TGFß1-Induced Fibrotic Responses of Conjunctival Fibroblasts through the Wnt/ß-Catenin/CRYAB Signaling Pathway.

Conjunctival fibrosis is a common postoperative complication of glaucoma filtration surgery, resulting in uncontrolled intraocular pressure and surgery failure. Therefore, it is urgent to understand the molecular mechanisms underlying conjunctival fibrosis and to explore novel pharmacologic anti-fibrosis therapies for glaucoma filtration surgery. The 4D-DIA quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/ß-catenin pathway in transforming growth factor (TGF)-ß1-induced human conjunctival fibroblasts (HConFs). Treatment with ICG-001, a Wnt/ß-catenin inhibitor, effectively inhibited cell proliferation and migration in TGFß1-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix proteins induced by TGFß1. In addition, ICG-001 reduced the expression level of α smooth muscle actin (α-SMA) and inhibited cell contractility in TGFß1-treated HConFs. Proteomics data further suggested that αB-crystallin (CRYAB) was a downstream target of Wnt/ß-catenin, which was up-regulated by TGFß1 and down-regulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and ß-catenin in TGFß1-treated HConFs, implying that CRYAB stabilized ß-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased extracellular matrix protein levels, and up-regulated α-SMA expression of HConFs under TGFß1 stimulation. CRYAB rescued TGFß1-induced fibrotic responses that were suppressed by ICG-001. In conclusion, this study elucidates the regulatory mechanism of the Wnt/ß-catenin/CRYAB pathway in conjunctival fibrosis, offering promising therapeutic targets for mitigating bleb scarring after glaucoma filtration surgery.

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

AIM: To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. METHODS: A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. RESULTS: A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. CONCLUSION: Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

Inhibiting the SARM1-NAD+ axis reduces oxidative stress-induced damage to retinal and nerve cells.

Retinal neurodegenerative diseases are a category of refractory blinding eye conditions closely associated with oxidative stress induced by mitochondrial dysfunction in retinal cells. SARM1, a core driver molecule leading to axonal degeneration, possesses NAD+ enzyme (NADase) activity. However, the role of the SARM1-NAD+ axis in oxidative stress-induced retinal cell death remains unclear. Here, we employed the SARM1 NADase inhibitor DSRM-3716 and established a glucose oxidase (GOx)-induced oxidative stress cell model. We found that compared to the GOx group, the DSRM-3716 pre-treated group reduced the hydrolysis of NAD+, inhibited the elevation of oxidative stress markers induced by GOx, decreased mitochondrial dysfunction, lowered the phosphorylation level of JNK, and attenuated the occurrence of pyroptosis in retinal and nerve cells, thereby providing protection for neurite growth. Further utilization of the JNK activator Anisomycin activated JNK, revealed that the JNK/c-Jun pathway down-regulated NMNAT2 expression. Consequently, it reduced cellular NAD+ synthesis, exacerbated mitochondrial dysfunction and cell pyroptosis, and reversed the protective effect of DSRM-3716 on cells. In summary, the inhibition of SARM1 NADase activity substantially mitigates oxidative damage to retinal cells and mitochondrial damage. Additionally, JNK simultaneously serves as both an upstream and downstream regulator in the SARM1-NAD+ axis, regulating retinal cell pyroptosis and neurite injury. Thus, this study provides new insights into the pathological processes of retinal cell oxidative stress and identifies potential therapeutic targets for retinal neurodegenerative diseases.

Epidemic features and megagenomic analysis of childhood Mycoplasma pneumoniae post COVID-19 pandemic: a 6-year study in southern China.

With the atypical rise of Mycoplasma pneumoniae infection (MPI) in 2023, prompt studies are needed to determine the current epidemic features and risk factors with emerging trends of MPI to furnish a framework for subsequent investigations. This multicentre, retrospective study was designed to analyse the epidemic patterns of MPI before and after the COVID-19 pandemic, as well as genotypes and the macrolide-resistance-associated mutations in MP sampled from paediatric patients in Southern China. Clinical data was collected from 1,33,674 patients admitted into investigational hospitals from 1 June 2017 to 30 November 2023. Metagenomic next-generation sequencing (mNGS) data were retrieved based on MP sequence positive samples from 299 paediatric patients for macrolide-resistance-associated mutations analysis. Pearson's chi-squared test was used to compare categorical variables between different time frames. The monthly average cases of paediatric common respiratory infection diseases increased without enhanced public health measures after the pandemic, especially for influenza, respiratory syncytial virus infection, and MPI. The contribution of MPI to pneumoniae was similar to that in the outbreak in 2019. Compared to mNGS data between 2019-2022 and 2023, the severity of MP did not grow stronger despite higher rates of macrolide-resistance hypervariable sites, including loci 2063 and 2064, were detected in childhood MP samples of 2023. Our findings indicated that ongoing surveillance is necessary to understand the impact of post pandemic on MP transmission disruption during epidemic season and the severity of clinical outcomes in different scenarios.

