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Abstract Background Wheat is one of major sources of human cadmium (Cd) intake. Reducing the grain Cd concentrations in wheat is urgently required to ensure food security and human health. In this study, we performed a field experiment at Wenjiang experimental field of Sichuan Agricultural University (Chengdu, China) to reveal the effects of FeCl3 and Fe2(SO4)3 on reducing grain Cd concentrations in dwarf Polish wheat (Triticum polonicum L., 2n = 4x = 28, AABB). Results Soil application of FeCl3 and Fe2(SO4)3 (0.04 M Fe3+/m2) significantly reduced grain Cd concentration in DPW at maturity by 19.04% and 33.33%, respectively. They did not reduce Cd uptake or root-to-shoot Cd translocation, but increased Cd distribution in lower leaves, lower internodes, and glumes. Meanwhile, application of FeCl3 and Fe2(SO4)3 up-regulated the expression of TpNRAMP5, TpNRAMP2 and TpYSL15 in roots, and TpYSL15 and TpZIP3 in shoots; they also downregulated the expression of TpZIP1 and TpZIP3 in roots, and TpIRT1 and TpNRAMP5 in shoots. Conclusions The reduction in grain Cd concentration caused by application of FeCl3 and Fe2(SO4)3 was resulted from changes in shoot Cd distribution via regulating the expression of some metal transporter genes. Overall, this study reports the physiological pathways of soil applied Fe fertilizer on grain Cd concentration in wheat, suggests a strategy for reducing grain Cd concentration by altering shoot Cd distribution.

Abstract This study aimed to unveil the central mechanism of moxibustion treating chronic inflammatory visceral pain (CIVP) from the angle of circRNA-miRNA-mRNA networks in the spinal cord. The rat CIVP model was established using a mixture of 5% (w/v) 2,4,6-trinitrobenzene sulfonic acid and 50% ethanol at a volume ratio of 2:1 via enema. Rats in the moxibustion group received herb-partitioned moxibustion at Tianshu (ST25, bilateral) and Qihai (CV6) points. The abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were adopted for pain behavior observation and pain sensitivity assessment. The circRNA, miRNA, and mRNA expression profiles were detected using the high-throughput sequencing technique. Relevant databases and bioinformatics analysis methods were used to screen for differentially expressed (DE) RNAs and build a circRNA-miRNA-mRNA (competing endogenous RNA) ceRNA regulatory network. The real-time quantitative PCR was employed to verify the sequencing result. CIVP rat models had a significantly higher AWR and lower TWL and MWT than normal rats. Between normal and model rats, there were 103 DE-circRNAs, 16 DE-miRNAs, and 397 DE-mRNAs in the spinal cord. Compared with the model group, the moxibustion group had a lower AWR and higher TWL and MWT; between these two groups, there were 118 DE-circRNAs, 15 DE-miRNAs, and 804 DE-mRNAs in the spinal cord. Two ceRNA networks were chosen to be verified. As a result, moxibustion’s analgesic effect on visceral pain in CIVP rats may be associated with regulating the circRNA_02767/rno-miR-483-3p/Gfap network in the spinal cord and improving central sensitization.

Abstract Background Cryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion. Methods We utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells. Results To illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum. Conclusions In this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum. Graphical Abstract

Abstract Background Intracellular DNA-sensing pathway cGAS-STING, inflammasomes and pyroptosis act as critical natural immune signaling axes for microbial infection, chronic inflammation, cancer progression and organ degeneration, but the mechanism and regulation of the crosstalk network remain unclear. Main body of the abstract Cellular stress disrupts mitochondrial homeostasis, facilitates the opening of mitochondrial permeability transition pore and the leakage of mitochondrial DNA to cell membrane, triggers inflammatory responses by activating cGAS-STING signaling, and subsequently induces inflammasomes activation and the onset of pyroptosis. Meanwhile, the inflammasome-associated protein caspase-1, Gasdermin D, the CARD domain of ASC and the potassium channel are involved in regulating cGAS-STING pathway. Importantly, this crosstalk network has a cascade amplification effect that exacerbates the immuno-inflammatory response, worsening the pathological process of inflammatory and autoimmune diseases. Given the importance of this crosstalk network of cGAS-STING, inflammasomes and pyroptosis in the regulation of innate immunity, it is emerging as a new avenue to explore the mechanisms of multiple disease pathogenesis. Therefore, efforts to define strategies to selectively modulate cGAS-STING, inflammasomes and pyroptosis in different disease settings have been or are ongoing. In this review, we will describe how this mechanistic understanding is driving possible therapeutics targeting this crosstalk network, focusing on the interacting or regulatory proteins, pathways, and a regulatory mitochondrial hub between cGAS-STING, inflammasomes, and pyroptosis. Short conclusion This review aims to provide insight into the critical roles and regulatory mechanisms of the crosstalk network of cGAS-STING, inflammasomes and pyroptosis, and to highlight some promising directions for future research and intervention.

Abstract Background Long noncoding RNAs (lncRNAs) play a key role in the occurrence and progression of myopia. However, the function of lncRNAs in retinal ganglion cells (RGCs) in the pathogenesis of myopia is still unknown. The aim of our study was to explore the lncRNA-mediated competing endogenous RNA (ceRNA) network in RGCs during the development of myopia. Methods RNA sequencing was performed to analyze lncRNA and mRNA expression profiles in RGCs between guinea pigs with form-deprived myopia (FDM) and normal control guinea pigs, and related ceRNA networks were constructed. Then, potentially important genes in ceRNA networks were verified by qRT‒PCR, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore biological functions in the RGCs of FDM guinea pigs. The important genes and related signaling pathways were further verified by qRT‒PCR, immunohistochemistry, immunofluorescence and Western blot in myopia in FDM guinea pigs, FDM mice, and highly myopic adults. Results The distribution of RGCs was uneven, the number of RGCs was decreased, and RGC apoptosis was increased in FDM guinea pigs. In total, 873 lncRNAs and 2480 mRNAs were determined to be differentially expressed genes in RGCs from normal control and FDM guinea pigs. Via lncRNA-mediated ceRNA network construction and PCR verification, we found that lncRNA-XR_002792574.1 may be involved in the development of myopia through the miR-760-3p/Adcy1 pathway in RGCs. Further verification in FDM guinea pigs, FDM mice, and highly myopic adults demonstrated that the lncRNA-XR_002792574.1/miR-760-3p/Adcy1 axis in RGCs might be related to cGMP/PKG, the apelin signaling pathway and scleral remodeling. Conclusion We demonstrated that the lncRNA-XR_002792574.1/miR-760-3p/Adcy1 axis in RGCs might be related to myopia. On the one hand, the lncRNA-XR_002792574.1/miR-760-3p/Adcy1 axis might inhibit the cGMP/PKG and apelin signaling pathways in RGCs, thereby causing RGC damage in myopia. On the other hand, the lncRNA-XR_002792574.1/miR-760-3p/Adcy1 axis may cause myopic scleral remodeling through the ERK-MMP-2 pathway. These findings may reveal novel potential targets in myopia and provide reference value for exploration and development of gene editing therapeutics for hereditary myopia.

