All-Cause and Cause-Specific Mortality among Users of Basal Insulins NPH,Detemir, and Glargine

Background

Insulin therapy in type 2 diabetes may increase mortality and cancer incidence, but the impact of different types of basal insulins on these endpoints is unclear. Compared to the traditional NPH insulin, the newer, longer-acting insulin analogues detemir and glargine have shown benefits in randomized controlled trials. Whether these advantages translate into lower mortality among users in real life is unknown.

Objective

To estimate the differences in all-cause and cause-specific mortality rates between new users of basal insulins in a population-based study in Finland.

Methods

23 751 individuals aged ≥40 with type 2 diabetes, who initiated basal insulin therapy in 2006–2009 were identified from national registers, with comprehensive data for mortality, causes of death, and background variables. Propensity score matching was performed on characteristics. Follow-up time was up to 4 years (median 1.7 years).

Results

2078 deaths incurred. With NPH as reference, the adjusted HRs for all-cause mortality were 0.39 (95% CI, 0.30–0.50) for detemir, and 0.55 (95% CI, 0.44–0.69) for glargine. As compared to glargine, the HR was 0.71 (95% CI, 0.54–0.93) among detemir users. Compared to NPH, the mortality risk for both cardiovascular causes as well as cancer were also significantly lower for glargine, and especially for detemir in adjusted analysis. Furthermore, the results were robust in various sensitivity analyses.

Conclusion

In real clinical practice, mortality was substantially higher among users of NPH insulin as compared to insulins detemir or glargine. Considering the large number of patients who require insulin therapy, this difference in risk may have major clinical and public health implications. Due to limitations of the observational study design, further investigation using an interventional study design is warranted.     

Detection of Brugia malayi in laboratory and wild-caught Mansonioides mosquitoes (Diptera: Culicidae) using Hha I PCR assay

An Hha 1 based polymerase chain reaction (PCR) assay developed for the detection of Brugia malayi, the causative agent of Brugian lymphatic filariasis, was evaluated for its sensitivity in the laboratory and for its usefulness in measuring changes in transmission of the disease in the field. Laboratory studies showed that the new assay was highly sensitive in comparison with the standard dissection and microscopy technique. The assay can detect as little as 4 pg of parasite DNA or a single microfilaria in pools of up to 100 mosquitoes. The optimum pool size for convenience was found to be 50 mosquitoes per pool. The efficacy of PCR assay was evaluated in filariasis control programmes in operation in endemic areas of Kerala State, South India. The infection rates obtained by the Hha I PCR assay and the conventional dissection and microscopy technique were 1.2% and 1.7% respectively in operational areas and 8.3% and 4.4% respectively, in check areas, which were not significantly different (P < 0.05). Thus, the Hha I PCR assay was found to be as sensitive as the conventional technique and hence it can be used as a new epidemiological tool for assessing parasite infection in field-collected mosquitoes.     

A single amino acid change (Asp 53 --> Ala53) converts Survivin from anti-apoptotic to pro-apoptotic

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. Recently, Survivin has attracted growing attention because of its tumor-specific expression and potential applications in tumor therapy. However, its inhibitory mechanism and subcellular localization remain controversial. Here, we report a novel Survivin mutant Surv-D53A, which displays a function opposite to Survivin and a distinctive subcellular distribution compared with its wild-type counterpart. Surv-D53A was shown to induce apoptosis in a p53-independent manner, indicating that tumor suppressor p53 is not involved in its apoptosis pathway. Surv-D53A was shown to markedly sensitize apoptosis induced by TRAIL, doxorubicin, and RIP3. We also demonstrated that similar to wild-type Survivin, Surv-D53A was localized in cytoplasm in interphase and to midbody at telophase. However, it fails to colocalize in chromosomes with Aurora-B in metaphase as wt-Survivin. Surv-D53A mutant is less stable than wt-Survivin and is degraded more rapidly by ubiquitin-proteasome pathway. Additionally, we found that Surv-D53A interacts with wt-Survivin to form heterodimer or with itself to form mutant homodimer, which may account for the loss of its antiapoptotic function. Finally, unlike Survivin*Survivin, neither Surv-D53A*Survivin nor Surv-D53A*Surv-D53A is able to bind to Smac/DIABLO, which may explain the underlying mechanism for its abolishment of antiapoptotic activity of Survivin.     

