Alteration of prothrombin time in Plasmodium falciparum and Plasmodium vivax infections with different levels of severity: a systematic review and meta-analysis.

Malaria infection leads to hematological abnormalities, including deranged prothrombin time (PT). Given the inconsistent findings regarding PT in malaria across different severities and between Plasmodium falciparum and P. vivax, this study aimed to synthesize available evidence on PT variations in clinical malaria. A systematic literature search was performed in PubMed, Embase, Scopus, Ovid, and Medline from 27 November 2021 to 2 March 2023 to obtain studies documenting PT in malaria. Study quality was evaluated using the Joanna Briggs Institute checklist, with data synthesized through both qualitative and quantitative methods, including meta-regression and subgroup analyses, to explore heterogeneity and publication bias. From 2767 articles, 21 studies were included. Most studies reported prolonged or increased PT in malaria patients compared to controls, a finding substantiated by the meta-analysis (P < 0.01, Mean difference: 8.86 s, 95% CI 5.32-12.40 s, I2: 87.88%, 4 studies). Severe malaria cases also showed significantly higher PT than non-severe ones (P = 0.03, Hedges's g: 1.65, 95% CI 0.20-3.10, I2: 97.91%, 7 studies). No significant PT difference was observed between P. falciparum and P. vivax infections (P = 0.88, Mean difference: 0.06, 95% CI - 0.691-0.8, I2: 65.09%, 2 studies). The relationship between PT and malaria-related mortality remains unclear, underscoring the need for further studies. PT is typically prolonged or increased in malaria, particularly in severe cases, with no notable difference between P. falciparum and P. vivax infections. The inconsistency in PT findings between fatal and non-fatal cases highlights a gap in current understanding, emphasizing the need for future studies to inform therapeutic strategies.

Dual Effects of 3-epi-betulin from Daphniphyllum glaucescens in Suppressing SARS-CoV-2-Induced Inflammation and Inhibiting Virus Entry.

The continuous emergence of SARS-CoV-2 variants has led to a protracted global COVID-19 pandemic with significant impacts on public health and global economy. While there are currently available SARS-CoV-2 vaccines and therapeutics, most of the FDA-approved antiviral agents directly target viral proteins. However, inflammation is the initial immune pathogenesis induced by SARS-CoV-2 infection, there is still a need to find additional agents that can control the virus in the early stages of infection to alleviate disease progression for the next pandemic. Here, we find that both the spike protein and its receptor CD147 are crucial for inducing inflammation by SARS-CoV-2 in THP-1 monocytic cells. Moreover, we find that 3-epi-betulin, isolated from Daphniphyllum glaucescens, reduces the level of proinflammatory cytokines induced by SARS-CoV-2, consequently resulting in a decreased viral RNA accumulation and plaque formation. In addition, 3-epi-betulin displays a broad-spectrum inhibition of entry of SARS-CoV-2 pseudoviruses, including Alpha (B.1.1.7), Eplison (B.1.429), Gamma (P1), Delta (B.1.617.2) and Omicron (BA.1). Moreover, 3-epi-betulin potently inhibits SARS-CoV-2 infection with an EC50 of <20 µM in Calu-3 lung epithelial cells. Bioinformatic analysis reveals the chemical interaction between the 3-epi-betulin and the spike protein, along with the critical amino acid residues in the spike protein that contribute to the inhibitory activity of 3-epi-betulin against virus entry. Taken together, our results suggest that 3-epi-betulin exhibits dual effect: it reduces SARS-CoV-2-induced inflammation and inhibits virus entry, positioning it as a potential antiviral agent against SARS-CoV-2.

The association between genetic variants at 3'-UTR and 5'-URR of HLA-G gene and the clinical outcomes of patients with leukemia receiving hematopoietic stem cell transplantation.

