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Abstract Objective To identify recurrent venous thromboembolism (VTE) after discontinuation of anticoagulation in patients with isolated distal deep vein thrombosis based on its anatomic localization (axial or muscular veins). Methods Data were sourced from PubMed, Embase, Cochrane Library, Web of Science, and ClinicalTrials.gov databases in the time period up to October 2023. The study followed PRISMA guidelines using a registered protocol (CRD42023443029). Studies reporting recurrent VTE in patients with axial or muscular DVT were included in the analysis. Results Five studies with a total of 1,403 participants were evaluated. The results showed a pooled odds ratio of 1.12 (95% confidence interval 0.77–1.63) between axial and muscular DVT. Heterogeneity was low (I2 = 0%, p = 0.91) and there was no significant difference in the rate of recurrent VTE between axial and muscular DVT in each subgroup. Conclusions Muscular and axial DVT showed comparable recurrent VTE rates after anticoagulation. However, uncertainties regarding the possibility of recurrence affecting the popliteal vein or resulting in pulmonary embolism following muscular DVT anticoagulation persisted. Randomized trials in patients with isolated distal DVT are still needed to clarify its prognosis for different anatomical thrombus locations.

Abstract Objective To compare the predictive efficacy of the PADUA and Caprini models for pulmonary embolism (PE) in gynecological inpatients, analyze the risk factors for PE, and validate whether both models can effectively predict mortality rates. Methods A total of 355 gynecological inpatients who underwent computed tomography pulmonary angiography (CTPA) were included in the retrospective analysis. The comparative assessment of the predictive capabilities for PE between the PADUA and Caprini was carried out using receiver operating characteristic (ROC) curves. Logistic regression analysis was used to identify risk factors associated with PE. Additionally, Kaplan–Meier survival analysis plots were generated to validate the predictive efficacy for mortality rates. Results Among 355 patients, the PADUA and Caprini demonstrated the area under the curve (AUC) values of 0.757 and 0.756, respectively. There was no statistically significant difference in the AUC between the two models (P = 0.9542). Multivariate logistic analysis revealed immobility (P

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype. Molecular stratification and target therapy bring clinical benefit for TNBC patients, but it is difficult to implement comprehensive molecular testing in clinical practice. Here, using our multi-omics TNBC cohort (N = 425), a deep learning-based framework was devised and validated for comprehensive predictions of molecular features, subtypes and prognosis from pathological whole slide images. The framework first incorporated a neural network to decompose the tissue on WSIs, followed by a second one which was trained based on certain tissue types for predicting different targets. Multi-omics molecular features were analyzed including somatic mutations, copy number alterations, germline mutations, biological pathway activities, metabolomics features and immunotherapy biomarkers. It was shown that the molecular features with therapeutic implications can be predicted including the somatic PIK3CA mutation, germline BRCA2 mutation and PD-L1 protein expression (area under the curve [AUC]: 0.78, 0.79 and 0.74 respectively). The molecular subtypes of TNBC can be identified (AUC: 0.84, 0.85, 0.93 and 0.73 for the basal-like immune-suppressed, immunomodulatory, luminal androgen receptor, and mesenchymal-like subtypes respectively) and their distinctive morphological patterns were revealed, which provided novel insights into the heterogeneity of TNBC. A neural network integrating image features and clinical covariates stratified patients into groups with different survival outcomes (log-rank P < 0.001). Our prediction framework and neural network models were externally validated on the TNBC cases from TCGA (N = 143) and appeared robust to the changes in patient population. For potential clinical translation, we built a novel online platform, where we modularized and deployed our framework along with the validated models. It can realize real-time one-stop prediction for new cases. In summary, using only pathological WSIs, our proposed framework can enable comprehensive stratifications of TNBC patients and provide valuable information for therapeutic decision-making. It had the potential to be clinically implemented and promote the personalized management of TNBC.

ABSTRACT: Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.

