Mutations in DHDPSL Are Responsible For Primary Hyperoxaluria Type III

Primary hyperoxaluria (PH) is an autosomal-recessive disorder of endogenous oxalate synthesis characterized by accumulation of calcium oxalate primarily in the kidney. Deficiencies of alanine-glyoxylate aminotransferase (AGT) or glyoxylate reductase (GRHPR) are the two known causes of the disease (PH I and II, respectively). To determine the etiology of an as yet uncharacterized type of PH, we selected a cohort of 15 non-PH I/PH II patients from eight unrelated families with calcium oxalate nephrolithiasis for high-density SNP microarray analysis. We determined that mutations in an uncharacterized gene, DHDPSL, on chromosome 10 cause a third type of PH (PH III). To overcome the difficulties in data analysis attributed to a state of compound heterozygosity, we developed a strategy of “heterozygosity mapping”—a search for long heterozygous patterns unique to all patients in a given family and overlapping between families, followed by reconstruction of haplotypes. This approach enabled us to determine an allelic fragment shared by all patients of Ashkenazi Jewish descent and bearing a 3 bp deletion in DHDPSL. Overall, six mutations were detected: four missense mutations, one in-frame deletion, and one splice-site mutation. Our assumption is that DHDPSL is the gene encoding 4-hydroxy-2-oxoglutarate aldolase, catalyzing the final step in the metabolic pathway of hydroxyproline.     

Lipolytic, Proteolytic, and Cholesterol-Degrading Bacteria from the Human Cerumen

Cerumen, also known as ear wax, is a yellowish waxy substance secreted from specialized glands in the ear canal of mammals. Human cerumen is rich in protein (mainly keratin), lipids (long-chain fatty acids), alcohols, squalene, and cholesterol. To-date the role of cerumen is not totally clear but it is believed to have antimicrobial properties. Here we describe the isolation of multiple bacterial species from human cerumen (among them many Staphylococcus spp. and, interestingly, multiple Bacillus spp.) showing that many of these bacteria harbor biochemical traits enabling them to utilize different cerumen components for their growth. We also suggest the existence of microbial consortia.     

Composites of peptide nucleic acids with ttitanium dioxide nanoparticles. I. Construction of nanocomposites containing DNA/PNA duplexes and their delivery into HeLa cells

In order to study the possibility of using titanium dioxide (TiO₂) nanoparticles to deliver peptide nucleic acids (PNA) in eukaryotic cells, a PNA oligomer was synthesized, and a method of PNA immobilization in the form of hybrid DNA/PNA duplexes on the surface of TiO₂ nanoparticles covered with polylysine (PL) was developed. The attachment of a DNA/PNA duplex to TiO₂ · PL nanoparticles occurs due to electrostatic interactions between the negatively charged DNA chain and the positively charged amino groups of PL. The binding of the PNA to the nanocomposite is achieved through noncovalent Watson-Crick interactions between PNA and complementary DNA. The capacity of the obtained TiO₂ · PL · DNA/PNA nano-composites depending on immobilization conditions was 10?C30 nmol PNA per 1 mg of TiO₂ particles, which corresponds to ??1?C3 PNA molecules per one TiO₂ particle with a size of 4?C6 nm. It was shown by confocal laser scanning microscopy that fluorescently-labeled PNA molecules in the TiO₂ · PL · DNA/^FluPNA nano-composites effectively penetrate into HeLa cells without transfection agents, electroporation, or other auxiliary procedures.     

