Gut microbiota and metabolites signatures of clinical response in anti-PD-1/PD-L1 based immunotherapy of biliary tract cancer.

BACKGROUND: Accumulating evidence suggests that the gut microbiota and metabolites can modulate tumor responses to immunotherapy; however, limited data has been reported on biliary tract cancer (BTC). This study used metagenomics and metabolomics to identify characteristics of the gut microbiome and metabolites in immunotherapy-treated BTC and their potential as prognostic and predictive biomarkers. METHODS: This prospective cohort study enrolled 88 patients with BTC who received PD-1/PD-L1 inhibitors from November 2018 to May 2022. The microbiota and metabolites significantly enriched in different immunotherapy response groups were identified through metagenomics and LC-MS/MS. Associations between microbiota and metabolites, microbiota and clinical factors, and metabolites and clinical factors were explored. RESULTS: Significantly different bacteria and their metabolites were both identified in the durable clinical benefit (DCB) and non-durable clinical benefit (NDB) groups. Of these, 20 bacteria and two metabolites were significantly associated with survival. Alistipes were positively correlated with survival, while Bacilli, Lactobacillales, and Pyrrolidine were negatively correlated with survival. Predictive models based on six bacteria, four metabolites, and the combination of three bacteria and two metabolites could all discriminated between patients in the DCB and NDB groups with high accuracy. Beta diversity between two groups was significantly different, and the composition varied with differences in the use of immunotherapy. CONCLUSIONS: Patients with BTC receiving immunotherapy have specific alterations in the interactions between microbiota and metabolites. These findings suggest that gut microbiota and metabolites are potential prognostic and predictive biomarkers for clinical outcomes of anti-PD-1/PD-L1-treated BTC.

EMS1/DLL4-Notch Signaling Axis Augments Cell Cycle-Mediated Tumorigenesis and Progress in Human Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant neoplasm that is prone to local invasion and metastasis. Meanwhile, overexpressed endothelial cell-specific molecule 1 (ESM1) is closely related to tumorigenesis of multitudinous tumors. However, the prognosis value and biological function of ESM1 in ACC remains undefined. In the current essay, the assessment in human ACC samples and multiple public cancer databases suggested that ESM1 was significantly overexpressed in ACC patients. The abnormal expression of ESM1 was evidently correlated with dismal overall survival (OS) in ACC patients. Then, the gene-set enrichment analysis (GSEA) was applied to unravel that ESM1 was mostly involved in cell cycle and Notch4 signaling pathway. Furthermore, in vitro experiment, RNA interference of ESM1 was carried out to state that ESM1 augments CDK1 and p21-mediated G2/M-phase transition of mitosis, cell proliferation via DLL4-Notch signaling pathway in human ACC cell line, SW13 cells. Additionally, two possible or available therapeutic strategies, including immunotherapy and chemotherapy, have been further explored. Immune infiltration analysis highlighted that no significant difference was found in ACC patients between EMS1high and EMS1low group for immune checkpoint-related genes. In addition, the overexpression of ESM1 might trigger the accumulation of tumor mutation burden (TMB) during the cell cycle of DNA replication in ACC. The gene-drug interaction network then indicated that ESM1 inhibitors, such as cisplatin, might serve as potential drugs for the therapy of ACC. Collectively, the results asserted that ESM1 and related regulators might act as underlying prognostic biomarkers or novel therapeutic targets for ACC.

What Causes Postpartum Depression and How to Cope with It: A Phenomenological Study of Mothers in China.

Culturally specific beliefs about mental illnesses influence people's experience of mental illnesses and their likelihood of seeking help. This study explores how mothers in China understand the causes of postpartum depression (PPD) and their preferred coping strategies through semi-structured interviews. It finds that these mothers often take a situational approach to PPD, attributing it to external factors that could be changed (e.g., lack of support from in-laws) or to internal factors that will disappear over time (e.g., hormonal fluctuation). As a result, these mothers often choose to cope with their PPD symptoms through self-help strategies such as talking to someone, enlisting help, and positive thinking. Seeking professional help is not a preferred coping strategy. Furthermore, participants often make different attributions about the PPD experiences of themselves and the experiences of others. In explaining other women's PPD symptoms, they are more likely to evoke the myths about gender and motherhood in the Chinese culture and blame PPD on these mothers' personality flaws, husbands' affairs, and financial hardships. Practically, our findings provide baseline data for the promotion of professional mental health care among mothers experiencing PPD in China.

