[Effect of FBXW7 gene mutation on the prognosis of immunotherapy in patients with non-small cell lung cancer].

Objective: To explore the effect of F-box and WD-40 domain protein 7 (FBXW7) gene mutation on the prognosis of immunotherapy in patients with non-small cell lung cancer (NSCLC). Methods: (1) The clinical data of 125 patients with advanced NSCLC in the First Affiliated Hospital of Nanjing Medical University from January 2019 to June 2021 were collected. There were 70 males and 55 females, with a median age [M (Q1, Q3)] of 64(57, 70) years. The correlation of FBXW7 mutation with immunotherapy efficacy and prognosis was analyzed. (2) The Data set of NSCLC patients treated with immunotherapy from cBioPortal database was downloaded. A total of 261 patients were included as immunotherapy group. There were 120 males and 141 females, with a median age of 66(57, 73) years. The association of FBXW7 mutation with clinical characteristics and prognosis of NSCLC patients was investigated. (3) The data of patients with NSCLC from the cancer genome atlas (TCGA) database were downloaded. A total of 1 030 patients were included as TCGA group. There were 633 males and 397 females, with a median age of 67(60, 73) years. The effect of FBXW7 mutation on the efficacy of immunotherapy was analyzed. The key molecules and their biological functions were also determined Results: Among the 125 NSCLC patients, the FBXW7 mutation rate was 5.6% (7/125). All FBXW7 mutation types was truncating mutation. In these FBXW7 mutation patients who received immunotherapy, 4 had partial response, 2 had stable disease, and 1 had progressive disease. The objective response rate (ORR) and disease control rate (DCR) were 4/7 and 6/7, respectively. The median progression-free survival (PFS) was 13.0 months (95%CI: 7.0-22.0 months) for patients with FBXW7 mutation and 4.0 months (95%CI: 2.0-11.5 months) for patients with FBXW7 wild type, with a statistically significant difference (P=0.046). The bioinformatics analysis indicated that patients with FBXW7 mutation have higher clinical benefits from immunotherapy. Moreover, patients with FBXW7 mutation in immunotherapy group had higher tumor mutational burden (TMB) [M (Q1, Q3): 17.8 (11.5, 29.3)/Mb] than those with FBXW7 wild type [5.7 (3.0, 10.4)/Mb, P=0.001]. The TMB of patients with FBXW7 mutation in TCGA group was 15.9 (4.2, 28.1)/Mb, insertion-deletion (Indel) neoantigen was 192.5 (70.8, 535.0) and single nucleotide variant (SNV) neoantigen was 363.0 (194.8, 534.8), which were significantly higher than those of patients with FBXW7 wild type [5.6 (3.2, 8.9)/Mb, 53.0 (12.0, 131.0), 83.5 (34.0, 178.0), respectively] (P=0.002, P=0.008, P<0.001). The results indicated that FBXW7 mutated tumors had stronger immunogenicity, which might generate anti-tumor immunity. FBXW7 mutation was also related to the activation of T cells (T lymphocyte receptor complexes and signaling). In addition, FBXW7 mutation was correlated with increased infiltration of CD8+T cells and M1 macrophages. CD8+T cells and M1 macrophages infiltration level was significantly up-regulated in FBXW7 mutation group than wild-type group [10% (8%, 14%) vs 7%(4%, 12%), P=0.049; 8%(4%, 11%) vs 4%(1%, 8%), P=0.046]. Conclusions: NSCLC patients with FBXW7 mutant have higher clinical benefits from immunotherapy. FBXW7 mutation has the potential as a predictive marker for the efficacy of immunotherapy in NSCLC patients.

Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose.

Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H2O2. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

Development of a sandwich ELISA for determining plasma prolactin concentration in domestic birds.

The present study was conducted to establish a sandwich ELISA for the determination of prolactin (PRL) concentrations in the plasma of domestic fowls. The assay uses a recombinant goose PRL as the reference standard, expressed in a eukaryotic system, and as the antigen for raising a polyclonal antibody in rabbit. This rabbit anti-goose PRL polyclonal antibody was used for coating the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and to catalyze the chromogenic reaction using 3,3',5,5'-tetramethylbenzidine as the substrate. The assay showed a linear relationship between the optical density and concentration of the standard PRL in the 0 to 12.5 ng/mL range, and the assay was sensitive to a concentration as low as 0.39 ng/mL. The intra- and inter-assay CVs were <7% and 11%, respectively. The response curves of the serially diluted plasma samples from goose, duck, and chicken exhibited similar parallel relationships to that observed for the reference standards. Consistent with previous findings, the assay effectively detected differences in PRL concentration in plasma samples from chicken, duck, and goose at various reproductive stages.

Dietary -carbamylglutamate and rumen-protected -arginine supplementation ameliorate fetal growth restriction in undernourished ewes.