In addition to the classical human leukocyte antigen (HLA) genes, the outcomes of post-hematopoietic stem cell transplantation (HSCT) are associated with human leukocyte antigen (HLA)-related genes and non-HLA genes involved in immune regulation. HLA-G gene plays an important role in immune tolerance, assisting immune escape of tumor cells, and decrease of transplant rejection. In this study, we explored the association of genetic variants at the 3'-untranslated region (3'-UTR) and 5'-upstream regulatory region (5'-URR) of HLA-G gene with the adverse outcomes of patients with leukemia receiving HSCT. The genomic DNAs of 164 patients who had acute leukemia and received HSCT were collected for analysis. Nine single nucleotide polymorphisms (SNPs) and six haplotypes in the 3'-UTR and 27 SNPs and 6 haplotypes in the 5'-URR were selected to investigate their relationship with the development of adverse outcomes for patients receiving HSCT, including mortality, relapse, and graft-versus-host disease. Our results revealed that two SNPs (rs371194629 and rs9380142) and one haplotype (UTR-3) located in the 3'-UTR and two SNPs (rs3823321 and rs1736934) and one haplotype (G0104a) located in the 5'-URR of HLA-G were associated with the occurrence of chronic GVHD or development of any forms of GVHD. No SNP was found to associate with the occurrence of mortality and relapse for patients receiving HSCT. These SNPs and haplotypes may play important roles in regulating immune tolerance of allografts post-HSCT that can be used to predict the risk of poor outcomes after receiving HSCT and giving preventive treatment to patients on time.

Melastoma malabathricum L. Suppresses Neutrophil Extracellular Trap Formation Induced by Synthetic Analog of Viral Double-Stranded RNA Associated with SARS-CoV-2 Infection.

Platelet hyper-reactivity and neutrophil extracellular trap (NET) formation contribute to the development of thromboembolic diseases for patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study investigated the pathophysiological effects of SARS-CoV-2 surface protein components and the viral double-stranded RNA (dsRNA) on platelet aggregation and NET formation. Traditional Chinese medicine (TCM) with anti-viral effects was also delineated. The treatment of human washed platelets with SARS-CoV-2 spike protein S1 or the ectodomain S1 + S2 regions neither caused platelet aggregation nor enhanced agonists-stimulated platelet aggregation. Moreover, NET formation can be induced by polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of viral dsRNA, but not by the pseudovirus composed of SARS-CoV-2 spike, envelope, and membrane proteins. To search for TCM with anti-NET activity, the plant Melastoma malabathricum L. which has anticoagulant activity was partially purified by fractionation. One of the fractions inhibited poly(I:C)-induced NET formation in a dose-dependent manner. This study implicates that SARS-CoV-2 structural proteins alone are not sufficient to promote NET and platelet activation. Instead, dsRNA formed during viral replication stimulates NET formation. This study also sheds new insight into using the active components of Melastoma malabathricum L. with anti-NET activity in the battle of thromboembolic diseases associated with SARS-CoV-2 infection.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the ßγ subunit of Gi-protein of fMLP receptor in human neutrophils.

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein ßγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 µM)-induced superoxide anion production (IC50:1.98 ± 0.14 µM), the release of cathepsin G (IC50:2.76 ± 0.15 µM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the ßγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCß by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the ßγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

Single nucleotide polymorphisms within HLA region are associated with the outcomes of unrelated cord blood transplantation.

Cord blood transplantation (CBT) provides a treatment scheme for hematologic diseases and leukemia in both children and adults. However, adverse reactions and transplantation-related death may still occur in patients receiving CBT even when donor and recipient have fully matched HLA in high-resolution HLA typing analysis. Single nucleotide polymorphisms (SNPs) of HLA-related and unrelated genes are known to associate with disease status of patients with unrelated stem cell transplantation. In this study, the genomic regions ranging from 500 base pairs upstream to 500 base pairs downstream of the eight SNPs that were reported as transplantation determinants by Petersdorf et al. were analyzed to evaluate whether genetic variants were associated with the survival status of patients, and the risk for severe (grades 3-4) graft-versus-host disease (GVHD) or cytomegalovirus (CMV) infection/reactivation. The analyses were performed in the mode of recipient genotype, donor genotype, and recipient-donor mismatching, respectively. By analysis of sixty-five patients and their HLA-matched unrelated donors, we found that five SNPs were associated with patient survival which included the recipient genotype with SNPs of rs107822 in the RING1 gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene; and the recipient-donor mismatching with SNPs of rs9282369 in HLA-DOA gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene. Five SNPs were associated with the risk for severe GVHD which included the donor genotype with SNPs of rs213210 and rs2523675; the recipient genotype with SNPs of rs9281491 in the HCP5 gene; and the recipient-donor mismatching with SNPs of rs209130 in the TRIM27 gene, and rs986522 in the COL11A2 gene. Six SNPs were related to the risk for CMV infection/reactivation which included the donor genotype with SNPs of rs435766, rs380924, and rs2523957; and the recipient-donor mismatching with SNPs of rs2070120, rs17220087, and rs17213693 in the HLA-DOB gene; and rs435766 and rs380924 in the MICD gene. This study provides the basis for larger analyses and if the results are confirmed, a way of selecting better unrelated CBT candidate donors.

