The predicting role of circulating tumor DNA landscape in gastric cancer patients treated with immune checkpoint inhibitors.

A more common and noninvasive predicting biomarker for programmed cell death 1 (PD-1) antibody remains to be explored. We assessed 46 patients with advanced gastric cancer who received PD-1 antibody immunotherapy and 425-genes next-generation sequencing (NGS) testing. Patients who had a > 25% decline in maximal somatic variant allelic frequency (maxVAF) had a longer progression free survival (PFS) and higher response rate than those who did not (7.3 months vs 3.6 months, p = 0.0011; 53.3% vs 13.3%, p = 0.06). The median PFS of patients with undetectable and detectable post-treatment circulating tumor DNA (ctDNA) was 7.4 months vs. 4.9 months (p = 0.025). Mutation status of TGFBR2, RHOA, and PREX2 in baseline ctDNA influenced the PFS of immunotherapy (p < 0.05). Patients with alterations in CEBPA, FGFR4, MET or KMT2B (p = 0.09) gene had greater likelihood of immune-related adverse events (irAEs). ctDNA can serve as a potential biomarker of the response to immunotherapy in advanced gastric cancers, and its potential role in predicting irAEs worth further exploration.

Gα13 Switch Region 2 Relieves Talin Autoinhibition to Activate αIIbß3 Integrin.

Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0-F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (Gα13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of Gα13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows Gα13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo Altogether, we present a mechanistic basis for the regulation of talin through Gα13.

[Fourier transform infrared spectral analysis on peanut (Arachis Hypogaea) plants under calcium deficiency stress].

The objective of the present study was to reveal different tolerance of peanut plants to Ca deficiency by determining Ca uptake and Fourier transform infrared spectral (FTIR) differences of two peanut cultivars grown in nutrition solution. Peanut cultivars LH11 and YZ9102 were selected. Seedlings at the first leaf stage were cultivated for 28 days in nutrient solution with 0, 0.01 and 2.0 mmol x L(-1) Ca treatments, respectively. The results showed that under 0 and 0.01 mmol x L(-1) Ca supply, YZ9102 did not show Ca deficiency symptoms and the plant biomass did not change, whereas LH11 exhibited shoot-tip necrosis, smaller plant size, more lateral branches, and plant dry matter weights decreased significantly. YZ9102 had higher plant Ca concentration and Ca accumulation than LH11. Besides, for LH11, Ca was mainly accumulated in roots, while for YZ9102 mainly in leaves. As compared with plants cultivated in 2.0 mol x L(-1) Ca nutrition, root, stem and leaf of LH11 plants under Ca deficiency stress showed higher transmittance at peaks 1 060, 1 380, 1 655, 2 922, and 3 420 cm(-1) in FTIR spectra, indicating that the contents of protein, sugar and lipid decreased obviously in LH11 plants in condition that Ca supply was limited. However, the FTIR spectra of YZ9102 were less affected by Ca deficiency. It is suggested that YZ9102 might be more tolerant to Ca deficiency.

HPLC-MTT assay: anticancer activity of aqueous garlic extract is from allicin.

A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

Genetically Predicted Causal Effects of Gut Microbiota and Gut Metabolites on Digestive Tract Cancer: A Two-Sample Mendelian Randomization Analysis.

Background: Evidence from numerous observational studies and clinical trials has linked gut microbiota and metabolites to digestive tract cancer. However, the causal effect between these factors remains uncertain. Methods: Data for this study were obtained from the MiBioGen, TwinsUK Registry, and FinnGen (version R8). Two-sample Mendelian randomization analysis with inverse variance weighting method was primarily used, and the results were validated by heterogeneity analysis, pleiotropy test, and sensitivity analysis. Results: At P < 5 × 10-8, our analysis identified four gut microbiotas as risk factors for digestive tract cancer and six as risk factors for colorectal cancer. Conversely, one gut microbiota exhibited protection against bile duct cancer, and two showed protective effects against stomach cancer. At P < 1 × 10-5, our investigation revealed five, six, three, eight, eight, and eight gut microbiotas as risk factors for esophageal, stomach, bile duct, liver, pancreatic, and colorectal cancers, respectively. In contrast, four, two, eight, two, two, and five gut microbiotas exhibited protective effects against these cancers. Additionally, GABA, a metabolite of gut microbiota, displayed a significant protective effect against colorectal cancer. Conclusion: In conclusion, specific gut microbiota and metabolites play roles as risk factors or protective factors for digestive tract cancer, and a causal relationship between them has been established, offering novel insights into gut microbiota-mediated cancer development.

2,4,6-Tris(2,4-dimethyl-phen-yl)-1,3,5-triazine.

