The SUMO E3 ligase activity of ORF45 determines KSHV lytic replication.

RSK1, an essential cellular kinase for Kaposi's sarcoma-associated herpesvirus (KSHV) replication, is highly phosphorylated and SUMOylated during KSHV lytic cycle, which determine the substrate phosphorylation and specificity of RSK1, respectively. However, the SUMO E3 ligase responsible for attaching SUMO to RSK1 has not yet been identified. By genome-wide screening, we found that KSHV ORF45 is necessary and sufficient to enhance RSK1 SUMOylation. Mechanistically, KSHV ORF45 binds to SUMOs via two classic SUMO-interacting motifs (SIMs) and functions as a SIM-dependent SUMO E3 ligase for RSK1. Mutations on these ORF45 SIMs resulted in much lower lytic gene expressions, viral DNA replication, and mature progeny virus production. Interestingly, KSHV ORF45 controls RSK1 SUMOylation and phosphorylation via two separated functional regions: SIMs and amino acid 17-90, respectively, which do not affect each other. Similar to KSHV ORF45, ORF45 of Rhesus Macaque Rhadinovirus has only one SIM and also increases RSK1 SUMOylation in a SIM-dependent manner, while other ORF45 homologues do not have this function. Our work characterized ORF45 as a novel virus encoded SUMO E3 ligase, which is required for ORF45-RSK1 axis-mediated KSHV lytic gene expression.

RSK1 SUMOylation is required for KSHV lytic replication.

RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1K110/335/421R expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (166IRVDV170), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.

Genetic variety of ORF3a shapes SARS-CoV-2 fitness through modulation of lipid droplet.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leads to the accumulation of lipid droplets (LD), the central hubs of the lipid metabolism, in vitro or in type II pneumocytes and monocytes from coronavirus disease 19 (COVID-19) patients and blockage of LD formation by specific inhibitors impedes SARS-CoV-2 replication. Here, we showed that ORF3a is necessary and sufficient to trigger LD accumulation during SARS-CoV-2 infection, leading to efficient virus replication. Although highly mutated during evolution, ORF3a-mediated LD modulation is conserved in most SARS-CoV-2 variants except the Beta strain and is a major difference between SARS-CoV and SARS-CoV-2 that depends on the genetic variations on the amino acid position 171, 193, and 219 of ORF3a. Importantly, T223I substitution in recent Omicron strains (BA.2-BF.8) impairs ORF3a-Vps39 association and LD accumulation, leading to less efficient replication and potentially contributing to lower pathogenesis of the Omicron strains. Our work characterized how SARS-CoV-2 modulates cellular lipid homeostasis to benefit its replication during virus evolution, making ORF3a-LD axis a promising drug target for the treatment of COVID-19.

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent for the coronavirus disease 2019 (COVID-19) pandemic and there is an urgent need to understand the cellular response to SARS-CoV-2 infection. Beclin 1 is an essential scaffold autophagy protein that forms two distinct subcomplexes with modulators Atg14 and UVRAG, responsible for autophagosome formation and maturation, respectively. In the present study, we found that SARS-CoV-2 infection triggers an incomplete autophagy response, elevated autophagosome formation but impaired autophagosome maturation, and declined autophagy by genetic knockout of essential autophagic genes reduces SARS-CoV-2 replication efficiency. By screening 26 viral proteins of SARS-CoV-2, we demonstrated that expression of ORF3a alone is sufficient to induce incomplete autophagy. Mechanistically, SARS-CoV-2 ORF3a interacts with autophagy regulator UVRAG to facilitate PI3KC3-C1 (Beclin-1-Vps34-Atg14) but selectively inhibit PI3KC3-C2 (Beclin-1-Vps34-UVRAG). Interestingly, although SARS-CoV ORF3a shares 72.7% amino acid identity with the SARS-CoV-2 ORF3a, the former had no effect on cellular autophagy response. Thus, our findings provide the mechanistic evidence of possible takeover of host autophagy machinery by ORF3a to facilitate SARS-CoV-2 replication and raise the possibility of targeting the autophagic pathway for the treatment of COVID-19.

