Fusobacterium nucleatum-induced imbalance in microbiome-derived butyric acid levels promotes the occurrence and development of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) ranks among the most prevalent malignant tumors globally. Recent reports suggest that Fusobacterium nucleatum (F. nucleatum) contributes to the initiation, progression, and prognosis of CRC. Butyrate, a short-chain fatty acid derived from the bacterial fermentation of soluble dietary fiber, is known to inhibit various cancers. This study is designed to explore whether F. nucleatum influences the onset and progression of CRC by impacting the intestinal metabolite butyric acid. AIM: To investigate the mechanism by which F. nucleatum affects CRC occurrence and development. METHODS: Alterations in the gut microbiota of BALB/c mice were observed following the oral administration of F. nucleatum. Additionally, DLD-1 and HCT116 cell lines were exposed to sodium butyrate (NaB) and F. nucleatum in vitro to examine the effects on proliferative proteins and mitochondrial function. RESULTS: Our research indicates that the prevalence of F. nucleatum in fecal samples from CRC patients is significantly greater than in healthy counterparts, while the prevalence of butyrate-producing bacteria is notably lower. In mice colonized with F. nucleatum, the population of butyrate-producing bacteria decreased, resulting in altered levels of butyric acid, a key intestinal metabolite of butyrate. Exposure to NaB can impair mitochondrial morphology and diminish mitochondrial membrane potential in DLD-1 and HCT116 CRC cells. Consequently, this leads to modulated production of adenosine triphosphate and reactive oxygen species, thereby inhibiting cancer cell proliferation. Additionally, NaB triggers the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, blocks the cell cycle in HCT116 and DLD-1 cells, and curtails the proliferation of CRC cells. The combined presence of F. nucleatum and NaB attenuated the effects of the latter. By employing small interfering RNA to suppress AMPK, it was demonstrated that AMPK is essential for NaB's inhibition of CRC cell proliferation. CONCLUSION: F. nucleatum can promote cancer progression through its inhibitory effect on butyric acid, via the AMPK signaling pathway.

Phosphatidylinositol-specific phospholipase C can decrease Müller cell viability and suppress its phagocytic activity by modulating PI3K/AKT signaling pathway.

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.

MAIT cells in bacterial infectious diseases: heroes, villains, or both?

Due to the aggravation of bacterial drug resistance and the lag in the development of new antibiotics, it is crucial to develop novel therapeutic regimens for bacterial infectious diseases. Currently, immunotherapy is a promising regimen for the treatment of infectious diseases. Mucosal-associated invariant T (MAIT) cells, a subpopulation of innate-like T cells, are abundant in humans and can mount a rapid immune response to pathogens, thus becoming a potential target of immunotherapy for infectious diseases. At the site of infection, activated MAIT cells perform complex biological functions by secreting a variety of cytokines and cytotoxic substances. Many studies have shown that MAIT cells have immunoprotective effects because they can bridge innate and adaptive immune responses, leading to bacterial clearance, tissue repair, and homeostasis maintenance. MAIT cells also participate in cytokine storm generation, tissue fibrosis, and cancer progression, indicating that they play a role in immunopathology. In this article, we review recent studies of MAIT cells, discuss their dual roles in bacterial infectious diseases and provide some promising MAIT cell-targeting strategies for the treatment of bacterial infectious diseases.

Inhibition of the Occurrence and Development of Inflammation-Related Colorectal Cancer by Fucoidan Extracted from Sargassum fusiforme.

Fucoidan has many biological activities, including the inhibitory effect on the development of various cancer types. This study showed that lipopolysaccharide-induced inflammation in FHC cells (normal human colonic epithelial cells) could be reversed using fucoidan at different concentrations. The fucoidan-induced anti-inflammatory effect was also confirmed through in vivo experiments in mice. Compared to the mice of the model group, the ratio of Firmicutes/Bacteroidetes in feces increased and the diversity of gut microbial composition was restored in mice after fucoidan intervention. In colorectal cancer (CRC) cells DLD-1 and SW480, fucoidan inhibited cell proliferation and promoted cell apoptosis. It also blocked the cell cycle of DLD-1 and SW480 at the G0/G1 phase. The animal model of inflammation-related CRC showed that the incidence of tumors in mice was significantly reduced by fucoidan intervention. Furthermore, the administration of fucoidan decreased the expression levels of inflammatory factors such as TNF-α IL-6 and IL-1ß in the colonic tissues. Therefore, fucoidan can effectively prevent the development of colitis-associated CRC.

