Characterizing the Gait of People With Different Types of Amputation and Prosthetic Components Through Multimodal Measurements: A Methodological Perspective

Prosthetic gait implies the use of compensatory motor strategies, including alterations in gait biomechanics and adaptations in the neural control mechanisms adopted by the central nervous system. Despite the constant technological advancements in prostheses design that led to a reduction in compensatory movements and an increased acceptance by the users, a deep comprehension of the numerous factors that influence prosthetic gait is still needed. The quantitative prosthetic gait analysis is an essential step in the development of new and ergonomic devices and to optimize the rehabilitation therapies. Nevertheless, the assessment of prosthetic gait is still carried out by a heterogeneous variety of methodologies, and this limits the comparison of results from different studies, complicating the definition of shared and well-accepted guidelines among clinicians, therapists, physicians, and engineers. This perspective article starts from the results of a project funded by the Italian Worker's Compensation Authority (INAIL) that led to the generation of an extended dataset of measurements involving kinematic, kinetic, and electrophysiological recordings in subjects with different types of amputation and prosthetic components. By encompassing different studies published along the project activities, we discuss the specific information that can be extracted by different kinds of measurements, and we here provide a methodological perspective related to multimodal prosthetic gait assessment, highlighting how, for designing improved prostheses and more effective therapies for patients, it is of critical importance to analyze movement neural control and its mechanical actuation as a whole, without limiting the focus to one specific aspect

Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S- HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states

Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S- HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states

Targeted therapies and complete responses in first line treatment of metastatic renal cell carcinoma. A meta-analysis of published trials

Antiangiogenic agents (AAs) have reported grater efficacy compared to interferon. Despite these advances, radiological complete response to therapy is rare. We meta-analyzed the incidence of complete response in patients treated with AAs and in controls in main randomized clinical trials for first-line therapy in metastatic renal cell carcinoma.PubMed was reviewed for phase II-III randomized clinical trials with AAs vs. non-AAs in patients with good or intermediate prognosis. We calculated the relative risk of events in patients assigned to AAs compared to control.Five RCTs were found; four were phase III and one was phase II. A total of 2747 patients was valuable for final analysis and randomized to receive AAs or control. Patients in the control-group had interferon (85%) or placebo (15%); patients in the AAs-group received bevacizumab (48%), sunitinib (26%), pazopanib (20%) or sorafenib (6%).The incidence of complete response in patients treated with AAs was 2.0% (95% CI, 1.2-2.8) compared to 1.4% (95% CI, 0.7-2.1) in the control arm. Comparing the different type of AAs, the incidence of complete response was 2.5% (95% CI, 1.2-3.8) in the bevacizumab group and 1.6% (95% CI, 0.1-2.5) in the TKIs group. The relative risk to have a complete response was 1.52 (95% CI, 0.85-2.73; p=0.16) in patients treated with AAs compared to controls; this was found higher in patients treated with TKIs compared to bevacizumab.The complete response is a rare event in metastatic kidney tumor, even if AAs reported greater efficacy in terms of progression-free survival and of overall response rate, they did not increase the curative rate of metastatic disease. Probably, some biologic factors other than angiogenesis may influence the complete response in this disease. © 2013 Elsevier Ltd

Global Muscle Coactivation of the Sound Limb in Gait of People with Transfemoral and Transtibial Amputation

The aim of this study was to analyze the effect of the level of amputation and various prosthetic devices on the muscle activation of the sound limb in people with unilateral transfemoral and transtibial amputation. We calculated the global coactivation of 12 muscles using the time-varying multimuscle coactivation function method in 37 subjects with unilateral transfemoral amputation (10, 16, and 11 with mechanical, electronic, and bionic prostheses, respectively), 11 subjects with transtibial amputation, and 22 healthy subjects representing the control group. The results highlighted that people with amputation had a global coactivation temporal profile similar to that of healthy subjects. However, amputation increased the level of the simultaneous activation of many muscles during the loading response and push-off phases of the gait cycle and decreased it in the midstance and swing subphases. This increased coactivation probably plays a role in prosthetic gait asymmetry and energy consumption. Furthermore, people with amputation and wearing electronic prosthesis showed lower global coactivation when compared with people wearing mechanical and bionic prostheses. These findings suggest that the global lower limb coactivation behavior can be a useful tool to analyze the motor control strategies adopted and the ability to adapt to the prosthetic device

Preanalytical Procedures for DNA Studies: The Experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Preanalytical variables, including the anticoagulants and stabilizing agents, time, storage temperature, and methods of DNA extraction applied to blood samples, may affect quality and quantity of isolated nucleic acids for future genomic applications. Considering the large number of collected samples, standard operating procedure optimization for whole blood preservation before DNA extraction is a crucial step in a biological repository. Moreover, the future application of the biological material may not be known subsequent to its extraction. To define standard operating procedures for whole blood preservation before DNA extraction, we aimed to determine whether different combinations of anticoagulants, blood storage temperatures, and time intervals before storage at -80 degrees C might have an impact on quality and quantity of subsequent extracted DNA. After spectrophotometer quantification, the quality and integrity of DNA were assessed by agarose gel electrophoresis, polymerase chain reaction, and real-time polymerase chain reaction methods. We observed that decrease in DNA recovery during blood storage time was more pronounced at room temperature than at 4 degrees C. Based on our experience, we recommend as anticoagulants of choice sodium citrate and ethylenediaminetetraacetate, whereas sodium citrate theophylline adenosine dipyridamole could represent an alternative choice, excluding a priori lithium heparin and Fluoride-Oxalate. Based on the overall evaluation criteria, we conclude that the procedures necessary to preserve the whole blood before the DNA extraction may have a significant impact on downstream molecular biological applications

An AT-rich region in the APC gene may cause misinterpretation of familial adenomatous polyposis molecular screening

Familial adenomatous polyposis (FAP) is an autosomal-dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism

DNA fingerprinting for sample authentication in biobanking: recent perspectives

The availability of biological samples has assumed a crucial role in the field of biomedical research in order to assess predisposition to complex diseases, identification and validation of new diagnostic biomarkers, drug targets, and improvement of monitoring strategies. To ensure correct collection, storage, and use of biological material, a biobank requires standard operating procedures, not only to guarantee the integrity of the sample, but also for the traceability and uniqueness of the same sample and to avoid potential contamination. Over the past three decades, researchers have increasingly used DNA polymorphisms for biological identification purposes, radically changing forensic investigations. This revolution has led to the birth of new scientific terms, such as “DNA fingerprinting” or “genetic profiling”, and a new field of legal medicine known as “forensic genetics”. Given the significant need for a biobank to devise a system of identification, authentication, and traceability of biological samples, many researchers are applying forensic genetics-based methodologies to the science of biobanking. This review recapitulates the main DNA polymorphisms presently known and the techniques most widely used for biological identification purposes, from standard methods based on short tandem repeats, to use of single nucleotide polymorphisms, through to the more recent insertion/deletion polymorphisms. The experience of several research groups who have applied classical or innovative methods for identification of biological samples in the context of a biorepository is also discussed. Finally, some technical issues are suggested that could facilitate decisions about the most appropriate markers and DNA fingerprint methodology for evaluation of identity and traceability in each disease-based biobank

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications