responding to low temperature were significantly annotated to glycometabolism, such as starch and sucrose metabolism and amino sugar and nucleotide sugar metabolism. Notably, the top 2 pathways of KEGG enrichment were phenylpropanoid biosynthesis and flavonoid biosynthesis. For the antioxidant system, POD in conjunction with CAT removed hydrogen peroxide, and CAT appeared to be more important. The up-regulated DEGs responding to low temperature and ABA were also found to be enriched in arginine and proline metabolism, starch and sucrose metabolism, and peroxisome. Moreover, ABA triggered the expression of P5CS and P5CR, but inhibited the ProDH expression, which might contribute to Pro accumulation. Interestingly, there was no significant change in malondialdehyde (MDA) content during the cold event (P > 0.05), suggesting foliar application of ABA effectively alleviated the adverse effects of freezing stress on K. obovata by activating the antioxidant enzyme activity and increasing osmolytes accumulation, such as Pro, and the outcome was proportional to ABA concentration. CONCLUSIONS: This study deepened our understanding of the physiological characters and molecular mechanisms underlying the response of K. obovata to natural frost conditions and exogenous ABA at the field level, which could provide a sound theoretical foundation for expanding mangroves plantations in higher latitudes, as well as the development coastal landscape.

A Study of Blended Learning Using the Smart Class Teaching Module on Psychosocial Dysfunction Course During the Training of Undergraduate Occupational Therapy Students in China.

BACKGROUND Online blended learning, also known as "smart classes", has benefits when compared with traditional teaching methods that use books and lectures. This study aimed to compare the use of the Smart Class teaching module with traditional teaching on the topic of psychosocial dysfunction during the training of undergraduate occupational therapy (OT) students in China. MATERIAL AND METHODS We recruited Grade 2017 OT students as the Smart Class teaching module group and Grade 2016 OT students as the Traditional Class teaching module group to participate in the study. The objective evaluation (assignment score, practical exam score, written exam score, and final score) and subjective evaluation (data from student questionnaires and information from interviews with the lead teacher and assistant teachers) were performed in both groups. RESULTS No significant difference was found in the final scores (P=0.874) and students' questionnaire results between the 2 groups. However, data from the student questionnaires and teacher interviews indicated a preference for combining the Smart Class teaching module and the Traditional Class teaching module. CONCLUSIONS The advantage of the Smart Class teaching module is that it can effectively integrate excellent teaching resources across geographical restrictions and it is conducive to promoting independent learning for students and all-around supervision for teaching. The Smart Class teaching module was comparable to traditional teaching methods for the training of undergraduate OT students in China, but was preferred by the students.

A method for estimating binding affinity from primary DEL selection data.

DEL selections are binding assays conducted with mixtures of chemically diverse DNA-tagged ligands and a screening target. DEL selections use DNA sequence counts to measure target binding, where ideally higher affinity ligands will have higher counts than weaker affinity ligands. However, there is not always a clear relationship between DNA sequence count (assay signal) and binding affinity. This disconnect may be due to the fidelity of library chemistry, where reactions often do not go to completion, and also to repetitive rounds of binding and elution that are standard practice in most DEL selection experiments. We describe here a strategy that addresses both of these issues and provides a means to calculate ligand affinity from primary selection data. The reaction yields of selected compounds during DEL library synthesis can also be predicted with this method.

A simple method for determining compound affinity and chemical yield from DNA-encoded library selections.

DNA-encoded libraries (DELs) can contain billions of unique chemical species; selecting against such large inputs, it is typical to find more candidate binders than is reasonable to pursue for follow-up synthesis and testing. Given this wealth of choices, common practice is to limit synthesis to only those compounds estimated to have the greatest chance of being high-affinity binders; of the many potential factors contributing to this estimation, the strength of the selection signal of a candidate binder is always important. We define here methods and equations which relate the theoretical selection signal of a compound to its affinity and chemical yield. Tests using known binders of BRD4 and ROCK2 support the theory backing these equations and suggest they should be of use for prospectively determining affinity and chemical yield from primary DEL selection data.

