Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.     

β-Glucan extracts inhibit the in vitro intestinal uptake of long-chain fatty acids and cholesterol and down-regulate genes involved in lipogenesis and lipid transport in rats

BackgroundDietary fiber reduces the intestinal absorption of nutrients and the blood concentrations of cholesterol and triglycerides.AimWe wished to test the hypothesis that high-viscosity (HV) and low-viscosity preparations of barley and oat β-glucan modify the expression of selected genes of lipid-binding proteins in the intestinal mucosa and reduce the intestinal in vitro uptake of lipids.MethodsFive different β-glucan extracts were separately added to test solutions at concentrations of 0.1–0.5% (wt/wt), and the in vitro intestinal uptake of lipids into the intestine of rats was assessed. An intestinal cell line was used to determine the effect of β-glucan extracts on the expression of intestinal genes involved in lipid metabolism and fatty acid transport.ResultsAll extracts reduced the uptake of 18:2 when the effective resistance of the unstirred water layer was high. When the unstirred layer resistance was low, the HV oat β-glucan extract reduced jejunal 18:2 uptake, while most extracts reduced ileal 18:2 uptake. Ileal 18:0 uptake was reduced by the HV barley extract, while both jejunal and ileal cholesterol uptakes were reduced by the medium-purity HV barley extract. The inhibitory effect of HV barley β-glucan on 18:0 and 18:2 uptake was more pronounced at higher fatty acid concentrations. The expression of genes involved in fatty acid synthesis and cholesterol metabolism was down-regulated with the HV β-glucan extracts. β-Glucan extracts also reduced intestinal fatty-acid-binding protein and fatty acid transport protein 4 mRNA.ConclusionsThe reduced intestinal fatty acid uptake observed with β-glucan is associated with inhibition of genes regulating intestinal uptake and synthesis of lipids. The inhibitory effect of β-glucan on intestinal lipid uptake raises the possibility of their selective use to reduce their intestinal absorption.     

Substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs as potent Kv1.3 ion channel blockers. Part 2

We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced further as potential tool compounds for in vivo validation studies.     

Structure-based design of novel human Pin1 inhibitors (II)

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge–charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand–protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.     

Effects of sieving, drying and rewetting upon soil bacterial community structure and respiration rates

Soil microcosm studies often require some form of soil homogenisation, such as sieving, to provide a representative sample. Frequently, soils are also homogenised following drying and are then rewetted, yet little research has been done to understand how these methods impact upon microbial communities. Here we compared the molecular diversity and functional responses of intact cores from a Scottish grassland soil with homogenised samples prepared by drying, sieving and rewetting or freshly sieving wet soils. Results showed that there was no significant difference in total soil CO₂-C efflux between the freshly sieved and intact core treatments, however, respiration was significantly higher in the dried and rewetted microcosms. Molecular fingerprinting (T-RFLP) of bacterial communities at two different time-points showed that both homogenisation methods significantly altered bacterial community structure with the largest differences being observed after drying and rewetting. Assessments of responsive taxa in each treatment showed that intact cores were dominated by Acidobacterial peaks whereas an increased relative abundance of Alphaproteobacterial terminal restriction fragments were apparent in both homogenised treatments. However, the shift in community structure was not as large in the freshly sieved soil. Our findings suggest that if soil homogenisation must be performed, then fresh sieving of wet soil is preferable to drying and rewetting in approximating the bacterial diversity and functioning of intact cores.     

Naphthoquinone-lactones and extended quinones from Ventilago calyculata

New quinones have been isolated from the root bark of Ventilago calyculata. Ventilatones A and B are benzisochromanquinones, related to the ventiloquinones, which have an additional fused lactone ring while ventileins A and B are benzisochroman dimers having a dihydroxy-peri-xanthenoxanthenequinone chromophore.     

Benzisochromanquinones in ventilago species

From the acetone extract of the root bark of Ventilago maderaspatana eight new benzisochromanquinones; ventiloquinones A, B, C, D, E, F, G and H, have been isolated. Ventiloquinones I, J and K are three more new benzisochromanquinones isolated from the root bark of V. calyculata. The majority are 3,4,5,10-tetrahydro-cis-1,3-dimethyl-1H-naphtho[2,3-c]pyran-5,10-quinones related to eleutherin, but F, H, I, J and K are 6,9-quinones related to ventilagone.     

Evaluation of culture methods to identify bovine feces with high concentrations of Escherichia coli O157

Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.     

Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice

LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 +/- 0.9 km/day for WT vs. 1.7 +/- 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency.