Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

Persistent SARS-CoV-2 infectivity greater than 50 days in a case series of allogeneic peripheral blood stem cell transplant recipients.

The coronavirus disease 19 (COVID-19) pandemic has infected tens of millions across the world, but there is a significant gap in our understanding about COVID-19 in the hematopoietic stem transplant (HSCT) recipient population. Prolonged viral shedding is frequently observed with severe acute respiratory syndrome coronavirus-2 (SARSCoV-2), but studies suggest viral loads decline 10 days after symptom onset. Current CDC guidance suggests that severely ill and immunocompromised hosts are no longer infectious after 20 days from symptom onset. Cycle threshold (Ct) values are inversely proportional to the viral load and are used to detect SARS-CoV-2 RNA concentration. Specimens with reverse transcriptase PCR (RT-PCR) Ct values > 33-34 have been associated with inability to culture virus, and have been used as a surrogate for diminished infectivity. We report two cases of  allogeneic peripheral blood stem cell transplant (PBSCT) recipients who had prolonged durations of infectivity with SARSCov-2, based on culture positivity and persistently low Ct values for greater than 50 days.

Comparison of the Quidel Sofia SARS FIA Test to the Hologic Aptima SARS-CoV-2 TMA Test for Diagnosis of COVID-19 in Symptomatic Outpatients.

The Quidel Sofia severe acute respiratory syndrome (SARS) fluorescent immunoassay (FIA) test (SOFIA) is a rapid antigen immunoassay for the detection of SARS coronavirus 2 (SARS-CoV-2) proteins from nasal or nasopharyngeal swab specimens. The purpose of this study was to compare the results of the SOFIA test to those of the Hologic Aptima SARS-CoV-2 TMA test (APTIMA TMA), a high-throughput molecular diagnostic test that uses transcription-mediated amplification (TMA) for the detection of SARS-CoV-2 nucleic acid from upper respiratory tract specimens. Three hundred forty-seven symptomatic patients from an urgent care center in an area with a high prevalence of SARS-CoV-2 infections were tested in parallel using nasal swabs for the SOFIA test and nasopharyngeal swabs for the APTIMA TMA test. The SOFIA test demonstrated a positive percent agreement (PPA) of 82.0% with the APTIMA TMA test for symptomatic patients tested ≤5 days from symptom onset and a PPA of 54.5% for symptomatic patients >5 days from symptom onset. The Cepheid Xpert Xpress SARS-CoV-2 reverse transcription-PCR (RT-PCR) test was used to determine the cycle threshold (CT ) value for any specimens that were discrepant between the SOFIA and APTIMA TMA tests. Using a CT value of ≤35 as a surrogate for SARS-CoV-2 culture positivity, we estimate that the SOFIA test detected 87.2% of symptomatic patients tested ≤5 days from symptom onset who were likely to be culture positive.

Attempted Isolation of Cryptococcus Species and Incidental Isolation of Exophiala dermatitidis from Human Oral Cavities.

Recent molecular studies suggest that Cryptococcus may inhabit the normal human mouth. We attempted to isolate Cryptococcus from 21 adult non-acutely ill patients and 40 volunteer medical and non-medical staff in Southeastern Wisconsin, USA. An upper lip sulcus culture and an oral rinse specimen were inoculated separately onto Staib (birdseed) agar containing chloramphenicol and incubated in gas impermeable zip lock bags at 35 °C. No cryptococci were grown from any of the 122 samples from the 61 subjects. Both specimens from a woman with no risk factors for fungal disease yielded a black yeast at 4 days on Staib agar. This isolate was shown to be Exophiala dermatitidis by colony and microscopic morphology, analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and sequencing through the internal transcribed spacer ribosomal RNA gene. This appears to be a novel isolation of E. dermatitidis from the oral cavity of a generally healthy human.

