Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny¹. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44⁺CD24^low/-)². Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues^3-5.Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD133^6-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH⁺ and CD44⁺CD24⁺ pancreatic CSCs are similarly tumorigenic, but ALDH⁺ cells are relatively more invasive⁸. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts⁹. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH¹⁰. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.     

Loss of the Mono-ADP-ribosyltransferase,Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp^−/− mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp^+/+ mice survived the 30-day treatment. Dioxin-treated Tiparp^−/− mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.     

Solid-phase microextraction and the human fecal VOC metabolome

The diagnostic potential and health implications of volatile organic compounds (VOCs) present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME) has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 μm CAR-DVB-PDMS, 85 μm CAR-PDMS, 65 μm DVB-PDMS, 7 μm PDMS, and 60 μm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein.     

Functional characterization of germin and germin-like protein genes in various plant species using transgenic approaches

The important role of germins (GER) and genes coding for germin-like proteins (GLP) in responses against various stresses in both homologous and heterologous systems is well validated. This review summarizes the work on their functional validation using various biotechnological approaches. The genes are widely expressed during a specific period of plant growth and development, and exhibit a pattern of evolutionary subfunctionalization at both the intracellular and whole plant level. Their applications against various biotic and abiotic stresses, especially against fungal pathogens, are enormous. Although the validation of these proteins against various stresses has led to the development of commercially and agronomically important transgenic plants, much work is still needed to exploit this ever-expanding repertoire of genes and deploy them for commercial use. Historical progress of genetic engineering in GERs and GLPs is reviewed, and future prospects for their potential role in crop improvement are highlighted.     

Genome-Wide Association of Stem Water Soluble Carbohydrates in Bread Wheat

Water soluble carbohydrates (WSC) in stems play an important role in buffering grain yield in wheat against biotic and abiotic stresses; however, knowledge of genes controlling WSC is very limited. We conducted a genome-wide association study (GWAS) using a high-density 90K SNP array to better understand the genetic basis underlying WSC, and to explore marker-based breeding approaches. WSC was evaluated in an association panel comprising 166 Chinese bread wheat cultivars planted in four environments. Fifty two marker-trait associations (MTAs) distributed across 23 loci were identified for phenotypic best linear unbiased estimates (BLUEs), and 11 MTAs were identified in two or more environments. Liner regression showed a clear dependence of WSC BLUE scores on numbers of favorable (increasing WSC content) and unfavorable alleles (decreasing WSC), indicating that genotypes with higher numbers of favorable or lower numbers of unfavorable alleles had higher WSC content. In silico analysis of flanking sequences of trait-associated SNPs revealed eight candidate genes related to WSC content grouped into two categories based on the type of encoding proteins, namely, defense response proteins and proteins triggered by environmental stresses. The identified SNPs and candidate genes related to WSC provide opportunities for breeding higher WSC wheat cultivars.     

Ceph-X: development and evaluation of 2D cephalometric system

Background

Cephalometric analysis and measurements of skull parameters using X-Ray images plays an important role in predicating and monitoring orthodontic treatment. Manual analysis and measurements of cephalometric is considered tedious, time consuming, and subjected to human errors. Several cephalometric systems have been developed to automate the cephalometric procedure; however, no clear insights have been reported about reliability, performance, and usability of those systems. This study utilizes some techniques to evaluate reliability, performance, and usability metric using SUS methods of the developed cephalometric system which has not been reported in previous studies.

Methods

In this study a novel system named Ceph-X is developed to computerize the manual tasks of orthodontics during cephalometric measurements. Ceph-X is developed by using image processing techniques with three main models: enhancements X-ray image model, locating landmark model, and computation model. Ceph-X was then evaluated by using X-ray images of 30 subjects (male and female) obtained from University of Malaya hospital. Three orthodontics specialists were involved in the evaluation of accuracy to avoid intra examiner error, and performance for Ceph-X, and 20 orthodontics specialists were involved in the evaluation of the usability, and user satisfaction for Ceph-X by using the SUS approach.

Results

Statistical analysis for the comparison between the manual and automatic cephalometric approaches showed that Ceph-X achieved a great accuracy approximately 96.6%, with an acceptable errors variation approximately less than 0.5 mm, and 1°. Results showed that Ceph-X increased the specialist performance, and minimized the processing time to obtain cephalometric measurements of human skull. Furthermore, SUS analysis approach showed that Ceph-X has an excellent usability user’s feedback.

Conclusions

The Ceph-X has proved its reliability, performance, and usability to be used by orthodontists for the analysis, diagnosis, and treatment of cephalometric.