The Association Between Single-Nucleotide Polymorphisms of Co-Stimulatory Genes Within Non-HLA Region and the Prognosis of Leukemia Patients With Hematopoietic Stem Cell Transplantation.

To avoid graft rejection, the hematopoietic stem cells with matched classical human leukocyte antigen (HLA) alleles are the primary choice for clinical allogeneic transplantation. However, even if the fully HLA-matched hematopoietic stem cells are used for transplantation, some patients still have poor prognosis after hematopoietic stem cell transplantation (HSCT), suggesting that the HLA system was not the only determinant of the outcomes of HSCT. In this study, we investigated whether the single-nucleotide polymorphisms (SNPs) of the co-stimulatory genes within non-HLA regions were related to the outcomes of HSCT. The genomic DNAs of 163 patients who had acute leukemia and received HSCT and their respective donors were collected for analysis. Thirty-four SNPs located in the four co-stimulatory genes including cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily 4 (TNFSF4), and programmed cell death protein 1 (PDCD1) were selected to explore their relationship with the adverse outcomes after transplantation, including mortality, cytomegalovirus infection, graft-versus-host disease, and relapse. Our results revealed that nine SNPs in the CTLA4 gene, five SNPs in the PDCD1 gene, two SNPs in the TNFSF4 gene, and four SNPs in the CD28 gene were significantly associated with the occurrence of adverse outcomes post-HSCT. These SNPs may play important roles in immune response to allografts post-HSCT and can be the targets for developing strategy to identify appropriate donors.

Carbonized Lysine-Nanogels Protect against Infectious Bronchitis Virus.

In this study, we demonstrate the synthesis of carbonized nanogels (CNGs) from an amino acid (lysine hydrochloride) using a simple pyrolysis method, resulting in effective viral inhibition properties against infectious bronchitis virus (IBV). The viral inhibition of CNGs was studied using both in vitro (bovine ephemeral fever virus (BEFV) and pseudorabies virus (PRV)) and in ovo (IBV) models, which indicated that the CNGs were able to prevent virus attachment on the cell membrane and penetration into the cell. A very low concentration of 30 µg mL-1 was found to be effective (>98% inhibition) in IBV-infected chicken embryos. The hatching rate and pathology of IBV-infected chicken embryos were greatly improved in the presence of CNGs. CNGs with distinctive virus-neutralizing activities show great potential as a virostatic agent to prevent the spread of avian viruses and to alleviate the pathology of infected avian species.

The anti-inflammatory effect of ε-viniferin by specifically targeting formyl peptide receptor 1 on human neutrophils.

The uncontrol respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of ε-viniferin, a lignan from the root of Vitis thunbergii var. thunbergii, inhibits N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP) induced respiratory burst by antagonizing formyl peptide receptor 1 in human neutrophils. Briefly, ε-viniferin specifically inhibited fMLP (0.1 µM: formyl peptide receptor 1 agonist or 1 µM: formyl peptide receptor 1, 2 agonist)-induced superoxide anion production in a concentration-dependent manner (IC50 = 2.30 ± 0.96 or 9.80 ± 0.21 µM, respectively) without affecting this induced by formyl peptide receptor 2 agonist (WKYMVM). ε-viniferin inhibited fMLP (0.1 µM)-induced phosphorylation of ERK, Akt, Src or intracellular calcium mobilization without affecting these caused by WKYMVM. The synergistic suppression of fMLP (1 µM)-induced superoxide anion production was observed only in the combination of ε-viniferin and formyl peptide receptor 2 antagonist (WRW4) but not in combination of ε-viniferin and formyl peptide receptor 1 antagonist (cyclosporine H). ε-viniferin inhibited FITC-fMLP binding to formyl peptide receptors. Moreover, the synergistic suppression of FITC-fMLP binding was observation only in the combination of ε-viniferin and WRW4 but not in other combinations. ATPγS induced superoxide anion production through formyl peptide receptor 1 in fMLP desensitized neutrophils and this effect was inhibited by ε-viniferin. The concentration-response curve of fMLP-induced superoxide anion was not parallel shifted by ε-viniferin. Furthermore, the inhibiting effect of ε-viniferin on fMLP-induced superoxide anion production was reversible. These results suggest that ε-viniferin is an antagonist of formyl peptide receptor 1 in a reversible and non-competitive manner.