Aberrant expression of thyroid transcription factor-1 in schwannomas.

Aberrant expression of thyroid transcription factor-1 (TTF-1) has been observed in tumors arising in locations other than thyroid gland, lung and ventral forebrain. However, TTF-1 expression in schwannomas has not yet been studied. Meanwhile, a few inconsistent changes in protein expression have been identified between schwannomas and other peripheral nerve sheath tumors. We evaluated TTF-1 expression in 161 schwannomas and 43 other peripheral nervous system lesions, including ganglioneuromas (n = 8), malignant peripheral nerve sheath tumors (MPNSTs) (n = 11), neurofibromas (n = 24), and traumatic neuromas (n = 9), using immunohistochemistry and verified it using quantitative real-time reverse-transcription polymerase chain reaction (qPCR) to explore TTF-1 expression in peripheral nervous system lesions. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained for both analyses. In this study, we observed nuclear TTF-1 staining in 109 (67.7%) schwannomas, including 102 of 131 (77.9%) conventional, 1 of 20 (5.0%) cellular and 6 of 10 (60.0%) plexiform schwannomas. Nuclear staining was not observed in normal peripheral nerves and non-schwannoma lesions. qPCR verified the aberrant expression and revealed a correlation between TTF-1 protein and mRNA levels (r = 0.633, P = .003). In conclusion, the data from our study show that TTF-1 is selectively expressed in the majority of schwannomas, particularly the conventional variants. Based on this observation, the TTF-1 protein and mRNA are specifically expressed in schwannomas. This highly aberrant expression of varying amounts of TTF-1 may provide new clues to reveal the pathogenesis of schwannoma.

Postpartum Depression and Social Support in China: A Cultural Perspective.

This study explored how Chinese culture affects the relationship between social support and postpartum depression. In-depth interviews with 38 mothers in mainland China showed that discrepancies between expected and perceived available social support and conflicts among social support providers are two major contributors to the stress associated with postpartum depression. These dynamics are deeply rooted in the context of Chinese culture with its distinctive gender roles and family dynamics. These cultural norms further prevent women from seeking social support.

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array.

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.

Effect of Annealing Temperature on the Performance of SnO2 Thin Film Transistors Prepared by Spray Pyrolysis.

We fabricated SnO2 thin film transistors on thermally oxidized p-type silicon substrates by low-cost spray pyrolysis. The effect of annealing temperatures on electrical characteristics of SnO2 thin film transistors were investigated. Thermal annealing at higher temperatures induced a negative shift of the threshold voltage (VT) and an increase in the saturation mobility. It was found that the device annealed at 450 °C exhibited a good electrical performance with the field-effect mobility of 0.19 cm2/Vs, the threshold voltage of 2.5 V, and the on/off current ratio of 10(3).

Tissue distribution of six major bio-active components after oral administration of Zhenqi Fuzheng capsules to rats using ultra-pressure liquid chromatography-tandem mass spectrometry.

Radix Astragali (Huangqi in Chinese) and Fructus Ligustri Lucidi (Nvzhenzi in Chinese) (2:1, w/w) are combined in an herbal formulation called Zhenqi Fuzheng capsules (ZFCs) for use in China to improve immunity, promote the recovery of normal functions after surgical operations, and as the most important adjuvant therapy in cancer. In this study, the tissue distribution profiles of the six major bio-active constituents (calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV and astragaloside II) were examined after oral administration of ZFCs to rats. All six constituents in each tissue were detected simultaneously using UPLC-ESI-MS, and the concentration of each constituent per gram of each tissue was determined. Quantification was performed using low-energy collision tandem mass spectrometry (CID-MS/MS) in multiple reaction monitoring (MRM) scan mode for the following precursor ion→product ion transitions at m/z 447.21→285.30 for calycosin-7-O-ß-D-glucoside, m/z 285.29→270.38 for calycosin, m/z 431→269 for ononin, m/z 269→237 for formononetin, m/z 807.40→627.50 for astragaloside IV, m/z 849.60→669.65 for astragaloside II and m/z 633.18→331.18 for the internal standard (hesperidin). The results showed that in general the tissue concentrations for all six constituents were in the following order: spleen>stomach>thymus>lung>liver>kidney>heart>testicle. The high levels in the spleen and thymus indicated that all six compounds accumulated in organs involved in the immune response, consistent with the immunity effects of ZFC. The high levels in the stomach were consistent with the oral administration of ZFC. This study was the first to compare the tissue distribution of calycosin-7-O-ß-D-glucoside with that of calycosin or of ononin with that of formononetin in rats. It was also the first study to examine the tissue distribution of astragaloside II, calycosin and formononetin following oral administration of ZFC to rats.

