Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its ...associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.
•Viral DNA replication promotes DNA-PK activation and genome concatenation during E4-infection.•The MRN complex is not essential for DNA-PK autophosphorylation at Ser2056.•Ku86 is transiently localized to viral replication centers, which is promoted by the MRN complex.
Background
Multi‐component, multi‐target approaches may be needed for treatment of Vascular Dementia (VaD). SaiLuoTong (SLT) consists of active ingredients derived from Ginkgo Biloba, Panax Ginseng ...and Crocus Sativus. A phase II, randomized, controlled, double‐blind study (NCT01978730) demonstrated the symptomatic efficacy of SLT in VaD, however, the slowing of disease progression remains unexplored. This exploratory analysis is to compare the Vascular Dementia Assessment Scale‐cognitive subscale (VaDAS‐cog) between early‐ and delayed‐starters over a 52‐week period so as to assess the disease‐modification effect of SLT.
Method
Subjects with probable mild to moderate VaD were randomized to SLT 360 or 240 mg (early‐starters), or placebo (delayed‐starters). Whilst maintaining blinding, all early‐starters received SLT for 52 weeks and delayed‐starters switched to SLT 360 or 240 mg from placebo at week‐26. Using a modified intention‐to‐treat population of 325 subjects, comparison of change in VaDAS‐cog from baseline between early‐starters and delayed‐starters was performed using linear mixed‐effects models for repeated measurements, with fixed effect of intervention (early‐starters vs. delayed‐starters), time, time and intervention interaction, baseline VaDAS‐cog and randomization stratification factors. An unstructured variance‐covariance structure was employed.
Result
Early‐starters compared to delayed‐starters showed statistically significant improvement in adjusted mean change from baseline in VaDAS‐cog score at all timepoints: (Adjusted mean difference (AMD) 95% CI: ‐1.53 ‐2.35, ‐0.71; P<0.001) at week‐13, (AMD 95% CI: ‐2.69 ‐3.69, ‐1.69; P<0.001) at week‐26, (AMD 95% CI: ‐2.15 ‐3.31, ‐0.98; P<0.001) at week‐39 and (AMD 95% CI: ‐1.94 ‐3.38, ‐0.51; P = 0.008) at week‐52. The largest difference (early‐starters vs delayed‐starters) was observed at week‐26.
Conclusion
In this analysis, the effect of SLT was observed at week‐13 in early‐starters and sustained until week‐52. Delayed‐starters did not catch up with early‐starters on mean change in VaDAS‐cog from baseline at week‐52, suggesting not only a symptomatic effect of SLT but also a possible disease‐modifying effect.
References: Jia J, Wei C, Chen S, et al. Efficacy and safety of the compound Chinese medicine SaiLuoTong in vascular dementia: A randomized clinical trial. Alzheimers Dement (N Y). 2018;4:108‐117. Published 2018 Mar 22. https://doi.org/10.1016/j.trci.2018.02.004
Plasma homocysteine levels are increasingly studied as a potential risk factor for dementia. Elevated homocysteine levels have been linked with gray and white matter volume reduction among ...individuals with mild cognitive impairment and Alzheimer's disease. However, the effects of homocysteine on brain changes in preclinical stages of dementia remain unexplored.
To examine the association of elevated homocysteine levels with markers of neurodegeneration, i.e., white and gray matter volume in an elderly population.
The study included 768 participants (mean age: 69.6±6.5 years, 51.3% women) from the Epidemiology of Dementia In Singapore study. Participants underwent a brain MRI scan and blood tests. Serum homocysteine was measured using competitive immunoassay. Cortical thickness and subcortical structural volume were quantified using FreeSurfer whereas white matter volume was quantified using a previous validated method.
Higher homocysteine levels were significantly associated with decreased global white matter volume mean difference (β) in volume (ml) per micromole per liter (μmol/l) increase in homocysteine levels: - 0.555, 95% Confidence Interval (CI): - 0.873; - 0.237, decreased parietal cortical thickness β in thickness (μm) per μmol/l increase in homocysteine levels:- 1.429, 95% CI: - 2.781; - 0.077, and smaller volumes of the thalamus β: - 0.017, 95% CI: - 0.026; - 0.008, brainstem β: - 0.037, 95% CI: - 0.058; - 0.016, and accumbens β: - 0.004, 95% CI: - 0.006; - 0.002.
Higher homocysteine levels were associated with cerebral atrophy. Further studies are required to assess whether lowering plasma homocysteine levels may prevent neurodegenerative changes or delay progression of clinical symptoms before the development of dementia.
