Promise and challenges of dystonia brain banking: establishing a human tissue repository for studies of X-Linked Dystonia-Parkinsonism.

X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.

Neuroinflammation and histone H3 citrullination are increased in X-linked Dystonia Parkinsonism post-mortem prefrontal cortex.

Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.

Verminous Arteritis Due to Crassicauda sp. in Cuvier's Beaked Whales (Ziphius Cavirostris).

The vascular system of Cuvier's beaked whales (CBW) (Ziphius cavirostris; family Ziphiidae), an extremely deep, prolonged-diving cetacean, is increasingly receiving anatomic and physiologic study due to possible anthropogenic interactions; however, vascular pathology rarely has been reported in this species. Thirteen CBW stranded in the Canary Islands from June 2008 to June 2014 were autopsied. A careful dissection of the thoracic and abdominal vasculature was performed on these animals. All had moderate to severe and extensive chronic fibrosing arteritis with aneurysms, hemorrhages, and thrombosis primarily involving the mesenteric and gastroepiploic arteries and the thoracic and abdominal aorta. Microscopically, the lesions varied from subacute subintimal hemorrhages and severe neutrophilic, eosinophilic, and histiocytic dissecting arteritis with intralesional nematode larvae to marked, chronic, fibrosing arteritis with thickening and distortion of the vascular wall with calcification and occasional cartilage metaplasia. In addition, adult nematodes in renal arteries and veins, renal parenchyma and/or ureter were identified morphologically as Crassicauda sp. Nucleic acid sequenced from renal nematodes from 2 animals yielded closest nucleotide identity to C. magna The pathogenesis is proposed to involve a host response to larval migration from the intestine to the kidney through the mesenteric arteries, abdominal aorta, and renal arteries. Severe consequences for such lesions are possible and could vary from reduced vascular compliance to chronic renal disease and predisposition to the development of disseminated intravascular coagulation and multiorgan failure. Severe chronic arteritis in CBW is associated with renal parasitism by Crassicauda spp.

Fatal Erysipelothrix rhusiopathiae septicemia in two Atlantic dolphins (Stenella frontalis and Tursiops truncatus).

We describe gross, histopathologic, ultrastructural, immunohistochemical, and microbiologic features of acute septicemia by Erysipelothrix rhusiopathiae in an Atlantic spotted dolphin Stenella frontalis and an Atlantic bottlenose dolphin Tursiops truncatus. Generalized lymphadenomegaly and widespread hemorrhages were the most consistent macroscopic findings. Tricavitary effusion and icterus were noted in one individual. Histologically, all organs examined showed numerous variably sized bacillary bacterial emboli (Gram-positive; Ziehl-Neelsen-negative), typically associated with systemic congestion, edema, hemorrhages, and fibrinocellular thrombi. These bacteria were frequently intravascular, either extracellular or intramonocytic/macrophagic, and to a lesser extent, free within the interstitium of parenchymal organs. In both cases, microbiological analysis yielded E. rhusiopathiae. A primary anti-E. rhusiopathiae antibody created in mice from one of the strains isolated allowed positive immunohistochemical detection. Electron microscopy and dual immunohistochemistry with lysozyme and MAC387 antibodies confirmed the intramacrophagic location of the bacilli. E. rhusiopathiae, a known multispecies and zoonotic agent, should be considered as a potential etiologic agent in septicemia cases in free-ranging individuals of these dolphin species.

Mixed testicular neoplasia in a short beaked common dolphin Delphinus delphis.

A diagnosis of mixed testicular neoplasia in a short beaked common dolphin Delphinus delphis involving a Sertoli cell tumor, an interstitial (Leydig) cell tumor and a seminoma is presented. Lymphatic spread of the Sertoli cell tumor to an adjacent retroperitoneal lymph node was observed. Testicular neoplasms have been infrequently reported in marine mammals. Demonstration of clinical signs and further health implications is extremely challenging when dealing with non accessible wildlife species, such as dolphins. However, metastatic potential for these neoplastic conditions should be considered.

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat.

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of ~ 20- > 100 repeats and contractions of ~ 20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntington's disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism.

X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34' was further investigated in cellular assays. Subsequently, microexon 34' incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34'. In addition, we show that (1) microexon 34'-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34' incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34' as the molecular basis for this disease.

SVA insertion in X-linked Dystonia Parkinsonism alters histone H3 acetylation associated with TAF1 gene.

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.

Correlating the immune response with the clinical-pathological course of persistent mastitis experimentally induced by Mycoplasma agalactiae in dairy goats.

