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BackgroundPancreatic cancer remains an extremely malignant digestive tract tumor, posing a significant global public health burden. Patients with pancreatic cancer, once metastasis occurs, lose all hope of cure, and prognosis is extremely poor. It is important to investigate liver metastasis of Pancreatic cancer in depth, not just because it is the most common form of metastasis in pancreatic cancer, but also because it is crucial for treatment planning and prognosis assessment. This study aims to delve into the mechanisms of pancreatic cancer liver metastasis, with the goal of providing crucial scientific groundwork for the development of future treatment methods and drugs.MethodsWe explored the mechanisms of pancreatic cancer liver metastasis using single-cell sequencing data (GSE155698 and GSE154778) and bulk data (GSE71729, GSE19279, TCGA-PAAD). Initially, Seurat package was employed for single-cell data processing to obtain expression matrices for primary pancreatic cancer lesions and liver metastatic lesions. Subsequently, high-dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify genes associated with liver metastasis. Machine learning algorithms and COX regression models were employed to further screen genes related to patient prognosis. Informed by both biological understanding and the outcomes of algorithms, we meticulously identified the ultimate set of liver metastasis-related gene (LRG). In the study of LRG genes, various databases were utilized to validate their association with pancreatic cancer liver metastasis. In order to analyze the effects of these agents on tumor microenvironment, we conducted an in-depth analysis, including changes in signaling pathways (GSVA), cell differentiation (pseudo-temporal analysis), cell communication networks (cell communication analysis), and downstream transcription factors (transcription factor activity prediction). Additionally, drug sensitivity analysis and metabolic analysis were performed to reveal the effects of LRG on gemcitabine resistance and metabolic pathways. Finally, functional experiments were conducted by silencing the expression of LRG in PANC-1 and Bx-PC-3 cells to validate its influence to proliferation and invasiveness on PANC-1 and Bx-PC-3 cells.ResultsThrough a series of algorithmic filters, we identified PAK2 as a key gene promoting pancreatic cancer liver metastasis. GSVA analysis elucidated the activation of the TGF-beta signaling pathway by PAK2 to promote the occurrence of liver metastasis. Pseudo-temporal analysis revealed a significant correlation between PAK2 expression and the lower differentiation status of pancreatic cancer cells. Cell communication analysis revealed that overexpression of PAK2 promotes communication between cancer cells and the tumor microenvironment. Transcription factor activity prediction displayed the transcription factor network regulated by PAK2. Drug sensitivity analysis and metabolic analysis revealed the impact of PAK2 on gemcitabine resistance and metabolic pathways. CCK8 experiments showed that silencing PAK2 led to a decrease in the proliferative capacity of pancreatic cancer cells and scratch experiments demonstrated that low expression of PAK2 decreased invasion capability in pancreatic cancer cells. Flow cytometry reveals that PAK2 significantly inhibited apoptosis in pancreatic cancer cell lines. Molecules related to the TGF-beta pathway decreased with the inhibition of PAK2, and there were corresponding significant changes in molecules associated with EMT.ConclusionPAK2 facilitated the angiogenic potential of cancer cells and promotes the epithelial-mesenchymal transition process by activating the TGF-beta signaling pathway. Simultaneously, it decreased the differentiation level of cancer cells, consequently enhancing their malignancy. Additionally, PAK2 fostered communication between cancer cells and the tumor microenvironment, augments cancer cell chemoresistance, and modulates energy metabolism pathways. In summary, PAK2 emerged as a pivotal gene orchestrating pancreatic cancer liver metastasis. Intervening in the expression of PAK2 may offer a promising therapeutic strategy for preventing liver metastasis of pancreatic cancer and improving its prognosis.

Prior studies reported inconsistent results on the altered gut microbial composition in patients with Crohn’s disease (CD), likely under the influences of many confounding factors including genetic, life style and environmental variations among different study cohorts. This study aims to examine the gut microbiota of CD patients with particular efforts to minimize the impact of the confounding factors. For this purpose, the healthy relatives of the patients were enrolled as control subjects so that the paired study subjects may have similar genetic background, dietary habits, and household environment. The fecal microbiota of the study subjects were examined by 16S rRNA sequencing. After the identification of the differential bacterial genera, multivariate regression analysis was performed to adjust the results for the impact of confounding factors. We found that the microbiota of the CD patients were featured with reduced short chain fatty acid (SCFA) producing bacteria and elevated opportunistic pathogen Escherichia-Shigella. Correlation analysis indicated that the elevation in Escherichia-Shigella and the reduction in SCFA-producing bacteria usually occur simultaneously. These differential genera exhibited a high capacity in distinguishing between CD and healthy controls achieving an area under curve of 0.89, and were correlated with the changes in inflammation related blood biochemical markers. Consistent with the reduction in SCFA-producing bacteria in CD, metabolomics analysis revealed decreased blood level of SCFAs in the patients. The differential genera identified in this study demonstrated outstanding capability to serve as diagnosis markers for CD and are potential targets for intervention.