Two virtually superimposable mol-ecules comprise the asymmetric unit of the title compound, C(27)H(27)N(3). The range of dihedral angles between the central 1,3,5-triazine ring and the attached benzene rings is 20.88 (14)-31.36 (14)°, and the shape of each mol-ecule is of a flattened bowl. The crystal packing features weak C-H⋯π bonds and π-π inter-actions between triazine and benzene rings [centroid-centroid separations = 3.7696 (17) and 3.7800 (18) Å] that result in the formation of supra-molecular layers in the ac plane. The crystal studied was a non-merohedral twin with a minor twin component of 20.7 (3)%.

Association of body composition with survival and inflammatory responses in patients with non-metastatic nasopharyngeal cancer.

OBJECTIVES: It is unknown whether or not the body composition is correlated with the prognosis and inflammatory response in patients with nasopharyngeal cancer (NPC). MATERIALS AND METHODS: This cohort included 1767 patients with NPC. Visceral, subcutaneous and intra muscular adipose tissues (VAT, SAT and IMAT), and skeletal muscle index were quantified with computed tomography. We used the optimal stratification to select cut points for VAT, SAT and IMAT. We defined sarcopenia according to a widely used cut-point. The primary endpoint was overall survival (OS). The association between body composition and inflammatory response was also examined. RESULTS: Low VAT, SAT, IMAT and sarcopenia were observed in 260 (14.7%), 451 (25.5%), 773 (43.7%) and 683 (38.7%) patients, respectively. Low VAT (P < 0.001, hazard ratio [HR], 1.884; 95% confidence interval [CI], 1.436-2.473,) and SAT (P = 0.022, HR, 1.334, 95%CI, 1.043-1.706) were both associated worse survival. IMAT and sarcopenia were not with prognostic value. In multivariate analysis, we found the prognostic value of the VAT (HR: 1.544, 95% CI: 1.128-2.114; P = 0.007) was independent of T stage, N stage, disease stage, lactic dehydrogenase, neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), the systemic immune-inflammation index (SII), EBV-DNA and body mass index. We observed higher NLR (P = 0.028) and PLR (P < 0.001) in patients with low SAT. Both low VAT (P = 0.009) and SAT (P = 0.005) were associated with decreased stromal lymphocyte infiltrating intensity. CONCLUSIONS: Among body composition parameters, VAT was an independent prognostic factor, especially in patients with locally advanced NPC.

Applications of Immobilized Metal Affinity Electrophoresis.

Immobilized metal affinity electrophoresis (IMAEP) is a technique in which metal ions are integrated in a localized polyacrylamide gel section and which possess an insignificant electrophoretic migration. IMAEP has been implemented into both native and sodium dodecyl sulfate (SDS) one-dimensional (1-D) and two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) systems. The gel piece containing metal ions is overlaid on top of an individual well in 1-D IMAEP, and gel strip is overlaid on top of the two-second dimensional polyacrylamide gel in 2-D IMAEP, respectively. This method is used to capture/enrich metal ion binding proteins. Due to the preferential binding between iron (III), manganese (II), or aluminum (III) ions and the phosphate group, these metal ions are used to capture/enrich phosphoproteins from a mixture of proteins. Data shows that SDS not only does not interfere with the extraction of phosphoproteins but also help unmasking phosphate groups by unfolding the phosphoproteins to facilitate metal ion-phosphate binding while supplying the protein with negative charges. In addition, IMAEP together with avidin functional affinity electrophoresis (AFAEP) and antibody affinity electrophoresis (AAEP) have been used to demonstrate the separation of proteins by their functionalities. AFAEP is used here to capture biotinylated proteins using avidin and AAEP is used here to capture protein G using IgG.

An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides.

We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.

Mass-dependent signaling between G protein coupled receptors.

The present study provides evidence that G protein coupled receptor (GPCR) signaling pathways participate in an interactive signaling network governed by the principles of mass action. Using an inducible thromboxane A2 receptor (TPR)/platelet activating factor receptor (PAFR) co-expressing cell model, TPR or PAFR expression was independently up-regulated. Immunostaining and radioligand binding experiments demonstrated that this receptor up-regulation resulted in increased GPCR:G protein mass ratios. This increase in mass ratio impacted both TPR and PAFR ligand affinity. Specifically, up-regulating TPR expression not only decreased TPR ligand affinity, but also decreased the ligand affinity of PAFRs. A similar effect on ligand affinities was observed when PAFRs were up-regulated. In addition, increasing the GPCR:G protein mass ratio for TPRs led to desensitization of the calcium mobilization response to PAFR activation, and increasing PAFR mass desensitized the TPR-mediated calcium response. Finally, it was observed that an increased TPR:G protein mass ratio was associated with a shift in the TPR signaling response, and revealed an additional TPR signaling pathway through G(S). Collectively, these results describe a novel mechanism, i.e., mass-dependent GPCR signaling, by which cells can modulate their GPCR signaling pathways and signaling priorities.