AAV9-Stathmin1 gene delivery improves disease phenotype in an intermediate mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating infantile genetic disorder caused by the loss of survival motor neuron (SMN) protein that leads to premature death due to loss of motor neurons and muscle atrophy. The approval of an antisense oligonucleotide therapy for SMA was an important milestone in SMA research; however, effective next-generation therapeutics will likely require combinatorial SMN-dependent therapeutics and SMN-independent disease modifiers. A recent cross-disease transcriptomic analysis identified Stathmin-1 (STMN1), a tubulin-depolymerizing protein, as a potential disease modifier across different motor neuron diseases, including SMA. Here, we investigated whether viral-based delivery of STMN1 decreased disease severity in a well-characterized SMA mouse model. Intracerebroventricular delivery of scAAV9-STMN1 in SMA mice at P2 significantly increased survival and weight gain compared to untreated SMA mice without elevating Smn levels. scAAV9-STMN1 improved important hallmarks of disease, including motor function, NMJ pathology and motor neuron cell preservation. Furthermore, scAAV9-STMN1 treatment restored microtubule networks and tubulin expression without affecting tubulin stability. Our results show that scAAV9-STMN1 treatment improves SMA pathology possibly by increasing microtubule turnover leading to restored levels of stable microtubules. Overall, these data demonstrate that STMN1 can significantly reduce the SMA phenotype independent of restoring SMN protein and highlight the importance of developing SMN-independent therapeutics for the treatment of SMA.

The use of substrate materials and topography to modify growth patterns and rates of differentiation of muscle cells.

Cells are cultured on platforms made of a variety of materials with selected topographies during studies of cell response and behavior. Understanding the effects of substrates is essential for such applications as developing effective interfaces between body cells and implanted materials and devices. In this study, the effects of substrate surface properties on cell differentiation and alignment on C2C12 myoblasts cultured on conventional or fabricated polymeric cell culture substrates were investigated. Comparisons were made between cells cultured on tissue culture grade polystyrene (TCPS), glass, Permanox, and cured polydimethylsiloxane (PDMS) substrates. Fluorescent immunohistochemistry of cell markers was used to analyse the extent of differentiation. Alignment and guidance of cell growth and spread were studied using patterned platforms. Gratings were made on polystyrene (PS) and PDMS and differentiation was facilitated after 5 days by media exchange. Differences in cell morphology were observed between cells cultured on TCPS and PDMS substrates. Fully differentiated myotubes were observed in highest numbers on TCPS substrates and were non-detectable on PDMS substrates in the time frame of 144 h. Muscle cell alignment and their differentiation followed along the grating patterns on PS and elongated along the pattern length. On the other hand, on PDMS cells formed sheets of tissue and peeled from the substrate. We have revealed the potential for the combinations of surface materials and topography on cell behavior to induce accelerated differentiation and coordinated alignment. The results demonstrate that culture environment can be designed or engineered to modify or regulate muscle cell functions. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1638-1645, 2016.

Comparison of equations for the calculation of LDL-cholesterol in hospitalized patients.

BACKGROUND: The Friedewald equation is widely used to calculate LDL-C for cardiovascular risk prediction but is less accurate with comorbidities and extreme lipid values. Several novel formulae have been reported to outperform the Friedewald formula. METHODS: We examined 14,219 lipid profiles and evaluated four formulae (Friedewald, Chen, de Cordova, Hattori) and compared these to direct measurement of LDL-C across various triglyceride (TG), total cholesterol (TC) and HDL-cholesterol (HDL-C) ranges using Beckman reagents and instruments. Linear regression and ROC analysis were performed. RESULTS: The de Cordova formula showed a high correlation with directly measured LDL-C (r=0.90, P<0.001), comparable to the Friedewald calculated values for directly measured LDL-C (r=0.95, P<0.001). The de Cordova formula was favorable in some ranges of HDL, TC and the lowest TG range (r=0.97, P<0.001) but performed least well in comparison with the three other LDL-C calculations (AUC=0.8331), demonstrating inconsistent bias. The Chen formula performed better than Friedewald (AUC=0.9049). The Hattori formula outperformed all formulae including Friedewald over various ranges of lipid values (AUC=0.9097). CONCLUSIONS: We observe favorable correlations of the de Cordova formula with Friedewald at low TG values. However, the Hattori formula appears to be best for application in hospitalized patients, even at extreme lipid values.

Using mouse cranial muscles to investigate neuromuscular pathology in vivo.

Neuromuscular pathology is a classic hallmark of many diseases such as muscular dystrophy, myasthenia gravis, amyotrophic lateral sclerosis and spinal muscular atrophy. It is also a feature of many congenital and acquired myopathies and neuropathies such as diabetic neuropathy and toxin-exposure. The availability of experimentally accessible nerve-muscle preparations from rodent models in which pathological events can be studied in nerve and muscle, as well as at the neuromuscular junction, is therefore of fundamental importance for investigating neuromuscular disease. The group of small cranial muscles, which move the ear in the mouse provide ideal experimental preparations for the study of neuromuscular disease in vivo, but information regarding their anatomical and functional characteristics is currently lacking. Here, we provide a detailed description of the levator auris longus, auricularis superior, abductor auris longus and interscutularis muscles. In addition, we briefly review their differential fibre type and developmental characteristics, which can be exploited to aid our understanding of neuromuscular vulnerability and to provide preferable alternatives to more traditional muscle preparations such as gastrocnemius, soleus and diaphragm.

