Proton spectroscopy in Alzheimer's disease and cognitive impairment not dementia: a community study

OBJECTIVE: To describe the proton magnetic resonance spectroscopy (1H-ERM) data in Alzheimer's disease (AD) and Cognitive Impairment Not Dementia (CIND) in a community sample. METHOD: We investigated subjects with AD (n=6), CIND (n=7) and normal control (n=7). 1H-ERM was performed with single voxel (8 cm3) placed in temporal, parietal and occipital regions and studied metabolites were: N-acetylaspartate (NAA), creatine (Cr), choline (Cho) and myo-inositol (mI). RESULTS: NAA concentration was higher in control subjects than AD and intermediated in CIND patients. Cho parietal plus occipital and Cr parietal plus Cho occipital classified correctly 92.3% of subjects Control vs AD. Temporal mI classified 78.6% of subjects between Control vs CIND. CONCLUSION: Spectroscopy can be used in the diagnosis and follow-up of individuals with cognitive impairment; evaluation of community subjects may show different patterns of brain metabolites distribution.     

Vascular pathology in Alzheimer disease: correlation of cerebral amyloid angiopathy and arteriosclerosis/lipohyalinosis with cognitive decline

Sporadic, late-onset Alzheimer disease (AD) constitutes the most frequent cause of dementia in the elderly population. AD-related pathology is often accompanied by vascular changes. The predominant vascular lesions in AD are cerebral amyloid angiopathy (CAA) and arteriosclerosis/lipohyalinosis (AS/LH). The present study was carried out to examine the coincidence of these small vessel pathologies during the development of cognitive deficits, amyloid beta-protein (A beta) deposition, and neurofibrillary tangle (NFT) formation in sporadic late-onset AD. We correlated the clinical dementia rating (CDR) score, the sequential extension of AD-related A beta deposition into different parts of the brain, and the extension of NFTs to involve more brain regions with the distribution of CAA and AS/LH in 52 human autopsy brains. The extension of CAA and AS/LH to involve different areas of the brain was associated with a rise of CDR scores and an increase in the extension of A beta deposition and NFT generation. AD cases showed a higher number of regions with CAA and AS/LH compared to nondemented patients with AD-related pathology and controls. Moreover, we demonstrated a hierarchical sequence in which the different regions of the brain exhibited CAA and AS/LH-affected vessels, allowing the distinction of 3 stages in the development of CAA and AS/LH. The first stage of CAA involved leptomeningeal and neocortical vessels. The second stage was characterized by additional A beta deposition in allocortical and midbrain vessels. Finally, in a third stage, CAA was observed in the basal ganglia, the thalamus, and in the lower brainstem. In contrast, AS/LH initially affected the basal ganglia in stage A. In stage B this pathology made inroads into the deep white matter, the leptomeningeal arteries of the cortex, the cerebellum, and into the thalamus. Stage C was characterized by AS/LH in brainstem vessels. Our results demonstrate widespread CAA and AS/LH to be associated with the development of cognitive deficits in AD. A combination of both CAA and AS/LH may, therefore, contribute to neurodegeneration in AD. These data also suggest that small vessel disease due to arteriosclerosis and fibrolipohyalinosis is a potential target for the treatment of AD.     

The association of multiple sclerosis and Hodgkin's disease: the role of Epstein-Barr virus infection

The aetiology of multiple sclerosis (MS) is still an unresolved question. Several recently reported studies are implicating Epstein-Barr virus (EBV) as one of the key players in MS pathogenesis. We present a 21-year-old male patient with a history of EBV-related infectious mononucleosis in puberty and Hodgkin's disease two years previously treated with irradiation and chemotherapy. The patient presented with spastic paraparesis that resolved promptly on corticosteroid treatment. There were no signs of Hodgkin's disease relapse. In the next three years he had three more bouts of the disease and he was diagnosed with relapse-remitting MS. There is evidence of EBV involvement in MS pathogenesis; the association of MS with EBV-related infectious mononucleosis and Hodgkin's disease being just one of them. Although there are many unanswered questions, it is clear that without EBV seropositivity MS in adults is extremely rare.     

POLR3A variants in hereditary spastic paraparesis and ataxia: clinical,genetic, and neuroradiological findings in a cohort of Italian patients

Neurological Sciences - Mutations in POLR3A are characterized by high phenotypic heterogeneity, with manifestations ranging from severe childhood-onset hypomyelinating leukodystrophic syndromes to...     

