DNA methylation profile of single in vitro matured bovine oocytes.

Somatic cell nuclear transfer (SCNT) is commercially used despite incomplete nuclear reprogramming of the somatic cell nucleus by the enucleated oocyte compromising its efficiency. Oocyte selection is a key factor in increasing this efficiency as its cytoplasm reprograms the differentiated cell. In this study, we adapted a methodology to characterize epialleles in potential epigenetic markers in single in vitro matured oocytes. Characterization of the regions that control the expression of imprinted genes, X-chromosome inactivation, and satellite I DNA (IGF2, ICR-H19, XIST, RepA, and SAT1) showed methylated and unmethylated alleles in the imprinted genes IGF2 and ICR-H19 while XIST-DMR1 and RepA showed hypermethylated alleles. There was great variation in methylation patterns for candidate regions which may be related to oocyte quality. Moreover, the identification of different epialleles in the same oocyte suggests that, at least for those loci, the epigenome of the metaphase plate and polar body is different. The single-cell bisulfite polymerase chain reaction technique can be used to improve the precision of selecting the best oocytes for SCNT procedures, thereby increasing its efficiency.

Recurrence rate of basal cell carcinoma among different micrographic surgery techniques: systematic review with meta-analysis.

In high-risk basal cell carcinomas (BCCs), micrographic surgery (MS) has high tissue preservation and low recurrence rates. The Mohs technique is the most commonly used technique, with limited use of other MS techniques. No studies have been designed to compare the MS methods. This review aimed to assess BCC recurrence rates of different MS techniques. A systematic review and meta-analysis were conducted to search for related studies in PubMed, LILACS, EMBASE, SCOPUS, WEB OF SCIENCE, CINHAL and COCHRANE until March 2021. Randomized clinical trials (RCTs) and observational studies involving patients with BCC and indications for different MS techniques were included. Study selection and data extraction were performed independently by three peer reviewers, as was the risk of bias assessment using the Joanna Briggs Institute tool. Pooled estimates were assessed using the random-effects model (Logit), and heterogeneity was assessed by the chi-squared test (χ2 ). Stata Software version 17.0 was used for analysis. Eighteen studies were included, two RCTs and sixteen observational studies. The overall recurrence rate was 2% (95% CI, 1.0-3.0%; χ2 = 46.2; P = 0.00; 18 studies, 10 424 BCCs). In studies using the Mohs technique, the recurrence rate was 3.0% (95% CI, 1.0-5.0%; χ2 = 11.0; P = 0.00; 6 studies; 1,582 BCCs), with the Munich technique 3.0% (95% CI, 2.0-5.0%; χ2 = 0.0; no heterogeneity; 3 studies; 404 BCCs), with Tubingen technique 1% (95% CI, 1.0-2.0%; χ2 = 12.1; P = 0.00; 8 studies; 8374 BCCs) and with the Muffin technique 0.0% (95% CI, 0.0-6.0%; 1 study; 64 BCCs). Relapse rates between MS techniques were low and appeared to be similar. However, the design of this review and the absence of primary studies that directly compare the techniques do not allow us to assert the superiority between them.

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures-surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations.

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir-Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.

Correlation of postural balance and knee muscle strength in the sit-to-stand test among women with and without postmenopausal osteoporosis.

