Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin.

Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.

Diverse heterochromatin states restricting cell identity and reprogramming.

Heterochromatin is defined as a chromosomal domain harboring repressive H3K9me2/3 or H3K27me3 histone modifications and relevant factors that physically compact the chromatin. Heterochromatin can restrict where transcription factors bind, providing a barrier to gene activation and changes in cell identity. While heterochromatin thus helps maintain cell differentiation, it presents a barrier to overcome during efforts to reprogram cells for biomedical purposes. Recent findings have revealed complexity in the composition and regulation of heterochromatin, and shown that transiently disrupting the machinery of heterochromatin can enhance reprogramming. Here, we discuss how heterochromatin is established and maintained during development, and how our growing understanding of the mechanisms regulating H3K9me3 heterochromatin can be leveraged to improve our ability to direct changes in cell identity.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes.

Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

p53 is essential for DNA methylation homeostasis in naïve embryonic stem cells, and its loss promotes clonal heterogeneity.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53-/-) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53-/- mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53-/- mESCs display increased cellular heterogeneity both in the "naïve" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.

Rothia in Nonsmall Cell Lung Cancer is Associated With Worse Survival.

INTRODUCTION: The microbiome is known to play a significant role in cancer biology; however, few studies have elucidated its relationship with Nonsmall Cell Lung Cancer (NSCLC) patient outcomes. We hypothesized that there are specific microorganisms that are closely related with NSCLC patient survival. METHODS: Total of 647 NSCLC (Adenocarcinoma and Squamous Cell Carcinoma combined) patients in The Cancer Genome Atlas (TCGA) were analyzed using the R software. RESULTS: A Volcano Plot was analyzed with the patients divided into Short and Long Survivors by overall survival of 0.9 years, and we found that a bacterium Rothia was significantly abundant in Short Survivors, and Blastococcus, Leptospira, and Haematobacter in Long Survivors, but presence of Rothia alone was associated with overall survival. The age, race, subtype, and sex were not significantly different by the presence of Rothia in NSCLC. Unexpectedly, Rothia-positive NSCLC was associated with less cell proliferation by gene set enrichment analysis, Mki67 expression, proliferation score, with less fraction altered and homologous recombination deficiency, and with high infiltration of stromal cells, indicating favorable oncological characteristics. Further, Rothia-positive tumors were associated with significantly higher infiltration of CD8 T cells, CD4 T cells, Monocytes, and NK cells, and high interferon-gamma response, T-cell receptor richness, cytolytic activity, indicating favorable tumor immune microenvironment. CONCLUSIONS: NSCLC with Rothia was associated with worse survival but also with favorable oncological characteristics such as less cell proliferation and favorable tumor immune microenvironment. We cannot help but speculate that Rothia in NSCLC is associated with mortality unrelated to oncological characteristics.

LncPRESS1 Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56.

Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.

The effect of attentional cues on mechanical efficiency and movement smoothness in running gait: An interdisciplinary investigation.

The aim was to examine the effect of focus of attention cues on foot angle for retraining movement purposes. Twenty (females: 8) rearfoot-striking recreational runners (mass: 72.5 ± 11.8 kg; height: 1.73 ± 0.09 m; age: 32.9 ± 11.3 years) were randomly assigned to an internal focus (IF) (n = 10) or external focus (EF) (n = 10) verbal cue group. Participants performed 5 × 6 minute blocks of treadmill running (control run, 3 × cued running, retention run) at a self-selected running velocity (9.4 ± 1.1 km∙h-1) during a single laboratory visit. Touchdown foot angle, mechanical efficiency, internal and external work were calculated and, centre of mass (COM) and foot movement smoothness was quantified. Linear-mixed effect models showed an interaction for foot angle (p < 0.001, ηp2 = 0.35) and mechanical efficiency (p < 0.001, ηp2 = 0.40) when comparing the control to the cued running. Only the IF group reduced foot angle and mechanical efficiency during cued running, but not during the retention run. The IF group produced less external work during the 1st cued run than the control run. COM and foot smoothness were unaffected by cueing. Only an IF produced desired technique changes but at the cost of reduced mechanical efficiency. Movement smoothness was unaffected by cue provision. Changes to foot angle can be achieved within 6 minutes of gait retraining.

Detecting Psychological Interventions Using Bilateral Electromyographic Wearable Sensors.

