Contrastive learning based method for X-ray and CT registration under surgical equipment occlusion.

Deep learning-based 3D/2D surgical navigation registration techniques achieved excellent results. However, these methods are limited by the occlusion of surgical equipment resulting in poor accuracy. We designed a contrastive learning method that treats occluded and unoccluded X-rays as positive samples, maximizing the similarity between the positive samples and reducing interference from occlusion. The designed registration model has Transformer's residual connection (ResTrans), which enhances the long-sequence mapping capability, combined with the contrast learning strategy, ResTrans can adaptively retrieve the valid features in the global range to ensure the performance in the case of occlusion. Further, a learning-based region of interest (RoI) fine-tuning method is designed to refine the misalignment. We conducted experiments on occluded X-rays that contained different surgical devices. The experiment results show that the mean target registration error (mTRE) of ResTrans is 3.25 mm and the running time is 1.59 s. Compared with the state-of-the-art (SOTA) 3D/2D registration methods, our method offers better performance on occluded 3D/2D registration tasks.

Flavobacterium adhaerens sp. nov. and Flavobacterium maritimum sp. nov., two novel flavobacteria isolated from the Pearl River Estuary.

Two strains, designated as SYSU M80004T and SYSU M80005T, were isolated from water sampled in the Pearl River Estuary, Guangzhou, Guangdong, PR China. The strains were Gram-stain-negative and aerobic. Strain SYSU M80004T could grow at pH 6.0-8.0 (optimum, pH 7.0), 22-30 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0 %). Strain SYSU M80005T could grow at pH 6.0-8.0 (optimum, pH 7.0), 4-37 °C (optimum, 28 °C) and in the presence of 0-1 % NaCl (w/v; optimum 0%). Both strains contained MK-6 as the predominant menaquinone. C16 : 0 and iso-C15 : 0 were identified as the major fatty acids (>10 %) of strain SYSU M80004T while strain SYSU M80005T contained iso-C15 : 0 and iso-C17 : 0 3-OH as major fatty acids. Phosphatidylethanolamine was present as the major polar lipid in both strains. The average nucleotide identity and digital DNA-DNA hybridization values between these two strains and their closest relatives were 73.5-79.3 % and 19.6-23.2 %, respectively. Phylogenetic analysis based on the 16S rRNA gene and genomic sequences indicated they belonged to the genus Flavobacterium. Therefore, on the basis of phenotypic, physiological, chemotaxonomic and genomic evidence, two novel species, Flavobacterium adhaerens sp. nov. (type strain=SYSU M80004T=CDMCC 1.4522T=KCTC 102268T) and Flavobacterium maritimum sp. nov. (type strain=SYSU M80005T=CGMCC 1.4523T= KCTC 102269T) are proposed.

Comparative metagenomic analysis of microbial community compositions and functions in cage aquaculture and its nearby non-aquaculture environments.

In the context of burgeoning global aquaculture, its environmental repercussions, particularly in marine ecosystems, have gained significant attentions. Cage aquaculture, a prominent method, has been observed to significantly influence marine environments by discharging substantial amounts of organic materials and pollutants. It is also one of the important reasons for water eutrophication. This study investigated the impacts of cage aquaculture on microbial diversity and functional potential using metagenomics. Specifically, a comparison was made of the physicochemical indicators and microbial diversity between three grouper aquaculture cage nets in Lingshui Xincun Port and three nearby non-aquaculture area surface waters. We found that compared to non-aquaculture areas, the eutrophication indicators in aquaculture environments significantly increased, and the abundances of Vibrio and Pseudoalteromonas in aquaculture environments significantly rose. Additionally, microbial functional genes related to carbon, nitrogen, and sulfur metabolisms were also found to be significantly affected by aquaculture activities. The correlation analysis between microbial populations and environmental factors revealed that the abundances of most microbial taxa showed positive correlations with dissolved inorganic nitrogen, soluble reactive phosphorus, NH4+, and negative correlations with dissolved oxygen. Overall, this study elucidated the significant impacts of aquaculture-induced eutrophication on the diversity and functions of planktonic bacterial communities.

