A method for the ultrastructural preservation of tiny percutaneous needle biopsy material from skeletal muscle

Skeletal muscle biopsies require transecting the muscle fibers resulting, in structural damage near the cut ends. Classically, the optimal ultrastructural preservation has been obtained by the use of relatively large biopsies in which the tissue fibers are restrained by ligating to a suitable retaining support prior to excision, and by examining regions at some distance from the cut ends. However, these methods require invasive surgical procedures. In the present study, we present and substantiate an alternative approach that allows for the excellent ultrastructural preservation of needle biopsy samples, even the very small samples obtained through tiny percutaneous needle biopsy (TPNB). TPNB represents an advantage, relative to standard muscle biopsy techniques and to other needle biopsies currently in use, as in addition to not requiring a skin incision, it leaves no scars in the muscle and requires an extremely brief recovery period. It is most appropriate for obtaining repeated samples in horizontal studies, e.g., in order to follow changes with athletic training and/or aging in a single individual and for studies of sarcopenic muscles in elderly patients. Due to the small size of the sample, TPNB may present limited usefulness for classical pathology diagnostics. However, it offers the major advantage of allowing multiple samples within a single session and this may be useful under specific circumstances

Tiny percutaneous needle biopsy: An efficient methodfor studying cellular and molecular aspectsof skeletal muscle in humans

Needle biopsy is widely used to obtain specimens for physiological, anatomical and biochemical studies of skeletal muscle (SM). We optimized a procedure which we termed tiny percutaneous needle biopsy (TPNB), to efficiently gather good numbers of human satellite cells and single dissociated fibers for the functional study of skeletal muscle; these samples permit isolation of high-quality RNA and sufficient amounts of proteins to allow molecular analysis. Moreover, TPNB showed a clear advantage in that the technique was easier than other procedures used on healthy volunteers in human trials. TPNB is a very safe minor surgical procedure. It is less traumatic than needle aspiration biopsy, and significant complications are improbable. TPNB should become established as an important tool in the investigation of SM and may be employed to study various physiological aspects of SM in human subjects. We suggest that TPNB should also be used in the study of muscle diseases and disorders including muscular dystrophy, congenital myopathy, and metabolic defects

Determination of blood lactate training zone boundaries with rating of perceived exertion in runners

This study aimed to determine the rating of perceived exertion (RPE) values corresponding to the blood lactate concentration (BLC) training zone boundaries (2 and 4 mmol.L-1) in moderately trained runners using the Borg CR-10 scale. Moderately trained runners (n = 95) performed a submaximal incremental test on a treadmill, recording BLC and RPE at every stage. Simple linear regression analysis was used to determine the RPE values corresponding to the BLC training zone boundaries, which revealed that RPE was significantly and strongly correlated with BLC (r= 0.821; P< 0.001; R2= 0.675; adjusted R2= 0.674; standard error of estimate= 1.18). The prediction equation (RPE= 1.092 7 BLC + 2.143) was obtained and RPE values at the BLC training zone boundaries of 2 mmol.L-1 and 4 mmol.L-1 calculated as 4.3 (95% CI, 3.9- 4.7) and 6.5 (95% CI, 6.0-7.1), respectively. In conclusion, the RPE values at the BLC training zone boundaries of 2 mmol.L-1 (4.3) and 4 mmol.L-1 (6.5) were adequately predicted. RPE (4.3 and 6.5) can be used as an affordable tool for controlling intensity in order to maintain the athletes in prescribed zones during training sessions

Hepatitis B virus DNA integration in tumour tissue of a non-cirrhotic HFE-haemochromatosis patient with hepatocellular carcinoma.

Co-existence of multiple causes of liver injury increases the risk of hepatocellular carcinoma (HCC) development. HCC usually develops in patients with cirrhosis although it may also occur in individuals with no or mild liver disease, in particular in cases with hepatitis B virus (HBV) infection. Here we report the case of a 43year-old man with HFE-haemochromatosis, seronegative for hepatitis B and C infections, who developed HCC in the absence of severe liver damage. Both tumoural and non-tumoural liver DNA extracts were tested by nested-PCR and primers specific for four different HBV genomic regions in order to evaluate the presence of occult HBV infection. Only X gene sequences were detected in tumour (but not in non-tumour) DNA extracts. HBV-Alu PCR showed a HBV integration involving a 5'-deleted X gene with an intact enhancer-II/basal-core promoter region. The viral-host junction sequencing revealed that this integrant was located upstream of the partitioning-defective-6-homolog-gamma gene (PARD6G) and real time-PCR quantification demonstrated that PARD6G was overexpressed in tumour compared to non-tumour liver tissues. In conclusion, the combination of HFE-haemochromatosis and occult HBV infection in this patient might have led to a sequel of cellular events that determined the development of HCC even in the absence of cirrhosis

Myomir dysregulation and reactive oxygen species in aged human satellite cells

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells. publisher: Elsevier articletitle: Myomir dysregulation and reactive oxygen species in aged human satellite cells journaltitle: Biochemical and Biophysical Research Communications articlelink: http://dx.doi.org/10.1016/j.bbrc.2016.03.030 content_type: article copyright: Copyright © 2016 The Authors. Published by Elsevier Inc. ispartof: Biochemical and Biophysical Research Communications vol:473 issue:2 pages:462-70 ispartof: location:United States status: publishe

Muscle Oxygen Delivery in the Forearm and in the Vastus Lateralis Muscles in Response to Resistance Exercise: A Comparison Between Nepalese Porters and Italian Trekkers

