Case Report: Clinical application of continuous arterial infusion chemotherapy in neoadjuvant therapy for locally advanced gastric cancer.

Platinum-fluorouracil combination chemotherapy is the standard neoadjuvant treatment for locally advanced gastric cancer in China, but it does not improve the survival benefit of patients. In recent years, the application of immune checkpoint inhibitors and/or targeted drugs in neoadjuvant therapy for gastric cancer has achieved certain efficacy, but the survival benefit of patients is still not obvious. Intra-arterial infusion chemotherapy, as a method of regional therapy, has been widely used in the treatment of many advanced tumors and achieved remarkable curative effect. The role of arterial infusion chemotherapy in neoadjuvant therapy for gastric cancer is not clear. We describe two patients with locally advanced gastric cancer treated with continuous arterial infusion neoadjuvant chemotherapy. Two patients received continuous arterial infusion of chemotherapy drugs for 50 hours, the drugs were pumped into the main feeding artery of the tumor through the arterial catheter. A total of 4 cycles were followed, then undergone surgical resection. The postoperative pathological pCR of two patients was 100%, TRG was 0 grade, and no further anti-tumor therapy was required after operation, achieving clinical cure. During the treatment period, no serious adverse events occurred in either patient. These results suggest that continuous arterial infusion chemotherapy may be a new adjuvant therapy for locally advanced gastric cancer.

Syndiospecific Copolymerization of Styrene with para-Methoxystyrene Catalyzed by Functionalized Fluorenyl-Ligated Rare-Earth Metal Complexes.

The development of efficient catalysts for the copolymerization of nonpolar monomers with polar monomers remains a great challenging task in polymer synthesis. A one-pot reaction of anhydrous LnCl3 with pyridyl-methylene-functionalized octamethylfluorenyl lithium OctFlu-CH2PyLi in a 1:1 molar ratio, followed by alkylation with 2 equiv of LiCH2SiMe3 in THF afforded the fluorenyl-ligated rare-earth metal bis(alkyl) complexes (OctFlu-CH2Py)Ln(CH2SiMe3)2(THF) [Ln = Sc (1), Y (2)]. Both complexes were isolated as neutral species and were characterized by NMR spectrum and elemental analysis. Complex 2 was subjected to single-crystal X-ray diffraction, which showed that the whole modified fluorenyl ligand was coordinated to Y3+ in the η5/κ1 mode to form a constrained geometry configuration. In the presence of excess AliBu3, and on activation with 1 equiv of [Ph3C][B(C6F5)4] in toluene, complexes 1 and 2 became active for both styrene (St) and para-methoxystyrene (pMOS) polymerization, giving polymers with high syndiotacticity (rrrr > 99%) without solvent extraction. Moreover, the ternary catalyst system composed of complex 2/AliBu3/[Ph3C][B(C6F5)4] was highly effective for the syndiospecific copolymerization of styrene with pMOS, producing random copolymers with high molecular weights and narrow molecular weight distributions. The contents of pMOS in the copolymers could be easily tuned in a wide range (11-93 mol %) by simply changing the pMOS-to-St feed ratios.

Integrin αV Mediates the Effects of Irisin on Human Mature Adipocytes.

Introdution and Aims: The myokine irisin is critical to modulating adipocytes thermogenesis and influence whole-body metabolism. However, whether there is difference in the effects of irisin on adipocytes derived from different depots remains unknown, and the receptor of irisin on adipocytes is still unclear. In this study, we determine the browning effect of irisin on adipocytes of subcutaneous and visceral human adipose tissue and explore the possibility that integrin αV was the receptor of irisin on human adipocytes. METHODS: Human adipose-derived stem cells were isolated from human subcutaneous and visceral white adipose tissues and induced to differentiate into mature adipocytes, and the expression of UCP1 and thermogenic genes in mature adipocytes were examined with or without irisin treatment and compared between groups of different adiposity and different spots. Immunoprecipitation analysis was used to detect the interaction between irisin and integrin αV on adipocytes, and the protein expression of integrin αV in adipocytes was also compared between groups of different adiposity and anatomic position. RESULTS: Irisin treatment could increase the expression level of beige adipocyte marker protein UCP1 and specific thermogenic genes in mature adipocytes derived from subcutaneous white adipose tissue but not in visceral adipose tissue. The results of immunoprecipitation showed that irisin could be attached to integrin αV on mature adipocytes, and there was no significant difference in the gene and protein expression of integrin αV in adipocytes, either derived from subcutaneous and visceral adipose tissue, or derived from obese and normal-weight individuals. CONCLUSION: The results of the present study indicated that irisin contributed to the transformation of mature white adipocytes to beige adipocytes in human subcutaneous adipose tissue but not in visceral adipose tissue. Integrin αV may mediate the browning effects of irisin on human mature adipocytes, which could provide the potential therapeutic targets for obesity and metabolic syndrome by promoting human brown adipose tissue activity.

