RESUMEN

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status.

Asunto(s)

Dermatitis Atópica , Humanos , Dermatitis Atópica/microbiología , Staphylococcus aureus/genética , ARN Ribosómico 16S/genética , Epidermis/microbiología , Piel/microbiología

RESUMEN

A 3-month-old female French Bulldog presented with hematuria, severe pollakiuria, and urinary incontinence lasting for 1.5 months. Broad-spectrum empirical antibiotic therapy and nonsteroidal anti-inflammatory drugs were initiated by the referring veterinarian. Due to a lack of improvement, the dog was referred. At referral examination, urinary clinical signs persisted (hematuria, severe pollakiuria) and a firm bladder was noted. Abdominal ultrasonography revealed severe, diffuse bladder wall thickening with a significant reduction in the bladder lumen. Urinary tract endoscopy showed whitish exophytic proliferations throughout the entire bladder wall. Histological bladder wall analysis led to a diagnosis of bladder malakoplakia. Prolonged antibiotic therapy with fluoroquinolones was prescribed and resulted in clinical remission despite persistent bacteria in the bladder wall. This report describes a case of successfully medically managed bladder malakoplakia, a very rare condition in veterinary medicine, well documented in humans.

Asunto(s)

Cistitis , Enfermedades de los Perros , Malacoplasia , Humanos , Perros , Femenino , Animales , Vejiga Urinaria/diagnóstico por imagen , Vejiga Urinaria/patología , Hematuria/tratamiento farmacológico , Hematuria/patología , Hematuria/veterinaria , Malacoplasia/diagnóstico , Malacoplasia/tratamiento farmacológico , Malacoplasia/veterinaria , Cistitis/diagnóstico , Cistitis/tratamiento farmacológico , Cistitis/veterinaria , Antibacterianos/uso terapéutico , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/patología

RESUMEN

The present study describes a new species of myxosporean, Auerbachia ignobili n. sp., infecting the hepatic bile ducts of Caranx ignobilis (Forsskål, 1775). Myxospores are club-shaped with a broad anterior region and a narrow, slightly curved and blunt caudal extension, measuring 17.4 ± 1.5 µm in length and 7.5 ± 7.4 µm in width. Shell valves asymmetrical, with a faint suture line, and enclosed a single, elongate-elliptical polar capsule with a ribbon-like polar filament, arranged in 5-6 coils. Developmental stages included early and late presporogonic stages, pansporoblast, and sporogonic stages with monosporic and disporic plasmodia. A. ignobili n. sp. differs from the other described species of Auerbachia in the shape and dimensions of the myxospores and polar capsules. The molecular analysis generated ∼1400 bp long SSU rDNA sequences and the present species exhibited a maximum similarity 94.04-94.91% with A. chakravartyi. Genetic distance analysis indicated the lowest interspecies divergence of 4.4% with A. chakravartyi. In phylogenetic analysis, A. ignobili n. sp. was positioned independently with a high bootstrap value (1/100) and appeared as sister to A. maamouni and A. chakravartyi. Fluorescent in situ hybridization and histology indicates that the parasite develops within the hepatic bile ducts. Histological studies did not reveal any pathological changes. Considering the morphological, morphometric, molecular, and phylogenetic differences coupled with the differences in host and geographic locations, the present myxosporean is treated as a new species and named A. ignobili n. sp.

Asunto(s)

Cnidarios , Enfermedades de los Peces , Myxozoa , Enfermedades Parasitarias en Animales , Animales , Myxozoa/genética , Cnidarios/genética , Filogenia , Hibridación Fluorescente in Situ , Enfermedades Parasitarias en Animales/parasitología , Peces , Enfermedades de los Peces/parasitología , ADN Ribosómico/genética