Review: neuromuscular synaptic vulnerability in motor neurone disease: amyotrophic lateral sclerosis and spinal muscular atrophy.

Amid the great diversity of neurodegenerative conditions, there is a growing body of evidence that non-somatic (that is, synaptic and distal axonal) compartments of neurones are early and important subcellular sites of pathological change. In this review we discuss experimental data from human patients, animal models and in vitro systems showing that neuromuscular synapses are targeted in different forms of motor neurone disease (MND), including amyotrophic lateral sclerosis and spinal muscular atrophy. We highlight important developments revealing the heterogeneous nature of vulnerability in populations of lower motor units in MND and examine how progress in our understanding of the molecular pathways underlying MND may provide insights into the regulation of synaptic vulnerability and pathology. We conclude that future experiments developing therapeutic approaches specifically targeting neuromuscular synaptic vulnerability are likely to be required to prevent or delay disease onset and progression in human MND patients.

1,9-Dimethylhypoxanthine from a southern Australian marine sponge spongosorites species.

A Spongosorites sp. collected off southern Australia has yielded 1, 9-dimethylhypoxanthine (4). The structure for 4 was solved by spectroscopic analysis.

Effects of lasalocid in milk replacer of calf starter on health and performance of calves challenged with Eimeria species.

Holstein bull calves (n = 48) were purchased from local sale barns at 3 to 7 d of age and were assigned randomly to a 2 x 2 factorial arrangement of lasalocid in milk replacer (0 or 80 mg/kg) and in calf starter (3 or 44 mg/kg of dry matter). On d 10 after arrival, calves were orally dosed with 100,000 Eimeria oocysts. Intakes of calf starter and milk replacer, body weight (BW), BW gain, excretion of fecal oocysts, and fecal scores were determined. Calves fed lasalocid in milk replacer consumed more calf starter, had greater BW gain, shed fewer oocysts in feces, and scoured less frequently and less severely than did calves fed no lasalocid or those fed lasalocid in calf starter alone. The combination of lasalocid in milk replacer and in calf starter did not improve performance above that of calves fed lasalocid in milk replacer alone. Low intake of calf starter prior to weaning may provide an insufficient amount of lasalocid to control effectively the effects of coccidiosis when calves are infected with Eimeria at an early age. Use of coccidiostats in milk replacers may reduce the effects of coccidiosis in young calves that are infected with Eimeria at an early age.

Body weight gain, feed efficiency, and fecal scores of dairy calves in response to galactosyl-lactose or antibiotics in milk replacers.

Holstein bull calves (n = 96) were purchased at approximately 7 d of age and were assigned to receive 400, 450, 500, and 550 g/d of milk replacer solids during wk 1 to 4, respectively. Treatments were nonmedicated milk replacer plus dried whey, medicated milk replacer (138 mg/kg of oxytetracycline and 276 mg/kg of neomycin) plus dried whey, or nonmedicated milk replacer plus whey processed with beta-galactosidase to contain 15% galactosyl-lactose. Processed whey was added to provide 1% of dry matter as galactosyl-lactose; an equivalent amount of dried whey was added to the other treatments to provide 6.3% of dry matter daily. Intake of milk replacer and fecal scores were measured daily. No calf starter or hay was fed. Body weights were measured weekly from arrival to 26 d. Serum immunoglobulin G, measured 1 d after arrival, averaged 18.3 g/L. Intake of milk replacer plus additive during the 26-d study was 445 g/d and did not vary with treatment. Body weight and body weight gain were increased by 52 and 72 g/d in response to antibiotics and galactosyl-lactose, respectively. Severity of scours and number of days scouring tended to be reduced when calves were fed milk replacer containing galactosyl-lactose or antibiotics.

A pragmatic approach to estimating total analytical error of immunoassays.

Several groups have recently commented on the need for more realistic information on analytical performance of laboratory tests. The term "total analytical error" is sometimes used in this context. However, differing opinions have been expressed on how best to obtain estimates of clinical assay error, as it would be perceived by clinicians. We suggest a pragmatic definition of total analytical error for immunoassays and describe our attempts to estimate it by simple designs in the internal quality-control process. We use results over 29 months from a total serum thyroxine RIA. The most important error sources were those related to calibration materials and operator effects, errors not usually captured by short-term or snapshot experiments.

Influence of specimen carryover on sensitive thyrotropin (TSH) assays: is there a problem?

