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In this paper, Ti6Al4V alloy and Ti6Al4V-SiCw composites were formed by selective laser melting (SLM) and electrochemical corrosion experiments were carried out. The effects of different SiCw contents and solid solution aging heat treatment on the corrosion properties of the composites were systematically studied, and the corrosion mechanism of Ti6Al4V-SiCw composites was revealed. The results show that Ti6Al4V-1SiCw composites have better corrosion resistance than Ti6Al4V alloy. After solution and aging heat treatment at 1050 °C, Ti6Al4V-1SiCw composites have better corrosion resistance. The main reasons for the improvement of the corrosion performance of the composites are as follows: (i) The high-density grain boundaries formed by grain refinement of the composites have higher energy and are more likely to react to form a stable passivation film. (ii) The microstructure of Ti6Al4V-SiCw composites is equiaxed α phase, which is easier to form stable passivation film than metastable α′-Ti phase. (iii) The presence of ceramic particles makes more microcells formed in the titanium matrix, which promotes the formation of soluble [TiCl6]2- complexes. The formation of passivation film on the surface of Ti6Al4V-SiCw composites was accelerated.

The hydrological dynamics and sources of karst tidal springs are difficult to capture and quantify due to fluctuations in their flow velocity. In this study, the Laolongshui (LLS) karst tidal spring in the Maocun underground river basin of Guilin City was taken as an example, with a 2-year continuous monitoring of the electrical conductivity, and water level, as well as water chemical analysis. The results showed the following: (1) discovered the variation pattern of LLS water level in different seasons, the water level fluctuates regularly on a daily scale, with a rise and fall time of 43.6 min after heavy rainfall in the rainy season, while in the dry season, it lasts for about 74–80 h. (2) Four peaks were extracted from the frequency distribution of electrical conductivity, representing the response of springwater under different rainfall conditions. (3) The annual average frequencies of the occurrence of P1, P2, P3, and P4 in terms of time are 53.82, 39.29, 6.18, and 0.72%, respectively. The results provide a new method for analyzing groundwater's source and dynamic changes in karst areas. HIGHLIGHTS Two years of automatic continuous monitoring illustrated the water level fluctuation of a typical karst tidal spring.; Explaining the forming mechanism of karst tidal springs.; The frequency distribution of conductivity quantified the source of karst water.; Providing a fast and low-cost new method for identifying karst water sources.; Enriching groundwater investigation methods and understanding of underground structures.;

Abstract Neuroblastoma (NB) is a challenging pediatric extracranial solid tumor characterized by a poor prognosis and resistance to chemotherapy. Identifying targets to enhance chemotherapy sensitivity in NB is of utmost importance. Increasing evidence implicates long noncoding RNAs (lncRNAs) play important roles in cancer, but their functional roles remain largely unexplored. Here, we analyzed our RNA sequencing data and identified the upregulated lncRNA ZNF674-AS1 in chemotherapy non-responsive NB patients. Elevated ZNF674-AS1 expression is associated with poor prognosis and high-risk NB. Importantly, targeting ZNF674-AS1 expression in NB cells suppressed tumor growth in vivo. Further functional studies have revealed that ZNF674-AS1 constrains cisplatin sensitivity by suppressing pyroptosis and promoting cell proliferation. Moreover, ZNF674-AS1 primarily relies on CA9 to fulfill its functions on cisplatin resistance. High CA9 levels were associated with high-risk NB and predicted poor patient outcomes. Mechanistically, ZNF674-AS1 directly interacted with the RNA binding protein IGF2BP3 to enhance the stability of CA9 mRNA by binding with CA9 transcript, leading to elevated CA9 expression. As a novel regulator of CA9, IGF2BP3 positively upregulated CA9 expression. Together, these results expand our understanding of the cancer-associated function of lncRNAs, highlighting the ZNF674-AS1/IGF2BP3/CA9 axis as a constituting regulatory mode in NB tumor growth and cisplatin resistance. These insights reveal the pivotal role of ZNF674-AS1 inhibition in recovering cisplatin sensitivity, thus providing potential therapeutic targets for NB treatment.

Abstract Recently, telomerase reverse transcriptase (TERT) gene rearrangements have been identified in neuroblastoma (NB), the typical pathological type of neuroblastic tumours (NTs); however, the prevalence of TERT rearrangements in other types of NT remains unknown. This study aimed to develop a practical method for detecting TERT defects and to evaluate the clinical relevance of TERT rearrangements as a biomarker for NT prognosis. A TERT break‐apart probe for fluorescence in situ hybridisation (FISH) was designed, optimised, and applied to assess the genomic status of TERT in Chinese children with NTs at the Beijing Children's Hospital from 2016 to 2019. Clinical, histological, and genetic characteristics of TERT‐rearranged NTs were further addressed. Genomic TERT rearrangements could be effectively detected by FISH and were mutually exclusive with MYCN amplification. TERT rearrangements were identified in 6.0% (38/633) of NTs overall, but 12.4% (31/250) in high‐risk patients. TERT rearrangements identified a subtype of aggressive NTs with the characteristics of Stage 3/4, high‐risk category, over 18 months old, and presenting all histological subtypes of NB and ganglioneuroblastoma nodular. Moreover, TERT rearrangements were significantly associated with elevated TERT expression levels and decreased survival chances. Multivariable analysis confirmed that it was an independent prognostic marker for NTs. FISH is an easily applicable method for evaluating TERT defects, which define a subgroup of NTs with unfavourable prognosis. TERT rearrangements would contribute to characterising NT molecular signatures in clinical practice.