NLRP3 inflammasome is implicated in the pathogenesis of inflammatory bowel diseases (IBD). Since guanylate-binding protein 5 (GBP5) induces the NLRP3 inflammasome activity, we aim to investigate the potential role of GBP5 in IBD pathogenesis. The expression of GBP5, NLRP3 inflammasome, and related cytokines and chemokines was examined in two cohorts of IBD patients and healthy controls, by microarray transcriptome analysis and quantitative real-time PCR. Cellular localization of GBP5 in colonic biopsies was examined by immunohistochemistry and immunofluorescence with confocal microscopy. For functional studies, GBP5 was induced by interferon γ or silenced by siRNA or CRISPR/CAS9 technique, and inflammatory activities were evaluated at mRNA and protein levels. We found that the expression of GBP5 was elevated in colonic mucosa in two geographically and culturally distinct IBD cohorts. In colonic tissues of IBD patients, GBP5 expression was mainly confined to immune cells and the levels of GBP5 expression were correlated with those of the inflammatory cytokines and chemokines. In cultured T and macrophage cells, the expression of proinflammatory cytokines and chemokines was increased when GBP5 was induced, while GBP5 deficiency leads to decreased expression of proinflammatory mediators including gasdermin D, caspase 1, cytokines, and chemokines. We conclude that GBP5 is required in the expression of many proinflammatory cytokines and chemokines in intestinal immune cells. In addition, GBP5 may upregulate inflammatory reactions through an inflammasome-mediated mechanism. Since GBP5 plays a proinflammatory role at the early steps of the inflammatory cascades of IBD pathogenesis, and is implicated in IBD patients of distinct genetic and environmental backgrounds, targeting GBP5 could be an effective strategy for the management of IBD.

BackgroundsAs a crucial enzyme in thyroid hormone synthesis, the genetic defective thyroid peroxidase (TPO) was one of the main genetic factors leading to congenital hypothyroidism (CH).MethodsMutations in the TPO gene were screened and identified in 219 patients with CH from northwest China by using high-throughput sequencing and bioinformatics analysis. The biological function of detected variants was studied by in vitro experiments and homology modeling.ResultsNineteen rare variants, including seven novel ones, were detected in 17 of 219 patients (7.8%). Most cases were detected with one single heterozygous variant, and only two patients were detected with multiple variants, i.e., compounds for (1) IVS7-1G>A, p.Ala443Val, and p.Arg769Trp and (2) p.Asn592Ser and p.Asn798Lys. The biological function of the four missense mutations (i.e., p.Ala443Val, p.Arg769Trp, p.Asn592Ser, and p.Asn798Lys) they carried were further studied. Experimental data showed that these four mutations did not affect the protein expression level of the TPO gene but remarkably reduced the peroxidase activity toward guaiacol oxidation, retaining 8–32% of activity of the wild-type protein. The comparison of the predicted 3-D structures of wild-type and mutant TPO proteins showed that these four amino acid substitutions changed the non-covalent interactions of studied residues that might alter the structure and function of the TPO protein.ConclusionThis study was the first to analyze the TPO mutation spectrum of patients with CH in northwest China. Our data indicated that the TPO mutation was not a common reason to cause CH in China. The functional data may help to clarify the structure-function relationship of the TPO protein and provide further evidence for the elucidation of the genetic etiology of CH.

Life-history theory predicts that increased resource allocation in current reproduction comes at the cost of survival and future reproductive fitness. In taxa with biparental care, each parent can adjust investment on current reproduction according to changes in their partner’s effort, but these adjustments may be different for males and females as they may have different reproductive strategies. Numerous theoretical and empirical studies have proposed the mechanism underlying such adjustments. In addition, the value of the brood or litter (brood size) has also been suggested to affect the amount of care through manipulation of brood size. While the two conditions have been studied independently, the impact of their interplay on potential sex-dependent future reproductive performance remains largely unknown. In this study, we simultaneously manipulated both care system (removal of either parent vs. no removal) and brood size in a burying beetle (Nicrophorus vespilloides) to understand their joint effect on reproductive allocation and trade-off between current and future reproduction. Our results show that males compensated for mate loss by significantly increasing the level of care regardless of brood size, while females exhibited such compensation only for small brood size. Additionally, with an increase in allocation to current reproduction, males showed decreased parental investment during the subsequent breeding event as a pair. These findings imply a dual influence of parental care system and brood size on allocation in current reproduction. Moreover, the impact of such adjustments on sex-dependent differences in future reproduction (parental care, larvae number, and average larval mass at dispersal) is also demonstrated. Our findings enhance the understanding of sex roles in parental investment and highlight their importance as drivers of reproductive allocation.