Abstract Background With the promotion of “One Health,” the health of animals and their impact on the environment have become major concerns recently. Widely distributed in China, the whooper swans (Cygnus cygnus) and black swans (Cygnus atratus) are not only important to the ecological environment, but they may also potentially influence public health security. The metagenomic approach was adopted to uncover the impacts of the gut microbiota of swans on host and public health. Results In this study, the intestinal microbiome and resistome of migratory whooper swans and captive-bred black swans were identified. The results revealed similar gut microbes and functional compositions in whooper and black swans. Interestingly, different bacteria and probiotics were enriched by overwintering whooper swans. We also found that Acinetobacter and Escherichia were significantly enriched in early wintering period swans and that clinically important pathogens were more abundant in black swans. Whooper swans and black swans are potential reservoirs of antibiotic resistance genes (ARGs) and novel ARGs, and the abundance of novel ARGs in whooper swans was significantly higher than that in black swans. Metagenomic assembly–based host tracking revealed that most ARG-carrying contigs originated from Proteobacteria (mainly Gammaproteobacteria). Conclusions The results revealed spatiotemporal changes in microbiome and resistome in swans, providing a reference for safeguarding public health security and preventing animal epidemics.

Abstract Background Cervical cancer is the fourth most common cancer among women worldwide. Genome-wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical cancer risk. Therefore, we aimed to investigate the correlation between single nucleotide polymorphisms (SNPs) of the CD40 gene and susceptibility to cervical squamous cell carcinoma (CSCC) in a population from the northeastern Han Chinese population. Methods The three SNPs (rs1800686, rs3765459, and rs4810485) of the CD40 gene were analyzed by multiplex polymerase chain reaction (PCR) combined with next-generation sequencing methods in 421 patients with CSCC, 594 patients with high-grade squamous intraepithelial lesions (HSIL), and 504 healthy females. Multivariate logistic regression analysis was used to analyze the potential relationship between CD40 gene polymorphisms and CSCC, or HSIL. Results Our research results showed the AA genotype of rs1800686 had a protective effect on CSCC in comparison to the GG genotype and AG+GG genotypes (AA vs. GG: p = 0.0389 and AA vs. AG+GG: p = 0.0280, respectively). After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively). Similarly, rs3765459 showed a reduced risk association for CSCC in the codominant and recessive models (AA vs. GG: p = 0.0286 and AA vs. AG+GG: p = 0.0222, respectively). Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively). However, these differences were no longer significant after the Bonferroni correction. In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC. The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC. The 3 SNPs genotypes of the CD40 gene were closely related to the squamous cell carcinoma antigen (SCC) of the patients with HSIL. Conclusions The CD40 gene may play a role in the occurrence and development of CSCC.

Abstract Background A close connection between a protozoan parasite and the balance of the other gut microbes of the host has been demonstrated. The calves may be naturally co-infected with many parasites, and the co-effects of parasites on other intestinal microbes of calves remain unclear. This study aims to preliminarily reveal the relationship between intestinal parasites and other intestinal microbes in calves. Methods Fecal samples were collected from four calves with bloody diarrhea, four calves with watery diarrhea, and seven normal calves, and the microbial flora of the samples were analyzed by whole-genome sequencing. Protozoal parasites were detected in the metagenome sequences and identified using polymerase chain reaction (PCR). Results Cryptosporidium, Eimeria, Giardia, Blastocystis, and Entamoeba were detected by metagenomic analysis, and the identified species were Giardia duodenalis assemblage E, Cryptosporidium bovis, Cryptosporidium ryanae, Eimeria bovis, Eimeria subspherica, Entamoeba bovis, and Blastocystis ST2 and ST10. Metagenomic analysis showed that the intestinal microbes of calves with diarrhea were disordered, especially in calves with bloody diarrhea. Furthermore, different parasites show distinct relationships with the intestinal microecology. Cryptosporidium, Eimeria, and Giardia were negatively correlated with various intestinal bacteria but positively correlated with some fungi. However, Blastocystis and Entamoeba were positively associated with other gut microbes. Twenty-seven biomarkers not only were significantly enriched in bloody diarrhea, watery diarrhea, and normal calves but were also associated with Eimeria, Cryptosporidium, and Giardia. Only Eimeria showed a distinct relationship with seven genera of bacteria, which were significantly enriched in the healthy calves. All 18 genera of fungi were positively correlated with Cryptosporidium, Eimeria, and Giardia, which were also significantly enriched in calves with bloody diarrhea. Functional genes related to parasites and diseases were found mainly in fungi. Conclusions This study revealed the relationship between intestinal protozoan parasites and the other calf gut microbiome. Different intestinal protozoan parasites have diametrically opposite effects on other gut microecology, which not only affects bacteria in the gut, but also is significantly related to fungi and archaea. Graphical Abstract

Abstract Background Cryptosporidium is second only to rotavirus as a cause of moderate-to-severe diarrhea in young children. There are currently no fully effective drug treatments or vaccines for cryptosporidiosis. MicroRNAs (miRNAs) are involved in regulating the innate immune response to Cryptosporidium parvum infection. In this study, we investigated the role and mechanism of miR-3976 in regulating HCT-8 cell apoptosis induced by C. parvum infection. Methods Expression levels of miR-3976 and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-3976 and B-cell lymphoma 2-related protein A1 (BCL2A1) was studied by luciferase reporter assay, RT-qPCR, and western blotting. Results Expression levels of miR-3976 were decreased at 8 and 12 h post-infection (hpi) but increased at 24 and 48 hpi. Upregulation of miR-3976 promoted cell apoptosis and inhibited the parasite burden in HCT-8 cells after C. parvum infection. Luciferase reporter assay indicated that BCL2A1 was a target gene of miR-3976. Co-transfection with miR-3976 and a BCL2A1 overexpression vector revealed that miR-3976 targeted BCL2A1 and suppressed cell apoptosis and promoted the parasite burden in HCT-8 cells. Conclusions The present data indicated that miR-3976 regulated cell apoptosis and parasite burden in HCT-8 cells by targeting BCL2A1 following C. parvum infection. Future study should determine the role of miR-3976 in hosts’ anti-C. parvum immunity in vivo. Graphical Abstract

Abstract There is strong evidence to support that mutations and dysregulation of miRNAs are associated with a variety of diseases, including cancer. However, the experimental methods used to identify disease-related miRNAs are expensive and time-consuming. Effective computational approaches to identify disease-related miRNAs are in high demand and would aid in the detection of lncRNA biomarkers for disease diagnosis, treatment, and prevention. In this study, we develop an ensemble learning framework to reveal the potential associations between miRNAs and diseases (ELMDA). The ELMDA framework does not rely on the known associations when calculating miRNA and disease similarities and uses multi-classifiers voting to predict disease-related miRNAs. As a result, the average AUC of the ELMDA framework was 0.9229 for the HMDD v2.0 database in a fivefold cross-validation. All potential associations in the HMDD V2.0 database were predicted, and 90% of the top 50 results were verified with the updated HMDD V3.2 database. The ELMDA framework was implemented to investigate gastric neoplasms, prostate neoplasms and colon neoplasms, and 100%, 94%, and 90%, respectively, of the top 50 potential miRNAs were validated by the HMDD V3.2 database. Moreover, the ELMDA framework can predict isolated disease-related miRNAs. In conclusion, ELMDA appears to be a reliable method to uncover disease-associated miRNAs.

