Dietary glycemic index and glycemic load predict longitudinal change in glycemic and cardio-metabolic biomarkers among old diabetic adults living in a resource-poor country.

This study aims to investigate longitudinal associations between the dietary glycemic index (GI) and glycemic load (GL) and changes in glycemic and cardio-metabolic outcomes. A 28-month retrospective cohort study included 110 Vietnamese diabetic patients, collecting their dietary GI and GL values along with blood biochemical data from baseline 24-h dietary recall and medical records. Latent class growth modelling identified three distinct HbA1c trajectories during the follow-up period, with 51% of patients achieving good glycemic control. The adjusted linear mixed-effect model showed that 1 unit increase in logarithms in dietary GL was associated with a 0.14% increase in the log-HbA1c. Among poorly controlled diabetic patients, baseline GL values were positively correlated with increases in HbA1c; GI showed effects on changes in fasting plasma glucose and the triglyceride-glucose (TyG) index. No significant association was observed in patients with good glycemic control.

Essential trace element and phosphatidylcholine remodeling: Implications for body composition and insulin resistance.

BACKGROUND: Recent studies indicated that bioactive lipids of phosphatidylcholines (PCs) and lysophosphatidylcholines (LysoPCs) predict unhealthy metabolic phenotypes, but results remain inconsistent. To fill this knowledge gap, we investigated whether essential trace elements affect PC-Lyso PC remodeling pathways and the risk of insulin resistance (IR). METHODS: Anthropometric and blood biochemical data (glucose, insulin, and lipoprotein-associated phospholipase A2 (Lp-PLA2)) were obtained from 99 adults. Blood essential/probably essential trace elements and lipid metabolites were respectively measured by inductively coupled plasma mass spectrometry (ICP-MS), and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). RESULT AND CONCLUSION: Except for LysoPC (O-18:0/0:0), an inverse V shape was observed between body weight and PC and LysoPC species. A Pearson correlation analysis showed that essential/probably-essential metals (Se, Cu, and Ni: r=-0.4∼-0.7) were negatively correlated with PC metabolites but positively correlated with LysoPC (O-18:0/0:0) (Se, Cu, and Ni: r=0.85-0.64). Quantile-g computation showed that one quantile increase in essential metals was associated with a 2.16-fold increase in serum Lp-PLA2 (ß=2.16 (95 % confidence interval (CI): 0.34, 3.98), p=0.023), which are key enzymes involved in PC/Lyso PC metabolism. An interactive analysis showed that compared to those with the lowest levels (reference), individuals with the highest levels of serum PCs (pooled, M2) and the lowest essential/probably essential metals (M1) were associated with a healthier body composition and had a 76 % decreased risk of IR (odds ratio (OR)=0.24 (95 % CI: 0.06, 0.90), p<0.05). In contrast, increased exposure to LysoPC(O-18:0/0:0) (M2) and essential metals (M2) exhibited an 8.22-times highest risk of IR (OR= 8.22 (2.07, 32.57), p<0.05) as well as an altered body composition. In conclusion, overexposure to essential/probably essential trace elements may promote an unhealthy body weight and IR through modulating PC/LysoPC remodeling pathways.

The survival outcome differs between left-sided colon cancer and middle/low rectal cancer after colorectal hepatic metastasectomy.

BACKGROUND: The clinical outcomes between left-sided colon cancer and middle/low rectal cancer seem to be different. This study aimed to examine the effect of primary tumor location regarding the left-sided colon and middle/low rectum on the overall survival (OS) of patients who underwent colorectal hepatic metastasectomy. METHODS: Patients who underwent colorectal hepatic metastasectomy were retrospectively enrolled. Patients were classified into 2 groups according to the primary tumor location (left-sided colon and middle/low rectum). Categorical variables were compared using the chi-square test or Fisher exact test, and continuous variables were analyzed using the Student t test. Survival was analyzed using the Kaplan-Meier method and log-rank test. The prognostic factors were analyzed by univariate and multivariate analyses using Cox proportional hazards regression models. RESULTS: Overall, 365 patients were enrolled. Patients with left-sided colon cancer had significantly better OS than those with middle/low rectal cancer (hazard ratio [HR], 0.725; P = .018), with median OS estimates of 48 and 38 months, respectively. In the subgroup analysis of RAS mutations, patients with left-sided colon cancer had significantly prolonged OS compared with those with middle/low rectum cancer (HR, 0.608; P = .034), with median OS estimates of 49 and 26 months, respectively. This observation was limited to patients with RAS mutations. CONCLUSION: According to our findings, patients with middle/low rectal cancer had poorer survival outcome and should not be categorized together with patients with left-sided colon cancer in terms of OS after colorectal hepatic metastasectomy.

Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.

RATIONALE: Chlorothalonil (CHT), a broad-spectrum fungicide, has been employed widely to control foliar diseases, whereas with a major metabolite of polar 4-hydroxychlorothalonil (CHT-4-OH), only an acceptable nonpolar CHT residue is allowed by most countries. This study involves the method development for CHT residue in vegetables/fruits using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a novel modified discharge-adaptor (DA) interface. METHODS: CHT residue was analyzed using LC-MS/MS with DA interface (LC-DA-MS/MS), developed in our previous works. A DA was placed on the electrospray tip to switch the ionization modes. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to extract CHT residue of vegetables/fruits efficiently with less sample preparation time and analysis cost. RESULTS: CHT and CHT-4-OH spiked in four different vegetables/fruits were extracted using the modified QuEChERS method. After LC with isocratic elution, CHT and CHT-4-OH were separated within 3 min. Using LC-DA-MS/MS, the ion signals of CHT were improved two to three times, and the limit of quantification of 5 ng/g and linearity (r2 > 0.99) in the range of 5-200 ng/g were achieved using 10 g of vegetables/fruits. The precision and accuracy were within 15% each. The modified QuEChERS and LC-DA-MS/MS were applied to examine eight field-grown vegetables/fruits; 9.5 and 2588.9 ng/g of CHT were detected in two vegetables/fruits. CONCLUSION: LC-DA-MS/MS combined with modified QuEChERS was successfully applied to determine CHT residue <10 ng/g in vegetables/fruits and with satisfied validation results. The developed method could reduce both analysis cost and time, attributing to simplifications in modified QuEChERS, isocratic elution, and DA interface in LC-DA-MS/MS.

Weight loss induced by a hypocaloric diet with or without fish oil supplementation re-established iron and omega-3 fatty acid homeostasis in middle-aged women with obesity: A post-hoc analysis of a randomized controlled trial.

OBJECTIVE: Middle-aged women with obesity are at increased risk of iron overload and iron disorder is known to disrupt n-3 polyunsaturated fatty acid homeostasis. We evaluated relationships between pretreatment hemoglobin and n-3 polyunsaturated fatty acid levels, and tested whether pretreatment hemoglobin contributed to inter-individual variability in weight loss with special focus on changes in body weight, iron and n-3 polyunsaturated fatty acid profiles. STUDY DESIGN: 117 middle and older aged women with obesity and more than two metabolic abnormalities were randomized to a 12-week hypocaloric diet without or with fish oil supplementation. Blood iron biomarker and erythrocyte membrane phospholipid profiles were evaluated. MAIN OUTCOME: The absolute change from baseline to week 12 in serum iron and erythrocyte n-3 polyunsaturated fatty acid levels according to pretreatment hemoglobin tertiles and fish oil supplementation. RESULTS: A Pearson correlation analysis showed that pretreatment hemoglobin levels were negatively correlated with linoleic acid (r = -0.231), α-linoleic acid (r = -0.279), and n-3 polyunsaturated fatty acid (r = -0.217) (all p < 0.05). Dietary weight loss markedly enhanced erythrocyte membrane lipids of linoleic acid, α-linoleic acid, and n-6 and n-3 polyunsaturated fatty acid only in those women with the highest pretreatment hemoglobin levels (tertile 3) (all p < 0.05). Fish oil supplementation increased bioavailable iron in women with moderate pretreatment hemoglobin levels (tertile 2) (p < 0.05) and, to a lesser extent, prevented a reduction in circulating iron in those with the lowest hemoglobin levels (tertile 1). CONCLUSION: Dietary weight loss is an effective treatment program to manage obesity-related iron and n-3 polyunsaturated fatty acid disorders, particularly for middle-aged women with obesity and iron overload.

Djulis Hull Enhances the Efficacy of Ferric Citrate Supplementation via Restoring Normal Iron Efflux through the IL-6-Hepcidin-Ferroportin Pathway in High-Fat-Diet-Induced Obese Rats.

