Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.

Retroviral vectors are silenced in embryonic stem (ES) cells by epigenetic mechanisms whose kinetics are poorly understood. We show here that a 3'D4Z4 insulator directs retroviral expression with persistent but variable expression for up to 5 months. Combining an internal 3'D4Z4 with HS4 insulators in the long terminal repeats (LTRs) shows that these elements cooperate, and defines the first retroviral vector that fully escapes long-term silencing. Using FLP recombinase to induce deletion of 3'D4Z4 from the provirus in ES cell clones, we established retroviral silencing at many but not all integration sites. This finding shows that 3'D4Z4 does not target retrovirus integration into favorable epigenomic domains but rather protects the transgene from silencing. Chromatin analyses demonstrate that 3'D4Z4 blocks the spread of heterochromatin marks including DNA methylation and repressive histone modifications such as H3K9 methylation. In addition, our deletion system reveals three distinct kinetic classes of silencing (rapid, gradual or not silenced), in which multiple epigenetic pathways participate in silencing at different integration sites. We conclude that vectors with both 3'D4Z4 and HS4 insulator elements fully block silencing, and may have unprecedented utility for gene transfer applications that require long-term gene expression in pluripotent stem (PS) cells.

Angiogenesis-associated pathways play critical roles in neonatal sepsis outcomes.

Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.

Adaptive immune responses to two-dose COVID-19 vaccine series in healthy Canadian adults ≥ 50 years: a prospective, observational cohort study.

To evaluate immune responses to COVID-19 vaccines in adults aged 50 years and older, spike protein (S)-specific antibody concentration, avidity, and function (via angiotensin-converting enzyme 2 (ACE2) inhibition surrogate neutralization and antibody dependent cellular phagocytosis (ADCP)), as well as S-specific T cells were quantified via activation induced marker (AIM) assay in response to two-dose series. Eighty-four adults were vaccinated with either: mRNA/mRNA (mRNA-1273 and/or BNT162b2); ChAdOx1-S/mRNA; or ChAdOx1-S/ChAdOx1-S. Anti-S IgG concentrations, ADCP scores and ACE2 inhibiting antibody concentrations were highest at one-month post-second dose and declined by four-months post-second dose for all groups. mRNA/mRNA and ChAdOx1-S/mRNA schedules had significantly higher antibody responses than ChAdOx1-S/ChAdOx1-S. CD8+ T-cell responses one-month post-second dose were associated with increased ACE2 surrogate neutralization. Antibody avidity (total relative avidity index) did not change between one-month and four-months post-second dose and did not significantly differ between groups by four-months post-second dose. In determining COVID-19 correlates of protection, a measure that considers both antibody concentration and avidity should be considered.

Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

Differential effects of bicarbonate on severe hypoxia- and hypercapnia-induced cardiac malfunctions in diverse fish species.

We tested in six fish species [Pacific lamprey (Lampetra richardsoni), Pacific spiny dogfish (Squalus suckleyi), Asian swamp eel (Monopterus albus), white sturgeon (Acipenser transmontanus), zebrafish (Danio rerio), and starry flounder (Platichthys stellatus)] the hypothesis that elevated extracellular [HCO3-] protects spontaneous heart rate and cardiac force development from the known impairments that severe hypoxia and hypercapnic acidosis can induce. Hearts were exposed in vitro to either severe hypoxia (~ 3% of air saturation), or severe hypercapnic acidosis (either 7.5% CO2 or 15% CO2), which reduced heart rate (in six test species) and net force development (in three test species). During hypoxia, heart rate was restored by [HCO3-] in a dose-dependent fashion in lamprey, dogfish and eel (EC50 = 5, 25 and 30 mM, respectively), but not in sturgeon, zebrafish or flounder. During hypercapnia, elevated [HCO3-] completely restored heart rate in dogfish, eel and sturgeon (EC50 = 5, 25 and 30 mM, respectively), had a partial effect in lamprey and zebrafish, and had no effect in flounder. Elevated [HCO3-], however, had no significant effect on net force of electrically paced ventricular strips from dogfish, eel and flounder during hypoxia and hypercapnia. Only in lamprey hearts did a specific soluble adenylyl cyclase (sAC) inhibitor, KH7, block the HCO3--mediated rescue of heart rate during both hypoxia and hypercapnia, and was the only species where we conclusively demonstrated sAC activity was involved in the protective effects of HCO3- on cardiac function. Our results suggest a common HCO3--dependent, sAC-dependent transduction pathway for heart rate recovery exists in cyclostomes and a HCO3--dependent, sAC-independent pathway exists in other fish species.

