Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.

Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil.

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

Comparative genomic analysis of Brazilian Leptospira kirschneri serogroup Pomona serovar Mozdok.

Leptospira kirschneri is one of the pathogenic species of the Leptospira genus. Human and animal infection from L. kirschneri gained further attention over the last few decades. Here we present the isolation and characterisation of Brazilian L. kirschneri serogroup Pomona serovar Mozdok strain M36/05 and the comparative genomic analysis with Brazilian human strain 61H. The M36/05 strain caused pulmonary hemorrhagic lesions in the hamster model, showing high virulence. The studied genomes presented high symmetrical identity and the in silico multilocus sequence typing analysis resulted in a new allelic profile (ST101) that so far has only been associated with the Brazilian L. kirschneri serogroup Pomona serovar Mozdok strains. Considering the environmental conditions and high genomic similarity observed between strains, we suggest the existence of a Brazilian L. kirschneri serogroup Pomona serovar Mozdok lineage that could represent a high public health risk; further studies are necessary to confirm the lineage significance and distribution.

Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2.

The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil.

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.

Velusetrag rescues GI dysfunction, gut inflammation and dysbiosis in a mouse model of Parkinson's disease.

In patients with Parkinson's disease (PD), constipation is common, and it appears in a prodromal stage before the hallmark motor symptoms. The present study aimed to investigate whether Velusetrag, a selective 5­HT4 receptor agonist, may be a suitable candidate to improve intestinal motility in a mouse model of PD. Five months old PrP human A53T alpha-synuclein transgenic (Tg) mice, which display severe constipation along with decreased colonic cholinergic transmission already at 3 months, were treated daily with the drug for 4 weeks. Velusetrag treatment reduced constipation by significantly stimulating both the longitudinal and circular-driven contractions and improved inflammation by reducing the level of serum and colonic IL1ß and TNF-α and by decreasing the number of GFAP-positive glia cells in the colon of treated mice. No significant downregulation of the 5-HT4 receptor was observed but instead Velusetrag seemed to improve axonal degeneration in Tgs as shown by an increase in NF-H and VAChT staining. Ultimately, Velusetrag restored a well-balanced intestinal microbial composition comparable to non-Tg mice. Based on these promising data, we are confident that Velusetrag is potentially eligible for clinical studies to treat constipation in PD patients.

Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum.

Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.

Molecular epidemiology of Leptospira noguchii reveals important insights into a One Health context.

Leptospirosis presents a complex and dynamic epidemiology. Bovine leptospirosis has been described as a major infectious disease impairing reproductive efficiency. Although infections by Leptospira interrogans, L. santarosai and L. borgpetersenii are frequently reported in cattle, the presence of L. noguchii in these animals should not be neglected. In this study, we describe serological (MAT) and molecular characterization (rrs and secY gene sequencing, multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]) of eight L. noguchii strains obtained from slaughtered cows. Intraspecific genetic diversity was evaluated, and haplotype networks were constructed based on hosts and geographical localizations. Strains were characterized as belonging to serogroups Australis, Autumnalis and Panama, and molecular characterization showed a high heterogeneity of these strains. Ten different STs were found (including nine new STs and 39 novel alleles) as well as nine different pulsotypes. Two clonal complexes were found. Phylogenetic trees based on secY locus and concatenated MLST loci showed two main clusters, with sequences from the present study included in the first. In general, there was no relationship between the geographical origin and the secY phylogenetic clusters, as well as between secY phylogenetic clusters and serogroups. Molecular diversity indexes confirmed a high variability (H > 0.8). This high intraspecific variation observed may be related to differences in virulence, pathogenicity and antigenicity or even adaptability of the strains. In addition, haplotype networks clearly demonstrated the circulation of genotypes between humans and animals, confirming the zoonotic potential. The present study provides relevant data for the study of leptospirosis in the One Health context, where human, animal and environmental health is closely connected.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen.

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10(-1) to 10(-4)) between the selective and non-selective media at different dilution factors. At 1/10(th) dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100(th) dilution, (3%, 2/80) and (49%, 39/ 80) and at 1/1,000(th) dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

Microbiome, Parkinson's Disease and Molecular Mimicry.

Parkinson's Disease (PD) is typically classified as a neurodegenerative disease affecting the motor system. Recent evidence, however, has uncovered the presence of Lewy bodies in locations outside the CNS, in direct contact with the external environment, including the olfactory bulbs and the enteric nervous system. This, combined with the ability of alpha-synuclein (αS) to propagate in a prion-like manner, has supported the hypothesis that the resident microbial community, commonly referred to as microbiota, might play a causative role in the development of PD. In this article, we will be reviewing current knowledge on the importance of the microbiota in PD pathology, concentrating our investigation on mechanisms of microbiota-host interactions that might become harmful and favor the onset of PD. Such processes, which include the secretion of bacterial amyloid proteins or other metabolites, may influence the aggregation propensity of αS directly or indirectly, for example by favoring a pro-inflammatory environment in the gut. Thus, while the development of PD has not yet being associated with a unique microbial species, more data will be necessary to examine potential harmful interactions between the microbiota and the host, and to understand their relevance in PD pathogenesis.