RESUMEN

Human epidermal growth factor receptor 2 (HER2) is a prognostic biomarker and therapeutic target in carcinomas of the breast, stomach and colon. In 2018, clinical trial data confirmed the prognostic and predictive role of HER2 in uterine serous carcinoma, with a demonstrated survival benefit from combined chemotherapy and anti-HER2 targeted therapy in patients with advanced or recurrent disease. Approximately one-third of uterine serous carcinomas demonstrate HER2 protein overexpression and/or gene amplification and HER2 immunohistochemistry, supplemented by in situ hybridisation in equivocal cases, is fast becoming a reflex ancillary test at time of diagnosis. The potential role of HER2 in gynaecological tumours other than uterine serous carcinoma is yet to be firmly established. With the advent of personalised medicine, routine tumour sequencing and pursuit of targeted therapies, this is a field currently under active investigation. Emerging data suggest triaging endometrial carcinomas for HER2 analysis based on molecular classification may be superior to histotype-based testing, with copy-number high/p53 mutant tumours enriched for HER2 overexpression or amplification. Accordingly, many carcinosarcomas and a subset of clear cell and high-grade endometrioid carcinomas may be eligible for HER2 targeted therapy, although any clinical benefit in this context is currently undefined. For ovarian carcinomas, combined data support the role of HER2 as a prognostic biomarker, however its use as a therapeutic target is yet to be elucidated through clinical trials. In the cervix, reported rates of HER2 overexpression vary and are generally low, and currently there is insufficient evidence to justify routine HER2 testing in this context. Limited data suggest HER2 holds promise as a prognostic and predictive biomarker in vulvar Paget disease. Future clinical trials, with pathologist input to develop and refine site-specific scoring criteria, are required to establish what role HER2 might play more broadly in gynaecological cancer care.

Asunto(s)

Adenocarcinoma Mucinoso , Biomarcadores de Tumor , Neoplasias Endometriales , Terapia Molecular Dirigida , Receptor ErbB-2 , Femenino , Humanos , Biomarcadores de Tumor/metabolismo , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/terapia , Receptor ErbB-2/metabolismo , Adenocarcinoma Mucinoso/tratamiento farmacológico , Adenocarcinoma Mucinoso/metabolismo

RESUMEN

Background: Turner syndrome (TS) is the most common chromosomal abnormality in females. The diagnosis of TS is based on karyotyping of 30 blood lymphocytes. This technique does not rule out tissue mosaicism or low-grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome. Aims: This study was set to determine the value of additional genetic studies such as fluorescent in situ hybridisation and the inclusion of buccal cells in search for mosaicism in TS patients. Settings and Design: This cross-sectional, descriptive study was performed in Human Genetics Department, Medical Research Institute, Alexandria University. Materials and Methods: Fluorescence in situ hybridisation technique was applied to lymphocyte cultures as well as buccal smears using centromeric probes for X and Y chromosomes. Genotype phenotype correlation was also evaluated. Statistical Analysis Used: Descriptive study where categorical variables were described using number and percentage and continuous variables were described using mean and standard deviation. Results: Fluorescence in situ hybridisation technique study detected hidden mosaicism in 60% of studied patients; 20% of patients had a cell line containing Y material, while 40% had variable degrees of X, XX mosaicism, and in the remaining 40% no second cell line was detected. Fluorescence in situ hybridisation study helped identify the origin of the marker to be Y in all patients. The introduction of an additional cell line helped in identifying mosaicism in patients with monosomy X. Virilisation signs were only observed among TS patients with Y cell line mosaicism. The clinical manifestations were more severe in patients with monosomy X than other mosaic cases. Conclusions: Molecular cytogenetic investigation for all suspected cases of TS should be considered for appropriate treatment plan and genetic counselling.

