Crossroads of assembling a moss genome: navigating contaminants and horizontal gene transfer in the moss Physcomitrellopsis africana.

The first chromosome-scale reference genome of the rare narrow-endemic African moss Physcomitrellopsis africana is presented here. Assembled from 73x nanopore long reads and 163x BGI-seq short reads, the 414 Mb reference comprises 26 chromosomes and 22,925 protein-coding genes (BUSCO: C:94.8%[D:13.9%]). This genome holds two genes that withstood rigorous filtration of microbial contaminants, have no homolog in other land plants and are thus interpreted as resulting from two unique horizontal gene transfers from microbes. Further, Physcomitrellopsis africana shares 176 of the 273 published HGT candidates identified in Physcomitrium patens, but lacks 98 of these, highlighting that perhaps as many as 91 genes were acquired in P. patens in the last 40 million years following its divergence from its common ancestor with P. africana. These observations suggest rather continuous gene gains via HGT followed by potential losses, during the diversification of the Funariaceae. Our findings showcase both dynamic flux in plant HGTs over evolutionarily "short" timescales, alongside enduring impacts of successful integrations, like those still functionally maintained in extant Physcomitrellopsis africana. Furthermore, this study describes the informatic processes employed to distinguish contaminants from candidate HGT events.

Investigating the effects of rare ginsenosides on hyperuricemia and associated sperm damage via nontargeted metabolomics and gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: In ancient times, ginseng was used for hyperuricemia treatment as described in the classic traditional Chinese medical text Shang Han Lun. Recent studies have shown that common ginsenosides and rare ginsenosides (RGS) are the main active compounds in ginseng. RGS have higher activity and are less studied in the treatment of hyperuricemia. AIM OF THE STUDY: To determine whether RGS prevents and ameliorates potassium oxonate(PO)-induced hyperuricemia and concomitant spermatozoa damage in mice and the possible underlying mechanisms. MATERIALS AND METHODS: Potassium oxonate (PO, 300 mg/kg) induced hyperuricemia in mice via the oral administration of RGS (50, 100, or 200 mg/kg) or allopurinol (ALL, 5 mg/kg) for 35 days. Uric acid (UA) and xanthine oxidase (XO) levels were measured to assess the degree of histopathological damage in the liver, kidney, and testis, and renal creatinine (CRE), urea nitrogen (BUN), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and inflammatory factor (IL-1ß) levels were measured to calculate the sperm density. Mechanisms were also explored based on blood and urine metabolomics and the gut microbiota. RESULTS: In this study, we demonstrated that RGS containing Rg3, Rk1, Rg6, and Rg5 could reduce serum UA levels, inhibit serum and hepatic XO activity, reduce renal CRE and BUN levels, further restore renal SOD and GSH activities, reduce the accumulation of MDA in the kidneys, and attenuate the production of renal IL-1ß. RGS was able to restore sperm density. Metabolomic analysis revealed that RGS improved sphingolipid metabolism, pyrimidine metabolism, and other metabolic pathways. 16S rDNA sequencing revealed that RGS could increase gut microbial diversity, restore the Firmicutes/Bacteroidetes (F/B) ratio, and adjust the intestinal microbial balance. Spearman's correlation analysis revealed a correlation between differentially metabolites and the gut microbiota. Lactobacillus and Akkermansia are the core genera. CONCLUSION: RGS can be a candidate for the prevention and amelioration of hyperuricemia and concomitant sperm damage. Its mechanism of action is closely related to sphingolipid metabolism, pyrimidine metabolism, and the modulation of gut microbiota, such as Lactobacillus and Akkermansia.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Inhibition of p38 MAPK/NF-κB p65 signaling pathway activity by rare ginsenosides ameliorates cyclophosphamide-induced premature ovarian failure and KGN cell injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C. A. Mey., one of the most used herbs in the world, shows effective treatment in reproductive injury. Recent studies have proven that the processed product, red ginseng, which is more active than ginseng itself. Therefore, it is speculated that its main functional component, rare ginsenosides (heat-transformed saponin, HTS), may be effective in treating premature ovarian failure (POF), but its efficacy has not yet been experimentally confirmed. AIM OF THE STUDY: To evaluate whether HTS could attenuate cyclophosphamide-induced inflammation and oxidative damage in POF model rats and the human granulosa-like KGN cell line and protect granulosa cell proliferation. MATERIAL AND METHODS: HTS were isolated from ginsenosides and high performance liquid chromatography (HPLC) analysis was used to analyze the HTS components. Cyclophosphamide (CP) was used to establish a POF rat model and KGN cell injury model. Reactive oxygen species (ROS) and antioxidant enzyme production was determined using specific assays, while inflammatory cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). The proliferative function of granulosa cells was assessed using high-content screening and immunohistochemistry to determine the Ki67 protein level. Protein expression in ovarian tissues and KGN cells was analyzed by Western blotting, quantitative real-time PCR (qRT-PCR) was used to determine the transcriptional changes in ovarian tissues and KGN cells. RESULTS: In CP-treated POF model rats, HTS significantly decreased malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels, increased glutathione oxidase (GSH) levels, and upregulated Ki67 expression in ovarian granulosa cells. In addition, HTS significantly increased cell survival and Ki67 expression levels in CP-treated cells, and superoxide dismutase (SOD) levels were significantly increased. HTS significantly downregulated IL-6, TNF-α, and interleukin-1ß (IL-1ß) mRNA expression and significantly inhibited nuclear factor kappa-B p65 (NF-κB p65) and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in POF model rats and KGN cells. Moreover, NF-κB p65 and p38 MAPK levels were significantly increased in ovarian granulosa cells. p65 and p38 protein and gene expression was significantly downregulated. CONCLUSION: HTS ameliorated CP-induced POF and human granulosa cell injury, possibly by inhibiting inflammation and oxidative damage mediated by the p38 MAPK/NF-κB p65 signaling pathway.