Hydroxyindole-O-methyltransferase (HIOMT) activity in the retina of melatonin-proficient mice.

Numerous pieces of evidence support the expression by the mammalian retina of Hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4), the enzyme directly responsible for the biosynthesis of the pineal chronobiotic hormone melatonin (MLT). However, conflicting results obtained so far by enzyme-kinetic and immune-detection techniques still make HIOMT presence and relevance in the eye a matter of debate. This work aimed at evaluating unambiguously HIOMT activity in the mouse retina, a valuable model for studying the effects of MLT variations on ocular pathophysiology. Since laboratory mouse strains can bear genetic polymorphisms yielding defective enzymes of MLT biosynthesis, retinas and control pineal glands used in this study were obtained in a MLT-proficient crossing of A/J mice, the A/J/C57BL/10 strain. To improve the radiochemical reference assay, we tested different homogenization procedures coupled with HPLC detection. Concomitantly, we quantified MLT, and its precursor N-acetyl-serotonin (NAS) by HPLC coupled to electrochemical detection in retinas isolated from either light- or dark-adapted mice. Results showed that the standard radio-chemical assay was successful for pineal HIOMT only, whereas specific homogenization buffers and HPLC were required to detect retinal activity, presumably due to interfering methyl-transferases inhibited by NAS. Under present conditions, retinal HIOMT Vmax accounted for by ≈ 40 fmol/h/mg protein, 2.6-hundreds-fold lower than the pineal counterpart, displaying equivalent KMs (≈10 µM). Moreover, NAS and MLT rapidly decreased in light-exposed isolated retinas, corroborating light-sensitive in-situ MLT formation. Conclusively, we measured mouse retinal HIOMT kinetics under basal conditions, a useful result to elucidate the regulatory patterns, the possible impact on eye health, and therapeutic approaches related to this enzyme.

Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil.

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers ≥ 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona.

Subcellular localization of alpha-synuclein aggregates and their interaction with membranes.

For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein (αS) oligomers and aggregates in α-synucleinopathies. These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions, have raised the question about where αS aggregation initiates. In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology. Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles, ER/Golgi and the mitochondria, while toxic species of αS have been shown to inhibit, among others, neurotransmission, protein trafficking and mitochondrial function. Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice. Thus, a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves, pointing out to αS functions that are primarily affected before onset of irreversible damage.

Draft Genome Sequence of Brazilian Leptospira interrogans Serovar Pomona Strain GR5, Isolated from Apparently Healthy Gilt.

Leptospira interrogans serovar Pomona is one of the most important serovars associated with worldwide porcine leptospirosis, and its infection is characterized by high antibody titers and the establishment of a renal carrier state. Here, we present the draft genome sequence of Leptospira interrogans serovar Pomona strain GR5 isolated from apparently healthy gilt in Brazil.

Social Cognition and Oxytocin in Huntington's Disease: New Insights.

This study is aimed at relating social cognition in Huntington's Disease (HD) to plasma levels of the social hormone oxytocin (OT). Indeed, HD patients commonly display reduced social skills and OT is involved in bonding behavior and improved recognition of facial emotions. Twelve mild-symptomatic HD patients (stage II Shoulson & Fahn) and 11 gender/age matched controls (healthy controls, HC), without concurrent psychiatric disorders, were investigated at baseline (T0) for OT plasma levels and social cognition through an extensive battery of neuropsychological tests. Social cognition was also re-examined after two years (T1) in 8 of the 12 patients. Results showed a trend for reduced T0-OT levels in HD vs. HC, mean ± stardard deviation: 6.5 ± 2.4 vs. 9.9 ± 7.2 pg/mL, without reaching statistical significance. At T0, patients showed significantly lower performances than controls at the "Faux-Pas" and "Strange Stories" tests (p < 0.05; p < 0.01); a reduced perception of visual emotions (p < 0.01) and verbal stimuli (p < 0.01) was also reported, involving anger, fear, and sadness (p < 0.05; p < 0.01). Additionally, in the HD population, OT concentrations positively correlated with T1-performances at Neutral\Faux-Pas test (p < 0.05), whereas the cognitive Montreal Cognitive Assessment (MoCA) and Mini Mental State Examination (MMSE) scores positively correlated with psychosocial perception at the "Strange Stories" and Karolinska Directed Emotional Faces (KDEF) tests (p < 0.05). This study, despite its limitations, supports correlations between OT and HD social cognition, suggesting a possible therapeutic use of this hormone. More subjects and additional body tissues/fluids, such as cerebrospinal fluid, should be investigated to confirm this hypothesis.

Efficacy of leptospiral commercial vaccines on the protection against an autochtonous strain recovered in Brazil.

