MultiPriDe: automated batch development of quantitative real-time PCR primers

Quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) is a commonly employed gene expression quantification technique. This requires the development of appropriately targeted oligonucleotide primers, which necessitates the identification of ideal amplicons, development of optimized oligonucleotide sequences under most favorable pre-determined reaction conditions, and management of the resultant target-oligonucleotide pair information for each gene to be studied. The Primer3 utility exists for development of oligonucleotide primers and fills that role effectively. However, the manual process of identifying target sites and individually generating primers is inefficient and prone to user-introduced error, especially when a large number of genes are to be examined. We have developed MultiPriDe (Multiple Primer Design), a Perl utility that accepts batch lists of Gene database identifiers, collects available intron and exon position data critical to qRT–PCR primer development, and supplies these sites as identified targets for the Primer3 utility. This automated ‘gene to primer’ procedure is coupled with a set of optimized hybridization conditions used by the Primer3 utility to maximize successful primer design. MultiPriDe and assembled repeat libraries are available upon request. Please direct requests to [email protected]

Uticaj transgenaze na kvalitet i prinos mesa kunića

In this paper results of the effect of transgenesis on quality and yield of rabbit meat are presented. During the trial body mass of transgenic progeny of F1 generation was monitored and compared to control group (nontransgenic animals of same age). Subsequent to slaughtering, meat yield, ratio between certain musculature parts and meat quality (proteins, lipids, water) were analyzed. Obtained data was compared to control group of animals of same age but standard genotype. Meat colour was evaluated on apparatus Specol 11 and expressed as percentage of remission on wave length of 540 μm. Content of elements in thigh muscle was established subsequent to dry mineralization in spectro-photometer UNICAM 939 Cambridge UK. Phosphorus content was measured spectro-photometrically on apparatus SPECOL 11. Subsequent to measuring and systematization, data was statistically analyzed and processed. Arithmetic mean values for certain groups of data were calculated, and their values compared using t-test (Hadživuković, 1991). Changes established in regard to content of water, lipids, energy and water binding capacity, were relative to changes in histological structure and level of metabolic processes. It is possible that these changes are result of pleiotropic effect of integrated gene. However, in order to confirm and interpret these changes, it is necessary to carry out further researches of the microscopic structure and metabolic processes of muscle tissues in transgenic rabbits.U radu su prikazani rezultati uticaja transgeneze na kvalitet i prinos mesa kunića. Ogled je vršen na komercijalnim tovnim hibridima nastalim ukrštanjem Novozelandskih belih i Kalifornijskih kunića. Dobijeni podaci upoređeni su sa kontrolnom grupom vršnjaka standardnog genotipa. Posmatrani su sledeći parametri kod obe grupe životinja: telesna masa (1, 2, 5, 10, 20 i 30- tog dana starosti), klanični podaci (w-težina pre žrtvovanja, dw- težina nakon iskrvarenja, s-težina kože, sp-težina distalnih delova zadnjih nogu, b- težina polutke sa kožom, hd-težina glave bez kože, fl- težina prednjih nogu, t- težina butova, r-težina rebara (grudi), bk - težina leđa, ht- težina srca, ky-težina bubrega, l- težina pluća, lvtežina jetre, git- težina stomaka i creva sa sadržajem, gite-težina praznog stomaka i creva, f- težina masnoće, bt- težina kostiju nogu, mt-težina mišića nogu, bf- težina kostiju prednjih nogu, c- prinos mesa (obraslost muskulaturom %); kvalitet mesa butova (cw- sadržaj vode, cp - sadržaj proteina, cf- sadržaj masti, ce- energija, pH, cc- boja, bw- kapacitet zadržavanja vode) i sadržaj mikroelemenata u mesu buta (Cu, Zn, Fe, K, Na, Mg, P, Ca). Posle merenja i sistematizacije podaci su statistički obrađeni. Izvršena su izračunavanja aritmetičkih sredina pojedinih grupa podataka, a zatim poređenje njihovih vrednosti t-testom (Hadživuković, 1991). Nizak nivo varijabiliteta u svim starosnim kategorijama u obe posmatrane grupe je jasno vidljiv iz vrednosti standardne greške aritmetičke sredine. To je manifestovano statistički značajnim uticajem procesa transgeneze na živu masu pri rođenju uprkos činjenici da je apsolutna razlika aritmetičkih sredina transgene i kontrolne grupe samo 0,005 kg (0,063±0,001 nasuprot 0,058±0,002) (tabela 1). Ovaj uticaj je na granici P=0,05 u tabeli analize varijanse (tabela 2). Razlika aritmetičkih sredina nije statistički značajna kod ostalih starosnih kategorija i ona se gubi već nakon 48 časova. Može se konstatovati da integrisani gen nema uticaja na porast transgenih kunića. Statistički značaj uticaja integrisanog gena je utvrđen kod parametara koji se odnose na masu distalnih delova transgenih i netransgenih kunića (0,062±0,001 nasuprot 0,069±0,001 kg), težine glave (0,119±0,003 nasuprot 0,128±0,003 kg) i težine butova (0,405±0,010 nasuprot 0,433±0,009 kg). Kod ostalih klaničnih karakteristika koje su testirane nisu utvrđene statistički značajne razlike koje bi bile uslovljene integracijom gena (tabele 3 i 4). Na uzorcima mesa nogu (butovi) praćene su vrednosti koje se odnose na kvalitet mesa (tabele 5 i 6). Dobijeni podaci koji se odnose na sastav mesa (tabela 5) ukazuju da je uticaj integracije gena bio statistički značajan (p (lt) 0,05) u grupi transgenih kunića u poređenju sa netransgenim u pogledu sledećih karakteristika: sadržaj proteina (74,03±0,26 nasuprot 74,84±0,28%), sadržaj masnoće (3,66±0,40 nasuprot 2,32±0,44%), sadržaja energije (495,43±11,81 nasuprot 458,07±12,94%), kapacitet zadržavanja vode (31,66±0,84 nasuprot 35,63±0,92%). Statistički značajne razlike kao posledica uticaja integrisanog gena nisu utvrđene kod ostalih posmatranih parametara (tabela 6). Srednje vrednosti sadržaja elemenata u tkivu mišića pokazale su najveće varijacije od svih posmatranih parametara. Najizraženije varijacije bile su u grupi netransgenih kunića (tabela 7). Broj životinja ili ponavljanja je igrao veliku ulogu. Za većinu posmatranih karakteristika može se reći da nisu pokazale uticaj integrisanog WAP- hFVIII gena u genotipu kunića. Značajnije razlike pojavile su se samo u okviru nekih parametara kvaliteta mesa (tabela 8). Rezultati ukazuju da nije utvrđeno ni prisustvo rhFVIII u skeletnim mišićima transgenih kunića

