Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites.

RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.

Enhancing SCI Care: Using Wearable Technologies for Physical Activity, Sleep, and Cardiovascular Health.

Wearable devices have the potential to advance healthcare by enabling real-time monitoring of biobehavioral data and facilitating the management of an individual's health conditions. Individuals living with spinal cord injury (SCI) have impaired motor function, which results in deconditioning and worsening cardiovascular health outcomes. Wearable devices may promote physical activity and allow the monitoring of secondary complications associated with SCI, potentially improving motor function, sleep, and cardiovascular health. However, several challenges remain to optimize the application of wearable technologies within this population. One is striking a balance between research-grade and consumer-grade devices in terms of cost, accessibility, and validity. Additionally, limited literature supports the validity and use of wearable technology in monitoring cardio-autonomic and sleep for individuals with SCI. Future directions include conducting performance evaluations of wearable devices to precisely capture the additional variation in movement and physiological parameters seen in those with SCI. Moreover, efforts to make the devices small, lightweight, and inexpensive for consumer ease of use may impact those with severe motor impairments. Overcoming these challenges holds the potential for wearable devices to help individuals living with SCI receive timely feedback to manage their health conditions and help clinicians gather comprehensive patient health information to aid in diagnosis and treatment.

The Clinical Relevance of Autonomic Dysfunction, Cerebral Hemodynamics, and Sleep Interactions in Individuals Living With SCI.

A myriad of physiological impairments is seen in individuals after a spinal cord injury (SCI). These include altered autonomic function, cerebral hemodynamics, and sleep. These physiological systems are interconnected and likely insidiously interact leading to secondary complications. These impairments negatively influence quality of life. A comprehensive review of these systems, and their interplay, may improve clinical treatment and the rehabilitation plan of individuals living with SCI. Thus, these physiological measures should receive more clinical consideration. This special communication introduces the under investigated autonomic dysfunction, cerebral hemodynamics, and sleep disorders in people with SCI to stakeholders involved in SCI rehabilitation. We also discuss the linkage between autonomic dysfunction, cerebral hemodynamics, and sleep disorders and some secondary outcomes are discussed. Recent evidence is synthesized to make clinical recommendations on the assessment and potential management of important autonomic, cerebral hemodynamics, and sleep-related dysfunction in people with SCI. Finally, a few recommendations for clinicians and researchers are provided.

The hypoxic ventilatory response and hypoxic burden are predictors of the magnitude of ventilatory long-term facilitation in humans.

Mild intermittent hypoxia initiates progressive augmentation (PA) and ventilatory long-term facilitation (vLTF) in humans. The magnitude of these forms of plasticity might be influenced by anthropometric and physiological variables, as well as protocol elements. However, the impact of many of these variables on the magnitude of respiratory plasticity has not been established in humans. A meta-analysis was completed using anthropometric and physiological variables obtained from 124 participants that completed one of three intermittent hypoxia protocols. Simple correlations between the aggregate variables and the magnitude of PA and vLTF standardized to baseline was completed. Thereafter, the variables correlated to PA or vLTF were input into a multilinear regression equation. Baseline measures of the hypoxic ventilatory response was the sole predictor of PA (R = 0.370, P = 0.012). Similarly, this variable along with the hypoxic burden predicted the magnitude of vLTF (R = 0.546, P < 0.006 for both variables). In addition, the magnitude of PA was strongly correlated to vLTF (R = 0.617, P < 0.001). Anthropometric measures do not predict the magnitude of PA and vLTF in humans. Alternatively, the hypoxic ventilatory response was the sole predictor of PA, and in combination with the hypoxic burden, predicted the magnitude of vLTF. These influences should be considered in the design of mild intermittent hypoxia protocol studies in humans. Moreover, the strong correlation between PA and vLTF suggests that a common mechanistic pathway may have a role in the initiation of these forms of plasticity. KEY POINTS: Mild intermittent hypoxia initiates progressive augmentation (PA) and ventilatory long-term facilitation (vLTF) in humans. Many of the anthropometric and physiological variables that could impact the magnitude of these forms of plasticity are unknown. Anthropometric and physiological variables were measured from a total of 124 participants that completed one of three distinct intermittent hypoxia protocols. The variables correlated to PA or vLTF were input into a multilinear regression analysis. The hypoxic ventilatory response was the sole predictor of PA, while this variable in addition to the average hypoxic burden predicted the magnitude of vLTF. A strong correlation between PA and vLTF was also revealed. These influences should be considered in the design of mild intermittent hypoxia protocol studies in humans. Moreover, the strong correlation between PA and vLTF suggests that a common mechanistic pathway may have a role in the initiation of these forms of plasticity.

Remote Ischemic conditioning as an emerging tool to improve corticospinal transmission in individuals with chronic spinal cord injury.

PURPOSE OF REVIEW: Remote ischemic conditioning (RIC) involves transient blood flow restriction to one limb leading to systemic tissue-protective effects. RIC shares some potential underlying mechanisms with intermittent hypoxia (IH), in which brief bouts of systemic hypoxia trigger increases in growth factor expression and neural plasticity. RIC has shown promise in acute myocardial infarction and stroke but may be applicable toward chronic neuropathology as well. Consequently, this review discusses similarities and differences between RIC and IH and presents preliminary and ongoing research findings regarding RIC. RECENT FINDINGS: Several publications demonstrated that combining RIC with motor training may enhance motor learning in adults with intact nervous systems, though the precise mechanisms were unclear. Our own preliminary data has found that RIC, in conjunction with task specific exercise, can increase corticospinal excitability in a subset of people without neurological injury and in those with chronic cervical spinal cord injury or amyotrophic lateral sclerosis. SUMMARY: RIC is a low-cost intervention easy to deliver in a clinical or home setting. Its potential application to facilitate neural plasticity and motor learning during rehabilitation training for individuals with chronic neurological disorders is a novel concept requiring further investigation to characterize mechanisms, safety, and efficacy.

Daily Exposure to Mild Intermittent Hypoxia Reduces Blood Pressure in Male Patients with Obstructive Sleep Apnea and Hypertension.

Rationale: Daily exposure to mild intermittent hypoxia (MIH) may elicit beneficial cardiovascular outcomes. Objectives: To determine the effect of 15 days of MIH and in-home continuous positive airway pressure treatment on blood pressure in participants with obstructive sleep apnea and hypertension. Methods: We administered MIH during wakefulness 5 days/week for 3 weeks. The protocol consisted of twelve 2-minute bouts of hypoxia interspersed with 2 minutes of normoxia. End-tidal carbon dioxide was maintained 2 mm Hg above baseline values throughout the protocol. Control participants were exposed to a sham protocol (i.e., compressed air). All participants were treated with continuous positive airway pressure over the 3-week period. Results are mean ± SD. Measurements and Main Results: Sixteen male participants completed the study (experimental n = 10; control n = 6). Systolic blood pressure at rest during wakefulness over 24 hours was reduced after 15 days of MIH (142.9 ± 8.6 vs. 132.0 ± 10.7 mm Hg; P < 0.001), but not following the sham protocol (149.9 ± 8.6 vs. 149.7 ± 10.8 mm Hg; P = 0.915). Thus, the reduction in blood pressure from baseline was greater in the experimental group compared with control (-10.91 ± 4.1 vs. -0.17 ± 3.6 mm Hg; P = 0.003). Modifications in blood pressure were accompanied by increased parasympathetic and reduced sympathetic activity in the experimental group, as estimated by blood pressure and heart rate variability analysis. No detrimental neurocognitive and metabolic outcomes were evident following MIH. Conclusions: MIH elicits beneficial cardiovascular and autonomic outcomes in males with OSA and concurrent hypertension. Clinical trial registered with www.clinicaltrials.gov (NCT03736382).

Is Sleep Disordered Breathing Confounding Rehabilitation Outcomes in Spinal Cord Injury Research?