Fusobacterium nucleatum Affects Cell Apoptosis by Regulating Intestinal Flora and Metabolites to Promote the Development of Colorectal Cancer.

Background/Aims: Intestinal flora, especially Fusobacterium nucleatum (Fn), can affect the development of colorectal cancer (CRC). In this study, we examined the composition of intestinal flora and their metabolites in the tissues, serum and feces of CRC patients. Materials and Methods: CRC tissues, adjacent normal colonic tissues, fecal and serum samples were collected from CRC patients who received surgical treatment between January 2018 and January 2020. Fecal and serum samples were collected from healthy individuals for comparison. In addition, fecal samples were collected from BALB/c female mice. SW480, a human CRC cell line, was utilized for in vitro studies. The experiments involved 1H-NMR-based metabolomics analysis, targeted and untargeted mass spectrometry analysis, and intestinal flora 16S rDNA V4 region sequencing. Results: The abundance of Bacteroides and propionic acid concentration were decreased and that of Lactobacillus and lactic acid concentration were increased in CRC tissues. In addition, the abundances of Ruminococcus, Prevotella, and Sutterell were decreased in CRC patients. The levels of leucine and isoleucine were decreased in the serum and tumor tissues of CRC patients. Aspartate, glutamate and glutathione levels were elevated in the tissues of CRC patients only. The serum glutamine, tyrosine, valine, alanine, and histidine levels were decreased significantly. Lactic acid inhibited and propionic acid promoted apoptosis among SW480 CRC cells. Conclusion: Fn affected the apoptosis of CRC cells and promoted the progression of CRC by affecting the distribution of intestinal flora, which altered the concentrations of metabolites such as lactic acid, propionic acid. Intestinal flora could regulate amino acid metabolism.

A Comparative Genomic Approach to Determine the Virulence Factors and Horizontal Gene Transfer Events of Clinical Acanthamoeba Isolates.

Acanthamoeba species are among the most ubiquitous protists that are widespread in soil and water and act as both a replicative niche and vectors for dispersal. They are the most important human intracellular pathogens, causing Acanthamoeba keratitis (AK) and severely damaging the human cornea. The sympatric lifestyle within the host and amoeba-resisting microorganisms (ARMs) promotes horizontal gene transfer (HGT). However, the genomic diversity of only A. castellanii and A. polyphaga has been widely studied, and the pathogenic mechanisms remain unknown. Thus, we examined 7 clinically pathogenic strains by comparative genomic, phylogenetic, and rhizome gene mosaicism analyses to explore amoeba-symbiont interactions that possibly contribute to pathogenesis. Genetic characterization and phylogenetic analysis showed differences in functional characteristics between the "open" state of T3 and T4 isolates, which may contribute to the differences in virulence and pathogenicity. Through comparative genomic analysis, we identified potential genes related to virulence, such as metalloprotease, laminin-binding protein, and HSP, that were specific to the genus Acanthamoeba. Then, analysis of putative sequence trafficking between Acanthamoeba and Pandoraviruses or Acanthamoeba castellanii medusaviruses provided the best hits with viral genes; among bacteria, Pseudomonas had the most significant numbers. The most parsimonious evolutionary scenarios were between Acanthamoeba and endosymbionts; nevertheless, in most cases, the scenarios are more complex. In addition, the differences in exchanged genes were limited to the same family. In brief, this study provided extensive data to suggest the existence of HGT between Acanthamoeba and ARMs, explaining the occurrence of diseases and challenging Darwin's concept of eukaryotic evolution. IMPORTANCEAcanthamoeba has the ability to cause serious blinding keratitis. Although the prevalence of this phenomenon has increased in recent years, our knowledge of the underlying opportunistic pathogenic mechanism maybe remains incomplete. In this study, we highlighted the importance of Pseudomonas in the pathogenesis pathway using comprehensive a whole genomics approach of clinical isolates. The horizontal gene transfer events help to explain how endosymbionts contribute Acanthamoeba to act as an opportunistic pathogen. Our study opens up several potential avenues for future research on the differences in pathogenicity and interactions among clinical strains.