     

Forest situation analysis and future forecasting of famous Upper Tanawal forests ecosystems on western banks of lesser Himalaya

The current work was carried out in Upper Tanawal which lies in the mid of Western part of lesser Himalaya. The area lies between 34°0.34.40′ N to 34°0.48.88′ N latitude and 72°0.84.27′ E to 73°0.10.50′ E longitude. Upper Tanawal is rich in biodiversity. No work regarding forest and forest situation is done previously in this area. (Farooq, et al., 2017). The area was once housing notable and thick forest strands famous for their habitat uniqueness to harbor number of pheasant and other wildlife species. Due to security reasons and worse situation of law and order compromising government writ the area remained scientifically as well departmentally neglected resulting rapid and vigorous ruthless deforestation. The area was extensively visited and sampled using Quadrate method for phytosociological data collection. Phytosociological attributes like density, cover, and frequency were also Calculated and recorded for all tree species of the area in each quadrat. Altitude, slope angle, atmospheric humidity and temperature were also considered as environmental variables for each sampling site. Results reflect that Upper Tanawal hosts 52 different tree species of 39 genera belonging to 25 tree families.The important forests of Upper Tanawal are under high anthropogenic and environmental disturbances. Some of them may easily be managed because of the presence of large number of seedling; however, some areas need serious attention. It was observed that Pinus roxburghii and Pinus wallichiana are the potential tree species for reforestation and afforestation in the area. This study will be the baseline for new researchers in the study area.     

Attenuation of DNA damage and mRNA gene expression in hypoxic rats using natural antioxidants

This study aimed to explore the efficiency of carnosine (Cs) and/or l ‐arginine (Agn) in the downregulation of apoptotic and inflammatory molecule expression and DNA damage caused hepatic injury in response to sodium nitrite (Sd)‐induced hypoxia in rats. Rats were injected with Sd; Agn or/and Cs were administrated prior to Sd intoxication. Sd significantly decreased hemoglobin concentration and Bcl‐2 mRNA expression, while increased expressions of apoptotic markers (Bax and caspase), tumor necrosis factor‐α, nuclear factor kappa B, and C‐reactive protein and the oxidative DNA damage in hepatic tissue. Moreover, administration of Agn or/and Cs exhibited a modulation of the previous parameters. However, concurrent treatment with the forementioned antioxidants modulated these levels. It was concluded that the treatment with the combination of Agn and Cs was the most effective regimen in ameliorating Sd toxicity accompanied by hypoxic stress.     

Genome-wide association study for agronomic and physiological traits in spring wheat evaluated in a range of heat prone environments

Key message

We identified 27 stable loci associated with agronomic traits in spring wheat using genome-wide association analysis, some of which confirmed previously reported studies. GWAS peaks identified in regions where no QTL for grain yield per se has been mapped to date, provide new opportunities for gene discovery and creation of new cultivars with desirable alleles for improving yield and yield stability in wheat.

Abstract

We undertook large-scale genetic analysis to determine marker-trait associations (MTAs) underlying agronomic and physiological performance in spring wheat using genome-wide association studies (GWAS). Field trials were conducted at seven sites in three countries (Sudan, Egypt, and Syria) over 2–3 years in each country. Twenty-five agronomic and physiological traits were measured on 188 wheat genotypes. After correcting for population structure and relatedness, a total of 245 MTAs distributed over 66 loci were associated with agronomic traits in individual and mean performance across environments respectively; some of which confirmed previously reported loci. Of these, 27 loci were significantly associated with days to heading, thousand kernel weight, grain yield, spike length, and leaf rolling for mean performance across environments. Despite strong QTL by environment interactions, eight of the loci on chromosomes 1A, 1D, 5A, 5D, 6B, 7A, and 7B had pleiotropic effects on days to heading and yield components (TKW, SM^?2, and SNS). The winter-type alleles at the homoeologous VRN1 loci significantly increased days to heading and grain yield in optimal environments, but decreased grain yield in heat prone environments. Top 20 high-yielding genotypes, ranked by additive main effects and multiplicative interaction (AMMI), had low kinship relationship and possessed 4–5 favorable alleles for GY MTAs except two genotypes, Shadi-4 and Qafzah-11/Bashiq-1–2. This indicated different yield stability mechanisms due to potentially favorable rare alleles that are uncharacterized. Our results will enable wheat breeders to effectively introgress several desirable alleles into locally adapted germplasm in developing wheat varieties with high yield stability and enhanced heat tolerance.

     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.