Obesity-related functional iron disorder remains a major nutritional challenge. We evaluated the effects of djulis hull (DH) on iron metabolism in 50% high-fat-diet-induced obese rats supplemented with ferric citrate (2 g iron/kg diet) for 12 weeks. DH supplementation (5, 10, 15% dry weight/kg diet) significantly increased serum and hepatic iron but decreased appetite hormones, body weight, hepcidin, and liver inflammation (all p < 0.05). The Spearman correlation showed that appetite hormones were negatively associated with iron but positively correlated with liver hepcidin (all p < 0.05). A Western blot analysis showed that DH significantly downregulated hepatic hepcidin through the IL-6-JAK-STAT3 and enhanced ferroportin (Fpn) via the Keap1-Nrf2 and PHD2-HIF-2α. An in vitro study revealed that major bioactive compounds of DH, hexacosanol, and squalene suppressed LPS-induced IL-6 and hepcidin but enhanced Fpn expression in activated THP-1 cells. In conclusion, DH may exert nutraceutical properties for the treatment of functional iron disorder and restoration of iron efflux may have beneficial effects on weight control.

The Antiviral Activity of Varenicline against Dengue Virus Replication during the Post-Entry Stage.

Dengue virus (DENV) poses a significant global health challenge, with millions of cases each year. Developing effective antiviral drugs against DENV remains a major hurdle. Varenicline is a medication used to aid smoking cessation, with anti-inflammatory and antioxidant effects. In this study, varenicline was investigated for its antiviral potential against DENV. This study provides evidence of the antiviral activity of varenicline against DENV, regardless of the virus serotype or cell type used. Varenicline demonstrated dose-dependent effects in reducing viral protein expression, infectivity, and virus yield in Vero and A549 cells infected with DENV-1 and DENV-2, with EC50 values ranging from 0.44 to 1.66 µM. Time-of-addition and removal experiments demonstrated that varenicline had a stronger inhibitory effect on the post-entry stage of DENV-2 replication than on the entry stage, as well as the preinfection and virus attachment stages. Furthermore, cell-based trans-cleavage assays indicated that varenicline dose-dependently inhibited the proteolytic activity of DENV-2 NS2B-NS3 protease. Docking models revealed the formation of hydrogen bonds and van der Waals forces between varenicline and specific residues in the DENV-1 and DENV-2 NS2B-NS3 proteases. These results highlight the antiviral activity and potential mechanism of varenicline against DENV, offering valuable insights for further research and development in the treatment of DENV infection.

Metabolic engineering of E. coli for ß-alanine production using a multi-biosensor enabled approach.

ß-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of ß-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo ß-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in ß-alanine yield (0.85 mol ß-alanine/mole glucose) and specific ß-alanine production (0.74 g/L/OD600), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol ß-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L ß-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of ß-alanine.

DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.

Prevention of needle-stick injury among nurses in an acute ward of a hospital: a best practice implementation project.

OBJECTIVES: This evidence implementation project aimed to identify barriers leading to needle-stick injuries (NSIs) and to develop implementation strategies to prevent NSIs in the acute ward of a hospital in central Taiwan. INTRODUCTION: The incidence rate of NSIs was 5.6% in the acute ward of a hospital in Taiwan. NSIs commonly occur during the drawing of blood, intravenous insertion, needle recapping, or performing any procedure involving sharp medical devices. NSIs are critical occupational risks among healthcare workers, possibly leading to transmission of infectious diseases, especially blood-borne viruses, such as HIV, hepatitis B, and hepatitis C. METHODS: A clinical audit was undertaken using the JBI Practical Application of Clinical Evidence System (PACES) and the Getting Research into Practice (GRiP) approach. Five audit criteria that represented best practice recommendations for prevention of NSIs were used. Baseline data were collected from 177 nurses in five acute wards, followed by the implementation of multiple strategies during a 20-week period of the project. Both baseline and postimplementation audits were undertaken to determine changes in practice. RESULTS: According to the pre-audit concerning the use of safety-engineered injection devices and safe use and disposal of needles, there was 14-15% compliance, which indicated poor compliance with current best-practice criteria. Following the project implementation, the nursing staff were educated about the well tolerated use and disposal of sharps and the improved compliance rate ranged from 40 to 96.6%, with safety needle use increasing from 16 to 95.5%, safety needle operation procedure awareness increasing from 14 to 96%, needles not recapped after use increasing from 47 to 85%, and placing used needles in the sharps collection box increasing from 75 to 80%. CONCLUSION: This article suggests that standardized puncture prevention education and training enhanced nurses' awareness in the acute ward.

Functional kupffer cells migrate to the liver from the intraperitoneal cavity.