Novel insights into clear cell renal cell carcinoma prognosis by comprehensive characterization of aberrant alternative splicing signature: a study based on large-scale sequencing data.

Clear cell renal cell carcinoma (ccRCC) is the most common type with poor prognosis in kidney tumor. Growing evidence has indicated that aberrant alternative splicing (AS) events are efficacious signatures for tumor prognosis prediction and therapeutic targets. However, the detailed roles of AS events in ccRCC are largely unknown. In our study, level 3 RNA-seq data was acquired from The Cancer Genome Atlas dataset and corresponding AS profiles were detected with the assistance of SpliceSeq software. A total of 2100 aberrant survival-associated AS events were identified via differential expression and univariate cox regression analysis. The final prognostic panel formed by 17 specific events was developed by stepwise least absolute shrinkage and selection operator (LASSO) penalty, with the area under curve (AUC) values of receiver operator characteristic (ROC) curves keeping above 0.7 spanning 1 year to 5 years. And the results from functional enrichment analyses are unanimous that autophagy could be a potential mechanism of splicing regulation in ccRCC. Furthermore, splicing regulatory network was constructed via Spearman correlation between splicing factors and AS events. Finally, unsupervised clustering analysis revealed three clusters with distinct survival patterns, and associated with specific clinicopathological phenotypes. In overall, we developed a robust and individualized predictive model based on large-scale sequencing data. The identified AS events and splicing network may be valuable in deciphering the crucial posttranscriptional mechanisms on tumorigenesis of ccRCC.

A preliminary study on the role of Bacteroides fragilis in stent encrustation.

OBJECTIVE: To preliminarily study the characteristics of bacterial flora distribution in the urine of ureteral stent encrustation patients as well as the relation between Bacteroides and stent encrustation. METHODS: Patients undergoing ureteral stenting were included in the study and divided into encrustation group and non-encrustation group based on the condition of stent encrustation. The urine of patients was collected to undergo 16s DNA test to compare the bacterial flora distribution characteristics of the two groups. The bacterial genus with highest abundance in the urine of encrustation group was used for animal experiment. A rat model with a foreign body in the bladder was created, in which the rats were injected with the aforesaid bacterial genus. A control group injected with normal saline was also formed. The incidence of foreign body tube encrustation between the two groups was compared. RESULTS: The urine collected from the patients in encrustation group contained a variety of bacteria, while dominant bacteria genera included g_Lactobacillus (23.1%), g_Bacteroides (18.8%) and g_norank_Bacteroides (17.1%). While the urine from the non-encrustation group was less diverse in bacteria flora, as the major bacteria genera were g_Escherichia-Shigella (32.2%), g_Enterococcus (24.9%) and g_Pseudomonas (18.2%). Bacteroidetes in the encrustation group were significantly higher, therefore Bacteroides fragilis in this genus was adopted for animal experiment, resulting in a higher incidence of foreign body tube encrustation in the bladder among rats. CONCLUSION: The present study enriches our knowledge about ureteral stent encrustation and reveals that the target regulation of urine bacteria is worth further research and clinical application.

Expression, purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243-368).

Zinc finger protein ZNF191(243-368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243-368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243-368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His6-tag system could be used to express ZNF191(243-368). The presence of the His6-tag at the N-terminus of ZNF191(243-368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His6-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.

Valproic acid sensitizes metformin-resistant human renal cell carcinoma cells by upregulating H3 acetylation and EMT reversal.