This study investigated the impact of auditory stimuli on muscular activation patterns using wearable surface electromyography (EMG) sensors. Employing four key muscles (Sternocleidomastoid Muscle (SCM), Cervical Erector Muscle (CEM), Quadricep Muscles (QMs), and Tibialis Muscle (TM)) and time domain features, we differentiated the effects of four interventions: silence, music, positive reinforcement, and negative reinforcement. The results demonstrated distinct muscle responses to the interventions, with the SCM and CEM being the most sensitive to changes and the TM being the most active and stimulus dependent. Post hoc analyses revealed significant intervention-specific activations in the CEM and TM for specific time points and intervention pairs, suggesting dynamic modulation and time-dependent integration. Multi-feature analysis identified both statistical and Hjorth features as potent discriminators, reflecting diverse adaptations in muscle recruitment, activation intensity, control, and signal dynamics. These features hold promise as potential biomarkers for monitoring muscle function in various clinical and research applications. Finally, muscle-specific Random Forest classification achieved the highest accuracy and Area Under the ROC Curve for the TM, indicating its potential for differentiating interventions with high precision. This study paves the way for personalized neuroadaptive interventions in rehabilitation, sports science, ergonomics, and healthcare by exploiting the diverse and dynamic landscape of muscle responses to auditory stimuli.

Characterizing Longitudinal Alterations in Postural Control Following Lower Limb Injury in Professional Rugby Union Players.

Assessment of player's postural control following a lower limb injury is of interest to sports medicine practitioners due to its fundamental role in daily tasks and sporting activities. The aim was to longitudinally monitor professional rugby union players' postural control during each phase of the rehabilitation program (acute, middle, and late) following a lower limb injury. Seven male rugby union players (height 1.80 [0.02] m; mass 100.3 [11.4] kg; age 24 [4] y) sustained a time loss, noncontact lower limb injury. Static postural control was assessed via sway path (in meters), and dynamic postural control was assessed via vertical postural stability index. Group differences (P < .05) were reported across the acute, middle, and late phase. Smaller magnitudes of sway path were observed for eyes-open sway path, and for the middle and late phase smaller magnitudes of vertical postural stability index (P < .05) at the end session compared with first session. Whereas larger magnitudes of vertical postural stability index were found between baseline and the last session (P < .05). Large interindividual and intraindividual variation was apparent across the 3 phases of rehabilitation. Postural control improvements were identified during rehabilitation. However, postural control did not return to baseline, with altered kinetics throughout each rehabilitation phase.

Wide-Area Debris Field and Seabed Characterization of a Deep Ocean Dump Site Surveyed by Autonomous Underwater Vehicles.

Disposal of industrial and hazardous waste in the ocean was a pervasive global practice in the 20th century. Uncertainty in the quantity, location, and contents of dumped materials underscores ongoing risks to marine ecosystems and human health. This study presents an analysis of a wide-area side-scan sonar survey conducted with autonomous underwater vehicles (AUVs) at a dump site in the San Pedro Basin, California. Previous camera surveys located 60 barrels and other debris. Sediment analysis in the region showed varying concentrations of the insecticidal chemical dichlorodiphenyltrichloroethane (DDT), of which an estimated 350-700 t were discarded in the San Pedro Basin between 1947 and 1961. A lack of primary historical documents specifying DDT acid waste disposal methods has contributed to the ambiguity surrounding whether dumping occurred via bulk discharge or containerized units. Barrels and debris observed during previous surveys were used for ground truth classification algorithms based on size and acoustic intensity characteristics. Image and signal processing techniques identified over 74,000 debris targets within the survey region. Statistical, spectral, and machine learning methods characterize seabed variability and classify bottom-type. These analytical techniques combined with AUV capabilities provide a framework for efficient mapping and characterization of uncharted deep-water disposal sites.

Movement strategy correspondence across jumping and cutting tasks after anterior cruciate ligament reconstruction.

There are currently a multitude of tests used to assess readiness to return to sport (RTS) following anterior cruciate ligament reconstruction (ACLR). The aim of this study was to establish the extent to which movement strategies transfer between three common assessment tasks to help improve design of athlete testing batteries following ACLR. A cohort of 127 male patients 8-10 months post-ACLR and 45 non-injured controls took part in the study. Three movement tasks were completed (unilateral and bilateral drop jump, and 90° pre-planned cut), while ground reaction forces and three-dimensional kinematics (250 Hz) were recorded. Compared to the bilateral drop jump and cut, the unilateral drop jump had a higher proportion of work done at the ankle (d = 0.29, p < 0.001 and d = -1.87, p < 0.001, respectively), and a lower proportion of work done at the knee during the braking phase of the task (d = 0.447, p < 0.001 and d = 1.56, p < 0.001, respectively). The ACLR group had higher peak hip moments than the non-injured controls, although the proportion of work done at the ankle, knee and hip joints were similar. Movement strategies were moderately and positively related at the ankle (rs  = 0.728, p < 0.001), knee (rs  = 0.638, p < 0.001) and hip (rs  = 0.593, p < 0.001) between the unilateral and bilateral drop jump, but there was no relationship at the ankle (rs  = 0.10, p = 0.104), knee (rs  = 0.106, p = 0.166) and hip (rs  = -0.019, p = 0.808) between the unilateral drop jump and the cut. Clinicians could therefore consider omitting one of the drop jumps from assessment batteries but should include both jumping and cutting tasks.