RESUMEN

Glioblastomas are frequent malignant brain tumours with a very poor prognosis and a need for new and efficient therapeutic strategies. With the approval of anti-TRK targeted therapies to treat patients with advanced NTRK-rearranged cancers, independent of the type of cancer, potential new treatment opportunities are available for the 0.5-5% of patients with NTRK-rearranged glioblastomas. Identification of these rare NTRK-rearranged glioblastomas requires efficient diagnostic tools and strategies which are evaluated in this study. We compared the results of NTRK1, NTRK2 and NTRK3 fluorescent in situ hybridisation (FISH) assays to those of pan-TRK immunohistochemistry (IHC) using two EPR17341 and A7H6R clones in a set of 196 patients with glioblastomas. Cases with at least 15% of positive nuclei using FISH analyses were further analysed using RNA sequencing. Above the 15% threshold, seven positive glioblastomas (3.57%) were identified by FISH assays (4 NTRK1, 3 NTRK2, no NTRK3). NTRK rearrangements were confirmed by RNA sequencing analyses in four cases [1 LMNA-NTRK1, 1 PRKAR2A-NTRK2, 1 SPECC1L-NTRK2 and 1 NACC2-NTRK2 fusions, i.e., 4/196 (2%) of NTRK-rearranged tumours in our series] but no rearrangement was detected in three samples with less than 30% of positive tumour nuclei as determined by NTRK1 FISH. Pan-TRK immunostaining showed major discrepancies when using either the EPR17341 or the A7H6R clones for the following criteria: main intensity, H-Score based scoring and homogeneity/heterogeneity of staining (Kappa values <0.2). This led to defining adequate criteria to identify NTRK-rearranged gliomas exhibiting strong and diffuse immunostaining contrasting to the variable and heterogeneous staining in non-NTRK-rearranged gliomas (p<0.0001). As assessing NTRK rearrangements has become crucial for glioma therapy, FISH seems to be a valuable tool to maximise access to TRK testing in patients with glioblastomas. In contrast to other cancers, pan-TRK IHC appears of limited interest in this field because there is no 'on/off' IHC positivity criterion to distinguish between NTRK-rearranged and non-NTRK-rearranged gliomas. RNA sequencing analyses are necessary in FISH positive cases with less than 30% positive nuclei, to avoid false positivity when scoring is close to the detection threshold.

Asunto(s)

Glioblastoma , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas Tirosina Quinasas Receptoras , Análisis de Secuencia de ARN , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Femenino , Reordenamiento Génico , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/terapia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor trkA/análisis , Receptor trkA/genética , Receptor trkC/análisis , Receptor trkC/genética , Adulto Joven

RESUMEN

Codeletion of chromosomal arms 1p and 19q, in conjunction with a mutation in the isocitrate dehydrogenase 1 or 2 gene, is the molecular diagnostic criterion for oligodendroglioma, IDH mutant and 1p/19q codeleted. 1p/19q codeletion is a diagnostic marker and allows prognostication and prediction of the best drug response within IDH-mutant tumours. We performed a Cochrane review and simple economic analysis to establish the most sensitive, specific and cost-effective techniques for determining 1p/19q codeletion status. Fluorescent in situ hybridisation (FISH) and polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) test methods were considered as reference standard. Most techniques (FISH, chromogenic in situ hybridisation [CISH], PCR, real-time PCR, multiplex ligation-dependent probe amplification [MLPA], single nucleotide polymorphism [SNP] array, comparative genomic hybridisation [CGH], array CGH, next-generation sequencing [NGS], mass spectrometry and NanoString) showed good sensitivity (few false negatives) for detection of 1p/19q codeletions in glioma, irrespective of whether FISH or PCR-based LOH was used as the reference standard. Both NGS and SNP array had a high specificity (fewer false positives) for 1p/19q codeletion when considered against FISH as the reference standard. Our findings suggest that G banding is not a suitable test for 1p/19q analysis. Within these limits, considering cost per diagnosis and using FISH as a reference, MLPA was marginally more cost-effective than other tests, although these economic analyses were limited by the range of available parameters, time horizon and data from multiple healthcare organisations.

Asunto(s)

Neoplasias Encefálicas , Glioma , Oligodendroglioma , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Aberraciones Cromosómicas , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 19/genética , Glioma/diagnóstico , Glioma/genética , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Mutación , Oligodendroglioma/diagnóstico , Oligodendroglioma/genética , Oligodendroglioma/patología

