Stability analysis of reference genes for RT-qPCR assays involving compatible and incompatible Ralstonia solanacearum-tomato 'Hawaii 7996' interactions.

Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of Ralstonia solanacearum with 'Hawaii 7996' (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of 'Hawaii 7996' with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms. TIP41 and EF1α (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and TIP41 (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were PDS and TIP41 (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and PDS/EXP (with BestKeeper). Global analyses on the basis of the three algorithms across 20 R. solanacearum-tomato experimental conditions identified UBI3, TIP41 and ACT as the best choices as reference tomato genes in this important pathosystem.

Green synthesis of silver nanoparticles using tomato leaf extract and their entrapment in chitosan nanoparticles to control bacterial wilt.

BACKGROUND: Silver nanoparticles (AgNPs), particularly those entrapped in polymeric nanosystems, have arisen as options for managing plant bacterial diseases. Among the biopolymers useful for the entrapment of AgNPs, chitosan is promising because of its low cost, good biocompatibility, antimicrobial properties and biodegradability. The present study aimed: (i) to greenly-synthesize AgNPs using different concentrations of aqueous extract of tomato leaves followed by entrapment of AgNPs with chitosan (CH-AgNPs); (ii) to characterize the optical, structural and biological properties of the nanosystems produced; (iii) to evaluate the antimicrobial activities of AgNPs and nanomaterials; and (iv) to assess the effectiveness of AgNPs and nanomaterials for controlling tomato bacterial wilt caused by Ralstonia solanacearum. RESULTS: Spherical and oval AgNPs had incipient colloidal instability, although the concentration of the tomato leaf extract influenced both size (< 87 nm) and the polydispersity index. Nanomaterials (< 271 nm in size) were characterized by a highly stable matrix of chitosan containing polydisperse AgNPs. Free AgNPs and CH-AgNPs were stable for up to 30 days, with no significant alteration in physicochemical parameters. The AgNPs and nanomaterials had antibacterial activity and decreased bacterial growth at micromolar concentrations after 48 h. Morphological changes in R. solanacearum cells were observed after treatment with CH-AgNPs. The application of CH-AgNPs at 256 µmol L-1 reduced the incidence of bacterial wilt in a partially resistant tomato genotype but not in the susceptible line. CONCLUSION: Greenly-synthesized chitosan-derived nanomaterials containing AgNPs produced with leaf extracts from their own species appear to comprise a promising and sustainable alternative in an integrated management approach aiming to reduce the yield losses caused by bacterial wilt. © 2019 Society of Chemical Industry.

History and Status of Selected Hosts of the Ralstonia solanacearum Species Complex Causing Bacterial Wilt in Brazil.

Bacterial wilt induced by the Ralstonia solanacearum species complex is endemic to Brazil, where it can cause variable losses in many hosts. Its economic importance, however, cannot be precisely measured due to Brazil's continental size, subject to variable weather conditions which directly affect disease expression. The objectives of this paper were (i) to gather scattered information on historical facts; (ii) to show the current distribution of the pathogen in the country, and (iii) to comment on future trends on the importance of the disease in economically important current and potential hosts, based on the pathogen's variability and the global climate change under way.

Genome Sequence of Ralstonia pseudosolanacearum Strains with Compatible and Incompatible Interactions with the Major Tomato Resistance Source Hawaii 7996.

We report here the complete genome sequences of two Ralstonia pseudosolanacearum strains, isolated from the warm northeast region of Brazil. They display divergent (compatible versus incompatible) interactions with the resistant tomato line Hawaii 7996. Polymorphisms were detected in a subset of effector genes that might be associated with these contrasting phenotypes.

A comparative analysis of oral and maxillofacial pathology over a 16-year period, in the north of Portugal.

