Cadherin Expression,Vectorial Active Transport,and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

Background

Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods

Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results

It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions

The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.     

Cry Protein Crystals: A Novel Platform for Protein Delivery

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.     

Impact of Community Based Peer Support in Type 2 Diabetes: A Cluster Randomised Controlled Trial of Individual and/or Group Approaches

Background

Diabetes peer support, where one person with diabetes helps guide and support others, has been proposed as a way to improve diabetes management. We have tested whether different diabetes peer support strategies can improve metabolic and/or psychological outcomes.

Methods

People with type 2 diabetes (n = 1,299) were invited to participate as either ‘peer’ or ‘peer support facilitator’ (PSF) in a 2x2 factorial randomised cluster controlled trial across rural communities (130 clusters) in England. Peer support was delivered over 8–12 months by trained PSFs, supported by monthly meetings with a diabetes educator. Primary end point was HbA1c. Secondary outcomes included quality of life, diabetes distress, blood pressure, waist, total cholesterol and weight. Outcome assessors and investigators were masked to arm allocation. Main factors were 1:1 or group intervention. Analysis was by intention-to-treat adjusting for baseline.

Results

The 4 arms were well matched (Group n = 330, 1:1(individual) n = 325, combined n = 322, control n = 322); 1035 (79•7%) completed the mid-point postal questionnaire and 1064 (81•9%) had a final HbA1c. A limitation was that although 92.6% PSFs and peers were in telephone contact, only 61.4% of intervention participants attended a face to face session. Mean baseline HbA1c was 57 mmol/mol (7•4%), with no significant change across arms. Follow up systolic blood pressure was 2•3mm Hg (0.6 to 4.0) lower among those allocated group peer-support and 3•0mm Hg (1.1 to 5.0) lower if the group support was attended at least once. There was no impact on other outcomes by intention to treat or significant differences between arms in self-reported adherence or medication.

Conclusions

Group diabetes peer support over 8–12 months was associated with a small improvement in blood pressure but no other significant outcomes. Long term benefits should be investigated.

Trial Registration

ISRCTN.com ISRCTN6696362166963621     

Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors—imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.     

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12–15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells.     

Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ¹³C and δ¹⁵N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ¹³C and δ⁴⁴Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ¹³C and low δ⁴⁴Ca values; however, the δ⁴⁴Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ⁴⁴Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ⁴⁴Ca and δ¹³C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius. Am J Phys Anthropol 154:633–643, 2014. © 2014 Wiley Periodicals, Inc.     

Diselenides as universal oxidative folding catalysts of diverse proteins

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides. Compared to the disulfide-linked dimer of glutathione (the most commonly used oxidant for in vitro protein folding), selenoglutathione provided markedly improved efficiencies in the folding of biotechnologically important proteins such as hirudin, lysozyme, human epidermal growth factor and interferon α-2a. Selenoglutathione also enhances the renaturation of more challenging targets such as bovine serum albumin, whose native state contains 17 disulfide bonds, and the Fab fragment of an antibody. In the latter case, micromolar amounts of selenoglutathione are able to match the modest yield provided by a previously optimized redox buffer, which contains millimolar levels of glutathione. Taken together, the folding reactions of these diverse proteins exemplify the advantages and limitations of diselenide catalysts.     

Linking monitoring and modelling: can long-term datasets be used more effectively as a basis for large-scale prediction?

Data from long-term monitoring sites are vital for biogeochemical process understanding, and for model development. Implicitly or explicitly, information provided by both monitoring and modelling must be extrapolated in order to have wider scientific and policy utility. In many cases, large-scale modelling utilises little of the data available from long-term monitoring, instead relying on simplified models and limited, often highly uncertain, data for parameterisation. Here, we propose a new approach whereby outputs from model applications to long-term monitoring sites are upscaled to the wider landscape using a simple statistical method. For the 22 lakes and streams of the UK Acid Waters Monitoring Network (AWMN), standardised concentrations (Z scores) for Acid Neutralising Capacity (ANC), dissolved organic carbon, nitrate and sulphate show high temporal coherence among sites. This coherence permits annual mean solute concentrations at a new site to be predicted by back-transforming Z scores derived from observations or model applications at other sites. The approach requires limited observational data for the new site, such as annual mean estimates from two synoptic surveys. Several illustrative applications of the method suggest that it is effective at predicting long-term ANC change in upland surface waters, and may have wider application. Because it is possible to parameterise and constrain more sophisticated models with data from intensively monitored sites, the extrapolation of model outputs to policy relevant scales using this approach could provide a more robust, and less computationally demanding, alternative to the application of simple generalised models using extrapolated input data.     

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1β, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.