Structure of the shutdown state of myosin-2.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads1, which prevents filament formation and inactivates the motors2. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.

Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

A multiscale approach reveals the molecular architecture of the autoinhibited kinesin KIF5A.

Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is ∼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled-coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled-coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative-stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.

All-Inside Lateral Meniscal Repair via Anterolateral Portal Increases Risk of Vascular Injury: A Cadaveric Study.

PURPOSE: To compare the distance from the device tip to the neurovascular structures during an all-inside medial and lateral meniscal repair using anteromedial and anterolateral portals in a fresh-frozen cadaveric study. METHODS: Ten fresh-frozen cadaveric knees were studied. The popliteal artery, popliteal vein, and tibial nerve were identified after dissection via a posterior approach. An all-inside meniscal repair device was set to a 20-mm depth limit and inserted into a fixed point in the posterior horn at the meniscocapsular junction. This was performed for medial and lateral menisci via anteromedial and anterolateral arthroscopic portals. The distances between the device tip and the neurovascular structures were measured. We performed t tests to determine statistical significance. RESULTS: The distance between the device and popliteal artery was significantly closer when aimed at the posterior horn of the lateral meniscus via the anterolateral portal (4.7 ± 2.3 mm) versus the anteromedial portal (13.0 ± 8.0 mm, P = .010). The distance to the popliteal vein was closer via the anterolateral portal (6.7 ± 2.9 mm) versus the anteromedial portal (13.9 ± 5.8 mm, P = .004). For medial meniscal repair, the distance to the popliteal artery was significantly closer via the anteromedial portal (12.8 ± 11.3 mm) versus the anterolateral portal (23.8 ± 7.7 mm, P = .022). The distance to the popliteal vein was closer via the anteromedial portal (16.5 ± 11.3 mm) versus the anterolateral portal (28.3 ± 8.2 mm, P = .017). No significant difference was found in the distance to the tibial nerve when aimed at either meniscus via either portal. CONCLUSION: For all-inside meniscal repair, the popliteal vein is at risk and the popliteal artery is at high risk of injury when the posterior horn of the lateral meniscus is repaired via an anterolateral working portal. CLINICAL RELEVANCE: The popliteal artery and vein are at risk of injury when the posterior horn of the lateral meniscus undergoes all-inside repair via the anterolateral portal. Surgeons need to be aware of the risks when performing this repair.

Salvage arthrodesis for failed first metatarsophalangeal joint arthroplasty: A network meta-analysis.

BACKGROUND: We aim to provide an evidence-based literature review of salvage arthrodesis for failed first metatarsophalangeal joint arthroplasty with a network meta-analysis. METHODS: A search of PubMed, Embase and Cochrane databases was conducted in December 2016 which identified 12 relevant articles out of 340 articles assessing the efficacy of salvage arthrodesis for failed joint arthroplasty of the first metatarsophalangeal joint. The 12 studies were assigned a level of evidence (I-V) and interventions were graded a level of recommendation (A-C, I) in support of or against the treatment modality. RESULTS: There is fair evidence (grade B) to support salvage arthrodesis with structural bone graft. There is poor evidence (grade C) for salvage arthrodesis without bone graft. There was no good evidence (grade A) to recommend either intervention. Meta-analysis showed that salvage arthrodesis resulted in improved functional outcome over time. CONCLUSIONS: Salvage arthrodesis showed good bone union rates and patient satisfaction. LEVEL OF CLINICAL EVIDENCE: III - Systematic Review of Level III studies.

The effect of interactive reminders on medication adherence: A randomized trial.

Expanding on evidence that interventions to improve health are more effective when informed by behavioral science, we explore whether reminders designed to harness behavioral science principles can improve medication adherence. We conducted a randomized controlled trial with 46,581 U.S. participants with commercial or Medicare Advantage insurance from Humana. Participants were randomly assigned to one of four experimental conditions. Participants in the usual care condition only received standard mailings that the insurer usually sends. In addition to the standard mailings, participants in the other three conditions also received (1) mailings that reminded them to take a target medication (basic reminder condition), (2) reminders that prompted them to predict their medication adherence in the next 30days (prediction condition), or (3) reminders that prompted them to commit to a self-determined level of adherence for the next 30days (commitment condition). We sent these mailings once a month for three months from November, 2014 through January, 2015, and tracked prescription refills. We find that, during the mailing period, reminders increased adherence by 0.95 percentage points (p<0.05), and this effect was driven by the prediction and commitment conditions; during the three-month post-mailing period, reminders increased adherence by 0.98 percentage points (p<0.05), and this effect was driven by the basic reminder and commitment conditions. The reminders increased medication adherence by 0.7 pills per dollar spent over our 181day study period. Trial registry name: Effect of Reminders on Adherence. Registration identification number: NCT02411006 URL for the registry: https://clinicaltrials.gov/ct2/show/NCT02411006.

Radiofrequency microtenotomy for plantar fasciitis: A systematic review and meta-analysis.