Klotho accelerates the progression of polycystic ovary syndrome through promoting granulosa cell apoptosis and inflammation.

The morbidity of polycystic ovary syndrome (PCOS) is in highly increasing rate nowadays. PCOS not only affects the fertility in women, but also threatens the health of whole life. Hence, to find the prognostic risk factors is of great value. However, the effective predictors in clinical practice of PCOS are still in blackness. In this study, we found Klotho was increased in FF (Follicular Fluid) and primary luteinized granulosa cells (GCs) from PCOS patients with hyperandrogenism. Furthermore, we found follicular Klotho was negatively correlated with numbers of mature oocytes, and positively correlated with serum testosterone, LH, and LH/FSH levels menstrual cycle and number of total antral follicles in PCOS patients. In primary luteinized GCs, the increased Klotho was accompanied with upregulation of cell apoptosis and inflammation-related genes. In ovaries of PCOS mice and cultured human KGN cell line, Klotho was up-regulated and accompanied by apoptosis, inflammation and mitochondrial dysfunction. Therefore, our findings suggest new mechanisms for granulosa cell injury and revealed to target inhibit Klotho maybe a new therapeutic strategy for treatment of PCOS.

Deciphering the pathogenic risks of microplastics as emerging particulate organic matter in aquatic ecosystem.

Microplastics are accumulating rapidly in aquatic ecosystems, providing habitats for pathogens and vectors for antibiotic resistance genes (ARGs), potentially increasing pathogenic risks. However, few studies have considered microplastics as particulate organic matter (POM) to elucidate their pathogenic risks and underlying mechanisms. Here, we performed microcosm experiments with microplastics and natural POM (leaves, algae, soil), thoroughly investigating their distinct effects on the community compositions, functional profiles, opportunistic pathogens, and ARGs in Particle-Associated (PA) and Free-Living (FL) bacterial communities. We found that both microplastics and leaves have comparable impacts on microbial community structures and functions, enriching opportunistic pathogens and ARGs, which may pose potential environmental risks. These effects are likely driven by their influences on water properties, including dissolved organic carbon, nitrate, DO, and pH. However, microplastics uniquely promoted pathogens as keystone species and further amplified their capacity as hosts for ARGs, potentially posing a higher pathogenic risk than natural POM. Our research also emphasized the importance of considering both PA and FL bacteria when assessing microplastic impacts, as they exhibited different responses. Overall, our study elucidates the role and underlying mechanism of microplastics as an emerging POM in intensifying pathogenic risks of aquatic ecosystems in comparison with conventional natural POM.

Macrophage colony-stimulating factor and its role in the tumor microenvironment: novel therapeutic avenues and mechanistic insights.

The tumor microenvironment is a complex ecosystem where various cellular and molecular interactions shape the course of cancer progression. Macrophage colony-stimulating factor (M-CSF) plays a pivotal role in this context. This study delves into the biological properties and functions of M-CSF in regulating tumor-associated macrophages (TAMs) and its role in modulating host immune responses. Through the specific binding to its receptor colony-stimulating factor 1 receptor (CSF-1R), M-CSF orchestrates a cascade of downstream signaling pathways to modulate macrophage activation, polarization, and proliferation. Furthermore, M-CSF extends its influence to other immune cell populations, including dendritic cells. Notably, the heightened expression of M-CSF within the tumor microenvironment is often associated with dismal patient prognoses. Therefore, a comprehensive investigation into the roles of M-CSF in tumor growth advances our comprehension of tumor development mechanisms and unveils promising novel strategies and approaches for cancer treatment.

An Hsp70 promoter-based mouse for heat shock-induced gene modulation.