Druse-Induced Morphology Evolution in Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a key site of pathogenesis for many retina diseases. The formation of drusen in the retina is characteristic of retinal degeneration. We investigate morphological changes in the RPE in the presence of soft drusen using an integrated experimental and modeling approach. We collect RPE flat mount images from donated human eyes and develop 1) statistical tools to quantify the images and 2) a cell-based model to simulate the morphology evolution. We compare three different mechanisms of RPE repair evolution, cell apoptosis, cell fusion, and expansion, and Simulations of our RPE morphogenesis model quantitatively reproduce deformations of human RPE morphology due to drusen, suggesting that a purse-string mechanism is sufficient to explain how RPE heals cell loss caused by drusen-damage. We found that drusen beneath tissue promote cell death in a number that far exceeds the cell numbers covering the drusen. Tissue deformations are studied using area distributions, Voronoi domains and a texture tensor.Fil: Mazzitello, Karina Irma. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Zhang, Qing. University of Emory; Estados UnidosFil: Chrenek, M. A.. University of Emory; Estados UnidosFil: Family, F.. University of Emory; Estados UnidosFil: Grossniklaus, H. E.. University of Emory; Estados UnidosFil: Nickerson, J. M.. University of Emory; Estados UnidosFil: Jiang, Y.. Georgia State University; Estados Unido

