Expanding individualized therapeutic options via genoproteomics.

Clinical next-generation sequencing (NGS) tests have enabled treatment recommendations for cancer patients with driver gene mutations. Targeted therapy options for patients without driver gene mutations are currently unavailable. Herein, we performed NGS and proteomics tests on 169 formalin-fixed paraffin-embedded (FFPE) samples of non-small cell lung cancers (NSCLC, 65), colorectal cancers (CRC, 61), thyroid carcinomas (THCA, 14), gastric cancers (GC, 2), gastrointestinal stromal tumors (GIST, 11), and malignant melanomas (MM, 6). Of the 169 samples, NGS detected 14 actionable mutated genes in 73 samples, providing treatment options for 43% of the patients. Proteomics identified 61 actionable clinical drug targets approved by the FDA or undergoing clinical trials in 122 samples, providing treatment options for 72% of the patients. In vivo experiments demonstrated that the Mitogen-Activated Protein Kinase (MEK) inhibitor could block lung tumor growth in mice with overexpression of Map2k1 protein. Therefore, protein overexpression is a potentially feasible indicator for guiding targeted therapies. Collectively, our analysis suggests that combining NGS and proteomics (genoproteomics) could expand the targeted treatment options to 85% of cancer patients.

The predictive value and correlation of ß-catenin, CMTM6, and PD-L1 expression in colorectal cancer.

CMTM6 is a major regulator of PD-L1 expression. Aberrant Wnt pathway signaling occurs in most sporadic colorectal cancers (CRC). However, the significance and correlation of ß-catenin, CMTM6, and PD-L1 immunohistochemical expression in CRC is still unknown and need to be further verified. We evaluated the expression levels of ß-catenin, CMTM6, PD-L1, and MMR (mismatch repair) proteins by immunohistochemistry in CRC tissue microarray (TMA), and evaluated the association among ß-catenin, CMTM6, PD-L1 expression, MMR status, and clinicopathological features in 704 CRC patients. Positive expression of PD-L1 in tumor cells (TC) is associated with more frequent dMMR (mismatch repair deficient) status, CMTM6 expression, right colon, and younger CRC patients. The expression of PD-L1 in tumor-infiltrating immune cells (IC) is associated with a higher frequency of adenocarcinoma, ß-catenin, and CMTM6 expression. In univariate analysis, age, histological subtype, histologic grade, lymphatic metastasis, TNM stage, MMR status, and expression of PD-L1 protein in IC were significantly associated with the overall survival. In multivariate analysis, age, histologic grade, TNM stage, MMR status, and expression of PD-L1 protein in IC were independent prognostic factors. The overall survival of the adjuvant chemotherapy group was significantly higher than those non-chemotherapy in TNM stage III-IV CRC patients, but no significant overall survival improvement was found in the positive PD-L1 in TC, positive PD-L1 in IC, positive CMTM6, low ß-catenin expression, or dMMR status subgroups. Expression of CMTM6 and PD-L1 in CRC are positively associated with ß-catenin and reliable biomarkers for the prediction of responding to chemotherapy. The expression of ß-catenin/CMTM6/PD-L1 and MMR status may be valuable biomarkers for guiding different treatment strategies in CRC patients.

Obesity and Heath-Carter Somatotyping of 3438 Adults in the Xinjiang Uygur Autonomous Region of China by Multivariate Analysis.

INTRODUCTION: This study aimed to investigate the somatotype and obesity of adults in the Xinjiang Uygur Autonomous Region of China and to explore multivariate path analysis for the feasibility and scientificity of using somatotypes to evaluate obesity. SUBJECTS AND METHODS: According to anthropometric methods, a cross-sectional study was performed on 10 indexes of 3438 adults (1690 men and 1748 women, aged > 20 years) living in the Xinjiang Uygur Autonomous Region of China (including Kazakh, Kyrgyz, Xibe, Uzbek, Tatar and Tajik). The Heath-Carter anthropometric method and body mass index (BMI) were used to evaluate somatotype and obesity, respectively. The feasibility and scientificity of using somatotypes to evaluate obesity were analysed by correspondence analysis. RESULTS: Among the six populations, the somatotypes were mainly distributed as endomorphic mesomorph, mesomorph-endomorph and mesomorphic endomorph populations, accounting for 66.5% of males and 78.8% of females. The obesity rate (27.4% in males, 27.8% in females) of the six populations in the Xinjiang Uygur Autonomous Region of China was much higher than the average Chinese adult obesity rate (12.1%) and the global adult obesity rate (male: 11%, female: 15%). The distribution of BMI was significantly different (male: P=0.000, female: P=0.033) in different populations, and the incidence of overweight and obesity in the Xinjiang Uygur Autonomous Region of China increased gradually. This study found that there were significant differences in somatotype distribution among different obesity groups in the Xinjiang Uygur Autonomous Region of China (P=0.000). There was a strong correlation between overweight or obesity and endomorph-mesomorph, endomorphic mesomorph and mesomorphic endomorph. Furthermore, this study indicated that using somatotypes to evaluate obesity was reliable and scientific. CONCLUSION: This study concluded that the somatotype of overweight or obese people was mainly related to endomorphic mesomorph, mesomorph-endomorph, and mesomorphic endomorph.