Physical therapy is extensively employed in clinical settings. Nevertheless, the absence of suitable animal models has resulted in an incomplete understanding of the in vivo mechanisms and cellular distribution that respond to physical stimuli. The objective of this research was to create a mouse model capable of indicating the cells affected by physical stimuli. In this study, we successfully established a mouse line based on the heat shock protein 70 (Hsp70) promoter, wherein the expression of CreERT2 can be induced by physical stimuli. Following stimulation of the mouse tail, ear, or cultured calvarias with heat shock (generated by heating, ultrasound, or laser), a distinct Cre-mediated excision was observed in cells stimulated by these physical factors with minimal occurrence of leaky reporter expression. The application of heat shock to Hsp70-CreERT2; FGFR2-P253R double transgenic mice or Hsp70-CreERT2 mice infected with AAV-BMP4 at calvarias induced the activation of Cre-dependent mutant FGFR2-P253R or BMP4 respectively, thereby facilitating the premature closure of cranial sutures or the repair of calvarial defects. This novel mouse line holds significant potential for investigating the underlying mechanisms of physical therapy, tissue repair and regeneration, lineage tracing, and targeted modulation of gene expression of cells in local tissue stimulated by physical factor at the interested time points. KEY MESSAGES: In the study, an Hsp70-CreERT2 transgenic mouse was generated for heat shock-induced gene modulation. Heat shock, ultrasound, and laser stimulation effectively activated Cre expression in Hsp70-CreERT2; reporter mice, which leads to deletion of floxed DNA sequence in the tail, ear, and cultured calvaria tissues of mice. Local laser stimuli on cultured calvarias effectively induce Fgfr2-P253R expression in Hsp70-mTmG-Fgfr2-P253R mice and result in accelerated premature closure of cranial suture. Heat shock activated AAV9-FLEX-BMP4 expression and subsequently promoted the repair of calvarial defect of Hsp70-CreERT2; Rosa26-mTmG mice.

Systemic loss of CD36 aggravates NAFLD-related HCC through MEK1/2-ERK1/2 signaling pathway.

BACKGROUND & AIMS: CD36, a membrane protein widely present in various tissues, is crucial role in regulating energy metabolism. The rise of HCC as a notable outcome of NAFLD is becoming more apparent. Patients with hereditary CD36 deficiency are at increased risk of NAFLD. However, the impact of CD36 deficiency on NAFLD-HCC remains unclear. METHODS: Global CD36 knockout mice (CD36KO) and wild type mice (WT) were induced to establish NAFLD-HCC model by N-nitrosodiethylamine (DEN) plus high fat diet (HFD). Transcriptomics was employed to examine genes that were expressed differentially. RESULTS: Compared to WT mice, CD36KO mice showed more severe HFD-induced liver issues and increased tumor malignancy. The MEK1/2-ERK1/2 pathway activation was detected in the liver tissues of CD36KO mice using RNA sequencing and Western blot analysis. CONCLUSION: Systemic loss of CD36 leaded to the advancement of NAFLD to HCC by causing lipid disorders and metabolic inflammation, a process that involves the activation of MAPK signaling pathway. We found that CD36 contributes significantly to the maintenance of metabolic homeostasis in NAFLD-HCC.

Fumarate Hydratase-Deficient Renal Cell Carcinoma With Predominant Tubulocystic Features Mimics Tubulocystic Renal Cell Carcinoma.

CONTEXT.­: Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) rarely exhibits a predominant tubulocystic architecture with few other components. RCC with pure tubules and cysts lined by eosinophilic tumor cells with prominent nucleoli would raise the diagnosis of tubulocystic RCC. It is important to differentiate the 2 entities because they lead to different outcomes. OBJECTIVE.­: To address the concern, a multicenter study was implemented to explore useful clinicopathologic features in differentiation between tubulocystic FH-deficient RCC and tubulocystic RCC. DESIGN.­: Clinical factors included age, sex, tumor size, and outcome. Morphologic factors included cell morphology, presence or absence of a nontubulocystic component, and stromal findings. Immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing were performed to explore the protein expression and molecular profiles of the 2 entities. RESULTS.­: We evaluated 6 patients with tubulocystic RCC and 10 patients with tubulocystic FH-deficient RCC. Tubulocystic RCC exhibited a small size (<4.0 cm, pT1a), low Ki-67 index (<5%), retained FH, and negative 2SC expression. Tubulocystic FH-deficient RCC had a relatively large size and a high Ki-67 index. Perinucleolar haloes, loss of FH, and 2SC positivity were always observed. Pure tubulocystic architecture was not observed in FH-deficient RCC, because focal nontubulocystic components can always be seen. CONCLUSIONS.­: We emphasized multiple sectioning to identify a nontubulocystic architecture to exclude tubulocystic RCC. Moreover, tumor size, FH/2SC staining, and the Ki-67 index can differentiate tubulocystic FH-deficient RCC from tubulocystic RCC. The diagnosis of tubulocystic RCC was not recommended in renal mass biopsy because of the limited tissues sampled.

