Cyanobacterial diversity and a new Acaryochloris-like symbiont from Bahamian sea-squirts

Symbiotic interactions between ascidians (sea-squirts) and microbes are poorly understood. Here we characterized the cyanobacteria in the tissues of 8 distinct didemnid taxa from shallow-water marine habitats in the Bahamas Islands by sequencing a fragment of the cyanobacterial 16S rRNA gene and the entire 16S-23S rRNA internal transcribed spacer region (ITS) and by examining symbiont morphology with transmission electron (TEM) and confocal microscopy (CM). As described previously for other species, Trididemnum spp. mostly contained symbionts associated with the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two unique clades. The first contained a novel cyanobacterial clade, while the second clade was closely associated with Acaryochloris marina. CM revealed the presence of chlorophyll d (chl d) and phycobiliproteins (PBPs) within these symbiont cells, as is characteristic of Acaryochloris species. The presence of symbionts was also observed by TEM inside the tunic of both the adult and larvae of L. fragile, indicating vertical transmission to progeny. Based on molecular phylogenetic and microscopic analyses, Candidatus Acaryochloris bahamiensis nov. sp. is proposed for this symbiotic cyanobacterium. Our results support the hypothesis that photosymbiont communities in ascidians are structured by host phylogeny, but in some cases, also by sampling location.

Bartonella apis sp. nov., a honey bee gut symbiont of the class Alphaproteobacteria.

Here, we report the culture and characterization of an alphaproteobacterium of the order Rhizobiales, isolated from the gut of the honey bee Apis mellifera. Strain PEB0122T shares >95 % 16S rRNA gene sequence similarity with species of the genus Bartonella, a group of mammalian pathogens transmitted by bloodsucking arthropods. Phylogenetic analyses showed that PEB0122T and related strains from the honey bee gut form a sister clade of the genus Bartonella. Optimal growth of strain PEB0122T was obtained on solid media supplemented with defibrinated sheep blood under microaerophilic conditions at 35-37 °C, which is consistent with the cultural characteristics of other species of the genus Bartonella. Reduced growth of strain PEB0122T also occurred under aerobic conditions. The rod-shaped cells of strain PEB0122T had a mean length of 1.2-1.8 μm and revealed hairy surface structures. Strain PEB0122T was positive for catalase, cytochrome c oxidase, urease and nitrate reductase. The fatty acid composition was comparable to those of other species of the genus Bartonella, with palmitic acid (C16 : 0) and isomers of 18- and 19-carbon chains being the most abundant. The genomic DNA G+C content of PEB0122T was determined to be about 45.5 mol%. The high 16S rRNA gene sequence similarity with species of Bartonella and its close phylogenetic position suggest that strain PEB0122T represents a novel species within the genus Bartonella, for which we propose the name Bartonella apis sp. nov. The type strain is PEB0122T ( = NCIMB 14961T = DSM 29779T).

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

Flavonóides, norisoprenóides e outros terpenos das folhas de Tapirira guianensis

From hexane fraction of methanol extract of leaves of Tapirira guianensis (Anacardiaceae) were obtained lupeol, 24-methylenecycloartan-3-ol, phytol, α-amyrin, β-amyrin, sitosterol, sitostenone, glycosyl sitosterol, as well as sitosterol esterified with palmitic and stearic acids. Phytol, α-amyrin and β-amyrin esterified with fatty acids were also identified from same extract. The EtOAc extract besides the norisoprenoids (6S,7E,9S)-6,9-dihydroxy-megastigma-4,7-dien -3-one 9-O-β-glucopyranoside and (6S,7E,9R)-6,9-dihydroxy-megastigma-4,7-dien-3-one 9-O-β-glucopyranoside also afforded kaempferol 3-O-rhamnoside, kaempferol 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, and kaempferol. The structural elucidation of isolated compounds were based on UV, IR, MS, ¹H and 13C NMR data analysis.

Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts

Using PCR-based assays with specific primers for amplification of the ribosomal DNA intergenic spacer region (IGS) and a portion of the mitochondrial DNA small subunit ribosomal RNA gene (mtDNA SSU rRNA), the genetic variability among Verticillium dahliae isolates from olive (Olea europaea) and other host species from Argentina and Brazil was estimated. The derived UPGMA-generated phenograms based upon the restriction fingerprinting data of rDNA IGS products revealed genetic differences, correlating with the host of origin. Isolates infecting olive genetically distinct from those from cocoa (Theobroma cacao) and sunflower (Helianthus annuus). Digestion of mitochondrial DNA SSU rRNA PCR products revealed less variability, distinguishing only one isolate from sunflower. Ribosomal DNA ITS restriction patterns were identical for all isolates of V. dahliae, irrespective of host of origin. These preliminary results may have relevance for Verticillium wilt control practices, possibly reflecting a different evolutionary origin, or reproductive isolation of the pathogen in olive, distinct from populations of other hosts.

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.

Caracterização espectral de estágios sucessionais de vegetação secundária arbórea em Altamira (PA), através de dados orbitais

Imagens TM/Landsat 5 (bandas TM1-TM5 e TM7) de Altamira, PA, de 07.02.1991 foram transformadas em reflectância de superfície usando o modelo 6S de correção atmosférica com o objetivo de caracterizar espectralmente formações florestais secundárias. Dados biofísicos (DAP, G e H) foram coletados de 16 parcelas em campo, os quais foram utilizados no cálculo da área basal total (GT) e da rugosidade do dossel (Rug) e no estabelecimento de correlações entre os valores de reflectância de superfície dessas mesmas parcelas. Os coeficientes de correlação mais elevados foram verificados nas relações com a banda TM7. Regressões lineares foram estabelecidas tendo como variável dependente os parâmetros biofísicos e como variável independente, os valores de reflectância de superfície da banda TM7, a partir das quais foram elaborados mapas temáticos representativos da distribuição espacial dos parâmetros biofísicos. A reflectância de superfície não foi sensível a valores pequenos de dados biofísicos. Apesar disso, a metodologia aqui empregada mostrou-se eficaz para estimativas remotas de dados dendrométricos.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli Campylobacters are a common cause of bacterial gastroenteritis worldwide, with Campylobacter jejuni and C. coli being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the C. jejuni and C. coli strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were in vitro highly effective towards Campylobacter species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected Campylobacter enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Controle da leptospirose em bovinos de leite com vacina autógena em Santo Antônio do Monte, Minas Gerais

Um surto de leptospirose foi observado em bovinos leiteiros em Santo Antônio do Monte, Minas Gerais. O rebanho apresentava reações positivas anti-leptospira sorovar Hardjo no teste de microaglutinação (MAT) e havia sido vacinado anteriormente com vacina experimental contendo a sorovariedade Hardjo. O MAT revelou 48,06% dos bovinos positivos para sorovariedade Hardjo genótipo Hardjobovis, 36,82% para sorovariedade Hardjo genótipo Hardjoprajitno. Os animais apresentavam aborto e mastite com presença de sangue no leite. A presente pesquisa teve como objetivos isolar as sorovariedades existentes a partir da urina de vacas sorologicamente positivas, elaborar uma vacina experimental com as sorovariedades isoladas no rebanho, avaliar a eficiência do programa de vacinação por um período de dois anos por meio da sorologia do rebanho. Foi isolada Leptospira spp. a partir da urina de duas vacas com sinais sugestivos da doença. As amostras isoladas foram identificadas pela sorologia com anticorpos monoclonais e seqüenciamento do gene 16S rRNA como pertencentes à espécie Leptospira interrogans, sorogrupo Sejroe, sorovariedade Hardjo e genótipo Hardjoprajitno. O uso da vacina autógena foi eficaz no controle da leptospirose no rebanho no período de dois anos. Os resultados da sorologia revelaram ausência de animais positivos na última prova realizada no rebanho.