Identifying Individuals Who Currently Report Feelings of Anxiety Using Walking Gait and Quiet Balance: An Exploratory Study Using Machine Learning.

Literature suggests that anxiety affects gait and balance among young adults. However, previous studies using machine learning (ML) have only used gait to identify individuals who report feeling anxious. Therefore, the purpose of this study was to identify individuals who report feeling anxious at that time using a combination of gait and quiet balance ML. Using a cross-sectional design, participants (n = 88) completed the Profile of Mood Survey-Short Form (POMS-SF) to measure current feelings of anxiety and were then asked to complete a modified Clinical Test for Sensory Interaction in Balance (mCTSIB) and a two-minute walk around a 6 m track while wearing nine APDM mobility sensors. Results from our study finds that Random Forest classifiers had the highest median accuracy rate (75%) and the five top features for identifying anxious individuals were all gait parameters (turn angles, variance in neck, lumbar rotation, lumbar movement in the sagittal plane, and arm movement). Post-hoc analyses suggest that individuals who reported feeling anxious also walked using gait patterns most similar to older individuals who are fearful of falling. Additionally, we find that individuals who are anxious also had less postural stability when they had visual input; however, these individuals had less movement during postural sway when visual input was removed.

Autonomous learning and interpretation of channel multipath scattering using braid manifolds in underwater acoustic communications.

In this work, we explore machine learning through a model-agnostic feature representation known as braiding, that employs braid manifolds to interpret multipath ray bundles. We generate training and testing data using the well-known BELLHOP model to simulate shallow water acoustic channels across a wide range of multipath scattering activity. We examine three different machine learning techniques-k-nearest neighbors, random forest tree ensemble, and a fully connected neural network-as well as two machine learning applications. The first application applies known physical parameters and braid information to determine the number of reflections the acoustic signal may undergo through the environment. The second application applies braid path information to determine if a braid is an important representation of the channel (i.e., evolving across bands of higher amplitude activity in the channel). Testing accuracy of the best trained machine learning algorithm in the first application was 86.70% and the testing accuracy of the second application was 99.94%. This work can be potentially beneficial in examining how the reflectors in the environment changeover time while also determining relevant braids for faster channel estimation.

Phase 1 study to evaluate safety, tolerability and pharmacokinetics of a novel intra-tympanic administered thiosulfate to prevent cisplatin-induced hearing loss in cancer patients.

Cisplatin is a widely used chemotherapy for the treatment of certain solid tumors. Ototoxicity and subsequent permanent hearing loss remain a serious dose-limiting side effect associated with cisplatin treatment. To date, no therapies have been approved to prevent or treat cisplatin-induced hearing loss (CIHL). Sodium thiosulfate effectively inactivates cisplatin through covalent binding and may provide protection against cisplatin-induced ototoxicity. DB-020 is being developed as a novel formulation of sodium thiosulfate pentahydrate in 1% sodium hyaluronate for intratympanic injection (IT), enabling the delivery of high concentrations of thiosulfate into the cochlea prior to cisplatin administration. In the DB-020-002 phase 1a single-ascending dose study, healthy volunteers were enrolled into 5 cohorts to receive different doses of DB-020 via IT injection. Cohorts 1-4 received unilateral injections while Cohort 5 received bilateral injections. Plasma thiosulfate pharmacokinetics was measured, and safety and audiometric data were collected throughout the study. This study has demonstrated that intratympanic administration of DB-020 results in nominal systemic increases in thiosulfate levels, hence it should not compromise cisplatin anti-tumor efficacy. Furthermore, DB-020 was safe and well tolerated with most adverse events reported as transient, of mild-to-moderate severity and related to the IT administration procedure. These results support the design and execution of the ongoing proof-of-concept study, DB-020-002, to assess otoprotection using DB-020 in cancer patients receiving cisplatin without negatively impacting cisplatin anti-tumor efficacy.

Inflammation-induced iron transport and metabolism by brain microglia.

