Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry

The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type

Linking Cellular and In Vitro Microtubule Behavior: Reconstitution Assays to the Rescue

The cytoskeleton is composed of intermediate filaments, the actin network and microtubules (MTs); it is a dynamic cellular web that serves multiple purposes which go beyond a mere structural function. The cytoskeleton allows complex morphological changes in cells, which are critical for cell specialization and functions. This thesis describes how MT behavior can be influenced by subtle changes in its building block, the tubulin protein, and by a subset of MT-associated proteins (the so-called “+end-tracking proteins”, or +TIPs), which associate to the dynamic, fast-growing ends of MTs. The studies presented in this thesis widen our knowledge on MTs, tubulin and +TIPs. Three major issues are discussed: 1) the discovery of novel tubulin-associated proteins (TAPs) as well as the effects of some tubulin mutations involved in neurodevelopmental disorders on the MT network stability in cells and on TAP binding; 2) the effects of these same mutations on tubulin structure within a protofilament, investigated in silico with molecular modelling and molecular dynamics simulations; 3) the regulation of MTs by +TIPs, in particular the cytoplasmic linker proteins (CLIPs). __Chapter 1__ provides an overview of MT function, structure and behavior, together with the structural features, isotypes and post-translational modifications of its building block, the tubulin dimer. Since regulation of MTs is carried out by +TIPs, a description of the structure and role of the main ones presented in this thesis is also provided. Finally, a brief introduction to molecular dynamics simulations is given to guide the reading of Chapter 3. __Chapter 2__ describes our strategy and studies centered on the identification of novel TAPs via purification of isotypes of α- and β-tubulin. We also report on the binding of TAPs to two tubulin mutants found in patients with neurodevelopmental disorders, and the effects of these mutations on the MT network in cells. __Chapter 3__ describes the effects of the tubulin mutations (discussed in Chapter 2) on tubulin and protofilament structure at the atomic level, by using molecular modelling and molecular dynamics simulations. The model proposed helps in understanding possible mechanisms leading to tubulin/MT dysfunction in cells (Chapter 2). __Chapter 4__ focuses on the roles in MT dynamics of the +TIP CLIP-115 compared to those of its close relative CLIP-170. These two proteins have always been considered redundant in function, but our studies suggest that they also have distinct properties. Finally, __Chapter 5__ discusses the major findings presented in the other chapters, and some aspects of current methodologies commonly used in the field, such as in vitro reconstitution assays; the implications of the studies presented in this thesis and future prospects are addressed

Exercise Guidelines for Gait Function in Parkinson's Disease: A Systematic Review and Meta-analysis

This systematic review and meta-analysis is to provide comprehensive evidence-based exercise recommendations targeting walking function for adults with Parkinson's disease. Fixed- or random-effect meta-analyses estimated standardized effect sizes (Hedge's g), comparing treatment effects from exercise with nonexercise and another form of exercise (non-EXE control and EXE control). Cuing and exercise duration were used as moderators for subanalyses. The 40 included randomized controlled trials comprised 1656 patients. The exercise group showed significantly superior performance in timed up-and-go ( g = -0.458; g = -0.390) compared with non-EXE control and EXE control; significantly greater improvement in comfortable walking speed ( g = 0.449), fast walking speed ( g = 0.430), and stride or step length ( g = 0.379) compared with non-EXE control; and significantly greater cadence ( g = 0.282) compared with EXE controls. No significant differences between intervention and control groups were observed for double-leg support time (DLST), dynamic gait index (DGI), 6-minute walk test, or freezing of gait questionnaire (FOG-Q). Notably, treatment effect from the exercise of interest compared with a standard exercise was greater than for nonexercise for cadence and FOG-Q. Moreover, EXE control was favored for DLST and DGI. Cuing had a significantly positive effect on stride length alone. Exercise duration significantly, but negatively, influenced the treatment effect on comfortable walking speed. Gait-specific training, rather than a general exercise program, should be emphasized if gait is the outcome of interest. Further investigation is needed on exercise dosage and its selective effect on more challenging walking tasks, endurance, and freezing of gait

Are Retinoids a Promise for Alzheimer's Disease Management?