Abstract Background The role of microRNA (miRNA) in modulating the function of cancer stem cells through diverse signaling pathway has been evidenced. We here identified a role of microRNA (miRNA) family, specifically miR-148/152, in gastric cancer and delineated its functional effects on gastric cancer stem cells. Methods Bioinformatics analysis was conducted to analyze expression of integrin α5 (ITGA5) which was verified through expression determination in clinical tissue samples. Next, the upstream regulatory factors of ITGA5 were determined. CD44+EpCAM (high) cells sorted from AGS cells subjected to gain-of-function experiments, followed by evaluation of their capacity of colony formation, generation of tumorosphere, cell migration and viability in vitro and xenograft tumor formation in vivo. Results ITGA5 was elevated in gastric cancer tissues and confirmed as a target gene of the miR-148/152 family members. The miR-148/152 family members were downregulated in gastric cancer tissues and cells. Decreased expression of miR-148/152 family members was also detected in gastric cancer stem cells. However, the raised expression led to reduced colony formation, tumorosphere, cell migration, cell viability, and drug resistance of CD44+EpCAM (high) AGS cells in vitro, and tumorigenesis in vitro. ITGA5 overexpression reversed the effect of the miR-148/152 family members. Conclusions This study demonstrates that the miR-148/152 family members may prevent gastric cancer stem cell-like properties by targeting ITGA5, which can serve as an appealing target for gastric cancer treatment.

Abstract Background The prevalence of preterm birth has been rising, and there is a paucity of nationwide data on the perinatal characteristics and neonatal outcomes of twin deliveries of very preterm infants (VPIs) in China. This study compared the perinatal characteristics and outcomes of singletons and twins admitted to neonatal intensive care units (NICUs) in China. Methods The study population comprised all infants born before 32 weeks in the Chinese Neonatal Network (CHNN) between January 2019 and December 2019. Three-level and population-average generalized estimating equation (GEE)/alternating logistic regression (ALR) models were used to determine the association of twins with neonatal morbidities and the use of NICU resources. Results During the study period, there were 6634 (71.2%) singletons and 2680 (28.8%) twins, with mean birth weights of 1333.70 g and 1294.63 g, respectively. Twins were significantly more likely to be delivered by caesarean section (p

Abstract Background Cryptosporidium species are zoonotic protozoans that are important causes of diarrhoeal disease in both humans and animals. Non-coding RNAs (ncRNAs) play an important role in the innate immune defense against Cryptosporidium infection, but the underlying molecular mechanisms in the interaction between human ileocecal adenocarcinoma (HCT-8) cells and Cryptosporidium species have not been entirely revealed. Methods The expression profiles of messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) in the early phase of infection of HCT-8 cells with Cryptosporidium parvum and at 3 and 12 h post infection were analyzed using the RNA-sequencing technique. The biological functions of differentially expressed RNAs (dif-RNAs) were discovered through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The targeting relationships between three ncRNAs and mRNAs were analyzed using bioinformatics methods, followed by building a competing endogenous RNA (ceRNA) regulatory network centered on miRNAs. Results After strictly filtering the raw data, our analysis revealed 393 dif-lncRNAs, 69 dif-miRNAs and 115 dif-mRNAs at 3 hpi, and 450 dif-lncRNAs, 129 dif-miRNAs, 117 dif-mRNAs and one dif-circRNA at 12 hpi. Of these, 94 dif-lncRNAs, 24 dif-miRNAs and 22 dif-mRNAs were detected at both post-infection time points. Eleven dif-lncRNAs, seven dif-miRNAs, eight dif-mRNAs and one circRNA were randomly selected and confirmed using the quantitative real-time PCR. Bioinformatics analyses showed that the dif-mRNAs were significantly enriched in nutritional absorption, metabolic processes and metabolism-related pathways, while the dif-lncRNAs were mainly involved in the pathways related to the infection and pathogenicity of C. parvum (e.g. tight junction protein) and immune-related pathways (e.g. cell adhesion molecules). In contrast, dif-miRNAs and dif-circRNA were significantly enriched in apoptosis and apoptosis-related pathways. Among the downregulated RNAs, the miRNAs has-miR-324-3p and hsa-miR-3127-5p appear to be crucial miRNAs which could negatively regulate circRNA, lncRNA and mRNA. Conclusions The whole transcriptome profiles of HCT-8 cells infected with C. parvum were obtained in this study. The results of the GO and KEGG pathway analyses suggest significant roles for these dif-RNAs during the course of C. parvum infection. A ceRNA regulation network containing miRNA at its center was constructed for the first time, with hsa-miR-324-3p and hsa-miR-3127-5p being the crucial miRNAs. These findings provide novel insights into the responses of human intestinal epithelial cells to C. parvum infection. Graphical Abstract

Abstract Background Necrotizing enterocolitis (NEC) is a disastrous gastrointestinal disease of newborns, and the mortality rate of infants with NEC is approximately 20%-30%. The exploration of pathogenic targets of NEC will be conducive to timely diagnosis of NEC. Methods The whole transcriptome RNA sequencing was performed on NEC samples to reveal the expression of lncRNAs, circRNAs, miRNAs and mRNAs. Using differential expression analysis, cross analysis, target prediction, enrichment analysis, the pathogenic ceRNA network and target was found. Results Preliminarily, 281 DEmRNAs, 21 DEmiRNAs, 253 DElncRNAs and 207 DEcircRNAs were identified in NEC samples compared with controls. After target prediction and cross analyses, a key ceRNA regulatory network was built including 2 lncRNAs, 4 circRNAs, 2 miRNAs and 20 mRNAs. These 20 mRNAs were significantly enriched in many carbohydrate metabolism related pathways. After cross analysis of hypoxia-, carbohydrate metabolism-related genes, and 20 core genes, one gene HK2 was finally obtained. Dendritic cells activated were significantly differentially infiltrated and negatively correlated with HK2 expression in NEC samples. Conclusions The promising pathogenic hypoxia-related gene HK2 has been firstly identified in NEC, which might also involve in the carbohydrate metabolism in NEC.

Abstract Background Cryptosporidium parvum is a zoonotic parasitic protozoan that can infect a variety of animals and humans and is transmitted between hosts via oocysts. The oocyst wall provides strong protection against hostile environmental factors; however, research is limited concerning the oocyst wall at the proteomic level. Methods A comprehensive analysis of the proteome of oocyst wall of C. parvum was performed using label-free qualitative high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based qualitative proteomics technologies. Among the identified proteins, a surface protein (CpSP1) encoded by the C. parvum cgd7_5140 (Cpcgd7_5140) gene was predicted to be located on the surface of the oocyst wall. We preliminarily characterized the sequence and subcellular localization of CpSP1. Results A total of 798 proteins were identified, accounting for about 20% of the CryptoDB proteome. By using bioinformatic analysis, functional annotation and subcellular localization of the identified proteins were examined for better understanding of the characteristics of the oocyst wall. To verify the localization of CpSP1, an indirect immunofluorescent antibody assay demonstrated that the protein was localized on the surface of the oocyst wall, illustrating the potential usage as a marker for C. parvum detection in vitro. Conclusion The results provide a global framework about the proteomic composition of the Cryptosporidium oocyst wall, thereby providing a theoretical basis for further study of Cryptosporidium oocyst wall formation as well as the selection of targets for Cryptosporidium detection. Graphical Abstract

Abstract Background Cancer cell membrane-camouflaged nanotechnology for metal complex can enhance its biocompatibility and extend the effective circulation time in body. The ruthenium polypyridyl complex (RuPOP) has extensive antitumor activity, but it still has disadvantages such as poor biocompatibility, lack of targeting, and being easily metabolized by the organism. Cancer cell membranes retain a large number of surface antigens and tumor adhesion molecules CD47, which can be used to camouflage the metal complex and give it tumor homing ability and high biocompatibility. Results Therefore, this study provides an electrostatic adsorption method, which uses the electrostatic interaction of positive and negative charges between RuPOP and cell membranes to construct a cancer cell membrane-camouflaged nano-platform (RuPOP@CM). Interestingly, RuPOP@CM maintains the expression of surface antigens and tumor adhesion molecules, which can inhibit the phagocytosis of macrophage, reduce the clearance rate of RuPOP, and increase effective circulation time, thus enhancing the accumulation in tumor sites. Besides, RuPOP@CM can enhance the activity of cellular immune response and promote the production of inflammatory cytokines including TNF-α, IL-12 and IL-6, which is of great significance in treatment of tumor. On the other hand, RuPOP@MCM can produce intracellular ROS overproduction, thereby accelerating the apoptosis and cell cycle arrest of tumor cells to play an excellent antitumor effect in vitro and in vivo. Conclusion In brief, engineering cancer cell membrane-camouflaged metal complex is a potential strategy to improve its biocompatibility, biological safety and antitumor effects.