Integrin αIIbß3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2.

BACKGROUND: Bidirectional integrin αIIbß3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function. RESULTS: An antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbß3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling. CONCLUSIONS: These findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbß3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

Synbiotics Alleviate the Gut Indole Load and Dysbiosis in Chronic Kidney Disease.

Chronic kidney disease (CKD) has long been known to cause significant digestive tract pathology. Of note, indoxyl sulfate is a gut microbe-derived uremic toxin that accumulates in CKD patients. Nevertheless, the relationship between gut microbiota, fecal indole content, and blood indoxyl sulfate level remains unknown. In our study, we established an adenine-induced CKD rat model, which recapitulates human CKD-related gut dysbiosis. Synbiotic treatment in CKD rats showed a significant reduction in both the indole-producing bacterium Clostridium and fecal indole amount. Furthermore, gut microbiota diversity was reduced in CKD rats but was restored after synbiotic treatment. Intriguingly, in our end-stage kidney disease (ESKD) patients, the abundance of indole-producing bacteria, Bacteroides, Prevotella, and Clostridium, is similar to that of healthy controls. Consistently, the fecal indole tends to be higher in the ESKD patients, but the difference did not achieve statistical significance. However, the blood level of indoxyl sulfate was significantly higher than that of healthy controls, implicating that under an equivalent indole production rate, the impaired renal excretion contributes to the accumulation of this notorious uremic toxin. On the other hand, we did identify two short-chain fatty acid-producing bacteria, Faecalibacterium and Roseburia, were reduced in ESKD patients as compared to the healthy controls. This may contribute to gut dysbiosis. We also identified that three genera Fusobacterium, Shewanella, and Erwinia, in the ESKD patients but not in the healthy controls. Building up gut symbiosis to treat CKD is a novel concept, but once proved effective, it will provide an additional treatment strategy for CKD patients.

Dual Effects of Let-7b in the Early Stage of Hepatitis C Virus Infection.

MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses, followed by various molecular and cellular assays using Huh7 cells expressing wild-type (WT) or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon-stimulated genes (ISGs). Let-7b augmented retinoic acid-inducible gene I (RIG-I) signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-ß. Let-7b directly targeted the ATG12 and IκB kinase alpha (IKKα) transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5' UTR of the HCV genome, and limits HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection.IMPORTANCE HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I interferon (IFN) signaling displays potent antiviral and immunomodulatory effects. Host factors, including microRNAs (miRNAs), play a role in upregulating IFN signaling to limit HCV replication. Let-7b is a liver-abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles against HCV replication by increasing the expression of IFN and interferon-sensitive response element (ISRE)-driven interferon-stimulated genes (ISGs) in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

Circulating tumor cell enumeration for improved screening and disease detection of patients with colorectal cancer.

