MORC3 represses the HCMV major immediate early promoter in myeloid cells in the absence of PML nuclear bodies.

Human cytomegalovirus (HCMV) can undergo either a latent or a lytic infection in cells of the myeloid lineage. Whilst the molecular mechanisms which determine the outcome of infection are far from clear, it is well established that a key factor is the differential regulation of the major immediate early promoter (MIEP) responsible for driving lytic immediate early gene expression. Using a myelomonocytic cell line stably transduced with a GFP reporter under the control of the MIEP, which recapitulates MIEP regulation in the context of virus infection, we have used an unbiased CRISPR-Cas9 sub-genomic, epigenetic library screen to identify novel cellular factors involved in MIEP repression during establishment and maintenance of latency in myeloid cells. One such cellular factor identified was MORC3. Consistent with MORC3 being a robust repressor of the MIEP, we show that THP1 cells devoid of MORC3 fail to establish latency. We also show that MORC3 is induced during latent infection, recruited to the MIEP and forms MORC3 nuclear bodies (MORC3-NBs) which, interestingly, co-localize with viral genomes. Finally, we show that the latency-associated functions of MORC3 are regulated by the deSUMOylase activity of the viral latency-associated LUNA protein likely to prevent untimely HCMV reactivation.

Incidence and profile of inpatient stroke-induced aphasia in Ontario, Canada.

OBJECTIVES: To determine the incidence rate of inpatient stroke-induced aphasia in Ontario, Canada, and to examine the demographic and clinical characteristics for stroke patients with and without aphasia. DESIGN: Age- and sex-specific incidence rates for aphasia in Ontario were calculated using the Ontario Stroke Audit. In addition, data collected from the Registry of the Canadian Stroke Network (RCSN) were used to determine the demographic and clinical characteristics for stroke patients with and without aphasia. SETTING: All hospitals and regional stroke centers in Ontario, Canada. PARTICIPANTS: The Ontario Stroke Audit is a representative weighted sample of more than 3000 stroke inpatients admitted to emergency departments in all hospitals in Ontario within the 2004/2005 fiscal year. RCSN data included a cohort of more than 15,000 consecutive patients presenting with stroke at 12 regional stroke centers in Ontario from 2003 to 2007. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Presence of aphasic symptoms on admission to hospital and at discharge, age and sex, stroke type and severity, severity of disability, services received in hospital, length of stay, and discharge destination. RESULTS: Thirty-five percent (1131/3207) of adult patients admitted with a diagnosis of stroke in the province of Ontario during the 2004 to 2005 Ontario Stroke Audit had symptoms of aphasia at the time of discharge. This amounts to an incidence rate of 60 per 100,000 persons per year. Risk of aphasia increased significantly with age. In comparison with nonaphasic stroke patients, patients with aphasia were older, presented with more severe strokes on admission, had more severe disability, and were more frequently discharged to long-term care and/or rehabilitation (unadjusted results). Adjusting for stroke severity, age, sex, comorbidity, and stroke subtype, the presence of aphasia was found to be an independent predictor of longer hospital stays, increased use of rehabilitation services, and higher rates of thrombolytic therapy. CONCLUSIONS: A significant number of people with stroke experience aphasia, with advancing age associated with a higher risk. The profile and patterns for stroke patients with aphasia differed significantly from those who did not experience aphasia as a residual disability after stroke, particularly in relation to service usage. Given the personal and system cost associated with aphasia, best practices in the area of stroke should include recommendations on how to best serve this population throughout the clinical pathway.

Distinct sarcomeric substrates are responsible for protein kinase D-mediated regulation of cardiac myofilament Ca2+ sensitivity and cross-bridge cycling.

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser(22)/Ser(23), reduces myofilament Ca(2+) sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser(22)/Ser(23) remains to be established. To determine the role of cTnI phosphorylation at Ser(22)/Ser(23) in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser(22)/Ser(23) are substituted by nonphosphorylatable Ala (cTnI-Ala(2)). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser(22)/Ser(23) and decreased the Ca(2+) sensitivity of force. In contrast, PKD had no effect on the Ca(2+) sensitivity of force in myocardium from cTnI-Ala(2) mice, in which Ser(22)/Ser(23) were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala(2) mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser(273), Ser(282), and Ser(302), and revealed that PKD phosphorylates only Ser(302). Furthermore, PKD phosphorylated Ser(302) selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala(2) mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca(2+) sensitivity through cTnI phosphorylation at Ser(22)/Ser(23) but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser(302), which may mediate the latter effect.

Protein kinase d in the cardiovascular system: emerging roles in health and disease.

The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases, within the protein kinase complement of the mammalian genome. Relative to their alphabetically superior cousins in the AGC group of kinases, namely the various isoforms of protein kinase A, protein kinase B/Akt, and protein kinase C, PKD family members have to date received limited attention from cardiovascular investigators. Nevertheless, increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. This review provides a primer on PKD signaling, using information gained from studies in multiple cell types, and discusses recent data that suggest novel functions for PKD-mediated pathways in the heart and the circulation.