RESUMEN

Nuclear ribosomal DNA (nrDNA) has displayed extraordinary dynamics during the evolution of plant species. However, the patterns and evolutionary significance of nrDNA array expansion or contraction are still relatively unknown. Moreover, only little is known of the fate of minority nrDNA copies acquired between species via horizontal transfer. The barley genus Hordeum (Poaceae) represents a good model for such a study, as species of section Stenostachys acquired nrDNA via horizontal transfer from at least five different panicoid genera, causing long-term co-existence of native (Hordeum-like) and non-native (panicoid) nrDNAs. Using quantitative PCR, we investigated copy number variation (CNV) of nrDNA in the diploid representatives of the genus Hordeum. We estimated the copy number of the foreign, as well as of the native ITS types (ribotypes), and followed the pattern of their CNV in relation to the genus' phylogeny, species' genomes size and the number of nrDNA loci. For the native ribotype, we encountered an almost 19-fold variation in the mean copy number among the taxa analysed, ranging from 1689 copies (per 2C content) in H. patagonicum subsp. mustersii to 31342 copies in H. murinum subsp. glaucum. The copy numbers did not correlate with any of the genus' phylogeny, the species' genome size or the number of nrDNA loci. The CNV was high within the recognised groups (up to 13.2 × in the American I-genome species) as well as between accessions of the same species (up to 4×). Foreign ribotypes represent only a small fraction of the total number of nrDNA copies. Their copy numbers ranged from single units to tens and rarely hundreds of copies. They amounted, on average, to between 0.1% (Setaria ribotype) and 1.9% (Euclasta ribotype) of total nrDNA. None of the foreign ribotypes showed significant differences with respect to phylogenetic groups recognised within the sect. Stenostachys. Overall, no correlation was found between copy numbers of native and foreign nrDNAs suggesting the sequestration and independent evolution of native and non-native nrDNA arrays. Therefore, foreign nrDNA in Hordeum likely poses a dead-end by-product of horizontal gene transfer events.

RESUMEN

BACKGROUND: The role of the gut microbiota in health and disease is becoming increasingly apparent. Faeces is the most accessible sample to collect from human volunteers for studying the gut microbiota. However, the impact of stool collection and storage conditions on microbial and metabolic profiles have not been fully evaluated. By understanding the effect of different stool collection and storage conditions on microbial and metabolic composition, we can consider these parameters in the design of in vitro fermentation studies. METHODS: Stool samples from 3 volunteers were stored under 5 different conditions to mimic methods that researchers may use to collect and store stool samples for study of the gut microbiota, including: fresh sample used within 10 min; stored on wet ice (4 °C) for 60 min; stored in an anaerobic chamber in a temperature-controlled bag (4 °C) for 60 min; freezing at -20 °C for 60 min and freezing at -20 °C for 60 min and then at -80 °C for 2 weeks. The stored samples were added to basal medium in batch culture fermenters alone (negative control) or with 5 g 2'-Fucosyllactose (2'FL) Human Milk Oligosaccharide (HMO) (as a positive fermentation control). Samples were collected at 3 timepoints (0, 12 and 24 h) for analysis by Flow Cytometry-Fluorescent In Situ Hybridisation (FC-FISH) and 1H-Nuclear Magnetic Resonance (NMR) spectroscopy to assess the impact on microbial and metabolic profiles, respectively. RESULTS: Freezing stool significantly impacted microbial numbers and activity during in vitro fermentations, whereas storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min had minimal effects on microbial and metabolic profiles throughout the 24 h batch culture fermentation experiments. DISCUSSION: For in vitro batch culture fermentation studies where it may not be practical or possible to use fresh stool, either storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min could be plausible alternatives to maintain microbial and metabolic profiles for analysis.

Asunto(s)

Heces/microbiología , Microbioma Gastrointestinal , Manejo de Especímenes/métodos , Adulto , Técnicas de Cultivo Celular por Lotes/métodos , Femenino , Fermentación , Citometría de Flujo/métodos , Congelación , Humanos , Hibridación Fluorescente in Situ/métodos , Espectroscopía de Resonancia Magnética/métodos , Metaboloma , Leche Humana , Temperatura