Albuca Bracteata Polysaccharides Attenuate AOM/DSS Induced Colon Tumorigenesis via Regulating Oxidative Stress, Inflammation and Gut Microbiota in Mice.

Inflammation is an important risk factor in the development of inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Accumulating evidence indicates that some phytochemicals have anti-cancer properties. Polysaccharides extracted from Albuca bracteata (AB) have been reported to possess anti-neoplastic activities on colorectal cancer (CRC) models. However, it is still unclear whether they exert therapeutic effects on colorectal cancer. In this study, we investigate the properties of polysaccharides of A. bracteate, named ABP. The average molecular weight of ABP was 18.3 kDa and ABP consisted of glucose, mannose, galactose, xylose, galacturonic acid, glucuronic acid at a molar ratio of 37.8:8:2.5:1.7:1:1. An Azoxymethane/Dextran sodium sulfate (AOM/DSS) induced CAC mouse model was established. The CAC mice treated with ABP showed smaller tumor size and lower tumor incidence than untreated ones. ABP increased anti-inflammatory cytokine IL-10, inhibited secretion of pro-inflammatory cytokines (IL-6, IFN-γ, and TNF-α), mitigated oxidative stress by increasing GSH and decreasing MDA levels, suppressed the activation of STAT3 and expressions of its related genes c-Myc and cyclin D1. Moreover, ABP treatment increased the relative abundance of beneficial bacteria (f_Ruminococcaceae, g_Roseburia, g_Odoribacter, g_Oscillospira, and g_Akkermansia) and the levels of fecal short-chain fatty acid (SCFA) in CAC model mice. In summary, our data suggest that ABP could be a potential therapeutic agent for treating CAC.

PDGFRB is a potential prognostic biomarker and correlated with immune infiltrates in gastric cancer.

Gastric cancer (GC) is a common cancer with high mortality and morbidity rates worldwide. Although medical and surgical treatments have improved, the mechanisms of the progression of GC remain unclear. Platelet-derived growth factor receptor-ß (PDGFRB) plays a pivotal role in angiogenesis and tumor cell proliferation and has been suggested as a prognostic marker of cancer. This study aimed to explore the relationship of PDGFRB expression with clinicopathologic characteristics, immune cell infiltration status, and prognosis in GC. In this study, we visualized the expression and prognostic values of PDGFRB in GC using the Oncomine, UALCAN, GEPIA, and Kaplan-Meier Plotter databases. And then we explored the potential relationships between PDGFRB expression and the levels of immune cell infiltration using the TIMER, GEPIA databases and CIBERSORT algorithm. Furthermore, LinkedOmics analysis was performed to explore the functions for PDGFRB. The results showed close correlations between PDGFRB and immune cell infiltration especially M2 Macrophage infiltration in GC. High PDGFRB expression was related to poor outcomes in GC. High PDGFRB expression can negatively affect GC prognosis by promoting angiogenesis and modulating the tumor immune microenvironment. These results strongly suggest that PDGFRB can be used as a prognostic biomarker of GC and provide novel insights into possible immunotherapeutic targets.

The distal arthrogryposis-linked p.R63C variant promotes the stability and nuclear accumulation of TNNT3.

BACKGROUND: Distal arthrogryposis (DA) is comprised of a group of rare developmental disorders in muscle, characterized by multiple congenital contractures of the distal limbs. Fast skeletal muscle troponin-T (TNNT3) protein is abundantly expressed in skeletal muscle and plays an important role in DA. Missense variants in TNNT3 are associated with DA, but few studies have fully clarified its pathogenic role. METHODS: Sanger sequencing was performed in three generation of a Chinese family with DA. To determine how the p.R63C variant contributed to DA, we identified a variant in TNNT3 (NM_006757.4): c.187C>T (p.R63C). And then we investigated the effects of the arginine to cysteine substitution on the distribution pattern and the half-life of TNNT3 protein. RESULTS: The protein levels of TNNT3 in affected family members were 0.8-fold higher than that without the disorder. TNNT3 protein could be degraded by the ubiquitin-proteasome complex, and the p.R63C variant did not change TNNT3 nuclear localization, but significantly prolonged its half-life from 2.5 to 7 h, to promote its accumulation in the nucleus. CONCLUSION: The p.R63C variant increased the stability of TNNT3 and promoted nuclear accumulation, which suggested its role in DA.