Autoantibodies targeting TRIM72 compromise membrane repair and contribute to inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM) involve chronic inflammation of skeletal muscle and subsequent muscle degeneration due to an uncontrolled autoimmune response; however, the mechanisms leading to pathogenesis are not well understood. A compromised sarcolemmal repair process could promote an aberrant exposure of intramuscular antigens with the subsequent initiation of an inflammatory response that contributes to IIM. Using an adoptive transfer mouse model of IIM, we show that sarcolemmal repair is significantly compromised in distal skeletal muscle in the absence of inflammation. We identified autoantibodies against TRIM72 (also known as MG53), a muscle-enriched membrane repair protein, in IIM patient sera and in our mouse model of IIM by ELISA. We found that patient sera with elevated levels of TRIM72 autoantibodies suppress sarcolemmal resealing in healthy skeletal muscle, and depletion of TRIM72 antibodies from these same serum samples rescues sarcolemmal repair capacity. Autoantibodies targeting TRIM72 lead to skeletal muscle fibers with compromised membrane barrier function, providing a continuous source of autoantigens to promote autoimmunity and further amplifying humoral responses. These findings reveal a potential pathogenic mechanism that acts as a feedback loop contributing to the progression of IIM.

Sensory focused exercise improves anxiety in Parkinson's disease: A randomized controlled trial.

Anxiety has been implicated as one of the greatest influences on quality of life in Parkinson's disease (PD). The etiology of anxiety is unclear, although previous work suggests that anxiety may be linked to sensory deficits that cause uncertainty in movement. Thus, the current study examined whether focusing attention on sensory feedback during goal-based exercise has the potential to provide benefits to anxiety in PD. Thirty-five participants with PD were randomized to either a Sensory Attention Focused Exercise (SAFEx) (i.e. internal focus of attention, n = 18) or Sham Exercise control (i.e. external focus of attention, n = 17) and completed 33 one-hour attention-based exercise sessions over 11-weeks. Before and after the program (pre and post), participants completed the Parkinson Anxiety Scale (PAS) questionnaire. The PAS includes three anxiety sections: persistent, episodic, and avoidance. Changes in the total PAS score and within each section of the PAS were subjected to two-factor mixed repeated measures ANCOVA. Significant group by time interactions demonstrated that from pre to post, total PAS scores (p = 0.007) and episodic anxiety scores (p = 0.010) significantly decreased in the SAFEx group only (ΔTotal PAS = -5.2, F(1,27) = 5.41, p = 0.028, ηp2 = 0.17; ΔEpisodic Score = -1.8, F(1,27) = 6.89, p = 0.014, ηp2 = 0.20). In conclusion, focusing attention on sensory feedback while completing goal-based exercises may provide significant benefits to improving anxiety in PD. As such, sensory attention focused exercise may be a critical adjunct therapy for improving anxiety, and ultimately quality of life in people with PD.

Strengthening Public Health in Wisconsin Through the Wisconsin Clinical Laboratory Network.

The Wisconsin Clinical Laboratory Network (WCLN) at the University of Wisconsin-Madison is a partnership of 138 clinical and public health laboratories (as of February 2019) coordinated by the Wisconsin State Laboratory of Hygiene. This article describes the WCLN, its current activities, and lessons learned through this partnership. A laboratory technical advisory group, which consists of representatives from clinical laboratories, provides clinical laboratory perspective to the WCLN and fosters communication among laboratories. Activities and resources available through the WCLN include annual regional meetings, annual technical workshops, webinars, an email listserv, laboratory informational messages, in-person visits by a WCLN coordinator to clinical laboratories, and laboratory-based surveillance data and summaries distributed by the Wisconsin State Laboratory of Hygiene. One challenge to maintaining the WCLN is securing continual funding for network activities. Key lessons learned from this partnership of more than 20 years include the importance of in-person meetings, the clinical perspective of the laboratory technical advisory group, and providing activities and resources to clinical laboratories to foster sharing of data and clinical specimens for public health surveillance and outbreak response.

Methylobacterium infection of an arthritic knee.