To correlate the clinical course of mycoplasma mastitis with its immune response, right mammary glands of 15 lactating goats were inoculating with 10(10) colony-forming units (cfu) of Mycoplasma agalactiae (Ma). Before sacrificing the animals at 5, 15 or 45 days post-inoculation (dpi), blood Ma antibody titres and milk mycoplasma colony and somatic cell counts were monitored. Ma colonised the mammary gland and milk counts increased to over 10(12)cfu/ml within 5 dpi. During this period, an innate immune response involving neutrophils and macrophages was observed, and Ma antigen appeared in the degenerated acinar epithelium. From 7 dpi, a specific antibody response coincided with reduced viable mycoplasmas in milk. The humoral immune response was limited; by 37 dpi, all animals scored negative for anti-Ma antibodies, and around 10(8)cfu/ml were shed. Results indicate an early immune response to Ma inoculation unable to control mycoplasmal invasion. An ensuing humoral response, despite reducing the mycoplasma burden, leads to chronic, persistent infection.

Malignant peripheral nerve sheath tumour (malignant schwannoma) in the diaphragm of a goat.

This report describes a malignant schwannoma in the diaphragm of a 2-year-old goat. The immunohistochemical and histological features indicated a diagnosis of malignant schwannoma rather than neurofibrosarcoma. The diaphragm represents an unusual location for neoplasia in both domestic animals and human beings. A possible anatomical origin from the phrenic nerve is discussed.

Impact assessment of the rational selection of training and test sets on the predictive ability of QSAR models.

This study performed an analysis of the influence of the training and test set rational selection on the quality and predictively of the quantitative structure-activity relationship (QSAR) model. The study was carried out on three different datasets of Influenza Neuraminidase (H1N1) inhibitors. The three datasets were divided into training and test sets using three rational selection methods: based on k-means, Kennard-Stone algorithm and Activity and the results were compared with Random selection. Then, a total of 31,490 mathematical models were developed and those models that presented a determination coefficient higher than: r2train > 0.8, r2loo > 0.7, r2test > 0.5 and minimum standard deviation (SD) and minimum root-mean square error (RMS) were selected. The selected models were validated using the internal leave-one-out method and the predictive capacity was evaluated by the external test set. The results indicate that random selection could lead to erroneous results. In return, a rational selection allows for obtaining more reliable conclusions. The QSAR models with major predictive power were found using the k-means algorithm and selection by activity.

Identification and prevalence of swine pasivirus 1 in eastern Romanian pig farms.

Swine pasivirus 1 (SPaV-1) was first detected in the feces of healthy pigs in France as a new species in family Picornaviridae. We investigated the presence, distribution, and genetic variability of this virus in 7 geographic areas with intensive pig breeding farms in eastern Romania. A total of 564 porcine specimens, including 82 fecal specimens and 482 pools of organs, were collected from healthy pigs in different stages of production from pathogen-free swine farming units. The virus was found in 6 of 7 areas investigated. Of the 564 samples analyzed, 218 were positive for SPaV-1, with the highest prevalence of the virus in organ homogenates (39% positive) followed by feces (37% positive). The highest susceptibility to infection was found in nurseries (50% positive in both the first and second months of feeding). Sequencing analysis of VP0 revealed 3 different Romanian sequences. The phylogenetic investigations suggest that the Romanian sequences cluster with other Pasivirus strains selected from the GenBank database, forming a separate clade from other Picornaviridae genera and defining the described Pasivirus.

Hepatosplenic large cell immunoblastic lymphoma in a bottlenose dolphin (Tursiops truncatus) with high levels of polychlorinated biphenyl congeners.

This report describes a large cell immunoblastic lymphoma in a bottlenose dolphin found stranded alive in Gran Canaria, Spain. Diffuse infiltration of round neoplastic cells was observed in the splenic cords and sinuses and in hepatic sinusoids, resulting in moderate organ enlargement. The tumour cells (immunophenotype IgG+ and CD3-) showed scant, lightly eosinophilic or basophilic cytoplasm, distinct cell boundaries and hyperchromatic nuclei, each with one or more nucleoli. Mitoses were common. On the basis of histopathological, immunohistochemical and ultrastructural features, the tumour was classified as an immunoblastic lymphoma. Eleven polychlorinated biphenyl (PCB) congeners, 23 organochlorine pesticides and 16 polycyclic aromatic hydrocarbons (PAHs) were detected in the blubber and liver. High concentrations of PCBs 153, 180, 138 and 187 found in the liver may have been associated with the hepatosplenic lymphoma.

Uterine Leiomyoma and Prolapse in a Live-stranded Atlantic Spotted Dolphin (Stenella frontalis).

A uterine prolapse associated with a leiomyoma (fibroid) was observed in a live-stranded Atlantic spotted dolphin (Stenella frontalis). A 7 cm segment of the reproductive tract including the cervix, uterine neck and caudal uterine body had intussuscepted and prolapsed into the cranial vaginal vault. In the leading edge of the intussuscepted/prolapsed uterine wall was a 6 × 3 × 3.5 cm leiomyoma expanding the myometrium. The leiomyoma and prolapse were associated with necrotizing exposure endometritis. This is the first report of a uterine prolapse associated with a leiomyoma in a cetacean. This lesion was believed to be the underlying cause of the live stranding.

