Identification of potentially pathogenic variants in candidate breast and ovarian cancer susceptibility genes in BRCAX patients

Mutations within the BRCA1 and BRCA2 genes account for approximately 20% of hereditary breast cancers, with a further 10%–15% being attributable to rare mutations in moderate-risk genes and common variants in low-risk genes. The genes harbouring mutations in the remaining ∼65% of hereditary breast cancers are unknown. The identification of mutation carriers in hereditary breast and ovarian cancer (hboc) families is critical for determining who is most at risk of developing the disease and therefore who should be offered risk-reducing procedures or more intensive screening, or both.

Many of the high- and moderate-risk genes for hereditary breast cancers encode proteins that work in concert to maintain genomic stability and in dna damage signalling and repair. A novel BRCA1 protein complex identified within the research group whose target genes are involved in dna repair provided novel candidates for hboc susceptibility genes. These 12 candidate genes were sequenced in a cohort of 675 affected individuals from the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) with hereditary breast or ovarian cancer, but with no mutations in known susceptibility genes (BRCAx patients). This analysis identified 20 individuals (each from a different BRCAx family) with different potentially pathogenic variants across 6 of the candidate hboc susceptibility genes. The family members of each BRCAx index case were tested for the presence of the specific mutation identified in the proband to examine segregation with disease. To further expand on the potential role of the novel candidate hboc susceptibility genes identified in this study, the genetic variation of a second cohort of 520 Northern Irish BRCAx patients is being characterized using a 61-gene panel.

A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic Escherichia coli in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for E. coli with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring E.coli levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Next-Generation Sequencing of Hereditary Hemochromatosis-Related Genes: Novel Likely Pathogenic Variants Found in the Portuguese Population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G>C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5'-UTR of HAMP gene (c.-25G>A). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Impact of Cryptosporidium spp. interaction with co-occurring microorganisms on moderate-to-severe diarrhea in the developing world

Diarrheal illness is responsible for over a quarter of all deaths in children under 5 years of age in sub-Saharan Africa and South Asia. Recent findings have identified the parasite Cryptosporidium as a contributor to enteric disease. We examined 9,348 cases and 13,128 controls from the Global Enteric Multicenter Study to assess whether Cryptosporidium interacted with co-occurring pathogens based on adjusted odds of moderate-to-severe diarrhea (MSD). Cryptosporidium was found to interact negatively with Shigella spp., with multiplicative interaction score of 0.16 (95% CI: 0.07 to 0.37, p-value=0.000), and an additive interaction score of -9.81 (95% CI: -13.61 to -6.01, p-value=0.000). Cryptosporidium also interacted negatively with Aeromonas spp., Adenovirus, Norovirus, and Astrovirus with marginal significance. Odds of MSD for Cryptosporidium co-infection with Shigella spp., Aeromonas spp., Adenovirus, Norovirus, or Astrovirus are lower than odds of MSD with either organism alone. This may reduce the efficacy of intervention strategies targeted at Cryptosporidium.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sectr in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi raising concerns as occupational hazards in cork industry. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city. The chosen fungal species are the ones most frequently associated with respiratory problems in workers from these industries.

Indução de resistência e tratamento de sementes de soja com fosfitos de potássio

The use of inputs containing phosphites have been presenting results in many studies, taking on importance to the control of diseases in some cultures and demonstrating the resistance induction in seedlings, with ability to activate defense mechanisms, conferring protection to plants against microorganisms. The soybean crop is recognized for its importance in providing grains and derivatives for human consumption, animal, production of biofuels, pharmaceuticals, among others. Positive results obtained through studies based on resistance inducers in some cultures arouse the interest for further study. The objective of this study was to evaluate the effect of potassium phosphites on the resistance induction and treatment of soybean seeds. Therefore were conducted four laboratory studies at the Federal Technological University of Paraná, Campus of Dois Vizinhos. In the first study it was evaluated the quality attributes of the seeds and the resistance induction as seed treatment. Then it was verified that phosphites have action upon the seedlings metabolism in due to seed treatment, having the phosphite Reforce® contributed to seed quality attributes and phosphites FitofosK® and Fitofos K Plus® induced the resistance increasing the activity of β-1,3-glucanase. In the second study it was evaluated the the resistance induction in soybean cotyledons, in which the phosphites demonstrated induction potential of phytoalexin gliceolin. In the third study It was evaluated the soybean seed health treated with potassium phosphites.. it was observed that the phosphites reduced the incidence of many fungi on seeds, especially of storage fungi like Aspergillus sp. and Fusarium semitectum. In the fourth study it was evaluated the in vitro effect of potassium phosphites on pathogenic fungi of the culture. And it was found direct action of phosphites on the mycelial growth of Fusarium semitectum, Pythium sp. and Sclerotinia sclerotiorum. Based on these results, we concluded that potassium phosphites have potential in seeds treatment, as resistance inducer and on in vitro control of phytopathogens.