Microglia are immune cells of the central nervous system and are implicated in brain inflammation. However, how brain microglia modulate transport and metabolism of the essential metal iron in response to pro- and anti-inflammatory environmental cues is unclear. Here, we characterized uptake of transferrin (Tf)-bound iron (TBI) and non-Tf-bound iron (NTBI) by immortalized microglial (IMG) cells. We found that these cells preferentially take up NTBI in response to the proinflammatory stimulus lipopolysaccharide (LPS) or ß-amyloid (Aß). In contrast, the anti-inflammatory cytokine interleukin 4 (IL-4) promoted TBI uptake. Concordant with these functional data, levels of the Tf receptor (TfR) in IMG cells were up-regulated in response to IL-4, whereas divalent metal transporter-1 (DMT1) and ferritin levels increased in response to LPS or Aß. Similar changes in expression were confirmed in isolated primary adult mouse microglia treated with pro- or anti-inflammatory inducers. LPS-induced changes in IMG cell iron metabolism were accompanied by notable metabolic changes, including increased glycolysis and decreased oxidative respiration. Under these conditions, the extracellular acidification rate was increased, compatible with changes in the cellular microenvironment that would support the pH-dependent function of DMT1. Moreover, LPS increased heme oxygenase-1 (HO1) expression in IMG cells, and iron released because of HO1 activity increased the intracellular labile free-iron pool. Together, this evidence indicates that brain microglia preferentially acquire iron from Tf or from non-Tf sources, depending on their polarization state; that NTBI uptake is enhanced by the proinflammatory response; and that under these conditions microglia sequester both extra- and intracellular iron.

MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296.

Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta.

BACKGROUND: Alzheimer's disease is associated with amyloid-beta (Aß)-induced microglia activation. This pro-inflammatory response promotes neuronal damage, and therapies are sought to limit microglial activation. Screening efforts to develop new pharmacological inhibitors require a robust in vitro cell system. Current models lack significant responses to Aß, and their use in examining age-related neurodegenerative diseases is questionable. For example, the commonly used BV-2 microglial line was derived from embryonic mononuclear cells and its activation by various stimuli is limited. To this end, we have established a new immortalized microglial (IMG) cell line from adult murine brain. The objective of this study was to characterize Aß-induced activation of IMG cells, and here, we demonstrate the ability of cannabinoids to significantly reduce this inflammatory response. METHODS: Microglial cells derived from adult murine brain were immortalized via infection with the v-raf/v-myc retrovirus under conditions that selectively promote microglia growth. The presence or absence of markers CD11b and F4/80 (microglial), NeuN (neuronal), and GFAP (astrocytic) was assessed by immunofluorescence microscopy and western blotting. Using IMG and BV-2 cells, levels of pro- and anti-inflammatory transcripts in response to extracellular stimuli were determined by quantitative PCR (qPCR). Phagocytosis of fluorescent beads and fluorescein isothiocyanate (FITC)-labeled Aß oligomers was assessed using flow cytometry and fluorescence microscopy. FITC-Aß uptake was quantified using a fluorescence plate reader. The ability of cannabinoids to mitigate Aß-induced expression of inducible nitric oxide synthase (iNOS) was evaluated. RESULTS: IMG cells express the microglial markers CD11b and F4/80 but not NeuN or GFAP. Relative to BV-2 cells, IMG cells increased iNOS (>200-fold) and Arg-1 (>100-fold) in response to pro- and anti-inflammatory stimuli. IMG cells phagocytose foreign particles and Aß oligomers, with the latter trafficked to phagolysosomes. Aß-induced activation of IMG cells was suppressed by delta-9-tetrahydrocannabinol and the CB2-selective agonist JWH-015 in a time- and concentration-dependent manner. CONCLUSIONS: IMG cells recapitulate key features of microglial cell activation. As an example of their potential pharmacological use, cannabinoids were shown to reduce activation of Aß-induced iNOS gene expression. IMG cells hold promising potential for drug screening, mechanistic studies, and functional investigations directed towards understanding how Aß interacts with microglia.

sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Iron transport across the blood-brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy.

There are two barriers for iron entry into the brain: (1) the brain-cerebrospinal fluid (CSF) barrier and (2) the blood-brain barrier (BBB). Here, we review the literature on developmental iron accumulation by the brain, focusing on the transport of iron through the brain microvascular endothelial cells (BMVEC) of the BBB. We review the iron trafficking proteins which may be involved in the iron flux across BMVEC and discuss the plausible mechanisms of BMVEC iron uptake and efflux. We suggest a model for how BMVEC iron uptake and efflux are regulated and a mechanism by which the majority of iron is trafficked across the developing BBB under the direct guidance of neighboring astrocytes. Thus, we place brain iron uptake in the context of the neurovascular unit of the adult brain. Last, we propose that BMVEC iron is involved in the aggregation of amyloid-ß peptides leading to the progression of cerebral amyloid angiopathy which often occurs prior to dementia and the onset of Alzheimer's disease.