A functional screening of the kinome identifies the Polo-like kinase 4 as a potential therapeutic target for malignant rhabdoid tumors, and possibly, other embryonal tumors of the brain.

PURPOSE: Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific inhibitors. METHODS: We individually mutated 160 kinases in a well-characterized rhabdoid tumor cell line (MON) using lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The kinase that most significantly impaired cell growth was further validated. Its expression was evaluated by microarray gene expression (GE) within 111 pediatric tumors, and functional assays were performed. A small molecule inhibitor was tested in multiple rhabdoid tumor cell lines and its toxicity evaluated in zebrafish larvae. RESULTS: The Polo-like kinase 4 (PLK4) was identified as the kinase that resulted in higher impairment of cell proliferation when mutated by CRISPR/Cas9. PLK4 CRISPR-mutated rhabdoid cells demonstrated significant decrease in proliferation, viability, and survival. GE showed upregulation of PLK4 in rhabdoid tumors and other embryonal tumors of the brain. The PLK4 inhibitor CFI-400945 showed cytotoxic effects on rhabdoid tumor cell lines while sparing non-neoplastic human fibroblasts and developing zebrafish larvae. CONCLUSIONS: Our findings indicate that rhabdoid tumor cell proliferation is highly dependent on PLK4 and suggest that targeting PLK4 with small-molecule inhibitors may hold a novel strategy for the treatment of MRT and possibly other embryonal tumors of the brain. This is the first time that PLK4 has been described as a potential target for both brain and pediatric tumors.

Histone deacetylase 1 deficiency impairs differentiation and electrophysiological properties of cardiomyocytes derived from induced pluripotent cells.

Epigenetic and chromatin modifications play particularly important roles in embryonic and induced pluripotent stem cells (ESCs and iPSCs) allowing for the cells to both differentiate and dedifferentiate back to a pluripotent state. We analyzed how the loss of a key chromatin-modifying enzyme, histone deacetylase 1 (HDAC1), affects early and cardiovascular differentiation of both ESCs and iPSCs. We also investigated potential differences between these two cell types when differentiation is induced. Our data indicate an essential role for HDAC1 in deacetylating regulatory regions of key pluripotency-associated genes during early differentiation. Although HDAC1 functions primarily as a HDAC, its loss also affects DNA methylation in ESCs and iPSCs both during pluripotency and differentiation. We show that HDAC1 plays a crucial, nonredundant role in cardiomyocyte differentiation and maturation. Our data also elucidate important differences between ESCs and iPSCs, when levels of this enzyme are reduced, that affect their ability to differentiate into functional cardiomyocytes. As varying levels of chromatin-modifying enzymes are likely to exist in patient-derived iPSCs, understanding the molecular circuitry of these enzymes in ESCs and iPSCs is critical for their potential use in cardiovascular therapeutic applications

Genome-wide quantitative assessment of variation in DNA methylation patterns.

Genomic DNA methylation contributes substantively to transcriptional regulations that underlie mammalian development and cellular differentiation. Much effort has been made to decipher the molecular mechanisms governing the establishment and maintenance of DNA methylation patterns. However, little is known about genome-wide variation of DNA methylation patterns. In this study, we introduced the concept of methylation entropy, a measure of the randomness of DNA methylation patterns in a cell population, and exploited it to assess the variability in DNA methylation patterns of Alu repeats and promoters. A few interesting observations were made: (i) within a cell population, methylation entropy varies among genomic loci; (ii) among cell populations, the methylation entropies of most genomic loci remain constant; (iii) compared to normal tissue controls, some tumors exhibit greater methylation entropies; (iv) Alu elements with high methylation entropy are associated with high GC content but depletion of CpG dinucleotides and (v) Alu elements in the intronic regions or far from CpG islands are associated with low methylation entropy. We further identified 12 putative allelic-specific methylated genomic loci, including four Alu elements and eight promoters. Lastly, using subcloned normal fibroblast cells, we demonstrated the highly variable methylation patterns are resulted from low fidelity of DNA methylation inheritance.