INTRODUCTION: Osteoarthritis (OA) is a common cause of knee pain in older adults. OA is primarily caused by deterioration of cartilage in the knee, which decreases the ability of synovial fluid to absorb shock and increases the opportunity for bones of the joint to rub together. Hylan G-F 20 (Synvisc-One) is a compound that can be injected directly into the knee to help combat the pain associated with OA by lubricating and cushioning the joint. CASE PRESENTATION: A 92-year-old male reported to his primary care provider with complaints of pain due to OA. An ultrasound-guided injection of Hylan G-F 20 was administered without complication; however, the patient presented to an emergency department approximately 10 h after the injection complaining of stabbing pain and swelling in the same knee. Specimens submitted for culture 12 h post-injection yielded a Methylobacterium spp. that was identified following biochemical testing, MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) MS analysis and bacterial sequencing. Interestingly, symptoms began to subside following aspiration of synovial fluid, and new cultures of synovial fluid collected 24 h post-Hylan G-F 20 injection were negative for the presence of Methylobacterium. The patient's knee returned to baseline with diminished pain due to OA approximately 1 week after the initial injection without antibiotic treatment. CONCLUSION: We report short-term complications following treatment of OA with a Methylobacterium-contaminated lot of Hylan G-F 20.

The effects of exercise on cognition and gait in Parkinson's disease: A scoping review.

Cognitive and gait deficits are two debilitating symptoms that occur in Parkinson's disease (PD). Importantly, a relationship between cognitive and gait deficits exists in PD, suggesting reliance on cognition is increased to compensate for gait deficits and/or deterioration of cognition and gait may share common mechanisms. Rehabilitation strategies targeting one factor could lead to the improvement of the other, presenting a unique opportunity to treat both simultaneously. Gold-standard pharmaceuticals partially alleviate these deficits with significant side effects, highlighting the importance of investigating adjunct therapies like exercise. We critically reviewed the influence of three exercise modalities (aerobic, resistance, and goal-based) on cognition and/or gait in PD. Most studies showed improvements in cognition or gait, yet, a limited number investigated them concurrently. This is the first review examining exercise for cognition and gait in PD. Key gaps in the literature are identified; potential exercise-driven mechanisms for enhancements in cognition and gait proposed, and suggestions for the design of future studies investigating the effects of exercise on cognition and gait in PD.

A Murine Model of Myocardial Ischemia-Reperfusion Injury.

Ligation of the left anterior descending (LAD) coronary artery in the mouse heart is a widely used model to simulate myocardial infarction and ischemia-reperfusion injury. Here we describe a ligation technique routinely performed in our laboratory to induce myocardial infarction that may be used to study ischemia-reperfusion injury in the myocardium. The methods described enhance location of the LAD coronary artery to allow for accurate ligation, thus increasing reproducibility of infarct size and location.

Non-contrast estimation of diffuse myocardial fibrosis with dual energy CT: A phantom study.

BACKGROUND: Estimation of diffuse myocardial fibrosis, substrate for adverse events such as heart failure and arrhythmias in patients with various cardiac disorders, is presently done by histopathology or cardiac magnetic resonance. We sought to develop a non-contrast method to estimate the amount of diffuse myocardial fibrosis leveraging dual energy computed tomography (DECT) in phantoms and a suitable small animal model. METHODS AND RESULTS: Phantoms consisted of homogenized bovine myocardium with varying amounts of Type 1 collagen. Fifteen mice underwent sham surgery, no procedure, or transverse aortic constriction (TAC) for 5 or 8 weeks to produce moderate or severe fibrosis, respectively. Phantoms and ex vivo mouse hearts were imaged on a single source, DECT scanner equipped with kVp switching. Monochromatic images were reconstructed at 40-140 keV. Linear discriminant analysis (LDA) was performed on mean myocardial CT numbers derived from single energy (70 keV) images as well as images reconstructed across multiple energies. Classification of myocardial fibrosis severity as low, moderate or severe was more often correct using the multi-energy CT/LDA approach vs. single energy CT/LDA in both phantoms (80.0% vs. 70.0%) and mice (93.3% vs. 33.3%). CONCLUSIONS: DECT myocardial imaging with multi-energy analysis better classifies myocardial fibrosis severity compared to a single energy-based approach. Non-contrast DECT can accurately and non-invasively estimate the extent of diffuse myocardial fibrosis in phantom and animal models. These data support further evaluation of this approach for in vivo myocardial fibrosis estimation.

Mobile Integrated Healthcare Intervention and Impact Analysis with a Medicare Advantage Population.