The purpose of this article is to highlight the importance of considering sleep-disordered breathing (SDB) as a potential confounder to rehabilitation research interventions in spinal cord injury (SCI). SDB is highly prevalent in SCI, with increased prevalence in individuals with higher and more severe lesions, and the criterion standard treatment with continuous positive airway pressure remains problematic. Despite its high prevalence, SDB is often untested and untreated in individuals with SCI. In individuals without SCI, SDB is known to negatively affect physical function and many of the physiological systems that negatively affect physical rehabilitation in SCI. Thus, owing to the high prevalence, under testing, low treatment adherence, and known negative effect on the physical function, it is contended that underdiagnosed SDB in SCI may be confounding physical rehabilitation research studies in individuals with SCI. Studies investigating the effect of treating SDB and its effect on physical rehabilitation in SCI were unable to be located. Thus, studies investigating the likely integrated relationship among physical rehabilitation, SDB, and proper treatment of SDB in SCI are needed. Owing to rapid growth in both sleep medicine and physical rehabilitation intervention research in SCI, the authors contend it is the appropriate time to begin the conversations and collaborations between these fields. We discuss a general overview of SDB and physical training modalities, as well as how SDB could be affecting these studies.

Exposure to mild intermittent hypoxia increases loop gain and the arousal threshold in participants with obstructive sleep apnoea.

KEY POINTS: Repeated daily mild intermittent hypoxia has been endorsed as a therapy to promote the recovery of respiratory and limb motor dysfunction. One possible side-effect of this therapy is an increase in apnoeic event number and duration, which is particularly relevant to participants with motor disorders coupled with an increased incidence of sleep apnoea. In this study, we report that increases in apnoeic event number and duration, following exposure to daily intermittent hypoxia, are the result of an increase in respiratory loop gain and the arousal threshold, in participants with obstructive sleep apnoea. Daily exposure to mild intermittent hypoxia also led to an increase in the ventilatory response to arousal. Accordingly, individuals with motor disorders receiving mild intermittent hypoxia as a therapy should be screened for the presence of sleep apnoea, and if present, administration of intermittent hypoxia during hours of wakefulness should be combined with continuous positive airway pressure treatment during sleep. ABSTRACT: We determined if exposure to mild intermittent hypoxia (MIH) causes an increase in loop gain (LG) and the arousal threshold (AT) during non-rapid eye movement (NREM) sleep. Male participants with obstructive sleep apnoea (apnoea-hypopnoea index > 5 events/h), matched for age, body mass index and race were divided into two groups (n = 13 in each group). Following a baseline sleep study, one group was exposed to twelve 4-min episodes of hypoxia each day for 10 days and the other group to a sham protocol (SP). On Days 1 and 10, a sleep study was completed following exposure to MIH or the SP. For each sleep study, LG and the AT were measured during NREM sleep, using a model-based approach, and expressed as a fraction of baseline measures. LG increased after exposure to MIH (Day 1: 1.11 ± 0.03, P = 0.002, Day 10: 1.17 ± 0.05, P = 0.001), but not after the SP (Day 1: 1.03 ± 0.04, P = 1.0, Day 10: 1.0 ± 0.02, P = 1.0). AT also increased after exposure to MIH (Day 1: 1.13 ± 0.05, P = 0.01, Day 10: 1.19 ± 0.08, P = 0.05) but not after the SP (Day 1: 1.04 ± 0.05, P = 0.6, Day 10: 0.96 ± 0.04, P = 1.0). Our results might account for increases in apnoea frequency and duration previously observed during NREM sleep following exposure to MIH. Our results also have implications for the use of MIH as a therapeutic modality.

Effects of Overground Locomotor Training on Walking Performance in Chronic Cervical Motor Incomplete Spinal Cord Injury: A Pilot Study.

OBJECTIVE: To determine the effects of a novel overground locomotor training program on walking performance in people with chronic cervical motor incomplete spinal cord injury (iSCI). DESIGN: Before-after pilot study. SETTING: Human performance research laboratory. PARTICIPANTS: Adults (N=6, age >18y) with chronic cervical iSCI with American Spinal Injury Association Impairment Scale grades C and D. INTERVENTIONS: Overground locomotor training included two 90-minute sessions per week for 12 to 15 weeks. Training sessions alternated between uniplanar and multiplanar stepping patterns. Each session was comprised of 5 segments: joint mobility, volitional muscle activation, task isolation, task integration, and activity rehearsal. MAIN OUTCOME MEASURES: Overground walking speed, oxygen consumption (V˙o2), and carbon dioxide production (V˙co2). RESULTS: Overground locomotor training increased overground walking speed (.36±.20 vs .51±.24 m/s, P<.001, d=.68). Significant decreases in V˙o2 (6.6±1.3 vs 5.7±1.4mL·kg·min, P=.038, d=.67) and V˙co2 (753.1±125.5 vs 670.7±120.3mL/min, P=.036, d=.67) during self-selected constant work rate treadmill walking were also noted after training. CONCLUSIONS: The overground locomotor training program used in this pilot study is feasible and improved both overground walking speed and walking economy in a small sample of people with chronic cervical iSCI. Future studies are necessary to establish the efficacy of this overground locomotor training program and to differentiate among potential mechanisms contributing to enhanced walking performance in people with iSCI after overground locomotor training.

Fatigability, oxygen uptake kinetics and muscle deoxygenation in incomplete spinal cord injury during treadmill walking.

PURPOSE: The purpose of the present study was to characterize hypothesized relationships among fatigability and cardiorespiratory fitness in individuals with chronic motor-incomplete SCI (iSCI) during treadmill walking. The theoretical framework was that exacerbated fatigability would occur concomitantly with diminished cardiorespiratory fitness in people with iSCI. METHODS: Subjects with iSCI (n = 8) and an able-bodied reference group (REF) (n = 8) completed a 6-min walking bout followed by a walking bout of 30-min or until volitional exhaustion, both at a self-selected walking speed. Fatigability was assessed using both perceived fatigability and performance fatigability measures. Pulmonary oxygen uptake kinetics (VO2 on-kinetics) was measured breath-by-breath and changes in deoxygenated hemoglobin/myoglobin concentration (∆[HHb]) of the lateral gastrocnemius was measured by near-infrared spectroscopy. Adjustment of VO2 and ∆[HHb] on-kinetics were modeled using a mono-exponential equation. RESULTS: Perceived fatigability and performance fatigability were 52% and 44% greater in the iSCI group compared to the REF group (p = 0.003 and p = 0.004). Phase II time constant (τp) of VO2 on-kinetics and ∆[HHb] ½ time during resting arterial occlusion were 55.4% and 16.3% slower in iSCI vs REF (p < 0.01 and p = 0.047, respectively). CONCLUSIONS: The results of the present study may suggest that compromised O2 delivery and/or utilization may have contributed to the severity of fatigability in these individuals with iSCI. The understanding of the extent to which fatigability and VO2 and Δ[HHb] on-kinetics impacts locomotion after iSCI will assist in the future development of targeted interventions to enhance function.

Assessment of femoral physeal activity after transitory hemiepiphysiodesis using screws and nonabsorbable filament.

BACKGROUND: Different techniques for gradual correction of angular deformities of lower limbs exist. Long-term behavior of the growth plate after hemiepiphysiodesis is not yet well understood. We assessed the restoration of normal physeal activity in New Zealand rabbits, after transitory hemiepiphysiodesis, using screws and nonabsorbable filament. METHODS: We performed a lateral distal femoral epiphysiodesis using nonabsorbable filament and screws, in the right knees of 14 New Zealand male rabbits, aged 11 weeks. Two groups were created: in group 1, the suture was cut after 1 month, and in group 2 it was left uncut. Simple plain radiographs were taken at the beginning and at 4, 8, and 12 weeks of follow-up to evaluate lower limb deformity rate and femoral length. The left knee of each rabbit served as controls. Angular deformity and femoral length were compared between the groups. RESULTS: Eleven rabbits completed the protocol. The control group had no modification in lower limb mechanical axis. On the fourth week, all operated limbs had a significant valgus tibiofemoral angle variation (mean 24 degrees, P<0.05). When the filament was cut, there was complete restoration of the mechanical axis. When the filament was kept, tibiofemoral angle increased its valgus deformity until the eighth week (mean 32 degrees) without changes thereafter. The final femoral length was shorter in group 2 compared with the other 2 groups (P<0.05), whereas group 1 was slightly longer than the control group (P>0.05). CONCLUSIONS: Lateral distal femoral hemiepiphysiodesis with nonabsorbable filament resulted in a valgus deformity on the femur. Once the filament is cut, the femur can restore its normal alignment, while maintaining longitudinal growth. Keeping the physeal tether increases the valgus achieved during the first 8 weeks, and remained stable throughout the study, shortening the bone. CLINICAL RELEVANCE: This is an effective alternative for the correction of angular limb deformities that maintains physeal function and may be useful for orthopedic surgeons. It could also represent an adequate model for the study of rebound effect.