Study of the gene expression and microRNA expression profiles of malignant rhabdoid tumors originated in the brain (AT/RT) and in the kidney (RTK).

PURPOSE: Malignant rhabdoid tumors (MRT) can occur in a variety of anatomical sites. The most frequent locations are the brain, where they are named atypical teratoid/rhabdoid tumors (AT/RT), and the kidney, where they are named rhabdoid tumors of the kidney (RTK). MRTs at all sites are recognized as the same entity due to their similar morphology, aggressive behavior, and a common genetic abnormality, an inactivating mutation of the SMARCB1/INI-1/hSNF5/BAF47 gene. We aim to investigate potential molecular differences between AT/RT and RTK. METHODS: We analyzed the microRNA (miRNA) and gene expression (GE) profiles of 10 RTK, 13 AT/RT, and 2 human MRT cell lines (G401-RTK and MON-AT/RT). Illumina V2 MicroRNA Chips (Illumina, Inc., CA, USA) were used for miRNA analysis, and Illumina HT-12 whole genome expression arrays were used for GE analysis. RESULTS: The distribution of p values from GE showed a significant difference between RTK and AT/RT, with 20 % of the genes having p values ≤0.05 and the principal component analysis of the GE data showed separation between RTK and AT/RT. However, the miRNA expression failed to identify the different tumor groups. Among the 122 genes significantly differentially expressed between AT/RT and RTK, we found both genes related to brain development (i.e., FABP7, 22-fold increase in AT/RT) and genes related to kidney development (i.e., TCF21, sixfold increase in RTK). CONCLUSION: Based on our results, we hypothesized that although MRT are indeed the same tumor, independent of the site of origin, the GE differences reflect the influence of microenvironment over tumor development.

Effects of platelet-rich plasma on corneal re-epithelization and metalloproteinase expression in the cornea of sheep with experimentally-induced infectious keratoconjunctivitis.

Background and Aim: Infectious bovine keratoconjunctivitis is the most crucial ophthalmic disease among ruminants worldwide. Moraxella is the bacteria generally associated with this disease and leads to keratitis, conjunctivitis, corneal ulcers, or blindness. Platelet-rich plasma (PRP) effects in corneal ulcers and different ocular superficial diseases in animals and humans are beneficial and enhance rapid healing and improvement, but the effects in infectious keratoconjunctivitis in ruminants are uncertain. This study aimed to examine the effect of PRP on re-epithelization, corneal tissue, clinical signs, and matrix metalloproteinase (MMP) expression in sheep with infectious keratoconjunctivitis. Materials and Methods: Eighteen sheep were divided into three groups and subjected to a disease-induction experiment. Group 1 (G1) was administered 1.0 mL PRP subconjunctivally, Group 2 (G2) was administered 1.0 mL PRP subconjunctivally and 50 µL gentamicin drops, and the control group (CG) was administered 50 µL saline solution topically every 12 h. Clinical ophthalmologic examination, fluorescein staining, and photography were carried out. Ulcerated areas were measured employing J-Image software. Five and eleven days following the procedure, half of the animals from each group were euthanized, and their corneas were evaluated by histopathology and zymography. Results: Control Group and G2 epithelialized more rapidly. The CG exhibited fewer clinical signs of ocular disease. In histopathological analysis, in G2, alterations were observed only in the epithelium. The CG and G1 exhibited alterations in the epithelium, stroma, and Descemet's membrane. In zymography, a decline in MMP-2 expression in the animals treated with PRP was detected. Matrix metalloproteinase-9 was significantly expressed in the animals treated with PRP monotherapy, whereas PRP + gentamicin and CG caused a decrease. Conclusion: Platelet-rich plasma alone did not demonstrate any beneficial effect on re-epithelialization, a decline in clinical signs, tissue alterations, and expression of metalloproteinases. Platelet-rich plasma combined with gentamicin was capable of suppressing MMPs, primarily MMP-9, but do not display positive effects in re-epithelization, reduction of clinical signs, or tissue effects. These outcomes are similar to those discovered in untreated animals, so the use of PRP in patients with infectious keratoconjunctivitis does not offer greater benefits in sheep. Additional research is required to validate the results of PRP use in natural disease presentation.