Total body water percentage and 3rd space water are novel risk factors for training-related lower extremity muscle injuries in young males.

PURPOSE: To identify the risk factors for training-related lower extremity muscle injuries in young males by a non-invasive method of body composition analysis. METHODS: A total of 282 healthy young male volunteers aged 18 - 20 years participated in this cohort study. Injury location, degree, and injury rate were adjusted by a questionnaire based on the overuse injury assessment methods used in epidemiological studies of sports injuries. The occurrence of training injuries is monitored and diagnosed by physicians and treated accordingly. The body composition was measured using the BodyStat QuadScan 4000 multifrequency Bio-impedance system at 5, 50, 100 and 200 kHz to obtain 4 impedance values. The Shapiro-Wilk test was used to check whether the data conformed to a normal distribution. Data of normal distribution were shown as mean ± SD and analyzed by t-test, while those of non-normal distribution were shown as median (Q1, Q3) and analyzed by Wilcoxon rank sum test. The receiver operator characteristic curve and logistic regression analysis were performed to investigate risk factors for developing training-related lower extremity injuries and accuracy. RESULTS: Among the 282 subjects, 78 (27.7%) developed training injuries. Lower extremity training injuries revealed the highest incidence, accounting for 23.4% (66 cases). These patients showed higher percentages of lean body mass (p = 0.001), total body water (TBW, p = 0.006), extracellular water (p = 0.020) and intracellular water (p = 0.010) as well as a larger ratio of basal metabolic rate/total weight (p = 0.006), compared with those without lower extremity muscle injuries. On the contrary, the percentage of body fat (p = 0.001) and body fat mass index (p = 0.002) were lower. Logistic regression analysis showed that TBW percentage > 65.35% (p = 0.050, odds ratio = 3.114) and 3rd space water > 0.95% (p = 0.045, odds ratio = 2.342) were independent risk factors for lower extremity muscle injuries. CONCLUSION: TBW percentage and 3rd space water measured with bio-impedance method are potential risk factors for predicting the incidence of lower extremity muscle injuries in young males following training.

A Novel Cargo Delivery System-AnCar-ExoLaIMTS Ameliorates Arthritis via Specifically Targeting Pro-Inflammatory Macrophages.

Macrophages are heterogenic phagocytic cells that play distinct roles in physiological and pathological processes. Targeting different types of macrophages has shown potent therapeutic effects in many diseases. Although many approaches are developed to target anti-inflammatory macrophages, there are few researches on targeting pro-inflammatory macrophages, which is partially attributed to their non-s pecificity phagocytosis of extracellular substances. In this study, a novel recombinant protein is constructed that can be anchored on an exosome membrane with the purpose of targeting pro-inflammatory macrophages via antigen recognition, which is named AnCar-ExoLaIMTS . The data indicate that the phagocytosis efficiencies of pro-inflammatory macrophages for different AnCar-ExoLaIMTS show obvious differences. The AnCar-ExoLaIMTS3 has the best targeting ability for pro-inflammatory macrophages in vitro and in vivo. Mechanically, AnCar-ExoLaIMTS3 can specifically recognize the leucine-rich repeat domain of the TLR4 receptor, and then enter into pro-inflammatory macrophages via the TLR4-mediated receptor endocytosis pathway. Moreover, AnCar-ExoLaIMTS3 can efficiently deliver therapeutic cargo to pro-inflammatory macrophages and inhibit the synovial inflammatory response via downregulation of HIF-1α level, thus ameliorating the severity of arthritis in vivo. Collectively, the work established a novel gene/drug delivery system that can specifically target pro-inflammatory macrophages, which may be beneficial for the treatments of arthritis and other inflammatory diseases.