OBJECTIVES: To determine the frequency and spectrum of oral and maxillofacial lesions biopsied in a hospital population in the northern region of Portugal. METHODS: We conducted descriptive analyses of pathology reports from biopsies of oral and maxillofacial lesions performed between 1990 and 2006, in Oporto Hospital Center. Information on gender and age of patient, location of the lesions and the histopathological diagnosis were analysed. RESULTS: The analyses revealed that 1,520 (47.7%) patients were male and 1,666 (52.3%) were female. They had a mean age ± standard deviation of 47.8 ± 18.6 years. The site most frequently biopsied was the labial mucosa (17.5%). A non-neoplastic diagnosis was established in 2,162 (63.3%) cases, potentially malignant disorders in 163 (5.1%) and neoplasms in 886 (27.6%) (403 benign and 483 malignant). The most commonly reported diagnosis was fibroepithelial polyp (n = 186; 15.9%), followed by squamous cell carcinoma (SCC) (n = 158; 13.6%). SCC was the lesion most commonly found in male patients (n = 279; 18.4%) whilst fibroepithelial polyp was the lesion most commonly found in female patients (n = 268; 16.1%). The most common lesion in patients 0-17 years of age was a follicular cyst (n = 25; 12.8%), whereas in patients 18-64 years of age it was a fibroepithelial polyp (n = 299; 13%). SCC was the most common type of lesion found in patients ≥ 65 years of age (n = 160; 24.6%). CONCLUSION: This large sample provides useful information about the incidence and distribution of oral biopsies over a period of 16 years, allowing valuable comparison with other countries. Non-neoplastic lesions were the types of lesion most commonly reported, with fibroepithelial polyp being most frequent. SCC was the second most common diagnosis.

Duodenal gastrointestinal stromal tumor and endoscopic ultrasound.

Duodenal gastrointestinal stromal tumors (GISTs) are a very rare condition. The pre-operative diagnosis can be a challenge but it is very important because GISTs have singularities that differ from other tumors and their location in the duodenum itself can have a major role in the choice of the surgical approach. We present two cases of duodenal GISTs where endoscopic ultrasound had a single role in their management, namely allowing the possibility to obtain material for immunocytochemical pre-operative diagnosis and regarding the precise relation to the papilla of Vater. The patients were operated and histological examination confirmed the diagnosis in both cases.

High CDC20 expression is associated with poor prognosis in oral squamous cell carcinoma.

OBJECTIVES: Human Cell Division Cycle 20 (CDC20) homolog is a crucial target of the spindle assembly checkpoint. It is an activator of the Anaphase-Promoting Complex/Cyclosome (APC/C) which promotes anaphase onset and mitotic exit through the ubiquitination of securin and cyclin B1. Overexpression of CDC20 was previously reported in oral squamous cell carcinoma (OSCC). Here, we propose to explore the clinicopathological significance of CDC20 overexpression and its potential use as a prognostic marker in OSCC. METHODS: Using tissue microarray technology, we analyzed CDC20 expression in 65 primary OSCC tissues by immunohistochemistry. Statistical analysis was performed to evaluate the clinicopathological and prognostic significance of CDC20 expression in OSCC. RESULTS: Of the 65 cases of patients with OSCC studied, 37 (56.9%) showed high CDC20 protein expression. No clinicopathological features were correlated with CDC20 expression. Importantly, in univariable analysis, OSCC patients with higher CDC20 protein expression showed significantly shorter cancer-specific survival rate (P = 0.018). Multivariable analysis identified high CDC20 expression as an independent prognostic factor (P = 0.032). CONCLUSION: High CDC20 expression is associated with poor prognosis in OSCC and may be used to identify high-risk OSCC patients and may serve as a therapeutic target.

Phenotype characterization of Staphylococcus species strains isolated from buffalo (Bubalus bubalis) milk.