Case report: Rubella virus-associated cutaneous granuloma in an adult with TAP1 deficiency.

Rubella virus-associated granulomas commonly occur in immunocompromised individuals, exhibiting a diverse range of clinical presentations. These manifestations can vary from predominantly superficial cutaneous plaques or nonulcerative nodules to more severe deep ulcerative lesions, often accompanied by extensive necrosis and significant tissue destruction. TAP1 deficiency, an exceedingly rare primary immune-deficiency disorder, presents with severe chronic sino-pulmonary infection and cutaneous granulomas. This report highlights the occurrence of rubella virus-associated cutaneous granulomas in patients with TAP1 deficiency. Notably, the pathogenic mutation responsible for TAP1 deficiency stems from a novel genetic alteration that has not been previously reported. This novel observation holds potential significance for the field of diagnosis and investigative efforts in the context of immunodeficiency disorders.

Alterations of lung microbiota in lung transplant recipients with pneumocystis jirovecii pneumonia.

BACKGROUND: Increasing evidence revealed that lung microbiota dysbiosis was associated with pulmonary infection in lung transplant recipients (LTRs). Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungal pathogen that frequently causes lethal pneumonia in LTRs. However, the lung microbiota in LTRs with P. jirovecii pneumonia (PJP) remains unknow. METHODS: In this prospective observational study, we performed metagenomic next-generation sequencing (mNGS) on 72 bronchoalveolar lavage fluid (BALF) samples from 61 LTRs (20 with PJP, 22 with PJC, 19 time-matched stable LTRs, and 11 from LTRs after PJP recovery). We compared the lung microbiota composition of LTRs with and without P. jirovecii, and analyzed the related clinical variables. RESULTS: BALFs collected at the episode of PJP showed a more discrete distribution with a lower species diversity, and microbiota composition differed significantly compared to P. jirovecii colonization (PJC) and control group. Human gammaherpesvirus 4, Phreatobacter oligotrophus, and Pseudomonas balearica were the differential microbiota species between the PJP and the other two groups. The network analysis revealed that most species had a positive correlation, while P. jirovecii was correlated negatively with 10 species including Acinetobacter venetianus, Pseudomonas guariconensis, Paracandidimonas soli, Acinetobacter colistiniresistens, and Castellaniella defragrans, which were enriched in the control group. The microbiota composition and diversity of BALF after PJP recovery were also different from the PJP and control groups, while the main components of the PJP recovery similar to control group. Clinical variables including age, creatinine, total protein, albumin, IgG, neutrophil, lymphocyte, CD3+CD45+, CD3+CD4+ and CD3+CD8+ T cells were deeply implicated in the alterations of lung microbiota in LTRs. CONCLUSIONS: This study suggests that LTRs with PJP had altered lung microbiota compared to PJC, control, and after recovery groups. Furthermore, lung microbiota is related to age, renal function, nutritional and immune status in LTRs.

The Role and Mechanism of Nicotinamide Riboside in Oxidative Damage and a Fibrosis Model of Trabecular Meshwork Cells.

Purpose: To investigate the potential effects and mechanism of nicotinamide riboside (NR) on the oxidative stress and fibrosis model of human trabecular meshwork (HTM) cell line cells. Methods: HTM cells were pretreated with NR, followed by the induction of oxidative injury and fibrosis by hydrogen peroxide (H2O2) and TGF-ß2, respectively. Cell viability was tested using Hoechst staining and MTT assays, cell proliferation was assessed by EdU assay, and cell apoptosis was detected by flow cytometry and western blotting. DCFH-DA and DHE probes were used to measure the level of reactive oxygen species (ROS), and MitoTracker staining was used to measure the mitochondrial membrane potential (MMP). Fibrotic responses, including cell migration and deposition of extracellular matrix (ECM) proteins, were detected via Transwell assays, qRT-PCR, and immunoblotting. Results: NR pretreatment improved the viability, proliferation, and MMP of H2O2-treated HTM cells. Compared to cells treated solely with H2O2, HTM cells treated with both NR and H2O2, exhibited a reduced rate of apoptosis and generation of ROS. Compared with H2O2 pretreatment, NR pretreatment upregulated expression of the JAK2/Stat3 pathway but inhibited mitogen-activated protein kinase (MAPK) pathway expression. Moreover, 10-ng/mL TGF-ß2 promoted cell proliferation and migration, which were inhibited by NR pretreatment. Both qRT-PCR and immunoblotting showed that NR inhibited the expression of fibronectin in a TGF-ß2-induced fibrosis model. Conclusions: NR has a protective effect on oxidative stress and fibrosis in HTM cells, which may be related to the JAK2/Stat3 pathway and MAPK pathway. Translational Relevance: Our research provides the ongoing data for potential therapy of NAD+ precursors in glaucoma.