Mobile Integrated Healthcare (MIH) is a patient-centered, innovative delivery model offering on-demand, needs-based care and preventive services, delivered in the patient's home or mobile environment. An interprofessional MIH clinical team delivered a care coordination program for a Medicare Advantage Preferred Provider Organization that was risk assigned prior to intervention to target the highest risk members. Using claims and eligibility data, 6 months of pre-program experience and 6 months of program-influenced experience from the intervention cohort was compared to a propensity score-matched comparison cohort to measure impact. The intervention led to a reduction in inpatient and emergency department utilization, resulting in net savings amount totals of $2.4 million over the 6 months of the program. After accounting for the costs of implementing the program, the intervention produced a return on investment of 2.97. Additionally, high patient activation and experience lend strength to this MIH intervention as a promising model to reduce utilization and costs while keeping patient satisfaction high.

Can Dual Task Walking Improve in Parkinson's Disease After External Focus of Attention Exercise? A Single Blind Randomized Controlled Trial.

BACKGROUND: It may be possible to use attention-based exercise to decrease demands associated with walking in Parkinson's disease (PD), and thus improve dual task walking ability. For example, an external focus of attention (focusing on the effect of an action on the environment) may recruit automatic control processes degenerated in PD, whereas an internal focus (limb movement) may recruit conscious (nonautomatic) control processes. Thus, we aimed to investigate how externally and internally focused exercise influences dual task walking and symptom severity in PD. METHODS: Forty-seven participants with PD were randomized to either an Externally (n = 24) or Internally (n = 23) focused group and completed 33 one-hour attention-based exercise sessions over 11 weeks. In addition, 16 participants were part of a control group. Before, after, and 8 weeks following the program (pre/post/washout), gait patterns were measured during single and dual task walking (digit-monitoring task, ie, walking while counting numbers announced by an audio-track), and symptom severity (UPDRS-III) was assessed ON and OFF dopamine replacement. Pairwise comparisons (95% confidence intervals [CIs]) and repeated-measures analyses of variance were conducted. RESULTS: Pre to post: Dual task step time decreased in the external group (Δ = 0.02 seconds, CI 0.01-0.04). Dual task step length (Δ = 2.3 cm, CI 0.86-3.75) and velocity (Δ = 4.5 cm/s, CI 0.59-8.48) decreased (became worse) in the internal group. UPDRS-III scores (ON and OFF) decreased (improved) in only the External group. Pre to washout: Dual task step time ( P = .005) and percentage in double support ( P = .014) significantly decreased (improved) in both exercise groups, although only the internal group increased error on the secondary counting task (ie, more errors monitoring numbers). UPDRS-III scores in both exercise groups significantly decreased ( P = .001). CONCLUSIONS: Since dual task walking improvements were found immediately, and 8 weeks after the cessation of an externally focused exercise program, we conclude that externally focused exercise may improve on functioning of automatic control networks in PD. Internally focused exercise hindered dual tasking ability. Overall, externally focused exercise led to greater rehabilitation benefits in dual tasking and motor symptoms compared with internally focused exercise.

Laboratory Focus on Improving the Culture of Biosafety: Statewide Risk Assessment of Clinical Laboratories That Process Specimens for Microbiologic Analysis.

The Wisconsin State Laboratory of Hygiene challenged Wisconsin laboratories to examine their biosafety practices and improve their culture of biosafety. One hundred three clinical and public health laboratories completed a questionnaire-based, microbiology-focused biosafety risk assessment. Greater than 96% of the respondents performed activities related to specimen processing, direct microscopic examination, and rapid nonmolecular testing, while approximately 60% performed culture interpretation. Although they are important to the assessment of risk, data specific to patient occupation, symptoms, and travel history were often unavailable to the laboratory and, therefore, less contributory to a microbiology-focused biosafety risk assessment than information on the specimen source and test requisition. Over 88% of the respondents complied with more than three-quarters of the mitigation control measures listed in the survey. Facility assessment revealed that subsets of laboratories that claim biosafety level 1, 2, or 3 status did not possess all of the biosafety elements considered minimally standard for their respective classifications. Many laboratories reported being able to quickly correct the minor deficiencies identified. Task assessment identified deficiencies that trended higher within the general (not microbiology-specific) laboratory for core activities, such as packaging and shipping, direct microscopic examination, and culture modalities solely involving screens for organism growth. For traditional microbiology departments, opportunities for improvement in the cultivation and management of highly infectious agents, such as acid-fast bacilli and systemic fungi, were revealed. These results derived from a survey of a large cohort of small- and large-scale laboratories suggest the necessity for continued microbiology-based understanding of biosafety practices, vigilance toward biosafety, and enforcement of biosafety practices throughout the laboratory setting.