We established a method of KC transplantation by intraperitoneal (i.p.) injection using EGFP-expressing cells (EGFP-KCs) and normal KCs. The novel method is easier and less invasive than conventional methods so that it is not only technically advantageous but also ethically preferable for experiments using animals. We demonstrated that KCs migrated to the liver following i.p. Injection. Engraftment in the liver was not observed for peritoneal macrophages (pMPs). This suggests that KCs migrate to the liver via a sorting mechanism. KC injection decreased the KC number at 24 h and then recovered the KCs at 10 days to a normal level. Additionally, recovery to the normal level by KC injection was observed in mice with KC depletion induced by GdCl3. These results suggest that a regulatory mechanism exists for controlling the number of KCs.

Bioproduced Proteins On Demand (Bio-POD) in hydrogels using Pichia pastoris.

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.

Association of Increased Risk of Pneumonia and Using Proton Pump Inhibitors in Patients With Type II Diabetes Mellitus.

BACKGROUND: This study explored the possible association between the use of proton pump inhibitors (PPIs) and the increased incidence of pneumonia in patients with type 2 diabetes mellitus (T2DM). METHODS: We selected 4940 patients with T2DM of whom 988 and 3952 were enrolled in PPI and propensity score-matched control cohorts, respectively. All patients were followed from the index date until admission with pneumonia, withdrawal from the National Health Insurance program or the end of 2013. The PPIs associated with risk of incident pneumonia were examined. Furthermore, we assessed the risk of pneumonia according to annual defined daily doses in the PPI cohort. RESULTS: After a 14-year follow-up, the cumulative incidence of pneumonia in the PPI users was 11.4% higher than that in the controls (30.3% vs 18.9%). Compared to the controls, the PPI users had a 1.70-fold higher risk of pneumonia in the Cox proportional hazards model after adjustment for matched pairs. The risk of pneumonia increased with the annual PPI defined daily dose. CONCLUSION: The results of this population-based retrospective cohort study suggest that PPI use increased the risk of pneumonia in patients with T2DM. The effects were more prominent in patients administered higher doses of PPIs.

Hypothyroidism is an independent risk factor for Menière's disease: A population-based cohort study.

The relationship of hypothyroidism and Menière's disease (MD) has been discussed before, yet not well documented. Our study aims to investigate the correlation of both diseases.This is a retrospective cohort study based on data from the LHID2000 (Longitudinal Health Insurance Database 2000), a subset of the Taiwan National Research Health Insurance Database that contains claims data for the 2000 to 2011 period. A total of 27,050 patients were included in this study, 5410 of whom had received a hypothyroidism diagnosis. The prevalence of MD was high in patients with hypothyroidism (95% confidence interval [CI]: 1.14-1.51), especially in those older than 50 years old (P < .001). Although comorbidities such as hypertension or cirrhosis are significant risk factors for Menière's disease (P < .001, P < .05), the incidence rate of Menière's disease in patients with hypothyroidism differs significantly between groups without these comorbidities (95% CI: 1.14-1.95). Regarding the timing for the occurrence of Menière's disease in patients with hypothyroidism, there was a significant time interval of <5 years (P < .05). The risk of MD decreased after treatment with thyroxine and did not differ from that of the nonhypothyroidism cohort (adjusted HR [aHR] = 0.85, 95% CI: 0.66-1.11).The study demonstrates a significant association between hypothyroidism and Menière's disease, especially in elderly female patients. Physicians should consider verifying the thyroid function when encountering these patients.

PARP-1-dependent recruitment of cold-inducible RNA-binding protein promotes double-strand break repair and genome stability.

Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events. Here we report cold-inducible RNA-binding protein (CIRBP) as a newly identified key regulator in DNA double-strand break (DSB) repair. On DNA damage, CIRBP temporarily accumulates at the damaged regions and is poly(ADP ribosyl)ated by poly(ADP ribose) polymerase-1 (PARP-1). Its dissociation from the sites of damage may depend on its phosphorylation status as mediated by phosphatidylinositol 3-kinase-related kinases. In the absence of CIRBP, cells showed reduced γH2AX, Rad51, and 53BP1 foci formation. Moreover, CIRBP-depleted cells exhibited impaired homologous recombination, impaired nonhomologous end-joining, increased micronuclei formation, and higher sensitivity to gamma irradiation, demonstrating the active involvement of CIRBP in DSB repair. Furthermore, CIRBP depleted cells exhibited defects in DNA damage-induced chromatin association of the MRN complex (Mre11, Rad50, and NBS1) and ATM kinase. CIRBP depletion also reduced phosphorylation of a variety of ATM substrate proteins and thus impaired the DNA damage response. Taken together, these results reveal a previously unrecognized role for CIRBP in DSB repair.