How COVID-19 elucidated challenges in the pedagogy of physiotherapy entry-level education in Brazil and directions for their remediation with special attention to digital teaching and learning.

INTRODUCTION: As experienced physiotherapy educators in Brazil, we observed that COVID-19 elucidated challenges in the pedagogy of entry-level education overall, and directions for their remediation. In this commentary, we describe our observations with particular attention to the opportunity for digital and distance teaching and learning in Brazil's exemplary middle-income country. BODY: First, the legislation in Brazil around health professional education, specifically entry-level physiotherapy education, is described concerning distanced learning. Then, we contrast such education before and during the COVID-19 pandemic, and in the aftermath of its peak. Our observations reinforce the need to preserve teaching and learning excellence in physiotherapy education with various approaches including distanced and digital learning; be aware of both advantages and disadvantages; and identify means of balancing these for optimal delivery and learner outcomes. Our collective experience and insights strongly support the need for change in the legislative document governing physiotherapy education in Brazil. CONCLUSION: We hope our experiences will enable other educators to evaluate their contexts, reflect on how best to deliver entry-level physiotherapy education in general and during a pandemic, and reinforce the essentiality of practical face-to-face classes in achieving physiotherapy competencies. Only in this way will global standards of practice be ensured, through quality professional education and the factors that inform and govern these.

Genetic and epigenetic variations contributed by Alu retrotransposition.

BACKGROUND: De novo retrotransposition of Alu elements has been recognized as a major driver for insertion polymorphisms in human populations. In this study, we exploited Alu-anchored bisulfite PCR libraries to identify evolutionarily recent Alu element insertions, and to investigate their genetic and epigenetic variation. RESULTS: A total of 327 putatively recent Alu insertions were identified, altogether represented by 1,762 sequence reads. Nearly all such de novo retrotransposition events (316/327) were novel. Forty-seven out of forty-nine randomly selected events, corresponding to nineteen genomic loci, were sequence-verified. Alu element insertions remained hemizygous in one or more individuals in sixteen of the nineteen genomic loci. The Alu elements were found to be enriched for young Alu families with characteristic sequence features, such as the presence of a longer poly(A) tail. In addition, we documented the occurrence of a duplication of the AT-rich target site in their immediate flanking sequences, a hallmark of retrotransposition. Furthermore, we found the sequence motif (TT/AAAA) that is recognized by the ORF2P protein encoded by LINE-1 in their 5'-flanking regions, consistent with the fact that Alu retrotransposition is facilitated by LINE-1 elements. While most of these Alu elements were heavily methylated, we identified an Alu localized 1.5 kb downstream of TOMM5 that exhibited a completely unmethylated left arm. Interestingly, we observed differential methylation of its immediate 5' and 3' flanking CpG dinucleotides, in concordance with the unmethylated and methylated statuses of its internal 5' and 3' sequences, respectively. Importantly, TOMM5's CpG island and the 3 Alu repeats and 1 MIR element localized upstream of this newly inserted Alu were also found to be unmethylated. Methylation analyses of two additional genomic loci revealed no methylation differences in CpG dinucleotides flanking the Alu insertion sites in the two homologous chromosomes, irrespective of the presence or absence of the insertion. CONCLUSIONS: We anticipate that the combination of methodologies utilized in this study, which included repeat-anchored bisulfite PCR sequencing and the computational analysis pipeline herein reported, will prove invaluable for the generation of genetic and epigenetic variation maps.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana.

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Occurrence and expression of p53 suppressor gene and c-Myc oncogene in dog eyelid tumors.