Immunohistochemical characterisation of parasitic pneumonias of dolphins stranded in the Canary Islands.

This is the first work that applies immunohistochemistry in the characterisation of the inflammatory infiltrate of verminous bronchopneumonia associated with lungworm parasites in stranded dolphins. Samples from three different species (Stenella coerulealba, Stenella frontalis and Delphinus delphis) stranded in the Canary Islands were used. The most common findings found in these animals varied from bronchitis to verminous bronchopneumonia with different degree of severity. The immunohistochemical study showed variable expressions of Lysozyme, MHC-II, iNOS and IgG. The main population presenting in the inflammatory infiltrates were CD3(+) lymphocytes. However, moderate number of Foxp3(+) lymphocytes was found in lymph nodes even though no Foxp3(+) cells were found in lung lesions in any of the samples analysed. This study revealed that lung lesions showed a chronic inflammatory infiltrate mainly composed by lymphocytes CD3(+). Deeper studies are needed in order to provide a more complete scope about the infiltrates involved in these types of lesions.

The importance of the proportion of heme/nonheme iron in the diet to minimize the interference with calcium, phosphorus, and magnesium metabolism on recovery from nutritional ferropenic anemia.

The digestive utilization of Fe and its nutritive interaction with Ca, P, and Mg were studied in rats with nutritional ferropenic anemia. The diet contained 80% ferric citrate and 20% heme iron (80/20 diet). The weight gain, digestive utilization of Fe, and regeneration efficiency of hemoglobin and seric Fe were higher in iron-deficient rats (ID) fed the 80/20 diet than in iron-deficient rats fed the 50/50 diet (Campos et al., 1996). The phospho-calcic metabolism, which is adversely affected in ferropenic anemia, returned to normal values when iron was added to the diet. The digestive utilization of Mg, which fell with the 50/50 diet (Campos et al., 1996), returned to normal values when the ferropenic anemia was reversed with the 80/20 diet. In a state of iron deficiency, certain parameters related to the glucose and lipid metabolism are affected; the glucose and triglycerides values return to a normal range with the 80/20 diet.

Immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mh) is the primary agent of porcine enzootic pneumonia (PEN), a chronic respiratory disease endemic to pig farms, and characterized histologically by infiltration of mononuclear cells in airways and prominent hyperplasia of the bronchus-associated lymphoid tissue (BALT). To gain further insight into the pathogenesis of PEN, cytokine expression in the lung, with particular attention to the BALT, was examined immunohistochemically in pigs naturally infected with Mh. An increase (P < 0.05) in proinflammatory and immunoregulatory cytokines (especially interleukin [IL]-2, IL-4 and tumour necrosis factor [TNF]-alpha, and to a lesser extent IL-1 [alpha and beta] and IL-6) was detected in the BALT, which showed intense lymphoid hyperplasia. IL-1beta and TNF-alpha were also detected in the bronchoalveolar exudate of infected pigs, and IL-6 and IL-8 were demonstrated in mononuclear cells of the alveolar septa. The results showed that in Mh infection, macrophage and lymphocyte activation results in the expression of a number of cytokines capable of inducing lung lesions and lymphoreticular hyperplasia of the BALT.

An in vitro study of the effect of restorative technique on marginal leakage in posterior composites.

The purpose of this in vitro study was to compare marginal leakage of class 2 light-cured composite resin restorations, with different insertion and polymerization techniques. Fifty sound premolars extracted for prosthetic purposes were used in the study. Conservative class 2 (MOD) preparations were made in all teeth, with one proximogingival margin on enamel and the other on cementum. All the preparations and restorations were performed by the same operator utilizing the same restorative materials. After restoration the teeth were thermocycled and their gingival margins exposed to a dye. Results showed that the kind of matrix system had no significant influence upon the degree of marginal leakage on enamel. On cementum, oblique and horizontal incremental insertion and polymerization techniques using the collimator cone exhibited the least leakage, while the clear matrix band/reflective wedge technique presented the greatest leakage.

Effect of pentobarbital or urethane on bile secretion and chemical composition of blood in the rabbit.

A study was made of the effects of anaesthesia with pentobarbital and urethane on the bile secretion and the chemical composition of the blood of New Zealand rabbits. Neither of the agents was observed to affect arterial pH or pO2, but with urethane pCO2 values decreased significantly. This was associated with a pronounced hyperglycaemia. Bile flow was significantly higher in pentobarbital-anaesthetized animals than in urethane-anaesthetized animals, a phenomenon that can be attributed to a different canalicular flow and that will not be related to differences in the bile-acid-dependent fraction of secretion. Under both anaesthetics, bile sodium concentrations were greater than those found in plasma, which can be explained by the formation of micelles with low osmotic activity. Bile bicarbonate concentrations proved to be greater than those observed in plasma. Plasma calcium concentrations were significantly lower with urethane than with pentobarbital, whereas in bile the situation was reversed; both these aspects are discussed.