STK4 deficiency and epidermodysplasia verruciformis-like lesions: A case report.

Serine/threonine kinase 4 deficiency (STK4 or MST1, OMIM:614868) is an autosomal recessive (AR) combined immunodeficiency that can present with skin lesions such as epidermodysplasia verruciformis-like lesions (EVLL). Herein, we describe a 17-year-old male patient born from consanguineous parents presenting with recurrent respiratory infections, verruciform plaques, poikiloderma, chronic benign lymphoproliferation, and Sjögren syndrome with suspected interstitial lymphocytic pneumonia.

Both subscales of the Keratoconus End-Points Assessment Questionnaire have excellent test-retest reliability.

Purpose: The keratoconus end-points assessment questionnaire (KEPAQ) is a disease-specific scale designed to evaluate the quality of life in keratoconus patients and provides the measurement of both functional and emotional compromise in keratoconus. It was previously developed, tested, and validated and now we want to evaluate the test-retest reliability of the KEPAQ, in an effort to contribute evidence on its internal consistency and capability of measuring clinical state with minimal inference of random chance. Methods: This is a prospective analytical study, designed to evaluate the test-retest reliability of the KEPAQ through the repeated application of the questionnaire to a group of clinically stable individuals. A number of patients with a confirmed diagnosis of keratoconus underwent double application of the KEPAQ, seven days apart. Mean KEPAQ score was obtained through Rasch analysis, while test-retest reliability was evaluated through Spearman rank-order correlation and intraclass correlation coefficient. Rasch analysis was performed in JMetrik version 4.1.1 (Psychomeasurement Systems LLC; Charlottesville, VA, USA) in a MacBook Air computer running macOS Catalina version 10.15.2 (Apple Inc.; Cupertino, CA, USA). Results: A total of 100 patients were included. For KEPAQ-E, Spearman correlation was R = 0.963 while ICC was 0.981 (95% confidence interval 0.972-0.987). For KEPAQ-F, Spearman correlation was R = 0.921 while ICC was 0.952 (95% confidence interval 0.929-0.968). Conclusion: The KEPAQ is a robust, well-developed, extremely reliable scale which can be confidently used for clinical and research endeavors.

Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration.

The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi.

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection.

BACKGROUND: Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. METHOD & MATERIAL: Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. RESULTS & DISCUSSION: For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.

Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency.

While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte's ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.

PURIFICACIÓN DEL ANTÍGENO 38 kDa DE Mycobacterium tuberculosis Y SU POTENCIAL USO EN DIAGNÓSTICO MEDIANTE INMUNOSENSORES PIEZOELÉCTRICOS / Mycobacterium tuberculosis 38 kDa Antigen Purification and Potential Diagnostic Use by Piezoelectric Immunosensors

Un paso crucial en el desarrollo de un inmunosensor piezoeléctrico para la detección de tuberculosis (TB), es la selección y obtención de los inmunoreactivos empleados en el inmunoensayo y la estrategia para la biofuncionalización del transductor. Diversos estudios han reportado el uso del antígeno proteico 38kDa (Ag38kDa) de Mycobacterium tuberculosis (Mtb) como un buen biomarcador de la enfermedad y el cumplimiento de las características físicas y bioquímicas para ser inmovilizado por monocapas autoensambladas (SAMs), en la superficie del electrodo de oro de cristales piezoeléctricos. Un inmunosensor piezoeléctrico desarrollado a partir de un antígeno nativo purificado de Mtb podría ser un método alternativo simple para la detección de Mtb con ventajas de rapidez y reusabilidad, contribuyendo al control y el tratamiento oportuno de la enfermedad. En este estudio se presenta el proceso de purificación del Ag38kDa a partir de proteínas de secreción filtradas de cultivo (CFP) de Mtb para ser usado como inmunoreactivo con potencial aplicación en la detección de Mtb con inmunosensores piezoeléctricos. Se obtuvieron cristales funcionalizados mediante la técnica modificada de monocapas autoensambladas (SAMs), con el antígeno nativo purificado y CFP. Las superficies biofuncionalizadas fueron caracterizadas cualitativamente con microscopía de fuerza atómica (AFM) para validar las condiciones de optimización del protocolo de inmovilización con antígenos de secreción de Mtb. Estos cristales modificados pueden ser acoplados a un sistema de caracterización de un inmunosensor piezoeléctrico para la detección de Mtb mediante un inmunoensayo competitivo directo.