Abstract Background MicroRNAs (miRNAs) are involved in the regulation of both the innate and adaptive immune response to Cryptosporidium parvum infection. We previously reported that C. parvum upregulated miR‑942‑5p expression in HCT‑8 cells via TLR2/TLR4‑NF‑κB signaling. In the present study, the role of miRNA-942-5p in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated HCT-8 cell apoptosis induced by C. parvum was investigated. Methods Quantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis. Results Forced expression of miRNA-942-5p resulted in decreased apoptosis and an increased C. parvum burden in HCT-8 cells. The opposite results were observed using the suppressed expression of miRNA-942-5p. The miRNA-942-5p led to the translational suppression of IFI27 gene through targeting the 3’-untranslated region of the IFI27 gene. Moreover, overexpression of the IFI27 gene produced a high apoptotic ratio and low C. parvum burden. In contrast, a low apoptotic ratio and a high C. parvum burden were observed following downregulation of the IFI27 gene. Both miR-942-5p and the IFI27 gene influenced TRAIL and caspase-8 expression induced by C. parvum in HCT-8 cells. Moreover, TRAIL promoted HCT-8 cell apoptosis in a concentration-dependent manner. Conclusions These data suggested that C. parvum induced the downregulation of IFI27 via relief of miR-942-5p-mediated translational suppression. IFI27 downregulation was affected the burden of C. parvum by regulating HCT-8 cell apoptosis through TRAIL-dependent pathways. Future studies should determine the mechanisms by which C. parvum infection increases miR-942-5p expression and the role of miR-942-5p in hosts' anti-C. parvum immunity in vivo. Graphical Abstract

Abstract Background The aim of this work was to screen and validate biomarkers of ovarian cancer-initiating cells to detect the mechanisms of recurrence of epithelial ovarian cancer (EOC). Methods Stably labelled the amino acid in side population (SP) cells of epithelial ovarian cancer which were rich in cancer-initiating cells and non-SP cells with isotope in culture and differentially expressed cellular membrane proteins in SP cells were identified through proteomics technology. The new candidate biomarker was screened and validated through RT-PCR and western blot. Both in cell lines and primary EOC, cancer-initiating biofunctions of CDC50A positive cells were validated. Moreover, the characteristics of mesenchymal transition (EMT) was also detected and the correlation between the biomarker and clinical prognosis was observed. Results Through proteomics technology, candidate protein CDC50A was screened, and its significantly differential expression in SP cells was validated. CDC50A-positive cells from cell lines and primary ovarian cancer tissues were validated to show characteristics of cancer-initiating cells both in vitro and in vivo, including sphere-forming, self-renewal, differentiation, tumor metastasis and tumorigenicity in mice. The relationship between CDC50A-positive cells from primary tissues and tumour metastasis was confirmed based on their mesenchymal transition characteristics. Among 16 high-grade ovarian serous cancer patients, a high ratio of CDC50A-positive cells in primary tumours was correlated with a shorter platinum-free interval (p = 0.031, HR 0.260, 95% CI 0.77 ~ 0.885). Conclusion CDC50A could be used to screen ovarian cancer-initiating cells and might be a new target to resolve tumour development in EOC patients.

Abstract Background Prostate cancer (PCa) is known to be suitable for hypofractionated radiotherapy due to the very low α/β ratio (about 1.5–3 Gy). However, several randomized controlled trials have not shown the superiority of hypofractionated radiotherapy over conventionally fractionated radiotherapy. Besides, in vivo and in vitro experimental results show that the linear-quadratic (LQ) model may not be appropriate for hypofractionated radiotherapy, and we guess it may be due to the influence of fractionation schedules on the α/β ratio. Therefore, this study attempted to estimate the α/β ratio in different fractionation schedules and evaluate the applicability of the LQ model in hypofractionated radiotherapy. Methods The maximum likelihood principle in mathematical statistics was used to fit the parameters: α and β values in the tumor control probability (TCP) formula derived from the LQ model. In addition, the fitting results were substituted into the original TCP formula to calculate 5-year biochemical relapse-free survival for further verification. Results Information necessary for fitting could be extracted from a total of 23,281 PCa patients. A total of 16,442 PCa patients were grouped according to fractionation schedules. We found that, for patients who received conventionally fractionated radiotherapy, moderately hypofractionated radiotherapy, and stereotactic body radiotherapy, the average α/β ratios were 1.78 Gy (95% CI 1.59–1.98), 3.46 Gy (95% CI 3.27–3.65), and 4.24 Gy (95% CI 4.10–4.39), respectively. Hence, the calculated α/β ratios for PCa tended to become higher when the dose per fraction increased. Among all PCa patients, 14,641 could be grouped according to the risks of PCa in patients receiving radiotherapy with different fractionation schedules. The results showed that as the risk increased, the k (natural logarithm of an effective target cell number) and α values decreased, indicating that the number of effective target cells decreased and the radioresistance increased. Conclusions The LQ model appeared to be inappropriate for high doses per fraction owing to α/β ratios tending to become higher when the dose per fraction increased. Therefore, to convert the conventionally fractionated radiation doses to equivalent high doses per fraction using the standard LQ model, a higher α/β ratio should be used for calculation.

Abstract Background Lignocellulosic feedstocks have attracted much attention as a potential carbon source for lactic acid (LA) production because of their ready availability, sustainability, and renewability. However, there are at least two major technical challenges to producing LA from lignocellulose. Inhibitors derived from lignocellulose pretreatment have a negative impact on the growth of cells producing LA. Furthermore, pentose sugars produced from the pretreatment are difficultly utilized by most LA producers, which is known as the carbon catabolite repression (CCR) effect. This complex feedstock can be utilized by a robust microbial consortium with high bioconversion efficiency. Results In this study, a thermophilic consortium DUT50 producing LA was enriched and employed to improve corn stover (CS) utilization. Enterococcus was the dominant family in the consortium DUT50, accounting for 93.66% of the total abundance, with Lactobacillus, Bacillus, Lactococcus, and Trichococcus accounted for the remaining 2.68%. This consortium could be resistant to inhibitors concentration up to 9.74 g/L (2.88 g/L acetic acid, 2.46 g/L furfural, 2.20 g/L 5-HMF, and 2.20 g/L vanillin derived from pretreatment of CS), and simultaneously metabolizes hexose and pentose without CCR effect. Based on the promising consortium features, an efficient process of simultaneous saccharification and co-fermentation (SSCF) was developed to produce LA from acid pretreated corn stover, in which solid–liquid separation and detoxification were avoided. The key influencing factors were investigated and optimized, including dry biomass and cellulase loading, corn steep liquor powder concentration, and the pre-hydrolysis time. The highest LA titer of 71.04 g/L with a yield of 0.49 g/g-CS was achieved at a dry biomass loading of 20% (w/v), which is the highest LA production from non-detoxified acid pretreated corn stover via the SSCF process without wastewater generation reported to date. The simultaneous metabolism of hexose and pentose revealed collaboration between Enterococcus in the consortium, whereas xylose may be efficiently metabolized by Lactobacillus and Bacillus with low abundance via the pentose phosphate pathway. Conclusions The experimental results demonstrated the potential advantage of symbiosis in microbial consortia used for LA production from lignocellulosic biomass.