Land use in karst areas affects soil properties, impacting carbon sinks. Accurate estimation of carbon sink flux in karst areas through zoning and classification is crucial for understanding global carbon cycling and climate change. The peak cluster depression is the largest continuous karst landform region in southern China, with the depressions primarily covered by farmland and influenced by agricultural activities. This study focused on the Guancun Underground River Basin, a typical peak cluster depression basin, where sampling and analysis were conducted during the agricultural period of 2021–2022. Using hydrochemical analysis and isotopic methods, the results indicated that: (1) The primary hydrochemical type in the Guancun Underground River Basin is HCO3-Ca, with hydrochemical composition mainly controlled by carbonate rock weathering. (2) The primary sources of Cl−, SO42−, and NO3− are agricultural activities, with agriculture contributing 0.68 mmol/L to dissolved inorganic carbon (DIC), accounting for about 13.86%, as confirmed by ion concentration analysis and isotope verification. (3) The size of the depression area is proportional to the contribution of agricultural activities to DIC, while also being influenced by dilution effects. A comparison was made regarding the contribution of other land use types to DIC. The impact of land use on DIC in karst processes should not be overlooked, and zoning and classification assessments of carbon sink flux under different influencing factors contribute to carbon peaking and carbon neutrality goals.

Abstract Wound healing is a common occurrence. However, delayed healing and aberrant scarring result in pathological wound healing. Accordingly, a scarless wound healing remains a significant clinical challenge. In this study, we constructed hyaluronic acid (HA)-modified and verteporfin (VP)-loaded polylactic acid (PLA) nanogels (HA/VP-PLA) to promote scarless wound healing by accelerating wound re-epithelialization and controlling scar formation. Owing to the unique structure of HA incorporating and coating in VP-loaded PLA nanoparticles, HA/VP-PLA could be topically applied on wound to achieve targeted delivery to fibroblasts. Then, HA/VP-PLA released HA and lactic acid (LA) to stimulate the proliferation and migration of fibroblasts, as well as VP to inhibit Yes-associated protein (YAP) expression and nuclear localization to suppress fibrosis. In vitro (skin fibroblasts) and in vivo (rat and rabbit models) experiments strongly suggested that HA/VP-PLA promoted scarless wound healing by accelerating wound re-epithelialization and controlling scar formation. Therefore, our work provides a feasible strategy for scarless wound healing, and the sophisticated HA/VP-PLA exhibit a great potential for clinical applications.

Abstract Background Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children, which is highly prone to bone marrow (BM) metastasis. BM can monitor early signs of mild disease and metastasis. Existing biomarkers are insufficient for the diagnosis and treatment of NB. Bromodomain PHD finger transcription factor (BPTF) is an important subunit of the chromatin-remodeling complex that is closely associated with tumors. Here, we evaluated whether BPTF in BM plays an important role in predicting NB progression, and explore the molecular mechanism of BPTF in NB. Methods The clinical relevance of the BPTF was predicted in the GEO (GSE62564) and TARGET database. The biological function of BPTF in NB was investigated by constructing cell lines and employing BPTF inhibitor AU1. Western blot was used to determine the changes of BPTF, TFAP4, PI3K/AKT signaling and Epithelial-mesenchymal transition (EMT) related markers. A total of 109 children with newly diagnosed NB in Beijing Children's Hospital from January 2018 to March 2021 were included in this study. RT-PCR was used to measure the BPTF and TFAP4 expression in BM. The cut-off level was set at the median value of BPTF expression levels. Results Databases suggested that BPTF expression was higher in NB and was significantly associated with stage and grade. Proliferation and migration of NB cells were slowed down when BPTF was silenced. Mechanistically, TFAP4 could positively regulate BPTF and promotes EMT process through activating the PI3K/AKT signaling pathway. Moreover, detection of the newly diagnosed BM specimens showed that BPTF expression was significantly higher in high-risk group, stage IV group and BM metastasis group. Children with high BPTF at initial diagnosis were considered to have high risk for disease progression and recurrence. BPTF is an independent risk factor for predicting NB progression. Conclusions A novel and convenient BPTF-targeted humoral detection that can prompt minimal residual and predict NB progression in the early stages of the disease were identified. BPTF inhibitor AU1 is expected to become a new targeted drug for NB therapy. It’s also reveal previously unknown mechanisms of BPTF in NB cell proliferation and metastasis through TFAP4 and PI3K/AKT pathways.