PURPOSE: To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c-Myc in eyelid tumors of dogs using the PCR, RT-PCR, PCR-ELISA and RT-PCR-ELISA techniques. These genes have not been described in dog eyelid tumors before. METHODS: Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin-eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR-ELISA and RT-PCR-ELISA were used to detect p53 and c-Myc genes. RESULTS: The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c-Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. CONCLUSIONS: Eyelid and third eyelid tumors of dogs express both the p53 and the c-Myc genes as shown by PCR and RT-PCR. However, PCR ELISA and RT-PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis.

Immediate effect of myofascial release on range of motion, pain and biceps and rectus femoris muscle activity after total knee replacement.

OBJECTIVES: The role of a myofascial release (MFR) on flexion contractures after total knee arthroplasty (TKA) has not yet been elucidated. Therefore, the purpose of this study was to determine its immediate effect on such patients. METHODS: In this A-B single subject experimental study, 33 TKA's patients with knee flexion contracture had their gluteal, posterior fascia lata, posterior crural and plantar fasciae released. Patients' knee range of motion (KROM), pain and muscle electric activity were assessed pre- and post-intervention. RESULTS: An increase in electric activity of the biceps femoris muscle was identified after treatment (pre RMS = 0.087 ± 0.066 V; post RMS = 0.097 ± 0.085 V; p = 0.037). Mean gain of KROM was 5.72 ± 6.27, correspondent to an 11.9% improvement (p = 0.01). Eight subjects had their pain decreased on 56.9% (p = 0.04). CONCLUSIONS: MFR increased muscle activity, reduced pain and improved the KROM of TKA patients. Thus, MFR is a useful resource of rehabilitation after TKA.

Conservation and development in Amazonian extractive reserves: the case of Alto Juruá.

Extractive reserves constitute an innovative approach to match conservation and development objectives, which were originally envisaged as part of a land struggle by forest dwellers in Brazil. In spite of the idea's popularity and the attempts to apply the concept to different tropical regions, there has been little analysis of the combined conservation and development performance of extractive reserve programs. We present a detailed analysis of deforestation and demographic and socioeconomic changes in Alto Juruá, the first extractive reserve created in Brazil in 1990. Forest cover has remained fairly stable. Population has declined slightly, with some internal displacements. The cash economy base has shifted from the original rubber production to a diversified portfolio of agriculture and livestock, and there has been a dramatic rise in nonagrarian income. We conclude that the Reserve represents a very dynamic setting with positive conservation and development outcomes during its first decade.

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix.

Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.

Use of (99m)Tc-anti-TNF-α scintigraphy in a patient with non-radiographic axial spondyloarthritis.

The aim of this study was to describe the use of (99m)Tc-anti-TNF-α scintigraphy for detecting inflammation of the sacroiliac joints in a patient with non-radiographic axial spondyloarthritis. A 47-year-old female patient, non-smoker and non-drinker, complained of a low back pain inflammation, which began 4 years before her condition have exacerbated to morning stiffness and anterior uveitis in the last 6 months. Initially diagnosed as mechanical low back pain, she irregularly took non-steroidal anti-inflammatory drugs and corticosteroids, without significant long-lasting results. Radiographic findings were negative. There was increased uptake of (99m)Tc-anti-TNF-α in an area corresponding to the topography of ileum and sacroiliac right joint upon (99m)Tc-anti-TNF-α scintigraphy. Magnetic resonance imaging (MRI), the most used image diagnosis tool, showed minimum impregnation of gadolinium in the right sacroiliac joint and at the iliac face of the inferior third of the right sacroiliac joint. We suggest that (99m)Tc-anti-TNF-α can facilitate early diagnosis of patients with non-radiographic axial spondyloarthritis. More studies are now ongoing.