The objective of the current study was to isolate and identify phenotypes of Staphylococcus spp. strains derived from buffalo (Bubalus bubalis) milk. A total of 548 milk samples from 137 buffalo were cultured in Columbia agar enriched with 5% defibrinated sheep blood. Determination of the capacity of Staphylococcus aureus to produce enterotoxins A-D and toxic shock syndrome toxin-1 (TSST-1) was achieved by reverse passive latex agglutination (RPLA). Antimicrobial sensitivity of S. aureus strains was evaluated using the disk diffusion technique, and ß-lactamase detection was achieved using the chromogenic test with paper discs impregnated with nitrocefin. From all the mammary quarters examined, 36 (10.8%) were positive for Staphylococcus spp., 83.3% were coagulase-negative staphylococcus (CNS), 11.1% were coagulase-positive staphylococcus (CPS), and 5.6% were of CPS+CNS positive. All isolates of S. aureus produced at least 1 toxin and 5 out of 6 isolates (83.0%) produced ß-lactamase. One hundred percent of S. aureus isolates were sensitive to methicillin and amoxicillin + clavulanic acid, and resistant to ampicillin, penicillin, and oxacillin. Analysis of the results obtained in the current study highlight the epidemiologic importance of buffalo milk regarding the production of enterotoxins and TSST-1 and the potential risk to public health.

Complex carcinomas of the mammary gland in cats: pathological and immunohistochemical features.

Biphasic epithelial myoepithelial (complex) carcinomas of the feline mammary gland are rare. This article describes the pathological and immunohistochemical features and clinical outcome of eight cases of feline mammary carcinomas displaying complex morphology. This tumour type is a low grade malignancy that shows histopathological features distinctive from more common feline mammary carcinomas and from complex mammary carcinomas of dogs. It appears to have a better overall survival than other carcinomas of the mammary gland of cats.

DNA study of bladder papillary tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in Fisher rats.

To examine DNA abnormalities in bladder papillary tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female rats, using image cytometric DNA analysis and cytogenetics. Thirty female rats were exposed to BBN in their drinking water for 20 weeks. One group of 10 animals served as controls. The animals exposed to BBN were killed at a rate of two per week, with the bladder being collected under aseptic conditions and those tumours with exophytic growth removed. The nuclear DNA content of the tumours was evaluated using image cytometric analysis. In two rats part of the tumour pieces was stipulated for culturing. Cytogenetic analysis was performed on at least 30 cells from each cell population and on both tumours. Papillary carcinomas were classified as low grade and high grade. DNA ploidy studies were carried out on 28 low-grade and 21 high-grade papillary carcinomas. Histograms obtained by image analysis showed that a normal urothelium was diploid; 28.6% and 100% of low-and high-grade papillary carcinomas were aneuploid respectively. Both tumours used for cell culture showed multiple numerical and structural chromosome alterations and several marker chromosomes. Image cytometric DNA analysis proved to be a good and reliable method for examining DNA alterations in papillary bladder carcinomas. The present findings establish that the DNA content is statistically different between low-grade and high-grade papillary carcinomas and that deviation from the diploid number is markedly higher in the high-grade ones. In addition, the occurrence of marker chromosomes seems to be related to the aggressiveness of the tumour.

Significância clínica de estafilococos coagulase-negativa isolados de recém-nascidos / Clinical significance of coagulase-negative staphylococci isolated from neonates