Comparative transcriptome analysis of nonchilled, chilled, and late-pink bud reveals flowering pathway genes involved in chilling-mediated flowering in blueberry.

BACKGROUND: Blueberry cultivars require a fixed quantity of chilling hours during winter endo-dormancy for vernalization. In this study, transcriptome analysis using RNA sequencing data from nonchilled, chilled, and late pink buds of southern highbush blueberry 'Legacy' was performed to reveal genes associated with chilling accumulation and bud break. RESULTS: Fully chilled 'Legacy' plants flowered normally whereas nonchilled plants could not flower. Compared to nonchilled flower buds, chilled flower buds showed differential expression of 89% of flowering pathway genes, 86% of MADS-box genes, and 84% of cold-regulated genes. Blueberry orthologues of FLOWERING LOCUS T (FT) did not show a differential expression in chilled flower buds (compared to nonchilled flower bud) but were up-regulated in late-pink buds (compared to chilled flower bud). Orthologoues of major MADS-box genes were significantly up-regulated in chilled flower buds and down-regulated in late-pink buds. Functional orthologues of FLOWERING LOCUS C (FLC) were not found in blueberry. Orthologues of Protein FD (FD), TERMINAL FLOWER 1 (TFL1), and LEAFY (LFY) were down-regulated in chilled flower buds and in late-pink buds compared to nonchilled flower bud. CONCLUSIONS: The changes from nonchilled to chilled and chilled to late-pink buds are associated with transcriptional changes in a large number of differentially expressed (DE) phytohormone-related genes and DE flowering pathway genes. The profile of DE genes suggests that orthologues of FT, FD, TFL1, LFY, and MADS-box genes are the major genes involved in chilling-mediated blueberry bud-break. The results contribute to the comprehensive investigation of the vernalization-mediated flowering mechanism in woody plants.

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.

Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.

Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.

In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.

[Epidemic condition and molecular subtyping of ciprofloxacin and cefotaxime co-resistant Salmonella Indiana isolated from retail chicken carcasses in six provinces, China].

OBJECTIVE: To elucidate the epidemic condition and molecular subtyping of ciprofloxacin and cefotaxime co-resistant Salmonella Indiana (S. Indiana) isolated from retail chicken carcasses in six provinces of China. METHODS: A total of 2 647 Salmonella strains isolated from retail chicken carcasses collected from six provinces of China were subjected to antimicrobial susceptibility testing. All Salmonella isolates co-resistant to ciprofloxacin and cefotaxime were further characterized by serotyping, extended-spectrum beta-lactamases (ESBLs) producing strains screening and pulsed field gel electrophoresis (PFGE) typing. RESULTS: Among 2 629 Salmonella isolates tested, 227 (8.52%) isolates were co-resistant to ciprofloxacin and ceftazidime/cefotaxime (Beijing: 11.67% (99/874), Jilin: 8.20% (60/726), Guangdong: 1.39% (7/502), Jiangsu: 15.61% (42/260), Shaanxi: 8.56% (16/186), Inner Mongolia: 0 (0/81)), and 224 of them were identified as S. Indiana. 213 (95.10%) isolates of S. Indiana were ESBLs producing strains. All ciprofloxacin and cefotaxime co-resistant S. Indiana isolates developed a multi-drug resistant profile and 17.86% (40/224) of them were resistant to all antibiotics tested except carbapenems, and 50.89% (114/224) of them resistant to 9 antibiotics, additionally, 25.45% (57/224) of them showed multi-drug resistance to 8 antibiotics. All ciprofloxacin and cefotaxime co-resistant S. Indiana isolates were divided into 32 PFGE clusters and 150 PFGE patterns. Strains of S. Indiana from same or different sampling site and time seemed to either share the same PFGE patterns or be differential to each other in different regions. CONCLUSION: The results indicated that chicken carcasses collected from parts of China were heavily contaminated by ciprofloxacin and cefotaxime co-resistant S. Indiana and could serve as an important reservoir of ciprofloxacin and cefotaxime co-resistant Salmonella. Molecular subtyping results indicated that cross contamination or common pollution source might be in these strains.