Prevalence and molecular characteristics of ceftazidime-avibactam resistance among carbapenem-resistant Pseudomonas aeruginosa clinical isolates.

OBJECTIVES: Resistance against ceftazidime-avibactam (CZA) in carbapenem-resistant Pseudomonas aeruginosa (CRPA) is emerging. This study was aimed at detecting the prevalence and molecular characteristics of CZA-resistant CRPA clinical isolates in Guangdong Province, China. METHODS: The antimicrobial susceptibility profile of these strains was determined. A subset of 16 CZA-resistant CRPA isolates was analysed by whole-genome sequencing (WGS). Genetic surroundings of carbapenem resistance genes and pan-genome-wide association analysis were further studied. RESULTS: Of the 250 CRPA isolates, CZA resistance rate was 6.4% (16/250). The minimum inhibitory concentration (MIC) of CZA range was from 0.25 to >256 mg/L. MIC50 and MIC90 were 2/4 and 8/4 mg/L, respectively. Among the 16 CZA-resistant CRPA strains, 31.3% (5/16) of them carried class B carbapenem resistance genes, including blaIMP-4, blaIMP-45, and blaVIM-2, located on IncP-2 megaplasmids or chromosomes, respectively. Pan-genome-wide association analysis of accessory genes for CZA-susceptible or -resistant CRPA isolates showed that PA1874, a hypothetical protein containing BapA prefix-like domain, was enriched in CZA-resistant group significantly. CONCLUSIONS: Class B carbapenem resistance genes play important roles in CZA resistance. Meanwhile, the PA1874 gene may be a novel mechanism involving in CZA resistance. It is necessary to continually monitor CZA-resistant CRPA isolates.

Mutation analysis of RHO in patients with non-syndromic retinitis pigmentosa.

PURPOSE: To identify RHO mutations in patients with non-syndromic retinitis pigmentosa (NS-RP). METHODS: A total of 143 probands (46 family history and 97 sporadic cases) with NS-RP were recruited from Southeast China. The coding exons and adjacent intronic regions of RHO were PCR-amplified and sequenced by Sanger sequencing. The candidate variant was evaluated by the guidelines of American College of Medical Genetics and further validated through co-segregation analysis within the family. RESULTS: Five heterozygous mutations in RHO were detected in 5 out of 143 probands, where the frequency of RHO mutations in our cohort was approximately 3.5% (5/143) and 10.8% (5/46) for probands and families with NS-RP, respectively. Three known disease-causing mutations including c.C1030T (p.Q344X), c.C173G (p.T58R), and c.G266A (p.G89D) were identified in three unrelated families. The other two previously unreported mutations c.557C>A (p.S186X) and c.944delA (p.N315TfsX43) were confirmed in Family RP-087 and Family RP-139, respectively. These mutations co-segregated with available affected individuals in each family were not observed in the unaffected family members or in the 112 unrelated controls. CONCLUSIONS: This report expands the mutational spectrum of RHO gene associated with NS-RP and demonstrates the frequency of RP RHO mutations in Southeast Chinese populations.

Novel Variants of HPS6 Cause Suspected Ocular Albinism: A Report of 2 Cases and the Profile of HPS6 Variants.