Programmed cell death (PCD) is a fundamental biological process for maintaining cellular equilibrium and regulating development, health, and disease across all living organisms. Among the various types of PCD, apoptosis plays a pivotal role in numerous diseases, notably cancer. Cancer cells frequently develop mechanisms to evade apoptosis, increasing resistance to standard chemotherapy treatments. This resistance has prompted extensive research into alternative mechanisms of programmed cell death. One such pathway is oncosis, characterized by significant energy consumption, cell swelling, dilation of the endoplasmic reticulum, mitochondrial swelling, and nuclear chromatin aggregation. Recent research suggests that oncosis can impact conditions such as chemotherapeutic cardiotoxicity, myocardial ischemic injury, stroke, and cancer, mediated by specific oncosis-related proteins. In this review, we provide a detailed examination of the morphological and molecular features of oncosis and discuss various natural or small molecule compounds that can induce this type of cell death. Additionally, we summarize the current understanding of the molecular mechanisms underlying oncosis and its role in both normal physiology and pathological conditions. These insights aim to illuminate future research directions and propose innovative strategies for leveraging oncosis as a therapeutic tool against human diseases and cancer resistance.

Abstract Background The gut microbiota is a critical factor in the regulation of host health, but the relationship between the differential resistance of hosts to pathogens and the interaction of gut microbes is not yet clear. Herein, we investigated the potential correlation between the gut microbiota of piglets and their disease resistance using single-cell transcriptomics, 16S amplicon sequencing, metagenomics, and untargeted metabolomics. Results Porcine epidemic diarrhea virus (PEDV) infection leads to significant changes in the gut microbiota of piglets. Notably, Landrace pigs lose their resistance quickly after being infected with PEDV, but transplanting the fecal microbiota of Min pigs to Landrace pigs alleviated the infection status. Macrogenomic and animal protection models identified Lactobacillus reuteri and Lactobacillus amylovorus in the gut microbiota as playing an anti-infective role. Moreover, metabolomic screening of the secondary bile acids’ deoxycholic acid (DCA) and lithocholic acid (LCA) correlated significantly with Lactobacillus reuteri and Lactobacillus amylovorus, but only LCA exerted a protective function in the animal model. In addition, LCA supplementation altered the distribution of intestinal T-cell populations and resulted in significantly enriched CD8+ CTLs, and in vivo and in vitro experiments showed that LCA increased SLA-I expression in porcine intestinal epithelial cells via FXR receptors, thereby recruiting CD8+ CTLs to exert antiviral effects. Conclusions Overall, our findings indicate that the diversity of gut microbiota influences the development of the disease, and manipulating Lactobacillus reuteri and Lactobacillus amylovorus, as well as LCA, represents a promising strategy to improve PEDV infection in piglets. Video Abstract

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 μM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 μM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Abstract Digital pathology allows computerized analysis of tumor ecosystem using whole slide images (WSIs). Here, we present single-cell morphological and topological profiling (sc-MTOP) to characterize tumor ecosystem by extracting the features of nuclear morphology and intercellular spatial relationship for individual cells. We construct a single-cell atlas comprising 410 million cells from 637 breast cancer WSIs and dissect the phenotypic diversity within tumor, inflammatory and stroma cells respectively. Spatially-resolved analysis identifies recurrent micro-ecological modules representing locoregional multicellular structures and reveals four breast cancer ecotypes correlating with distinct molecular features and patient prognosis. Further analysis with multiomics data uncovers clinically relevant ecosystem features. High abundance of locally-aggregated inflammatory cells indicates immune-activated tumor microenvironment and favorable immunotherapy response in triple-negative breast cancers. Morphological intratumor heterogeneity of tumor nuclei correlates with cell cycle pathway activation and CDK inhibitors responsiveness in hormone receptor-positive cases. sc-MTOP enables using WSIs to characterize tumor ecosystems at the single-cell level.

Abstract The molecular heterogeneity and distinct features of HER2-low breast cancers, particularly in the Chinese population, are not well understood, limiting its precise management in the era of antibody‒drug conjugates. To address this issue, we established a cohort of 434 Chinese patients with HER2-low breast cancer (433 female and one male) and integrated genomic, transcriptomic, proteomic, and metabolomic profiling data. In this cohort, HER2-low tumors are more distinguished from HER2-0 tumors in the hormone receptor–negative subgroup. Within HER2-low tumors, significant interpatient heterogeneity also exists in the hormone receptor–negative subgroup: basal-like tumors resemble HER2-0 disease, and non-basal-like HER2-low tumors mimic HER2-positive disease. These non-basal-like HER2-low tumors are enriched in the HER2-enriched subtype and the luminal androgen receptor subtype and feature PIK3CA mutation, FGFR4/PTK6/ERBB4 overexpression and lipid metabolism activation. Among hormone receptor–positive tumors, HER2-low tumors show less loss/deletion in 17q peaks than HER2-0 tumors. In this work, we reveal the heterogeneity of HER2-low breast cancers and emphasize the need for more precise stratification regarding hormone receptor status and molecular subtype.