Exploring the role of psychological assistance hotlines in improving mental health problems among Chinese adult women: A perspective based on social expectations and gender roles.

This paper looks at how social expectations and gender roles affect the mental health of Chinese women. Traditional Chinese culture, influenced by Confucianism and patriarchy, still has a negative impact on women's mental well-being, despite efforts for gender equality. Women's mental health is vital for both individuals and society, and this study aims to understand these issues better to help shape policies and interventions.

Monogenic Causes Identified in 23.68% of Children with Steroid-Resistant Nephrotic Syndrome: A Single-Centre Study.

Introduction: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of end-stage kidney disease in children, mostly associated with focal segmental glomerulosclerosis (FSGS). Advances in genomic science have enabled the identification of causative variants in 20-30% of SRNS patients. Methods: We used whole exome sequencing to explore the genetic causes of SRNS in children. Totally, 101 patients with SRNS and 13 patients with nephrotic proteinuria and FSGS were retrospectively enrolled in our hospital between 2018 and 2022. For the known monogenic causes analysis, we generated a known SRNS gene list of 71 genes through reviewing the OMIM database and literature. Results: Causative variants were identified in 23.68% of our cohort, and the most frequently mutated genes in our cohort were WT1 (7/27), NPHS1 (3/27), ADCK4 (3/27), and ANLN (2/27). Five patients carried variants in phenocopy genes, including MYH9, MAFB, TTC21B, AGRN, and FAT4. The variant detection rate was the highest in the two subtype groups with congenital nephrotic syndrome and syndromic SRNS. In total, 68.75% of variants we identified were novel and have not been previously reported in the literature. Conclusion: Comprehensive genetic analysis is key to realizing the clinical benefits of a genetic diagnosis. We suggest that all children with SRNS undergo genetic testing, especially those with early-onset and extrarenal phenotypes.

Molecular mechanism of salinity and waterlogging tolerance in mangrove Kandelia obovata.

Mangrove forests are colloquially referred to as "Earth's kidneys" and serve many important ecological and commercial functions. Salinity and waterlogging stress are the most important abiotic stressors restricting the growth and development of mangroves. Kandelia obovata (K. obovata) is the greatest latitudinally-distributed salt mangrove species in China.Here, morphology and transcriptomics were used to study the response of K. obovata to salt and waterlogging stress. In addition, weighted gene co-expression network analysis of the combined gene expression and phenotypic datasets was used to identify core salinity- and waterlogging-responsive modules. In this study, we observed that both high salinity and waterlogging significantly inhibited growth and development in K. obovata. Notably, growth was negatively correlated with salt concentration and positively correlated with waterlogging duration, and high salinity was significantly more inhibitive than waterlogging. A total of 7, 591 salt-responsive and 228 waterlogging-responsive differentially expressed genes were identified by RNA sequencing. Long-term salt stress was highly correlated with the measured physiological parameters while long-term waterlogging was poorly correlated with these traits. At the same time, 45 salinity-responsive and 16 waterlogging-responsive core genes were identified. All 61 core genes were mainly involved in metabolic and biosynthesis of secondary metabolites pathways. This study provides valuable insight into the molecular mechanisms of salinity and waterlogging tolerance in K. obovata, as well as a useful genetic resource for the improvement of mangrove stress tolerance using molecular breeding techniques.

Integrating single-cell and spatial transcriptomics to elucidate the crosstalk between cancer-associated fibroblasts and cancer cells in hepatocellular carcinoma with spleen-deficiency syndrome.