Background Epidemiological characteristics of paediatric burn can help to protect children from burn injuries. Most of the previous studies have been conducted on small scale and single centre in China. This study analysed the epidemiological characteristics of paediatric burn using a large-scale and multicentre database from 23 children’s hospitals in China to increase child protection against burn injuries, improve the quality of care and reduce hospitalisation costs.Methods Excerpted information from medical records of 6741 paediatric burn cases was extracted from the Futang Research Center of Pediatric Development database from 2016 to 2019. The epidemiological characteristics of patients, including gender, age, cause of burn injuries, complication, the timing of hospitalisation (season and month) and hospitalisation length and cost, were collected.Results Male gender (63.23%), aged 1–2 years (69.95%), and hydrothermal scald (80.57%) were significantly dominant among cases. Furthermore, complications were significantly different among groups with different age of patients. Pneumonia was the most common complication (21%). Meanwhile, most paediatric burn occurred in spring (26.73%), and hospitalisation length and cost significantly depended on the cause of the burn injuries and surgical intervention.Conclusions This large-scale epidemiological study of paediatric burn in China revealed that boys aged 1–2 years with higher activity and lack of self-awareness are more likely to suffer from burn injuries by hydrothermal scald. In addition, complications, especially pneumonia, need attention and early prevention in paediatric burn.

ABSTRACT The 2022 World Health Organization guidelines recommend use of two core anti-tuberculosis (TB) drugs, bedaquiline (BDQ) and clofazimine (CFZ), for treatment of drug-resistant (DR)-TB. However, several mutated Mycobacterium tuberculosis (MTB) genes, conferring BDQ and CFZ resistance, have been reported that predominantly arose from sporadic mutations that have not been comprehensively characterized. Herein, MTB clinical isolates collected from drug-susceptible (DS)-, multidrug-resistant (MDR)-, and extensively drug-resistant (XDR)-TB patients were cultured in vitro with BDQ or CFZ to generate progeny strains with resistance to these drugs. Progeny strains exposed to CFZ exhibited increased CFZ minimum inhibitory concentrations (MICs) that exceeded MIC increases of BDQ-exposed progeny strains. Notably, mmpR and pepQ mutations accounted for 83% and 17% of BDQ-induced spontaneous gene mutations, respectively, and 86% and 14% of CFZ-induced spontaneous gene mutations, respectively. Analyses of predicted mutation-induced changes in amino acid sequences and structures of MmpR and PepQ mutants revealed several point mutations affected sequence conversation and functionality as an underlying mechanism for observed acquired BDQ/CFZ resistance. Moreover, our results revealed differences in patterns of BDQ- and CFZ-induced acquired spontaneous mutations that may enhance our understanding of MTB BDQ/CFZ-resistance mechanisms. IMPORTANCE This study of MTB drug resistance mechanisms revealed patterns of spontaneous MTB mutations associated with acquired BDQ and CFZ resistance that arose after clinical MTB isolates were cultured in vitro with BDQ or CFZ. Results of protein sequence and structural analyses provided insights into potential mechanisms underlying associations between MTB gene mutations and DR phenotypes. Taken together, these results revealed differences in acquired BDQ and CFZ resistance mechanisms as a new perspective that may enhance our understanding of BDQ/CFZ resistance mechanisms and facilitate the development of new methods for detecting MTB drug resistance genes.

Pediatric tuberculosis (TB) is a serious infectious disease that affects many children worldwide and is more likely to be extrapulmonary than adult TB. However, the clinical and epidemiological profile, and cost burden of pediatric extrapulmonary TB (EPTB) in China remain unknown. Here, we conducted a descriptive, multicenter study of pediatric TB patients from 22 hospitals across all six regions in China from October 2015 to December 2018. Of 4,654 patients, 54.23% (2,524) had pulmonary TB (PTB), 17.76% (827) had EPTB, and 28.00% (1,303) had concurrent extrapulmonary and pulmonary TB (combined TB). Compared with PTB, EPTB and combined TB were associated with lower hospitalization frequency (2.43 and 2.21 vs. 3.16 times), longer length of stay (10.61 and 11.27 vs. 8.56 days), and higher rate of discharge against medical advice (8.46% and 9.44% vs. 5.67%). EPTB was associated with higher mortality (0.97% vs. 0.24% and 0.31%), higher rate of low birth weight (17.69% vs. 6.79% and 6.22%), worse diagnosis at the first visit (21.16% vs. 34.67% and 44.47%), and worse hospitalization plan situation (4.35% vs. 7.81% and 7.44%), compared with PTB and combined TB. EPTB and combined TB had higher financial burdens (17.67% and 16.94% vs. 13.30%) and higher rates of catastrophic expenditure (8.22% and 9.59% vs. 5.03%), compared with PTB. Meningitis TB (34.18%) was the most frequent form of total extrapulmonary infection and had the highest cost burden and rate of catastrophic expenditure. In conclusion, improved screening approaches for pediatric EPTB are needed to reduce diagnostic challenges and financial burden.