Objetivo: avaliar a significância clínica de estafilococos coagulase-negativa (ECN) isolados de processos infecciosos em recém-nascidos da unidade neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu.Método: as linhagens de ECN isoladas foram identificadas eclassificadas em significativas e contaminantes, com base em uma série de dados clínicos e laboratoriais obtidos dos prontuários dos pacientes internados na unidade neonatal. Foram pesquisados os dados referentes a fatores perinatais de risco para infecção, evolução clínica, alterações do hemograma e/ou positividade de proteína C-reativa e antibioticoterapia.Resultados: das 117 linhagens de ECN isoladas, 60 (51,3por cento)foram classificadas como significativas, e 57 (48,7por cento) como contaminantes. Das 54 crianças com infecção por ECN, 43 (79,6por cento) eram prematuras, e 27 (50,0por cento) tiveram peso ao nascimento < 1.500g. A maioria das crianças com infecção por ECN estava submetida a dois ou mais procedimentos invasivos (77,8por cento), incluindo o uso de cateter(88,9por cento), nutrição parenteral (64,8por cento) e ventilação mecânica (61,1por cento).O S.epidermidis foi a espécie mais freqUentemente isolada (77,8por cento), e mais associada com infecção (86,7por cento) do que com contaminação (68,4por centi). Outras espécies de ECN, incluindo duas linhagens de s.haemolyticus, três linhagens de s. lugdunensis, uma linhagem de S. simulans, uma de S. warneri e uma linhagem de S. xylosus também foram isoladas de crianças com evidência clínica de pneumonia, nterocolite necrosante e sepse.Conclusão: a maioria dos recém-nascidos com infecção por ECN apresentou...

[Clinical significance of coagulase-negative staphylococci isolated from neonates]. / Significância clínica de estafilococos coagulase-negativa isolados de recém-nascidos.

OBJECTIVE: To evaluate the clinical significance of coagulase-negative staphylococci (CNS) isolated from newborns' infections at Neonatal Unit of Hospital das Clínicas da Faculdade de Medicina de Botucatu. METHODS: The CNS strains isolated were identified and classified as clinically significant and contaminant, based on a series of clinical and laboratory data obtained from patients who stayed in the Neonatal Unit. The following data were analyzed: risk factors for infections, clinical evolution, abnormal blood cell counts and/or C-reactive protein and antibiotic therapy. RESULTS: Among the 117 CNS strains isolated, 60 (51.3%) were classified as significant and 57 (48.7%) as contaminant. Among the 54 infants infected by CNS, 43 (79.6%) presented very low birthweight (<1,500 g). Most of the infants infected by CNS were submitted to two or more invasive procedures (77.8%), including use of catheter (88.9%), parenteral nutrition (64.8%) and mechanical ventilation (61.1%). Staphylococcus epidermidis was the most frequently isolated species (77.8%) and more often associated with infection (86.7%) than with contamination (68.4%). Other species of CNS, including two strains of S. haemolyticus, three strains of S. lugdunensis, one strain of S. simulans, one strain of S. warneri and one strain of S. xylosus were also isolated from infants with clinical evidence of pneumonia, necrotizing enterocolitis and sepsis. CONCLUSIONS: Most newborns infected by CNS presented important risk factors for infection onset, including birthweight <1,500 g, foreign body presence and previous use of antibiotics. The identification of CNS species constitutes a useful marker of infection, since S. epidermidis was the species more frequently associated with infection.

Occurrence of Bacillus cereus in Meat Products, Raw Meat and Meat Product Additives.

One thousand nine hundred and sixty three samples of meat products, raw meat and meat product additives from different slaughterhouses, meat processing factories and retail meat shops in six prefectures of Japan, were examined for the presence and number of Bacillus cereus . Although B. cereus was found in meat products (18.3%) and raw meat (6.6%), the contamination levels were generally lower than 102 per gram. In contrast, meat product additives showed contamination levels ranging from 102 to 104/g with the highest values (104/g) in samples of spices and animal proteins. On the basis of these results, we suggest that the main source of B. cereus contamination in meat products is contaminated meat product additives.

Enumeration of Aerobic Spore-Formers and Bacillus cereus in Meat Product Additives.

Microbial populations in 225 samples of meat product additives including spices, seasoning, proteins, starch, salt, sugar and colorants, were enumerated by means of aerobic plate counts (APC), aerobic spore counts (ASC), Bacillus cereus total counts (BcTC), and B. cereus spore counts (BcSC). Our data indicate that meat product additives with the exception of sugar and salt were contaminated mainly by aerobic spore-formers including B. cereus under spore-forming conditions. To improve the quality of meat products, it will be necessary to develop methods to reduce the bacterial contamination of meat product additives.