Artificial intelligence in the risk prediction models of cardiovascular disease and development of an independent validation screening tool: a systematic review.

BACKGROUND: A comprehensive overview of artificial intelligence (AI) for cardiovascular disease (CVD) prediction and a screening tool of AI models (AI-Ms) for independent external validation are lacking. This systematic review aims to identify, describe, and appraise AI-Ms of CVD prediction in the general and special populations and develop a new independent validation score (IVS) for AI-Ms replicability evaluation. METHODS: PubMed, Web of Science, Embase, and IEEE library were searched up to July 2021. Data extraction and analysis were performed for the populations, distribution, predictors, algorithms, etc. The risk of bias was evaluated with the prediction risk of bias assessment tool (PROBAST). Subsequently, we designed IVS for model replicability evaluation with five steps in five items, including transparency of algorithms, performance of models, feasibility of reproduction, risk of reproduction, and clinical implication, respectively. The review is registered in PROSPERO (No. CRD42021271789). RESULTS: In 20,887 screened references, 79 articles (82.5% in 2017-2021) were included, which contained 114 datasets (67 in Europe and North America, but 0 in Africa). We identified 486 AI-Ms, of which the majority were in development (n = 380), but none of them had undergone independent external validation. A total of 66 idiographic algorithms were found; however, 36.4% were used only once and only 39.4% over three times. A large number of different predictors (range 5-52,000, median 21) and large-span sample size (range 80-3,660,000, median 4466) were observed. All models were at high risk of bias according to PROBAST, primarily due to the incorrect use of statistical methods. IVS analysis confirmed only 10 models as "recommended"; however, 281 and 187 were "not recommended" and "warning," respectively. CONCLUSION: AI has led the digital revolution in the field of CVD prediction, but is still in the early stage of development as the defects of research design, report, and evaluation systems. The IVS we developed may contribute to independent external validation and the development of this field.

Open-DPSM: An open-source toolkit for modeling pupil size changes to dynamic visual inputs.