Objectives: To estimate the impact of nonpharmaceutical interventions (NPIs) targeted at the COVID-19 pandemic on the admission number of respiratory diseases, including pneumonia, acute bronchitis & bronchiolitis, and acute upper respiratory infections (AURIs) for children in China. Methods: Continuous hospitalization records aged 0-18 years from January 1, 2016, to December 31, 2020, were collected from 26 tertiary children's hospitals. Interrupted time series analysis with a quasi-Poisson model was conducted with the start time of the COVID-19 pandemic as the interrupted timepoint and the weekly admission numbers of all-cause respiratory disease, pneumonia, acute bronchitis & bronchiolitis, and AURI as the outcome measures. Hospitalizations of childhood neoplasms were analyzed as the reference group. Results: The reduction in admission numbers following NPIs was -55.0% (-57.9 to -51.9%) for all-cause respiratory diseases, -62.7% (-65.7 to -59.5%) for pneumonia, -48.1% (-53.3 to -42.3%) for bronchitis & bronchiolitis, and -24.3% (-28.6 to -19.8%) for AURI. The effect estimates of NPIs on childhood neoplasms was -29.1% (-33.6 to -24.4%). Stratification analysis showed the reduction was most drastic for children at 4-6 and 7-12 years. Conclusion: The admission number for respiratory diseases among children in China decreased drastically after the implementation of NPIs. NPIs with low socio-economic burdens should be suggested even outside the COVID-19 pandemic.

Carbonate weathering is an important part of the global carbon cycle, acts as an atmospheric and soil CO2 sink. Driven by ecosystem factors and affected by global change, carbon sinks in karst areas have the characteristics of surface and underground carbon sinks.This work briefly introduces the relationship between the karst carbon cycle and global change, discusses the related scientific issues and main progress of karst carbon sinks, analyzes the potential of karst sink increases and land use changes, and further proposes a conceptual model of carbonate weathering processes based on the concept of karst critical zones.Carbon sink generated by weathering of carbonate rocks may contribute to the "missing sink" of the global carbon cycle and play a role in mitigating the release of soil CO2 to the atmosphere, thus becoming an important regulator of the item of "land use change" (ELUC) in the global carbon cycle model.The carbonate weathering process responds quickly to short-time scale environmental factors, which is the core driving mechanism that connects biological, hydrological, as well as geochemical processes in karst critical zones.The karst carbon cycle can be regarded as an extension or lateral component of the soil-ecosystem carbon cycle, which together constitute a complete terrestrial shallow surface carbon cycle system in karst areas.The negative feedback effect of carbonate weathering on the increase of atmospheric CO2 concentration and the continuous promotion of rock desertification control and ecological restoration contain huge potential for karst carbon sequestration and sink increase.The monitoring and research on seasonal and regional variations in soil CO2 should be strengthened, and an inverse model based on the correlation between soil CO2 and the watershed hydrochemistry index should be constructed to estimate the background of the regional carbon sink and evaluate the annual carbon sink increment and potential, thus providing a clearer and effective scheme and approach for karst sink increase.

Abstract The determination of length of time from the last drinking is critical for cases like drunk driving, sexual assault victims, and also postmortem suspected poisoning cases. The study was aimed to established a method of estimating the time of last drinking through the pharmacokinetic study of conjugation metabolites of alcohol in blood after a single oral dose. Twenty-six volunteers (14 males) consumed alcohol with food at a fixed dose of 0.72 g/kg after fasting for 12 h. Five milliliters of blood were collected 120 h after the start of drinking, and all samples were analyzed with headspace-gas chromatography and high-performance liquid chromatography–tandem mass spectrometry. The time point of last drinking was estimated through the relationship between the concentration ratio of ethyl glucuronide to ethyl sulphate and the length of time after drinking. Pharmacokinetic parameters were analyzed by a pharmacokinetic software DAS according to the non-compartment model. A good correlation model was obtained from the relationship between concentration ratio of ethyl glucuronide to ethyl sulphate in blood and the time of alcohol use, and the margin of error was mostly lower than 10%. The time of maximum concentration, maximum concentration, and elimination half-life of ethyl glucuronide in blood were 4.12 ± 1.07 h, 0.31 ± 0.11 mg/L and 2.56 ± 0.89 h; the time of maximum concentration, maximum concentration, and elimination half-life of ethyl sulphate in blood were 3.02 ± 0.70 h, 0.17 ± 0.04 mg/L, and 2.04 ± 0.76 h. The study established a potential method to estimate the length of time after a moderate oral dose, and provided pharmacokinetic parameters of ethyl glucuronide and ethyl sulphate in Chinese population.

