Survey of bovine mycotic mastitis in dairy herds in the State of São Paulo,Brazil

The purpose of this study was to isolate fungi from the quarter milk of cow udders from several dairy herds and to identify the different genera and species involved in mastitis. A total of 2078 milk samples from normal, clinical and subclinical mastitis quarters from 22 dairy herds of 16 districts in the State of São Paulo, Brazil was utilized in this survey. Two hundred and fifty one (12.07%) fungi were isolated from the samples. Two hundred and eight of these (82.86%) were yeasts and 30 (11.95%) were moulds. The fungi were isolated in pure culture (24.77%) or in cultures mixed with bacteria (72.22%). The yeasts isolated were:Cryptococcus spp. (71 strains),Rhodotorula spp. (40),Candida spp. (68),Trichosporon cutaneum (21),Aureobasidium pullulans (7), andPichia ohmeri (1). Moulds classified in following genera were also isolated:Aspergillus (3),Penicillium (3),Alternaria (3),Phoma (3),Epicoccum (2), andGeotrichum (16).     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Breast pathology and reduction mammaplasty

Breast cancer is the tumor with the highest prevalence and incidence in women. Reduction mammaplasty is one of the most common procedures performed in Brazil by the plastic surgeon, and it is not uncommon for the surgeon to find a breast tumor during the operation or afterward, when the histopathological report is received. In this study, 2488 patient files were reviewed retrospectively. All patients had undergone reduction mammaplasty at the senior author's private clinic (the Ivo Pitanguy Clinic) between January of 1957 and December of 2002. Resected breast tissue was examined histopathologically. The objective of this study was to verify the occurrence of breast carcinoma found accidentally postoperatively. The senior author's team performed all of the operations and the same pathologist performed every histopathological examination. The histopathological test results were divided into two groups: benign lesions and tumors. The highest frequency of breast pathology was benign lesions, and of them, 80.8 percent involved fibrocystic changes and fibroadiposity. The tumor group was subdivided into benign tumors and malignant tumors. Among the benign tumors, fibroadenoma was the one most common, in 2.2 percent. The frequency of malignant tumors was 0.5 percent of all patients. Most of the histopathological lesions were found in patients between 30 and 50 years of age. A reduced number of patients had no lesions (3.7 percent). Lack of a pathological investigation or a cursory or hurried examination of any mammary tissue by the pathologist may overlook important lesions. In the analysis of these statistics, the concept of normal breast tissue was questioned.     

Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance

Passive surveillance of infectious diseases with a high percentage of asymptomatic cases or long incubation periods, such as acquired immunodeficiency syndrome (AIDS), does not reflect the current transmission dynamics. Thus, a multi-strategic surveillance, such as the human immunodeficiency virus (HIV) sentinel surveillance proposed by the World Health Organization (WHO), is necessary. The Brazilian HIV sentinel surveillance was started in May 1992 with this purpose. The objectives of this study were to evaluate the feasibility and costs of HIV and hepatitis C virus (HCV) surveillance using dried blood spots (DBS) collected for neonatal screening of metabolic diseases in the state of Minas Gerais, Brazil. This was accomplished through the comparison of HIV and HCV seroprevalence with previous Brazilian studies. From December 2001 to June 2002, 24,905 newborns were tested for HIV and 4211 for HCV. HIV seroprevalence was 0.25% and the 95% confidence interval (CI) was 0.18, 0.31%; and HCV seroprevalence was 0.71% and the 95% CI was 0.46, 0.97%. These numbers are similar to previous Brazilian studies. Cost in this study was approximately USD 3.10 per sample, which was roughly one third of the cost of the same exam at the Brazilian HIV sentinel surveillance. We conclude that it is possible and more cost-effective to use DBS for infectious diseases surveillance, albeit it is still necessary to compare these results with the usual sentinel methodology in a concomitant trial.     

A shell finite element model of the pelvic floor muscles

The pelvic floor gives support to the organs in the abdominal cavity. Using the dataset made public in (Janda et al. J. Biomech. (2003) 36(6), pp. 749-757), we have reconstructed the geometry of one of the most important parts of the pelvic floor, the levator ani, using NURB surfaces. Once the surface is triangulated, the corresponding mesh is used in a finite element analysis with shell elements.Based on the 3D behavior of the muscle we have constructed a shell that takes into account the direction of the muscle fibers and the incompressibility of the tissue. The constitutive model for the isotropic strain energy and the passive strain energy stored in the fibers is adapted from Humphrey's model for cardiac muscles. To this the active behavior of the skeletal muscle is added.We present preliminary results of a simulation of the levator ani muscle under pressure and with active contraction. This research aims at helping simulate the damages to the pelvic floor that can occur after childbirth.     

Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia)

We used mitochondrial DNA control region sequences to examine phylogeography and population differentiation of the endangered Amazonian manatee Trichechus inunguis. We observe lack of molecular differentiation among localities and we find weak association between geographical and genetic distances. However, nested clade analysis supports restricted gene flow and/or dispersal with some long-distance dispersal. Although this species has a history of extensive hunting, genetic diversity and effective population sizes are relatively high when compared to the West Indian manatee Trichechus manatus. Patterns of mtDNA haplotype diversity in T. inunguis suggest a genetic disequilibrium most likely explained by demographic expansion resulting from secession of hunting and enforcement of conservation and protective measures. Phylogenetic analysis of T. manatus and T. inunguis haplotypes suggests that T. inunguis is nested within T. manatus, effectively making T. manatus a paraphyletic entity. Paraphyly of T. manatus and recent divergence times of T. inunguis and the three main T. manatus lineages suggest a possible need for a taxonomic re-evaluation of the western Atlantic Trichechus.     

Microsatellite DNA analysis shows that greater sage grouse leks are not kin groups

The spectacular social courtship displays of lekking birds are thought to evolve via sexual selection, but this view does not easily explain the participation of many males that apparently fail to mate. One of several proposed solutions to this 'lek skew paradox' is that kin selection favours low-ranking males joining leks to increase the fitness of closely related breeders. We investigated the potential for kin selection to operate in leks of the greater sage grouse, Centrocercus urophasianus, by estimating relatedness between lekking males using microsatellite DNA markers. We also calibrated these estimates using data from known families. Mean relatedness within leks was statistically indistinguishable from zero. We also found no evidence for local clustering of kin during lek display, although males tended to range closer to kin when off the lek. These results make kin selection an unlikely solution to the lek skew paradox in sage grouse. Together with other recent studies, they also raise the question of why kin selection apparently promotes social courtship in some lekking species, but not in others.     

Exploring the genome of Trypanosoma vivax through GSS and in silico comparative analysis

A survey of the Trypanosoma vivax genome was carried out by the genome sequence survey (GSS) approach resulting in 1,086 genomic sequences. A total of 455 high-quality GSS sequences were generated, consisting of 331 non-redundant sequences distributed in 264 singlets and 67 clusters in a total of 135.5 Kb of the T. vivax genome. The estimation of the overall G+C content, and the prediction of the presence of ORFs and putative genes were carried out using the Glimmer and Jemboss packages. Analysis of the obtained sequences was carried out by BLAST programs against 12 different databases and also using the Conserved Domain Database, InterProScan, and tRNAscan-SE. Along with the existing 23 T. vivax entries in the GenBank, the 32 putative genes predicted and the 331 non-redundant GSS sequences reported herein represent new potential markers for the development of PCRbased assays for specific diagnosis and typing of Trypanosoma vivax.     

Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains

Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains.     

Molecular cytogenetic analysis of recently evolved Tragopogon (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors

Tragopogon mirus and T. miscellus (both 2n = 4x = 24) are recent allotetraploids derived from T. dubius × T. porrifolius and T. dubius × T. pratensis (each 2n = 2x = 12), respectively. The genome sizes of T. mirus are additive of those of its diploid parents, but at least some populations of T. miscellus have undergone genome downsizing. To survey for genomic rearrangements in the allopolyploids, four repetitive sequences were physically mapped. TPRMBO (unit size 160 base pairs [bp]) and TGP7 (532 bp) are tandemly organized satellite sequences isolated from T. pratensis and T. porrifolius, respectively. Fluorescent in situ hybridization to the diploids showed that TPRMBO is a predominantly centromeric repeat on all 12 chromosomes, while TGP7 is a subtelomeric sequence on most chromosome arms. The distribution of tandem repetitive DNA loci (TPRMBO, TGP7, 18S-5.8S-26S rDNA, and 5S rDNA) gave unique molecular karyotypes for the three diploid species, permitting the identification of the parental chromosomes in the polyploids. The location and number of these loci were inherited without apparent changes in the allotetraploids. There was no evidence for major genomic rearrangements in Tragopogon allopolyploids that have arisen multiple times in North America within the last 80 yr.