Ocorrência da infecção por Cryptosporidium spp. em cabritos (Capra hircus)

O presente estudo teve como objetivo determinar a ocorrência da infecção por Cryptosporidium spp. em cabritos de Quixadá, Ceará, Brasil. Participaram do estudo 400 cabritos, com idade entre três e 360 dias, de ambos os sexos, com e sem padrão racial definido, procedentes de 25 estabelecimentos rurais distribuídos em três circuitos. As fezes foram cadastradas de acordo com o aspecto e cor, distribuídas em tubos tipo "eppendorf®" e congeladas in natura a -20°C, até o momento das extrações de DNA genômico do parasito com auxílio de kit comercial. Para amplificação de fragmentos da subunidade 18S do RNA ribossômico (rRNA) foi utilizada a "Nested"-PCR. A ocorrência de Cryptosporidium spp em cabritos de Quixadá foi de 7,50% (30/400). A frequência no período seco e no chuvoso foi de 9,55% (19/199) e 5,47% (11/201), respectivamente (χ²=2,39 e P>0,05). Amostras positivas foram identificadas em 64,00% (16/25) das propriedades estudadas e dessas amostras 50,00% (15/30) e 70,00% (21/30) tinham as fezes com aspecto e cor normais, respectivamente, sugerindo que cabritos assintomáticos estão eliminando oocistos. Não foi observada positividade para Cryptosporidium spp. em animais com 301 a 360 dias, demonstrando que animais mais velhos apresentam menos possibilidade de se infectarem com o parasito.

Research of Klebsiella pneumoniae in dairy herds

Klebsiella pneumoniae is a common environmental agent of clinical and subclinical mastitis affecting dairy herds, and may be present in the final product decreasing its quality. Mastitis caused by K. pneumoniae is even more severe due to its poor response to antibiotic therapy, rapid evolution to toxic shock and death of the animal. This paper aimed to study the prevalence of this pathogen among dairy herds in ten farms located in different municipalities of São Paulo State based on size and use of milking technology. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20mL) was collected from all CMT-positive quarters and bulk tanks, whereas swab samples were collected from feces, hind limbs of the animals, bedding and milking parlor. Identification of K. pneumoniae was performed using conventional microbiology culture, biochemical assay and Polimerase Chain Reaction. The primers were designed and tested at the Laboratory of Molecular Biology applied to Zoonoses (FMVZ, Unesp-Botucatu) targeting the 16S rRNA gene. This study included 1067 animals. Six cases of intramammary infection by K. pneumoniae were detected in six different cows in two farms. Moreover, K. pneumoniae was isolated in 77 swabs (34 from bedding in 9 farms, 7 from waiting rooms in 5 farms, 6 from milking parlors in 4 farms, 11 from rectums in six farms, and 19 from hindlimbs in 7 farms. Molecular analysis confirmed the agent was K. pneumoniae. At least one strain of the agent was identified in a certain site in all farms, showing the need of maintaining the hygiene in dairy farms.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somniwith sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.

The nucleolus: a paradigm for cell proliferation and aging

The nucleolus is the cellular site of ribosome biosynthesis. At this site, active ribosomal DNA (rDNA) genes are rapidly transcribed by RNA polymerase I (pol I) molecules. Recent advances in our understanding of the pol I transcription system have indicated that regulation of ribosomal RNA (rRNA) synthesis is a critical factor in cell growth. Importantly, the same signaling networks that control cell growth and proliferation and are deregulated in cancer appear to control pol I transcription. Therefore, the study of the biochemical basis for growth regulation of pol I transcription can provide basic information about the nuclear signaling network. Hopefully, this information may facilitate the search for drugs that can inhibit the growth of tumor cells by blocking pol I activation. In addition to its function in ribosome biogenesis, recent studies have revealed the prominent role of the nucleolus in cell senescence. These findings have stimulated a new wave of research on the functional relationship between the nucleolus and aging. The aim of this review is to provide an overview of some current topics in the area of nucleolus biology, and it has been written for a general readership.