Accurate estimate of carbonate weathering and the related carbon sink flux induced by anthropogenic H2SO4 is of great significance for improving understanding of the hydrogeochemical evolution and the global carbon cycle. Here, to quantitatively evaluate the influence of anthropogenic H2SO4 on different lithological carbonate weathering and the related carbon sink budget, karst spring water in the typical limestone and mixed limestone–dolomite catchments in Yaji and Beidiping affected by acid precipitation in southwest China were sampled monthly for the analysis of hydrochemical and δ13CDIC characteristics. Results show for the period of sampling (August 2013 to December 2014) that the average contribution rates of atmospheric inputs and carbonate weathering to total dissolved cations are 2.24% and 97.8%, and 3.09% and 96.9% in Yaji and Beidiping, respectively. The δ13CDIC values (−17.0% to −14.7‰) and the [Ca2+ + Mg2+]/[HCO3−] (0.98 to 1.25) and [Ca2+ + Mg2+]/[HCO3− + SO42−] (approximately 1) equivalent ratios of samples prove that H2CO3 and H2SO4 simultaneously participate in carbonate weathering. The contribution rates of H2SO4 to [Ca2+ + Mg2+] and [HCO3−] produced by carbonate weathering in Yaji and Beidiping are 0–30% and 0–18%, and 0–37% and 0–23%, with average values of 14% and 7%, and 19% and 11%, respectively, suggesting that the influence of H2SO4 on different lithological carbonate weathering is different. H2SO4 precipitation participating in carbonate weathering increases the weathering rate by 14–19%, whereas it decreases the flux of karst carbon sink by 7–11% in Southwest China. Therefore, anthropogenic acids have influenced the global carbon cycle and climate change by carbonate weathering due to the large karst areas in the world, and their influences on different lithological carbonate weathering should not be ignored in the regional and global carbon cycles in future studies.

ABSTRACT Introduction Cardiac neoplasms are particularly rare in children, and the majority of these tumors are benign. Approximately 10% of cardiac neoplasms are malignant, including soft tissue sarcomas and lymphomas. Cardiac tumors could also be metastases. Primitive EWSR1‐negative round or spindle cell undifferentiated sarcoma harboring CIC gene translocation is a highly aggressive malignancy mainly occurring in soft tissues. However, it has not yet been described in the heart. Case presentation We report a sarcoma that arose from the right ventricle in a 1‐year‐old girl. Histologically, it was composed of closely arranged small round or oval undifferentiated cells with fibrovascular separation, hyaline degeneration, and geographical necrosis. Immunohistochemically, the neoplastic cells exhibited focal membrane positivity for CD99 and diffuse positivity for WT1 and ETV4. Fluorescent in situ hybridization analysis showed EWSR1‐negative but CIC‐positive split signals. The breakpoint was also confirmed by whole genome sequencing. Conclusion Based on morphological, immunohistochemical and molecular findings, this cardiac mass was diagnosed as CIC‐rearranged sarcoma.

ABSTRACT Importance First branchial cleft anomalies (FBCAs) are rare congenital malformations, accounting for < 8% of all branchial cleft anomalies. However, little is currently known about the cause of FBCAs at the molecular level. Objective To identify genomic alterations related to the genetic etiology of FBCAs in Chinese children. Methods We performed whole‐exome sequencing of samples from 10 pediatric patients with FBCAs. Data analysis was carried out using the Burrow‐Wheeler Alignment software package, and the dbSNP database for comparisons. Rare variants were further validated by Sanger sequencing. Insertion/deletions (indels) were examined using the Genome Analysis Toolkit. Results We identified 14 non‐synonymous mutations in seven potential FBCA‐susceptibility genes (TRAPPC12, NRP2, NPNT, SH3RF2, RHPN1, TENM4, and ARMCX4). We also detected 133 shared small indels in 125 genes. Gene Ontology analysis indicated that most of the identified genes played critical roles in development and differentiation pathways involved in regulating organ development. Interpretation We characterized the mutational landscape in pathways involved in development and differentiation in Chinese children with FBCA. The results identified potential pathogenic genes and mutations related to FBCA, and provide molecular‐level support for the branchial theory of FBCA pathogenesis.

ABSTRACT Importance Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. More than 90% of cases are classified as embryonic RMS (ERMS) or alveolar RMS (ARMS). ERMS has a worse prognosis than ARMS. Early differential diagnosis is of paramount importance for optimization of treatment. Objective To identify genes that are differentially expressed between ARMS and ERMS, which can be used for accurate rhabdomyosarcoma classification. Methods Three Gene Expression Omnibus datasets composed of ARMS and ERMS samples were screened and 35 differentially expressed genes (DEGs) were identified. Receiver operating characteristic curve analysis and area under the curve analysis was performed for these 35 DEGs and seven candidate genes with the best differential expression scores between ARMS and ERMS were determined. The expression of these seven candidate genes was validated by immunohistochemical analysis of pre‐chemotherapy ARMS and ERMS specimens. Results The levels of DCX and CRABP2 were confirmed to be remarkably different between paraffin‐embedded ARMS and ERMS tissues, while EGFR abundance was only marginally different between these two RMS subtypes. Interpretation DCX and CRABP2 are potential biomarkers for distinguishing ARMS from ERMS in pre‐chemotherapy pediatric patients.