Urinary exosomal long noncoding RNAs serve as biomarkers for early detection of non-small cell lung cancer.

OBJECTIVE: Increasing the efficiency of early diagnosis using noninvasive biomarkers is crucial for enhancing the survival rate of lung cancer patients. We explore the differential expression of non-small cell lung cancer (NSCLC)-related long noncoding RNAs (lncRNAs) in urinary exosomes in NSCLC patients and normal controls to diagnose lung cancer. METHODS: A differential expression analysis between NSCLC patients and healthy controls was performed using microarrays. Gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to predict potential functions of lncRNAs in NSCLC. quantitative real-time PCR (QT-PCR) was used to verify microarray results. RESULTS: A total of 640 lncRNAs (70 up- and 570 down-regulated) were differentially expressed in NSCLC patients in comparison to healthy controls. Six lncRNAs were detected by QT-PCR. GO term and KEGG pathway analyses showed that differential lncRNAs were enriched in cellular component organization or biogenesis, as well as other biological processes and signaling pathways, such as the PI3K-AKT, FOXO, p53, and fatty acid biosynthesis. CONCLUSIONS: The differential lncRNAs in urinary exosomes are potential diagnostic biomarkers of NSCLC. The lncRNAs enriched in specific pathways may be associated with tumor cell proliferation, tumor cell apoptosis, and the cell cycle involved in the pathogenesis of NSCLC.

Peptide Presentations of Marsupial MHC Class I Visualize Immune Features of Lower Mammals Paralleled with Bats.

Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus-host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum-specific insertions, the 3-aa Ile52Glu53Arg54 and 1-aa Arg154 insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen-host interactions. Residue Arg154 insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.

Albuca Bracteate Polysaccharides Synergistically Enhance the Anti-Tumor Efficacy of 5-Fluorouracil Against Colorectal Cancer by Modulating ß-Catenin Signaling and Intestinal Flora.

The first-line treatment for colorectal cancer (CRC) is 5-fluorouracil (5-FU). However, the efficacy of this treatment is sometimes limited owing to chemoresistance as well as treatment-associated intestinal mucositis and other adverse events. Growing evidence suggests that certain phytochemicals have therapeutic and cancer-preventing properties. Further, the synergistic interactions between many such plant-derived products and chemotherapeutic drugs have been linked to improved therapeutic efficacy. Polysaccharides extracted from Albuca bracteata (Thunb.) J.C.Manning and Goldblatt (ABP) have been reported to exhibit anti-oxidant, anti-inflammatory, and anti-tumor properties. In this study, murine CRC cells (CT26) and a murine model of CRC were used to examine the anti-tumor properties of ABP and explore the mechanism underlying the synergistic interactions between ABP and 5-FU. Our results revealed that ABP could inhibit tumor cell proliferation, invasion, and migratory activity in vitro and inhibited tumor progression in vivo by suppressing ß-catenin signaling. Additionally, treatment with a combination of ABP and 5-FU resulted in better outcomes than treatment with either agent alone. Moreover, this combination therapy resulted in the specific enrichment of Ruminococcus, Anaerostipes, and Oscillospira in the intestinal microbiota and increased fecal short-chain fatty acid (SCFA) levels (acetic acid, propionic acid, and butyric acid). The improvement in the intestinal microbiota and the increase in beneficial SCFAs contributed to enhanced therapeutic outcomes and reduced the adverse effects of 5-FU. Together, these data suggest that ABP exhibits anti-neoplastic activity and can effectively enhance the efficacy of 5-FU in CRC treatment. Therefore, further research on the application of ABP in the development of novel anti-tumor drugs and adjuvant compounds is warranted and could improve the outcomes of CRC patients.