RESUMEN

OBJECTIVES: To evaluate human epidermal growth factor receptor 2 (HER2) protein overexpression by immunohistochemistry (IHC) and gene amplification by fluorescent in situ hybridisation (FISH) in urothelial non-muscle-invasive bladder carcinoma (NMIBC), as HER2 is a potential therapeutic target in muscle-invasive bladder carcinoma (MIBC) and HER2 expression and gene amplification in low/high-grade and pTa/pT1 NMIBC is not clear. PATIENTS AND METHODS: The study included 93 bladder cancers; 25 MIBC and 68 NMIBC (37 low- and 31 high-grade). All HER2 positive (3+) and equivocal (2+) cases were subjected to FISH using a HER2/CEN 17 dual-colour probe kit. IHC and FISH were scored as per the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2013 Guidelines for breast cancers. Based on the number of signals/nuclei, amplification was categorised as low (≥6-10) and high-level (≥10). RESULTS: HER2 2-3+ expression was seen in 29% of NMIBCs (10.8% low- and 51.6% high-grade). HER2 3+ expression was seen in high-grade NMIBC (nine of 31; 29%) and MIBC (nine of 25; 36%). In all, 87% of high-grade NMIBCs were lamina invasive (pT1). Gene amplification was found in 45% (eight of 18) of 3+ tumours. None of the HER2 2+ tumours showed gene amplification. IHC and FISH results were in closest agreement when ≥50% of tumour cells showed 3+ expressions. High-level amplification correlated with increased gene expression on reverse transcriptase-polymerase chain reaction. On multivariate analysis, lower  stage, grade, and HER2 expression significantly correlated with progression-free survival. HER2 3+ expression in NMIBC correlated significantly with time to recurrence and progression. CONCLUSION: Our present results show that HER2 FISH should not be performed for HER2 2 + and low-grade NMIBC. This contrasts with breast cancers where it is recommended for equivocal 2+ tumours. About 50% of HER2 3+ MIBC and high-grade NMIBC show HER2 gene amplification and can be potential candidates for HER2-targeted therapy. ABBREVIATIONS: ASCO/CAP: American Society of Clinical Oncology/College of American Pathologists; DAB: 3,3'-diaminobenzidine; FISH: fluorescent in situ hybridisation; HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry;(N)MIBC: (non-) muscle-invasive bladder carcinoma; MPUC: micropapillary variant of urothelial bladder cancer; PFS: progression-free survival; TURBT: transurethral resection of bladder tumour.

RESUMEN

JUSTIFICATION: The prevalence of gastric cancer (GC) with increased expression of the HER2 oncoprotein shows important variations worldwide. Incidence and mortality rates of GC in Costa Rica are among the highest in Latin America and the world; however, the prevalence of HER2-positive cases in this country is unknown. Evaluation of this parameter is important to decide the therapeutic approach for GC patients. The aim of this study was to provide an estimation of the prevalence of GC patients overexpressing the HER2 oncogene in Costa Rica. METHODS: The investigation was carried out in two phases. The first one consisted of a retrospective review of 331 clinical records of patients diagnosed with advanced or metastatic GC from January 2010 to January 2012 in four hospitals in Costa Rica. In the second phase, immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) analyses were performed in formalin-fixed and paraffin-embedded (FFPE) surgical samples from 50 patients diagnosed with GC between 2012 and 2015. RESULTS: Of the 331 clinical files reviewed, the assessment of HER2 status was carried out in 62 patients (18.7%), of which only five (8%) were HER2-positive. In the 50 surgical specimens in which IHC and FISH analyses were performed, two of them (4%) presented overexpression and amplification of the HER2 oncogene. CONCLUSION: This study suggests that the prevalence of GC cases overexpressing the HER2 oncogene in Costa Rica is less than 8%. This is the first attempt ever undertaken to estimate the prevalence of HER2-positivity in GC in Costa Rica.

