Myoelectric interface for neurorehabilitation conditioning to reduce abnormal leg co-activation after stroke: a pilot study.

BACKGROUND: The ability to walk is an important factor in quality of life after stroke. Co-activation of hip adductors and knee extensors has been shown to correlate with gait impairment. We have shown previously that training with a myoelectric interface for neurorehabilitation (MINT) can reduce abnormal muscle co-activation in the arms of stroke survivors. METHODS: Here, we extend MINT conditioning to stroke survivors with leg impairment. The aim of this pilot study was to assess the safety and feasibility of using MINT to reduce abnormal co-activation between hip adductors and knee extensors and assess any effects on gait. Nine stroke survivors with moderate to severe gait impairment received 6 h of MINT conditioning over six sessions, either in the laboratory or at home. RESULTS: MINT participants completed a mean of 159 repetitions per session without any adverse events. Further, participants learned to isolate their muscles effectively, resulting in a mean reduction of co-activation of 70% compared to baseline. Moreover, gait speed increased by a mean of 0.15 m/s, more than the minimum clinically important difference. Knee flexion angle increased substantially, and hip circumduction decreased. CONCLUSION: MINT conditioning is safe, feasible at home, and enables reduction of co-activation in the leg. Further investigation of MINT's potential to improve leg movement and function after stroke is warranted. Abnormal co-activation of hip adductors and knee extensors may contribute to impaired gait after stroke. Trial registration This study was registered at ClinicalTrials.gov (NCT03401762, Registered 15 January 2018, https://clinicaltrials.gov/study/NCT03401762?tab=history&a=4 ).

A rare anaerobic cause of vertebral osteomyelitis and psoas abscess: A case study.

Parvimonas micra is an obligate anaerobe that forms part of the normal gastrointestinal flora. The advent of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) and 16s ribosomal RNA gene sequencing has led to increased detection of many rare anaerobic isolates, including Parvimonas micra. Typical risk factors for Parvimonas micra bacteremia include dental procedures or spinal instrumentation. Here, we report a case of Parvimonas micra spondylodiscitis and psoas abscess in a patient with no obvious antecedent risk factors and explore the challenges in isolation of the organism from tissue samples.

Myoelectric interface for neurorehabilitation conditioning to reduce abnormal leg co-activation after stroke: a pilot study.

Background: The ability to walk is an important factor in quality of life after stroke. Co-activation of hip adductors and knee extensors has been shown to correlate with gait impairment. We have shown previously that training with a myoelectric interface for neurorehabilitation (MINT) can reduce abnormal muscle co-activation in the arms of stroke survivors. Methods: Here, we extend MINT conditioning to stroke survivors with leg impairment. The aim of this pilot study was to assess the safety and feasibility of using MINT to reduce abnormal co-activation between hip adductors and knee extensors and assess any effects on gait. Nine stroke survivors with moderate to severe gait impairment received six hours of MINT conditioning over six sessions, either in the laboratory or at home. Results: MINT participants completed a mean of 159 repetitions per session without any adverse events. Further, participants learned to isolate their muscles effectively, resulting in a mean reduction of co-activation of 70% compared to baseline. Moreover, gait speed increased by a mean of 0.15 m/s, more than the minimum clinically important difference. Knee flexion angle increased substantially, and hip circumduction decreased. Conclusion: MINT conditioning is safe, feasible at home, and enables reduction of co-activation in the leg. Further investigation of MINT's potential to improve leg movement and function after stroke is warranted. Abnormal co-activation of hip adductors and knee extensors may contribute to impaired gait after stroke. Trial registration: This study was registered at ClinicalTrials.gov (NCT03401762, Registered 15 January 2018, https://clinicaltrials.gov/study/NCT03401762?tab=history&a=4).

Serum potassium changes during hypothermia and rewarming: a case series and hypothesis on the mechanism.

Introduction: Hypokalemia is known to occur in association with therapeutically induced hypothermia and is usually managed by the administration of potassium (K+). Methods: We reviewed data from 74 patients who underwent a therapeutic hypothermia protocol at our medical institution. Results: In four patients in whom data on serum K+ and temperature were available, a strong positive correlation between serum K+ and body temperature was found. Based on the close positive relationship between serum K+ and total body temperature, we hypothesize that serum K+ decreases during hypothermia owing to decreased activity of temperature-dependent K+ exit channels that under normal conditions are sufficiently active to match cellular K+ intake via sodium/K+/adenosine triphosphatase. Upon rewarming, reactivation of these channels results in a rapid increase in serum K+ as a result of K+ exit down its concentration gradient. Conclusion: Administration of K+ during hypothermia should be done cautiously and avoided during rewarming to avoid potentially life-threatening hyperkalemia. K+ exit via temperature-dependent K+ channels provides a logical explanation for the rebound hyperkalemia. K+ exit channels may play a bigger role than previously appreciated in the regulation of serum K+ during normal and pathophysiological conditions.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Wearable myoelectric interface enables high-dose, home-based training in severely impaired chronic stroke survivors.

BACKGROUND: High-intensity occupational therapy can improve arm function after stroke, but many people lack access to such therapy. Home-based therapies could address this need, but they don't typically address abnormal muscle co-activation, an important aspect of arm impairment. An earlier study using lab-based, myoelectric computer interface game training enabled chronic stroke survivors to reduce abnormal co-activation and improve arm function. Here, we assess feasibility of doing this training at home using a novel, wearable, myoelectric interface for neurorehabilitation training (MINT) paradigm. OBJECTIVE: Assess tolerability and feasibility of home-based, high-dose MINT therapy in severely impaired chronic stroke survivors. METHODS: Twenty-three participants were instructed to train with the MINT and game for 90 min/day, 36 days over 6 weeks. We assessed feasibility using amount of time trained and game performance. We assessed tolerability (enjoyment and effort) using a customized version of the Intrinsic Motivation Inventory at the conclusion of training. RESULTS: Participants displayed high adherence to near-daily therapy at home (mean of 82 min/day of training; 96% trained at least 60 min/day) and enjoyed the therapy. Training performance improved and co-activation decreased with training. Although a substantial number of participants stopped training, most dropouts were due to reasons unrelated to the training paradigm itself. INTERPRETATION: Home-based therapy with MINT is feasible and tolerable in severely impaired stroke survivors. This affordable, enjoyable, and mobile health paradigm has potential to improve recovery from stroke in a variety of settings. Clinicaltrials.gov: NCT03401762.

Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (KmAPP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.

Structural and functional studies of gpX of Escherichia coli phage P2 reveal a widespread role for LysM domains in the baseplates of contractile-tailed phages.

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the "minimal" myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.

Use of prophage free host for achieving homogenous population of bacteriophages: new findings.

We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation.

Antistaphylococcal activity of bacteriophage derived chimeric protein P128.

BACKGROUND: Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b) of lysostaphin.Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. RESULTS: Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA), and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 µg/mL) were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature.In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h.In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. CONCLUSIONS: The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for clearance of S. aureus nasal colonization and MRSA infection. The protein is active against globally prevalent antibiotic-resistant clinical isolates and other clinically significant staphylococcal species including S. epidermidis. The P128 hydrogel formulation was bactericidal against Staphylococci including S. aureus recovered from the nares of 31 healthy people, demonstrating its in situ efficacy.

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein.

BACKGROUND: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. RESULTS: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. CONCLUSIONS: We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection.

BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.