Immune Checkpoint Axes Are Dysregulated in Patients With Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. The immune system in patients with AH is hyperactive and yet dysfunctional. Here, we investigated whether this immune-dysregulated state is related to the alcoholic impact on immune checkpoints (ICPs). We used multiplex immunoassays and enzyme-linked immunosorbent assay to quantify plasma levels of 18 soluble ICPs (sICPs) from 81 patients with AH, 65 heavy drinkers without liver diseases (HDCs), and 39 healthy controls (HCs) at baseline, 33 patients with AH and 32 HDCs at 6-month follow-up, and 18 patients with AH and 29 HDCs at 12-month follow-up. We demonstrated that baseline levels of 6 sICPs (soluble T-cell immunoglobulin and mucin domain 3 [sTIM-3], soluble cluster of differentiation [sCD]27, sCD40, soluble Toll-like receptor-2 [sTLR-2], soluble herpesvirus entry mediator [sHVEM], and soluble lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells [sLIGHT]) were up-regulated, while 11 sICPs (soluble B- and T-lymphocyte attenuator [sBTLA], sCD160, soluble cytotoxic T-lymphocyte-associated protein 4 [sCTLA-4], soluble lymphocyte-activation gene 3 [sLAG-3], soluble programmed death 1 [sPD-1], sPD ligand 1 [sPD-L1], sCD28, soluble glucocorticoid-induced tumor necrosis factor receptor-related protein [sGITR], sGITR ligand [sGITRL], sCD80, and inducible T-cell costimulator [sICOS]) were down-regulated in patients with AH compared to HDCs. The up-regulated sICPs except sLIGHT and down-regulated sCD80, sCD160, sCTLA-4, and sLAG-3 correlated positively or negatively with AH disease severity, bacterial translocation, and inflammatory factors. At follow-up, abstinent patients with AH still had higher levels of several sICPs compared to HDCs. We also compared expression of 10 membrane-bound ICPs (mICPs) on peripheral blood mononuclear cells (PBMCs) from patients with AH and HCs by flow cytometry and found that several mICPs were dysregulated on blood cells from patients with AH. The function and regulation of sICPs and mICPs were studied using PBMCs from patients with AH and HCs. Recombinant sHVEM affected tumor necrosis factor (TNF)-α and interferon-γ production by T cells from patients with AH and HCs. Conclusion: Both sICPs and mICPs were dysregulated in patients with AH, and alcohol abstinence did not fully reverse these abnormalities. The HVEM axis plays a role in regulating T-cell function in patients with AH.

Fusobacterium nucleatum Promotes the Progression of Colorectal Cancer Through Cdk5-Activated Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: Growing evidence supports the direct link of Fusobacterium nucleatum with colorectal cancer (CRC). However, to date, the underlying mechanism of action remains poorly understood. In this study, we examined the effects of F. nucleatum on the progression of CRC and investigated whether cyclin-dependent kinase 5 (Cdk5) is involved in the effect through activating the Wnt/ß-catenin signaling pathway. MATERIALS AND METHODS: CRC tissues and matched histologically normal specimens were collected from patients who were diagnosed with CRC and underwent surgical treatment in our hospital between January 2018 and January 2019. Two human CRC cell lines, including DLD-1 and SW480, were utilized mainly for in vitro mechanistic investigations. RESULTS: The abundance of F. nucleatum was significantly greater in CRC tissues than in cancer-free specimens, which was significantly correlated with the progression of CRC. In vitro investigations revealed that F. nucleatum significantly enhanced the proliferation and migration of CRC cells. Furthermore, F. nucleatum significantly induced the expression of Cdk5 and activation of the Wnt/ß-catenin signaling pathway. Notably, knockdown of Cdk5 significantly abrogated the effects of F. nucleatum on cellular processes and Wnt/ß-catenin signaling in relation to the progression of CRC. CONCLUSION: The results of this study demonstrate that F. nucleatum orchestrates a molecular network involving the direct role of Cdk5 in activating Wnt/ß-catenin signaling to modulate CRC progression. Thus, in-depth investigations of F. nucleatum-associated molecular pathways may offer valuable insight into the pathogenesis of CRC, which may help further the development of treatment for this disease.

The Role of TSC1 in the Macrophages Against Vibrio vulnificus Infection.

Vibrio vulnificus (V. vulnificus) is an estuarine bacterium that is capable of causing rapidly fatal infection in humans. Proper polarization and bactericidal activity of macrophages play essential roles in defending against invading pathogens. How macrophages limit V. vulnificus infection remains not well understood. Here we report that tuberous sclerosis complex 1 (TSC1) is crucial for the regulation of V. vulnificus-induced macrophage polarization, bacterial clearance, and cell death. Mice with myeloid-specific deletion of TSC1 exhibit a significant reduction of survival time after V. vulnificus infection. V. vulnificus infection induces both M1 and M2 polarization. However, TSC1 deficient macrophages show enhanced M1 response to V. vulnificus infection. Interestedly, the absence of TSC1 in myeloid cells results in impaired bacterial clearance both in vivo and in vitro after V. vulnificus infection. Inhibition of the mammalian target of rapamycin (mTOR) activity significantly reverses V. vulnificus-induced hypersensitive M1 response and resistant bactericidal activity both in wild-type and TSC1-deficient macrophages. Moreover, V. vulnificus infection causes cell death of macrophages, possibly contributes to defective of bacterial clearance, which also exhibits in a mTORC1-dependent manner. These findings highlight an essential role for the TSC1-mTOR signaling in the regulation of innate immunity against V. vulnificus infection.

