Flickering of fetal erythrocytes membrane under gestational diabetes observed with dual time resolved membrane fluctuation spectroscopy.

The membrane flickering of human fetal red blood cells (RBCs) affected by gestational diabetes mellitus (GDM) was studied with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of RBCs at two distant membrane points simultaneously. The micro-rheological parameters were obtained from direct membrane flickering measurements, followed by Fourier decomposition and cell membrane model adjustment. Our results show a significant decrease of 6.01 ± 1.19 nm in membrane fluctuations amplitude in healthy fetal, compared with healthy adult RBCs, meanwhile the amplitude in GDM cells increased 3.22 ± 1.10 nm compared with healthy fetal RBCs. Between GDM and healthy fetal RBCs, there are significant differences, especially in the bending modulus. Considering the mean of the two membrane points measured, the tension for GDM RBCs increased by 6.431 ± 3.57 (10-7 [N/m]) compared with healthy fetal RBCs, meanwhile, the bending was increased by 2.483 ± 0.58 (10-19 [J]) in GDM compared with healthy fetal RBCs. These results showed significant increment of 1.23 ± 0.07-fold and 3.29 ± 0.36-fold in tension and bending modulus in GDM, respectively. The strong impact of GDM on bending modulus could be associated with oxidative stress and lipid peroxidation, previously reported in fetal plasma of GDM cases.

Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

Simultaneous and Spatially-Resolved Analysis of T-Lymphocytes, Macrophages and PD-L1 Immune Checkpoint in Rare Cancers.

Penile, vulvar and anal neoplasms show an incidence lower than 0.5% of the population per year and therefore can be considered as rare cancers but with a dramatic impact on quality of life and survival. This work describes the experience of a Chilean cancer center using multiplexed immunofluorescence to study a case series of four penile cancers, two anal cancers and one vulvar cancer and simultaneous detection of CD8, CD68, PD-L1, Cytokeratin and Ki-67 in FFPE samples. Fluorescent image analyses were performed using open sources for automated tissue segmentation and cell phenotyping. Our results showed an objective and reliable counting of objects with a single or combined labeling or within a specific tissue compartment. The variability was below 10%, and the correlation between analytical events was 0.92-0.97. Critical cell phenotypes, such as TILs, PD-L1+ or proliferative tumor cells were detected in a supervised and unsupervised manner with a limit of detection of less than 1% of relative abundance. Finally, the observed diversity and abundance of the different cell phenotypes within the tumor microenvironment for the three studied tumor types confirmed that our methodology is useful and robust to be applicable for many other solid tumors.

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores.

BACKGROUND: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. MATERIALS & METHODS: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. RESULTS: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. CONCLUSION: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin.

Clinical parameters and biomarkers for anti-TNF treatment prognosis in rheumatoid arthritis patients.

Tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). This finding has led to the development of TNF blockers for RA treatment. However, response to these therapies is heterogeneous with success in only two thirds of patient. Some clinical aspects useful in the attempt to predict the response to TNF inhibitors is the promptness and the magnitude of the response at the first weeks and a low basal disease activity, while comorbidities, tobacco, glucocorticoids treatment, and high basal radiological score correlate with a poorer response. The role of TNF promoter polymorphisms in clinical response to anti-TNF therapies is controversial. A correlation between the presence of high baseline titers of rheumatoid factor (RF) and decreased response to anti-TNF treatment has been reported. Most studies show decreased RF titers during anti-TNF treatment mainly in patients who responded to treatment. There is no consensus about the usefulness of basal anti-citrullinated protein antibodies (ACPA) levels, and a decrease in ACPA titers as predictor of clinical response to anti-TNF therapy. Despite some promising markers identified to fulfill this role, currently the predictive value of single markers seems not strong enough to predict treatment response in an individual RA patient.

Real-Time PCR in faecal samples of Triatoma infestans obtained by xenodiagnosis: proposal for an exogenous internal control.

