High Occurrence of Zoonotic Subtypes of Cryptosporidiumparvum in Cypriot Dairy Farms

Cryptosporidium parvum is one of the major causes of neonatal calf diarrhoea resulting in reduced farm productivity and compromised animal welfare worldwide. Livestock act as a major reservoir of this parasite, which can be transmitted to humans directly and/or indirectly, posing a public health risk. Research reports on the prevalence of Cryptosporidium in ruminants from east Mediterranean countries, including Cyprus, are limited. This study is the first to explore the occurrence of Cryptosporidium spp. in cattle up to 24 months old on the island of Cyprus. A total of 242 faecal samples were collected from 10 dairy cattle farms in Cyprus, all of which were screened for Cryptosporidium spp. using nested-PCR amplification targeting the small subunit of the ribosomal RNA (18S rRNA) gene. The 60 kDa glycoprotein (gp60) gene was also sequenced for the samples identified as Cryptosporidium parvum-positive to determine the subtypes present. The occurrence of Cryptosporidium was 43.8% (106/242) with at least one positive isolate in each farm sampled. Cryptosporidium bovis, Cryptosporidium ryanae and C. parvum were the only species identified, while the prevalence per farm ranged from 20–64%. Amongst these, the latter was the predominant species, representing 51.8% of all positive samples, followed by C. bovis (21.7%) and C. ryanae (31.1%). Five C. parvum subtypes were identified, four of which are zoonotic—IIaA14G1R1, IIaA15G1R1, IIaA15G2R1 and IIaA18G2R1. IIaA14G1R1 was the most abundant, representing 48.2% of all C. parvum positive samples, and was also the most widespread. This is the first report of zoonotic subtypes of C. parvum circulating in Cyprus. These results highlight the need for further research into the parasite focusing on its diversity, prevalence, host range and transmission dynamics on the islan

Genetics and physical mapping studies on mouse chromosome 2

This thesis describes two genetic maps of mouse chromosome 2 (MMU2), genetic maps relative to the wasted (wst) and Ragged (Ra) mutations on distal MMU2 and a physical map of the region likely to contain the latter two genes. The first two maps include 18 new PCR markers which were isolated from a subgenomic library constructed from a mouse-hamster somatic cell hybrid line whose main mouse-genome component was MMU2. Fourteen of these loci define microsatellite sequences and four represent randomly chosen DNA sequences. The maps were constructed using two interspecific backcrosses established using a laboratory mouse strain and mice from the related species Mus spretus. The inheritance pattern on MMU2 reveals loci that exhibit significant segregation distortion (SD) in males. The genes responsible for the wasted (wst) and ragged (Ra) phenotypes are very closely linked (0.2 cM) on the extreme end of MMU2. Two interspecific backcrosses segregating wst and Ra were set up in order to define the genetic position of these loci relative to currently available molecular markers. In total, 167 wst/wst and 329 Ra/+ or +/+ mice were phenotyped for 5 microsatellites and two genes which, on the basis of the consensus map, would be expected to map near wst and Ra. The gene order established is: D2Ucl1, Gnas- 1.8 cM ± 1.0, D2Mit200- 1.8 cM ± 1.0, D2Mit230 0.6 cM ± 0.6, D2Mit74- 0.6 cM ± 0.6, Acra4- 0.6 cM ± 0.6, D2Mit266, wst D2Ucl1, Gnas- 1.2 cM ± 0.6, D2Mit200- 0.6 cM ± 0.4, D2Mit230- 0.6 cM ± 0.4, D2Mit74, Acra4, D2Mit266, Ra In an attempt to isolate flanking markers for both mutations, four overlapping yeast artificial chromosomes (YACs) were isolated and analysed by way of fluorescence in situ hybridisation (FISH), Southern blot and PCR analysis. These clones span a region of at least 360 kb and encompass the markers D2Mit74, Acra4 and D2Mit266\ the contig extends at least 200 kb proximal and 160 kb distal to Acra4. In the context of this work, a framework physical map extending 200 kb proximal and 310 kb distal to this gene was also established. Two YACs were found to span the T(2;16)28H translocation breakpoint. On the basis of these results, D2Mit74 is tentatively proposed to reside proximal to the breakpoint. All the genes which map to human chromosome 20 also map to distal MMU2 and the order appears to be conserved. The human homologues of Ra and wst would be expected to map on 20q13.1→ qter. The human homologue of the rat gene for vitamin D3 24-hydroxylase (CYP24) maps to 20q13.1→ qter. In this study, the mouse homologue of this gene (Cyp24) was localised to distal MMU29 cM proximal to Ra

Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

Studies proposed a model for embryonic neurogenesis where the expression levels of the SOXB2 and SOXB1 factors regulate the differentiation status of the neural stem cells. However, the precise role of the SOXB2 genes remains controversial. Therefore, this study aims to investigate the effects of individual deletions of the SOX21 and SOX14 genes during the development of the dorsal midbrain. We show that SOX21 and SOX14 function distinctly during the commitment of the GABAergic lineage. More explicitly, deletion of SOX21 reduced the expression of the GABAergic precursor marker GATA3 and BHLHB5 while the expression of GAD6, which marks GABAergic terminal differentiation, was not affected. In contrast deletion of SOX14 alone was sufficient to inhibit terminal differentiation of the dorsal midbrain GABAergic neurons. Furthermore, we demonstrate through gain-of-function experiments, that despite the homology of SOX21 and SOX14, they have unique gene targets and cannot compensate for the loss of each other. Taken together, these data do not support a pan-neurogenic function for SOXB2 genes in the dorsal midbrain, but instead they influence, sequentially, the specification of GABAergic neurons

C1q ablation exacerbates amyloid deposition: A study in a transgenic mouse model of ATTRV30M amyloid neuropathy.

ATTRV30M amyloid neuropathy is a lethal autosomal dominant sensorimotor and autonomic neuropathy, caused by deposition of amyloid fibrils composed of aberrant transthyretin (TTR). Ages of onset and penetrance exhibit great variability and genetic factors have been implicated. Complement activation co-localizes with amyloid deposits in amyloidotic neuropathy and is possibly involved in the kinetics of amyloidogenesis. A candidate gene approach has recently identified C1q polymorphisms to correlate with disease onset in a Cypriot cohort of patients with ATTRV30M amyloid neuropathy. In the current study we use a double transgenic mouse model of ATTRV30M amyloid neuropathy in which C1q is ablated to elucidate further a possible modifier role for C1q. Amyloid deposition is found to be increased by 60% in the absence of C1q. Significant up regulation is also recorded in apoptotic and cellular stress markers reflecting extracellular toxicity of pre-fibrillar and fibrillar TTR. Our data further indicate that in the absence of C1q there is marked reduction of macrophages in association with amyloid deposits and thus less effective phagocytosis of TTR

Pax6 is expressed in subsets of V0 and V2 interneurons in the ventral spinal cord in mice.

The embryonic spinal cord in mice is organized into eleven progenitor domains. Cells in each domain first produce neurons and then switch to specifying glia. Five of these domains known as p3, pMN, p2, p1 and p0 are located in the ventral spinal cord and each expresses a unique code of transcription factors (TFs) that define the molecular profile of progenitor cells. This code is largely responsible for determining the subtype specification of neurons generated from each domain. Pax6 codes for a homedomain-containing TF that plays a central role in defining the molecular boundaries between the two ventral-most domains, p3 and pMN. Using fate mapping and gene expression studies we show that PAX6, in addition to each patterning function, is expressed in a group of late born interneurons that derive from the p2 and p0 domains. The p2-derived neurons represent a subset of late born V2b interneurons and their specification depends on Notch signaling. The V0 neurons represent V0v ventral neurons expressing Pax2. Our data demonstrate that interneuron diversity in the ventral spinal cord is more complex than originally appreciated and point to the existence of additional mechanisms that determine interneuron diversity, particularly in the p2 domain

Intraperitoneal melatonin is not neuroprotective in the G93ASOD1 transgenic mouse model of familial ALS and may exacerbate neurodegeneration