Pupil size change is a widely adopted, sensitive indicator for sensory and cognitive processes. However, the interpretation of these changes is complicated by the influence of multiple low-level effects, such as brightness or contrast changes, posing challenges to applying pupillometry outside of extremely controlled settings. Building on and extending previous models, we here introduce Open Dynamic Pupil Size Modeling (Open-DPSM), an open-source toolkit to model pupil size changes to dynamically changing visual inputs using a convolution approach. Open-DPSM incorporates three key steps: (1) Modeling pupillary responses to both luminance and contrast changes; (2) Weighing of the distinct contributions of visual events across the visual field on pupil size change; and (3) Incorporating gaze-contingent visual event extraction and modeling. These steps improve the prediction of pupil size changes beyond the here-evaluated benchmarks. Open-DPSM provides Python functions, as well as a graphical user interface (GUI), enabling the extension of its applications to versatile scenarios and adaptations to individualized needs. By obtaining a predicted pupil trace using video and eye-tracking data, users can mitigate the effects of low-level features by subtracting the predicted trace or assess the efficacy of the low-level feature manipulations a priori by comparing estimated traces across conditions.

Chromosome-level genome assembly of Niphotrichum japonicum provides new insights into heat stress responses in mosses.

With a diversity of approximately 22,000 species, bryophytes (hornworts, liverworts, and mosses) represent a major and diverse lineage of land plants. Bryophytes can thrive in many extreme environments as they can endure the stresses of drought, heat, and cold. The moss Niphotrichum japonicum (Grimmiaceae, Grimmiales) can subsist for extended periods under heat and drought conditions, providing a good candidate for studying the genetic basis underlying such high resilience. Here, we de novo assembled the genome of N. japonicum using Nanopore long reads combined with Hi-C scaffolding technology to anchor the 191.61 Mb assembly into 14 pseudochromosomes. The genome structure of N. japonicum's autosomes is mostly conserved and highly syntenic, in contrast to the sparse and disordered genes present in its sex chromosome. Comparative genomic analysis revealed the presence of 10,019 genes exclusively in N. japonicum. These genes may contribute to the species-specific resilience, as demonstrated by the gene ontology (GO) enrichment. Transcriptome analysis showed that 37.44% (including 3,107 unique genes) of the total annotated genes (26,898) exhibited differential expression as a result of heat-induced stress, and the mechanisms that respond to heat stress are generally conserved across plants. These include the upregulation of HSPs, LEAs, and reactive oxygen species (ROS) scavenging genes, and the downregulation of PPR genes. N. japonicum also appears to have distinctive thermal mechanisms, including species-specific expansion and upregulation of the Self-incomp_S1 gene family, functional divergence of duplicated genes, structural clusters of upregulated genes, and expression piggybacking of hub genes. Overall, our study highlights both shared and species-specific heat tolerance strategies in N. japonicum, providing valuable insights into the heat tolerance mechanism and the evolution of resilient plants.

Subcutaneous Rapid Dissolution Microneedle Patch Integrated with CuO2 and Disulfiram for Augmented Antimelanoma Efficacy through Multimodal Synergy of Photothermal Therapy, Chemodynamic Therapy, and Chemotherapy.

Melanoma is a malignancy of the skin that is resistant to conventional treatment, necessitating the development of effective and safe new therapies. The percutaneous microneedle (MN) system has garnered increasing interest as a viable treatment option due to its high efficacy, minimal invasiveness, painlessness, and secure benefits. In this investigation, a sensitive MN system with multiple functions was created to combat melanoma effectively. This MN system utilized polyvinylpyrrolidone (PVP) as microneedle substrates and biocompatibility panax notoginseng polysaccharide (PNPS) as microneedle tips, which encapsulated PVP-stabilized CuO2 nanoparticles as a therapeutic agent and disulfiram-containing F127 micelles to enhance the tumor treatment effect. The MN system had sufficient mechanical properties to pierce the skin, and the excellent water solubility of PNPS brought high-speed dissolution properties under the bio conditions, allowing the MNs to effectively penetrate the skin and deliver the CuO2 nanoparticles as well as the drug-loaded micelles to the melanoma site. CuO2 nanoparticles released by the MN system generated Cu2+ and H2O2 in the tumor acidic environment to achieve self-supply of hydrogen peroxide to chemodynamic therapy (CDT). In addition, Cu2+ was chelated with disulfiram to produce CuET, which killed tumor cells. And the MN system had excellent near-infrared (NIR) photothermal properties due to the loading of CuO2 nanoparticles and induced localized thermotherapy in the melanoma region to further inhibit tumor growth. Thus, the designed MN system accomplished effective tumor suppression and minimal side effects in vivo via combined therapy, offering patients a safe and effective option for melanoma treatment.