Abstract Background Septic shock is the most severe complication of sepsis, and is a major cause of childhood mortality, constituting a heavy public health burden. Methods We analyzed the gene expression profiles of septic shock and control samples from the Gene Expression Omnibus (GEO). Four differentially expressed genes (DEGs) from survivor and control groups, non-survivor and control groups, and survivor and non-survivor groups were selected. We used data about these genes to establish a logistic regression model for predicting the survival of septic shock patients. Results Leave-one-out cross validation and receiver operating characteristic (ROC) analysis indicated that this model had good accuracy. Differential expression and Gene Set Enrichment Analysis (GSEA) between septic shock patients stratified by prediction score indicated that the systemic lupus erythematosus pathway was activated, while the limonene and pinene degradation pathways were inactivated in the high score group. Conclusions Our study provides a novel approach for the prediction of the severity of pathology in septic shock patients, which are significant for personalized treatment as well as prognostic assessment.

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Abstract Background Accumulating evidence implies that autophagy plays a critical role in breast cancer development and progression. It is crucial to screen out autophagy-related encoding genes (ARGs) with prognostic value in breast cancer and reveal their biological properties in the aggressiveness of breast cancer. Methods Univariate and multivariate Cox proportional hazards analyses were used to identify a prognostic risk model of ARGs from The Cancer Genome Atlas (TCGA). Kaplan–Meier analysis, univariate and multivariate Cox regression analyses and receiver operating characteristic (ROC) curve analysis were performed to validate the risk model. Western blot and immunohistochemistry (IHC) were conducted to assess the expression of VPS35 (one of ARGs in risk model). CCK8, Colony formation assay, Transwell migration/invasion assays and autophagy flux assay were used to confirm biological function of VPS35 in breast cancer. Results In this study, the prognostic risk model consisting of six ARGs (VPS35, TRIM21, PRKAB2, RUFY4, MAP1LC3A and LARP1) in breast cancer were identified. The risk model was further verified as a novel independent prognostic factor for breast cancer patients. We also clarified that vacuolar protein sorting-associated protein 35 (VPS35), one of ARGs in the risk model, was upregulated in breast cancer samples and cell lines. VPS35 overexpression was correlated with more aggressive phenotype of breast cancer and indicated worse prognosis in both progression-free survival and overall survival analyses. Meanwhile, VPS35 knockdown inhibited breast cancer cell proliferation, migration and invasion, suggesting that VPS35 promoted the progression of breast cancer. VPS35 silence also influenced autophagy process, indicating that VPS35 was essential for autophagy completion. Conclusion Taken together, the six ARGs risk model has a remarkably prognostic value for breast cancer. Among them, VPS35 might exert as a significant oncogenic and prognostic factor for breast cancer and could be a promising autophagy-related therapeutic target in clinical practice.

Abstract Background Cryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity. Methods By integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor. Results Our ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa. Conclusions This study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment. Graphic Abstract

Abstract Background Nanoparticles assembled from food-grade calcium carbonate have attracted attention because of their biocompatibility, digestibility, particle and surface features (such as size, surface area, and partial wettability), and stimuli-responsiveness offered by their acid-labile nature. Results Herein, a type of edible oil-in-water Pickering emulsion was structured by calcium carbonate nanoparticles (CaCO3 NPs; mean particle size: 80 nm) and medium-chain triglyceride (MCT) for delivery of lipophilic drugs and simultaneous oral supplementation of calcium. The microstructure of the as-made CaCO3 NPs stabilized Pickering emulsion can be controlled by varying the particle concentration (c) and oil volume fraction (φ). The emulsification stabilizing capability of the CaCO3 NPs also favored the formation of high internal phase emulsion at a high φ of 0.7–0.8 with excellent emulsion stability at room temperature and at 4 °C, thus protecting the encapsulated lipophilic bioactive, vitamin D3 (VD3), against degradation. Interestingly, the structured CaCO3 NP-based Pickering emulsion displayed acid-trigged demulsification because of the disintegration of the CaCO3 NPs into Ca2+ in a simulated gastric environment, followed by efficient lipolysis of the lipid in simulated intestinal fluid. With the encapsulation and delivery of the emulsion, VD3 exhibited satisfying bioavailability after simulated gastrointestinal digestion. Conclusions Taken together, the rationally designed CaCO3 NP emulsion system holds potential as a calcium-fortified formulation for food, pharmaceutical and biomedical applications.

Abstract Background Abscisic acid (ABA) plays an important role in plant abiotic stress responses, and ABA INSENSITIVE 4 (ABI4) is a pivotal transcription factor in the ABA signaling pathway. In Arabidopsis, ABI4 negatively regulates salt tolerance; however, the mechanism through which ABI4 regulates plant salt tolerance is poorly understood. Our previous study showed that ABI4 directly binds to the promoter of the VITAMIN C DEFECTIVE 2 (VTC2) gene, inhibiting the transcription of VTC2 and ascorbic acid (AsA) biosynthesis. Results In the present study, we found that treatment with exogenous AsA could alleviate salt stress sensitivity of ABI4-overexpressing transgenic plants. The decreased AsA content and increased reactive oxygen species (ROS) levels in ABI4-overexpressing seedlings under salt treatment indicated that AsA-promoted ROS scavenging was related to ABI4-mediated salt tolerance. Gene expression analysis showed that ABI4 was induced at the early stage of salt stress, giving rise to reduced VTC2 expression. Accordingly, the abundance of the VTC2 protein decreased under the same salt stress conditions, and was absent in the ABI4 loss-of-function mutants, suggesting that the transcriptional inhibition of ABI4 on VTC2 resulted in the attenuation of VTC2 function. In addition, other encoding genes in the AsA biosynthesis and recycling pathways showed different responses to salt stress, demonstrating that AsA homeostasis is complicated under salinity stress. Conclusions This study elucidates the negative modulation of ABI4 in salt stress tolerance through the regulation of AsA biosynthesis and ROS accumulation in plants.