RESUMEN

Probiotic administration may favour caries prevention, as recent research has shown. This in vitro study aimed to investigate the growth of Lactobacillus rhamnosus GG (LGG) in experimental biofilms exposed to various carbohydrates, and also to assess its cariogenic potential. Multispecies experimental oral biofilms with or without LGG were grown with a sole-carbohydrate source (fructose/glucose/lactose/sorbitol/sucrose). The viable cells of LGG and structure of the biofilms were examined after 64.5 h of incubation, and pH values of spent media were measured at 16.5, 40.5, and 64.5 h. Fermentation profiles of LGG in biofilm media were assessed with study carbohydrate as the sole energy source. Our results showed that LGG reached higher viable cell numbers with glucose and sucrose in 64.5-h multispecies experimental oral biofilms compared to other carbohydrates. When LGG was incorporated in biofilms, no distinct pH changes at any time points were observed under any of the carbohydrates used; the pH values of spent media at each time point were lower when lactose was used, compared to other carbohydrates. The fermentation profiles of LGG in biofilm media were similar to its growth in MRS (no obvious growth with lactose or sucrose). In conclusion, LGG in our in vitro multispecies experimental oral biofilms was capable of surviving and growing well in each carbohydrate source. LGG might not have harmful effects on dental hard tissues. Another finding from our study was that the lowest pH values were observed in the presence of lactose, and the thickest biofilms were in sucrose.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Carbohidratos/farmacología , Lacticaseibacillus rhamnosus/crecimiento & desarrollo , Adulto , Carga Bacteriana , Biopelículas/efectos de los fármacos , Medios de Cultivo , Humanos , Concentración de Iones de Hidrógeno , Hibridación Fluorescente in Situ , Técnicas In Vitro , Lacticaseibacillus rhamnosus/efectos de los fármacos , Masculino , Saliva/metabolismo

RESUMEN

Infectious diarrhoea is a worldwide problem in newborns. Optimal bacterial colonisation may enhance gut maturation and protect against pathogenic bacteria after birth. We hypothesised that lactic acid bacteria (LAB) administration prevents pathogen-induced diarrhoea in formula-fed newborns. Newborn caesarean-delivered, colostrum-deprived term piglets on parenteral nutrition for the first 15 h, were used as models for sensitive newborn infants. A commercially available probiotic strain, Lactobacillus paracasei F19 (LAP, 2·6×108 colony-forming units (CFU)/kg per d) and a novel LAB isolate, Pediococcus pentosaceus (PEP, 1·3×1010 CFU/kg per d), were administered for 5 d with or without inoculation of the porcine pathogen, Escherichia coli F18 (F18, 1010 CFU/d). This resulted in six treatment groups: Controls (n 9), LAP (n 10), PEP (n 10), F18 (n 10), F18-LAP (n 10) and F18-PEP (n 10). The pathogen challenge increased diarrhoea and density of F18 in the intestinal mucosa (P<0·05). LAB supplementation further increased the diarrhoea score, relative to F18 alone (P<0·01). Intestinal structure and permeability were similar among groups, whereas brush border enzymes were affected in variable intestinal regions with decreased activities in most cases after F18 and LAB inoculation. Bacterial density in colon mucosa increased after F18 inoculation (P<0·05) but was unaffected by LAB supplementation. In colon contents, acetic and butyric acids were increased by PEP (P<0·05). The LAB used in this study failed to reduce E. coli-induced diarrhoea in sensitive newborn pigs. In vulnerable newborns there may be a delicate balance among bacterial composition and load, diet and the host. Caution may be required when administering LAB to compromised newborns suffering from enteric infections.

Asunto(s)

Animales Recién Nacidos/microbiología , Diarrea/veterinaria , Infecciones por Escherichia coli/veterinaria , Lacticaseibacillus paracasei , Pediococcus pentosaceus , Enfermedades de los Porcinos/microbiología , Ácido Acético/análisis , Animales , Ácido Butírico/análisis , Colon/química , Colon/microbiología , Recuento de Colonia Microbiana , Diarrea/microbiología , Diarrea/prevención & control , Dieta/veterinaria , Suplementos Dietéticos , Modelos Animales de Enfermedad , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/complicaciones , Mucosa Intestinal/microbiología , Intestinos/microbiología , Probióticos/uso terapéutico , Sus scrofa , Porcinos