UNLABELLED: The task of standing up from a chair forms a part of daily life for all independent individuals. However, this task becomes more difficult with advancing age. Women with postmenopausal osteoporosis presented diminished knee extensor and flexor muscle strength. There was a weak correlation between knee muscle strength (greater with extensor strength) and postural balance during the act of standing up. INTRODUCTION: This study aims to evaluate postural balance during the transition from sitting to standing and its relationship with knee extensor and flexor strength among women with and without postmenopausal osteoporosis. METHODS: Assessments were made on 126 women (aged 55-65 years), divided into osteoporosis and control groups according to lumbar bone density. Their balance during the task of standing up from a chair was evaluated using the Balance Master® device. Knee muscle strength was evaluated using an isokinetic dynamometer (Biodex®), in concentric/concentric mode, at a velocity of 60°/s. Spearman's correlation between the variables of muscle strength and postural balance was evaluated. Subsequently, to evaluate the association of each balance variable with the group and with muscle strength, multiple linear regression models were fitted. The significance level was set at 0.05. RESULTS: There was a difference in knee muscle strength between the groups (P < 0.05). However, independent of the muscle strength values, there were no differences in relation to weight transfer times (P = 0.556) or center of gravity sway velocity (P = 0.952). Transfer time diminished with increasing extensor strength (P = 0.025). The center of gravity sway velocity tended to increase with increasing extensor strength (P = 0.013) and was the same in the two groups (P = 0.264). CONCLUSION: Women with postmenopausal osteoporosis presented diminished knee extensor and flexor muscle strength. There was a weak correlation between knee muscle strength (greater with extensor strength) and postural balance during the act of standing up.

Epigenetic Reprogramming and Somatic Cell Nuclear Transfer.

Epigenetics is an area of genetics that studies the heritable modifications in gene expression and phenotype that are not controlled by the primary sequence of DNA. The main epigenetic mechanisms are DNA methylation, post-translational covalent modifications in histone tails, and non-coding RNAs. During mammalian development, there are two global waves of epigenetic reprogramming. The first one occurs during gametogenesis and the second one begins immediately after fertilization. Environmental factors such as exposure to pollutants, unbalanced nutrition, behavioral factors, stress, in vitro culture conditions can negatively affect epigenetic reprogramming events. In this review, we describe the main epigenetic mechanisms found during mammalian preimplantation development (e.g., genomic imprinting, X chromosome inactivation). Moreover, we discuss the detrimental effects of cloning by somatic cell nuclear transfer on the reprogramming of epigenetic patterns and some molecular alternatives to minimize these negative impacts.

Low levels of sulfur and cobalt during the pre- and periconceptional periods affect the oocyte yield of donors and the DNA methylome of preimplantation bovine embryos.

Maternal nutrition is critical in mammalian development, influencing the epigenetic reprogramming of gametes, embryos, and fetal programming. We evaluated the effects of different levels of sulfur (S) and cobalt (Co) in the maternal diet throughout the pre- and periconceptional periods on the biochemical and reproductive parameters of the donors and the DNA methylome of the progeny in Bos indicus cattle. The low-S/Co group differed from the control with respect to homocysteine, folic acid, B12, insulin growth factor 1, and glucose. The oocyte yield was lower in heifers from the low S/Co group than that in the control heifers. Embryos from the low-S/Co group exhibited 2320 differentially methylated regions (DMRs) across the genome compared with the control embryos. We also characterized candidate DMRs linked to the DNMT1 and DNMT3B genes in the blood and sperm cells of the adult progeny. A DMR located in DNMT1 that was identified in embryos remained differentially methylated in the sperm of the progeny from the low-S/Co group. Therefore, we associated changes in specific compounds in the maternal diet with DNA methylation modifications in the progeny. Our results help to elucidate the impact of maternal nutrition on epigenetic reprogramming in livestock, opening new avenues of research to study the effect of disturbed epigenetic patterns in early life on health and fertility in adulthood. Considering that cattle are physiologically similar to humans with respect to gestational length, our study may serve as a model for studies related to the developmental origin of health and disease in humans.

Phosphorylation of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 facilitates cation-induced conformational changes and actin assembly.

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study.

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of ß-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.

Identification of novel GH-regulated genes in C2C12 cells.

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Abnormal expression of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele.

