High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C(1)-BODIPY-C(12)). The library used consisted of 2,080 compounds with known biological activities. Of these, approximately 1.8% reduced cell-associated C(1)-BODIPY-C(12) fluorescence and were selected as potential inhibitors of human FATP2-mediated fatty acid uptake. Based on secondary screens, 28 compounds were selected as potential fatty acid uptake inhibitors. Some compounds fell into four groups with similar structural features. The largest group was structurally related to a family of tricyclic, phenothiazine-derived drugs used to treat schizophrenia and related psychiatric disorders, which are also known to cause metabolic side effects, including hypertriglyceridemia. Potential hit compounds were studied for specificity of interaction with human FATP and efficacy in human Caco-2 cells. This study validates this screening system as useful to assess the impact of drugs in preclinical screening for fatty acid uptake.     

Retrospective review of pelvic malignancies undergoing total pelvic exenteration

ABSTRACT: BACKGROUND: In patients with locally advanced or recurrent pelvic malignancies, total pelvic exenteration (TPE) may be necessary for curative treatment. Despite improvements in mortality rates since TPE was first described, morbidity rates remain high due to the extensive resection and the aggressiveness of these tumors. We have studied the outcomes of TPE surgery performed at our institution. METHODS: Fifty-three patients with various pelvic pathologies underwent TPE between 2004 and 2010. Patients were divided into two groups based on pathology: colorectal (n =36) versus noncolorectal (n =17) malignancies. Demographics, operative reports, pathology reports, periprocedural events, and outcomes were analyzed. Comparison of the two groups was performed using student'st-test and Fisher's exact test. Survival curves were constructed using the Kaplan-Meier method and compared using the log rank test. RESULTS: The colorectal and non-colorectal groups were similar in demographics, operative times, length of stay, estimated blood loss, and rates of preoperative and intraoperative radiation use. Chemotherapy use was increased in the colorectal group compared with the noncolorectal group (55.6% vs. 23.5%, P =0.04). Complication rates were similar: 86% in the colorectal group and 76% in the non-colorectal group. In the colorectal group, 27.8% of patients developed perineal abscesses, whereas no patients developed these complications in the non-colorectal group (P =0.02). No survival difference was seen in primary versus recurrent colorectal tumors; however, within the colorectal group there was a survival advantage when comparing R0 resection to R1 and R2 resection combined. Median survival rates were 27.3 months for R0 resection and 10.7 months for R1 and R2 resection combined. The median survival was 21.4 months for the colorectal group and 6.9 months for the noncolorectal group (P =0.002). CONCLUSIONS: Patients undergoing TPE for colorectal tumors have improved survival when compared with patients undergoing exenteration for pelvic malignancies of other origins. Within the colorectal group, the extent of resection demonstrated a significant survival benefit of an R0 resection compared with R1 and R2 resections. Despite TPE carrying a high morbidity rate, mortality rates have improved and careful patient selection can optimize outcomes.     

Regulation of perforin activation and pre‐synaptic toxicity through C‐terminal glycosylation

Perforin is a highly cytotoxic pore‐forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca²⁺‐rich endoplasmic reticulum, remains unknown. Here, we show that N‐linked glycosylation of the perforin C‐terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C‐terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C‐terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post‐translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.     

Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.rRNA genes are widely used for estimation of evolutionary history and taxonomic assignment of individual organisms (14, 26, 50-52). The choice of rRNA genes as optimal tools for such purposes is based on both observations and assumptions of ribosomal conservation (13, 50). rRNA genes are essential components of the ribosome, which consists of >50 proteins and three classes of RNA molecules; precise spatial relationships may be essential for assembly of functional ribosomes, constraining rRNA genes from drastic change (9, 13). In bacteria, the three rRNA genes are organized into a gene cluster which is expressed as single operon, which may be present in multiple copies in the genome. In organisms with multiple rRNA gene operons, the gene sequences tend to evolve in concert. It is generally believed that copies of rRNA genes within an organism are subject to a homogenization process through homologous recombination, also known as gene conversion (18), a form of concerted evolution that maintains their fit within the ribosome. The homogenization process may involve short domains without affecting the entire sequence of each gene (8).However, significant differences between copies of rRNA genes in single organisms, albeit few, have been discovered in all three domains of life and in all three classes of rRNA genes. The amphibian Xenopus laevis and the loach Misgurnus fossilis have two types of 5S rRNA genes that are specific to either somatic or oocyte ribosomes (30, 48). The parasite Plasmodium berghei contains two types of 18S rRNA genes that differ at 3.5% of the nucleotide positions and are life cycle stage specific (17). The metazoan Dugesia mediterranea possesses two types of 18S rRNA genes with 8% dissimilarity (6). The archaeon Haloarcula marismortui contains two distinct types of 16S rRNA genes that differ by 5% (32, 33). In the domain Bacteria, the actinomycete Thermobispora bispora contains two types of 16S rRNA genes that differ by 6.4% (47). Copies of the 16S rRNA genes and 23S rRNA genes of the actinomycete Thermospora chromogena differ by approximately 6 and 10%, respectively (54). Paralogous copies of rRNA genes with different sequences may have functionally distinct roles.Divergent evolution between rRNA genes in the same genome may corrupt the record of evolutionary history and obscure the true identity of an organism. Substantial variation, if it occurs, may lead to the artificial classification of an organism into more than one species. For a cultivable organism, this problem can be resolved by cloning rRNA genes from a pure culture of the organism to identify the degree of variation. However, most environmental surveys and the recently initiated Human Microbiome Project (HMP) (http://nihroadmap.nih.gov/hmp/) (34) use cultivation-independent techniques to examine microbiomes that contain mixed species. In the case of the HMP, it is hoped that this approach may identify some idiopathic diseases that are caused by alterations in the microbiome in humans. In this type of study, it may be impossible to trace all rRNA genes observed back to their original host. For example, in the phylum TM7, multiple 16S rRNA gene sequences have been reported (21), but it is not known whether they belong to multiple species or to the same bacterium with a high degree of intragenomic variation among rRNA gene paralogs. Due to the limited number of microorganisms for which nucleotide sequences are available for all copies of the rRNA genes, intragenomic variation among 16S rRNA genes, and the likelihood of pyrosequencing errors (25, 40), the potential to overestimate the diversity of a microbiome exists.Coenye et al. analyzed 55 bacterial genomes and found the intragenomic heterogeneity between multiple 16S rRNA genes in these genomes was below the common threshold (1 to 1.3%) for distinguishing species (44) and was unlikely to have a profound effect on the classification of taxa (10). The analysis of 76 whole genomes by Acinas et al. revealed the extreme diversity (11.6%) of 16S rRNA genes in Thermoanaerobacter tengcongensis (2). These early analyses of intragenomic variation of 16S rRNA genes were limited to a small number of available whole genomes. With the increasing number of whole microbial genomes available from the National Center for Biotechnology Information (NCBI), the extent of diversity among the paralogous 16S rRNA genes within single organisms can now be more thoroughly assessed. In the present study, we (i) addressed the theory of 16S rRNA conservation by systematic evaluation of intragenomic diversity of 16S rRNA sequences in completely sequenced prokaryotic genomes to assess its effect on the accuracy of 16S rRNA-based molecular taxonomy and (ii) examined whether previously observed ribosomal constraints on conservation of 2° structures are uniformly applicable at the intragenomic level.     

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

Stable carbon and oxygen isotopes in human tooth enamel: Identifying breastfeeding and weaning in prehistory. Am. J. Phys. Anthropol. 106: 1–18.

ERRATUM: Wright LE and Schwarcz HP (1998) Stable Carbon and Oxygen Isotopes in Human Tooth Enamel: Identifying Breastfeeding and Weaning in Prehistory. Am. J. Phys. Anthropol. 106: 1–18. Isotopic ratios were incorrectly printed as percentages (%) rather than units permil (‰). Wherever “breast feeding” and “breast fed” occur, the words should be combined into “breastfeeding” and “breastfed” respectively. The correct information on isotopic ratios is the following: p. 1, Abstract: all % signs should be “‰”; p. 2, column two, last line: “… δ¹³C values¹, have been …”; p. 2, Footnote 1 should read: “Isotope ratios of carbon and oxygen are expressed in δ notation as follows, δ = [(R_sample/R_standard) − 1] × 1000, where R = ¹³C/¹²C for δ¹³C, and R = ¹⁸O/¹⁶O for δ¹⁸O, and are in units permil, ‰.”; p. 3, column two: all % signs should be “‰”, except for the 23rd line from the bottom, which reads: “… provided 95% of water intake by all infants …”; p. 5: all % signs should be “‰”; p. 6, column one: 8th and 9th line from bottom should be “… mean deviations were 0.029‰ for δ¹³C and 0.024‰ for δ¹⁸O …”; p. 6, column one: 2nd line from bottom should be “… only 0.208‰ for δ¹³C and only 0.091‰ for δ¹⁸O …”; p. 6, column two: top line: “… of 0.5‰ in δ¹³C and 0.2‰ in δ¹⁸O …”; p. 8, 9 and 10: all % signs should be “‰”; p. 11, column two: 7th line from top: “… the lipids are 4-6‰ lighter …”; p. 12, 13 and 14: all % signs should be “‰.”