In swine and bovines, leptospirosis prevention and control is carried out via vaccination of susceptible animals using bacterins. However, the efficiency of leptospirosis vaccines has been questioned. This work aimed to investigate the potency of five leptospirosis vaccines sold commercially in Brazil, challenging the animals with one autochthonous strain of Leptospira, Canicola serovar, denoted LO4, isolated from swine. The standard protocol was followed, and renal carriers of Leptospira were identified among the surviving animals by culture and PCR. Of the five vaccines tested, only two proved effective. None of the surviving animals was positive by culture; however, one animal was positive by PCR. Three of the five vaccines sold commercially in Brazil for the immunization of swine or bovines failed the test of the efficacy to protect the vaccinated animals following challenge with an autochthonous Leptospira strain, Canicola serovar. The two vaccines provided protection against the renal carrier state in the surviving animals. The criteria used to produce leptospirosis bacterins sold commercially in Brazil must be reviewed. The industry should support researches on leptospiral vaccinology to improve the quality of the present vaccines and discover new immunogenic strains, because it is known that vaccination is one of the most important tools to increase the reproduction rates in livestock.

Genomic characterization and comparative analysis of Leptospira interrogans serogroup Australis isolated from swine.

The Leptospira interrogans serogroup Australis is traditionally described as pathogenic for swine although animals usually present low serologic response and mild clinical signs. The first isolation of a Leptospira interrogans serogroup Australis strain from swine, in Brazil, was recently described. Herein we present the genomic characterization of this strain (SU5) and further comparison with the Leptospira serovars reference genomes available in the GenBank database. The SU5 strain was characterized with sequence types previously described in a serogroup Australis isolated from human and presented a new ST98 that is thus far exclusively of the Brazilian porcine L. interrogans serogroup Australis. Even though the SU5 strain presented higher similarity with the American porcine serogroup Australis strain PigK151, as expected, it also presented sequences blocks in both chromosomes which are absent in the genomes of the studied Leptospira serovars. These regions are flanked by insertion sequences and transposases genes, suggesting the existence of inter- and intra-serogroup genomic variability due to mobile elements transferring. This genomic plasticity has already been demonstrated for pathogenic Leptospira species; nevertheless, there is still a limited understanding of the relationship between genome organization and content and the divergence of Leptospira serogroups and serovars that needs to be further elucidated.

Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing--a continuous attempt at species and serovar differentiation.

Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.

Characterization of Leptospira santarosai Serogroup Grippotyphosa Serovar Bananal Isolated from Capybara ( Hydrochaeris hydrochaeris ) in Brazil.

Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Rodents appear to be the most important reservoirs of infection. They contaminate the environment and food and can transmit the pathogen when they are consumed by carnivores. Capybara ( Hydrochaeris hydrochaeris ) are efficient reservoirs of Leptospira, and because they are in close contact with farm animals and are found in semiurban areas, they represent a risk to public health. We isolated five Leptospira strains from capybara kidneys in Sao Paulo State, Brazil, in 2001 and typed them using serologic and molecular techniques. These strains include the Leptospira santarosai serogroup Grippotyphosa serovar Bananal. Pulsed field gel electrophoresis resulted in a unique pattern distinct from the reference strains, and the isolates clustered with greater than 85% similarity. The isolates also presented higher growth rates than other Leptospira serovars, with high minimal inhibitory concentration values for most of the tested antibiotics, with the exception of penicillin and ampicillin. This isolation and characterization of the L. santarosai serogroup Grippotyphosa serovar Bananal from capybara, highlights the importance of wild and sinantropic rodents as carriers of pathogenic leptospires.

Parkinson's disease and alpha-synucleinopathies: from arising pathways to therapeutic challenge.

Parkinson's Disease (PD) and alpha synucleinopathies are multifactorial disorders, which manifest through motor symptoms and non-motor symptoms involving the Central Nervous System (CNS), the Peripheral Nervous System (PNS) and, recently, also the Enteric Nervous System (ENS). The typical hallmarks of alpha synucleinopathies are proteinaceous inclusions of alpha synuclein (αS). In PD they are known as Lewy Bodies (LBs) and Lewy Neurites (LNs), discovered in dopaminergic neurons of substantia nigra (pars compacta) as well as in other regions of the central and peripheral nervous systems. Despite the clear causes which lead to LBs/LNs are still unknown, according to Braak's theory, these inclusions appear first in PNS to spread, following neuronal innervation, towards the CNS in a spatio- temporal dissemination described in a staging procedure. In line with these observations, several animal models have been used with the purpose to reproduce PD as well as to propose new therapeutic approaches. Different pathways can cooperate to neurodegeneration in PD such as genetic mutations of αS gene, mitochondrial dysfunctions, neuroinflammation. The present review highlights αS as the key-word for PD pathology and alpha synucleinopathies and a main target in PD research. Several therapeutic approaches can be proposed, however all of them are addressed in advanced stages of the pathology. Our focus will be the alteration of αS physiological pathway, which allows to address therapy in early stages at intracellular or extracellular level, such as the use of anti ER-stress compounds and innovative immunotherapy, which could be promising tools to reduce neuronal degeneration and to halt PD progression.