Divergent Peptide Presentations of HLA-A*30 Alleles Revealed by Structures With Pathogen Peptides.

Human leukocyte antigen (HLA) alleles have a high degree of polymorphism, which determines their peptide-binding motifs and subsequent T-cell receptor recognition. The simplest way to understand the cross-presentation of peptides by different alleles is to classify these alleles into supertypes. A1 and A3 HLA supertypes are widely distributed in humans. However, direct structural and functional evidence for peptide presentation features of key alleles (e.g., HLA-A*30:01 and -A*30:03) are lacking. Herein, the molecular basis of peptide presentation of HLA-A*30:01 and -A*30:03 was demonstrated by crystal structure determination and thermostability measurements of complexes with T-cell epitopes from influenza virus (NP44), human immunodeficiency virus (RT313), and Mycobacterium tuberculosis (MTB). When binding to the HIV peptide, RT313, the PΩ-Lys anchoring modes of HLA-A*30:01, and -A*30:03 were similar to those of HLA-A*11:01 in the A3 supertype. However, HLA-A*30:03, but not -A*30:01, also showed binding with the HLA*01:01-favored peptide, NP44, but with a specific structural conformation. Thus, different from our previous understanding, HLA-A*30:01 and -A*30:03 have specific peptide-binding characteristics that may lead to their distinct supertype-featured binding peptide motifs. Moreover, we also found that residue 77 in the F pocket was one of the key residues for the divergent peptide presentation characteristics of HLA-A*30:01 and -A*30:03. Interchanging residue 77 between HLA-A*30:01 and HLA-A*30:03 switched their presented peptide profiles. Our results provide important recommendations for screening virus and tumor-specific peptides among the population with prevalent HLA supertypes for vaccine development and immune interventions.

Target-catalyzed hairpin structure-mediated padlock cyclization for ultrasensitive rolling circle amplification.

Because STAT3 is a potent proto-oncogene, screening STAT3 gene has potential for use in tumor diagnosis, classification of subtypes, and molecular target therapy. Thus, in this study, using STAT3 gene as the model molecule, we developed a novel amplification strategy, ultrasensitive rolling circle amplification (THP-RCA) based on target-catalyzed hairpin structure-mediated padlock cyclization, for the ultrasensitive detection of human proto-oncogenes in a homogenous solution. In this system, HP1 was designed as the cyclization template and RCA reaction primer, while HP2 was the padlock probe. The two probes can fold into a hairpin structure via the self-hybridization and thus lock the signaling process in the absence of target species. The hybridization of HP2 with HP1 in an end-to-end fashion occurs with the help of target DNA. Subsequently, HP2 is cyclized by ligase on HP1 template. Interestingly, during the hybridization and enzymatic cyclization of HP2, the target DNA only serves as the catalytic probe and is not exhausted. The cyclized HP2 enables the rolling circle amplification, generating a long tandem single-stranded (ss) DNA product that is capable of hybridizing with considerable quantity of molecular beacons (MBs). As a result, the dramatically amplified fluorescence value is achieved for the ultrasensitive detection of the STAT3 gene. As a result, target DNA is able to be quantified down to 100 fM with a high specificity towards wild-type target DNA. Moreover, the sensing system is suitable for the target detection in human serum. The novel sensing strategy shows tremendous prospect for application in tumor diagnosis and clinical therapy guidance.

Effects of ocean acidification and solar ultraviolet radiation on physiology and toxicity of dinoflagellate Karenia mikimotoi.