INTRODUCTION: Hermansky-Pudlak syndrome (HPS) is a rare autosomal-recessive disease characterized by ocular albinism (OA) or oculocutaneous albinism (OCA), platelet dysfunction, and other symptoms. This study aimed to analyze the molecular defect in two Chinese families with suspected OA, as well as to investigate the profile of HPS6 variants and their genotype-phenotype correlations. METHODS: Seven members from two families were recruited and underwent clinical ophthalmologic examinations. The genomic DNA was extracted from peripheral blood leukocytes. Whole-exome sequencing was performed on the proband of family JX. The single coding exon of HPS6 was directly Sanger sequenced based on PCR amplification in all available family members. An additional 46 probands from families or sporadic cases with the pathogenic variants of HPS6 reported in the literature were reviewed. RESULTS: We identified two different compound heterozygous truncating variants of HPS6 in probands with suspected OA from two independent families. The proband of family JX had c.1674dup and c.503-504del variants, and the other proband from family CZ had a nonsense variant of c.1114C>T and a frameshift variant of c.1556del. Among them, c.1674dup and c.1556del variants in HPS6 have not been reported previously. Therefore, our patients were diagnosed as HPS6 disease by molecular diagnostics. In the retrospective cohort of HPS6 patients, we delineated the profile of HPS6 variants and revealed a significant overlap between CpG islands and the variants of HPS6, suggesting a potential link between DNA methylation and HPS6 variants. We also observed a spatial aggregation of the variants in 3D structure of HPS6 protein, implying the possible functional significance of these structural regions. In addition, we did not find any significant genotype-phenotype correlation of HPS6, and neither did we observe a correlation between the truncation length of the HPS6 protein and the phenotype of HPS6 disease. CONCLUSION: Our research expands the spectrum of HPS6 variants, providing a comprehensive delineation of their profile and systematically investigating genotype-phenotype correlations in HPS6. These findings could offer potentially valuable clues for investigating the molecular mechanism underlying HPS6 pathogenesis, as well as aiding the clinical diagnosis of HPS6 patients and improving disease prognosis.

A novel single domain bispecific antibody targeting VEGF and TNF-α ameliorates rheumatoid arthritis.

Anti-TNF-α therapy fails in 30% of patients, where TNF-α may not be the key causative factor in these patients. We developed a bispecific single-domain antibody block TNF-α and VEGF (V5-3).The experiments showed that V5-3 effectively activated proliferation and migration of RA-FLS and HUVEC, tube-forming role of HUVEC, and expression of inflammatory factors in vitro. Besides, the experiments indicated that the anti-RA activity of V5-3 was superior to Anbainuo in vivo. Application of V5-3 reduced the expression of inflammatory factors, extent of synovial inflammation and angiogenesis and attenuated the severity of autoimmune arthritis in collagen-induced arthritis (CIA) mice. Mechanistically, V5-3 suppressed p65, AKT and VEGFR2 phosphorylation, as well as production of TNF-α and VEGF in joint tissues. These results demonstrated that V5-3 displayed a superior effect of anti-RA, may be a new therapy to overcome the limitations of anti-TNF-α monoclonal antibody.

System analysis of Huang-Lian-Jie-Du-Tang and their key active ingredients for overcoming CML resistance by suppression of leukemia stem cells.

BACKGROUND: BCR-ABL1-based resistance to imatinib, mainly resulting from BCR-ABL1 mutations, is largely solved after second- and third-generation tyrosine kinase inhibitors (TKIs) are discovered. Nonetheless, imatinib resistance without BCR-ABL1 mutations, including intrinsic resistance induced by stem cells within chronic myeloid leukemia (CML), remains the major clinical challenge for many patients. PURPOSE: To study the key active ingredients and corresponding target proteins in Huang-Lian-Jie-Du-Tang (HLJDT) against BCR-ABL1-independent CML resistance to therapeutics, and then explore its mechanism of against CML drug resistance. METHODS: Cytotoxicity of HLJDT and its active ingredients in BCR-ABL1-independent imatinib resistance cells was analyzed through MTT assay. The cloning ability was measured through soft agar assay. Monitoring therapeutic effect on Xenografted mice CML model by in vivo imaging technology and mice survival time. Predicting the potential target protein binding sites by the technology of photocrosslinking sensor chip, molecular space simulation docking, and use Surface Plasmon Resonance (SPR) technology . Flow cytometry to detect the ratio of stem progenitor cells (CD34+). Constructing bone marrow transplantation mice CML leukemia model, detect the effects on leukemia stem cells LSK (Lin-\ Sca-1+ \C-kit+) self-renewal. RESULTS: Treatment with HLJDT, berberine and baicalein inhibited cell viability and colony formation of BCR-ABL1-independent imatinib-resistant cells in vitro while prolonging survival in mouse with CML xenografts and transplatation CML-like mouse models in vivo. JAK2 and MCL1were identified as targets of berberine and baicalein. JAK2 and MCL1 are involved in multi-leukemia stem cell-related pathways. Moreover, the ratio of CD34+ cells in resistant CML cells is higher than in treatment-sensitive CML cells. Treatment with BBR or baicalein partially suppressed CML leukemic stem cells (LSCs) self-renewal in vitro and in vivo. CONCLUSION: From the above, we concluded that HLJDT and its key active ingredients (BBR and baicalein) allowed to overcome imatinib resistance with BCR-ABL1 independent by eradication of LSCs by targeting the JAK2 and MCL1 protein levels. Our results lay the foundation for applying HLJDT in patients with TKI-resistant CML.