Immobilization of Alginate-Encapsulated Bacillus thuringiensis var. israelensis Containing Different Multivalent Counterions for Mosquito Control

Immobilized techniques have been used widely for the controlled release formulation of mosquitoes. Among the microbial formulations, polymeric matrices play an important role in the controlled release of microbial pesticide at rates sufficiently effective to kill mosquitoes in the field. The advantage of these matrices is that they enhance the stability of both spores and toxin against pH, temperature variations, and UV irradiation. The disadvantage of using calcium alginate beads is that they are unstable upon contact with phosphate of potassium or sodium ions rich in the mosquito habitats. To overcome these problems, attempts were made to encapsulate Bacillus thuringiensis var. israelensis within alginate by using different multivalent counterions, namely, calcium chloride, zinc sulfate, copper sulfate, cobalt chloride, and ferric chloride, and the beads formed were tested for its mosquito larvicidal activity. Among all the beads tested, zinc alginate beads resulted in maximum larvicidal activity of 98% (+/-1.40 SE) against Culex quinquefasciatus IIIrd instar larvae and maximum spore count of 3.36 x 10(5) (+/-5291.50 SE) CFU/ml. Zinc alginate beads maintained their structure for up to 48 h when shaken vigorously on a rotary shaker at 180 rpm in the presence of 10 mM potassium phosphate buffer (pH 6.8 +/- 0.1). In conclusion, our results suggest that the use of zinc sulfate as counterions to encapsulate B. thuringiensis var. israelensis within alginate may be a potent mosquito control program in the habitats where more phosphate ions are present.     

Evaluation of Ssp I polymerase chain reaction assay in the detection of Wuchereria bancrofti infection in field-collected Culex quinquefasciatus and its application in the transmission studies of lymphatic filariasis

Abstract: Ssp I polymerase chain reaction (PCR) assay, developed for Wuchereria bancrofti, was evaluated for its sensitivity in detecting infection in the vector, Culex quinquefasciatus, in the field. The evaluation of the assay was carried out using pools of vector mosquitoes collected from areas under filariasis survey and control trial projects, in comparison with the standard dissection and microscopy technique. In the filariasis survey area the infection rate as determined by the dissection and microscopy technique was 0.89% whereas it was 1.7% by PCR assay. In the Bacillus sphaericus trial area the infection rates as assessed by the conventional technique were 6.6 and 4.5% in the treated and check areas, respectively, whereas those obtained by the PCR assay were 4.7 to 2.2%. Although the infection rates determined by the PCR assay are slightly higher or lower than the rates obtained by the conventional technique, the difference was not statistically significant (P=0.451 for filariasis survey area, and P=0.203 and 0.161 for B. sphaericus trial area). When the pool size of Cx. quinquefasciatus was increased to 50 the sensitivity of the PCR was affected. The changes in infection rates as influenced by the antifilarial chemotherapy were similar when determined by PCR assay and the standard method. The major advantage of the PCR assay over the conventional technique is that thousands of mosquitoes can be processed within a short duration and this attribute has potential application in rapid assessment of disease prevalence and monitoring of the transmission dynamics.     