Interface Bonding With Corneal Crosslinking (CXL) After LASIK Ex Vivo.

Purpose: Interface bonding with corneal crosslinking (CXL) after LASIK using two different photosensitizers was studied ex vivo. Methods: A LASIK flap was created in enucleated rabbit eyes using a femtosecond laser. After the dissection, CXL was performed to seal the interface. In one group interface CXL was performed using rose bengal and green light, whereas in a second group riboflavin and UV-A light was used. In both groups irradiance, radiant exposure, dye concentration, and imbibition time was varied. In a control group, LASIK only was performed. After the procedures, the maximal shear-force required to separate the flap from the stroma was measured. Additionally, corneal transmission spectra were recorded. Results: Optimized parameters for rose bengal/green-light bonding lead to a 2.1-fold increase in shear-force compared with untreated control eyes (P < 0.01). The optimal parameter combination was: irradiance of 180 mW/cm2 for 14 minutes (total radiant exposure 150 J/cm2), rose bengal concentration 0.1%, and an imbibition time of 2 minutes. Optimized riboflavin/UV-A light parameters were 0.5% for 2 minutes with a radiant exposure of 8.1 J/cm2 obtained by an irradiance of 30 mW/cm2 for 4.5 minutes. These optimized parameters lead to a 2-fold increase compared with untreated control eyes (P < 0.01). Optical transmission experiments suggest safety for more posterior structures. Conclusions: Based on ex-vivo results, interface bonding after LASIK using crosslinking with either rose bengal or riboflavin increases the adhesion between flap and stromal bed. In vivo trials are needed to evaluate the temporal evolution of the effect.

Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B.

Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca2+-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysftm1Kcam/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca2+-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair.

Innovative Care Model to Improve Clinical Quality and Safety of Transitional Care: Early Outcomes.

One in five elderly patients returns to the hospital within 30 days of leaving. These rehospitalizations are a common and costly occurrence. A program developed to address problems in post-acute transitional care seems to be effective in reducing 30-day readmission rates for some Medicare fee-for-service beneficiaries.

Flexible Optical Waveguides for Uniform Periscleral Cross-Linking.

Purpose: Scleral cross-linking (SXL) with a photosensitizer and light is a potential strategy to mechanically reinforce the sclera and prevent progressive axial elongation responsible for severe myopia. Current approaches for light delivery to the sclera are cumbersome, do not provide uniform illumination, and only treat a limited area of sclera. To overcome these challenges, we developed flexible optical waveguides optimized for efficient, homogeneous light delivery. Methods: Waveguides were fabricated from polydimethylsiloxane elastomer. Blue light (445 nm) is coupled into the waveguide with an input fiber. Light delivery efficiency from the waveguide to scleral tissue was measured and fit to a theoretical model. SXL was performed on fresh porcine eyes stained with 0.5% riboflavin, using irradiances of 0, 25, and 50 mW/cm2 around the entire equator of the eye. Stiffness of scleral strips was characterized with tensiometry. Results: Light delivery with a waveguide of tapered thickness (1.4-0.5 mm) enhanced the uniformity of light delivery, compared to a flat waveguide, achieving a coefficient of variation of less than 10%. At 8% strain, sclera cross-linked with the waveguides at 50 mW/cm2 for 30 minutes had a Young's modulus of 10.7 ± 1.0 MPa, compared to 5.9 ± 0.5 MPa for no irradiation, with no difference in stiffness between proximally and distally treated halves. The stiffness of waveguide-irradiated samples did not differ from direct irradiation at the same irradiance. Conclusions: We developed flexible waveguides for periscleral cross-linking. We demonstrated efficient and uniform stiffening of a 5-mm-wide equatorial band of scleral tissue.