Experiencia clínica del uso de la tepoxalina en el tratamiento conservativo de la ruptura del ligamento cruzado craneal en perros / Clinical trial of tepoxalin use in cranial cruciate ligament ruptura conservative treatment in dogs

La ruptura de ligamento cruzado craneal (RLCC) se caracteriza por la laceración parcial o total del ligamento cruzado craneal. Pacientes con laceraciones agudas del ligamento (parcial o total) presentan claudicación aguda con incapacidad total o parcial de sustentación del peso en el miembro afectado. La terapia clínica con principios conservadores debe ser empleada previamente al tratamiento quirúrgico. La tepoxalina es un antiinflamatorio no esteroidal, indicado en la reducción de procesos inflamatorios y alivio del dolor causado por perturbaciones musculo-esqueléticas agudas y crónicas. Este medicamento promueve el bloqueo balanceado de la ruptura del ácido araquidónico por las vías de la lipoxigenasa y cicloxigenasa de la cascada de la inflamación. El presente trabajo tiene por objetivo evaluar la eficacia clínica del uso de la tepoxalina en el tratamiento conservativo de la RLLC, por medio de test clínicos específicos, examen radiográfico y análisis citológico del líquido articular. Diez perros con RLCC, macos y hembras, fueron incluidos en este estudio y recibieron dosis diarias de 10mg/kg de tepoxalina durante veintiocho días. La evaluación de la claudicación, los test de arco de movimiento en extensión máxima y digito-presión, así como el análisis del liquido articular después del periodo de tratamiento, permitieron concluir la efeicacia de la tepoxalina en el tratamiento conservativo de la RLCC. Su uso posibilitó el retorno a la deambulación normal en todos los casos tratados, siendo considerada una opción más de AINE para el tratamiento conservativo de esta artropatía...

Standardization of the values of the "Schirmer Tear Test" modificated and of the "Schiötz Tonometry Indentation" from dogs of the region of Jaboticabal - SP Brazil / Padronização dos valores do Teste da Lágrima de Schirmer modificado e da Tonometria de Indentação pelo método de Schiötz, em cães da região de Jaboticabal - SP Brasil

The keratoconjutivitis "sicca and the glaucoma could he diagnosticated easily through the Schirmer Tear and Schiötz Tonometry" respectively. To reach these objectives it"s important to know the intrinsic values of each geographic region, because they are affected by environmental changes. Fifty adult dogs were studied, with or without breed, healthy, at the Ophthalmology Service of the Veterinary Hospital UNESP - Jaboticabal. For the "Schirmer Test" the technic was based on the application of a filter paper ribbon (MelittaR) in the inferior conjunctival sac for 1 minute. For the "Schiötz Test" the technique included the appropriate contention and colocation of the Schiötz equipment upon the center of the cornea with a previous anesthesia. The results showed median values of 17,01 mm ± 4.28 mm for the "Schirmer Tear Test" and 29,15 mmHg ±4,60 for the "Schiötz Tonometry.

A ceratoconjuntivite seca (KCS) e o glaucoma constituem afecções freqüentes em Medicina Veterinária e podem ser diagnosticadas através do "Teste da Lágrima de Schirmer" e da "Tonometria de Schiötz, respectivamente. Para tal, necessita-se conhecer os valores próprios de cada região, uma vez que os mesmos eslão sujeitos a interferências ambientais. Estudaram-se 50 cães adultos, com e sem raça definida, considerados sadios, junto ao Serviço de Oftalmologia do Hospital Veterinário da FCAV - UNESP de Jaboticabal. Para o Teste da Lágrima de Schirmer", os procedimentos consistiram na aplicação de tira de papel de filtro (MelittaR) junto ao saco conjuntival inferior por 1 minuto e avaliação em régua milimetrada do segmento da tira hidratada pela lágrima. Para o procedimento restante, as manobras incluíram contenção apropriada e aplicação do tonômetro de Schiöt sobre o centro da córnea, previamente dessensibilizada com colírio anestésico. Os resultados mostraram valores médios de 17,01 mm ± 4,28 para o "Teste da Lágrima de Schirmer", 29,15 mmHg ± 4,60 para a "Tonometria de Schiötz".