In this paper, the cause and mechanism of local corrosion perforation under the bottom water regulator of an offshore water injection pipe string were analyzed by means of macro analysis, physical and chemical properties analysis, metallographic analysis, XRD analysis, SEM micro morphology analysis, energy spectrum analysis, water content and bacteria test, and finite element analysis and other methods, combined with the actual site conditions. Results showed that the internal corrosion perforation of the oil pipe was caused by bacterial corrosion under the scale, and the high Cl- in the residual acid after acidizing operation accelerated the local corrosion perforation. Based on this, it was suggested to regularly add chemicals and use corrosion-resistant pipes to prevent early corrosion perforation failure of oil pipes.

Abstract Background Immune checkpoint inhibitors had a great effect in triple-negative breast cancer (TNBC); however, they benefited only a subset of patients, underscoring the need to co-target alternative pathways and select optimal patients. Herein, we investigated patient subpopulations more likely to benefit from immunotherapy and inform more effective combination regimens for TNBC patients. Methods We conducted exploratory analyses in the FUSCC cohort to characterize a novel patient selection method and actionable targets for TNBC immunotherapy. We investigated this in vivo and launched a phase 2 trial to assess the clinical value of such criteria and combination regimen. Furthermore, we collected clinicopathological and next-generation sequencing data to illustrate biomarkers for patient outcomes. Results CD8-positivity could identify an immunomodulatory subpopulation of TNBCs with higher possibilities to benefit from immunotherapy, and angiogenesis was an actionable target to facilitate checkpoint blockade. We conducted the phase II FUTURE-C-Plus trial to assess the feasibility of combining famitinib (an angiogenesis inhibitor), camrelizumab (a PD-1 monoclonal antibody) and chemotherapy in advanced immunomodulatory TNBC patients. Within 48 enrolled patients, the objective response rate was 81.3% (95% CI, 70.2–92.3), and the median progression-free survival was 13.6 months (95% CI, 8.4–18.8). No treatment-related deaths were reported. Patients with CD8- and/or PD-L1- positive tumors benefit more from this regimen. PKD1 somatic mutation indicates worse progression-free and overall survival. Conclusion This study confirms the efficacy and safety of the triplet regimen in immunomodulatory TNBC and reveals the potential of combining CD8, PD-L1 and somatic mutations to guide clinical decision-making and treatments. Trial registration ClinicalTrials.gov: NCT04129996 . Registered 11 October 2019.

Abstract African classical swine fever virus (ASFV) has spread seriously around the world and has dealt with a heavy blow to the pig breeding industry due to the lack of vaccines. In this study, we produced recombinant Lactobacillus plantarum (L. plantarum) expressing an ASFV p54 and porcine IL-21 (pIL-21) fusion protein and evaluated the immune effect of NC8-pSIP409-pgsA'-p54-pIL-21 in a mouse model. First, we verified that the ASFV p54 protein and p54-pIL-21 fusion protein were anchored on the surface of L. plantarum NC8 by flow cytometry, immunofluorescence and Western blotting. Then, the results were verified by flow cytometry, ELISA and MTT assays. Mouse-specific humoral immunity and mucosal and T cell-mediated immune responses were induced by recombinant L. plantarum. The results of feeding mice recombinant L. plantarum showed that the levels of serum IgG and mucosal secreted IgA (SIgA), the number of CD4 and CD8 T cells, and the expression of IFN-γ in CD4 and CD8 T cells increased significantly, and lymphocyte proliferation occurred under stimulation with the ASFV p54 protein. Our data lay a foundation for the development of oral vaccines against ASFV in the future.