Multi-Wavelength Selective and Broadband Near-Infrared Plasmonic Switches in Anisotropic Plasmonic Metasurfaces.

Anisotropic plasmonic metasurfaces have attracted broad research interest since they possess novel optical properties superior to natural materials and their tremendous design flexibility. However, the realization of multi-wavelength selective plasmonic metasurfaces that have emerged as promising candidates to uncover multichannel optical devices remains a challenge associated with weak modulation depths and narrow operation bandwidth. Herein, we propose and numerically demonstrate near-infrared multi-wavelength selective passive plasmonic switching (PPS) that encompasses high ON/OFF ratios and strong modulation depths via multiple Fano resonances (FRs) in anisotropic plasmonic metasurfaces. Specifically, the double FRs can be fulfilled and dedicated to establishing tailorable near-infrared dual-wavelength PPS. The multiple FRs mediated by in-plane mirror asymmetries cause the emergence of triple-wavelength PPS, whereas the multiple FRs governed by in-plane rotational asymmetries avail the implementation of the quasi-bound states in the continuum-endowed multi-wavelength PPS with the ability to unfold a tunable broad bandwidth. In addition, the strong polarization effects with in-plane anisotropic properties further validate the existence of the polarization-resolved multi-wavelength PPS. Our results provide an alternative approach to foster the achievement of multifunctional meta-devices in optical communication and information processing.

LmNaTx15, a novel scorpion toxin, enhances the activity of Nav channels and induces pain in mice.

Polypeptide toxins are major bioactive components found in venomous animals. Many polypeptide toxins can specifically act on targets, such as ion channels and voltage-gated sodium (Nav) channels, in the nervous, muscle, and cardiovascular systems of the recipient to increase defense and predation efficiency. In this study, a novel polypeptide toxin, LmNaTx15, was isolated from the venom of the scorpion Lychas mucronatus, and its activity was analyzed. LmNaTx15 slowed the fast inactivation of Nav1.2, Nav1.3, Nav1.4, Nav1.5, and Nav1.7 and inhibited the peak current of Nav1.5, but it did not affect Nav1.8. In addition, LmNaTx15 altered the voltage-dependent activation and inactivation of these Nav channel subtypes. Furthermore, like site 3 neurotoxins, LmNaTx15 induced pain in mice. These results show a novel scorpion toxin with a modulatory effect on specific Nav channel subtypes and pain induction in mice. Therefore, LmNaTx15 may be a key bioactive component for scorpion defense and predation. Besides, this study provides a basis for analyzing structure-function relationships of the scorpion toxins affecting Nav channel activity.

Prone versus lateral position in acute hypoxemic respiratory failure patients with HFNO therapy: study protocol for a multicentre randomised controlled open-label trial.

BACKGROUND: High-flow nasal oxygen (HFNO) therapy is a leading treatment technique for acute hypoxemic respiratory failure (AHRF), but its treatment failure rate remains high. The awake prone position (APP) has been proven to increase oxygenation and reduce the endotracheal intubation rate in patients with COVID-19-induced AHRF. However, the APP is poorly tolerated in patients, and its performance in improving prognoses is controversial. The lateral position has a similar mechanism and effect to the prone position, but it is more tolerable than the prone position. Therefore, it is worth exploring whether the lateral position is better for awake patients with AHRF. METHODS: This is a protocol for a three-arm parallel-group multicentre randomised controlled open-label exploratory trial. A total of 583 patients from two hospitals in Chongqing, China, will be randomised to take the semi-recumbent position, lateral position, or prone position at a ratio of 1:1:1. Patients are all diagnosed with AHRF secondary to non-COVID-19 pneumonia or lung infection and receiving HFNO therapy. The primary outcome is ventilator-free days in 28 days. The secondary outcomes are the 28-day intubation rate, 28-day all-cause mortality, total position change time, the incidence of adverse events, number of hours using HFNO therapy, length of hospital and intensive care unit (ICU) stay, and others. We will conduct subgroup analyses on the arterial partial pressure of oxygen to the fraction of inspiration oxygen (PaO2/FiO2) ratio (> 200 mmHg or ≤ 200 mmHg), time from admission to intervention implementation (< 24 h or ≥ 24 h), position changing time, and different diagnoses. DISCUSSION: This trial will explore the prognostic effects of the APP with that of the lateral position in awake patients with non-COVID-19AHRF and compare the differences between them. To provide evidence for clinical decision-making and further research on position management. TRIAL REGISTRATION: This trial was registered in the Chinese Clinical Trial Registry. The registration number is ChiCTR2200055822 . Registered on January 20, 2022.