Optimization of green deep eutectic solvent (DES) extraction of Chenopodium quinoa Willd. husks saponins by response surface methodology and their antioxidant activities.

Quinoa saponins have outstanding activity, and there are an increasing number of extraction methods, but there are few research programs on green preparation technology. The extraction conditions of quinoa saponins with deep eutectic solvents (DESs) were optimized by single-factor experiments combined with response surface methodology. The antioxidant capacity of saponins extracted by DESs and traditional methods was evaluated by the DPPH clearance rate, iron ion chelation rate and potassium ferricyanide reducing power. The results show that the optimal DES is choline chloride: 1,2-propylene glycol (1 : 1), and its water content is 40%. The optimal extraction conditions were as follows: the solid-to-solvent ratio was 0.05 g mL-1, the extraction time was 89 min, and the extraction temperature was 75 °C. Under these conditions, the extraction of quinoa saponins by DES was more effective than the traditional extraction methods. The saponins extracted by DES and traditional methods were analyzed by UPLC-MS, and five main saponins were identified. Quantitative analysis by HPLC-UV showed that Q1 (m/z = 971) and Q2 (m/z = 809) had higher contents of saponins. In vitro antioxidant experiments showed that all DES saponin extracts showed good antioxidant capacity. This study provides new insight into the development and utilization of quinoa saponins.

Entropy-Stabilized Layered K0.6Ni0.05Fe0.05Mg0.05Ti0.05Mn0.725O2 as a High-Rate and Stable Cathode for Potassium-Ion Batteries.

Mn-based layered oxides have been considered the most promising cathode candidates for cost-effective potassium-ion batteries (PIBs). Herein, equiatomic constituents of Ni, Fe, Mg, and Ti have been introduced into the transition metal layers of Mn-based layered oxide to design a high-entropy K0.6Ni0.05Fe0.05Mg0.05Ti0.05Mn0.0725O2 (HE-KMO, S = 1.17R). Consequently, the experimental results manifest that the layered structure of HE-KMO is more stable than conventional low-entropy K0.6MnO2 (LE-KMO, S = 0.66R) during successive cycling and even upon exposure to moisture. Diffraction and electrochemical measurements reveal that HE-KMO undergoes a solid-solution mechanism, contrary to the multistage phase transition processes typically exemplified in K0.6MnO2. Benefiting from the stabilized high-entropy layered framework and the solid-solution K+ storage mechanism, the entropy-stabilized HE-KMO not only demonstrates exceptional rate capability but also shows excellent cyclic stability. Notably, a capacity retention ratio of 86% after 3000 cycles can still be sustained at a remarkable current density of 5000 mA g-1.

SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways.

Allergic asthma is characterized by goblet cell metaplasia and subsequent mucus hypersecretion that contribute to the morbidity and mortality of this disease. Here, we explore the potential role and underlying mechanism of protein SUMOylation-mediated goblet cell metaplasia. The components of SUMOylaion machinery are specifically expressed in healthy human bronchial epithelia and robustly upregulated in bronchial epithelia of patients or mouse models with allergic asthma. Intratracheal suppression of SUMOylation by 2-D08 robustly attenuates not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Phosphoproteomics and biochemical analyses reveal SUMOylation on K1007 activates ROCK2, a master regulator of goblet cell metaplasia, by facilitating its binding to and activation by RhoA, and an E3 ligase PIAS1 is responsible for SUMOylation on K1007. As a result, knockdown of PIAS1 in bronchial epithelia inactivates ROCK2 to attenuate IL-13-induced goblet cell metaplasia, and bronchial epithelial knock-in of ROCK2(K1007R) consistently inactivates ROCK2 to alleviate not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Together, SUMOylation-mediated ROCK2 activation is an integral component of Rho/ROCK signaling in regulating the pathological conditions of asthma and thus SUMOylation is an additional target for the therapeutic intervention of this disease.