A relatively slow transition has occurred from so-called 1st-generation thyrotropin (TSH) assays (e.g., RIAs) through 2nd-generation assays (e.g., IRMAs) to 3rd-generation assays (e.g., immunochemiluminometric assays). Analysis of data from a modified internal quality-control design, followed up by a computer simulation, showed that specimen carryover has minimal effect on 2nd-generation TSH assays. However, extension of the simulation to a 3rd-generation assay showed the possibility of substantial effects in the subnormal region. Carryover of 1:1250 (0.08%), for example, may reduce the theoretical 10-fold precision improvement claimed for 3rd-generation assays to nearer fourfold. Simulation results suggest maximum allowable specimen carryover of approximately 1:10,000 (approximately 0.01%) for 3rd-generation TSH assays. We suggest that when automated specimen handling is used in a TSH assay, a well-designed carryover experiment should become a routine part of reports that claim 3rd-generation (or better) performance characteristics.

Hemolytic anemia following a presumptive brown recluse spider bite.

This case report describes a systemic reaction occurring in a 12-year-old female following presumed envenomation by a brown recluse spider (Loxosceles reclusa). The systemic reaction included self-limited hemolysis necessitating blood transfusion. The clinical course and management are described and compared with those of previously reported cases of systemic loxoscelism.

Minimum distinguishable difference in concentration: a clinically oriented translation of assay precision summaries.

A trend to market-driven health care in many parts of the world is focusing increasing attention on getting maximum value from available resources. Laboratories are not exempted. Well-informed clinical input has a potentially valuable role in any laboratory rationalization process. However, a communication difficulty exists in the sense that, although laboratory workers, commercial developers, regulatory bodies, etc., are thoroughly conditioned to using assay coefficient of variation as a general performance measure (for excellent reasons), this is not necessarily the most intuitive or informative scale from a clinician's perspective. Here we use routine clinical data from an immunoradiometric assay of thyrotropin to illustrate, first, a general approach to estimation and prediction of reproducibility, and second, an alternative summary that expresses the discriminatory power of an assay. This latter measure, our experience suggests, is more suited to the way clinicians perceive assays and assay results. The overall aim is improved clinician/laboratory communication.

Development of thyroid disease during therapy of chronic viral hepatitis with interferon alfa.

Six of 237 (2.5%) patients with chronic viral hepatitis who were treated with recombinant interferon alfa developed thyroid disease while on treatment. Three patients developed hyperthyroidism, two of whom developed detectable levels of thyroid-stimulating immunoglobulin; three patients developed hypothyroidism in association with high titers of antithyroglobulin and/or antimicrosomal antibodies. The thyroid disease did not remit when interferon therapy was stopped, and all six patients required definitive therapy for the thyroid disease. These findings suggest that a small proportion of patients treated with interferon alfa develop autoimmune reactions and can develop autoimmune thyroid disease.

Clinical course and predictors of length of stay in hospitalized patients with rheumatoid arthritis.

Patients admitted for therapy of active rheumatoid arthritis were prospectively followed throughout their hospital stay. Average length of stay was 17.1 days. Serial global assessments, whether determined by rheumatologist, physiotherapist or patient appeared to improve linearly until at least hospital day 21. From admission to discharge, mean global assessment scores improved by about one third. Poor global assessment, high disability index, and the presence of comorbid disease and anemia on admission, as well as admission late in the week, were predictive of prolonged hospital stay.

Antiproliferative effects of methotrexate on peripheral blood mononuclear cells.

Methotrexate was added to cultured mononuclear cells from the peripheral blood of normal individuals and patients with rheumatoid arthritis (RA) to study the drug's effects on mononuclear cell proliferation and antibody synthesis. In the presence of methotrexate, marked antiproliferative effects (to levels less than 15% of baseline) were seen with 3H-deoxyuridine, but not with 3H-thymidine, as the marker of cell division. This difference was not due to altered kinetics of proliferation or the presence of salvage nucleotides in the culture medium. The absence of suppression of antibody production preactivated by pokeweed mitogen in vitro and the low levels of suppression of spontaneous IgM rheumatoid factor production by blood mononuclear cells from RA patients suggested a relative resistance of activated cells to the effects of methotrexate. The effects of methotrexate on both cell proliferation and antibody synthesis were completely reversed by the addition of high concentrations of exogenous folinic acid. The results suggest that methotrexate has effects on immunocompetent cells that may contribute to the efficacy of this drug in the treatment of RA and other autoimmune diseases.

A comparison of lecture-discussion and self-study methods in nursing education.

Three groups of students in a Nurse Practitioner Program were subjected to a multiple-choice test based upon two articles from the Nurse Practitioner. Prior to testing, one group read and studied the articles for one hour with no discussion permitted. The other two groups did not read the articles but participated in a lecture-discussion on their contents. The testing followed each learning session. Overall, the results support strongly the hypothesis that the Lecture-Discussion method is superior to the Reading Only method. The fact that the Lecture-Discussion method utilized two different instructors suggests that this finding is not based on some unique personality characteristic of one particular faculty member. Naturally, these findings are limited to this particular subject matter with nurse practitioner students. It is possible that the Reading Only method would be equal to or superior to the Lecture-Discussion method with other topics or with other student bodies. In any case, the present results run counter to the current wave of enthusiasm for the Self-study method in nursing education. These results suggest that caution be employed in a wholesale shift to autotutorial results. As Hogan states: "Self-instructional materials are not replacements for . . . teacher-centered instruction. Instead they are supplements that encourage individual initiative, autonomy, and responsibility for students who can use and wish to use them."