Rising temperatures in most agricultural regions of the world are associated with a higher incidence of extreme weather events such as heat waves. We performed an experiment to mitigate the impact of ...heat waves and exposure of berries in grapevine (
Vitis vinifera
cv. “Cabernet Sauvignon”) with untreated vines (Exposed) or with fruit-zone partial shading (Shaded) under 40 and 80% replacement of crop evapotranspiration (ET
c
) with sustained deficit irrigation in a factorially arranged experiment. The trial was performed in a vineyard with vertically shoot positioned trellis with a row orientation that concentrated solar radiation exposure on the southwest aspect of the fruit zone. Leaf stomatal conductance (
g
s
) and net carbon assimilation (
A
N
) were significantly lower in shaded leaves under partial fruit-zone shading that resulted in lower pruning mass for Shaded treatments. Stem water potential (Ψ
stem
) responded to a large extent to increased irrigation. However, grapevines with partial fruit-zone shading had transiently better water status under 40% ET
c
. Cluster maximum temperatures were 3.9°C greater in Exposed grapevines. Exposed clusters had transiently lower acidity and higher pH. However, Exposed clusters on 40% ET
c
had higher total soluble solids (TSS). The experimental vineyard suffered a 4-day heat wave 21 days before harvest, resulting in 25% of the clusters being damaged in Exposed treatment, regardless of irrigation amount. Furthermore, berries in Exposed treatments suffered a great loss of anthocyanins and flavonols even if they were not damaged by direct solar exposure. The pre-planting decision of using a vertically shoot positioned trellis that concentrated solar radiation on the Southwest aspect offered mild protection in a hot climate region with a sunny growing season with extreme heat events during the execution of study. The extreme conditions under which this study was conducted are not unusual, and have become more expected. Our work provided evidence of the vulnerability of grape berry to heat waves and exposure during heat wave events and possible protection methods to mitigate these effects
in situ
in context of climate change.
Despite recent interest in community-based screening programs to detect undiagnosed cognitive disorder, little is known about whether screening leads to further diagnostic evaluation, or the effects ...of such programs in terms of actual changes in patient or caregiver behavior. This study followed up informants of older adults (i.e. caregivers of patients who completed informant-based screening regarding the patient) following participation in a study screening for undiagnosed memory problems, to explore uptake of further diagnostic evaluation or treatment, advance planning or preparations, lifestyle changes, medication adherence, and use of support services.
A total of 140 informants of older adult patients were surveyed four to fifteen months following participation in a cognitive screening study. The informants were interviewed with a study-specific survey about cognitive assessment, advance planning, lifestyle changes, and use of support services and general medication adherence.
A minority of patients and informants had engaged in advance planning or made relevant lifestyle changes following cognitive screening. Those assessed as being at higher risk of memory problems were more likely to have attended a full diagnostic evaluation, engaged in support services and experienced medication adherence difficulties.
Only a small proportion of patients participating in cognitive screening subsequently engaged in diagnostic evaluation, advance planning, or lifestyle changes. However, those with higher risk of cognitive impairment were generally more likely to take some action following cognitive screening. Those at higher risk were also more vulnerable due to greater difficulties with medication adherence.
We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate ...diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.
Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been ...their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs.
First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect.
Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to β-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.
An approach is described for controlling the spatial organization of mammalian cells using ferromagnetic nanowires in conjunction with patterned micromagnet arrays. The nanowires are fabricated by ...electrodeposition in nanoporous templates, which allows for precise control of their size and magnetic properties. The high aspect ratio and large remanent magnetization of the nanowires enable suspensions of cells bound to Ni nanowires to be controlled with low magnetic fields. This was used to produce one- and two-dimensional field-tuned patterning of suspended 3T3 mouse fibroblasts. Self-assembled one-dimensional chains of cells were obtained through manipulation of the wires' dipolar interactions. Ordered patterns of individual cells in two dimensions were formed through trapping onto magnetic microarrays of ellipsoidal permalloy micromagnets. Cell chains were formed on the arrays by varying the spacing between the micromagnets or the strength of fluid flow over the arrays. The positioning of cells on the array was further controlled by varying the direction of an external magnetic field. These results demonstrate the possibility of using magnetic nanowires to organize cells.
Cortical atrophy is a key neuroimaging feature of dementia. However, the role of subcortical gray matter reduction in cognitive impairment has not been explored extensively.
We examined the risk ...factors of subcortical structures on neuroimaging and their association with cognitive impairment and dementia.
Data from two studies were used: a subsample from the Epidemiology of Dementia in Singapore (EDIS) study of non-demented community-dwelling subjects (n = 550) and a case-control study. Subjects underwent similar neuropsychological tests and brain MRI. Subcortical volumes of accumbens, amygdala, caudate, pallidum, putamen, thalamus, hippocampus, and brainstem were measured. Cognitive impairment no dementia (CIND), dementia and its subtypes, vascular cognitive impairment (VCI), were defined using accepted criteria. Cognitive function was also expressed as both composite and domain-specific Z-scores.
In the EDIS study, age, female gender, Malay ethnicity, diabetes, lacunar-infarcts, and white matter lesions were the most important risk factors for subcortical atrophy. Moreover, smaller volumes of accumbens, amygdala, caudate, thalamus, and brainstem were significantly associated with lower cognitive composite Z-scores. With respect to clinical outcomes in the case-control study, structures such as the accumbens, caudate, putamen, and hippocampus were associated with both CIND and dementia. Smaller caudate and pallidum volumes were related to VCI whereas amygdalar atrophy was only associated with non-VCI. Furthermore, subcortical atrophy was related to both VCI and non-VCI.
Subcortical gray matter atrophy is not only observed in dementia, but also in the preclinical stages of cognitive impairment. Furthermore, besides VCI, subcortical structures were also related to non-VCI.
Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have ...uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence
RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.
B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.
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