Molecular genetic analysis of positional candidate genes for mammary gland characteristics in pigs

Inverted teat reduce the number of functional mammary complexes in affected sows leading for reduction of piglet productivity and litter growth during the lactation period. This detect occures in commercial pig breeds with frequencies between 8 to 30%. Due to selection of affected animals economic consequences in the breeding programm are considerate. The study aims to identify single nucleotide polymorphism (SNPs) in the positional candidate genes for the inverted teat defect in porcine, and to determine the linkage and physical location of TGFB1, RLN and PTHLH gene. Moreover the association of TGFB1, RLN and PTHLH gene on inverted teat trait were evaluated. The transforming growth factor beta 1 gene (TGFB1) and relaxin gene (RLN) were proposed to be tested for association in a positional candidate gene approach which combines linkage information for inverted teat defect and mapping information of a candidate gene. Both genes mapped in QTL regions for inverted teats discovered in the Bonn-Berlin resource family and involved in proliferation and differentiation processes of mammary gland. The family-based association test (FBAT) was used for allelic association between inverted teat defect and genotype. Highly significant evidence for association of RLN and the inverted teat defect affection in the Bonn-Berlin DUMI resource population was found. No significant effects of TGFB1 on affection status were detected. In addition, parathyroid hormone like hormone gene (PTHLH) was proposed as a functional candidate gene that regulates epithelial mesenchymal interactions during the formation of mammary gland. It could also been shown to be highly significantly associated with affection status. Highly significant association was also found between TGFB1, RLN and PTHLH loci and number of teats and number of inverted teats. Further confirmation of these results in independent samples of other populations will be conducted.Die molekular-genetischen Analysen von positionellen Kandidatengenen für Zitzenmerkmale beim Schwein Stülpzitzen reduzieren die Anzahl funktionierender Zitzen in betroffenen Sauen und führen zum Rückgang der Produktivität der Ferkelerzeugung und des Ferkelwachstums während der Laktation. Stülpzitzen findet man in kommerziellen Schweinerassen mit Frequenzen von 8 bis 30 %. Da von Defekten betroffene Tiere selektiert werden, wird also auch eine wirtschaftliche Produktion in Zuchtprogrammen berücksichtigt. Die vorliegende Arbeit zielt darauf ab, Punktmutationen (SNPs) in den positionellen Kandidatengenen für den Gendefekt Stülpzitze im Schwein zu finden und die physikalische Lokalisation der Gene TGFB1, RLN und PTHLH zu untersuchen bzw. den Genort über Kopplung zu bestimmen. Weiterhin wurde die Assoziation der Gene TGFB1, RLN und PTHLH zum Merkmal Stülpzitze errechnet. Das Transforming Growth Faktor Beta 1 Gen (TGFB1) und das Relaxin Gen (RLN) wurden mit dem positionellen Kandidatengenansatz, welcher die Kopplungsinformation für den Stülpzitzendefekt und die Kartierungsinformation eines Kandidatengens verknüpft, auf Assoziation getestet. Beide Gene, welche in den QTL Regionen für Stülpzitze kartieren, die in der Bonn-Berlin Ressourcen Familie gefunden wurden, sind an Proliferations- und Differenzierungsprozessen der Milchdrüsen beteiligt. Der Familien-basierte Assoziationstest (FBAT) wurde verwendet, um die allele Assoziation zwischen dem Defekt Stülpzitze und den Genotypen zu untersuchen. Die hohe Signifikanz belegt die Assoziation zwischen der Affektion des Defektes Stülpzitze und des Genotyps von RLN in der Bonn-Berlin Dumi Ressource Population. Es wurden keine signifikanten Effekte von TGFB1 auf den Defektstatus gefunden. Des weiteren wurde das Parathyroid Hormon Like Hormon Gen (PTHLH) als ein funktionelles Kandidatengen erwählt, das Epithel - Mesenchymale Interaktionen während der Bildung der Milchdrüsen reguliert. Es wurde ebenfalls eine hoch signifikante Assoziation mit dem Defektstatus gezeigt. Eine hoch signifikante Assoziation wurde ebenfalls zwischen den Genorten TGFB1, RLN und PTHLH und der Anzahl Zitzen und Anzahl Stülpzitzen gefunden

