Spatio-Temporal Patterns of Demyelination and Remyelination in the Cuprizone Mouse Model

Cuprizone administration in mice provides a reproducible model of demyelination and spontaneous remyelination, and has been useful in understanding important aspects of human disease, including multiple sclerosis. In this study, we apply high spatial resolution quantitative MRI techniques to establish the spatio-temporal patterns of acute demyelination in C57BL/6 mice after 6 weeks of cuprizone administration, and subsequent remyelination after 6 weeks of post-cuprizone recovery. MRI measurements were complemented with Black Gold II stain for myelin and immunohistochemical stains for associated tissue changes. Gene expression was evaluated using the Allen Gene Expression Atlas. Twenty-five C57BL/6 male mice were split into control and cuprizone groups; MRI data were obtained at baseline, after 6 weeks of cuprizone, and 6 weeks post-cuprizone. High-resolution (100μm isotropic) whole-brain coverage magnetization transfer ratio (MTR) parametric maps demonstrated concurrent caudal-to-rostral and medial-to-lateral gradients of MTR decrease within corpus callosum (CC) that correlated well with demyelination assessed histologically. Our results show that demyelination was not limited to the midsagittal line of the corpus callosum, and also that opposing gradients of demyelination occur in the lateral and medial CC. T₂-weighted MRI gray/white matter contrast was strong at baseline, weak after 6 weeks of cuprizone treatment, and returned to a limited extent after recovery. MTR decreases during demyelination were observed throughout the brain, most clearly in callosal white matter. Myelin damage and repair appear to be influenced by proximity to oligodendrocyte progenitor cell populations and exhibit an inverse correlation with myelin basic protein gene expression. These findings suggest that susceptibility to injury and ability to repair vary across the brain, and whole-brain analysis is necessary to accurately characterize this model. Whole-brain parametric mapping across time is essential for gaining a real understanding of disease processes in-vivo. MTR increases in healthy mice throughout adolescence and adulthood were observed, illustrating the need for appropriate age-matched controls. Elucidating the unique and site-specific demyelination in the cuprizone model may offer new insights into in mechanisms of both damage and repair in human demyelinating diseases.     

Understanding and modeling retention of mammalian cells in fluidized bed centrifuges

Within the last decade, fully disposable centrifuge technologies, fluidized‐bed centrifuges (FBC), have been introduced to the biologics industry. The FBC has found a niche in cell therapy where it is used to collect, concentrate, and then wash mammalian cell product while continuously discarding centrate. The goal of this research was to determine optimum FBC conditions for recovery of live cells, and to develop a mathematical model that can assist with process scaleup. Cell losses can occur during bed formation via flow channels within the bed. Experimental results with the kSep400 centrifuge indicate that, for a given volume processed: the bed height (a bed compactness indicator) is affected by RPM and flowrate, and dead cells are selectively removed during operation. To explain these results, two modeling approaches were used: (i) equating the centrifugal and inertial forces on the cells (i.e., a force balance model or FBM) and (ii) a two‐phase computational fluid dynamics (CFD) model to predict liquid flow patterns and cell retention in the bowl. Both models predicted bed height vs. time reasonably well, though the CFD model proved more accurate. The flow patterns predicted by CFD indicate a Coriolis‐driven flow that enhances uniformity of cells in the bed and may lead to cell losses in the outflow over time. The CFD‐predicted loss of viable cells and selective removal of the dead cells generally agreed with experimental trends, but did over‐predict dead cell loss by up to 3‐fold for some of the conditions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1520–1530, 2016     

Cystathionine-γ-lyase gene silencing with siRNA in monocytes/macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H₂S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H₂S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.     

Sirtuin 1 and 7 mediate resveratrol-induced recovery from hyper-anxiety in high-fructose-fed prediabetic rats

Hyperglycaemia in diabetes is either caused by reduced availability of insulin (type 1 diabetes, T1D) or insulin resistance to the cells (type 2 diabetes, T2D). In recent years, the prevalence of T2D has increased to an alarming proportion, encompassing 95% of the total diabetic burden, probably due to economy-driven changes in lifestyle. Recent epidemiological studies show comorbid depression, anxiety and related mental illness. To explore the molecular mechanisms underlying this comorbid conditions, we used Sprague–Dawley rats on high-fructose diet for 8 weeks to induce prediabetic condition. Rats with this metabolic syndrome also showed hyper-anxiety when they were subjected to anxiety-related behavioural assays. Rats were administered with resveratrol, an activator of sirtuins, and metformin, a standard antidiabetic drug, simultaneously with fructose. We observed that resveratrol was more effective in protecting from both the metabolic (prediabetic) and affective (anxiety) disorders than metformin. Molecular studies showed that recovery was associated with the upregulation of few nuclear sirtuins that act epigenetically – Sirt 1 and 7, which were significantly attenuated in the striatum of prediabetic rats. In conclusion, our study showed that hyper-anxiety associated with prediabetic condition is ameliorated by resveratrol through modulation of sirtuins, which is more or less similar to metformin.     