Influenza is a serious respiratory disease that continues to threaten global health. Mucosa-associated invariant T (MAIT) cells use T-cell receptors (TCRs) that recognize microbial riboflavin derived intermediates presented by the major histocompatibility complex (MHC) class I-like protein MR1. Riboflavin synthesis is broadly conserved, but the roles or mechanisms of riboflavin in MR1–/– mouse influenza infection are not well understood. In our study, immunofluorescence techniques were applied to analyze the number and distribution of viruses in lung tissue. The amount of cytokine expression was assessed by flow cytometry (FCM), ELISA, and qPCR. The changes in the fecal flora of mice were evaluated based on amplicon sequencing of the 16S V3-V4 region. Our study showed that MAIT cell deficiency increased mortality and that riboflavin altered these effects in microbiota-depleted mice. The oral administration of riboflavin inhibited IL-1β, IL-17A, and IL-18 production but significantly increased the expression of IFN-γ, TNF-α, CCL2, CCL3, and CCL4 in a mouse model. The analysis of the mouse flora revealed that riboflavin treatment significantly increased the relative abundance of Akkermansia and Lactobacillus (p < 0.05) and decreased that of Bacteroides. In contrast, MR1–/– mice exhibited a concentrated aggregation of Bacteroides (p < 0.01), which indicated that MAIT cell deficiency reduced the diversity of the bacterial population. Our results define the functions of MAIT cells and riboflavin in resistance to influenza virus and suggest a potential role for riboflavin in enhancing MAIT cell immunity and the intestinal flora diversity. Gut populations can be expanded to enhance host resistance to influenza, and the results indicate novel interactions among viruses, MAIT cells, and the gut microbiota.

There are some limitations of traditional influenza vaccines concerning novel mutant strains. Therefore, it is particularly important to develop preventive means for antigen-unrelated types of influenza viruses. Recent studies have shown that probiotics can modulate the immune system and reduce the severity of viral infections. In this study, we investigated the potential of Lactiplantibacillus plantarum 0111 against influenza virus H9N2. Challenge experiments showed that L. plantarum 0111 pretreatments could effectively improve mice’s survival rate and weight loss and reduce the inflammatory cytokines IL-6 and TNF-α in the lungs and bronchoalveolar lavage fluid (BALF) along with the degree of lung and intestinal injury. FMT experiment demonstrates that the protective effect produced by L. plantarum 0111 is associated with gut microorganisms. In addition, 16S high-throughput sequencing of the mouse intestinal microbiota showed that L. plantarum 0111 remodeled the intestinal microbiota after H9N2 infection and maintained the gut microbiota balance. In a mouse model, the oral administration of L. plantarum 0111 increased IFN-β expression in the serum and BALF. At the same time, the transcript levels of IFN-β and related ISGs in the intestine and lungs of mice were also increased. In addition, the activation and polarization of T cells in mesenteric lymph nodes (MLNs) and the spleen were detected by flow cytometry, and the results showed that L. plantarum 0111 modulated cytokines in T cells and increased IgA expression in B cells in the MLNs and spleen. Thus, L. plantarum 0111 may improve gut microbiota-mediated immune responses and thus, resist infection by the influenza virus, and it could be used as an effective preventive measure against the influenza virus.

Abstract Background Loaches of Cobitinae, widely distributed in Eurasian continent, have high economic, ornamental and scientific value. However, the phylogeny of Cobitinae fishes within genera or family level remains complex and controversial. Up to now, about 60 Cobitinae mitogenomes had been deposited in GenBank, but their integrated characteristics were not elaborated. Results In this study, we sequenced and analyzed the complete mitogenomes of a female Cobits macrostigma. Then we conducted a comparative mitogenome analysis and revealed the conserved and unique characteristics of 58 Cobitinae mitogenomes, including C. macrostigma. Cobitinae mitogenomes display highly conserved tRNA secondary structure, overlaps and non-coding intergenic spacers. In addition, distinct base compositions were observed among different genus and significantly negative linear correlation between AT% and AT-skew were found among Cobitinae, genus Cobitis and Pangio mitogenomes, respectively. A specific 3 bp insertion (GCA) in the atp8-atp6 overlap was identified as a unique feature of loaches, compared to other Cypriniformes fish. Additionally, all protein coding genes underwent a strong purifying selection. Phylogenetic analysis strongly supported the paraphyly of Cobitis and polyphyly of Misgurnus. The strict molecular clock predicted that Cobitinae might have split into northern and southern lineages in the late Eocene (42.11 Ma), furthermore, mtDNA introgression might occur (14.40 Ma) between ancestral species of Cobitis and ancestral species of Misgurnus. Conclusions The current study represents the first comparative mitogenomic and phylogenetic analyses within Cobitinae and provides new insights into the mitogenome features and evolution of fishes belonging to the cobitinae family.