Articular Cartilage Gene Expression after Coxofemoral Joint Luxation in the Dog

This study examined the relationship between days of hip luxation and the expression of various mRNA. Twenty-six articular cartilages were used in the experiment: 3 samples were from normal dogs and 23 samples were collected from the femoral heads of hips that had been luxated for different lengths of time. Ten mRNA, including nonapoptotic genes (AGG, COL2A1, MMP-3, HAS-1, HAS-2, and TIMP-1) and apoptotic genes (BAX, BCL-2, CAS-3, and CAS-9), were studied for their expression using real-time PCR. We found very high correlation between expression level and luxation days (r2>0.9) in COL2A1, MMP-3, HAS-1, HAS-2, TIMP-1, BAX, and CAS-9, while the others (AGG, BCL-2, and CAS-3) also showed high correlation (r2=7–9). And we found a significant difference (P<0.05) in the expression of transcripts depending on the number of luxation days. In conclusion, a delay in joint reduction may increase the chances of development of osteoarthritis

Molecular phylogeny of trematodes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI)

AbstractObjectiveTo analyze a phylogenetic tree for understanding the molecular systematic of trematode in Family Heterophyidae, which are highly distributed in Thailand.MethodsBased on thirteen sequences of mitochondrial cytochrome c oxidase subunit I (mCOI) gene from six genera of heterophyid trematodes, viz. Haplorchis, Stellantchasmus, Centrocestus, Metagonimus, Pygidopsis, and Haplorchoides were aligned automatically using the Clustal × 2.0 program. A phylogenetic tree was constructed by maximum likeinghood (ML) and neighbor-joining (NJ) methods, with 1 000 bootstrap using the 5.0 program.ResultsThe phylogenetic relationship from both methods was similar and separated into three groups consisting of Haplorchoides pumilio group, Haplorchoides taichui group and another heterophyid genera.ConclusionsThe sequence data of mtCOI can be used to investigate the phylogenetic relationships of trematodes at the genus level. Each clade of different genera of heterophyid trematodes can be separated into sister groups that correlated with the morphological characteristic, kind of secondary intermediate host and geographic distribution

Determination of two fluoroquinolones and their combinations with hyaluronan effect in in vitro canine cartilage explants

Background Previous studies reported the effect of enrofloxacin (Enro) and marbofloxacin (Mar) on cell death and alteration of the key genes involved in catabolic and anabolic processes and demonstrated the beneficial effects of hyaluronan (HA) combined with fluoroquinolones (FQs) on primary canine chondrocytes. This study further determines the effects of these treatments on canine cartilage explants in both normal and interleukin-1 beta (IL-1β)-stimulated conditions. Methods We examined sulfate glycosaminoglycan (s-GAG) release, uronic acid (UA) content, and safranin-O staining, as well as the expression patterns of inflammatory, extracellular matrix (ECM) component and enzymes. Results Enro treatment alone effectively stimulated proteoglycan anabolism by increasing UA content and glycosaminoglycans (GAGs) in normal and pre-IL-1β-stimulated explant, whereas Mar showed opposite results. The combination of HA and FQs increased s-GAG release and UA content in normal explants in addition to effective down-regulated expression of MMP3. HA reduced the adverse effects of Mar by enhancing UA and GAG contents in both normal and pre-IL-1β-explants. Moreover, HA effectively induced HAS1and ACANup-regulation and reduced MMP9, TNF, PTGS2,and NFKB1 expression for a long term. Discussion Our results suggest the direct effects of Enro and Mar may selectively stimulate the conditioned explants to express MMP-codinggenes and promote gene expression involved in matrix production, pro-inflammatory cytokines, and cell degradation in different directions. HA successfully reduced the adverse effects of FQs by enhancing s-GAG and UA contents and down-regulated expression of MMPs

A new species of the Cyrtodactylus brevipalmatus group (Squamata, Gekkonidae) from the uplands of western Thailand

An integrative systematic analysis recovered a new species of the Cyrtodactylus brevipalmatus group from the uplands of Thong Pha Phum National Park, Kanchanaburi Province in western Thailand. Cyrtodactylus thongphaphumensis sp. nov. is deeply embedded within the brevipalmatus group, bearing an uncorrected pairwise sequence divergence of 7.6–22.3% from all other species based on a 1,386 base pair segment of the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs. It is diagnosable from all other species in the brevipalmatus group by statistically significant mean differences in meristic and normalized morphometric characters as well as differences in categorical morphology. A multiple factor analysis recovered its unique and non-overlapping placement in morphospace as statistically significantly different from that of all other species in the brevipalmatus group. The description of this new species contributes to a growing body of literature underscoring the high degree of herpetological diversity and endemism across the sky-island archipelagos of upland montane tropical forest habitats in Thailand, which like all other upland tropical landscapes, are becoming some of the most imperiled ecosystems on the planet

