Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.     

Betacyanin concentrations in young cell cultures from Portulaca grandiflora — an analysis of variation

From seedlings of two violet flowering, inbred lines of Portulaca grandiflora hypocotyls were isolated and then halved by cross sections. On a modified Murashige-Skoog medium the pairs of segments frequently yielded quartetts of primary calli from the four cut surfaces. After some subcultivations of these groups, the betacyanin concentrations of cell cultures were measured and subjected to several statistical methods.In 5.0 per cent of all cell cultures belonging to line L and in 2.1 per cent of those derived from line H extremely high and low concentrations were caused by the in vitro conditions. Some other ones are obviously due to the character of seedlings used for experiment L 2.Furthermore it is statistically detectable that the cell culture coming from the basipetal cut of a hypocotyl segment is superior to the cell culture of acropetal origin with regard to betacyanin concentration. These differences indicate the polarization of physiological conditions in the hypocotyl segments.Abbreviations DW dry weight - FW fresh weight     

Long insert whole genome sequencing for copy number variant and translocation detection

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.     

A novel index to aid in prioritizing habitats for site‐based conservation

Funding biodiversity conservation strategies are usually minimal, thus prioritizing habitats at high risk should be conducted. We developed and tested a conservation priority index (CPI) that ranks habitats to aid in prioritizing them for conservation. We tested the index using 1897 fish species from 273 African inland lakes and 34 countries. In the index, lake surface area, rarity, and their International Union for Conservation of Nature (IUCN) Red List status were incorporated. We retrieved data from the Global Biodiversity Information Facility (GBIF) and IUCN data repositories. Lake Nyasa had the highest species richness (424), followed by Tanganyika (391), Nokoué (246), Victoria (216), and Ahémé (216). However, lakes Otjikoto and Giunas had the highest CPI of 137.2 and 52.1, respectively. Lakes were grouped into high priority (CPI > 0.5; n = 56) and low priority (CPI < 0.5; n = 217). The median surface area between priority classes was significantly different (W = 11,768, p < .05, effect size = 0.65). Prediction accuracy of Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) for priority classes were 0.912 and 0.954, respectively. Both models exhibited lake surface area as the variable with the highest importance. CPI generally increased with a decrease in lake surface area. This was attributed to less ecological substitutability and higher exposure levels of anthropogenic stressors such as pollution to a species in smaller lakes. Also, the highest species richness per unit area was recorded for high‐priority lakes. Thus, smaller habitats or lakes may be prioritized for conservation although larger waterbodies or habitats should not be ignored. The index can be customized to local, regional, and international scales as well as marine and terrestrial habitats.     

Pectic oligosaccharides from agricultural by-products: production,characterization and health benefits

Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited.     

Synthesis of fluorine substituted pyrazolopyrimidines as potential leads for the development of PET-imaging agents for the GABAA receptors

Neuroimaging of GABA(A) receptors offers the potential for a better diagnosis of diseases related to a dysfunction of the GABAergic neurotransmission. A series of potent fluorinated analogues of the pyrazolopyrimidine Indiplon has been synthesized and evaluated in vitro as potential agents for imaging the GABA(A) receptor by means of positron emission tomography (PET). The most promising compound N-(3-fluoropropyl)-N-[3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]-phenyl]-acetamide (5b) showed an IC(50) value of 2.78+/-0.63 nM comparable to the lead compound Indiplon (IC(50) 3.29+/-0.37 nM), thus making it an interesting candidate for further investigations. In addition to the fluorinated reference compounds, suitable precursors for (18)F-radiolabelling studies have been synthesized.     

The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice

The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.     

Estimating Kidney Function in HIV-Infected Adults in Kenya: Comparison to a Direct Measure of Glomerular Filtration Rate by Iohexol Clearance

Background

More than two-thirds of the world''s HIV-positive individuals live in sub-Saharan Africa, where genetic susceptibility to kidney disease is high and resources for kidney disease screening and antiretroviral therapy (ART) toxicity monitoring are limited. Equations to estimate glomerular filtration rate (GFR) from serum creatinine were derived in Western populations and may be less accurate in this population.

Methods

We compared results from published GFR estimating equations with a direct measure of GFR by iohexol clearance in 99 HIV-infected, ART-naïve Kenyan adults. Iohexol concentration was measured from dried blood spots on filter paper. The bias ratio (mean of the ratio of estimated to measured GFR) and accuracy (percentage of estimates within 30% of the measured GFR) were calculated.

Results

The median age was 35 years, and 60% were women. The majority had asymptomatic HIV, with median CD4+ cell count of 355 cells/mm³. Median measured GFR was 115 mL/min/1.73 m². Overall accuracy was highest for the Chronic Kidney Disease Epidemiology Consortium (CKD-EPI) equation. Consistent with a prior report, bias and accuracy were improved by eliminating the coefficient for black race (85% of estimates within 30% of measured GFR). Accuracy of all equations was poor in participants with GFR 60–90 mL/min/1.73 m² (<65% of estimates within 30% of measured GFR), although this subgroup was too small to reach definitive conclusions.

Conclusions

Overall accuracy was highest for the CKD-EPI equation. Eliminating the coefficient for race further improved performance. Future studies are needed to determine the most accurate GFR estimate for use in individuals with GFR <90 mL/min/1.73 m², in whom accurate estimation of kidney function is important to guide drug dosing. Direct measurement of GFR by iohexol clearance using a filter paper based assay is feasible for research purposes in resource-limited settings, and could be used to develop more accurate GFR estimates in African populations.