Intermittent hypoxia: a call for harmonization in terminology.

Mild intermittent hypoxia may be a potent novel strategy to improve cardiovascular function, motor and cognitive function, and altitude acclimatization. However, there is still a stigma surrounding the field of intermittent hypoxia (IH). Major contributors to this stigma may be due to the overlapping terminology, heterogeneous methodological approaches, and an almost dogmatic focus on different mechanistic underpinnings in different fields of research. Many clinicians and investigators explore the pathophysiological outcomes following long-term exposure to IH in an attempt to improve our understanding of sleep apnea (SA) and develop new treatment plans. However, others use IH as a tool to improve physiological outcomes such as blood pressure, motor function, and altitude acclimatization. Unfortunately, studies investigating the pathophysiology of SA or the potential benefit of IH use similar, unstandardized terminologies facilitating a confusion surrounding IH protocols and the intentions of various studies. In this perspective paper, we aim to highlight IH terminology-related issues with the aim of spurring harmonization of the terminology used in the field of IH research to account for distinct outcomes of hypoxia exposure depending on protocol and individuum.

Racial differences in upper airway collapsibility and loop gain in young adult males.

STUDY OBJECTIVES: Previous studies reported that the apnea-hypopnea index was similar in young adult Black and White participants. However, whether this similarity reflects an analogous combination of apneas and hypopneas is unknown. Likewise, the physiological mechanisms underlying this similarity has not been explored. METHODS: 60 Black and 48 White males completed the study. After matching for age and body mass index, 41 participants remained in each group. All participants completed a sleep study. Subsequently, standard sleep indices along with loop gain and the arousal threshold were determined. In addition, airway collapsibility (24 of 60 and 14 of 48 participants) and the hypoxic ventilatory response during wakefulness (30 of 60 and 25 of 48 participants) was measured. RESULTS: The apnea-hypopnea index was similar in Blacks and Whites (p = .140). However, the index was comprised of more apneas (p = .014) and fewer hypopneas (p = .025) in Black males. These modifications were coupled to a reduced loop gain (p = .0002) and a more collapsible airway (p = .030). These differences were independent of whether or not the groups were matched. For a given hypoxic response, loop gain was reduced in Black compared to White males (p = .023). CONCLUSIONS: Despite a similar apnea-hypopnea index, more apneas and fewer hypopneas were evident in young adult Black compared to White males. The physiological mechanisms that contribute to these events were also different between groups. Addressing these differences may be important when considering novel therapeutic approaches to eliminate apnea in Black and White participants.

Divergent Ventilatory and Blood Pressure Responses are Evident Following Repeated Daily Exposure to Mild Intermittent Hypoxia in Males with OSA and Hypertension.

Introduction: Resting minute ventilation and ventilation during and following hypoxia may be enhanced following daily exposure to mild intermittent hypoxia (MIH). In contrast, resting systolic blood pressure (SBP) is reduced following daily exposure to MIH. However, it is presently unknown if the reduction in resting SBP following daily exposure, is coupled with reduced SBP responses during and after acute exposure to MIH. Methods: Participants with obstructive sleep apnea (OSA) and hypertension (n = 10) were exposed to twelve 2-min bouts of MIH (oxygen saturation-87%)/day for 15 days. A control group (n = 6) was exposed to a sham protocol during which compressed air (i.e., FIO2 = 0.21) was inspired in place of MIH. Results: The hypoxic ventilatory response (HVR) and hypoxic systolic blood pressure response (HSBP) increased from the first to the last hypoxic episode on the initial (HVR: 0.08 ± 0.02 vs. 0.13 ± 0.02 L/min/mmHg, p = 0.03; HSBP: 0.13 ± 0.04 vs. 0.37 ± 0.06 mmHg/mmHg, p < 0.001) and final (HVR: 0.10 ± 0.01 vs. 0.15 ± 0.03 L/min/mmHg, p = 0.03; HSBP: 0.16 ± 0.03 vs. 0.41 ± 0.34 mmHg/mmHg, p < 0.001) day. The magnitude of the increase was not different between days (p ≥ 0.83). Following exposure to MIH, minute ventilation and SBP was elevated compared to baseline on the initial (MV: 16.70 ± 1.10 vs. 14.20 ± 0.28 L/min, p = 0.01; SBP: 167.26 ± 4.43 vs. 151.13 ± 4.56 mmHg, p < 0.001) and final (MV: 17.90 ± 1.25 vs. 15.40 ± 0.77 L/min, p = 0.01; SBP: 156.24 ± 3.42 vs. 137.18 ± 4.17 mmHg, p < 0.001) day. The magnitude of the increases was similar on both days (MV: 3.68 ± 1.69 vs. 3.22 ± 1.27 L/min, SBP: 14.83 ± 2.64 vs. 14.28 ± 1.66 mmHg, p ≥ 0.414). Despite these similarities, blood pressure at baseline and at other time points during the MIH protocol was reduced on the final compared to the initial day (p ≤ 0.005). Conclusion: The ventilatory and blood pressure responses during and following acute MIH were similar on the initial and final day of exposure. Alternatively, blood pressure was down regulated, while ventilation was similar at all time points (i.e., baseline, during and following MIH) after daily exposure to MIH.