Abstract Background Lymphatic malformations (LMs) are benign congenital malformations that stem from the abnormal development of the lymphatic vessels during early embryogenesis. Somatic PIK3CA gene mutations are conventional cause leading to LMs. Both macrocystic and microcystic LMs arise due to lymphatic endothelial cell-autonomous defects, depending on the time in development at which PIK3CA gene mutation occurs. Recent study finds a PIK3CA mutation in 79% of LMs. However, discovering new genetic events in this disease is crucial to identify the molecular mechanism of the pathogenesis and further develop new targeted therapies. Results Here, we initially performed whole-exome sequencing in six children with LMs to find a new causal gene. Somatic mutations in PIK3CA (c.1633G > A [p. E545K] and PIK3CD (c.1997T > C [p.L666P]) were discovered in two different individuals. In vitro functional studies were conducted to demonstrate the pathogenicity of the novel mutation c.1997T > C in PIK3CD. We found that L666P promoted the cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) and induced hyperactivation of the mTOR pathway. These findings indicate that the PIK3CD mutation affects downstream signalling in endothelial cells, which may impair normal lymphangiogenesis. Conclusions This study reveals a novel candidate gene associated with the development of LMs, which is consistent with previous researches. These findings in our study may offer a novel gene target for developing therapies, which acts in tight interaction with the previously known PIK3CA.

Bovine herpesvirus type 1 (BHV-1) is a neurotropic herpesvirus that causes infectious rhinotracheitis and vulvovaginitis in cattle. The virion host shutoff protein encoded by the BHV-1 UL41 gene is highly conserved in the Alphaherpesvirinae subfamily. This protein can degrade viral and host messenger RNA (mRNA) to interrupt host defense and facilitate the rapid proliferation of BHV-1. However, studies on the BHV-1 UL41 gene are limited, and BHV-1 defective virus construction using the CRISPR/Cas9 system is somewhat challenging. In this study, we rapidly constructed a BHV-1 UL41-deficient strain using the CRISPR/Cas9 system in BL primary bovine-derived cells. BHV-1 UL41-defective mutants were screened by Western blot analysis using specific polyclonal antibodies as the primary antibodies. During the isolation and purification of the defective strain, a mixed virus pool edited by an efficient single-guide RNA (sgRNA) showed a plaque number reduction. Viral growth property assessment showed that BHV-1 UL41 was dispensable for replication, but the UL41-defective strain exhibited early and slowed viral replication. Furthermore, the BHV-1 UL41-deficient strain exhibited enhanced sensitivity to temperature and acidic environments. The BHV-1 UL41-deficient strain regulated viral and host mRNA levels to affect viral replication.

ABSTRACT Obstructive sleep apnea (OSA) is a common sleep‐disordered breathing disease that often leads to many comorbidities (e.g., cognitive dysfunction), which adversely affect the quality of life for patients with OSA. Thus far, the underlying mechanisms of this dysfunction remain unclear. Many studies have focused on OSA‐related characteristics, including intermittent hypoxemia and sleep fragmentation. There is increasing emphasis on neuroimaging studies to explore underlying relationships between neuropathological changes and cognitive dysfunction. This article reviews recent research progress concerning cognitive dysfunction associated with OSA to reveal potential mechanisms that contribute to this dysfunction.

Type III interferons (IFN–λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn–λ (boifn–λ) gene were synthetized after codon optimization. The boifn–λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN–λ3V18M. The recombinant plasmid pPICZαA–boIFN–λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high–level extracellular soluble form. Dominant expression strains of boIFN–λ3/λ3V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN–λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN–λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN–λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN–λ3 and boIFN–λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN–λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN–λs and provide material for therapeutic development.

Abstract Background Eradication of intramucosal Escherichia coli correlates with remission of periodic acid‐Schiff‐positive E coli‐associated granulomatous colitis (GC). Treatment failures attributed to multidrug resistant (MDR) bacteria necessitate alternative approaches. Hypothesis/objectives Determine clinical outcome of E coli‐associated GC in dogs treated based on antimicrobial susceptibility profiling and characterize E coli phylogeny and resistance mechanisms. Animals Twenty Boxers and 4 French Bulldogs with E coli‐associated GC. Methods Culture, antimicrobial susceptibility profiling, and molecular characterization of E coli were performed and response to treatment was evaluated. Results Initial biopsy sample culture yielded fluoroquinolone‐sensitive (FQ‐S) E coli from 9/24 dogs and fluoroquinolone‐resistant (FQ‐R) E coli from 15/24. All but 1 FQ‐R E coli were MDR with susceptibility to macrophage‐penetrating antimicrobials restricted to carbapenems in 13/15 dogs. Of 22/24 treated based on susceptibility profiling, 8/9 FQ‐S dogs had complete initial clinical response (CR) during fluoroquinolone (FQ) treatment, whereas 9/13 FQ‐R dogs had complete or partial response (PR) during meropenem or doxycycline treatment. In 5/9 FQ‐S and 12/13 FQ‐R dogs with follow‐up ≥3 months, CR was sustained in 5/5 FQ‐S (median, 25 months; range, 4‐46) whereas 6/12 FQ‐R had long‐term CR (median, 59 months; range 15‐102), 4/12 PR (median, 19 months; range, 5‐65), and 2/12 had no response (NR). Four dogs with long‐term follow‐up died within 4 years of diagnosis, including 2 euthanized for refractory colitis. Escherichia coli were genetically diverse. Fluoroquinolone resistance was associated with mutations in gyrA and parC, with plasmid‐mediated resistance less common. Conclusions and Clinical Importance Antimicrobial treatment guided by susceptibility profiling was associated with positive long‐term outcomes in >80% of cases. Fluoroquinolone‐resistance was widespread and not clonal. Further study is required to optimize treatment for dogs with MDR E coli‐associated GC.

Abstract Background Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous autosomal recessive disorder characterized by an almost total lack of adipose tissue in the body. Mutations in the AGPAT2, BSCL2, CAV1 and PTRF genes define I-IV subtype of BSLC respectively and clinical data indicate that new causative genes remain to be discovered. Here, we retrieved 341 cases from 60 BSCL-related studies worldwide and aimed to explore genotype-phenotype correlations based on mutations of AGPAT2 and BSCL2 genes from 251 cases. We also inferred new candidate genes for BSCL through protein-protein interaction and phenotype-similarity. Results Analysis results show that BSCL type II with earlier age of onset of diabetes mellitus, higher risk to suffer from premature death and mental retardation, is a more severe disorder than BSCL type I, but BSCL type I patients are more likely to have bone cysts. In BSCL type I, females are at higher risk of developing diabetes mellitus and acanthosis nigricans than males, while in BSCL type II, males suffer from diabetes mellitus earlier than females. In addition, some significant correlations among BSCL-related phenotypes were identified. New candidate genes prediction through protein-protein interaction and phenotype-similarity was conducted and we found that CAV3, EBP, SNAP29, HK1, CHRM3, OBSL1 and DNAJC13 genes could be the pathogenic factors for BSCL. Particularly, CAV3 and EBP could be high-priority candidate genes contributing to pathogenesis of BSCL. Conclusions Our study largely enhances the current knowledge of phenotypic and genotypic heterogeneity of BSCL and promotes the more comprehensive understanding of pathogenic mechanisms for BSCL.

High-entropy alloys (HEAs) with high hardness are promising materials for advanced industrial manufacturing. In this study, the AlCrFeNiV HEA was designed and successfully prepared using a plasma instantaneous process. The hardness test showed that AlCrFeNiV had a high hardness of 1076 ± 15 HV, which was much higher than those reported in the literature. The microstructure of AlCrFeNiV was composed of two different types of body-centered cubic (BCC) structures, BCC1 (Al, Cr, Fe, and Ni) and BCC2 (enriched V and Cr). A mixture of different BCC systems produced solution strengthening, which was responsible for the superior hardness. Moreover, the reciprocating sliding wear behavior of HEA against Al2O3 balls under dry and lubricated conditions at ambient temperature was investigated. The wear rates of AlCrFeNiV against Al2O3 under dry wear and lubrication were 17.2 × 10−5 mm3 N−1·m−1 and 12.4 × 10−5 mm3N−1·m−1, respectively, which were of the same order of magnitude as the wear rates of BCC HEAs. Regardless of the dry wear or wear with lubrication, the wear mechanism of the HEA was abrasive and delamination wear.

Echinochloa crus-galli var. zelayensis is a variant of E. crus-galli (L) Beauv, and it is the most pernicious weed in the east of China. Quinclorac, as synthetic auxin herbicide, could control this kind of weed effectively. In this study, two populations were used to further research the mechanism of quinclorac resistance, and the EcABCB1, EcABCB4 and EcABCB19 was functionally characterized to determine their roles in quinclorac resistance. It was found that root growth of quinclorac-resistant biotype SSXB-R was less inhibited by quinclorac at 5 μM and 50 μM when compared with the susceptible biotype JNNX-S. The results show that the IAA variations in root tip of JNNX-S were significantly higher than SSXB-R at 12 h after treatment with quinclorac (50 μM) and 1-N-naphthylthalamic acid (100 μM). There are no significant differences in IAA variations of the basal part of the root between susceptible and resistant biotypes after treatment with quinclorac and 1-N-naphthylthalamic acid (NPA). The transcript level of EcABCB1 and EcABCB19 in the root of JNNX-S showed down-regulated and up-regulated after treatment with quinclorac (TWQ) at 6 h in susceptible and resistant biotypes compared with control, respectively. The transcript level for EcABCB4 in the root showed up-regulated after TWQ at 12 h only in susceptible biotypes compared with control. It was found that the IC50 to quinclorac of AtABCB4 and AtABCB19 mutants were significantly higher than the parent line Col-0.

γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis. [ABSTRACT FROM AUTHOR]

ObjectivesTo investigate the genetic variants that are responsible for peripheral neuroblastic tumors (PNTs) oncogenesis in one family case.Materials and MethodsOne family was recruited, including the healthy parents, sister affected by neuroblastoma (NB), and brother who suffered from ganglioneuroma (GN). Whole-genome sequencing (WGS) of germline DNA from all the family members and RNA-seq of tumor RNA from the siblings were performed. Mutants were validated by Sanger sequencing and co-IP was performed to assess the impact of the mutant on chemosensitivity in the SH-SY5Y cell line.ResultsA novel compound heterozygous mutation of BRCA2 was locked as the cause of carcinogenesis. One allele was BRCA2-S871X (stop-gain) from the siblings’ mother, the other was BRCA2-N372H (missense) from their father. This novel compound heterozygous mutations of the BRCA2 gene associated with PNTs by disordering DNA damage and response (DDR) signal pathway. Moreover, chemosensitivity was reduced in the NB cell line due to the BRCA2-N372H mutant.ConclusionIn summary, these results revealed a novel germline compound heterozygous mutation of the BRCA2 gene associated with familial PNTs.