Gut bacterial community plays a key role in maintaining host health. The Tibetan pig (Sus scrofa), an ancient breed in China, has been known for its high adaptability to harsh environments and for its meat quality. To understand the underlying mechanisms facilitating to shape these unique features, in this study, 16S rRNA sequencing using pigs feces and subsequent bacterial functional prediction were performed. Also, the gut bacteria of two other breeds of pigs, Barkshire and Landrace, were examined for comparison. It was revealed that the structure of bacterial community in Tibetan pigs appeared to be more complex; the relative abundances of dominant bacterial families varied inversely with those of the other pigs, and the proportion of Firmicutes in Tibetan pigs was lower, but Bacteroides, Fibrobacterota, Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae were higher. Bacterial functional prediction revealed that the dominant flora in the Tibetan pigs was more correlated with functions regulating the hosts’ immune and inflammatory responses, such as NOD-like_receptor_signaling_pathway and vitamin metabolism. In addition, in Tibetan pigs, the taxonomic relationships in the gut bacteria on day 350 were closer than those on earlier stages. Furthermore, gender played a role in the composition and function of bacterial inhabitants in the gut; for boars, they were more correlated to drug resistance and xenobiotics metabolism of the host compared to the sows. In sum, our preliminary study on the gut bacterial composition of the Tibetan pigs provided an insight into the underlying host–microorganism interactions, emphasizing the role of intestinal bacteria in the context of modulating the host’s immune system and host development.

Abstract Background The RNA-binding protein Musashi-2 (MSI2) has been implicated in the tumorigenesis and tumor progression of some human cancers. MSI2 has also been reported to suppress tumor epithelial-to-mesenchymal transition (EMT) progression in breast cancer, and low MSI2 expression is associated with poor outcomes for breast cancer patients; however, the underlying mechanisms have not been fully investigated. This study investigated the expression and phenotypic functions of two major alternatively spliced MSI2 isoforms (MSI2a and MSI2b) and the potential molecular mechanisms involved in triple-negative breast cancer (TNBC) progression. Methods The Illumina sequencing platform was used to analyze the mRNA transcriptomes of TNBC and normal tissues, while quantitative reverse transcription-polymerase chain reaction and immunohistochemistry validated MSI2 isoform expression in breast cancer tissues. The effects of MSI2a and MSI2b on TNBC cells were assayed in vitro and in vivo. RNA immunoprecipitation (RIP) and RNA sequencing were performed to identify the potential mRNA targets of MSI2a, and RIP and luciferase analyses were used to confirm the mRNA targets of MSI2. Results MSI2 expression in TNBC tissues was significantly downregulated compared to that in normal tissues. In TNBC, MSI2a expression was associated with poor overall survival of patients. MSI2a overexpression in vitro and in vivo inhibited TNBC cell invasion as well as extracellular signal-regulated kinase 1/2 (ERK1/2) activity. However, MSI2b overexpression had no significant effects on TNBC cell migration. Mechanistically, MSI2a expression promoted TP53INP1 mRNA stability by its interaction with the 3′-untranslated region of TP53INP1 mRNA. Furthermore, TP53INP1 knockdown reversed MSI2a-induced suppression of TNBC cell invasion, whereas ectopic expression of TP53INP1 and inhibition of ERK1/2 activity blocked MSI2 knockdown-induced TNBC cell invasion. Conclusions The current study demonstrated that MSI2a is the predominant functional isoform of MSI2 proteins in TNBC, that its downregulation is associated with TNBC progression and poor prognosis and that MSI2a expression inhibited TNBC invasion by stabilizing TP53INP1 mRNA and inhibiting ERK1/2 activity. Overall, our study provides new insights into the isoform-specific roles of MSI2a and MSI2b in the tumor progression of TNBC, allowing for novel therapeutic strategies to be developed for TNBC.