Sphingosine-1-phosphate (S1P) is a phospholipid that regulates pleiotropic biological activities and exerts extracellular functions by binding to five specific G-protein-coupled receptors, S1P receptors (S1PR) 1–5. When activated by S1P, S1PR promote the proliferation and invasion of tumor cells by inducing the formation of new blood vessels. We developed and assessed a new monoclonal antibody imaging probe 99mTc-HYNIC-S1PR1mAb, to explore the feasibility of targeting the S1PR1 in vitro and in vivo. S1PR1mAb was prepared and followed by technetium-99m labeling with succinimidyl 6-hydraziniumnicotinate hydrochloride. Cell uptake and blocking studies were performed to investigate the binding specificity of 99mTc-HYNIC-S1PR1mAb in vitro. 99mTc-HYNIC-S1P1mAb was also tested in vivo in mice xenografted with SK-HEP-1 (high-expression of S1PR1) and MCF-7 (low-expression of S1PR1) using single-photon emission-computed tomography (SPECT). Ex vivo gamma counting of tissues from tumor-bearing mice was used to evaluate 99mTc-HYNIC-S1PR1mAb biodistribution. The biodistribution study results showed significantly higher uptake in SK-HEP-1 tumors than in MCF-7 tumors (P < 0.001). Reduced uptake of 99mTc-HYNIC-S1PR1mAb in SK-HEP-1 was observed in tumor-bearing nude mice pretreated with fingolimod, which binds competitively to the receptors, especially S1PR1. 99mTc-HYNIC-S1PR1mAb can be synthesized and specifically targeted to S1PR1 in vitro and in vivo, allowing S1PR1 expression assessment with SPECT imaging.

There are many and diverse intestinal microbiota, and they are closely related to various physiological functions of the body. They directly participate in the host's food digestion, nutrient absorption, energy metabolism, immune response, and many other physiological activities and are also related to the occurrence of many diseases. The intestinal microbiota are extremely important for maintaining normal physical health. In order to explore the composition and differences of the intestinal microbiota of whooper swans in different wintering areas, we collected fecal samples of whooper swans in Sanmenxia, Henan, and Rongcheng, Shandong, and we used the Illumina HiSeq platform to perform high-throughput sequencing of bacterial 16S rRNA genes. Comparison between Sanmenxia and Rongcheng showed no significant differences in ACE, Chao 1, Simpson, and Shannon indices (p > 0.05). Beta diversity results showed significant differences in bacterial communities between two groups [analysis of similarity (ANOSIM): R = 0.80, p = 0.011]. Linear discriminant analysis effect size (LEfSe) analysis showed that at the phylum level, the relative abundance of Actinobacteria was significantly higher in Sanmenxia whooper swans than Rongcheng whooper swans. At the genus level, the amount of Psychrobacter and Carnobacterium in Sanmenxia was significantly higher in Rongcheng, while the relative abundance Catellicoccus and Lactobacillus was significantly higher in Rongcheng than in Sanmenxia. This study analyzed the composition, characteristics, and differences of the intestinal microbiota of the whooper swans in different wintering environments and provided theoretical support for further exploring the relationship between the intestinal microbiota of the whooper swans and the external environment. And it played an important role in the overwintering physiology and ecology, population management, and epidemic prevention and control of whooper swans.

Systemic sclerosis (SSc) is a poorly understood heterogeneous condition with progressive multi-organ fibrosis. Recent genetic and genomic evidence suggest a pathogenic role for dysregulated innate immunity and toll-like receptor (TLR) activity in SSc. Levels of both TLR4, as well as certain endogenous TLR ligands, are elevated in skin and lung tissues from patients with SSc and correlate with clinical disease parameters. Conversely, genetic targeting of TLR4 or its endogenous “damage-associated” ligands ameliorates progressive tissue fibrosis. Targeting TLR4 signaling therefore represents a pharmacological strategy to prevent intractable fibrosis. We examined the effect of TAK242, a small molecule TLR4 inhibitor, in preclinical fibrosis models and in SSc fibroblasts. TAK242 treatment prevented, promoted regression of, bleomycin-induced dermal and pulmonary fibrosis, and reduced the expression of several pro-fibrotic mediators. Furthermore, TAK242 ameliorated peritoneal fibrosis and reduced spontaneous hypodermal thickness in TSK/+ mice. Importantly, TAK242 abrogated collagen synthesis and myofibroblasts differentiation in explanted constitutively active SSc fibroblast. Altogether, these findings identify TAK242 as an anti-fibrotic agent in preclinical models of organ fibrosis. TAK242 might potentially represent a novel strategy for the treatment of SSc and other fibrotic diseases.