This study was conducted with an ovine intrauterine growth restriction (IUGR) model to test the hypothesis that dietary -carbamylglutamate (NCG) and rumen-protected -Arg (RP-Arg) supplementation are effective in ameliorating fetal growth restriction in undernourished ewes. Beginning on d 35 of gestation, ewes were fed a diet providing 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations (50% NRC), 50% of NRC recommendations supplemented with 20 g/d RP-Arg (providing 10 g/d of Arg), and 50% of NRC recommendations supplemented with 5 g/d NCG product (providing 2.5 g/d of NCG). On d 110, maternal, fetal, and placental tissues and fluids were collected and weighed. Ewe weights were lower ( < 0.05) in nutrient-restricted ewes compared with adequately fed ewes. Maternal RP-Arg or NCG supplementation did not alter ( = 0.26) maternal BW in nutrient-restricted ewes. Weights of most fetal organs were increased ( < 0.05) in RP-Arg-treated and NCG-treated underfed ewes compared with 50% NRC-fed ewes. Supplementation of RP-Arg or NCG reduced ( < 0.05) concentrations of ß-hydroxybutyrate, triglycerides, and ammonia in serum of underfed ewes but had no effect on concentrations of lactate and GH. Maternal RP-Arg or NCG supplementation markedly improved ( < 0.05) concentrations of AA (particularly arginine-family AA and branched-chain AA) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids within nutrient-restricted ewes. These novel results indicate that dietary NCG and RP-Arg supplementation to underfed ewes ameliorated fetal growth restriction, at least in part, by increasing the availability of AA in the conceptus and provide support for its clinical use to ameliorate IUGR in humans and sheep industry production.

Trace element concentrations and distributions in the main body tissues and the net requirements for maintenance and growth of Dorper × Hu lambs.

A comparative slaughter trial was conducted to estimate the trace element concentrations and distributions in the main body tissues and the net requirements for maintenance and growth of Dorper × Hu crossbred lambs. Thirty-five lambs of each gender (19.2 ± 0.36 kg initial BW) were used. Seven lambs of each gender were randomly chosen and slaughtered at approximately 20 kg BW as the baseline group for measuring initial body composition. Another 7 lambs of each gender were also randomly chosen and offered a pelleted mixed diet for ad libitum intake and slaughtered at approximately 28 kg BW. The remaining 21 sheep of each gender were randomly divided into 3 groups with 7 sheep each and assigned to ad libitum or 40 or 70% of ad libitum intake of a pelleted mixed diet (42:58 concentrate:roughage, DM basis). The 3 groups of each gender were slaughtered when the sheep fed ad libitum reached approximately 35 kg BW. Empty body (head + feet, hide, viscera + blood, and carcass) trace element contents were determined after slaughter. The results showed that the trace elements were mainly distributed in viscera (blood included), except for Zn, which was mainly distributed in the muscle and bone tissues. The net requirements were calculated using the comparative slaughter technique. For males and females, the daily net trace element requirements for maintenance were 356.1 and 164.1 µg Fe, 4.3 and 3.4 µg Mn, 42.0 and 29.8 µg Cu, and 83.5 and 102.0 µg Zn per kilogram empty body weight (EBW), respectively. Net requirements for growth decreased from 65.67 to 57.27 mg Fe, 0.35 to 0.25 mg Mn, and 3.45 to 2.82 mg Cu and increased from 26.36 to 26.65 mg Zn per kilogram EBW gain (EBWG) for males. Net requirements for growth decreased from 30.66 to 22.14 mg Fe, 0.43 to 0.32 mg Mn, 2.86 to 2.18 mg Cu, and 27.71 to 25.83 mg Zn per kilogram EBWG for females from 20 to 35 kg BW. This study indicated that the net trace element requirements for Dorper × Hu crossbred lambs may be different from those of purebred or other genotypes, and more data are needed for sheep in general.

The effect of monosialotetrahexosylganglioside (GM1) in prevention of oxaliplatin induced neurotoxicity: a retrospective study.

Oxaliplatin is a key agent in the treatment of colorectal cancer. However, peripheral neuropathy markedly limits the use of oxaliplatin. This retrospective study was performed to assess the efficacy of monosialotetrahexosylganglioside (GM1) for preventing oxaliplatin induced neurotoxicity. Patients with colorectal cancer treated with oxaliplatin based chemotherapy (FOLFOX or XELOX) were retrospectively divided into two groups according to the use of GM1. The severity of neurotoxicity and efficacy of oxaliplatin were evaluated. A total of 278 cases were included, 114 in GM1 group and 164 in control group. A significantly lower incidence of grade 1-3 acute neurotoxicity (81% vs 92%, p=0.006), grade 2 acute neurotoxicity (26% vs 45%, p=0.002) was observed in GM1 group. Similarly, incidence of grade 1-3 (30% vs 48%, p=0.003) and grade 3 chronic neurotoxicity (4% vs 13%, p=0.021) was also lower in GM1 group. No difference was detected in objective response rate, progress free survival, and median overall survival between GM1 group and control group. The retrospective study demonstrated that GM1 significantly reduced the incidence of oxaliplatin induced neuropathy, especially severe neuropathy, without impairment of efficacy. Prospective trials of GM1 as neuroprotective of oxaliplatin treatment in colorectal cancer are warranted.

Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene.

BACKGROUND: Because of increased resistance to apoptosis in tumor cells, inhibition of specific anti-apoptotic factors may provide a rational approach for the development of novel therapeutic strategies. Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC - 7901 cell by shRNA-mediated silencing of the livin gene. METHODS: The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay. The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method. RESULTS: The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P<0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P<0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P<0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P<0.01). CONCLUSIONS: Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis. Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.