Abstract Background Eimeria spp. are responsible for chicken coccidiosis which is the most important enteric protozoan disease resulting in tremendous economic losses in the poultry industry. Understanding the interaction between the avian cecal microbiota and coccidia is of interest in the development of alternative treatments that do not rely on chemotherapeutics and do not lead to drug resistance. Methods We utilized 16S rRNA gene sequencing to detect the dynamics of the cecal microbial community in AA broilers challenged with Eimeria tenella. Histopathological analysis of the cecum was also conducted. Results We found that microbial shifts occur during the infection. Lactobacillus, Faecalibacterium, Ruminococcaceae UCG-013, Romboutsia and Shuttleworthia decreased in abundance. However, the opportunistic pathogens Enterococcus and Streptococcus increased in abundance over time in response to the infection. Conclusions Eimeria tenella disrupts the integrity of the cecal microbiota and could promote the establishment and growth of potentially pathogenic bacteria. Defining bacterial populations affected by coccidial infection might help identify bacterial markers for intestinal disease as well as populations or species that could be beneficial in maintaining and restoring gut homeostasis during and after infection with E. tenella.

Purpose: The proliferation marker Ki67 has prognostic and predictive values in breast cancer, and the cutoff of the Ki67 label index (LI) is a key index for chemotherapy. However, poor interobserver consistency in Ki67 assessment has limited the clinical use of Ki67, especially in luminal cancers. Here, we reported a modified Ki67 assessment method, size-set semiautomatic counting (SSSAC) and investigated its interobserver reproducibility. Methods: One hundred invasive breast cancer tissues were set immunostained for Ki67 in one laboratory, scanned as digital slides, and sent to 41 pathologists at the laboratories of 16 hospitals for Ki67 LI assessment using size-set semiautomatic counting (SSSAC), size-set visual assessment (SSVA) and size-set digital image analysis (SSDIA) with a specific image viewing software (Aperio Image Scope, Leica, Germany). The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate interobserver reproducibility. The Wilcoxon signed-rank test was used to analyze the difference in the Ki67 values assessed by SSSAC and SSDIA. Results: SSSAC demonstrated better interobserver reproducibility (ICC = 0.942) than SSVA (ICC = 0.802). The interobserver reproducibility was better in Ki67 homogeneously stained slides and centralized hot-spot slides than in scattered hot-spot slides. The Ki67 value assessed with SSSAC was obviously higher than that assessed with SSDIA (negative ranks (SSDIA SSSAC): N = 17, sum of ranks = 478.50; Z = −6.837; P

Avian influenza viruses can be efficiently transmitted through mucous membranes, and conventional vaccines are not effective in protecting against mucosal infection by influenza viruses. To induce multiple immune responses in an organism, we constructed a recombinant Lactobacillus plantarum expressing the influenza virus antigen HA1 with the adjuvant dendritic cell-targeting peptide (DCpep). The recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep were used to immunize mice via oral administration, and the humoral, cellular and mucosal immune responses were evaluated. In addition, the serum levels of specific antibodies and hemagglutination inhibition (HI) levels were also measured. Our results showed that recombinant L. plantarum activated dendritic cells in Peyer’s patches (PPs), increased the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes (MLNs), and affected the ability of CD4+ and CD8+ cells to proliferate in the spleen and MLNs. Additionally, recombinant L. plantarum increased the number of B220+IgA+ cells in PPs and the level of IgA in the lungs and different intestinal segments. In addition, specific IgG, IgG1 and IgG2a antibodies were induced at high levels in the mice serum, specific IgA antibodies were induced at high levels in the mice feces, and HI potency was significantly increased. Thus, the recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep have potential as vaccine candidates for avian influenza virus.

Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.