Simultaneous determination of 10 components in Bu-Zhong-Yi-Qi Wan by solid phase extraction-high performance liquid chromatography with diode array detection and evaporative light scattering detection.

An effective, accurate and reliable method for the simultaneous separation and determination of 10 major components in Chinese medicine Bu-Zhong-Yi-Qi Wan (BZYQW) was developed and validated using solid phase extraction column-high performance liquid chromatography-diode array detection-evaporative light scattering detection (SPE-HPLC-DAD-ELSD). The chromatographic separation was performed on a Spursil™ C18 column (250 mm × 4.6 mm, 5 µm) at 30°C with an acetonitrile-water gradient as mobile phase. The DAD detection wavelength 254 nm was utilized for the quantitative analysis. The drift tube temperature and the carrier gas flow rate of the ELSD detection was set at 110.5°C and 3.1 mL/min. The total run time is 103 min, these determined components peak out within 81 min. Excellent linear behaviors over the investigated concentration ranges were observed with the values of r(2) higher than 0.9990 for all the analytes. The Linear range over hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I is 4.50-94.50 µg/mL, 22.75-364.00 µg/mL, 2.30-45.00 µg/mL, 11.76-125.14 µg/mL, 4.62-50.35 µg/mL, 1.90-28.93 µg/mL, 1.29-159.00 µg/mL, 2.90-36.00 µg/mL, 35.40-192.40 µg/mL, 41.40-96.60 µg/mL, respectively. The method was validated by its repeatability [relative standard deviation (RSD) < 3.54%] and intra-day (RSD < 2.11%) and inter-day precision (RSD < 3.45%). The limits of detection and quantification of each component were in the ranges of 0.04-10.24 and 0.12-39.22 µg/mL, respectively. The average recovery yields of the 10 compounds ranged from 95.79 to 101.25%. The validated method was successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW from different manufacturers.

Neoadjuvant chemotherapy with FOLFOX4 regimen to treat advanced gastric cancer improves survival without increasing adverse events: a retrospective cohort study from a Chinese center.

BACKGROUND/AIM: To evaluate the clinical efficacy of FOLFOX4 (5-fluomumcil/leucovorin combined and oxaliplatin) neoadjuvant chemotherapy for advanced gastric cancer (AGC). PATIENTS AND METHODS: Fifty-eight AGC patients were enrolled in this retrospective cohort study, 23 in the neoadjuvant group and 35 in the adjuvant group. R0 resection, survival, and adverse events were compared. RESULTS: The two groups were well-matched, with no significant differences in R0 resection rate (82.6% versus 82.0%) and number of lymph nodes dissection (16 (0-49) versus 13 (3-40)) between the two groups (P > 0.05). The number of lymph node metastases in the neoadjuvant group (3 (0-14)) was significantly fewer than that in the adjuvant group (6 (0-27)) (P = 0.04). The neoadjuvant group had significantly better median overall survival (29.0 versus 22.0 months) and 3-year survival rate (73.9% versus 40.0%) than the adjuvant group (P = 0.013). The positive expression rate of Ki-67 in the neoadjuvant group (40.0%, 8/20) was lower than that in the adjuvant group (74.2%, 23/31; P = 0.015). CONCLUSION: The FOLFOX4 neoadjuvant chemotherapy could improve survival without increasing adverse events in patients with AGC.

Simultaneous determination of calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of Radix Astragali extraction for their pharmacokinetic studies by ultra-pressure liquid chromatography with tandem mass spectrometry.

A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC-MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters Oasis(TM) HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC™ BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r (2) > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-ß-D-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-ß-D-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.

Component analysis and structure identification of active substances for anti-gastric ulcer effects in Radix Astragali by liquid chromatography and tandem mass spectrometry.