BACKGROUND: Currently, there is limited evidence on outcomes for plantar fascia radiofrequency microtenotomy. An evidence-based systematic review and meta-analysis for outcomes of radiofrequency microtenotomy for the treatment of plantar fasciitis was conducted. METHODS: A comprehensive evidence-based literature review of PubMed and Cochrane Databases was conducted in March 2019, which identified 11 relevant articles assessing the efficacy of plantar fascia radiofrequency microtenotomy. The studies were then assigned to a level of evidence (I-IV). Individual studies were reviewed to provide a grade of recommendation (A-C, I) according to the Wright classification in support of or against endoscopic plantar fascia release. Meta-analysis was performed for 7 of the studies that measured AOFAS scores. RESULTS: Based on the results of this evidence-based review, there was fair (grade B) evidence to support plantar fascia radiofrequency microtenotomy. There was a statistically significant mean increase of 40.9 in AOFAS scores post procedure. CONCLUSION: There was fair (grade B) evidence to recommend radiofrequency microtenotomy for plantar fasciitis. There is a need for more high quality level I randomized controlled trials with validated outcome measures to allow for stronger recommendations to be made. LEVEL OF EVIDENCE: Level II, systematic review of level II studies.

All-inside Techniques for Meniscal Radial Root Tear Repair.

Meniscal radial root tears can disrupt the load-bearing function of the meniscus and worsen instability in anterior cruciate ligament-deficient knees. Paracentral radial tears adjacent to the root (types 1, 2, and 4) repaired with a transtibial pullout suture technique can lead to over-medialization of the meniscal root and a high-tension nonanatomic repair. We propose 2 all-inside techniques for anatomic repair of medial and lateral meniscal radial root tears with (1) an all-inside meniscal repair device and (2) an antegrade suture passer. We present the technical requirements and tips for these techniques. For lateral meniscal radial root repair with an all-inside meniscal repair device, ideal viewing is from an anterolateral portal with device entry from an anteromedial portal to reduce the risk of vascular injury. We recommend at least 2 stitches across the tear, with the depth setting limited to 18 to 20 mm for a central stitch and 16 mm or less for a peripherally placed stitch. For root repair with an antegrade suture passer, viewing should be from an anteromedial portal with the passer entering from an anterolateral portal. At least 2 stitches should be placed across the tear, with 1 central and 1 peripheral or 1 superior and 1 inferior.

Endoscopic plantar fasciotomy for plantar fasciitis: A systematic review and network meta-analysis of the English literature.

BACKGROUND: Currently, there is limited evidence on outcomes for endoscopic plantar fasciotomy. OBJECTIVES: An evidence-based literature review for outcomes of endoscopic plantar fasciotomy for the treatment of plantar fasciitis is provided. METHODS: A comprehensive evidence-based literature review of PubMed and Cochrane databases was conducted on 9th March 2019, which identified 12 relevant articles assessing the efficacy of endoscopic plantar fasciotomy. The studies were then assigned to a level of evidence (I-IV). Individual studies were reviewed to provide a grade of recommendation (A-C, I) according to the Wright classification in support of or against endoscopic plantar fascia release. RESULTS: Based on the results of this evidence-based review, there is poor evidence (grade C) to support endoscopic plantar fascia release. Release of the medial 2/3 of the plantar fascia in endoscopic plantar fasciotomy was associated with higher AOFAS score. CONCLUSION: Although the majority of the level of evidence was low (level IV) and grade of recommendation was poor (grade C), there seemed to be good outcomes for endoscopic plantar fasciotomy. There is a need for more high quality level I randomized controlled trials with validated outcome measures to allow for stronger recommendations to be made.

Optimization of Membrane Protein Production Using Titratable Strains of E. coli.

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

A report of nontraumatic cortical subarachnoid hemorrhage and subsequent management.

AIM: Report a case of cortical subarachnoid hemorrhage (cSAH) and discuss its management. PATIENT & METHODS: A 66-year-old woman presents with acute onset left arm numbness and weakness. Initial head CT shows small hyperdensity in sulci typical for cSAH. Extensive workup with MRI, lumbar puncture and blood tests is performed. No signs of infection, vascular malformations, thrombosis or cancer are found. At outpatient follow-up, she is diagnosed with cSAH secondary to amyloid angiopathy. She is treated with gabapentin. RESULTS & CONCLUSION: Diagnosis of cSAH is challenging given its subtle findings, and management is empiric as there are only a few case series in literature.

Trigger finger release with stepwise preservation of the A1 pulley: a functional pulley-preserving technique.

The first annular (A1) pulley is an important structure of the hand, providing a biomechanical support to the metacarpophalangeal joint and maintaining joint stability and flexor tendon alignment. Albeit uncommon, disruption of this pulley can result in dislocation or ulnar drift of the digit, particularly pronounced in patients with rheumatoid arthritis. Despite this, the A1 pulley is commonly divided without reconstruction in trigger finger. Several annular pulley reconstructive techniques have been developed to preserve its function. However, development of recurrent triggering has been observed due to fibrosis, largely due to inadequate release of the pulley. We have developed a technique to increase the volume within the flexor sheath while preserving the A1 pulley by way of stepwise lengthening. This has enabled an increase in the diameter of the pulley to 4 times its original size. A prospective study was performed comprising 10 trigger finger releases with stepwise lengthening of the A1 pulley. In all patients, there were no complications, and good hand function was achieved with no recurrence of triggering at 6 weeks of follow-up. This technique can thus safely achieve trigger release without sacrifice of the function of the A1 pulley.