RESUMEN

Increasing evidence within the literature has identified the presence of biofilms in chronic wounds and proposed that they contribute to delayed wound healing. This research aimed to investigate the presence of biofilm in diabetic foot ulcers (DFUs) using microscopy and molecular approaches and define if these are predominantly mono- or multi-species. Secondary objectives were to correlate wound observations against microscopy results in ascertaining if clinical cues are useful in detecting wound biofilm. DFU tissue specimens were obtained from 65 subjects. Scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridisation (PNA-FISH) techniques with confocal laser scanning microscopy (CLSM) were used to visualise biofilm structures. Next-generation DNA sequencing was performed to explore the microbial diversity. Clinical cues that included the presence of slough, excessive exudate, a gel material on the wound bed that reforms quickly following debridement, poor granulation and pyocyanin were correlated to microscopy results. Of the 65 DFU specimens evaluated by microscopy, all were characterised as containing biofilm (100%, P < 0·001). The presence of both mono-species and multi-species biofilms within the same tissue sections were detected, even when DNA sequencing analysis of DFU specimens revealed diverse polymicrobial communities. No clinical correlations were identified to aid clinicians in identifying wound biofilm. Microscopy visualisation, when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and form either mono- or multi-species biofilms. Clinical cues to aid clinicians in detecting wound biofilm are not accurate for use in DFUs. A paradigm shift of managing DFUs needs to consider anti-biofilm strategies.

Asunto(s)

Biopelículas , Pie Diabético/microbiología , Pie Diabético/patología , Anciano , Pie Diabético/diagnóstico por imagen , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Microscopía Confocal , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Ácidos Nucleicos de Péptidos , Estudios Prospectivos

RESUMEN

AIM: This study compared the faecal microbial composition of formula-fed infants who did and did not have colic. METHODS: Faecal samples from formula-fed infants under 16 weeks of age with (n = 38) and without (n = 39) colic were collected at Department of Pediatrics in Turin, Italy, between February 2014 and October 2015. The pH and faecal ammonia were determined and total bacteria, bifidobacteria, lactic acid bacteria and coliforms were quantified by fluorescent in situ hybridisation (FISH). RESULTS: Faecal ammonia was significantly higher in the colicky infants than in the controls (483 vs. 216 µg/g, p < 0.05). The FISH counts of total bacteria were lower in colicky infants (1.8E10 ± 1.5E10) than in the controls (3.4E10 ± 3.0E10) (p < 0.05). The relative abundance of coliform bacteria was significantly higher in colicky infants (p < 0.05). No differences were observed for the bifidobacteria and lactic acid bacteria counts between the two groups. CONCLUSION: Our comparison of formula-fed infants with and without colic revealed significant differences in total bacteria, Enterobacteriaceae and faecal ammonia. This study provides the stimulus for further studies of the gut microbiome, using new methods of analysis such as 16S metagenomics sequencing in order to lead to more tailored dietary approaches.

Asunto(s)

Cólico/microbiología , Amoníaco/análisis , Bifidobacterium/aislamiento & purificación , Estudios de Casos y Controles , Estudios Transversales , Enterobacteriaceae/aislamiento & purificación , Heces/microbiología , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Recién Nacido , Ácido Láctico/metabolismo , Masculino

RESUMEN

BACKGROUND: Killian-Pallister syndrome (KPS) is a rare form of chromosomal mosaicism and is defined by the existence of an extra chromosome 12 in some cell lines in one individual. The degree of mosaicism varies among tissues and dictates the clinical presentation of the syndrome. The clinical features of Killian-Pallister syndrome include mental retardation, typical facial dysmorphism and pigmentation defects. CASE PRESENTATION: We present a rare case of Killian-Pallister syndrome with severe form of the disease associated with isolated growth hormone deficiency and low-rate mosaicism on buccal smear. The absence of a marker chromosome 12p in lymphocyte cultures and the low degree of mosaicism lead to frequent misdiagnosis of this condition. CONCLUSIONS: The selection of tissue sampling is crucial in establishing the diagnosis of Killian-Pallister syndrome. Fluorescent in situ hybridisation on buccal smear remains the golden standard as a screening method if a suspicion of the syndrome exists.

RESUMEN

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR-, C- and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the 'Paranense Sea', probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.