BACKGROUND: Metformin (Met) is a widely available diabetic drug and shows suppressed effects on renal cell carcinoma (RCC) metabolism and proliferation. Laboratory studies in RCC suggested that metformin has remarkable antitumor activities and seems to be a potential antitumor drug. But the facts that metformin may be not effective in reducing the risk of RCC in cancer clinical trials made it difficult to determine the benefits of metformin in RCC prevention and treatment. The mechanisms underlying the different conclusions between laboratory experiments and clinical analysis remains unclear. The goal of the present study was to determine whether long-term metformin use can induce resistance in RCC, whether metformin resistance could be used to explain the disaccord in laboratory and clinical studies, and whether the drug valproic acid (VPA), which inhibits histone deacetylase, exhibits synergistic cytotoxicity with metformin and can counteract the resistance of metformin in RCC. METHODS: We performed CCK8, transwell, wound healing assay, flow cytometry and western blotting to detect the regulations of proliferation, migration, cell cycle and apoptosis in 786-O, ACHN and metformin resistance 786-O (786-M-R) cells treated with VPA, metformin or a combination of two drugs. We used TGF-ß, SC79, LY294002, Rapamycin, protein kinase B (AKT) inhibitor to treat the 786-O or 786-M-R cells and detected the regulations in TGF-ß /pSMAD3 and AMPK/AKT pathways. RESULTS: 786-M-R was refractory to metformin-induced antitumor effects on proliferation, migration, cell cycle and cell apoptosis. AMPK/AKT pathways and TGF-ß/SMAD3 pathways showed low sensibilities in 786-M-R. The histone H3 acetylation diminished in the 786-M-R cells. However, the addition of VPA dramatically upregulated histone H3 acetylation, increased the sensibility of AKT and inhibited pSMAD3/SMAD4, letting the combination of VPA and metformin remarkably reappear the anti-tumour effects of metformin in 786-M-R cells. CONCLUSIONS: VPA not only exhibits synergistic cytotoxicity with metformin but also counteracts resistance to metformin in renal cell carcinoma cell. The re-sensitization to metformin induced by VPA in metformin-resistant cells may help treat renal cell carcinoma patients.

Unmet needs in health training among nurses in rural Chinese township health centers: a cross-sectional hospital-based study.

PURPOSE: Maintaining a sufficient and competent rural nursing workforce is an important goal of the Chinese health delivery system. However, few studies have investigated the health training status or conducted a needs assessment of rural Chinese nurses during this time of great transformations in health policy. This study was conducted to explore the current health training status of nurses working in rural Chinese township health centers (THCs) and to ascertain their perceived needs. METHODS: A cross-sectional survey using a self-administered structured questionnaire was conducted among 240 THC nurses in Guangxi Zhuang Autonomous Region, China from March 2014 to August 2014. The survey questionnaire was adapted from the Second Chinese Survey of Demographic Data and Training Demand for Health Professionals in THCs developed by the Ministry of Education. RESULTS: The nurses in THCs were young, with a low educational level. Their perceived needs for health training included further clinical studies at city-level hospitals to improve their skills and theoretical studies at medical universities in emergency medicine and general practice. Overall, 71.9% of the nurses with a secondary technical school background expected to pursue junior college studies, and 68.5% of the nurses with a junior college education expected to pursue a bachelor's degree. A decentralized program with theoretical studies at medical universities and practical studies at county hospitals was regarded as feasible by 66.9% of the respondents. CONCLUSION: Health-training programs for nurses in Chinese THCs must be improved in terms of coverage, delivery mode, and content. A decentralized degree-linked training program in which medical universities and city hospitals collaborate would be an appropriate mode of delivery.

[Investigation of Lung Cancer Patients Complicated with Chronic Obstructive Pulmonary Disease in Thoracic Surgical Department].

BACKGROUND: Lung cancer is an important complication of chronic obstructive pulmonary disease (COPD), and even significantly affects the prognosis of patients with COPD. COPD also affects the postoperative complications and recurrence in patients with lung cancer. This study aims to investigate lung cancer patients complicated with COPD in thoracic surgical department. METHODS: All medical records of lung cancer patients discharged from the Department of Thoracic Surgery of People's Hospital, Peking University during January 2015 and December 2015 were reviewed, including gender, age, tobacco smoke history, harmful occupational exposure, clinic symptom, chest computed tomography (CT) scanning, postoperative pathology result report, discharged diagnosis and spirometry [All patients underwent pulmonary function test are received bronchial dilation test if the based predicted value of forced expiratory volume in one second (FEV1) <70%]. RESULTS: A full set of lung function test was measured in 703 lung cancer patients. Bronchial dilation test was finished in 67 patients. 62 (92.5%) patients were diagnosed as COPD. 677 cases with lung cancer were received surgery. Bronchial dilation test was measured in 42 cases. Of them 38 (92.7%) patients were diagnosed as COPD. It was found that the patients with lung cancer and COPD was more frequent in males, elders (≥65 yr), smokers, non-adenocarcinoma patients than those of patients without COPD (P<0.05). The males and the elders (≥65 yr) were more likely to suffer from COPD (OR: 2.374-2.807, 95%CI: 1.101-7.157)(P<0.05). Only 3 patients (4.3‰) were diagnosed as COPD and received standard treatment before admission. And only 5 patients (7.1‰) were diagnosed as COPD as discharged. CONCLUSIONS: The routine pulmonary function as well as bronchial dilation test are helpful for screening the patients with COPD. At present, the diagnosis and treatment of lung cancer combined with COPD is a serious problem, which needs to be paid attention to by thoracic surgeons and to join hands with physicians in order to improve the diagnosis level of COPD.

Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo.

Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear. Here fluorophore FlAsH-EDT2 and fluorescent proteins were employed to study the NO-induced sGC activation. FlAsH-EDT2 labeling study revealed that NO binding to the H-NOX domain of sGC increased the distance between H-NOX and PAS domain and the separation between H-NOX and coiled-coil domain. The heme pocket conformation changed from "closed" to "open" upon NO binding. In addition, the NO-induced conformational change of sGC was firstly investigated in vivo through fluorescence lifetime imaging microscopy. The results both in vitro and in vivo indicated the conformational change of the catalytic domain of sGC from "open" to "closed" upon NO binding. NO binding to the heme of H-NOX domain caused breaking of Fe-N coordination bond, initiated the domain moving and conformational change, induced the allosteric effect of sGC to trigger the NO-signaling from H-NOX via PAS &coiled-coil to the catalytic domain, and ultimately stimulates the cyclase activity of sGC.

Valproic acid induces autophagy by suppressing the Akt/mTOR pathway in human prostate cancer cells.

Previous studies have demonstrated that the chronic administration of valproic acid (VPA) suppresses angiogenesis in vivo; however, the mechanisms implicated in VPA-induced autophagy remain unclear. The current study aimed to assess VPA-induced autophagy in three prostate cancer cell lines (PC3, DU145 and LNCaP), in addition to analyzing the Akt/mammalian target of rapamycin (mTOR) signal pathway. Prostate cancer cell lines were cultured with various doses of VPA. Cell cycle was analyzed using flow cytometry, and autophagy markers [1A/1B-light chain 3 (LC3)-II and Beclin-1] were examined using transmission electron microscopy, fluorescent microscopy and western blotting. Activation of the Akt/mTOR signal pathway was also assessed by western blotting. The results demonstrated that VPA induced autophagosomes and suppressed the Akt/mTOR signal pathway. This was confirmed by detection of increased LC3-II and Beclin-1 in VPA-treated cells compared with untreated controls. Phosphorylated forms of Akt (PC3, P=0.048; DU145, P=0.045; LNCaP, P=0.039) and mTOR (PC3, P=0.012; DU145, P=0.41; LNCaP, P=0.35) were significantly reduced following VPA treatment. These results suggest that VPA may function as a histone deacetylase inhibitor, suppressing the growth of prostate cancer cells by modulating autophagy pathways, including inhibition of the Akt/mTOR pathway. Further experiments are required to determine the significance of all involved pathways regarding VPA-induced growth inhibition.

Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368).

Zinc finger proteins are associated with hereditary diseases and cancers. To obtain an adequate amount of zinc finger proteins for studying their properties, structure, and functions, many protein expression systems are used. ZNF191(243-368) is a zinc finger protein and can be fused with His-tag to generate fusion proteins such as His6-ZNF191(243-368) and ZNF191(243-368)-His8. The purification of His-tag protein using Ni-NTA resin can overcome the difficulty of ZNF191(243-368) separation caused by inclusion body formation. The influences of His-tag on ZNF191(243-368) properties and structure were investigated using spectrographic techniques and hydrolase experiment. Our findings suggest that insertion of a His-tag at the N-terminal or C-terminal end of ZNF191(243-368) has different effects on the protein. Therefore, an expression system should be considered based on the properties and structure of the protein. Furthermore, the hydrolase activity of ZNF191(243-368)-His8 has provided new insights into the design of biological functional molecules.

The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change.

Heme oxidation and loss of soluble guanylate cyclase (sGC) is thought to be an important contributor to the development of cardiovascular diseases. Nevertheless, it remains unknown why the heme loses readily in oxidized sGC. In the current study, the conformational change of sGC upon heme oxidation by ODQ was studied based on the fluorescence resonance energy transfer (FRET) between the heme and a fluorophore fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of sGC ß1. This study provides an opportunity to monitor the domain movement of sGC relative to the heme. The results indicated that heme oxidation by ODQ in truncated sCC induced the heme-associated αF helix moving away from the heme, the Per/Arnt/Sim domain (PAS) domain moving closer to the heme, but led the helical domain going further from the heme. We proposed that the synergistic effect of these conformational changes of the discrete region upon heme oxidation forces the heme pocket open, and subsequent heme loss readily. Furthermore, the kinetic studies suggested that the heme oxidation was a fast process and the conformational change was a relatively slow process. The kinetics of heme loss from oxidized sGC was monitored by a new method based on the heme group de-quenching the fluorescence of FlAsH-EDT2.