A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction.

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism.

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity.

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection.

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV was replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.

Structural insight into the substrate specificity of DNA Polymerase mu.

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins.

DNA polymerase zeta (pol zeta) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol zeta alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol zeta. Pol zeta is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol zeta is substantially lower than that of homologous B family pols alpha, delta and epsilon. Pol zeta is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol zeta in vitro is consistent with Pol zeta-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol zeta contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.

[Changes of somatostatin (SS) in stomach and duodenum of rats after +Gz exposure].

OBJECTIVE: To study effects of +Gz exposure on the change of somatostatin (SS) in stomach and duodenum of rats. METHOD: Forty male Wistar rats were randomly divided into +1 Gz control group, +5 Gz x 5 min group, +10 Gz x 5 min group and repeated exposure [(+5 Gz x 2 min) + (+10 Gz x 2 min) + (+5 Gz x 2 min)] group. After +Gz exposure, the rats in each group were anesthesized by aether, and gastric and duodenal mucosa were taken immediately. Levels of SS were assayed using radioimmunoassay (RIA) methods. RESULTS: Gastric and duodenal mucosa of the control group and +5 Gz group were intact and smooth. Under naked eye and light microscope, scattered hemorrhagic spoto, small ulcers, submucosal hyperemia, and erosion of mucosa were found in gastric antrum and duodenum in +10 Gz group rats. Diffuse hyperemia, erosion and small ulcers were found in mucosa of gastric fundus, gastric antrum and duodenum of repeated exposure group. Exposure to +Gz can increase somatostatin content in gastric antrum and duodenum (P<0.01). CONCLUSION: Increase of SS content may play an important role in the prevention of pathogenesis of stomach and duodenum after +G exposure.

Clinical significance of plasma D-dimer and von Willebrand factor levels in patients with ulcer colitis.

AIM: To investigate the levels of D-dimer(DD) and von Willebrand factor(vWF) and the relationship between DD and vWF in ulcerative colitis(UC) patients. METHODS: A total of 29 plasma specimens were obtained from patients with ulcerative colitis (male 13, female 16) aged 21-47 years (33+/-11). Disease activity was assessed by Truelove-Writeria. Patients with a score of above 5 were regarded as having active colitis. Twenty healthy people(male 12, female 8) aged 19-53 years(31+/-14) served as normal controls. Blood samples were taken from an antecubital vein puncture. Blood(1.8 mL) was injected into the tubes containing sodium citrate (0.13 mmol/L). The plasma was obtained by centrifugation at 3000 r.min(-1) for 10 min, and stored at -80 degrees until assayed by ELISA. RESULTS: The mean plasma levels of DD and vWF in active UC patients were significantly higher than those of the controls (0.69+/-0.41 vs 0.27+/-0.11, P<0.01 143+/-46 vs 103+/-35, P<0.01). The mean plasma levels of DD in the patients with active disease were higher than those with inactive disease(0.69+/-0.41 vs 0.48+/-0.29 P<0.05). The levels of vWF were not different between active and inactive patients. DD levels were positively related to vWF levels( r =0.574, P<0.01). There was no significant difference between levels of DD and vWF and the scope of disease and sex of the patients. CONCLUSION: vWF is an important feature and a good marker of UC intravascular thrombus and endothelial cell dysfunction were found in UC patients and the combined test of DD and vWF is helpful to distinguish the activity of the UC patients.