This study provided a comprehensive component analysis and structure identification of active substances for the anti-gastric ulcer effects of Radix Astragali. The data were generated by organically combining the results from in vivo pharmacodynamic experiments, a cell growth-promoting assay, structure identification, content determination, fingerprinting, and correlation analyses. The fingerprints from high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and from HPLC coupled with evaporative light scattering detectors (ELSD) from 95% ethanol extracts of Radix Astragali (ERA) were determined using HPLC-DAD-ELSD. The structures of 16 compounds were identified using ultra-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The contents of these 16 compounds were simultaneously determined in a single run using HPLC-DAD-ELSD. The strength of the anti-ulceration effect of each of the 16 compounds was correlated to its content in the HPLC spectrum using gray relation statistics. The sequence of the contribution from each of the 16 compounds to the anti-gastric ulcer effect was determined. The results showed that ononin, astragalosideIII, and astragalosideIV contributed most to the observed anti-gastric ulcer effects and that these three compounds also exhibited strong growth-promoting effects in cultured GES-1 cells. The results of this study can be used to evaluate the quality of Radix Astragali and to provide a theoretical foundation for its further study.

A Novel Phosphopeptide Microarray Based Interactome Map in Breast Cancer Cells Reveals Phosphoprotein-GRB2 Cell Signaling Networks.

The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.

[Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)].

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P < 0.01 or P < 0.05), but the content of lactic acid in serum of plateau pika was significantly higher than that of plateau zokor (P < 0.01); (2) MDH1 and MDH2 mRNA levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P < 0.01 or 0.05), AGC mRNA level of the zokor was significantly higher than that of the pika (P < 0.01), while no difference was found at OMC mRNA level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P < 0.01), MDH1 mRNA level of plateau zokor was markedly higher than that of MDH2 (P < 0.01), but the activities had no difference between MDH1 and MDH2 in liver of the zokor (P > 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen.

[Gene coding and mRNA expression of vascular endothelial growth factor as well as microvessel density in brain of plateau zokor: comparison with other rodents].

Vascular endothelial growth factor (VEGF) plays an important role in tissues angiogenesis. The adaptation of animals to hypoxic environment is relative to the microvessel density (MVD) in tissues. To further explore the adaptation mechanisms of plateau zokor (Myospalax baileyi) to the hypoxic-hypercapnic burrows, the VEGF mRNA and the MVD in cerebral tissues of the plateau zokor were studied. Total RNA was isolated from liver, and VEGF cDNA was obtained by RT-PCR, then the VEGF cDNA was cloned and sequenced. The coding sequence of plateau pika (Ochotona curzniae), rat (Rattus norvegicus) and mouse (Mus musculus) VEGF cDNA are obtained from GenBank, and the nucleotide and amino acid sequence homology of plateau zokor VEGF cDNA coding sequence with that of plateau pika, rat and mouse were analyzed and compared by using of bioinformatics software. The VEGF mRNA was detected by real-time PCR, and the MVDs in cerebral tissues of the plateau zokor, plateau pika and Sprague-Dawley (SD) rat were measured by immunohistochemical staining. The results showed that the open reading frame of the plateau zokor VEGF was 645 bp, and the coding sequence of the plateau zokor VEGF cDNA shared 92.1%, 93.6% and 93.8% nucleotide sequence homology to that of the plateau pika, rat and mouse, respectively. The deduced amino acid sequence of the plateau zokor VEGF cDNA was composed of 188 amino acids and the amino acids from 1 to 26 were signal peptide sequence. The plateau zokor VEGF188 was 90.2%, 94.9% and 94.4% homologous to that of plateau pika, rat and mouse. The level of VEGF mRNA in brain of the plateau zokor was significantly lower than that of SD rat, but there was no obvious difference in VEGF mRNA level between plateau zokor and plateau pika. The MVD in brain of the plateau zokor was markedly higher than that of plateau pika and SD rat. In conclusion, plateau zokor enhances its adaptation to the hypoxic environment by increasing the MVD. The level of VEGF mRNA in the brain of plateau zokor is lower than that of SD rat, which may be as a result of inhibition by the higher concentration of carbon dioxide in the burrow.

[Study on mitochondrial DNA damage in peripheral blood nucleate cells of the workers exposed to acrylonitrile].

OBJECTIVE: To study the potential aging effect on workers exposed to acrylonitrile (ACN). METHODS: The deletion rates of mitochondrial DNA (mtDNA) in peripheral blood nucleate cells of 47 exposed workers and 47 non-exposed workers (as control), as well as 12 old people and 12 young people were measured with polymerase chain reaction (PCR). RESULTS: The positive rates of mtDNA deletion in peripheral blood nucleate cells were 17.02% in the workers exposed to ACN and 25.00% in group of old people. However, the mtDNA deletion was not detected in the control group and young people. CONCLUSIONS: ACN could induce mtDNA deletion in peripheral blood nucleate cells of the exposed workers. There may be a potential molecular effect of occupational ACN exposure on workers' aging.