Walking endurance, muscle oxygen extraction, and perceived fatigability after overground locomotor training in incomplete spinal cord injury: A pilot study.

Objective: The purpose of this study was to examine the effects of overground locomotor training (OLT) on walking endurance and gastrocnemius oxygen extraction in people with chronic cervical motor-incomplete spinal cord injury (SCI).Design: Prospective single-arm pre-post pilot study.Setting: Human Performance Research Laboratory.Participants: Adult men with traumatic chronic cervical SCI (n = 6; age = 30.8 ± 12.5).Intervention: Twenty-four sessions of structured OLT.Outcome measures: Walking endurance was determined during a constant work-rate time-to-exhaustion treadmill test. Normalized perceived fatigability was calculated by dividing subjective ratings of tiredness by walking time. Cardiorespiratory outcomes and muscle oxygen extraction were analyzed using breath-by-breath gas-exchange and near-infrared spectroscopy.Results: OLT resulted in large effects on walking endurance (1232 ± 446 s vs 1645 ± 255 s; d = 1.1; P = 0.045) and normalized perceived fatigability (5.3 ± 1.5 a.u. vs 3.6 ± 0.9 a.u.; d = 1.3; P = 0.033). Small-to-medium effects on absolute (2.8 ± 2.5 a.u. vs 4.2 ± 3.5 a.u.; d = 0.42; P = 0.035) and isotime (2.8 ± 2.5 a.u. vs 3.8 ± 3.0 a.u.; d = 0.33; P = 0.023) muscle oxygen extraction were also observed after OLT.Conclusion: These findings provide preliminary data supporting the potential for improved walking endurance, enhanced muscle O2 extraction, and reduced perceived fatigability in people with chronic cervical motor-incomplete SCI following the OLT program described in this study.

A Ge.F.I. - ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method.

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists - International Society of Forensic Genetics (Ge.F.I. - ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Platelet rich plasma-derived microvesicles increased in vitro wound healing.

OBJECTIVE: Platelet rich plasma (PRP) is a haemoderivative used in clinical practice for the treatment of hard-to-heal wounds. Platelet (PLT) activation is a key factor in the wound healing process leading to the production of extracellular vesicles. We obtained PRP and PRP-derived microvesicles (PLT-MVs) from healthy donors and compared their pro-healing efficacy in an in vitro wound model using human keratinocytes. MATERIALS AND METHODS: We evaluated PLT-MVs' direct effect on an in vitro model of wound healing. PRP, PRP activated using calcimycin, and PLT-MVs separated by high speed centrifugation were added to scratched keratinocyte monolayers. Fluorescein diacetate was used in flow cytometry to distinguish PLTs and PLT-MVs from debris, and then, PLT-MVs were quantified on the basis of relative dimensions (Forward Scatter signals). RESULTS: Wound areas were measured at time 0 and after 24 hours and they were healed by 24.80 ± 4.28% in control conditions, while PRP, activated PRP, and PLT-MVs increased closure of 62.94 ± 0.96%, 52.69 ± 17.20% and 52.76 ± 9.44%, respectively. CONCLUSIONS: Our results indicate that PRP pro-healing effects were fully replicable by PLT-MVs, suggesting a key role of microvesicles in the healing process and a possible clinical use as an alternative to PRP.