Acinetobacter baumannii is one of the main causes of nosocomial infections. Increasing numbers of multidrug-resistant Acinetobacter baumannii cases have been reported in recent years, but its antibiotic resistance mechanism remains unclear. We studied 9 multidrug-resistant (MDR) and 10 drug-susceptible Acinetobacter baumannii clinical isolates using Label free, TMT labeling approach and glycoproteomics analysis to identify proteins related to drug resistance. Our results showed that 164 proteins exhibited different expressions between MDR and drug-susceptible isolates. These differential proteins can be classified into six groups: a. proteins related to antibiotic resistance, b. membrane proteins, membrane transporters and proteins related to membrane formation, c. Stress response-related proteins, d. proteins related to gene expression and protein translation, e. metabolism-related proteins, f. proteins with unknown function or other functions containing biofilm formation and virulence. In addition, we verified seven proteins at the transcription level in eight clinical isolates by using quantitative RT-PCR. Results showed that four of the selected proteins have positive correlations with the protein level. This study provided an insight into the mechanism of antibiotic resistance of multidrug-resistant Acinetobacter baumannii.

Introduction. Acute pancreatitis (AP) is a sudden inflammatory process in the pancreas with variable involvement of nearby organs or other organ systems, and it is a common cause for hospitalization of gastrointestinal origin. Early prediction of the prognosis of patients with AP is important to help physicians triage the patients and decrease mortality. Red cell distribution width (RDW) and total serum calcium (TSC) have been reported to be useful predictors of the severity of AP, but if these parameters are associated with the prognosis of AP is unknown. The objective of the study was to evaluate whether RDW/TSC can be used to predict the prognosis of patients with AP at an early stage. Methods. We retrospectively enrolled AP patients admitted to the emergency department of West China Hospital of Sichuan University from January 1, 2016, to June 30, 2016. According to the prognosis, AP patients were divided into ICU group and non-ICU group, surgery group and nonsurgery group, and hospital survival group and hospital death group. Demographic information and clinical and laboratory parameters of all enrolled patients after being admitted to ED were compared between the groups. The receiver operator characteristic (ROC) curves were used to evaluate the prognostic values of RDW, TSC, and RDW/TSC in patients with AP. Results. A total of 666 AP patients were enrolled in this study, with an average age of 47.99±14.11 years, including 633 patients who survived to discharge and 33 patients who died during hospitalization. The areas under the curve (AUC) of RDW and RDW/TSC predict that patients need to be admitted to ICU (0.773 vs. 0.824 vs. 0.723), patients need surgery treatment (0.744 vs. 0.768 vs. 0.690), and patients survived to hospital discharge (0.809 vs. 0.855 vs. 0.780) were greater than that of TSC, with RDW/TSC being the greatest. Conclusions. RDW/TSC may be a new method to identify the AP patients who need to be transferred to the ICU, accompanying complications which need surgery treatment, or may be died in hospital at an early stage, and we should pay more attention to RDW/TSC in patients with AP, for they may have a worse prognosis.

Lactic acid bacteria (LAB) are the primary genera of the intestinal flora and have many probiotic functions. In the present study, Lactobacillus rhamnosus GG (LGG) ATCC 53103 was used to treat BALB/c mice. After LGG intervention, both low and high LGG doses were shown to improve the observed OTU, Chao1, ACE, and Shannon indices, while the Simpson index decreased, demonstrating that LGG can promote intestinal microbiota abundance and diversity. Furthermore, LGG treatment increased the abundances of intestinal Firmicutes, Bacteroides and Actinomycetes while reducing that of Proteobacteria. In addition to its effect on gut the microbiota, LGG could also regulate the host immune system. In the present study, we showed that LGG could affect the percentage of CD3+ T lymphocytes in the spleens (SPLs), mesenteric lymph nodes (MLNs), Peyer’s patches (PPs) and lamina propria lymphocytes (LPLs) of mice, including total CD3+ T, CD3+CD4+ T, and CD3+CD8+ T lymphocytes. Furthermore, LGG could effectively increase the expression of Th1-type cytokines (IFN-γ) and Th2 cytokines (IL-4) in CD4+ T cells, indicating that the proportion of Th1 and Th2 cells in mice with LGG treatment was in a high equilibrium state compared to the control group. In addition, the IFN-γ/IL-4 ratio was greater than 1 in mice with LGG intervention, suggesting that LGG tends to mediate the Th1 immune response. The results of the present study also showed that LGG upregulated the expression of IL-17 in CD4+ T cells and regulated the percentage of CD4+CD25+Foxp3+ Treg cells in various secondary immunological organs, indicating that LGG may promote the balance of Th-17 and Treg cells.