Recognition Code of ZNF191(243-368) and Its Interaction with DNA.

ZNF191(243-368) is the C-terminal region of ZNF191 which contains a putative DNA-binding domain of four Cys2His2 zinc finger motifs. In this study, an expression vector of a fusion protein of ZNF191(243-368) with glutathione-S-transferase (GST) was constructed and transformed into Escherichia coli BL21. The fusion protein GST-ZNF191(243-368) was expressed using this vector to investigate the protein-DNA binding reaction through an affinity selection strategy on the basis of the binding quality of the zinc finger domain. Results showed that ZNF191(243-368) can selectively bind with sequences and react with genes which contain an AGGG core. However, the recognition mechanism of Cys2His2 zinc finger proteins to DNA warrants further investigation.

Structural basis for cytochrome c Y67H mutant to function as a peroxidase.

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe(3+) ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.

Role of SMAD4 in the mechanism of valproic acid's inhibitory effect on prostate cancer cell invasiveness.

PURPOSE: To investigate the influence of the histone deacetylase inhibitor valproic acid (VPA) on SMAD4 expression and invasive ability of prostate cancer cell lines. METHODS: DU145 and PC3 cell lines were treated with 0, 2, and 5 mMol/l of VPA; invasion of DU145 and PC3 cells were then examined by transwell assay. Immunohistochemistry and Western blot were used to examine SMAD4 protein expression in DU145 and PC3 cells. RESULTS: Compared with controls, VPA significantly suppressed invasiveness in both PC3 and DU145 cells in a dose-dependent way (P < 0.05). VPA also inhibited AKT protein (which was regarded as an effective indicator here), and meanwhile, SMAD4 expression was down-regulated after VPA treatment in a dose-dependent manner in both DU145 (P < 0.05) and PC3 (P < 0.01) cells. CONCLUSIONS: Valproic acid could suppress invasiveness of prostate cancer cell lines PC3 and Du145, possibly through multiple pathways other than the SAMD4 pathway. This implies that VPA treatment combined with other SMAD4 enhancers could form a basis for a novel prostate cancer treatment.

Probing the metal specificity mechanism of superoxide dismutase from human pathogen Clostridium difficile.

The molecular mechanism of the metal specificity to superoxide dismutase from human pathogen C. difficile (SODcd) was investigated by X-ray crystallography, spectroscopy, SOD activity assay, electrochemistry, and DFT calculations, and the results indicate that the cognate metal characters tuned by the metal micro-environment dominate the metal specificity of the SODcd.

Exploring the differences between mouse mAß(1-42) and human hAß(1-42) for Alzheimer's disease related properties and neuronal cytotoxicity.

The differences between mouse mAß(1-42) and human hAß(1-42), explored using CD and fluorescence spectroscopy, transmission electron microscopy, ROS fluorescent assay, and neuronal cell viability, revealed that mAß(1-42) as a three-site mutant (R5G, Y10F and H13R) of hAß(1-42) altered the metal (copper and zinc) binding sites, reduced the proneness to form ß-sheet structures and aggregated fibrils, alleviated the generation of ROS, and decreased the cytotoxicity, in contrast to hAß(1-42).

Generation of novel functional metalloproteins via hybrids of cytochrome c and peroxidase.

The continued interest in protein engineering has led to intense efforts in developing novel stable enzymes, which could not only give boost to industrial and biomedical applications, but also enhance our understanding of the structure-function relationships of proteins. We present here the generation of three hybrid proteins of cytochrome c (cyt c) and peroxidase via structure-based rational mutagenesis of cyt c. Several residues (positions 67, 70, 71 and 80) in the distal heme region of cyt c were mutated to the highly conserved amino acids in the heme pocket of peroxidases. The multiple mutants were found to exhibit high peroxidase activity and conserve the impressive stability of cyt c. We expect that this strategy could be extended to other cases of metalloprotein engineering, and lead to the development of stable and active biocatalysts for industrial uses. Besides, this study also provides insight into the structure-function relationships of hemoproteins.