Retinoids regulate several physiological and pathological processes through the interaction with nuclear receptors. Retinoid-associated signaling which plays an essential role in neurodevelopment appears to remain active in the adult central nervous system (CNS), thus assuming a high significance in the context of neurodegeneration, and indeed retinoid analogs are thought to be promising therapeutic agents for the treatment of neurodegenerative disorders. The ability of retinoids to exert antioxidant effects, inhibit amyloid-beta(A beta) deposits and likely A beta-induced mitochondrial dysfunction, tau hyperphosphorylation, A beta-induced IL6 production and neuroinflammation, increase survival in hippocampal neurons, and reverse cognitive deficits in animal models of Alzheimer's disease (AD) is discussed. Although retinoids with their multi-target activity are revealing to be promising for management of AD which is a multifaceted biochemical phenomenon, timing as well as appropriate dosage and safety remain, however, a challenge. The end-stage lesions, namely senile plaques and neurofibrillary tangles, are, at present, considered an adaptive response to oxidative stress underlying AD, thus paradoxically late administration of retinoids could even suppress a protective mechanism by inhibiting A beta deposits

Chronic low dose ethanol intake: Biochemical characterization of liver mitochondria in rats

Liver mitochondria were isolated from male rats exposed for 2 months to low doses of ethanol (3% v/v in drinking water), a condition not associated with tolerance or dependence. The results show no significant changes in the content of reduced or oxidized glutathione in the liver mitochondria of ethanol treated rats with respect to controls. However, a slight but significant increase in lipid peroxidation, accompanied by an increased content of oxidized proteins, was found in ethanol exposed animals. Mitochondrial content of cytochrome complexes was not significantly affected by ethanol intake. The specific enzymatic activity of cytochrome oxidase showed, however, a significant decrease in ethanol-treated rats. The slight mitochondrial alterations found in the liver of rats exposed chronically to low doses of ethanol might represent the beginning of a more extensive damage previously observed in rats exposed to high doses of this substance

Yoga Meditation Enhances Proprioception and Balance in Individuals Diagnosed With Parkinson's Disease

This study compared the effectiveness of two proprioceptive exercise programs for persons diagnosed with Parkinson's disease (PD). Thirty-three patients with mild to moderate PD were randomly assigned to a yoga meditation program (YoMed) or to an established proprioceptive training program (PRO). Both interventions included twice weekly sessions (45 minutes each), spanning a 12-week period. Outcome measures included: joint position sense (JPS °, JPS °, JPS °) and joint kinesthesia (JK and JK ), the Tinetti Balance Assessment Tool (TIN), Falls Efficacy Scale (FES), Balance Error Scoring System (BESS), dynamic posturography (DMA and TIME) and the Timed Up-and-Go Test (TUG). Test administrators were blinded to group affiliation. Significant between-group differences favoring the YoMed group were observed for TIN (  = 0.01,  = 0.77) and JK (  = 0.05,  = -0.72). DMA and TIME scores significantly improved for both groups, and no adverse events were reported. These findings indicate that the YoMed program is safe and effective for patients with PD. Researchers should continue to examine the clinical efficacy of mind-body techniques to improve movement control and body awareness in this population

Comparative Impacts of Tai Chi, Balance Training, and a Specially-Designed Yoga Program on Balance in Older Fallers

To compare the effect of a custom-designed yoga program with 2 other balance training programs. Randomized controlled trial. Research laboratory. A group of older adults (N=39; mean age, 74.15±6.99y) with a history of falling. Three different exercise interventions (Tai Chi, standard balance training, yoga) were given for 12 weeks. Balance performance was examined during pre- and posttest using field tests, including the 8-foot up-and-go test, 1-leg stance, functional reach, and usual and maximal walking speed. The static and dynamic balances were also assessed by postural sway and dynamic posturography, respectively. Training produced significant improvements in all field tests (P<.005), but group difference and time × group interaction were not detected. For postural sway, significant decreases in the area of the center of pressure with eyes open (P=.001) and eyes closed (P=.002) were detected after training. For eyes open, maximum medial-lateral velocity significantly decreased for the sample (P=.013). For eyes closed, medial-lateral displacement decreased for Tai Chi (P<.01). For dynamic posturography, significant improvements in overall score (P=.001), time on the test (P=.006), and 2 linear measures in lateral (P=.001) and anterior-posterior (P<.001) directions were seen for the sample. Yoga was as effective as Tai Chi and standard balance training for improving postural stability and may offer an alternative to more traditional programs

A Study of Drug Repurposing to Identify SARS-CoV-2 Main Protease (3CLpro) Inhibitors

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wreaked havoc all over the world. Although vaccines for the disease have recently become available and started to be administered to the population in various countries, there is still a strong and urgent need for treatments to cure COVID-19. One of the safest and fastest strategies is represented by drug repurposing (DRPx). In this study, thirty compounds with known safety profiles were identified from a chemical library of Phase II-and-up compounds through a combination of SOM Biotech&rsquo;s Artificial Intelligence (AI) technology, SOMAIPRO, and in silico docking calculations with third-party software. The selected compounds were then tested in vitro for inhibitory activity against SARS-CoV-2 main protease (3CLpro or Mpro). Of the thirty compounds, three (cynarine, eravacycline, and prexasertib) displayed strong inhibitory activity against SARS-CoV-2 3CLpro. VeroE6 cells infected with SARS-CoV-2 were used to find the cell protection capability of each candidate. Among the three compounds, only eravacycline showed potential antiviral activities with no significant cytotoxicity. A further study is planned for pre-clinical trials