This study aimed to compare the performance of MilliSect dissection and manual dissection. Twenty-five formalin-fixed paraffin-embedded (FFPE) breast cancer tissue blocks were selected for comparison. Specific areas of interest (AOIs) in invasive carcinoma on tissue sections were transferred to dissection slides by manual macrodissection or the MilliSect instrument. The comparison criteria were 1) the time required for dissection; 2) RNA concentration and purity; 3) RNA quantity of 5 housekeeping genes (by RT-qPCR); and 4) ER, PR, HER2, Ki-67 and recurrence score (RS) values (by the 21-gene assay). Then, tumor-adjacent tissues, including fibrocollagenous and epithelial tissues, from the same selected tissue blocks of 8 of 25 patients were scraped using the mesodissection method, and their RS values were assessed to evaluate the influence of tumor-adjacent tissues on the target AOIs. Ultimately, 4 AOIs of invasive ductal carcinoma (IDC) from 1 tissue block of another 4 patients with lymph node (LN) metastases each, LN tissue and a mixture of IDC and LN tissue from the other tissue block of the same 4 patients were mesodissected to evaluate the influence of infiltrating lymphocyte levels on the RS values of AOIs. In our experience, the MilliSect instrument, which provides process management documentation, required more time than manual macrodissection (on average, approximately 9.1 min per sample versus 5.8 min per sample, respectively). The RNA yield and quality of the dissected tissues were comparable for the 2 methods. However, the tumor-adjacent tissues of the AOIs may influence the RS to some extent. Tumor-infiltrating lymphocytes (TILs) can dramatically increase RSs, far exceeding the influence of tumor-adjacent fibrocollagenous and epithelial tissues. In conclusion, MilliSect mesodissection is comparable to manual dissection. This mesodissection tool may facilitate AOI alignment and the dissection process for the 21-gene RS assay. Samples whose adjacent tissues are intermixed with TILs warrant special attention.

Abstract Background Nuclear forkhead box protein P1 (N-FOXP1) expression in invasive breast cancer has been documented in the literature. However, the FOXP1 expression patterns at different stages of breast cancer progression are largely unknown, and the significance of cytoplasmic FOXP1 (C-FOXP1) expression in breast cancer has not been well illustrated. The aims of this study were to investigate FOXP1 expression patterns in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), atypical ductal hyperplasia (ADH) and usual ductal hyperplasia (UDH), and to analyze the clinicopathological relevance of C-FOXP1 and its prognostic value in IDC. Methods N-FOXP1 and C-FOXP1 expression in cases of IDC, DCIS, ADH and UDH was determined using immunohistochemistry. The correlation between C-FOXP1 expression and clinicopathological parameters as well as the overall survival (OS) and disease-free survival (DFS) rates of patients with IDC were analyzed. Results Exclusive N-FOXP1 expression was found in 85.0% (17/20), 40.0% (8/20), 12.2% (5/41) and 10.8% (9/83) of UDH, ADH, DCIS, and IDC cases, respectively, and exclusive C-FOXP1 expression was observed in 0% (0/20), 0% (0/20), 4.9% (2/41), and 31.3% (26/83) of the cases, respectively. Both N- and C-FOXP1 staining were observed in 15.0% (3/20), 60.0% (12/20), 82.9% (34/41) and 48.2% (40/83) of the above cases, respectively, while complete loss of FOXP1 expression was observed in only 9.6% (8/83) of IDC cases. Estrogen receptor (ER) expression in C-FOXP1-positive IDC cases (31/66, 47.0%) was significantly lower than that in C-FOXP1-negative cases (13/17, 76.5%) (p = 0.030). Calpain II expression was observed in 83.3% (55/66) of C-FOXP1-positive IDC cases, which was significantly higher than that in C-FOXP1-negative cases (9/17, 52.9%) (p = 0.007). Calpain II was significantly associated with pAKT (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p

The PI3K pathway is altered across various cancer types. Here the authors use amplicon exon sequencing to analyze the landscape of somatic mutations affecting the PI3K pathway specifically in breast cancer patients in China.