BACKGROUND: In some cases, a definitive confirmation of dysferlinopathy cannot be achieved by DNA test, because the mutation is detected in one allele only. PATIENTS AND METHODS: DYSFERLIN expression in skeletal muscle and peripheral blood monocytes (PBM) was studied by Western blot in two unrelated adult patients. The comparative C(T) method (ΔΔC(T) ) was used to calculate relative changes in dysferlin mRNA determined from real-time quantitative PCR experiments. The dysferlin gene was studied by direct sequencing of cDNA and genomic DNA and by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: A comparable severe reduction in dysferlin was demonstrated in both skeletal muscle and PBM. The expression of dysferlin mRNA was significantly reduced. A novel mutation in exon 47 (c.5289G>C) of the dysferlin gene in the heterozygous state, causing an amino acid change (p.Glu1763Asp), was detected in both patients. The MLPA analysis did not reveal any deletion or duplication. CONCLUSIONS: Dysferlin and/or dysferlin mRNA abnormalities are diagnostic for dysferlinopathy when mutational analysis detects a mutation in one allele only. Analysis of dysferlin mRNA can be helpful for distinguishing symptomatic heterozygotes from such patients.

Interactions of intrinsically disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes - synergistic effects of lipid composition and temperature on secondary structure.

Dehydrins are intrinsically disordered (unstructured) proteins that are expressed in plants experiencing stressful conditions such as drought or low temperature. Dehydrins are typically found in the cytosol and nucleus, but also associate with chloroplasts, mitochondria, and the plasma membrane. Although their role is not completely understood, it has been suggested that they stabilize proteins or membrane structures during environmental stress, the latter association mediated by formation of amphipathic α-helices by conserved regions called the K-segments. Thellungiella salsuginea is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We have cloned and expressed in Escherichia coli two dehydrins from this plant, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). Here, we show using transmission-Fourier transform infrared (FTIR) spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. Moreover, this induced folding is enhanced at low temperatures, lending credence to the hypothesis that dehydrins stabilize plant outer and organellar membranes in conditions of cold.

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status of In Vitro Cultured Bovine Skin Fibroblasts.

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH (p = 0.0495) and by the combination of SAH and procaine (p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH (p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine (p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine (p = 0.0007), SAH (p = 0.0060), and SAH/procaine (p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine (p = 0.0291), SAH (p = 0.0373), and procaine/SAH (p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

A new phenotype of dysferlinopathy with congenital onset.

We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.

Ciclo estral de Alouatta palliata y Alouatta seniculus: una revisión / Alouatta palliata and Alouatta seniculus oestrus: a review

RESUMEN El mono aullador rojo colombiano (A. seniculus) y mono aullador de manto (A. palliata) son representantes del género Alouatta en Colombia y, según análisis de la Unión Internacional para la Conservación de la Naturaleza (UICN), sus poblaciones tienden a decrecer por múltiples factores. Esto sugiere la necesidad de orientar acciones para su conservación, para ello se requiere conocimiento de sus dinámicas reproductivas como la identificación y caracterización del ciclo estral. Se examinaron documentos bajo los criterios Alouatta seniculus, Alouatta paliatta, Alouatta, ciclo estral y reproducción en Scielo, Google Scholar, PubMed, Web of Science, Wiley, Science Direct y SpringerLink. Se encontraron quince artículos, cuatro libros, cuatro tesis de maestría y una monografía; en estos estudios se abordaban aspectos sobre comportamientos sexuales, cambios en los genitales y medición de hormonas. Los estudios han establecido una duración promedio del ciclo para A. seniculus de 17 y de 29,5 ±1,5 días, lo que muestra un rango amplio en comparación con lo hallado para A. palliata, donde los estudios evidencian una duración de 16,3; 15,52 ±4,88 y 16,08 ±4,27 días. Se concluye que algunos aspectos comunes entre las especies son la estacionalidad de los nacimientos durante la época seca, la relevancia del rango social del macho en las solicitudes de cópula por parte de las hembras en estro y algunos comportamientos como el olfateo y el lamido de genitales durante la receptividad sexual. Asimismo, es necesario conducir estudios en las especies de Alouatta en Colombia que, además de abordar las dinámicas reproductivas, consideren la influencia de factores medioambientales y sociales sobre estas.