BACKGROUND: The immunochemical fecal occult blood test (iFOBT) for colorectal cancer (CRC) screening and the serum carcinoembryonic antigen (CEA) assay for disease detection of CRC is associated with a high false-positive rate and a low detection sensitivity, respectively. There is an unmet need to define additional modalities to complement these assays. Different subsets of circulating tumor cells (CTCs) are present in the peripheral blood of cancer patients. Whether or not CTCs testing supplements these clinical assays and is valuable for patients with CRC was investigated. METHODS: CTCs were enriched from pre-operative patients with CRC (n = 109) and the non-cancerous controls (n = 65). CTCs expressing either epithelial cell adhesion molecule (EpCAM) or podoplanin (PDPN, the marker associated with poor cancer prognosis) were defined by immunofluorescence staining and were analyzed alone or in combination with iFOBT or serum CEA. RESULTS: Patients with early or advanced stage of CRC can be clearly identified and differentiated from the non-cancerous controls (p < 0.001) by EpCAM+-CTC or PDPN+-CTC count. The sensitivity and specificity of EpCAM+-CTCs was 85.3% and 78.5%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL of blood; and the sensitivity and specificity of PDPN+-CTCs was 78.0% and 75.4%, respectively, when the cutoff value was 7 PDPN+-CTCs/mL of blood. Combined analysis of iFOBT with the EpCAM+-CTC and PDPN+-CTC count reduced the false-positive rate of iFOBT from 56.3% to 18.8% and 23.4%, respectively. Combined analysis of serum CEA with the EpCAM+-CTC and PDPN+-CTC count increased the disease detection rate from 30.3% to 89.9% and 86.2%, respectively. CONCLUSION: CTC testing could supplement iFOBT to improve CRC screening and supplement serum CEA assay for better disease detection of patients with CRC.

Multi-omics profiling reveals microRNA-mediated insulin signaling networks.

BACKGROUND: MicroRNAs (miRNAs) play a key role in mediating the action of insulin on cell growth and the development of diabetes. However, few studies have been conducted to provide a comprehensive overview of the miRNA-mediated signaling network in response to glucose in pancreatic beta cells. In our study, we established a computational framework integrating multi-omics profiles analyses, including RNA sequencing (RNA-seq) and small RNA sequencing (sRNA-seq) data analysis, inverse expression pattern analysis, public data integration, and miRNA targets prediction to illustrate the miRNA-mediated regulatory network at different glucose concentrations in INS-1 pancreatic beta cells (INS-1), which display important characteristics of the pancreatic beta cells. RESULTS: We applied our computational framework to the expression profiles of miRNA/mRNA of INS-1, at different glucose concentrations. A total of 1437 differentially expressed genes (DEGs) and 153 differentially expressed miRNAs (DEmiRs) were identified from multi-omics profiles. In particular, 121 DEmiRs putatively regulated a total of 237 DEGs involved in glucose metabolism, fatty acid oxidation, ion channels, exocytosis, homeostasis, and insulin gene regulation. Moreover, Argonaute 2 immunoprecipitation sequencing, qRT-PCR, and luciferase assay identified Crem, Fn1, and Stc1 are direct targets of miR-146b and elucidated that miR-146b acted as a potential regulator and promising target to understand the insulin signaling network. CONCLUSIONS: In this study, the integration of experimentally verified data with system biology framework extracts the miRNA network for exploring potential insulin-associated miRNA and their target genes. The findings offer a potentially significant effect on the understanding of miRNA-mediated insulin signaling network in the development and progression of pancreatic diabetes.

Cotargeting CHK1 and PI3K Synergistically Suppresses Tumor Growth of Oral Cavity Squamous Cell Carcinoma in Patient-Derived Xenografts.

Oral cavity squamous cell carcinomas (OSCCs) are aggressive tumors, and their recurrence leads to poor prognosis and reduced survival rates. This study aimed to identify therapeutic targets and to evaluate the efficacy of targeted inhibitors in OSCC patient-derived xenograft (PDX) models. Herein, we reported that OSCC PDXs recapitulated the genomic signatures of their paired primary tumors and the expression of CHEK1, PIK3CA, and PIK3CD was significantly upregulated in OSCC. The antitumor efficacy of CHK1 inhibitors (PF477736, AZD7762, LY2606368) and PI3K inhibitors (BYL719, GDC0941, GSK1059615) was investigated in OSCC cell lines and PDX models. Targeting either CHK1 or PI3K effectively inhibited cell proliferation and colony formation by inducing cell cycle arrest and apoptosis in in vitro cell-based assays. Cisplatin-based chemotherapy combined with CHK1 inhibitor treatment synergistically inhibited cell proliferation by suppressing CHK1 phosphorylation and inducing PARP cleavage. Furthermore, compared with monotherapy, cotreatment with CHK1 and PI3K inhibitors exerted synergistic anticancer effects by suppressing CHK1, AKT, and 4E-BP1 phosphorylation. In summary, our study identified CHK1 and PI3K as promising targets, especially in a dual treatment strategy combining a CHK1 inhibitor with cisplatin or a PI3K inhibitor as a novel therapeutic approach for OSCC patients with aberrant cell cycle regulation and PI3K signaling activation.