Intercoupling surface plasmon resonance and diffusion reflection measurements for real‐time cancer detection

Spatial diffusion reflection (DR) measurements of gold nanorods (GNR) were recently suggested as a simple and highly sensitive non‐invasive and non‐ionizing method for real‐time cancer detection. In this paper we demonstrate that wavelength dependent DR measurements enable the spectral red‐shift observation of highly concentrated GNR. By conjugating targeting moieties to the GNR, large density of GNR can specifically home onto cancer cells. The inter‐particle plasmon resonance pattern of the highly concentrated GNR leads to an extension and a red‐shift (Δλ) in the absorption spectrum of the concentrated GNR. Dark‐field microscopy was used in order to measure the expected Δλ in different GNR concentrations in vitro. Double‐wavelength DR measurements of tissue‐like phantoms and tumor bearing mice containing different GNR concentrations are presented. We show that the DR profile of the highly concentrated GNR directly correlate with the spectral extension and red‐shift. This presented work suggests that wavelength dependent DR method can serve as a promising tool for real‐time superficial tumor detection. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

BL-7010 Demonstrates Specific Binding to Gliadin and Reduces Gluten-Associated Pathology in a Chronic Mouse Model of Gliadin Sensitivity

Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.     

Second-Line Treatment of Her2-Positive Metastatic Breast Cancer: Trastuzumab beyond Progression or Lapatinib? A Population Based Cohort Study

Background

The relative efficacy of lapatinib vs. continuing trastuzumab beyond progression (TBP) in HER2-positive metastatic breast cancer (MBC) patients, who progressed on first-line trastuzumab, is still unclear. The objective of this population based cohort study was to compare outcomes of lapatinib vs. TBP in daily practice.

Methods

All HER2-positive MBC patients who began second-line anti HER2 therapy between 1st January 2010 and 30th August 2013 were selected from Clalit Health Services’ (CHS) electronic database. Available data on patient and disease characteristics and treatments were analyzed. The primary endpoint was overall survival (OS). Outcomes were compared using the Kaplan-Meier (log-rank) method and Cox proportional hazards model.

Results

64 patients received second-line lapatinib and 93 TBP. The two treatment groups were similar in age and co-morbidity rates, but differed in proportion of prior adjuvant trastuzumab (lapatinib: 29.7%, TBP: 16.1%, P = 0.043) and rates of prior brain metastases (lapatinib: 32.8%, TBP: 10.8%, P = 0.01). Lapatinib median OS was 13.0 months (95% CI: 9.5–16.5) vs. 31.0 for TBP (95% CI: 20.6–41.4), P<0.001. On multivariate analysis, longer OS was preserved for TBP, after controlling for differences in age, adjuvant trastuzumab, duration of first-line trastuzumab therapy, brain metastases, visceral metastases and hormonal treatment [Hazard Ratio (HR) = 0.63, 95% CI: 0.40–0.99, P = 0.045].

Conclusion

In this comparative cohort study, OS of HER2-positive MBC patients treated with TBP was significantly longer than with lapatinib. These results might be especially relevant in settings where ado-trastuzumab-emtansine (TDM-1), the current preferred agent in this setting, is not available yet for patients.     

Mass spectrometry‐based methods to study protein architecture and dynamics

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post‐translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini‐review highlights several popular mass spectrometry‐based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry.     

Differential role of the carboxy-terminus of the A2B adenosine receptor in stimulation of adenylate cyclase, phospholipase Cβ, and interleukin-8

In human mast cells and microvascular endothelial cells, the A_2B adenosine receptor controls at least three independent signaling pathways, i.e., Gs-mediated stimulation of adenylate cyclase, Gq-mediated stimulation of phospholipase Cβ, and Gs/Gq-independent upregulation of IL-8. Functional analysis of cells transfected with full-length and truncated receptor constructs revealed that the A_2B receptor C-terminus is important for coupling to Gs and Gq proteins. Removal of the entire cytoplasmic portion in the A_2B receptor C-terminus rendered it incapable of stimulating adenylate cyclase and phospholipase Cβ. Conversely, removal of the distal 16 amino acids facilitated signal transduction from the receptor to the downstream Gs but not Gq proteins. However, the A_2B receptor C-terminus is not essential for upregulation of IL-8. Analysis of chimeric A_2A/A_2B receptors demonstrated that only chimeras containing the third intracellular loop of the A_2B receptor mediated agonist-dependent IL-8 reporter stimulation, suggesting that this domain is important for upregulation of IL-8.