Background and aim: Most patients with hepatocellular carcinoma (HCC) in China have been diagnosed with spleen deficiency syndrome (SDS), which accelerates the progression of HCC by disrupting the tumor microenvironment homeostasis. This study aimed to investigate the intercellular crosstalk in HCC with SDS. Experimental procedure: An HCC-SDS mouse model was established using orthotopic HCC transplantation based on reserpine-induced SDS. Single-cell data analysis and cancer cell prediction were conducted using Seurat and CopyKAT package, respectively. Intercellular interactions were explored using CellPhoneDB and CellChat and subsequently validated using co-culture assays, ELISA and histological staining. We performed pathway activity analysis using gene set variation analysis and the Seurat package. The extracellular matrix (ECM) remodeling was assessed using a gel contraction assay, atomic force microscopy, and Sirius red staining. The deconvolution of the spatial transcriptomics data using the "CARD" package based on single-cell data. Results and conclusion: We successfully established the HCC-SDS mouse model. Twenty-nine clusters were identified. The interactions between cancer cells and cancer-associated fibroblasts (CAFs) were significantly enhanced via platelet-derived growth factor (PDGF) signaling in HCC-SDS. CAFs recruited in HCC-SDS lead to ECM remodeling and the activation of TGF-ß signaling pathway. Deconvolution of the spatial transcriptome data revealed that CAFs physically surround cancer cells in HCC-SDS. This study reveals that the crosstalk of CAFs-cancer cells is crucial for the tumor-promoting effect of SDS. CAFs recruited by HCC via PDGFA may lead to ECM remodeling through activation of the TGF-ß pathway, thereby forming a physical barrier to block immune cell infiltration under SDS.

CYP2C9*3(1075A > C), ABCB1 and SLCO1B1 genetic polymorphisms and gender are determinants of inter-subject variability in pitavastatin pharmacokinetics.

A pharmacokinetics study was conducted in 12 Chinese volunteers following a single dose of 1 mg, 2 mg and 4 mg of pitavastatin calcium in an open-label, randomized, three-period crossover design. Plasma concentrations of pitavastatin acid and pitavastatin lactone were determined by a HPLC method. Single-nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, SLCO1B1, CYP2C9 and CYP3A5 were determined by TaqMan (MGB) genotyping assay. An analysis was performed on the relationship between the aforementioned SNPs and dose-normalized (based on 1 mg) area under the plasma concentration-time curve extrapolated to infinity [AUC(0-infinity)] and peak plasma concentration (Cmax) values of the acid and lactone forms of pitavastatin. Pitavastatin exhibited linear pharmacokinetics and great inter-subject variability. Compared to CYP2C9*1/*1 carriers, CYP2C9*1/*3 carriers had higher AUC(0-infinity) and Cmax of pitavastatin acid and AUC(0-infinity) of pitavastatin lactone (P<0.05). With respect to ABCB1 G2677T/A, non-G carriers had higher Cmax and AUC(0-infinity) of pitavastatin acid, and Cmax of pitavastatin lactone compared to GT, GA or GG genotype carriers (P<0.05). Gene-dose effects of SLCO1B1 c.521T> C and g.11187G > A on pharmacokinetics of the acid and lactone forms were observed. Compared to non-SLCO1B1*17 carriers, SLCO1B1*17 carriers had higher Cmax and AUC(0-infinity) of the acid and lactone forms (P<0.05). Significant sex difference was observed for pharmacokinetics of the lactone. Female SLCO1B1 521TT subjects had higher Cmax and AUC(0-infinity) of pitavastatin lactone compared to male 521TT subjects, however, such gender difference disappeared in 521 TC and 521CC subjects. Pitavastatin pharmacokinetics was not significantly affected by ABCB1 C1236T, ABCB1C3435T, CYP3A5*3, ABCG2 c.34G > A, c.421C > A, SLCO1B1 c.388A>G, c.571T>C and c.597C>T. We conclude that CYP2C9*3, ABCB1 G2677T/A, SLCO1B1 c.521T>C, SLCO1B1 g.11187G > A, SLCO1B1*17 and gender contribute to inter-subject variability in pitavastatin pharmacokinetics. Personalized medicine should be necessary for hypercholesterolaemic patients receiving pitavastatin.