Altitude ascending represents an intriguing experimental model reproducing physiological and pathophysiological conditions sharing hypoxemia as the denominator. The aim of the present study was to investigate fractional oxygen extraction and blood dynamics in response to hypobaric hypoxia and to acute resistance exercises, taking into account several factors including different ethnic origin and muscle groups. As part of the “Kanchenjunga Exploration & Physiology” project, six Italian trekkers and six Nepalese porters took part in a high altitude trek in the Himalayas. The measurements were carried out at low (1,450 m) and high altitude (HA; 4,780 m). Near-infrared spectroscopy (NIRS)-derived parameters, i.e., Tot-Hb and tissue saturation index (TSI), were gathered at rest and after bouts of 3-min resistive exercise, both in the quadriceps and in the forearm muscles. TSI decreased with altitude, particularly in forearm muscles (from 66.9 to 57.3%), whereas the decrement was less in the quadriceps (from 62.5 to 57.2%); Nepalese porters were characterized by greater values in thigh TSI than Italian trekkers. Tot-Hb was increased after exercise. At altitude, such increase appeared to be higher in the quadriceps. This effect might be a consequence of the long-term adaptive memory due to the frequent exposures to altitude. Although speculative, we suggest a long-term adaptation of the Nepalese porters due to improved oxygenation of muscles frequently undergoing hypoxic exercise. Muscle structure, individual factors, and altitude exposure time should be taken into account to move on the knowledge of oxygen delivery and utilization at altitude

Effects of endurance, resistance and neuro-muscular electrical stimulation trainings to the anthropometric and functional mobility domains in elderly

Background and aims There\u2019s the need to increase physical activity engagement to promote healthy ageing. Different training protocols elicit different morpho-functional effects: the comprehension of the related assessment tests is a key to improve the specific proposals and to monitor adequately the adaptations. We aimed to identify the functional adaptation processes basing on different training protocols. Methods 40 healthy elderly (28 males and 12 females, 70.7 \ub1 4.39 y) were randomly divided into 4 groups: endurance, resistance, Neuro-Muscular Electrical Stimulation and control, trained for 12 weeks andex-post evaluated on anthropometric and functional domains. Results We found: significant effect for gender, time and time 7 protocols for Five Times Sit-to-Stand Test and Timed Up-and-Go test. Post-hoc analyses revealed effect for resistance and Neuro-Muscular Electrical Stimulation on Five Times Sit-to-Stand Test, and for endurance and Neuro-Muscular Electrical Stimulation on Timed Up-and-Go test. Correlations and factorial analysis linked Five Times Sit-to-Stand Test and Timed Up-and-Go test on the functional domain. Conclusions Medium-term physical interventions significantly modified functional characteristics of elderly. We found no ex-post effect on anthropometric parameters. The two functional tests are based on different underlying domains, our data therefore suggest to use both of them to specifically evaluate the training-induced functional adaptations in elderly. Our results promote the usefulness of evidence-based trainin

Signal transduction events induced by extracellular guanosine 5′triphosphate in excitable cells

A better understanding of the physiological effects of guanosine-based purines should help clarify the complex subject of purinergic signalling. We studied the effect of extracellular guanosine 5′triphosphate (GTP) on the differentiation of two excitable cell lines that both have specific binding sites for GTP: PC12 rat pheochromocytoma cells and C2C12 mouse skeletal muscle cells. PC12 cells can be differentiated into fully functional sympathetic-like neurons with 50′00 ng ml−1 of nerve growth factor, whereas serum starvation causes C2C12 cells to differentiate into myotubes showing functional excitation–contraction coupling, with the expression of myosin heavy chain proteins. Our results show that GTP enhances the differentiation of both of these excitable cell lines. The early events in guanosine-based purine signal transduction appear to involve an increase in intracellular Ca2+ levels and membrane hyperpolarization. We further investigated the early activation of extracellular-regulated kinases and phosphoinositide 3-kinase in GTP-stimulated PC12 and C2C12 cells, respectively. We found that GTP promotes the activation of both kinases. Together, our results suggest that, even if there are some differences in the signalling pathways, GTP-induced differentiation in both cell lines is dependent on an increase in intracellular Ca2+

Molecular and clinical correlates in iron overload associated with mutations in ferroportin

Mutations in ferroportin (Fpn) result in iron overload. We correlate the behavior of three Fpn mutants in vitro with patients' phenotypes. Patients with Fpn mutations A77D or N174I showed macrophage iron loading. In cultured cells, FpnA77D did not reach the cell surface and cells did not export iron. Fpn mutant N1741 showed plasma membrane and intracellular localization, and did not transport iron. Fpn mutation G80S was targeted to the cell surface and was transport competent, however patients showed macrophage iron. We suggest that FpnG80S represents a class of Fpn mutants whose behavior in vitro does not explain the patients' phenotype

Prohepcidin Levels in Refractory Anaemia Caused by Lead Poisoning

Recent research evidence suggests a central role for hepcidin in iron homeostasis. Hepcidin is a hormone synthesized in the liver. Hepcidin is also thought to play a vital role in the pathogenic mechanism of anaemia in patients with inflammation or chronic disease. A 38-year-old female who presented with recurrent abdominal pain was found to have raised urinary porphyrins and a blood lead level of 779 μg/l. Her haemoglobin level was 8.3 g/dl. Her MCV was normal. Serum ferritin, B12 and folate were normal. Her serum prohepcidin level was 2,489 ng/ml (normal <450 ng/ml). To our knowledge, this is the first report of raised prohepcidin levels in a patient with anaemia of chronic disease resulting from lead poisoning