A batch culture experiment was conducted to study the interactive effects of ocean acidification (OA) and solar ultraviolet radiation (UVR, 280-400 nm) on the harmful dinoflagellate Karenia mikimotoi. Cells were incubated in 7-days trials under four treatments. Physiological (growth, pigments, UVabc) and toxicity (hemolytic activity and its toxicity to zebrafish embryos) response variables were measured in four treatments, representing two factorial combinations of CO2 (400 and 1000 µatm) and solar irradiance (with or without UVR). Toxic species K. mikimotoi showed sustained growth in all treatments, and there was not statistically significant difference among four treatments. Cell pigment content decreased, but UVabc and hemolytic activity increased in all HC treatments and PAB conditions. The toxicity to zebrafish embryos of K. mikimotoi was not significantly different among four treatments. All HC and UVR conditions and the combinations of HC*UVR (HC-PAB) positively affected the UVabc, hemolytic activity in comparison to the LC*P (LC-P) treatment, and negatively affected the pigments. Ocean acidification (OA) was probably the main factor that affected the chlorophyll-a (Chl-a) and UVabc, but UVR was the main factor that affected the carotenoid (Caro) and hemolytic activity. There were no significant interactive effects of OA*UVR on growth, toxicity to zebrafish embryos. If these results are extrapolated to the natural environment, it can be hypothesized that this strain (DP-C32) of K. mikimotoi cells have the efficient mechanisms to endure the combination of ocean acidification and solar UVR. It is assumed that this toxic strain could form harmful bloom and enlarge the threatening to coastal communities, marine animals, even human health under future conditions.

Knock-Down of HOXB8 Prohibits Proliferation and Migration of Colorectal Cancer Cells via Wnt/ß-Catenin Signaling Pathway.

BACKGROUND There has been no research on the mechanism of HOXB8 action on colorectal cancer so far. This study was designed to investigate the mechanism of HOXB8 regulating colorectal cancer cell proliferation and invasion in vivo and in vitro. MATERIAL AND METHODS HOXB8 shRNA, HOXB8 overexpression, and negative control vector were designed and stably transfected into HCT116 cells. MTT assays were performed to detect cell proliferation. Western blot was utilized to detect HOXB8 expression level in HCT116 stable cells. The invasive and migration abilities were detected by Transwell assay and wound-healing assay. RESULTS HOXB8 knockdown inhibited cell proliferation. The invasiveness of HCT116 cells was significantly reduced following HOXB8 depletion compared with that in the shRNA control group, whereby the rates were reduced by 67% in HOXB8 knockdown group. The wound-healing rate of HOXB8 over-expression cells was significantly increased comparing with that of cells in the blank control group (P<0.05). HOXB8 knockdown promotes apoptosis of HCT116 cells. The expression of E-cadherin was restrained in the HOXB8 over-expression group and increased in the HOXB8 knockdown group. CONCLUSIONS Knock-down of HOXB8 prohibits the proliferation and migration of colorectal cancer cells via the Wnt/ß-catenin signaling pathway and the downregulation of various factors, such as MMP2, c-Myc, CyclinD1,and vimentin. Our data suggested that HOXB8 has great potential to be developed as a novel therapeutic agent for the treatment of human colorectal cancer.

Talin2 regulates breast cancer cell migration and invasion by apoptosis.

Talin is a key component molecule of the extracellular matrix-integrin-cytoskeleton. It serves an important role in the activation of integrin, which, in turn, is known to mediate physiological and pathological processes, including cell adhesion, growth, tumorigenesis, and metastasis. In vertebrates, there are two Talin genes, Talin1 and Talin2. Talin1 is known to regulate focal adhesion dynamics, cell migration and cell invasion; however, the precise role of Talin2 in cancer remains unclear. In the present study, the functional role of Talin2 was examined in the MDA-MB-231 breast cancer cell line. Talin2 knockdown significantly inhibited growth, migratory capacity and invasiveness of MDA-MB-231 cells, and promoted apoptosis. The expression levels of Talin2 in breast cancer cells and in the peritumoral normal breast tissues were also determined by immunohistochemistry. Talin2 was identified to be overexpressed in breast cancer tissues compared with that in the peritumoral breast tissues. In addition, the knockdown of Talin2 by specific RNA interference markedly inhibited cell growth, and caused the downregulation of the apoptotic markers, cleaved Caspase-3 and phosphorylation of poly ADP-ribose polymerase. These findings demonstrate that Talin2 expression is upregulated in human breast cancer and that downregulation of Talin2 may serve as a useful therapeutic target in patients with breast cancer.