Abstract Background With the extended life expectancy of the Chinese population and improvements in surgery and anesthesia techniques, the number of aged patients undergoing surgery has been increasing annually. However, safety, effectiveness, and quality of life of aged patients undergoing surgery are facing major challenges. In order to standardize the perioperative assessment and procedures, we have developed a perioperative evaluation and auxiliary decision-making system named “Aged Patient Perioperative Longitudinal Evaluation–Multidisciplinary Trial (APPLE-MDT)”. Methods We will conduct a perioperative risk evaluation and targeted intervention, with follow-ups at 1, 3, and 6 months after surgery. The primary objective of the study is to evaluate the effectiveness of the “Aged Patient Perioperative Longitudinal Evaluation-Multiple Disciplinary Trial Path” (hereinafter referred to as the APPLE-MDT path) in surgical decision-making for aged patients (≥75 years) undergoing elective surgery under non-local anesthesia in the operating room. The secondary objectives of the study are to evaluate the postoperative outcome and health economics of the APPLE-MDT path applied to the surgical decision-making of aged patients (≥75 years) undergoing elective surgery under non-local anesthesia and to optimize intervention strategies for aged patients undergoing surgery to reduce the occurrence of postoperative complications and improve the quality of life after surgery. Discussion It is necessary to formulate a reliable, effective, and concise evaluation tool, which can effectively predict the perioperative complications and mortality of aged patients, support targeted intervention strategies, and allow for a more comprehensive risk and benefit analysis, thereby forming an effective senile perioperative surgery management path. It is expected that the implementation of this protocol can reduce the occurrence of postoperative complications, improve the postoperative quality of life, shorten hospital stay, reduce hospitalization expenses, reduce social burden, and allow the elderly to have a good quality of life after surgery. Trial registration ChiCTR, ChiCTR1800020363 , Registered 15 December 2018.

Abstract Background Long noncoding RNAs (lncRNAs) are emerging as crucial contributors to the development of breast cancer and are involved in the stemness regulation of breast cancer stem cells (BCSCs). LncRNAs are closely associated with the prognosis of breast cancer patients. It is critical to identify BCSC-related lncRNAs with prognostic value in breast cancer. Methods A co-expression network of BCSC-related mRNAs-lncRNAs from The Cancer Genome Atlas (TCGA) was constructed. Univariate and multivariate Cox proportional hazards analyses were used to identify a stemness risk model with prognostic value. Kaplan–Meier analysis, univariate and multivariate Cox regression analyses and receiver operating characteristic (ROC) curve analysis were performed to validate the risk model. Principal component analysis (PCA) and Gene Set Enrichment Analysis (GSEA) functional annotation were conducted to analyze the risk model. Results In this study, BCSC-related lncRNAs in breast cancer were identified. We evaluated the prognostic value of these BCSC-related lncRNAs and eventually obtained a prognostic risk model consisting of 12 BCSC-related lncRNAs (Z68871.1, LINC00578, AC097639.1, AP003119.3, AP001207.3, LINC00668, AL122010.1, AC245297.3, LINC01871, AP000851.2, AC022509.2 and SEMA3B-AS1). The risk model was further verified as a novel independent prognostic factor for breast cancer patients based on the calculated risk score. Moreover, based on the risk model, the low- risk and high-risk groups displayed different stemness statuses. Conclusions These findings suggested that the 12 BCSC-related lncRNA signature might be a promising prognostic factor for breast cancer and can promote the management of BCSC-related therapy in clinical practice.

Abstract Purpose This study aims to identify predictive local recurrence risk factors and site-specific local recurrence pattern of upper tract urothelial carcinoma (UTUC) with different primary tumor locations. Methods Three hundred and eighty-nine UTUC patients with radical nephroureterectomy were included in this study. Univariate and multivariate Cox proportional hazards regressions were performed to measure the risk of local recurrence. We also mapped the position of local recurrence sites stratified by primary tumor locations. Results A total of 73 patients (18.7%) developed local recurrence within a median follow-up of 41 months (range, 3-80 months). For patients with local recurrence, the median interval of local recurrence was 9 months. Ureter tumor, multifocality, T stage, G grade, lymph node metastasis (LNM), lymph node dissection (LND), and lymph vascular invasion (LVI) were all significantly associated with increased local recurrence by univariable analyses (P < 0.05). Only multifocality, T3–4, G3, and LNM remained independent predictors of increased local recurrence by multivariable analyses. Adjuvant radiotherapy could reduce the local recurrence (HR = 0.177; 95% CI 0.064-0.493, P = 0.001). Patients with local recurrence had poorer cancer-specific survival (4-year cancer-specific survival rate 36 ± 7.5% vs 88.4 ± 2.2%, P = 0.000). We evaluated local recurrence pattern stratified by tumor locations. Para-aortic lymph node region was the most common recurrence area for all the patients. Left-sided UTUC had more than 70% recurrent lymph nodes in the left para-aortic region (LPA). For right-sided UTUC patients, recurrent para-aortic lymph nodes distributed in the LPA (33.3%), aortocaval (AC) (41.5%), and right paracaval (RPC) (25.2%) regions. Recurrence in the internal and external iliac regions was only found in the distal ureter group (P < 0.05). Renal pelvic fossa recurrence was only found in renal pelvic tumor (22.2%, P = 0.007). The ureter tumor bed recurrence rate was higher for ureter patients (P = 0.001). Conclusions Multifocality, T3–4, G3, and LNM are predictors of higher local recurrence rate of UTUC. Adjuvant radiotherapy can reduce local recurrence rate. Local recurrence patterns are different according to primary tumor locations.

Abstract Background Necrotizing enterocolitis (NEC) is the leading cause of death among preterm infants born at

Abstract Background Our objective was to study the phenotype of and molecular genetic mechanisms underlying congenital protein C (PC) deficiency in Chinese neonates. We report the case of a neonate who presented 4 h after birth with purpura fulminans of the skin and thrombosis in the kidney. We also carried out a through literature review to study the genotype and phenotype, relevance, diagnosis, management, and prognosis of neonates with congenital PC deficiency in China. Case presentation and literature review Following a septic work-up and check of PC and protein S (PS) levels that showed PC deficiency, we investigated the patient’s and her parents’ genotypes. Our patient was found to have a plasma PC level of 0.8%. Molecular testing revealed a compound heterozygous mutation of the PROC gene: From the father, a c._262 G > T p. ASP88Tyr mutation in exon 4; from the mother, a C. 400 + 5G mutation in intron 5 that had been previously reported as likely pathogenic. Both parents were found to have heterozygous mutations for PC deficiency. In China, 5 other cases of congenital PC deficiency in the neonatal period were reported in the literature. In those cases, purpura fulminans and thrombosis were the main symptoms, and homozygous or compound heterozygous mutations of the PROC gene were identified. Conclusion Congenital PC deficiency should be ruled out for neonates presenting with purpura fulminans and thrombosis.

Abstract Background This study aimed to examine the performance of the four risk of malignancy index (RMI) in discriminating borderline ovarian tumors (BOTs) and benign ovarian masses in daily clinical practice. Methods A total of 162 women with BOTs and 379 women with benign ovarian tumors diagnosed at the Second Affiliated Hospital of Harbin Medical University from January 2012 to December 2016 were enrolled in this retrospective study. Also, we classified these patients into serous borderline ovarian tumor (SBOT) and mucinous borderline ovarian tumor (MBOT) subgroup. Preoperative ultrasound findings, cancer antigen 125 (CA125) and menopausal status were reviewed. The area under the curve (AUC) of receiver operator characteristic curves (ROC) and performance indices of RMI I, RMI II, RMI III and RMI IV were calculated and compared for discrimination between benign ovarian tumors and BOTs. Results RMI I had the highest AUC (0.825, 95% CI: 0.790–0.856) among the four RMIs in BOTs group. Similar results were found in SBOT (0.839, 95% CI: 0.804–0.871) and MBOT (0.791, 95% CI: 0.749–0.829) subgroups. RMI I had the highest specificity among the BOTs group (87.6, 95% CI: 83.9–90.7%), SBOT (87.6, 95% CI: 83.9–90.7%) and MBOT group (87.6, 95% CI: 83.9–90.7%). RMI II scored the highest overall in terms of sensitivity among the BOTs group (69.75, 95% CI: 62.1–76.7%), SBOT (74.34, 95% CI: 65.3–82.1%) and MBOT (59.18, 95% CI: 44.2–73.0%) group. Conclusion Compared to other RMIs, RMI I was the best-performed method for differentiation of BOTs from benign ovarian tumors. At the same time, RMI I also performed best in the discrimination SBOT from benign ovarian tumors.