Meditation-induced neuroplastic changes of the prefrontal network are associated with reduced valence perception in older people.

BACKGROUND: Neuroplastic underpinnings of meditation-induced changes in affective processing are largely unclear. METHODS: We included healthy older participants in an active-controlled experiment. They were involved a meditation training or a control relaxation training of eight weeks. Associations between behavioral and neural morphometric changes induced by the training were examined. RESULTS: The meditation group demonstrated a change in valence perception indexed by more neutral valence ratings of positive and negative affective images. These behavioral changes were associated with synchronous structural enlargements in a prefrontal network involving the ventromedial prefrontal cortex and the inferior frontal sulcus. In addition, these neuroplastic effects were modulated by the enlargement in the inferior frontal junction. In contrast, these prefrontal enlargements were absent in the active control group, which completed a relaxation training. Supported by a path analysis, we propose a model that describes how meditation may induce a series of prefrontal neuroplastic changes related to valence perception. These brain areas showing meditation-induced structural enlargements are reduced in older people with affective dysregulations. CONCLUSION: We demonstrated that a prefrontal network was enlarged after eight weeks of meditation training. Our findings yield translational insights in the endeavor to promote healthy aging by means of meditation.

Potential therapeutic effects of meditation for treating affective dysregulation.

Affective dysregulation is at the root of many psychopathologies, including stress induced disorders, anxiety disorders, and depression. The root of these disorders appears to be an attenuated, top-down cognitive control from the prefrontal cortices over the maladaptive subcortical emotional processing. A form of mental training, long-term meditation practice can trigger meditation-specific neuroplastic changes in the brain regions underlying cognitive control and affective regulation, suggesting that meditation can act as a kind of mental exercise to foster affective regulation and possibly a cost-effective intervention in mood disorders. Increasing research has suggested that the cultivation of awareness and acceptance along with a nonjudgmental attitude via meditation promotes adaptive affective regulation. This review examined the concepts of affective regulation and meditation and discussed behavioral and neural evidence of the potential clinical application of meditation. Lately, there has been a growing trend toward incorporating the "mindfulness" component into existing psychotherapeutic treatment. Promising results have been observed thus far. Future studies may consider exploring the possibility of integrating the element of "compassion" into current psychotherapeutic approaches.

Environmental drivers of small scale spatial variation in the reproductive schedule of a commercially important bivalve mollusc.

Understanding variability in reproductive schedules is essential to the management of recruitment limited fisheries such as that of Pecten maximus. Small scale (<5 km) variation in gonad condition and the onset of spawning of P. maximus were found among commercial scallop grounds in Isle of Man waters. Environmental and fishing drivers of these spatial patterns were investigated using a generalised additive model. Rate of change in temperature over the month prior to sampling was identified as the short term driver of gonad weight associated with the autumn spawning event. Long term drivers were average annual chlorophyll a concentration, scallop density, stratification index and shell size. The model explained 42.8% of deviance in gonad weight. Within site variation in gonad condition was high, indicating a "bet hedging" reproductive strategy which may decrease the chance of fertilisation especially at low densities. Therefore, areas protected from fishing, where scallop densities can increase may help buffer against reproductive failure. An increase in shell length from 100 mm to 110 mm equated to an increase of approximately 20% in gonad weight. Protecting scallops from fishing mortality until 110 mm (age four) compared to 100 mm (age three) may lead to an overall increase in lifetime reproductive output by a factor of 3.4.

Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.