BACKGROUND: The polymerase chain reaction (PCR) has proved to be a sensitive technique to detect Trypanosoma cruzi in the chronic phase of Chagas disease, which is characterized by low and fluctuating parasitemia. Another technique proposed for parasitological diagnosis in this phase of infection combines a microscopic search for motile trypomastigote forms in faecal samples (FS) obtained by xenodiagnosis (XD) with conventional PCR (XD-PCR). In this study we evaluate the use of human blood DNA as an exogenous internal control (EIC) for real time PCR (qPCR) combined with XD (XD-qPCR) using chromosome 12 (X12) detection. FINDINGS: None of the FS-XD evaluated by qPCR amplified for X12. Nevertheless, all the EIC-FS-XD mixtures amplified for X12. CONCLUSIONS: We determined that X12 is useful as an EIC for XD-qPCR because we showed that the FS-XD does not contain human DNA after 30 or more days of XD incubation. This information is relevant for research on T. cruzi by XD-qPCR since it allows ruling out inhibition and false negative results due to DNA loss during the process of extraction and purification.

Linfoma testicular: incidencia, descripción epidemiológica y manejo durante el período 2005-2011 / Testicular lymphoma: incidence, epidemiologic description and management during the 2005-2011 period

El linfoma testicular es una patología infrecuente, correspondiendo al 9 por ciento de los cánceres testiculares, presentándose más frecuentemente entre los 60 a 80 años (25-50 por ciento). La presentación clínica más frecuente es el aumento de volumen unilateral e indo/oro. El tipo histológico más común es linfoma difuso de células grandes B (60-90 por ciento). La orquidectomía radical asociada a quimioterapia y radioterapia es la primera línea de tratamiento para los pacientes con enfermedad limitada. Material y método: Estudio retrospectivo descriptivo. Se revisó y filtró la lista de pacientes ingresados al SIGGES como tumor testicular entre enero 2005 a abril 2011. De los pacientes con diagnóstico histológico e inmunohistoquímico compatible, se registraron las características epidemiológicas, estudio, manejo y sobrevida. Posteriormente se realizó un análisis de la base de datos con el programa estadístico SPSS 13. 0. Resultados: De un total de 299 pacientes con el diagnóstico histológico de cáncer testicular, 8 pacientes fueron diagnosticados como linfoma testicular confirmado por histología e inmunohistoquímica. El promedio y mediana de edad fue 52 años y 63 años (18-73) respectivamente. Tres casos (37,5 por ciento) correspondieron a presentaciones secundarias. En 6 de los casos (75 por ciento) el testículo afectado fue el derecho. Histológicamente, el 63 por ciento correspondió a Linfoma difuso de células grande B. Clínicamente, el todos los casos se presentaron con aumento de volumen y con marcadores en rango normal. En 7 casos (8 7, 5 por ciento) el diagnóstico y manejo inicial fue mediante orquidectomía radical, y en un caso por biopsia testicular, con orquidectomía posterior 3 casos presentaron diseminación...

esticular lymphoma is a rare disease, happening in 9 percent of testicular cancers, most commonly between the ages 60 to 80 years (25 percent-50 percent). The most common presentation is unilateral indolent testicular growth. Histology shows a diffuse big B cell lymphoma in most of the cases (60 percent-90 percent). Radical orchiectomy, chemotherapy and radiation are the first line therapy for patients with limited disease. Materials and methods: Retrospective clinical study. We included and filtered the SIGGES list of patients admitted for Testicular Tumor from January 2005 to April 2011. Patients with a compatible diagnosis were analyzed, using SPSS 13.0® as statistical software. Result: Of a total number of 299 testicular cancer patients 8 presented with a histological and inmunnohistochemical testicular lymphoma. Mean age was 52 years and the median 63 years (18-73). ln three cases (37.5 percent) it was a secondary localization. ln 6 cases ( 75 percent) the affected testicle was the right one. 63 percent corresponded to a diffuse big cell B cell Lymphoma. All patients presented normal tumor markers. ln 7 (87,5 percent) cases the initial treatment was radical orchiectomy in one patient the diagnosis was don through a testicular biopsy, and the orchidectomy was differed. 3 cases presented dissemination. In 7 patients adjuvant chemotherapy was performed. Mortal/ty was 38 percent with a 1 7-month follow-up. Conclusion: Testicular lymphoma is a rare condition with bad prognosis. Histology is fundamental for treatment, an in this sense inmunohystochemcal analysis is especially helpful...

Relación entre el efecto Mozart y la prueba de solución de problemas misioneros y caníbales / Relationship between Mozart effect and the Missionaries and Cannibals problem solving task

La presente investigación exploró la relación que existe entre el efecto Mozart y la prueba de solucion de problemas "Misioneros y caníbales" en estudiantes de género femenino cuyas edades oscilaron entre los 17 y 20 años de edad