Abstract The Colombian red howler monkey (A. seniculus) and mantled howler monkey (A. palliata) are representatives of the genus Alouatta in Colombia and according to IUCN (for its name in Spanish) analysis their populations tend to decrease due to multiple factors. This suggests the need to guide actions for their conservation, which requires knowledge of their reproductive dynamics, such as the identification and characterization of the estrous cycle. Papers were examined under the criteria Alouatta seniculus, Alouatta paliatta, Alouatta, estrus cycle, and reproduction in Scielo, Google Scholar, PubMed, Web of Science, Wiley, Science Direct, and SpringerLink. 15 articles, 4 books, 4 master's theses and 1 monograph were found; these studies addressed aspects of sexual behaviors, changes in genitalia and hormone measurement. The studies have established an average cycle length for A. seniculus of 17 and 29.5 ±1.5 days, thus showing a wide range compared to what was found for A. palliata where the studies evidenced a cycle length of 16.3, 15.52 ±4.88 and 16.08 ±4.27 days. The conclusion is that some common aspects among the species are the seasonality of births during the dry season, the relevance of the male's social rank in copulation requests by females in estrus and some behaviors such as sniffing and genital licking during sexual receptivity. Likewise, it is necessary to conduct studies on Alouatta species in Colombia that not only address reproductive dynamics but also consider the influence of environmental and social factors on them.

Treadmill training in Parkinson's patients after deep brain stimulation: Effects on gait kinematic.

OBJECTIVE: The purpose of this study was to evaluate the effect of treadmill training with body weight support on gait kinematics parameters in patients with PD using DBS. DESIGN: Twelve patients completed the protocols (age: 60.9±10.6 years; disease duration: 20±7 years; and time since DBS surgery: 20±4 months). The same set of patients underwent two trainings protocols and four gait analyses (before and after each training). They received eight weeks of treadmill training without body weight support (16 sessions) in conjunction with physiotherapy program followed by six weeks of wash out period, followed by eight weeks of body-weight-supported treadmill training in conjunction with a same physiotherapy program. The Gait Kinematic Analysis involved eight infrared cameras that detected 19 reflective spherical markers attached in limb lower of patients. Statistical analysis used the Wilcoxon test (p≤0.05). RESULTS: Both the training no showed significant differences in linear variables. As the angular variables, only training with support showed significant increase of ranges of motion: pelvis tilt, obliquity and rotation amplitude; hip adduction-abduction and rotation amplitude; percentage of peak flexion in swing phase; foot progression amplitude. CONCLUSION: The body weight supported treadmill training may promote increase of mobility of lower limbs during gait and it could be a targeted intervention for PD patients treated with DBS.

Dysferlin expression in monocytes: a source of mRNA for mutation analysis.

Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.

Role of perfusion CT in the evaluation of functional primary tumour response after radiochemotherapy in head and neck cancer: preliminary findings.