Asunto(s)

Cromosomas de las Plantas/genética , Evolución Molecular , Cariotipo , Solanaceae/genética , Teorema de Bayes , Bandeo Cromosómico , Heterocromatina/genética , Hibridación Fluorescente in Situ , Cariotipificación , México , Océanos y Mares , Filogenia , América del Sur

RESUMEN

This placebo-controlled, randomised, double-blind, cross-over human feeding study aimed to determine the prebiotic effect of agave fructans. A total of thirty-eight volunteers completed this trial. The treatment consisted of 3 weeks' supplementation with 5 g/d of prebiotic agave fructan (Predilife) or equivalent placebo (maltodextrin), followed by a 2-week washout period following which subjects were crossed over to alternate the treatment arm for 3 weeks followed by a 2-week washout. Faecal samples were collected at baseline, on the last day of treatment (days 22 and 58) and washout (days 36 and 72), respectively. Changes in faecal bacterial populations, SCFA and secretory IgA were assessed using fluorescent in situ hybridisation, GC and ELISA, respectively. Bowel movements, stool consistencies, abdominal comfort and mood changes were evaluated by a recorded daily questionnaire. In parallel, the effect of agave fructans on different regions of the colon using a three-stage continuous culture simulator was studied. Predilife significantly increased faecal bifidobacteria (log10 9·6 (sd 0·4)) and lactobacilli (log10 7·7 (sd 0·8)) compared with placebo (log10 9·2 (sd 0·4); P = 0·00) (log10 7·4 (sd 0·7); P = 0·000), respectively. No change was observed for other bacterial groups tested, SCFA, secretory IgA, and PGE2 concentrations between the treatment and placebo. Denaturing gradient gel electrophoresis analysis indicated that bacterial communities were randomly dispersed and no significant differences were observed between Predilife and placebo treatments. The in vitro models showed similar increases in bifidobacterial and lactobacilli populations to that observed with the in vivo trial. To conclude, agave fructans are well tolerated in healthy human subjects and increased bifidobacteria and lactobacilli numbers in vitro and in vivo but did not influence other products of fermentation.

RESUMEN

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

Asunto(s)

Células Epiteliales/química , Genética Forense/métodos , Hibridación Fluorescente in Situ/métodos , Captura por Microdisección con Láser/métodos , Repeticiones de Microsatélite , Análisis para Determinación del Sexo/métodos , Delitos Sexuales/legislación & jurisprudencia , ADN/análisis , ADN/genética , Células Epiteliales/citología , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodos

RESUMEN

The human oro-gastrointestinal (GI) tract is a complex system, consisting of oral cavity, pharynx, oesophagus, stomach, small intestine, large intestine, rectum and anus, which all together with the accessory digestive organs constitute the digestive system. The function of the digestive system is to break down dietary constituents into small molecules and then absorb these for subsequent distribution throughout the body. Besides digestion and carbohydrate metabolism, the indigenous microbiota has an important influence on host physiological, nutritional and immunological processes, and commensal bacteria are able to modulate the expression of host genes that regulate diverse and fundamental physiological functions. The main external factors that can affect the composition of the microbial community in generally healthy adults include major dietary changes and antibiotic therapy. Changes in some selected bacterial groups have been observed due to controlled changes to the normal diet e.g. high-protein diet, high-fat diet, prebiotics, probiotics and polyphenols. More specifically, changes in the type and quantity of non-digestible carbohydrates in the human diet influence both the metabolic products formed in the lower regions of the GI tract and the bacterial populations detected in faeces. The interactions between dietary factors, gut microbiota and host metabolism are increasingly demonstrated to be important for maintaining homeostasis and health. Therefore the aim of this review is to summarise the effect of diet, and especially dietary interventions, on the human gut microbiota. Furthermore, the most important confounding factors (methodologies used and intrinsic human factors) in relation to gut microbiota analyses are elucidated.

Asunto(s)

Dieta , Tracto Gastrointestinal/microbiología , Microbiota/fisiología , Adulto , Heces/microbiología , Tracto Gastrointestinal/metabolismo , Humanos , Fenómenos Fisiológicos de la Nutrición