Effects of temperature changes in the transcriptional profile of the emerging fish pathogen Francisella noatunensis subsp. orientalis.

One of the major challenges in Nile tilapia (Oreochromis niloticus L.) farming is the occurrence of bacterial infections, and the Francisella noatunensis subsp. orientalis (FNO) is an important pathogen that has emerged in last decades. Francisellosis outbreaks have been reported in the literature as occurring seasonally when water temperature is below 24 °C. The aim of this study was to quantify the median lethal doses (LD50) of FNO in experimental challenges at 28 °C and 22 °C, and to investigate the impact of temperature changes in whole genome expression using microarray technology. The LD50 for Nile tilapia at 28 °C was ∼105.7, whereas at 22 °C, the LD50 was ∼102.2, showing that the decrease in temperature enhanced disease outcome. Out of 1917 genes screened, a total of 31 and 19 genes were down- and up-regulated at 22 °C, respectively. These genes were grouped by orthology into functional categories of: amino acid, inorganic ion, and carbohydrate transport and metabolism; transcription; and posttranslational modification, protein turnover, and chaperones. Expression of genes related to metabolism, oxidative stress, and thermal shock were regulated by temperature changes, reflecting an ability of FNO to adapt to the environment. Expression of virulence genes usually required for the Francisella genus was not changed between tested temperatures, including that of genes located on the Francisella Pathogenicity Island.

Complete genome sequencing of sixteen Francisella noatunensis subsp. orientalis isolates: A genomic approach for molecular characterization and spread dynamics of this clonal population.

Francisella noatunensis subsp. orientalis (FNO) is an important emerging pathogen associated with disease outbreaks in farm-raised Nile tilapia. FNO genetic diversity using PCR-based typing, no intra-species discrimination was achieved among isolates/strains from different countries, thus demonstrating a clonal behaviour pattern. In this study, we aimed to evaluate the population structure of FNO isolates by comparing whole-genome sequencing data. The analysis of recombination showed that Brazilian isolates group formed a clonal population; whereas other lineages are also supported by this analysis for isolates from foreign countries. The whole-genome multilocus sequence typing (wgMLST) analysis showed varying numbers of dissimilar alleles, suggesting that the Brazilian clonal population are in expansion. Each Brazilian isolate could be identified as a single node by high-resolution gene-by-gene approach, presenting slight genetic differences associated to mutational events. The common ancestry node suggests a single entry into the country before 2012, and the rapid dissemination of this infectious agent may be linked to market sales of infected fingerlings.

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

A MLST Clade 2 Clostridium difficile strain with a variant TcdB induces severe inflammatory and oxidative response associated with mucosal disruption.

The epidemiology of Clostridium difficile infections is highly dynamic as new strains continue to emerge worldwide. Here we present a detailed analysis of a new C. difficile strain (ICC-45) recovered from a cancer patient in Brazil that died from severe diarrhea. A polyphasic approach assigned a new PCR-ribotype and PFGE macrorestriction pattern to strain ICC-45, which is toxigenic (tcdA(+), tcdB(+) and ctdB(+)) and classified as ST41 from MLST Clade 2 and toxinotype IXb. Strain ICC-45 encodes for a variant TcdB that induces a distinct CPE in agreement with its toxinotype. Unlike epidemic NAP1/027 strains, which are also classified to MLST Clade 2, strain ICC-45 is susceptible to fluoroquinolones and does not overproduce toxins TcdA and TcdB. However, supernatants from strain ICC-45 and a NAP1/027 strain produced similar expression of pro-inflammatory cytokines, epithelial damage, and oxidative stress response in the mouse ileal loop model. These results highlight inflammation and oxidative stress as common features in the pathogenesis of C. difficile Clade 2 strains. Finally, this work contributes to the description of differences in virulence among various C. difficile strains.

TNF-alpha and melphalan modulate a specific group of early expressed genes in a murine melanoma model.

BACKGROUND: Cutaneous melanoma displays high morbidity and mortality rates. Isolated limb perfusion with melphalan (Mel) is used for the treatment of non-resectable, locally advanced extremity melanomas. When combined with tumor necrosis factor alpha (TNF-alpha) treatment, the complete response varies between 70% and 90%. The mechanisms underlying the effects of Mel and TNF-alpha are not completely understood. We evaluated the impact of systemic Mel and TNF-alpha administration on tumor growth, analyzed the morphological changes promoted by each treatment, and identified early expressed genes in response to Mel and TNF-alpha treatment, either alone or in combination, in a murine melanoma model. METHODS: Six- to eight-week-old male mice were subcutaneously inoculated with B16F10 melanoma cells and then intravenously injected with TNF-alpha, melphalan or a combination of both drugs when the tumors reached 1.0 cm(2). Tumor growth was monitored every other day, and histological analysis was performed when the tumors reached 3.0 cm(2). Total RNA was extracted from the resected tumors and submitted to amplification, labeling and hybridization on an oligonucleotide microarray (Fox Chase Cancer Center). Tumor growth and histological parameters were compared using ANOVA. Survival curves were calculated using the Kaplan-Meier method. Two-way ANOVA was used to identify differentially expressed genes among the various treatments, and Dunn's test was used for pair-wise comparisons. RESULTS: Systemic administration of Mel impaired tumor growth (p<0.001), improved animal survival (p<0.001), and decreased mitotic rate (p=0.049). Treatment with TNF-alpha alone had no impact, neither on tumor growth, nor on survival, but it increased necrosis (p<0.024) and decreased mitotic rates (p=0.001) in the tumors. Combined treatment with Mel and TNF-alpha had similar effects in tumor growth, survival, necrosis and mitotic rate as observed with individual treatments. Moreover, 118 genes were found differentially expressed by microarray analysis and 10% of them were validated by RT- real time PCR. In our model we found that the treatments regulate genes that play important roles in tumorigenesis such as cell adhesion (Pard3, Pecam1, Ilk, and Dlg5), proliferation (Tcfe3 and Polr1e), cell motility (Kifap3, Palld, and Arhgef6), apoptosis (Bcl2l11), and angiogenesis (Flt1 and Ptprj). CONCLUSIONS: Our data reproduces, in mice, some of the features observed in melanoma patients treated with the combination of Mel and TNF-alpha. The identification of genes with altered expression by these drugs both individually and in combination might help in the understanding of their mechanism of action and, as a consequence, improved strategies that could impact their clinical application.

SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation.

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants-Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients' follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Complete genome sequences of Francisella noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190: a fish pathogen with genomic clonal behavior.

The genus Francisella is composed of Gram-negative, pleomorphic, strictly aerobic and non-motile bacteria, which are capable of infecting a variety of terrestrial and aquatic animals, among which Francisella noatunensis subsp. orientalis stands out as the causative agent of pyogranulomatous and granulomatous infections in fish. Accordingly, F. noatunensis subsp. orientalis is responsible for high mortality rates in freshwater fish, especially Nile Tilapia. In the current study, we present the genome sequences of F. noatunensis subsp. orientalis strains FNO12, FNO24 and FNO190. The genomes include one circular chromosome of 1,859,720 bp, consisting of 32 % GC content, 1538 coded proteins and 363 pseudogenes for FNO12; one circular chromosome of 1,862,322 bp, consisting of 32 % GC content, 1537 coded proteins and 365 pseudogenes for FNO24; and one circular chromosome of 1,859,595 bp, consisting of 32 % GC content, 1539 coded proteins and 362 pseudogenes for FNO190. All genomes have similar genetic content, implicating a clonal-like behavior for this species.

Complete genome sequence of Streptococcus agalactiae strain GBS85147 serotype of type Ia isolated from human oropharynx.

Streptococcus agalactiae, also referred to as Group B Streptococcus, is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. The pathogen can also infect adults with underlying disease, particularly the elderly and immunocompromised ones. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. This study provides valuable structural, functional and evolutionary genomic information of a human S. agalactiae serotype Ia (ST-103) GBS85147 strain isolated from the oropharynx of an adult patient from Rio de Janeiro, thereby representing the first human isolate in Brazil. We used the Ion Torrent PGM platform with the 200 bp fragment library sequencing kit. The sequencing generated 578,082,183 bp, distributed among 2,973,022 reads, resulting in an approximately 246-fold mean coverage depth and was assembled using the Mira Assembler v3.9.18. The S. agalactiae strain GBS85147 comprises of a circular chromosome with a final genome length of 1,996,151 bp containing 1,915 protein-coding genes, 18 rRNA, 63 tRNA, 2 pseudogenes and a G + C content of 35.48 %.

Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common tumor of the oral cavity and has been associated with poor prognosis. Scarce prognostic markers are available for guiding treatment and/or sub-classifying patients. This study aims to identify biomarkers by searching for genes whose expression is increased or decreased during tumor progression (through T1 to T4 stages). Thirty-six samples from all tumor size stages (from T1 to T4) were analyzed using cDNA microarrays. Selected targets were analyzed by immunohistochemistry and in circulating tumor cells by immunofluorescence and Nanostring. Correlation was shown between PD-L1 and tumor size and lymph node metastasis, HOXB9 and tumor size, BLNK and perineural invasion, and between ZNF813 and perineural invasion. PD-L1 positivity was an independent prognostic factor in this cohort (p = 0.044, HH = 0.426). In CTCs from patients with locally advanced OSCC, we found a strong cytoplasmatic expression of PD-L1. PD-L1 is a ligand of PD-1 and is believed to limit T cell activity in inflammatory responses and limit autoimmune diseases. We demonstrated an important role for PD-L1 in primary tumors according to tumor size, and in disease specific survival. Therefore, we could further determine individuals with PD-L1+ CTCs, and possibly follow treatment using CTCs.

Comprehensive analysis of BRCA1, BRCA2 and TP53 germline mutation and tumor characterization: a portrait of early-onset breast cancer in Brazil.

Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most important disease-causing issues in young breast cancer patients worldwide. The specific defective biological processes that trigger germline mutation-associated and -negative tumors remain unclear. To delineate an initial portrait of Brazilian early-onset breast cancer, we performed an investigation combining both germline and tumor analysis. Germline screening of the BRCA1, BRCA2, CHEK2 (c.1100delC) and TP53 genes was performed in 54 unrelated patients <35 y; their tumors were investigated with respect to transcriptional and genomic profiles as well as hormonal receptors and HER2 expression/amplification. Germline mutations were detected in 12 out of 54 patients (22%) [7 in BRCA1 (13%), 4 in BRCA2 (7%) and one in TP53 (2%) gene]. A cancer familial history was present in 31.4% of the unrelated patients, from them 43.7% were carriers for germline mutation (37.5% in BRCA1 and in 6.2% in the BRCA2 genes). Fifty percent of the unrelated patients with hormone receptor-negative tumors carried BRCA1 mutations, percentage increasing to 83% in cases with familial history of cancer. Over-representation of DNA damage-, cellular and cell cycle-related processes was detected in the up-regulated genes of BRCA1/2-associated tumors, whereas cell and embryo development-related processes were over-represented in the up-regulated genes of BRCA1/2-negative tumors, suggesting distinct mechanisms driving the tumorigenesis. An initial portrait of the early-onset breast cancer patients in Brazil was generated pointing out that hormone receptor-negative tumors and positive familial history are two major risk factors for detection of a BRCA1 germline mutation. Additionally, the data revealed molecular factors that potentially trigger the tumor development in young patients.