Aerobic exercise reduces hippocampal ERK and p38 activation and improves memory of middle‐aged rats

Aging is often accompanied by cognitive decline, memory impairment, and an increased susceptibility to neurodegenerative disorders. Although the physiological processes of aging are not fully understood, these age‐related changes have been interpreted by means of various cellular and molecular theories. Among these theories, alterations in the intracellular signaling pathways associated with cell growth, proliferation, and survival have been highlighted. Based on these observations and on recent evidence showing the beneficial effects of exercise on cognitive function in the elderly, we investigated the cell signaling pathways in the hippocampal formation of middle‐aged rats (18 months old) submitted to treadmill exercise over 10 days. To do this, we evaluated the hippocampal activation of intracellular signaling proteins linked to cell growth, proliferation, and survival, such as Akt, mTOR, p70S6K, ERK, CREB, and p38. We also explored the cognitive performance (inhibitory avoidance) of middle‐aged rats. It was found that physical exercise reduces ERK and p38 activation in the hippocampal formation of aged rats, when compared to the control group. The hippocampal activation and expression of Akt, mTOR, p70S6K, and CREB were not statistically different between the groups. It was also observed that aged rats from the exercise group exhibited better cognitive performance in the inhibitory avoidance task (aversive memory) than aged rats from the control group. Our results indicate that physical exercise reduces intracellular signaling pathways linked to inflammation and cell death (i.e., ERK and p38) and improves memory in middle‐aged rats.     

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.New therapeutic strategies are needed to treat complex human diseases. Because disease phenotypes are often regulated by interwoven genetic networks, exploiting combination therapy to target multiple pathways, as opposed to only single ones, can enhance treatment efficacy (1). However, discovering effective combination therapies for human diseases is challenging with existing methods, due to the cost, effort, and labor required to construct and analyze each combination (2). For example, the National Cancer Institute tested ∼5,000 pairwise combinations of 100 cancer drugs against the NCI-60 panel in a study that took 2 y and cost about USD $4 million (3). Thus, there is a need for technological advances to accelerate the identification of effective combinatorial therapies. Here, we used our combinatorial genetics en masse (CombiGEM)-CRISPR platform to perform rapid pooled screening of pairwise genetic knockouts against genes coding for epigenetic regulators and then translated our screen hits into drug combinations against human ovarian cancer cells.CRISPR-Cas9 technology has been used for large-scale genetic perturbation screens with single-guide RNA (sgRNA) libraries for gene knockouts (4–7), repression, and activation (8, 9). Despite its simplicity for multiplexed genetic perturbations (10–12), new methods are needed to enable high-throughput CRISPR-Cas9–based screening with combinatorial sets of guide RNAs (gRNAs), which would be broadly useful for studying combinatorial gene functions in multigenic phenotypes and diseases. By using CombiGEM-based DNA assembly (13, 14), we developed a strategy for the simple and efficient assembly of barcoded combinatorial gRNA libraries. These libraries can be delivered into human cells by lentiviruses to create genetically ultradiverse cell populations harboring unique gRNA combinations that can be tracked via barcode sequencing in pooled assays. This strategy, termed CombiGEM-CRISPR, uses one-pot cloning steps to enable the assembly of combinatorial gRNA libraries, thus simplifying and accelerating the workflow toward systematic analysis of combinatorial gene functions.     