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.Androgen is important for the development, function, and proliferation of both normal and cancerous prostate cells (1). At the earliest stage of prostate cancer, prostate cancer cells are dependent on the presence of androgen, and androgen-deprivation therapy (ADT)¹ is used to treat prostate cancer (2). However, cells become androgen-independent as a result of androgen deprivation therapy, and they become more aggressive. This results in androgen-independent remission of prostate cancer (3). LNCaP and PC3 cell lines have been widely used as models of prostate cancer. LNCaP is an androgen-dependent cancer cell line, whereas PC3 is an androgen-independent cell line. The LNCaP cell line is less aggressive as compared with PC3 cells that have a high metastatic potential. LNCaP and PC3 cells have been previously studied by genomics and proteomics approaches to understand the mechanism(s) responsible for the aggressive and metastatic nature of prostate cancer (4–8).Post-translational modifications (PTMs) such as phosphorylation are important in the function of the androgen-dependent pathway. Androgen receptors bind to androgen and are then phosphorylated before translocating into the nucleus (3). However, protein PTMs cannot be directly inferred from gene expression. Glycosylation is an abundant PTM and most cell surface or secreted proteins are expected to be glycosylated (9). Glycosylation is one of the more complex PTMs because of the fact that different glycosylation machineries are present in different cells, multiple glycosylation sites exist on many glycoproteins and each glycosylation site can be modified by several different glycans (10, 11). Such microheterogeneity of glycan structures at each glycosylation site with different site occupancy significantly increases the structural diversity of each glycoprotein that is specific to the microenvironment of the cells where each glycoprotein is produced. Although these characteristics of protein glycosylation pose considerable challenges to the structural and functional analyses of glycoproteins, we expect that cell and cell microenvironment-specific glycoproteins differ according to the physiological and pathological states of the cells. Aberrant glycosylation is the result of alterations in glycosylation genes that may lead to the development of cancer. A systematic approach to analyze proteins, glycoproteins, and glycosylation is expected to permit the identification of the glycoprotein alterations that are specific to each cell state and aid the understanding of the functions of glycosylation because alterations in glycosylation can affect glycoprotein abundance or function (12, 13). A detailed analysis of glycoproteins in cancer cells with different functions is needed to understand tumor biology and how glycoproteins can function as therapeutic targets or diagnostic biomarkers (14, 15).In this study, a comprehensive proteomic and glycoproteomic platform was designed to investigate the differences in proteins, glycoproteins, and site-specific glycosylation forms of glycoproteins between LNCaP and PC3 cells (Fig. 1). To our knowledge, this is the first report to characterize glycoproteins with respect to protein abundance, glycosylation occupancy, and glycosylation heterogeneity at specific glycosites. These altered glycosylation patterns among proteins between LNCaP and PC3 cell lines have a significant potential to aid our understanding of the altered glycoprotein expression in prostate cancer cells, thus leading to novel specific methods to detect aggressive prostate cancer.Open in a separate windowFig. 1.Schematic representation of the workflow for the integrated analysis of glycosite-containing peptides, global protein expression, and intact glycopeptides. Proteins were obtained from LNCaP and PC3 cell lines followed by tryptic digestion and iTRAQ labeling. Labeled peptide samples were then combined and separated into two aliquots. One aliquot was enriched for glycosite-containing peptides using Solid Phase Extraction of Glycopeptides (SPEG) and the other aliquot was used for bRPLC separation followed by the analysis of global proteins and intact glycopeptides. Finally, peptides were analyzed using LC-MS/MS.     

Advanced backcross-QTL analysis in spring barley (H. vulgare ssp. spontaneum) comparing a REML versus a Bayesian model in multi-environmental field trials

A common difficulty in mapping quantitative trait loci (QTLs) is that QTL effects may show environment specificity and thus differ across environments. Furthermore, quantitative traits are likely to be influenced by multiple QTLs or genes having different effect sizes. There is currently a need for efficient mapping strategies to account for both multiple QTLs and marker-by-environment interactions. Thus, the objective of our study was to develop a Bayesian multi-locus multi-environmental method of QTL analysis. This strategy is compared to (1) Bayesian multi-locus mapping, where each environment is analysed separately, (2) Restricted Maximum Likelihood (REML) single-locus method using a mixed hierarchical model, and (3) REML forward selection applying a mixed hierarchical model. For this study, we used data on multi-environmental field trials of 301 BC₂DH lines derived from a cross between the spring barley elite cultivar Scarlett and the wild donor ISR42-8 from Israel. The lines were genotyped by 98 SSR markers and measured for the agronomic traits “ears per m2,” “days until heading,” “plant height,” “thousand grain weight,” and “grain yield”. Additionally, a simulation study was performed to verify the QTL results obtained in the spring barley population. In general, the results of Bayesian QTL mapping are in accordance with REML methods. In this study, Bayesian multi-locus multi-environmental analysis is a valuable method that is particularly suitable if lines are cultivated in multi-environmental field trials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimisation of an asymmetric polymerase chain reaction assay for the amplification of single-stranded DNA from Wuchereria bancrofti for electrochemical detection

Single-stranded DNA (ssDNA) is a prerequisite for electrochemical sensor-based detection of parasite DNA and other diagnostic applications. To achieve this detection, an asymmetric polymerase chain reaction method was optimised. This method facilitates amplification of ssDNA from the human lymphatic filarial parasite Wuchereria bancrofti. This procedure produced ssDNA fragments of 188 bp in a single step when primer pairs (forward and reverse) were used at a 100:1 molar ratio in the presence of double-stranded template DNA. The ssDNA thus produced was suitable for immobilisation as probe onto the surface of an Indium tin oxide electrode and hybridisation in a system for sequence-specific electrochemical detection of W. bancrofti. The hybridisation of the ssDNA probe and target ssDNA led to considerable decreases in both the anodic and the cathodic currents of the system''s redox couple compared with the unhybridised DNA and could be detected via cyclic voltammetry. This method is reproducible and avoids many of the difficulties encountered by conventional methods of filarial parasite DNA detection; thus, it has potential in xenomonitoring.     

Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing

Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.