OBJECTIVE: To report the initial results of a prospective study aimed at evaluating the CT perfusion parameter changes (∆PCTp) of the primary tumour after radiochemotherapy (RCT) in head and neck cancer (HNC) and to correlate with positron emission tomography (PET)/CT response. METHODS: Eligibility criteria included HNC (Stage III-IV) candidates for RCT. Patients underwent perfusion CT (PCT) at baseline and at 3 weeks and 3 months after treatment. Blood volume, blood flow, mean transit time (MTT) and permeability surface (PS) product were computed. Moreover, PET/CT was performed at baseline and 3 months after treatment. The ∆PCTp were evaluated between baseline and 3-week/3-month evaluations, whereas PET/CT response was based on the maximum standardized uptake value changes according to the European Organization for Research and Treatment of Cancer criteria. RESULTS: Between July 2012 and July 2015, 25 patients were enrolled. A significant reduction of all CT tumour perfusion parameters (PCTp) was observed from the baseline to after RCT (p < 0.001). Specifically, a significant reduction was shown at 3 weeks for all PCTp except MTT (from 6.18 to 5.14 s; p = 0.722). Differently, a significant reduction of all PCTp (p < 0.001) including MTT (from 6.18 to 2.24 s; p = 0.001) was shown at 3 months. Moreover, the reduction of PS resulted in a significant prediction of PET/CT response at 3 months (p = 0.037) with the trend also at 3 weeks (p = 0.099) at the multivariate analysis. CONCLUSION: Our preliminary findings seem to show that almost all PCTp are significantly reduced after RCT, whereas PS seems to come out as the strongest factor in predicting the PET/CT response. ADVANCES IN KNOWLEDGE: This article provides information on the potential useful role of PCT in evaluating tumour response after both early and late RCT.

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta / Caracterização epigenética do locus H19/IGF2 na placenta de bezerros clones

Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals' placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.(AU)

A Transferência Nuclear de Células Somáticas (TNCS-Clonagem) é uma técnica promissora em várias áreas, e se baseia na produção de indivíduos geneticamente idênticos. No entanto, sua eficiência é baixa. Estudos sugerem que a principal causa seja uma reprogramação epigenética inadequada. O objetivo desse trabalho é caracterizar o padrão de metilação da região éxon 10 do gene IGF2 e da Região Controladora de Imprinting (ICR) do gene H19 na placenta de bezerros clonados. Para a execução do trabalho foram selecionados clones bovinos fêmeas e machos, apresentando diferentes fenótipos. O DNA da placenta desses animais foi extraído, e em seguida foi tratado com bissulfito de sódio e amplificado para os loci ICR/H19 e IGF2. Os produtos da PCR foram clonados em bactérias competentes e, por fim, sequenciados. Foi encontrada uma diferença significativa entre os controles e os clones com fenótipos saudáveis para a região da ICR/H19. Nesta região, os controles tiveram um padrão hemimetilado, como previsto pela literatura, devido essa região ser imprinted. Enquanto os clones encontravam-se menos metilados. Para a região do éxon 10 do IGF2, não foi encontrada diferença significativa entre controles e clones. Estes resultados sugerem que as diferentes regiões do genoma podem se reprogramar independente umas das outras e que falhas na reprogramação do padrão de metilação do DNA de genes imprinted podem ser uma das causas da baixa eficiência da TNCS.(AU)

Inflammation in dysferlin myopathy: immunohistochemical characterization of 13 patients.

Inflammation was detected in 9 of 13 patients with different phenotypes of dysferlin myopathy. Endomysial or perivascular infiltrates consisted of 11.1% +/- 6.6% CD8(+) cells, 40.6% +/- 22.8% CD4(+) cells, 36.7% +/- 23.7% macrophages, and no B cells. Major histocompatibility complex class I was not upregulated in normal muscle fibers. In young patients with sporadic proximal weakness, very high creatine kinase levels, necrotic fibers and inflammation in the muscle biopsy, a diagnosis of dysferlin myopathy should be considered.

Antiganglioside antibodies in acute self-limiting ataxic neuropathy: incidence and significance.

Antidisialosyl antibodies have been previously associated to chronic and acute ataxic neuropathies. We studied the presence of these antibodies in nine patients with acute self-limiting ataxic neuropathy (ASLAN) using ELISA and TLC immunodetection. One patient showed serum IgG immunoreactivity against gangliosides GD3 and GQ1b. The patient's IgG was able to bind to the nodes of Ranvier on frozen human dorsal root. Our studies confirmed that antidisialosyl reactivity is associated to ataxic neuropathy and its specific binding to the dorsal root could explain the predominant sensory involvement. Nevertheless, the low incidence of this reactivity indicates that a different pathogenic mechanism should be involved in most ASLAN patients.