Abstract Background Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco). Results Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium. Conclusions This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.

Abstract Background The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased worldwide. Metformin decreases triglyceride (TG) accumulation in hepatocytes in vivo and in vitro. Stearyl-coenzyme A desaturase 1 (SCD1) knockout mice also show decreased liver TG accumulation; however, whether SCD1 plays a role in the effect of metformin on TG accumulation is unknown. Therefore, the aim of this study was to investigate whether SCD1 mediated the effect of metformin on TG accumulation. Methods HepG2 and AML12 cells were exposed to high glucose and high insulin with or without metformin. An adenovirus was used for the SCD1 knockdown and overexpression. The triglyceride level in cells was detected. The expression of related genes was detected by Western blot and quantitative real-time PCR. A dual-luciferase reporter assay was used to determine the effect of metformin on the transcriptional activity of the SCD1 promoter. Results Metformin decreased TG accumulation to normal level in HepG2 cells exposed to high glucose and high insulin. The expression of SCD1 and fatty acid synthetase (FAS) was also decreased to normal level by metformin. Knockdown of SCD1 mimicked the effect of metformin on decreasing TG levels in AML12 cells, and the overexpression of SCD1 attenuated the effect of metformin on decreasing TG accumulation in HepG2 cells. The dual-luciferase reporter assay showed that the transcriptional activity of the SCD1 promoter (− 550/+ 199) after metformin treatment was 2-fold lower compared to control group in HepG2 cells. Additionally, the phosphorylation of AMPK after metformin treatment was 2-fold higher, and the expression of sterol regulatory element-binding protein-1c (SREBP-1c) after metformin treatment was about 2-fold lower compared to high glucose and high insulin group in HepG2 cells. Conclusions Together, these results reveal that metformin reduces TG accumulation in HepG2 cells via inhibiting the expression of SCD1.

Abstract Background Xiamen is a pilot city in China for hierarchical diagnosis and treatment reform of non-communicable diseases, especially diabetes. Since 2012, Xiamen has implemented a program called the “three-in-one”, a team-based care model for the treatment of diabetes, which involves collaboration between diabetes specialists, general practitioners, and health managers. In addition, the program provides financial incentives to improve care, as greater accessibility to medications through community health care centers (CHCs). The aim of this study was to evaluate the effectiveness of these policies in shifting visits from general hospitals to CHCs for the treatment of type 2 diabetes mellitus (T2DM). Method and materials A retrospective observational cohort study was conducted using Xiamen’s regional electronic health record (EHR) database, which included 90% of all patients registered since 2012. Logistic regression was used to derive the adjusted odds ratio (OR) for patients shifting from general hospitals to CHCs. Among patients treated at hospitals, Kaplan-Meier(KM) curves were constructed to evaluate the time from each policy introduction until the switch to CHCs. A k-means clustering analysis was conducted to identify patterns of patient care-seeking behavior. Results In total, 89,558 patients and 2,373,524 visits were included. In contrast to increased outpatient visits to general hospitals in China overall, the percentage of visits to CHCs in Xiamen increased from 29.7% in 2012 to 66.5% in 2016. The most significant and rapid shift occurred in later periods after full policy implementation. Three clusters of patients were identified with different levels of complications and health care-seeking frequency. All had similar responses to the policies. Conclusions The “three-in-one” team-based care model showed promising results for building a hierarchical health care system in China. These policy reforms effectively increased CHCs utilization among diabetic patients.

Abstract Background Obesity and metabolic syndrome (MetS) have become urgent worldwide health problems, predisposing patients to unfavorable myocardial status and thyroid dysfunction. Low-carbohydrate diet (LCD) and time-restricted eating (TRE) have been confirmed to be effective methods for weight management and improving MetS, but their effects on the myocardium and thyroid are unclear. Methods We conducted a secondary analysis in a randomized clinical diet-induced weight-loss trial. Participants (N = 169) diagnosed with MetS were randomized to the LCD group, the 8 h TRE group, or the combination of the LCD and TRE group for 3 months. Myocardial enzymes and thyroid function were tested before and after the intervention. Pearson’s or Spearman’s correlation was assessed between functions of the myocardium and thyroid and cardiometabolic parameters at baseline. Results A total of 162 participants who began the trial were included in the intention-to-treat (ITT) analysis, and 57 participants who adhered to their assigned protocol were involved in the per-protocol (PP) analysis. Relative to baseline, lactate dehydrogenase, creatine kinase MB, hydroxybutyrate dehydrogenase, and free triiodothyronine (FT3) declined, and free thyroxine (FT4) increased after all 3 interventions (both analyses). Creatine kinase (CK) decreased only in the TRE (− 18 [44] U/L, P

Abstract Background In the USA, the prolonged effective survival of cancer population has brought significant attention to the rising risk of cardiometabolic morbidity and mortality in this population. This heightened risk underscores the urgent need for research into effective pharmacological interventions for cancer survivors. Notably, metformin, a well-known metabolic regulator with pleiotropic effects, has shown protective effects against cardiometabolic disorders in diabetic individuals. Despite these promising indications, evidence supporting its efficacy in improving cardiometabolic outcomes in cancer survivors remains scarce. Methods A prospective cohort was established using a nationally representative sample of cancer survivors enrolled in the US National Health and Nutrition Examination Survey (NHANES), spanning 2003 to 2018. Outcomes were derived from patient interviews, physical examinations, and public-access linked mortality archives up to 2019. The Oxidative Balance Score was utilized to assess participants’ levels of oxidative stress. To evaluate the correlations between metformin use and the risk of cardiometabolic diseases and related mortality, survival analysis of cardiometabolic mortality was performed by Cox proportional hazards model, and cross-sectional analysis of cardiometabolic diseases outcomes was performed using logistic regression models. Interaction analyses were conducted to explore the specific pharmacological mechanism of metformin. Results Among 3995 cancer survivors (weighted population, 21,671,061, weighted mean [SE] age, 62.62 [0.33] years; 2119 [53.04%] females; 2727 [68.26%] Non-Hispanic White individuals), 448 reported metformin usage. During the follow-up period of up to 17 years (median, 6.42 years), there were 1233 recorded deaths, including 481 deaths from cardiometabolic causes. Multivariable models indicated that metformin use was associated with a lower risk of all-cause (hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.47–0.81) and cardiometabolic (HR, 0.65; 95% CI, 0.44–0.97) mortality compared with metformin nonusers. Metformin use was also correlated with a lower risk of total cardiovascular disease (odds ratio [OR], 0.41; 95% CI, 0.28–0.59), stroke (OR, 0.44; 95% CI, 0.26–0.74), hypertension (OR, 0.27; 95% CI, 0.14–0.52), and coronary heart disease (OR, 0.41; 95% CI, 0.21–0.78). The observed inverse associations were consistent across subgroup analyses in four specific cancer populations identified as cardiometabolic high-risk groups. Interaction analyses suggested that metformin use as compared to non-use may counter-balance oxidative stress. Conclusions In this cohort study involving a nationally representative population of US cancer survivors, metformin use was significantly correlated with a lower risk of cardiometabolic diseases, all-cause mortality, and cardiometabolic mortality.