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E. coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E. coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Analyses of the community compositions of root rot pathogenic fungi in the soybean rhizosphere soil

Soybean ( Glycine max [L.] Merr.) root rot is an important disease of soybean under continuous cropping, and root rot is widely distributed throughout the world. This disease is extremely harmful, and it is difficult to prevent and control. The study aimed to elucidate the composition of root rot pathogenic fungal communities in the continuous cropping of soybean. In this study, we employed PCRDGGE technology to analyze the communities of root rot pathogenic fungi in soybean rhizosphere soil subjected to continuous cropping during a season with a high incidence of root rot in Heilongjiang province, China, the main soybean producing area in China. The results of 13 DGGE bands were analyzed by phylogenetic revealed that the predominant root rot pathogenic fungi in rhizosphere soil in the test area were Pythium ultimum and Fusarium species. The results of cluster analysis showed that the duration of continuous cropping, the soybean variety and the plant growth stage all had significant effects on the diversity of root rot pathogenic fungi in rhizosphere soil.

Next-generation sequencing of hereditary hemochromatosis-related genes: novel likely pathogenic variants found in the Portuguese population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1GNC)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5′-UTR of HAMP gene(c.-25GNA). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Prevalence and characterization of Salmonella spp. in three shellfish-harvesting areas in France

Shellfish farming is an important economic activity in the Brittany and Normandy regions. However, a part of the production sites corresponds to relatively sensitive areas where the presence of faecal microorganisms is a major concern for shellfish and constitutes a possible health risk. Indeed, shellfish bioaccumulates in their tissues pathogenic contaminants present in water and can cause food-borne diseases such as salmonellosis. During a two-year study, we evaluated the presence of faecal indicators, measured the prevalence of Salmonella spp., isolated and characterized Salmonella spp. from three French shellfish-harvesting areas (shellfish and sediment) and their watersheds (from river water samples).

Candida duobushaemulonii: an emerging rare pathogenic yeast isolated from recurrent vulvovaginal candidiasis in Brazil

The aim of this study was to identify Candida species isolated from women diagnosed with recurrent vulvovaginal candidiasis (RVVC) and their partners; and to evaluate the fluconazole (FLZ) susceptibility of the isolates. In a period of six years, among 172 patients diagnosed with vulvovaginal candidiasis, 13 women that presented RVVC and their partners were selected for this investigation. The isolates were obtained using Chromagar Candida medium, the species identification was performed by phenotypic and molecular methods and FLZ susceptibility was evaluated by E-test. Among 26 strains we identified 14 Candida albicans , six Candida duobushaemulonii, four Candida glabrata , and two Candida tropicalis . Agreement of the isolated species occurred in 100% of the couples. FLZ low susceptibility was observed for all isolates of C. duobushaemulonii (minimal inhibitory concentration values from 8-> 64 μg/mL), two C. glabrata isolates were FLZ-resistant and all C. albicans and C. tropicalis isolates were FLZ-susceptible. This report emphasises the importance of accurate identification of the fungal agents by a reliable molecular technique in RVVC episodes besides the lower antifungal susceptibility profile of this rare pathogen C. duobushaemulonii to FLZ.

Antiadhesive activity of poly-hydroxy butyrate biopolymer from a marine Brevibacterium casei MSI04 against shrimp pathogenic vibrios

Vibrio pathogens are causative agents of mid-culture outbreaks, and early mortality syndrome and secondary aetiology of most dreadful viral outbreaks in shrimp aquaculture. Among the pathogenic vibrios group, Vibrio alginolyticus and V. harveyi are considered as the most significant ones in the grow-out ponds of giant black tiger shrimp Penaeus monodon in India. Use of antibiotics was banned in many countries due to the emergence of antibiotic-resistant strains and accumulation of residual antibiotics in harvested shrimp. There is an urgent need to consider the use of alternative antibiotics for the control of vibriosis in shrimp aquaculture. Biofilm formation is a pathogenic and/or establishment mechanism of Vibrio spp. This study aims to develop novel safe antibiofilm and/ or antiadhesive process using PHB to contain vibrios outbreaks in shrimp aquaculture.

Trichosporon spp.: an emerging fungal pathogen

Trichosporon spp. has gained importance as the cases of immunosuppressed patients increase. The genus Trichosporon includes 6 species of clinical relevance that may cause supericial infections, such as white piedra and onychomycosis, or deep and invasive infections with high mortality rates. These microorganisms have a broad geographical distribution and some species are resistant to antifungal drugs in vitro. The present paper is a review on the virulence factors, associated infections, and in vitro susceptibility of the species with the highest incidence as pathogenic agents in humans.

Ultrastructure of mycoparasitism of Trichoderma koningiopsis 4.24 and effects of its metabolic products on pathogenic fungi.

2009