INCREASED SATELLITE CELL APOPTOSIS IN VASTUS LATERALIS MUSCLE AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION

ObjectiveRecovery of the quadriceps femoris muscle after anterior ligament reconstruction is impaired. The aim of this study was to investigate satellite cell content and function of the vastus lateralis muscle after anterior ligament reconstruction.MethodsBiopsies were obtained from the vastus lateralis muscle of 16 recreational athletes immediately before and again 12 weeks after anterior ligament reconstruction. Total satellite cell number (Pax7⁺), activated (Pax7⁺/MyoD⁺), differentiating (Pax7^–/MyoD⁺), and apoptotic (Pax7⁺/TUNEL⁺) satellite cells, myofibers expressing myosin heavy chain (MHC) I and II, and neonatal MHC (MHCneo) were determined immunohistochemically.ResultsAfter anterior ligament reconstruction, the number of apoptotic satellite cells was significantly (p = 0.019) increased, concomitant with a significant (p < 0.001) decrease in total satellite cell number, with no change in activated and differentiating satellite cell number. MHCneo⁺ myofibers tended towards an increase.CONCLUSIONSatellite cell apoptosis and the reduction in the satellite cell pool might provide an explanation for prolonged quadriceps muscle atrophy after anterior ligament reconstruction.LAY ABSTRACTProtracted muscle atrophy is common after anterior ligament reconstruction, even if athletes adhere to a structured rehabilitation programme. Satellite cells, the stem cells of skeletal muscle, play an important role in recovery of an atrophied muscle. Exercise can activate satellite cells, induce their proliferation, and probably also differentiation of these stem cells. The current study evaluated satellite cell content and function in biopsies from the vastus lateralis muscle of 16 recreational athletes immediately before and 12 weeks after anterior ligament reconstruction. After anterior ligament reconstruction, an increased number of satellite cells showed signs of apoptosis (cell death). Furthermore, total satellite cell number was decreased, with no change in the numbers of activated and differentiating satellite cells. The number of regenerating myofibers expressing neonatal myosin tended to increase. In conclusion, satellite cell apoptosis and the reduced satellite cell number might provide an explanation for the impaired muscle recovery after anterior ligament reconstruction.Key words: satellite cells, apoptosis, muscle regeneration, developmental myosin heavy chain, muscular atrophy, quadriceps muscle

Protracted atrophy and weakness of the quadriceps muscle are common after anterior cruciate ligament (ACL) injury and/or anterior cruciate ligament reconstruction (ACL-R), even if the patients undergo guided rehabilitation programmes (1–4). Muscle recovery is compromised due to negative changes in the knee extensor muscles, most likely due to impaired neuromuscular function (5), post-surgery inflammation (6) and immobilization (7). After ACL injury, fibrogenic alterations were observed in biopsies obtained from the vastus lateralis muscle of the injured leg (8, 9). Satellite cell (SC) abundance was also reduced compared with biopsies taken from the vastus lateralis muscle of the uninjured leg (8, 10). Furthermore, there was a surprising lack of increase in SC number after regular rehabilitation training (10), as well as after 12 weeks of supervised quadriceps strength training during rehabilitation after ACL-R (3).SCs play an important role in skeletal muscle growth and regeneration (11, 12). Increases in SC number occur after 11–12 weeks of quadriceps strength training in healthy subjects (13–15). The role of SCs in atrophy of human skeletal muscle, however, has scarcely been investigated. In the very few studies on the role of SCs in atrophy of human skeletal muscle (8, 10, 16), loss of SCs with atrophy is not a consistent finding. However, there is some evidence that a particularly severe atrophic environment, as is found, for example, after severe burn injury, has a negative impact on SC number and SC function and can induce SC apoptosis (17). With regard to findings in animal studies, it has been hypothesized that muscle wasting in old age (sarcopaenia) could at least partly be explained by SC dysfunction with increased SC apoptosis due to chronic low-grade systemic inflammation (18). The significantly reduced SC number in biopsies from the vastus lateralis muscle after ACL injury (8, 10) and the lack of increase in SC number after resumption of muscular training after ACL-R (3, 10) suggest that ACL injury and/or ACL-R with quadriceps tendon or semitendinosus tendon autografts, respectively, might generate a severe atrophic environment with negative effects on SC number and function.The primary aim of this study was to further investigate the effects of ACL-R on SCs and to determine whether the previously described reduction in SC number might be due to SC apoptosis. The study analysed muscle biopsies from the vastus lateralis muscle of recreational athletes immediately before ACL surgery and again after 12 weeks of early rehabilitation.     

Ultra-Deep Genomic Sequencing of HCV NS5A Resistance-Associated Substitutions in HCV/HIV Coinfected Patients

Background and Aims

The prevalence of naturally occurring HCV-NS5A resistance-associated substitutions (RAS) to DAA drugs might affect the response to treatment in HCV/HIV coinfected subjects. There are limited data on the frequency of HCV-NS5A naturally occurring drug-RAS at baseline in HCV/HIV coinfected patients when ultra-deep sequencing methodologies are applied.

Methods

HCV-NS5A-RAS were evaluated among 25 subjects in each group. Patients were matched by age, gender, and hepatic fibrosis stage category to control for selection bias.

Results

Within subtype 1a, RAS were observed in 28% of HCV monoinfected and 48% of HCV/HIV coinfected subjects. More patients in the HCV/HIV coinfected group had clinically relevant mutations to DAA directed at NS5A.

Conclusion

While the clinical significance of this observation may be limited in highly drug adherent populations, some HCV/HIV coinfected persons may be at greater risk of viral resistance if suboptimal dosing occurs.