qPCR

Autophagy is a metabolic process that degrades and recycles intracellular organelles and proteins with many connections to human disease and physiology. We studied the role of autophagy during hepatic stellate cell (HSC) activation, a key event in liver fibrogenesis. Analysis of the autophagic flux during in vitro activation of primary mouse HSCs was performed using a DsRed-GFP-LC3B encoding plasmid. The effect of autophagy inhibition by bafilomycin A1 on the in vitro activation process of human and mouse HSCs was examined by measuring proliferation, presence of activation markers by RT-qPCR, immunofluorescence, and Western blotting. Analysis of lipid droplet and microtubule-associated protein light chain 3 beta (LC3B) colocalization in the presence of PDGF-BB was investigated by immunocytochemistry. A significant increased autophagic flux was observed during culture induced mouse HSC activation. Treatment of mouse HSCs and human HSCs with autophagy inhibitor bafilomycin A1 results in a significant decreased proliferation and expression of activation markers. In addition, lipid droplets and LC3B colocalization was increased after PDGF-BB treatment in quiescent HSCs. During HSC activation, autophagic flux is increased. The demonstration of partly inhibition of in vitro HSC activation after treatment with an autophagy inhibitor unveils a potential new therapeutic strategy for liver fibrosis.

Wild aquatic birds, predominantly duck, geese and shorebirds, are natural reservoir of all subtypes of influenza A viruses. Avian influenza viruses are classified into two pathogenic types: low pathogenic avian influenza (LPAI) virus and highly pathogenic avian influenza (HPAI) virus. To date, LPAI has been isolated from migratory waterfowls, and the adaption of these viruses to land fowls might lead to the generation of HPAI which is a public health concern. The avian influenza virus is shed in large amounts in the feces, and can spread between birds by the fecaloral route and cause asymptomatic or low pathogenic infection. Alabama and Georgia are located in the flyways of wild migrating birds during the winter season, AIV surveillance of the samples from these states will be of great interest for the study of the epidemiology of LPAI virus. In the present study we analyzed the prevalence of AIV circulating in Alabama and Georgia, Real time RT-PCR was performed on 360 fecal samples. An overall AIV prevalence of 5% was detected with a peak during the spring and winter; resulting PCR product was further confirmed by sequencing, in which H1N1 subtype was identified. Our results support fecal sampling as an appropriate method for large-scale LPAI virus surveillance programs.

El personal del Ejército Nacional de Colombia se expone continuamente a contraer leishmaniasis debido a las operaciones que tienen lugar donde este parásito es endémico, lo cual ocasiona altos costos por tratamientos médicos. El objetivo de este estudio fue cuantificar la carga parasitaria mediante la amplificación del minicírculo del ADNk por PCR en tiempo real e identificar molecularmente la especie de Leishmania spp. en pacientes con diagnóstico para leishmaniasis cutánea, a partir de amplificación por PCR y secuenciación de los marcadores moleculares HSP70 y MPI, para monitorear y determinar la efectividad al tratamiento con antimoniato de meglumina frente al tratamiento con isetionato de pentamidina, así como su relación con otros datos clínico-epidemiológicos. Además, se demuestra la eficacia de los tratamientos con sus características, se identificó el riesgo de futuros brotes de leishmaniasis mucocutánea en ciertos casos y se refieren las diferencias encontradas entre las variables clínico-epidemiológicas explicadas multifactorialmente.

We test whether host quality drives mycorrhizal colonization strategies.We assess competitive dynamics between two fungi in the same microcosm using qPCR.We find that host quality affects competitive interactions among fungi.We conclude that plant quality can mediate fungal competition.Arbuscular mycorrhizal fungi exchange soil nutrients for carbon from plant hosts. Empirical works suggests that hosts may selectively provide resources to different fungal species, ultimately affecting fungal competition. However, fungal competition may also be mediated by colonization strategies of the fungi themselves. To test whether host quality drives fungal colonization strategies, we allowed competing fungi access to the roots of plants that varied in quality (manipulated by shading). We used quantitative PCR and microscopy to assess fungal competitive dynamics and found that shaded plants were not left as an open niche for less competitive fungi. However, while competitive fungi outcompeted less competitive fungi, the intensity of this effect depended on the quality of the host, with the strongest differences found on low-quality (shaded) hosts. Our results suggest that environmental conditions for the host aboveground play a role in the competitive interactions among fungi belowground.

Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.