Cir-DNA Sequencing Revealed the Landscape of Extrachromosomal Circular DNA in Articular Cartilage and the Potential Roles in Osteoarthritis.

OBJECTIVE: Extrachromosomal circular DNA (eccDNA) has been shown to be involved in several physiological and pathological processes including immunity, inflammation, aging, and tumor. However, the expression of eccDNA in cartilage has not been reported until now. In this study, we aimed to investigate the landscape of eccDNA in articular cartilage and analyze the potential roles in osteoarthritis (OA). METHODS: The samples of articular cartilage were obtained from total knee arthroplasty (TKA) donors with OA. The mitochondrial DNA (mtDNAs) and the linear DNAs from chondrocytes of articular cartilage were removed. Then the eccDNAs were enriched for cir-DNA sequencing. After quality control evaluation, we systematically revealed the identified eccDNA data including size distribution, the size range, and sequence pattern. Moreover, we explored and discussed the potential roles of eccDNA in OA via motif analysis and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. RESULTS: The chondrocytes from OA cartilage contained an abundance of eccDNAs, which was termed as OC-eccDNAs (OA cartilage-derived eccDNA). The characteristics of OC-eccDNAs were tissue-specific, including the distribution, the size range, and sequence pattern. Moreover, the functional analysis indicated that eccDNA may be involved in the homeostasis maintenance of chondrocytes and participated in the process of OA. CONCLUSIONS: Our data first showed the landscape of eccDNA in articular cartilage and preliminarily indicated the potential roles of eccDNA in OA.

Alsobacter ponti sp. nov., a novel denitrification and sulfate reduction bacterium isolated from Pearl River sediment.

A Gram-staining negative, aerobic, motile, and short rods strain, designated SYSU M60028T, was isolated from a Pearl River sediment sample in Guangzhou, Guangdong, China. The isolate could be able to grow at pH 6.0-8.0 (optimum, pH 7.0), 25-37 °C (optimum, 28 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0% NaCl). The cellular polar lipids of this strain were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and three unidentified lipids. The respiratory quinone of SYSU M60028T was found to be Q-10. The major fatty acids (> 5% of total) were summed feature 8, C16:0, and C18:1 ω7c 11-methy1. The genomic DNA G + C content was 69.9%. Phylogenetic analyses based on 16S rRNA gene sequences and core genes indicated that strain SYSU M60028T belonged to the genus Alsobacter and had the highest sequences similarities to Alsobacter metallidurans SK200a-9T (96.87%) and Alsobacter soli SH9T (96.87%). Based on the phenotypic, genotypic, and phylogenetic data, strain SYSU M0028T should be considered to represent a novel species of the genus Alsobacter, for which the name Alsobacter ponti sp. nov. is proposed. The type strain is SYSU M60028T (= CGMCC 1.19341T = KCTC 92046T).

Transcriptome analysis reveals the peptide toxins diversity of Macrothele palpator venom.