Podoplanin promotes cancer-associated thrombosis and contributes to the unfavorable overall survival in an ectopic xenograft mouse model of oral cancer.

BACKGROUND: Podoplanin (PDPN) is a transmembrane glycoprotein that mediates tumor cell-induced platelets aggregation in different cancer types. Emerging data indicate that PDPN is a marker for poor prognosis of human oral squamous cell carcinoma (OSCC). However, the functional impacts of PDPN on cancer formation and disease progression of OSCC remain to be elucidated. METHODS: The sublines of the OECM-1 oral cancer cells with PDPN knockdown or overexpression were established. The cellular characteristics and the ability to induce platelet aggregation of these cells lines were analyzed. An ectopic xenograft animal model by inoculating cancer cells into the anterior neck region of nude mice was established to investigate the functional impact of PDPN on disease progression and cancer-associated thrombosis of OSCC. RESULTS: PDPN promoted OSCC cell migration and invasion, but had no effect on cell proliferation in vitro and tumor growth in vivo. Co-incubation of PDPN-positive (PDPN+) OSCC cells with platelets induced platelet activation and aggregation. The mice bearing PDPN+ tumor had a decrease in overall survival despite that there was no gross appearance of distant metastasis. A speckled immunofluorescence staining pattern of platelet marker mCD41 was defined in the PDPN+ tumor sections and the intensity was greater than in the PDPN-low or negative tumor sections. Co-immunofluorescence staining of the tumor sections with mCD41 and the endothelial cell marker mCD31 further demonstrated that platelet aggregates were located in the lumen of blood vessel and were also distributed intratumorally in the mice bearing PDPN+ tumors. CONCLUSIONS: These data demonstrated that PDPN expression in the cancer cells is associated with high risk of thrombosis, leading to unfavorable overall survival of the mice. This study provides new insights into the functions of PDPN in cancer-associated thrombosis and in the pathophysiology of OSCC.

Sequential Circulating Tumor Cell Counts in Patients with Locally Advanced or Metastatic Hepatocellular Carcinoma: Monitoring the Treatment Response.

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD, n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD; n = 44 for PR + SD, p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21-139) and 15 (interquartile range 4-41) cells/mL of blood, respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC, in particular for the AFP-low cases.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2.

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Association between single nucleotide polymorphisms within HLA region and disease relapse for patients with hematopoietic stem cell transplantation.

Disease relapse occurs in patients with leukemia even hematopoietic stem cell transplantation (HSCT) was performed with human leukocyte antigen (HLA)-matched donors. As revealed previously by Petersdorf et al., there are nine single nucleotide polymorphisms (SNPs) located in the HLA region that potentially modulate the efficacy of HSCT. In this study, we investigated whether or not the genomic variants 500 base pairs flanking the nine transplantation-related SNPs were related to the risk of post-HSCT relapse for patients with leukemia (n = 141). The genomic DNAs collected from 85 patients with acute myeloid leukemia (AML), 56 patients with acute lymphocytic leukemia (ALL), and their respective HLA-matched donors were subject to SNPs analysis, conferred by the mode of mismatch between donor-recipient pair or by recipient or donor genotype analysis. Seven SNPs were revealed to associate with the risk of relapse post-HSCT. For patients with AML, the increased risk of post-HSCT relapse was associated with the donor SNP of rs111394117 in the intron of NOTCH4 gene, and the recipient SNPs of rs213210 in the ring finger protein 1 (RING1) gene promoter, and rs17220087 and rs17213693 in the intron of HLA-DOB gene. For patients with ALL, the increased risk of post-HSCT relapse was associated with the donor SNP of rs213210 in the RING1 gene promoter, and the recipient SNPs of rs79327197 in the HLA-DOA gene promoter, rs2009658 in the telomeric end of lymphotoxin-alpha (LTA) gene, rs17220087 and rs17213693 in the intron of HLA-DOB gene, and rs2070120 in the 3'-UTR of HLA-DOB gene. This study sheds new insight into selecting better candidate donors for performing HSCT in patients with AML and ALL.

A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction.

Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.