THE EFFECT OF YUCCA SCHIDIGERA EXTRACT IN DIET OF RABBITS ON NUTRIENT DIGESTIBILITY AND QUALITATIVE PARAMETERS IN CAECUM

ABSTRACT The aim of this study was to determine the effects of yucca (Yucca schidigera) powder extract added to the rabbit feed mixtures. The animals of experimental group (EG1) were fed diet enriched with supplement of Yucca schidigera dry extract 5g/100 kg feed; rabbits of EG2 had diet enriched with yucca dry extract 20g/100 kg feed for 42 days. The control group (CG) was fed untreated pellet diet. The experiment was carried out on commercial hybrid Hycole rabbits. Between 65 and 70 days of age, 3 male rabbits from each group were selected for digestibility tests using the balance method. The faeces were collected individually during 5 consecutive days according to the European reference method for rabbit digestion trials. Chemical analysis of the diets and faeces for dry matter, crude protein, crude fibre, crude fat, nitrogen free extract, organic matter and starch was carried out according to the same method. The caecal samples from each of three slaughtered rabbits were collected for analysis; pH, VFA, ammonia-N and lactic acid were determined. The rabbits in our experiment showed the lowest coefficients of digestibility of crude fibre, and higher digestibility of crude protein, crude fat and organic matter, but the differences were not statistically significant comparing with control diet. The effect of yucca extract addition is manifested in reducing ammonia levels in the caecum of rabbits

Effects of dietary supplementation of nickel and nickel-zinc on femoral bone structure in rabbits

<p>Abstract</p> <p>Background</p> <p>Nickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits.</p> <p>Methods</p> <p>One month-old female rabbits were divided into three groups of five animals each. Group 1 rabbits were fed a granular feed mixture with addition of 35 g NiCl₂per 100 kg of mixture for 90 days. In group 2, animals were fed a mixture containing 35 g NiCl₂and 30 g ZnCl₂per 100 kg of mixture. Group 3 without administration of additional Ni or Zn served as control. After the 90-day experimental period, femoral length, femoral weight and histological structure of the femur were analyzed and compared.</p> <p>Results</p> <p>The results did not indicate a statistically significant difference in either femoral length or weight between the two experimental groups and the control group. Also, differences in qualitative histological characteristics of the femora among rabbits from the three groups were absent, except for a fewer number of secondary osteons found in the animals of groups 1 and 2. However, values for vascular canal parameters of primary osteons were significantly lower in group 1 than in the control one. Peroral administration of a combination of Ni and Zn (group 2) led to a significant decreased size of the secondary osteons.</p> <p>Conclusions</p> <p>The study indicates that dietary supplementation of Ni (35 g NiCl₂per 100 kg of feed mixture) and Ni-Zn combination (35 g NiCl₂and 30 g ZnCl₂per 100 kg of the mixture) affects the microstructure of compact bone tissue in young rabbits.</p

Carcinoma Matrix Controls Resistance to Cisplatin through Talin Regulation of NF-kB

Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin β1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin β1, talin and FAK pathways that regulate NF-kB nuclear activity

MultiPriDe: automated batch development

of quantitative real-time PCR primer

Novel regulators of rabbit reproductive functions

This is a review of original data concerning new extra- and intracellular regulators of rabbit ovarian functions. Effects of some hormones including leptin, ghrelin, oxytocin, arginine-vasotocin, endothelin (ET-1), gonadotropin releasing hormone (GnRH), adrenocorticotropic hormone (ACTH), growth factors such as insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), pharmacological regulators of some protein kinases such as protein kinase A (PKA), mitogen-activated protein (MAP) kinase, cell division cycle protein 2 homolog (CDC2 kinase, CDK), tyrosine kinases), and plant molecules (resveratrol, rapamycin) on the functions of ovarian cells (proliferation, apoptosis, secretory activity, expression of some protein kinases) and reproductive end points (blood level of reproductive hormones, ovarian morphology, number of ovulations, embryo yield and quality, number and viability of offspring), and their possible interrelationships and practical application in rabbit breeding are reviewed