Natural mycotoxin contamination in dog food: A review on toxicity and detoxification methods.

Nowadays, the companion animals (dogs or other pets) are considered as members of the family and have established strong emotional relationships with their owners. Dogs are long lived compared to food animals, so safety, adequacy, and efficacy of dog food is of great importance for their health. Cereals, cereal by-products as well as feedstuffs of plant origin are commonly employed food resources in dry food, yet are potential ingredients for mycotoxins contamination, so dogs are theoretically more vulnerable to exposure when consumed daily. Aflatoxins (AF), deoxynivalenol (DON), fumonisins (FUM), ochratoxin A (OTA), and zearalenone (ZEA) are the most frequent mycotoxins that might present in dog food and cause toxicity on the growth and metabolism of dogs. An understanding of toxicological effects and detoxification methods (physical, chemical, or biological approaches) of mycotoxins will help to improve commercial ped food quality, reduce harm and minimize exposure to dogs. Herein, we outline a description of mycotoxins detected in dog food, toxicity and clinical findings in dogs, as well as methods applied in mycotoxins detoxification. This review aims to provide a reference for future studies involved in the evaluation of the risk, preventative strategies, and clear criteria of mycotoxins for minimizing exposure, reducing harm, and preventing mycotoxicosis in dog.

Multifunctional zwitterionic microneedle dressings for accelerated healing of chronic infected wounds in diabetic rat models.

Diabetic infected wounds are one of the major threats to public health but traditional wound dressings always have poor therapeutic efficacy influenced by the single treatment principle and limited penetration depth. Herein, we developed a novel kind of multifunctional degradable and removable zwitterionic microneedle dressings that could achieve multi-effective treatment of diabetic chronic wounds with a single dressing application. The substrates of microneedle dressings are composed of zwitterionic polymer polysulfobetaine methacrylate (PSBMA) and photothermal hair particles (HMPs), which can absorb wound exudate, form a barrier to the bacterial environment for the wound and exhibit an excellent photothermal bactericidal effect to promote wound healing. By loading zinc oxide nanoparticles (ZnO NPs) and asiaticoside in needle tips, drugs could diffuse in the wound area as the tips degrade to achieve highly effective antibacterial and anti-inflammatory effects and promote deep wound healing and tissue regeneration. The microneedles (MNs) were applied in diabetic rats with Staphylococcus aureus-infected wounds to demonstrate that the combination of drug and photothermal multi-treatment has accelerated tissue regeneration and collagen deposition and significantly promoted wound healing.

Sesamin Induces the Transdifferentiation of Type II Alveolar Epithelial Cells via AnnexinA1 and TRPV1.

PURPOSE: Acute lung injury (ALI) with high rates of morbidity is often accompanied by the apoptosis in the type I alveolar epithelial cells (ATIs). Thus, the transdifferentiation of type II alveolar epithelial cells (ATIIs) into ATIs is crucial for the maintenance of alveolar epithelial functions. We aimed to elucidate the role of sesamin in the transdifferentiation of ATIIs to ATIs and the involvement of the TRPV1/AKT pathway. METHODS: In vivo, the mouse model of ALI was simulated by intraperitoneal and intratracheal injections of lipopolysaccharide (LPS), respectively. The protective effects of sesamin on ALI were investigated using the survival rate, lung/body weight ratio, histological analysis of lung with HE staining, and mRNA levels of inflammatory factors. Western blot analysis and immunofluorescence detection of ATIs marker AQP5 were used to evaluate the protective effect of sesamin on ATIs. Western blot, EdU, and qPCR analyses were applied to detect changes in apoptosis, proliferation, and transdifferentiation markers of ATII A549 cell lines. Small interfering RNA (siRNA) was used to detect the involvement and relationships between the sesamin receptors (ANXA1 and TRPV1) and the AKT pathway in transdifferentiation. RESULTS: Sesamin (200 mg/kg) significantly improved LPS-induced ALI and inhibited LPS-induced ATIs reduction. A low concentration of sesamin (20 µM) promoted the transdifferentiation of ATIIs to ATIs. Both ANXA1 and TRPV1 were involved in sesamin-promoted transdifferentiation, while the P-AKT (S473) level was down-regulated by TRPV1 siRNA. CONCLUSION: Sesamin may promote transdifferentiation of ATII to ATI to ultimately rescue ALI, with TRPV1/AKT pathway involved in this transdifferentiation. This study revealed a novel role of sesamin in promoting the transdifferentiation of ATIIs to ATIs, providing experimental supports for the potential targets of ALI therapy.

