mRNA stability and antibody production in CHO cells: Improvement through gene optimization

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.     

Reliable gas chromatographic–mass spectrometric method combined with a headspace autosampler for isoflurane determination in blood

Isoflurane is a nonflammable, liquid, volatile inhalation anesthetic administered by vaporizing. Although it is now commonly used, fatal cases resulting from its abuse or misuse have been reported. A combined system of a gas chromatograph–mass spectrometer and a headspace autosampler is therefore proposed for the detection of blood isoflurane. This analytic method showed sharp and well separated peaks, and revealed a good linear relationship (r=0.9994) with a function of y=7.3768x−0.0222 at concentrations between 18.7 and 299.2 μg/ml. The limits of detection and quantitation of this method were 1.2 and 4.7 μg/ml, respectively. The within- and between-run precision for spiked samples, assessed by the coefficient of variations, ranged from 1.7 to 10.0% and from 4.1 to 12.8%, respectively. The within- and between-run accuracy, assessed by errors from theoretical values, were 2.2–7.8% and 2.4–9.6%, respectively. In addition, practical sample analysis showed a good applicability, with a within-run precision rate of 5.6 to 7.7% and a between-run precision rate of 5.2–10.6%. In summary, the present work presents a valid alternative for blood isoflurane analysis.     

Xenin-25 Potentiates Glucose-dependent Insulinotropic Polypeptide Action via a Novel Cholinergic Relay Mechanism

The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, “GIP/DT” animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the β-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in β-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.     

Effect of ration and body size on the energy budget of juvenile white sturgeon

Growth and energy budget were measured for three sizes (2.4, 11.1 and 22.5 g) of juvenile white sturgeon Acipenser transmontanus held at 18.5° C and fed tubificid worms at different levels ranging from starvation to ad libitum . For each size-class, specific growth rate increased linearly with increasing ration, and conversion efficiency was highest at the maximum ration. Growth rate decreased with increasing fish size at the maximum ration, but increased with size at each restricted ration. Conversion efficiency increased with increasing ration for each size-class and was usually highest at the maximum ration. Faecal production accounted for 3.2–5.2% of food energy. The proportion of food energy lost in nitrogenous excretion decreased with increasing ration. With increases in ration, the allocation of metabolizable energy to metabolism decreased, while that to growth increased. Fish size had no significant effect on the allocation of metabolizable energy to metabolism or growth. At the maximum ration, on average 64.9% of metabolizable energy was spent on metabolism, and 35.1% on growth.     

A Clinical Algorithm to Identify HIV Patients at High Risk for Incident Active Tuberculosis: A Prospective 5-Year Cohort Study

Background

Predicting the risk of tuberculosis (TB) in people living with HIV (PLHIV) using a single test is currently not possible. We aimed to develop and validate a clinical algorithm, using baseline CD4 cell counts, HIV viral load (pVL), and interferon-gamma release assay (IGRA), to identify PLHIV who are at high risk for incident active TB in low-to-moderate TB burden settings where highly active antiretroviral therapy (HAART) is routinely provided.

Materials and Methods

A prospective, 5-year, cohort study of adult PLHIV was conducted from 2006 to 2012 in two hospitals in Taiwan. HAART was initiated based on contemporary guidelines (CD4 count < = 350/μL). Cox regression was used to identify the predictors of active TB and to construct the algorithm. The validation cohorts included 1455 HIV-infected individuals from previous published studies. Area under the receiver operating characteristic (ROC) curve was calculated.

Results

Seventeen of 772 participants developed active TB during a median follow-up period of 5.21 years. Baseline CD4 < 350/μL or pVL ≥ 100,000/mL was a predictor of active TB (adjusted HR 4.87, 95% CI 1.49–15.90, P = 0.009). A positive baseline IGRA predicted TB in patients with baseline CD4 ≥ 350/μL and pVL < 100,000/mL (adjusted HR 6.09, 95% CI 1.52–24.40, P = 0.01). Compared with an IGRA-alone strategy, the algorithm improved the sensitivity from 37.5% to 76.5%, the negative predictive value from 98.5% to 99.2%. Compared with an untargeted strategy, the algorithm spared 468 (60.6%) from unnecessary TB preventive treatment. Area under the ROC curve was 0.692 (95% CI: 0.587–0.798) for the study cohort and 0.792 (95% CI: 0.776–0.808) and 0.766 in the 2 validation cohorts.

Conclusions

A validated algorithm incorporating the baseline CD4 cell count, HIV viral load, and IGRA status can be used to guide targeted TB preventive treatment in PLHIV in low-to-moderate TB burden settings where HAART is routinely provided to all PLHIV. The implementation of this algorithm will avoid unnecessary exposure of low-risk patients to drug toxicity and simultaneously, reduce the burden of universal treatment on the healthcare system.     

Thiazolidinediones and Risk of Long-Term Dialysis in Diabetic Patients with Advanced Chronic Kidney Disease: A Nationwide Cohort Study

Thiazolidinediones (TZDs) reduce urinary albumin excretion and proteinuria in diabetic nephropathy. The effect of TZDs on hard renal outcome in diabetic patients with chronic kidney disease (CKD) is unknown. We investigate the association of TZDs and risk of long-term dialysis or death in diabetic patients with advanced CKD. The nationwide population-based cohort study was conducted using Taiwan’s National Health Insurance Research Database. From January 2000 to June 2009, 12350 diabetic patients with advanced CKD (serum creatinine levels greater than 6 mg/dL but not yet receiving renal replacement therapy) were selected for the study. We used multivariable Cox regression models and a propensity score-based matching technique to estimate hazard ratios (HRs) for development of long-term dialysis and the composite outcome of long-term dialysis or death for TZD users (n=1224) as compared to nonusers (n=11126). During a median follow-up of 6 months, 8270 (67.0%) patients required long-term dialysis and 2593 (21.0%) patients died before starting long-term dialysis. Using propensity score matched analysis, we found TZD users were associated with a lower risk for long-term dialysis (HR, 0.80; 95% confidence interval [CI], 0.74-0.86) and the composite outcome of long-term dialysis or death (HR, 0.85; 95% CI, 0.80-0.91). The results were consistent across most patient subgroups. Use of TZDs among diabetic patients with advanced CKD was associated with lower risk for progression to end-stage renal disease necessitating long-term dialysis or death. Further randomized controlled studies are required to validate this association.