The selection and procurance of the immunoreagents used in the immunoassay and biofunctionalisation transducer strategy, are a key in the piezoelectric immunosensor development for the detection of tuberculosis (TB). Many have reported the use of 38kDa protein antigen (Ag38kDa) from Mycobacterium tuberculosis (Mtb) such as good biomarker of TB disease and compliance with physical and biochemical characteristics to be immobilized by self-assembled monolayers (SAMs), in the gold electrode of piezoelectrics crystals surfaces. A piezoelectric immunosensor developed from purified native antigens of Mtb may be an alternative simple method for detection of Mtb with speed and reusable advantages, contributing to the control and early treatment of disease. In this paper, the purification process of Ag38kDa Mtb from secretory proteins filtered culture (CFP) from Mtb is presented as an immunoreactive with potential application in the detection of Mtb by piezoelectric immunosensors. Functionalized crystals were obtained by using the modified self-assembled monolayers (SAMs) technique, with purified native antigen and CFP. The functionalized surfaces were qualitatively characterized using atomic force microscopy (AFM) in order to validate the immobilization protocol optimal conditions for secretion antigens from Mtb. These modified crystals may be coupled to piezoelectric immunosensor characterization system for detecting of Mtb by a direct competition immunoassay.

Genetic variability of Senepol cattle in Colombia using molecular markers / Variabilidad genética de bovinos Senepol en Colombia por marcadores moleculares / Variabilidade genética de gado Senepol na Colômbia por marcadores moleculares

The Senepol beef cattle breed was introduced into Colombia through the use of artificial insemination and embryo transfer from a small nucleus of animals. Objective: to estimate the genetic variability of Senepol cattle in Colombia by heterologous microsatellites and to estimate gene and genotypic frequencies of single nucleotide polymorphic markers through calpastatin (CAST1), calpain (CALP316), and leptin (PB) genes. Methods: 412 blood samples from 28 herds were genotyped for population genetic structure with the STR: INRA32, BM2113, ETH10, BM1824, INRA037, ETH225, INRA064, SPS115, TGLA126, and TGLA122 microsatellite markers. Three SNPs of calpastatin, calpain, and leptin genes were used. Results: all microsatellites and SNP markers were polymorphic. The number of alleles ranged from 4 (BM1824) to 11 (INRA37), and the observed heterozygosity varied between 0.21 (INRA64) and 0.89 (BM2113). Combined probability of exclusion for the microsatellites was higher than 99.99%, indicating the usefulness of this set of markers for parentage testing in Senepol. Conclusions: despite being a small and closed population, this nucleus presents high genetic variability and low inbreeding.

El ganado Senepol fue introducido en Colombia mediante el uso de la inseminación artificial y transferencia de embriones de un pequeño núcleo de los animales. Objetivo: estimar la variabilidad genética del ganado Senepol de Colombia por medio de marcadores microsatélites y estimar las frecuencias alélicas y genotípicas de SNPs en los genes que codifican para la calpastatina (CAST1), calpaína (CALP316) y leptina (PB). Métodos: 412 muestras de sangre de animales pertenecientes a 28 fincas fueron analizados para los STRs: INRA32, BM2113, ETH10, BM1824, INRA037, ETH225, INRA064, SPS115, TGLA126 y TGLA122 y los tres SNPs. Resultados: los microsatélites y los SNPs fueron polimórficos. El número de alelos de los microsatélites variaron entre 4 (BM1824) y 11 (INRA37), la heterocigosidad observada varió entre 0.21 (INRA64) y 0.89 (BM2113). La probabilidad de exclusión para el total de microsatélites fue mayor que 99.99%, indicando que el conjunto de microsatélites pueden ser usados para pruebas de filiación. Conclusiones: a pesar de ser una población pequeña y cerrada, este núcleo presenta una alta variabilidad genética y baja consanguinidad.

O gado Senepol foi introduzido na Colômbia mediante o uso da inseminação artificial e a transferência de embriões de um núcleo pequeno de animais. Objetivo: estimar a variabilidade genética do gado Senepol da Colômbia mediante marcadores microsatélites e estimar as frequências alélicas e genotípicas dos SNPs dos genes de calpastatina (CAST1), calpaina (CALP316) e leptina (PB). Métodos: 412 amostras de sangue de animais pertencentes a 28 rebanhos foram analisadas para os STRs INRA32, BM2113, ETH10, BM1824, INRA037, ETH225, INRA064, SPS115, TGLA126 e TGLA122 e os três SNPs. Resultados: os microsatélites e os SNPs foram polimórficos. O número de alelos dos microsatélites variaram entre 4 (BM1824) e 11 (INRA37), a heterocigosidade observada variou entre 0,21 (INRA64) e 0,89 (BM2113). A probabilidade de exclusão para o total de microsatélites polimórficos foi maior que 99.99%, indicando que o conjunto de microsatélites podem ser usados para testes de filiação. Conclusões: embora seja uma população pequena e fechada, o núcleo apresenta uma alta variabilidade genética e baixa consanguinidade.