In amyotrophic lateral sclerosis (ALS) reactive oxygen species and apoptosis are implicated in disease pathogenesis. Melatonin with its anti-oxidant and anti-apoptotic properties is expected to ameliorate disease phenotype. The aim of this study was to assess possible neuroprotection of melatonin in the G93A-copper/zinc superoxide dismutase (G93ASOD1) transgenic mouse model of ALS. Four groups of mice, 14 animals each, were injected intraperitoneally with 0 mg/kg, 0.5 mg/kg, 2.5 mg/kg and 50 mg/kg of melatonin from age 40 days. The primary end points were; disease onset, disease duration, survival and rotarod performance. No statistically significant difference in disease onset between the four groups was found. Survival was significantly reduced with the 0.5 mg/kg and 50 mg/kg doses and tended to be reduced with the 2.5 mg/kg dose. Histological analysis of spinal cords revealed increased motoneuron loss in melatonin treated mice. Melatonin treated animals were associated with increased oxidative stress as assessed with 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation. Histochemistry and Western blot data of spinal cord from melatonin treated mice revealed upregulation of human SOD1 compared to untreated mice. In addition, real-time PCR revealed a dose dependent upregulation of human SOD1 in melatonin treated animals. Thus, intraperitoneal melatonin, at the doses used, does not ameliorate and perhaps exacerbates phenotype in the G93ASOD1 mouse ALS model. This is probably due to melatonin's effect on upregulating gene expression of human toxic SOD1. This action presumably overrides any of its direct anti-oxidant and anti-apoptotic properties.Fil: Dardiotis, Efthimios . The Cyprus Institute of Neurology and Genetics; ChipreFil: Panayiotou, Elena . The Cyprus Institute of Neurology and Genetics; ChipreFil: Feldman, Mariana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Hadjisavvas, Andreas . The Cyprus Institute of Neurology and Genetics; ChipreFil: Malas, Stavros . The Cyprus Institute of Neurology and Genetics; ChipreFil: Vonta, Ilia. National Technical University of Athens; GreciaFil: Hadjigeorgiou, Georgios . Centre for Research and Technology-Thessaly; GreciaFil: Kyriakou, Kyriakos . The Cyprus Institute of Neurology and Genetics; ChipreFil: Kyriakides, Theodoros . The Cyprus Institute of Neurology and Genetics; Chipr

Data_Sheet_1_Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain.pdf

<p>Studies proposed a model for embryonic neurogenesis where the expression levels of the SOXB2 and SOXB1 factors regulate the differentiation status of the neural stem cells. However, the precise role of the SOXB2 genes remains controversial. Therefore, this study aims to investigate the effects of individual deletions of the SOX21 and SOX14 genes during the development of the dorsal midbrain. We show that SOX21 and SOX14 function distinctly during the commitment of the GABAergic lineage. More explicitly, deletion of SOX21 reduced the expression of the GABAergic precursor marker GATA3 and BHLHB5 while the expression of GAD6, which marks GABAergic terminal differentiation, was not affected. In contrast deletion of SOX14 alone was sufficient to inhibit terminal differentiation of the dorsal midbrain GABAergic neurons. Furthermore, we demonstrate through gain-of-function experiments, that despite the homology of SOX21 and SOX14, they have unique gene targets and cannot compensate for the loss of each other. Taken together, these data do not support a pan-neurogenic function for SOXB2 genes in the dorsal midbrain, but instead they influence, sequentially, the specification of GABAergic neurons.</p

hTTR levels in serum and stomach.

<p>hTTR immunoblotting exhibiting a single band at 14KDa (<i>A</i>). hTTR levels measured via immunoblotting in serum of V30M and V30M C1q KO animals of all three age groups indicating higher hTTR in the V30M mice and a gradual decrease in the amount of hTTR present as the animals age (<i>B</i>). hTTR levels measured via immunoblotting in stomach tissue of the V30M and V30M C1q KO animals exhibit a severe decrease in the second age group (<i>C</i>). <i>B&C</i> n = 15/age group/line. Data presented as mean ± 1SD.</p

Model of observed C1q ablation effects.

<p>Classical and alternative pathway activation lead to the hydrolysis of C3 initiating the formation of the membrane attack complex (MAC) and production of the chemoattractant C5a. C1q and C5a recruit and activate phagocytic cells such as macrophages through receptors located on their surfaces (<i>A</i>). In the absence of C1q, properdin expression increases along with the entire alternative pathway and the terminal MAC complex. Concurrently, the presence of CD68 positive phagocytes was decreased along with the expression of C5a anaphylatoxin and its receptor CD88 (<i>B</i>). As a result, amyloid deposition increases following C1q ablation (<i>b</i>) versus the original transgenic model (<i>a</i>).</p