Effect of Fuzheng formulas on the level of soluble immune checkpoint protein molecules in peripheral blood of patients with advanced NSCLC and its clinical significance / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：观察扶正方药对晚期三线非小细胞肺癌（NSCLC）患者外周血中12种可溶性免疫检查点分子水平的影响，并分析可溶性免疫检查点基线水平与NSCLC患者无进展生存期（PFS）的关系。方法：纳入2020年10月至2023年4月在龙华医院肿瘤科接受三线治疗（扶正方药联合标准西医方案治疗）的72例晚期NSCLC患者，采用LEGENDplex™多因子试剂盒检测患者治疗前及治疗4疗程后可溶性免疫检查点分子的表达水平，分析基线水平与PFS预后的相关性。结果：治疗4个疗程后，sCD137、sTGF-β1、sPD-L1、sPD-L2等指标均明显下降（P<0.05或P<0.01），Kaplan-Meier生存分析表明sPD-L2高水平患者的PFS短于sPD-L2低水平患者的PFS（P<0.05），COX多因素分析表明sPD-L2水平是晚期三线NSCLC患者PFS的独立影响因子（P<0.05）。结论：扶正方药对外周血可溶性免疫检查点蛋白表达具有一定的调节作用，高水平的sPD-L2预示晚期NSCLC患者较短的PFS，sPD-L2可能是晚期三线治疗NSCLC患者PFS的独立影响因子。

Head-to-head comparison of 99mTc-PSMA and 99mTc-MDP SPECT/CT in diagnosing prostate cancer bone metastasis: a prospective, comparative imaging trial.

The most common site of metastasis of prostate cancer (PCa) is bone. Skeletal-related events can increase the risk of death in patients with PCa by 28%. Due to the low detection rate of lesions in patients with low prostate-specific antigen (PSA) levels, the value of 99mTc methylene diphosphonate (99mTc-MDP) bone scintigraphy is limited. Prostate-specific membrane antigen (PSMA) is a small molecular probe that can efficiently and specifically detect PCa lesions. This prospective study aimed to evaluate the difference between 99mTc-PSMA single-photon emission computed tomography (SPECT)/CT and 99mTc-MDP SPECT/CT in the detection of bone metastasis in PCa. A total of 74 men with pathologically confirmed PCa from October 2019 to November 2021 were prospectively enrolled in this study. The median age was 70 (range, 55-87) years. All patients underwent both 99mTc-PSMA SPECT/CT and 99mTc-MDP SPECT/CT at an average interval of 12.1 (range, 1-14) days. The detected imaging-positive bone lesions were scored as "typical metastasis" or "equivocal metastasis" by a standard reporting schema. Subsequent therapy modality details were observed through follow-up. Twenty-five of the 74 patients were diagnosed with bone metastases. 99mTc-PSMA SPECT/CT and 99mTc-MDP SPECT/CT detected 20 and 18 bone metastases, with sensitivities of 80.0% (20/25) and 72.0% (18/25), specificities of 100.0% (49/49) and 81.3% (40/49), and AUCs of 88.0% and 84.9%, respectively. There was a significant difference in the AUC between the two imaging methods (P < 0.001). In an analysis of the number of bone metastasis lesions, the proportion of "typical metastasis" versus "equivocal metastasis" detected by the two imaging methods was 26.3:1 (PSMA) and 2.9:1 (MDP), and the difference was statistically significant (P = 0.005). There was a significant difference in the detection of bone metastatic lesions by 99mTc-PSMA and 99mTc-MDP when the maximum diameter of the lesions was ≤ 0.6 cm (P < 0.05). The optimal cut-off value for PSA was 2.635 ng/mL (PSMA) and 15.275 ng/mL (MDP). 99mTc-PSMA SPECT/CT led to a change in management to a more individualized therapy modality for 11 of 74 men (14.9%). 99mTc-PSMA SPECT/CT was superior to 99mTc-MDP SPECT/CT in the detection of bone metastases in PCa, especially for small lesions and in patients with low PSA levels, and demonstrated an additional benefit of providing information on extraskeletal metastases. With regard to therapy, 99mTc-PSMA scans might have utility in improving the subsequent therapy modality.

Vitamin C deficiency induces hypoglycemia and cognitive disorder through S-nitrosylation-mediated activation of glycogen synthase kinase 3ß.

Vitamin C (VC, l-ascorbic acid) is an essential nutrient that plays a key role in metabolism and functions as a potent antioxidant in regulating the S-nitrosylation and denitrosylation of target proteins. The precise function of VC deprivation in glucose homeostasis is still unknown. In the absence of L-gulono-1,4-lactone oxidoreductase, an essential enzyme for the last step of VC synthesis, VC deprivation resulted in persistent hypoglycemia and subsequent impairment of cognitive functions in female but not male mouse pups. The cognitive disorders caused by VC deprivation were largely reversed when these female pups were given glucose. VC deprivation-induced S-nitrosylation of glycogen synthase kinase 3ß (GSK3ß) at Cys14, which activated GSK3ß and inactivated glycogen synthase to decrease glycogen synthesis and storage under the feeding condition, while VC deprivation inactivated glycogen phosphorylase to decrease glycogenolysis under the fasting condition, ultimately leading to hypoglycemia and cognitive disorders. Treatment with Nω-Nitro-l-arginine methyl ester (l-NAME), a specific inhibitor of nitric oxide synthase, on the other hand, effectively prevented S-nitrosylation and activation of GSK3ß in female pups in response to the VC deprivation and reversed hypoglycemia and cognitive disorders. Overall, this research identifies S-nitrosylation of GSK3ß and subsequent GSK3ß activation as a previously unknown mechanism controlling glucose homeostasis in female pups in response to VC deprivation, implying that VC supplementation in the prevention of hypoglycemia and cognitive disorders should be considered in the certain groups of people, particularly young females.

Dysregulated adipose tissue expansion and impaired adipogenesis in Prader-Willi syndrome children before obesity-onset.