Spider venom is a large pharmacological repertoire of different bioactive peptide toxins. However, obtaining crude venom from some spiders is challenging. Thus, studying individual toxins through venom purification is a daunting task. In this study, we constructed the cDNA library and transcriptomic sequencing from the Macrothele palpator venom glands. Subsequently, 718 high-quality expressed sequence tags (ESTs) were identified, and grouped into three categories, including 449 toxin-like (62.53 %), 136 cellular component (18.94 %) and 133 non-matched (18.52 %) based on the gene function annotation. Additionally, 112 non-redundant toxin-like peptides were classified into 13 families (families A-M) based on their sequence homology and cysteine framework. Bioinformatics analysis revealed a high sequence similarity between families A-J and the toxins from Macrothele gigas in the NR database. In contrast, families K-M had a generally low sequence homology with known spider peptide toxins and unpredictable biological functions. Taken together, this study adds many new members to the spider toxin superfamily and provides a basis for identifying various potential biological tools in M. palpator venom.

Cyclophosphamide induced intestinal injury is alleviated by blocking the TLR9/caspase3/GSDME mediated intestinal epithelium pyroptosis.

OBJECTIVES: Cyclophosphamide (CYC) was commonly used to treat autoimmune disorders, and it could also cause side effects such as intestinal damage. This study aimed to explore the mechanism of CYC-induced intestinal cytotoxicity and provide evidence for protecting from intestinal damage by blocking TLR9/caspase3/GSDME mediated pyroptosis. METHODS: Intestinal epithelial cells (IEC-6) were treated with 4-hydroxycyclophosphamide (4HC), a key active metabolite of CYC. The pyroptotic rate of IEC-6 cells was detected by Annexin V/PI-Flow cytometry, microscopy imaging, and PI staining. The expression and activation of TLR9, caspase3 and GSDME in IEC-6 cells were detected by western blot and immunofluorescence staining. In addition, hydroxychloroquine (HCQ) and ODN2088 were used to inhibit TLR9 to investigate the role of TLR9 on caspase3/GSDME-mediated pyroptosis. Finally, mice lacking Gsdme or TLR9 or pretreating with HCQ were injected intraperitoneally with CYC, and the incidence and severity of intestinal damage were assessed. RESULTS: CYC induced lytic cell death in IEC-6 cells and increased the expression of TLR9, activated caspase3, and GSDME-N. Besides, both ODN2088 and HCQ could inhibit CYC-induced pyroptosis in IEC-6 cells. In vivo, CYC-induced intestinal injury was characterized by a large amount of intestinal villi abscission and structural disordered. Gsdme or TLR9 deficiency, or pretreatment of HCQ effectively attenuated intestinal damage in CYC-induced model mice. CONCLUSIONS: These results indicate an alternative mechanism for CYC-induced intestinal damage, which actives TLR9/caspase3/GSDME signaling pathway, leading to pyroptosis of intestinal epithelial cells. And targeting pyroptosis might be a potential therapeutic approach for CYC-induced intestinal damage.

Emerging roles of i-motif in gene expression and disease treatment.

As non-canonical nucleic acid secondary structures consisting of cytosine-rich nucleic acids, i-motifs can form under certain conditions. Several i-motif sequences have been identified in the human genome and play important roles in biological regulatory functions. Due to their physicochemical properties, these i-motif structures have attracted attention and are new targets for drug development. Herein, we reviewed the characteristics and mechanisms of i-motifs located in gene promoters (including c-myc, Bcl-2, VEGF, and telomeres), summarized various small molecule ligands that interact with them, and the possible binding modes between ligands and i-motifs, and described their effects on gene expression. Furthermore, we discussed diseases closely associated with i-motifs. Among these, cancer is closely associated with i-motifs since i-motifs can form in some regions of most oncogenes. Finally, we introduced recent advances in the applications of i-motifs in multiple areas.

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing of bacterial and eukaryotic microbial communities.

Sequencing of hypervariable regions as well as internal transcribed spacer regions of ribosomal RNA genes (rDNA) is broadly used to identify bacteria and fungi, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length using high throughput, cost-efficient second-generation sequencing. We developed a method to obtain nearly full-length rDNA by assembling single DNA molecules combining DNA co-barcoding with single-tube long fragment read technology and second-generation sequencing. Benchmarking was performed using mock bacterial and fungal communities as well as two forest soil samples. All mock species rDNA were successfully recovered with identities above 99.5% compared to the reference sequences. From the soil samples we obtained good coverage with identification of more than 20,000 unknown species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.