PPFIA1-targeting miR-181a mimic and saRNA overcome imatinib resistance in BCR-ABL1-independent chronic myeloid leukemia by suppressing leukemia stem cell regeneration.

A large proportion of patients with chronic myeloid leukemia (CML; 20%-50%) develop resistance to imatinib in a BCR-ABL1-independent manner. Therefore, new therapeutic strategies for use in this subset of imatinib-resistant CML patients are urgently needed. In this study, we used a multi-omics approach to show that PPFIA1 was targeted by miR-181a. We demonstrate that both miR-181a and PPFIA1-siRNA reduced the cell viability and proliferative capacity of CML cells in vitro, as well as prolonged the survival of B-NDG mice harboring human BCR-ABL1-independent imatinib-resistant CML cells. Furthermore, treatment with miR-181a mimic and PPFIA1-siRNA inhibited the self-renewal of c-kit+ and CD34+ leukemic stem cells and promoted their apoptosis. Small activating (sa)RNAs targeting the promoter of miR-181a increased the expression of endogenous primitive miR-181a (pri-miR-181a). Transfection with saRNA 1-3 inhibited the proliferation of imatinib-sensitive and -resistant CML cells. However, only saRNA-3 showed a stronger and more sustained inhibitory effect than the miR-181a mimic. Collectively, these results show that miR-181a and PPFIA1-siRNA may overcome the imatinib resistance of BCR-ABL1-independent CML, partially by inhibiting the self-renewal of leukemia stem cells and promoting their apoptosis. Moreover, exogenous saRNAs represent promising therapeutic agents in the treatment of imatinib-resistant BCR-ABL1-independent CML.

Carbon Mitigation and Environmental Co-Benefits of a Clean Energy Transition in China's Industrial Parks.

Industrial parks are emerging priorities for carbon mitigation. Here we analyze air quality, human health, and freshwater conservation co-benefits of decarbonizing the energy supply of 850 China's industrial parks. We examine a clean energy transition including early retirement of coal-fired facilities and subsequent replacement with grid electricity and onsite energy alternatives (municipal solid waste-to-energy, rooftop photovoltaic, and distributed wind power). We find that such a transition would reduce greenhouse gas emissions by 41% (equal to 7% of 2014 national CO2 equivalent emissions); emissions of SO2 by 41%, NOx by 32%, and PM2.5 by 43% and freshwater consumption by 20%, relative to a 2030 baseline scenario. Based on modeled air pollutant concentrations, we estimate such a clean energy transition will result in ∼42,000 avoided premature deaths annually due to reduced ambient PM2.5 and ozone exposure. Costs and benefits are monetized including technical costs of changes in equipment and energy use and societal benefits resulting from improvements in human health and reductions of climate impacts. We find that decarbonizing industrial parks brings annual economic benefits of US$30-156 billion in 2030. A clean energy transition in China's industrial parks thus provides both environmental and economic benefits.

Application of next-generation sequencing on diagnosis of bloodstream infection caused by Mycoplasma hominis in a patient with ANCA-associated vasculitis.

BACKGROUND: Mycoplasma hominis is one of the main opportunistic pathogenic mycoplasmas in humans which has a major impact on patients with bloodstream infections. Because it is difficult to detect or isolate, rapid and accurate diagnosis using improved methods is essential and still challenging for patients with bloodstream infection. CASE PRESENTATION: In this case, we reported the application of next -generation sequencing for the diagnosis of bloodstream infection caused by Mycoplasma hominis in a patient with Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. After 9 days of combined treatment with levofloxacin, polymyxin B and meropenem, the patient's condition was gradually controlled and he was discharged without further complications. During the three-month outpatient follow-up, no recurrence of symptoms or clinical signs was reported. CONCLUSIONS: This successful application of next generation sequencing assisted the rapid diagnosis of Mycoplasma hominis bloodstream infection, provided a new perspective in the clinical approach and highlighted the potential of this technique in rapid etiological diagnosis.

Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of metagenomic next-generation sequencing (mNGS) for the diagnosis of Pneumocystis jirovecii Pneumonia (PCP) in critically pediatric patients. METHODS: Seventeen critically pediatric patients with PCP and sixty patients diagnosed with non-PCP pneumonia who were admitted in pediatric intensive care unit between June 2018 and July 2021 were enrolled. Conventional methods and mNGS for detecting Pneumocystis jirovecii (P. jirovecii) were compared. The patients' demographics, comorbidities, laboratory test results, antibiotic treatment response and 30 day mortality were analyzed. RESULT: The mNGS showed a satisfying diagnostic performance with a sensitivity of 100% in detecting P. jirovecii compared with Gomori methenamine silver staining (5.9%), serum (1,3)-ß-D-glucan (86.7%) and and LDH (55.6%). The diagnostic specificity of mNGS for PCP was higher than that of serum BDG (56.7%) and LDH (71.4%). In PCP group, over one thirds' cases had mixed infections. Compared with survivors, non-survivors had higher stringently mapped read numbers (SMRNs) in bronchoalveolar lavage fluid (BALF) sample (P < 0.05), suggesting SMRNs were closely associated with the severity of response. The detection for P. jirovecii by mNGS both in BALF and blood samples reached a concordance rate of 100%, and the SMRNs in the BALF were remarkably higher than that in blood samples. Initial antimicrobial treatment was modified in 88.2% of PCP patients based on the mNGS results. CONCLUSION: The mNGS is a potential and efficient technology in diagnosing PCP and shows a satisfying performance in the detection of co-pathogens. Both blood and BALF samples for mNGS are suggested for the presumptive diagnosis of PCP.

Diagnosis by metagenomic next-generation sequencing of invasive pulmonary aspergillosis in an infant with chronic granulomatous disease.

Invasive pulmonary aspergillosis (IPA) is a serious fungal infection, with a high degree of mortality in immunocompromised individuals. Diagnosis of IPA is challenging in that clinical manifestations are not specific, with sensitivity of traditional detection procedures low. We report a case of IPA in a chronic granulomatous disease (CGD) infant who was initially suspected to have a lung tumor. Aspergillus fumigatus was identified as the pathogen in bronchoalveolar lavage fluid (BALF) by next-generation sequencing (mNGS). The patient recovered rapidly following a change of appropriate antifungal treatment and was discharged. This case highlights the additional value of BALF-mNGS for the diagnosis of pediatric invasive pulmonary fungal infection in immune-deficient children.

Prediction of noninvasive ventilation failure using the ROX index in patients with de novo acute respiratory failure.

BACKGROUND: The ratio of SpO2/FiO2 to respiratory rate (ROX) index is commonly used to predict the failure of high-flow nasal cannula. However, its predictive power for noninvasive ventilation (NIV) failure is unclear. METHODS: This was a secondary analysis of a multicenter prospective observational study, intended to update risk scoring. Patients with de novo acute respiratory failure were enrolled, but hypercapnic patients were excluded. The ROX index was calculated before treatment and after 1-2, 12, and 24 h NIV. Differences in predictive power for NIV failure using the ROX index, PaO2/FiO2, and PaO2/FiO2/respiratory rate were tested. RESULTS: A total of 1286 patients with de novo acute respiratory failure were enrolled. Of these, 568 (44%) experienced NIV failure. Patients with NIV failure had a lower ROX index than those with NIV success. The rates of NIV failure were 92.3%, 70.5%, 55.3%, 41.1%, 35.1%, and 29.5% in patients with ROX index values calculated before NIV of ≤ 2, 2-4, 4-6, 6-8, 8-10, and > 10, respectively. Similar results were found when the ROX index was assessed after 1-2, 12, and 24 h NIV. The area under the receiver operating characteristics curve was 0.64 (95% CI 0.61-0.67) when the ROX index was used to predict NIV failure before NIV. It increased to 0.71 (95% CI 0.68-0.74), 0.74 (0.71-0.77), and 0.77 (0.74-0.80) after 1-2, 12, and 24 h NIV, respectively. The predictive power for NIV failure was similar for the ROX index and for the PaO2/FiO2. Likewise, no difference was found between the ROX index and the PaO2/FiO2/respiratory rate, except at the time point of 1-2 h NIV. CONCLUSIONS: The ROX index has moderate predictive power for NIV failure in patients with de novo acute respiratory failure.