OBJECTIVE: Prader-Willi syndrome (PWS) is a rare genetic imprinting disorder resulting from the expression loss of genes on the paternally inherited chromosome 15q11-13. Early-onset life-thriving obesity and hyperphagia represent the clinical hallmarks of PWS. The noncoding RNA gene SNORD116 within the minimal PWS genetic lesion plays a critical role in the pathogenesis of the syndrome. Despite advancements in understanding the genetic basis for PWS, the pathophysiology of obesity development in PWS remains largely uncharacterized. Here, we aimed to investigate the signatures of adipose tissue development and expansion pathways and associated adipose biology in PWS children without obesity-onset at an early stage, mainly from the perspective of the adipogenesis process, and further elucidate the underlying molecular mechanisms. METHODS: We collected inguinal (subcutaneous) white adipose tissues (ingWATs) from phase 1 PWS and healthy children with normal weight aged from 6 M to 2 Y. Adipose morphology and histological characteristics were assessed. Primary adipose stromal vascular fractions (SVFs) were isolated, cultured in vitro, and used to determine the capacity and function of white and beige adipogenic differentiation. High-throughput RNA-sequencing (RNA-seq) was performed in adipose-derived mesenchymal stem cells (AdMSCs) to analyze transcriptome signatures in PWS subjects. Transient repression of SNORD116 was conducted to evaluate its functional relevance in adipogenesis. The changes in alternative pre-mRNA splicing were investigated in PWS and SNORD116 deficient cells. RESULTS: In phase 1 PWS children, impaired white adipose tissue (WAT) development and unusual fat expansion occurred long before obesity onset, which was characterized by the massive enlargement of adipocytes accompanied by increased apoptosis. White and beige adipogenesis programs were impaired and differentiated adipocyte functions were disturbed in PWS-derived SVFs, despite increased proliferation capacity, which were consistent with the results of RNA-seq analysis of PWS AdMSCs. We also experimentally validated disrupted beige adipogenesis in adipocytes with transient SNORD116 downregulation. The transcript and protein levels of PPARγ, the adipogenesis master regulator, were significantly lower in PWS than in control AdMSCs as well as in SNORD116 deficient AdMSCs/adipocytes than in scramble (Scr) cells, resulting in the inhibited adipogenic program. Additionally, through RNA-seq, we observed aberrant transcriptome-wide alterations in alternative RNA splicing patterns in PWS cells mediated by SNORD116 loss and specifically identified a changed PRDM16 gene splicing profile in vitro. CONCLUSIONS: Imbalance in the WAT expansion pathway and developmental disruption are primary defects in PWS displaying aberrant adipocyte hypertrophy and impaired adipogenesis process, in which SNORD116 deficiency plays a part. Our findings suggest that dysregulated adiposity specificity existing at an early phase is a potential pathological mechanism exacerbating hyperphagic obesity onset in PWS. This mechanistic evidence on adipose biology in young PWS patients expands knowledge regarding the pathogenesis of PWS obesity and may aid in developing a new therapeutic strategy targeting disturbed adipogenesis and driving AT plasticity to combat abnormal adiposity and associated metabolic disorders for PWS patients.

Afatinib 30 mg in the treatment of common and uncommon EGFR-mutated advanced lung adenocarcinomas: a retrospective, single-center, longitudinal study.

Background: Afatinib 30 mg has been proved to be with comparable efficacy but more tolerable than the dose of 40 mg for Asian patients with non-small cell lung cancer (NSCLC). This study aimed to investigate the clinical outcomes of afatinib at 30 mg/d in the treatment of advanced lung adenocarcinomas (LAD) with common and uncommon epidermal growth factor receptor (EGFR) mutations. Methods: EGFR-mutated advanced LAD patients receiving afatinib (30 mg/d) from January 2017 to November 2021 were retrospectively included. EGFR status was classified into three subtypes, namely common mutations including exon 19 deletions (19del) and exon 21 L858R (21L858R), uncommon mutations including G719X, L861Q, S768I, and complex mutations, and separately exon 20 insertions (20ins). Progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR) and adverse events (AEs) were analyzed during regular follow-up. Results: The overall median PFS of totally 58 included patients was 9.83 [95% confidence index (CI): 5.76-13.91] months. The number of patients with common, uncommon, and 20ins mutations was 32 (55.2%), 19 (32.8%) and 7 (12.1%), respectively. Baseline characteristics did not differ significantly among the three subtypes. The corresponding median PFS was 13.97 (12.06-15.89), 8.48 (0.32-16.64), and 3.78 (1.93-5.64) months, respectively (P=0.002). In the first-line setting, patients with common and uncommon mutations had a significantly longer PFS compared to those with 20ins [14.53 (13.53-15.53) vs. 10.39 (4.87-15.91) vs. 2.37 (0.00-5.11) months, P<0.001]. The first-line ORR showed significant differences among the three subtypes (60% vs. 80% vs. 0.0%, P=0.023). All-grade AEs occurred in 22 patients (37.9%). AEs ≥ grade 3 mainly included diarrhea (8.6%), and none of the patients discontinued treatment due to severe AEs. Conclusions: Afatinib at 30 mg/d is associated with a favorable efficacy and tolerability in the treatment of advanced LAD with common and major uncommon EGFR mutations except 20ins. Further large-scale prospective studies are warranted to confirm our findings.