This study revealed the microbial compositions in children with bacterial meningitis who were treated with antibiotics and made a comprehensive comparison between blood and CSF specimens for risk and prognosis assessment. We found that microbiome signatures could distinguish patient subsets and were correlated with the CRP level in blood and granulocyte percentage in CSF.

Objectives To investigate the genetic causes of hearing loss with enlarged vestibular aqueduct (EVA) in two children from unrelated two Chinese families. Methods Sanger sequencing of all coding exons in SLC26A4 (encoding Pendrin protein) was performed on the two patients, their sibling and parents respectively. To predict and visualize the potential functional outcome of the novel variant, model building, structure analysis, and in silico analysis were further conducted. Results The results showed that the proband from family I harbored a compound heterozygote of SLC26A4 c.1174A>T (p.N392Y) mutation and c.1181delTCT (p.F394del) variant in exon 10, potentially altering Pendrin protein structure. In family II, the proband was identified in compound heterozygosity with a known mutation of c.919-2A>G in the splice site of intron 7 and a novel mutation of c.1023insC in exon 9, which results in a frameshift and translational termination, consequently leading to truncated Pendrin protein. Sequence homology analysis indicated that all the mutations localized at high conservation sites, which emphasized the significance of these mutations on Pendrin spatial organization and function. Conclusion In summary, this study revealed two compound heterozygous mutations (c.1174A>T/c.1181delTCT; c.919- 2A>G/c.1023insC) in Pendrin protein, which might account for the deafness of the two probands clinically diagnosed with EVA. Thus this study contributes to improve understanding of the causes of hearing loss associated with EVA and develop a more scientific screening strategy for deafness.

Objective: The aim of this study was to explore the diagnostic value of gastroscopy under local anesthesia for congenital pyriform sinus fistula (CPSF).Methods: This research was a diagnostic study. Patients received gastroscopy under local anesthesia to diagnose CPSF, and suspension laryngoscopy under general anesthesia was performed 2 days after gastroscopy. Various conditions of the internal opening of CPSF were then recorded. Patients were grouped according to lesion sides, age, time after the inflammation subsided, and history of previous surgery. The sensitivity, specificity, area under the receiver operating characteristic curve (AUC), accuracy, and positive and negative predictive values of gastroscopy were compared between the groups.Results: A total of 48 patients were recruited in this study, and no patients had severe gastroscopy-related complications. The diagnostic values of gastroscopy in 41 cases (85.4%) were consistent with suspension laryngoscopy. The sensitivity of gastroscopy was 86.4%, the specificity was 75%, the AUC was 0.807, the positive prediction rate was 97.4%, the negative prediction rate was 33.3%, the accuracy rate was 85.4%, and the diagnostic odds ratio (DOR) was 2.1. The kappa consistency test results had statistical significance (P = 0.0026, kappa = 0.3913). The diagnostic value of gastroscopy was better for the patients with inflammation subsiding for more than 4 weeks (P < 0.0001).Conclusion: Gastroscopy under local anesthesia is a safe, effective, reliable and novel diagnostic method for CPSF, and it is especially recommended as a diagnostic method for the patients with inflammation subsiding for more than 4 weeks.

Interferon-chi (IFN-χ) is a type of function-unknown IFN. IFN-χ in bovines (BoIFN-χ) has evolved as a multigene family. This family comprises four IFN-χ subtypes, two of which are functional genes, which we demonstrated to (i) have antiviral and antiproliferative activities, (ii) be highly sensitive to trypsin, and (iii) remain stable with changes in pH and temperature. BoIFN-χ is a key intermediate in antiviral response, PAbs against BoIFN-χs could downregulate the transcriptional activation of ISGs induced by poly(I:C), and BoIFN-χs could be induced upon virus infection at the early and late phase. Additionally, BoIFN-χs bind with type-I IFN receptors, induce transcription of interferon regulatory factor 7 (IRF7), interferon-stimulated genes (ISGs), and type-I IFNs as well as myxovirus resistance protein 1 (Mx1) expression. Expression of ISGs and activation of IFN-stimulated response element (ISRE) induced with BoIFN-χs could be downregulated significantly by the Janus kinase (JAK) 1 and signal transducers and activators of transcription (STAT) 1 inhibitor. The promoters of BoIFN-β, nuclear factor-kappa B, and ISRE could be activated with BoIFN-χs, and the BoIFN-χ promoter could be activated by other type-I IFNs. Overall, BoIFN-χ could be induced with virus infection and signal through the JAK-STAT pathway to form a positive-feedback regulation of IFN production. These findings may facilitate further research on the role of IFN-χ in innate immune responses.