Natural Progression of Canine Glycogen Storage Disease Type IIIa

Glycogen storage disease type IIIa (GSD IIIa) is caused by a deficiency of glycogen debranching enzyme activity. Hepatomegaly, muscle degeneration, and hypoglycemia occur in human patients at an early age. Long-term complications include liver cirrhosis, hepatic adenomas, and generalized myopathy. A naturally occurring canine model of GSD IIIa that mimics the human disease has been described, with progressive liver disease and skeletal muscle damage likely due to excess glycogen deposition. In the current study, long-term follow-up of previously described GSD IIIa dogs until 32 mo of age (n = 4) and of family-owned GSD IIIa dogs until 11 to 12 y of age (n = 2) revealed that elevated concentrations of liver and muscle enzyme (AST, ALT, ALP, and creatine phosphokinase) decreased over time, consistent with hepatic cirrhosis and muscle fibrosis. Glycogen deposition in many skeletal muscles; the tongue, diaphragm, and heart; and the phrenic and sciatic nerves occurred also. Furthermore, the urinary biomarker Glc₄, which has been described in many types of GSD, was first elevated and then decreased later in life. This urinary biomarker demonstrated a similar trend as AST and ALT in GSD IIIa dogs, indicating that Glc₄ might be a less invasive biomarker of hepatocellular disease. Finally, the current study further demonstrates that the canine GSD IIIa model adheres to the clinical course in human patients with this disorder and is an appropriate model for developing novel therapies.Abbreviations: CCR, curly-coated retriever; CPK, creatine phosphokinase; GSD IIIa, glycogen storage disease type IIIa; Glc₄, Glcα1-6Glcα1-4Glcα1-4GlcGlycogen storage disease type IIIa (GSD IIIa; OMIM, 232400) is an autosomal recessive disorder caused by mutations in the glycogen debranching enzyme gene (AGL), leading to various clinical signs. The tissues mainly affected are liver, heart, and skeletal muscle. Clinical manifestations include hypoglycemia, elevated serum concentrations of liver and muscle enzymes, hepatomegaly, growth retardation, muscle weakness, cardiac hypertrophy with arrhythmia risk, polycystic ovaries and neuropathy.^15,17,29 Current treatments are mainly symptomatic and are not curative. The most frequently used therapies are dietary, such as providing uncooked corn starch to prevent hypoglycemia at young ages and high-protein diets, which have been shown to reverse the extent of cardiomyopathy associated with GSD IIIa.^7,8,30,37 In addition, the use of medium-chain triglycerides has shown positive therapeutic effects in patients with GSD Ia and GSD IIIa.^11,22 However, dietary therapies do not prevent the long-term complications of GSD IIIa, including hepatic cirrhosis, hepatocellular adenoma, hepatocellular carcinoma, cardiomyopathy, neuropathy, and myopathy.³¹An appropriate animal model is necessary to test novel therapies and address the long-term effects of GSD IIIa. Recently a mouse model for GSD III has been described that may prove beneficial in testing new therapies.¹⁹ However, the limitations of mouse models include a short lifespan that curtails the study of the long-term effects of novel treatments. In addition, a large animal model often mimics human disease more closely than do mouse models, as occurs in GSD type Ia dog models, which exhibit lactic acidosis similar to human patients, a characteristic that mouse models of GSD Ia lack.¹⁶ Therefore a naturally occurring large animal model for GSD IIIa may be more effective in terms of the development of new treatments than are available mouse models.GSD IIIa (OMIA, 001577) has been reported to occur in curly-coated retriever dogs (CCR) and is caused by a naturally occurring homozygous frameshift mutation in exon 32 that leads to the deletion of 126 amino acids at the C-terminus of glycogen debranching enzyme.^12,40 The dogs in these previous studies proved to have abnormalities similar to those seen in humans affected with the disorder, namely progressive glycogen accumulation in muscle and liver, elevated liver and muscle enzymes (ALP, AST, creatine phosphokinase [CPK], and ALT), and eventual liver fibrosis. However, these animals were not followed beyond 16 mo of age in the earlier studies.^12,40 The goal of the current study is to provide biochemical follow-up on these animals and analyze more extensively other tissues and organs involved in GSD IIIa in the dog model. A brief analysis of the naturally high protein diets of GSD IIIa dogs, as well as the effects of an increased protein diet in 2 dogs for the last few months of life, is included.We also include the analysis of a urinary biomarker, Glcα1–6Glcα1– 4Glcα1–4Glc (Glc₄), which is a breakdown product of glycogen by α-amylase and neutral α-1,4-glucosidase.³² Elevated levels of Glc₄ have been found in urine from patients with GSD types II, III, IV, VI, and IX and may correlate with disease advancement.^1,18,24,32 To our knowledge, Glc₄ has not been evaluated previously in dogs; we therefore here evaluated the utility of Glc₄ as a biomarker of canine GSD IIIa. A correlation of Glc₄ levels with liver enzyme concentrations in blood might indicate a role of Glc₄ as a less-invasive biomarker for determining the advancement of liver disease in human and canine patients.     