Abstract Background Existing researches on nurses’ work engagement and job burnout have mostly stayed at the individual level, and limited researches test the cross-level effects of psychosocial safety climate (PSC). The study aimed to explore the cross-level mediating effect of organization-based self-esteem (OBSE) and the moderating effect of psychological detachment between the relationship of PSC and work engagement and job burnout in nurses. Methods The cross-sectional study was conducted during November to December 2022 at a tertiary hospital in a northeastern province of China. Data was collected from 1832 nurses through an online questionnaire. Correlation analyses and hierarchical linear modeling were used to test study hypotheses. Results The results showed that PSC was positively associated with work engagement, and negatively associated with job burnout. OBSE mediated the effect of PSC on work engagement, as well as job burnout. Additionally, psychological detachment played a moderating role between PSC and work engagement, but no moderating effect was found between PSC and job burnout. Conclusions PSC at the organizational level increases work engagement and reduces job burnout by stimulating nurses’ high levels of OBSE. Psychological detachment, as a situational factor, enhances the positive influence of PSC on work engagement. The implementation of measures to improve the PSC levels of the organization, and the levels of OBSE and psychological detachment among nurses could help to promote their good work performance.

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Abstract Purpose The aim of this study is to find a new method for femoral side preservation positioning in anterior cruciate ligament (ACL) reconstruction and test the accuracy and precision of this method. Method Fifty patients with isolated ACL rupture (42 males and 8 females) who underwent single-bundle ACL reconstruction in our hospital between July 2022 and July 2023 were included. The lowest point of the cartilage margin of the lateral wall of the intercontinental fossa and the tibial plateau plumb line at 120° of knee flexion were used as the anatomical landmarks for positioning of the femoral tunnel for ACL reconstruction surgery. Femoral side remnant preservation was performed in all cases. Three-dimensional CT was performed 3 days postoperatively to collect the data, which were analyzed using Mimics 21.0 software. We measured the posterior cortical distance of the femoral condyle at 90° of knee flexion and the vertical distance from the center of the bone tunnel to the cortical extension line behind the femur. All femoral tunnel positions were marked on a 4 × 4 grid and visualized using the quadrant method. Results Using the new positioning method in 50 knees, the average distance of x was 25.26 ± 2.76% of t and the average distance of y was 23.69 ± 6.19% of h. This is close to the results of previous studies, where x was 24.2 ± 4.0% of t and the average distance of y was 21.6 ± 5.2% of h. Most femoral tunnel positions were located in the same area. The D values were distributed as follows: 60% in the range of 0 to 2 mm, 24% in the range of 2 to 4 mm, and 16% more than 4 mm. The E values were distributed as follows: 80% in the range of 0 to 4 mm and 20% more than 4 mm. Conclusion In arthroscopic ACL reconstruction, the knee was flexed at 120° and the lowest point of the cartilage edge of the lateral wall of the intercondylar fossa and the tibial plateau plumb line were used as anatomical landmarks for the positioning of the femoral bone tunnel, which resulted in more accurate femoral bone tunnel positioning, better reproducibility, and better preservation of the femoral stump compared to traditional positioning methods.

Abstract Non-coding RNAs (ncRNAs) draw much attention from studies widely in recent years because they play vital roles in life activities. As a good complement to wet experiment methods, computational prediction methods can greatly save experimental costs. However, high false-negative data and insufficient use of multi-source information can affect the performance of computational prediction methods. Furthermore, many computational methods do not have good robustness and generalization on different datasets. In this work, we propose an effective end-to-end computing framework, called GDCL-NcDA, of deep graph learning and deep matrix factorization (DMF) with contrastive learning, which identifies the latent ncRNA-disease association on diverse multi-source heterogeneous networks (MHNs). The diverse MHNs include different similarity networks and proven associations among ncRNAs (miRNAs, circRNAs, and lncRNAs), genes, and diseases. Firstly, GDCL-NcDA employs deep graph convolutional network and multiple attention mechanisms to adaptively integrate multi-source of MHNs and reconstruct the ncRNA-disease association graph. Then, GDCL-NcDA utilizes DMF to predict the latent disease-associated ncRNAs based on the reconstructed graphs to reduce the impact of the false-negatives from the original associations. Finally, GDCL-NcDA uses contrastive learning (CL) to generate a contrastive loss on the reconstructed graphs and the predicted graphs to improve the generalization and robustness of our GDCL-NcDA framework. The experimental results show that GDCL-NcDA outperforms highly related computational methods. Moreover, case studies demonstrate the effectiveness of GDCL-NcDA in identifying the associations among diversiform ncRNAs and diseases.

Abstract Background Gut microbiota plays a significant role in the development and treatment of gouty arthritis. Simiao decoction has been shown to alleviate gouty arthritis by inhibiting inflammation, regulating NLRP3 inflammasome, and altering gut microbiota. However, there is no evidence to prove whether gut microbiota directly mediates the therapeutic efficiency of Simiao decoction in treating gout arthritis. Methods In this study, fecal microbiota transplantation (FMT) was used to transfer the gut microbiota of gout arthritis mice treated with Simiao decoction or allopurinol to blank gout arthritis mice, in order to investigate whether FMT had therapeutic effects on gout arthritis. Results Both Simiao decoction and allopurinol effectively reduced the levels of serum uric acid, liver XOD activity, foot thickness, serum IL-1β, and G-CSF in gout arthritis mice. However, Simiao decoction also had additional benefits, including raising the pain threshold, reducing serum TNF-α and IL-6, alleviating gut inflammation, and repairing intestinal pathology, which were not observed with allopurinol treatment. Moreover, Simiao decoction had a greater impact on gut microbiota than allopurinol, as it was able to restore the abundance of phylum Proteobacteria and genus Helicobacter. After transplantation into gout arthritis mice, gut microbiota altered by Simiao decoction exhibited similar therapeutic effects to those of Simiao decoction, but gut microbiota altered by allopurinol showed no therapeutic effect. Conclusions These findings demonstrates that Simiao decoction can alleviate gout arthritis symptoms by regulating gut microbiota.

Abstract Background & aims The individual effect of working schedule on survival in the hypertensive population has not been adequately studied. Shiftworkers are also prone to unhealthy lifestyles like pro-inflammatory diet. Therefore, we assessed the effect of shift work and its joint association with dietary inflammatory potential on mortality risk among the large US nationally representative sample of adult hypertensive population. Methods Data were from a nationally representative prospective cohort among US hypertensive population (n = 3680; weighted population, 54,192,988). The participants were linked to the 2019 public-access linked mortality archives. The working schedule were self-reported using the Occupation Questionnaire Section. Dietary inflammatory index (DII) scores were equally calculated using the 24-hour dietary recall (24 h) interviews. Multivariable Cox proportional hazards regression models were used to estimate hazard ratio and 95% confidence intervals (95%CI) for survival of hypertension individuals by work schedule and dietary inflammatory potential. The joint effect of work schedule and dietary inflammatory potential was then examined. Results Among the 3680 hypertension individuals (39.89% female [n = 1479] and 71.42% white [n = 1707]; weighted mean [SE] age, 47.35 [0.32] years), 592 individuals reported shift work status. 474 (10.76%) reported shift work status with pro-inflammatory dietary pattern (DII scores > 0). 118 (3.06%) reported shift work status with anti-inflammatory dietary pattern (DII scores