Genetic assessment of three Fagaceae species in forest restoration trials

Restoring isolated patches of forest ecosystems in degraded landscapes could potentially lead to genetic loss and inbreeding. Therefore, this study determined the occurrence of genetic diversity among the tree species Castanopsis tribuloides, C. calathiformis, and Lithocarpus polystachyus all of which were proven previously to be effective native tree species in the restoration of upland evergreen forests in northern Thailand when using the seed sample collection method. We tested our hypothesis as to whether the genetic diversity of a plant population that had been planted from the seeds of 4–6 adult trees would be lower and whether incidences of fixation index (Fis) would be higher among the second generation seedlings of these three Fagaceae species in isolated forest restoration trial plots. Microsatellite primers were selected from the entire genome sequence of C. tribuloides and the genetic sequences of C. tribuloides, L. polystachyus, and C. calathiformis were analyzed. Our results indicated a high degree of genetic diversity (He) in C. tribuloides (0.736) and C. calathiformis (0.481); however, a low level of genetic diversity was observed in L. polystachyus (0.281) within the restored forest. The fixation index for the second generation of L. polystachyus and C. calathiformis in the restored forest showed evidence of inbreeding. These results imply the efficiency of the seed sample collection method and verify that it does not reduce the level of genetic diversity in C. tribuloides and C. calathiformis. However, it may result in incidences of an inbreeding phenomena, suggesting the need to increase the number of adult trees used at the seed collection stage

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the <it>MMP-3 </it>gene in <it>in vitro </it>human chondrosarcoma using siRNA.</p> <p>Methods</p> <p>Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and <it>MMP-3 </it>siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).</p> <p>Results</p> <p>In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (<it>p </it>< 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (<it>p </it>< 0.05). <it>MMP-3 </it>gene expression was downregulated significantly (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p><it>MMP-3 </it>specific siRNA can inhibit the expression of <it>MMP-3 </it>in chondrosarcoma. This suggests that <it>MMP-3 </it>siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.</p

Intrinsic Cellular Responses of Human Wharton’s Jelly Mesenchymal Stem Cells Influenced by O2-Plasma-Modified and Unmodified Surface of Alkaline-Hydrolyzed 2D and 3D PCL Scaffolds

Polycaprolactone (PCL), a hydrophobic-degradable polyester, has been widely investigated and extensively developed, to increase the biocompatibility for tissue engineering. This research was the first trial to evaluate the intrinsic biological responses of human Wharton&rsquo;s Jelly Mesenchymal Stem Cells (hWJMSCs) cultured on alkaline hydrolysis and low-pressure oxygen plasma modified 2D and 3D PCL scaffolds, without adding any differentiation inducers; this has not been reported before. Four types of the substrate were newly established: 2D plasma-treated PCL (2D-TP), 2D non-plasma-treated PCL (2D-NP), 3D plasma-treated PCL (3D-TP), and 3D non-plasma-treated PCL (3D-NP). Physicochemical characterization revealed that only plasma-treated PCL scaffolds significantly increased the hydrophilicity and % oxygen/carbon ratio on the surfaces. The RMS roughness of 3D was higher than 2D conformation, whilst the plasma-treated surfaces were rougher than the non-plasma treated ones. The cytocompatibility test demonstrated that the 2D PCLs enhanced the initial cell attachment in comparison to the 3Ds, indicated by a higher expression of focal adhesion kinase. Meanwhile, the 3Ds promoted cell proliferation and migration as evidence of higher cyclin-A expression and filopodial protrusion, respectively. The 3Ds potentially protected the cell from apoptosis/necrosis but also altered the pluripotency/differentiation-related gene expression. In summary, the different configuration and surface properties of PCL scaffolds displayed the significant potential and effectiveness for facilitating stem cell growth and differentiation in vitro. The cell&ndash;substrate interactions on modified surface PCL may provide some information which could be further applied in substrate architecture for stem cell accommodation in cell delivery system for tissue repair

A new species of the Cyrtodactylus brevipalmatus group (Squamata, Gekkonidae) from Tak Province, northwestern Thailand

An integrative taxonomic analysis was used to delimit and diagnose a new species of the Cyrtodactylus brevipalmatus group from Tak Province in western Thailand. Although Bayesian phylogenetic analyses place C. denticulatus sp. nov. within the brevipalmatus group, the new species is neither nested within nor is it the sister species of any other species in the brevipalmatus group. Furthermore, based on the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs, it bears an uncorrected pairwise sequence divergence of 7.87–21.94% from all other species in the brevipalmatus group. Cyrtodactylus denticulatus sp. nov. is differetiated from all other species in the brevipalmatus group by having a number of unique charateristics such as denticulate ventrolateral body folds and ventrolateral subcaudal ridges, characters not seen in any other species of the group (n = 51 individuals). Additionally, based on a multiple factor anlaysis, C. denticulatus sp. nov. does not overlap with any other species in multivariate space. The discovery of C. denticulatus sp. nov. underscores the unrealized diversity of upland ecosystems across Thailand and the urgent need for increased exploration and conservation of these unique imperiled montane refugia, especially in this era of climate change