Interaction of Catechu Dye with DNA: Spectroscopic and In Silico Approach

Catechin, a yellow colored molecule obtained from the wood of Acacia catechu was analyzed for its interaction with synthetic DNA duplexes using spectroscopic analysis. UV-Visible spectroscopic analysis revealed the non-intercalative binding mode. Fourier Transform Infrared spectroscopy (FTIR) analysis expose chemical shift indicated by various vibrational stretches and an increase in the intensity of base stacking was observed by Circular Dichroism (CD), respectively. This inference was further confirmed through nuclear staining technique and also in electrophoretic technique; the dye quenches the fluorescent intensity of ethidium bromide. The result of fluorescence spectroscopy was in concordance with the electrophoretic technique. In addition, the spectroscopic results were in accordance with the molecular docking studies of specific catechin compound from the catechu dye with CT-DNA. This kind of site specificity is a gain in the medicinal field as the drug can be DNA targeted for cancer therapeutics. The present work reveals that catechu dye has a noteworthy application in the field of medical bioscience.     

An Ultra‐Mechanosensitive Visco‐Poroelastic Polymer Ion Pump for Continuous Self‐Powering Kinematic Triboelectric Nanogenerators

A mechanosensitive, visco‐poroelastic polymer ion pump that can rapidly establish a dense electrical double layer via mechanical pressure, thereby significantly enhancing output performance of an ionic triboelectric nanogenerator (iTENG), is described. A working mechanism of an iTENG using a highly mechanosensitive, visco‐poroelastic ion pump is suggested and the optimal characteristics of the polymer ion pump are reported by investigating optical, mechanical, electrical, and electrochemical properties. Surprisingly, the pressure sensitivity of the iTENG reaches 23.3 V kPa^?1, which is tens of times the record value. To achieve controlled high‐frequency pulses from an iTENG, kinematic systems using a gear train and a cam are integrated with a single grounded iTENG, which produces a maximum of 600 V and 22 mA (≈2.2 W cm^?2) at an input frequency of 1.67 Hz; after power transforming, those values are converted to 1.42 V and 225 mA. A capacitor of 1 mF can be fully charged to 2 V in only 60 s, making it possible to continuously operate a wireless‐communicating self‐powered humidity sensor. Also, due to the high transparency and deformability of the polymer ion pump, a self‐powered transparent tactile sensor is successfully assembled using a 5 × 5 iTENG array.     

Melanin–Perovskite Composites for Photothermal Conversion

Biomacromolecular pigments, such as melanin, play an essential role in the survival of all living beings. Melanin absorbs sunlight and transforms it into heat, which is crucial for avoiding damage to skin cells. Light absorption produces excited electrons, which could either fall back to ground states by releasing the heat (photothermal effect) and/or light (photoluminescence), or stay at higher energy levels within its lifetime period, which can be captured through external electronic circuitry (photovoltaic effect). In this study, it is demonstrated that the combination of melanin with halide perovskite light absorber in the form of a composite exhibits high absorbance from the UV to NIR region in the solar spectrum. And the composite displays significantly reduced photoluminescence and minimized density of residual excited states (verified by photovoltaic measurement) owing to the significantly enhanced nonradiant quenching by the melanin. As a result, the composite shows an ultrahigh solar‐thermal quantum yield of 99.56% and solar‐thermal conversion efficiency of ≈81% under one‐sun illumination (AM1.5), which is superior to typical carbon materials such as graphene (≈70%). By coating the photothermal composite film on the hot‐side of thermoelectric devices, a 7000% increase in output power as compared to the blank device under illumination is observed.