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5.

BACKGROUND: An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. METHOD: A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. RESULT: Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. CONCLUSION: Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. GENERAL SIGNIFICANCE: The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

Severe hypertriglyceridemia secondary to venlafaxine use in an older adult on dialysis -case report.

BACKGROUND: Although the prescribing information for Venlafaxine extended release includes a discussion about possible increases in total cholesterol and triglycerides (TG) seen in healthier adult patients during premarketing clinical trials, no post-marketing studies or case reports, that discuss the effects of venlafaxine on TG in elderly patients with chronic kidney disease. CASE PRESENTATION: We report a 71 year-old male patient with end-stage renal disease on hemodialysis, with a history of coronary artery disease, mild hyperlipidemia, and hypertension. This patient twice demonstrated the severe rises in triglycerides while taking the antidepressant, i.e., venlafaxine, and discontinuing the long-term use of fenofirate. The adverse drug reaction sub-committee at the hospital rated the second event as a "probable reaction" using the Naranjo nomogram, accordingly. CONCLUSIONS: This case demonstrates the risk of changes in lipid profiles while taking venlafaxine and receiving on and off fenofibrate therapy in the older adult patient with chronic kidney disease and under hemodialysis. Regular monitoring for lipid changes after starting venlafaxine is strongly advised for patients with existing risk factors.

Protein Arginine Methyltransferase 8: Tetrameric Structure and Protein Substrate Specificity.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of various proteins, and their dysregulations often correlate with tumorigenesis or developmental deficiency. Recent studies have focused on the in vivo substrate identification and the enzyme mechanism with peptide substrates. However, how PRMTs recognize substrates at the protein level remains unknown. PRMT8 is one of the least characterized type I PRMTs, and its crystal structure has not been reported. Here, we report the crystal structure of the PRMT8:SAH complex, identify a new non-histone protein substrate NIFK, and uncover a previously unknown regulatory region specifically required for recognizing NIFK. Instead of the canonical dimeric structure for other type I PRMTs, PRMT8 exists as a tetramer in solution. Using X-ray crystallography in combination with small-angle X-ray scattering experiments, the dimer of dimers architecture in which two PRMT8 dimers are held together mainly by ß strand interactions was proposed. Mutation of PRMT8-ß15 impedes the methylation of NIFK but still allows methylation of the histone H2A/H2B dimer or a peptide substrate, suggesting a possible structural basis for recognition of protein substrates. Lastly, we observed two PRMT8 dimer orientations resulting in open (without SAH) and closed (with SAH bound) conformations. The comparison between open and closed conformations may provide useful information for PRMT1/8 inhibitor design.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an independent prognostic marker in wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy.

BACKGROUND: The impact of KRAS signaling on cancerous inhibitor of protein phosphatase 2A (CIP2A) expression has not yet been explored. We investigated the impact of KRAS on CIP2A expression in colorectal cancer patients after colorectal liver metastasectomy. METHODS: We examined CIP2A expression by immunohistochemistry (IHC) and used direct sequencing to identify the mutational status of KRAS exon 2 (codon 12 and 13). The association between CIP2A expression, KRAS genotype, clinicopathological parameters and survival were examined by the Kaplan-Meier method and the Cox proportional hazards model. A combination of immunoblotting and proliferation assays were employed to elucidate the role of CIP2A in signal transduction pathways in wild-type KRAS Caco-2 cells. RESULTS: A total of 220 colorectal cancer patients who had undergone colorectal liver metastasectomy were included in the study. The mutant KRAS genotype was associated with CIP2A overexpression. CIP2A expression was an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy (relative risk = 1.873, P = 0.019). Targeted silencing of CIP2A in Caco-2 cells (wild-type KRAS) led to decreased expression of pERK/ERK and decreased cell proliferation. Overexpression of mutant KRAS G12D in Caco-2 cells led to an increase in CIP2A expression and cell proliferation. In Caco-2 cells with the KRAS G12D, KRAS overexpression preserved the regulation effect of CIP2A in KRAS and abrogated the impact of CIP2A regulation on pERK/ERK and cell proliferation. CIP2A inhibition also increased the efficacy of cetuximab in Caco-2 cells. CONCLUSIONS: CIP2A is an independent prognostic marker in patients with wild-type KRAS metastatic colorectal cancer after colorectal liver metastasectomy.

Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.