Effectiveness of a Two-Tier Family-Oriented Intervention in Enhancing the Family Functioning and Care Capacity of the Family Caregivers of Stroke Survivors: Protocol for a Randomized Controlled Trial.

BACKGROUND: Stroke has profound impacts on families. Often, family members, including stroke survivors and the person who takes up the role of the primary caregiver, would encounter demands on finances, rehabilitation arrangement, and even conflicts. Hence, a family-oriented intervention is expected to enable families to rebuild internal and external resources to achieve optimal rehabilitation and community reintegration. OBJECTIVE: This study aims to describe a design of a two-tier family-oriented care management intervention for enhancing the family functioning and care capacity of the caregivers of stroke survivors. METHODS: The two-tier care management intervention was guided by a standardized protocol conducted by trained professional care managers (first tier) with the support of trained volunteers (second tier), which lasted for 8-12 weeks. Participants were recruited through collaborating hospitals according to inclusion and exclusion criteria. In order to examine the effectiveness and cost-effectiveness of the two-tier care management intervention, a two-arm randomization multicenter study was designed, including an active comparison group, which was guided by a standardized protocol conducted by trained volunteers. Dyadic participants, including both stroke survivors and their primary caregivers for both groups, were invited to participate in a questionnaire survey using standardized and purposefully developed measures 3 times: before the intervention, immediately after the intervention, and 2 months after the intervention. The primary outcome was family functioning measured by the Family Role Performance Scale and Family Assessment Device-General Functioning Scale. The secondary outcomes included caregiving burden, depressive symptoms, care management strategies, and the incremental cost-effectiveness ratio. RESULTS: Recruitment began in January 2017 and was completed at the end of April 2019. Data collection was completed at the end of March 2020. As of March 2020, enrollment has been completed (n=264 stroke caregivers). A total of 200 participants completed the baseline questionnaires. We aim to publish the results by mid-2021. CONCLUSIONS: This study successfully developed a two-tier care management protocol that aims to enhance the family functioning of the caregivers of stroke survivors. Guided by a standardized protocol, this family-oriented two-tier intervention protocol was found to be feasible among Chinese families. TRIAL REGISTRATION: ClinicalTrials.gov NCT03034330; https://ichgcp.net/clinical-trials-registry/NCT03034330. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/16703.

Second-trimester serum cytokines in women who develop spontaneous preterm labor at less than 28 weeks' gestation versus term labor.

We sought to determine if there is a relationship between serum concentrations of cytokines and the development of preterm labor. A panel of 28 cytokines was measured using the multiplex assay in serum samples collected between 15 and 18 weeks' gestation from women who developed spontaneous preterm labor and delivered between 24 and 28 weeks' gestation (N = 25) and from women who delivered at term (>or=37 weeks; N = 25). Sixteen of the 28 cytokines measured were detected. Except for vascular endothelial growth factor, which showed a trend toward a significant increase in patients who developed preterm labor, there was no difference in cytokine levels between groups in preterm labor and in term labor. Serum cytokine changes in women who develop spontaneous preterm labor possibly occur in the period between 18 weeks' gestation and the onset of labor.

An Assessment of Randomized Controlled Trial Quality in The Journal of Bone & Joint Surgery: Update from 2001 to 2013.

BACKGROUND: The quality of reporting of randomized controlled trials (RCTs) published in The Journal of Bone & Joint Surgery (JBJS) from 1988 to 2000 was previously analyzed. The purpose of this current study was to analyze the quality of reporting of RCTs published in JBJS from 2001 to 2013 to identify trends over time and potential areas of improvement for future clinical trials. METHODS: A manual search of the JBJS database identified RCTs published between January 2001 and December 2013. Quality assessments, using the Detsky quality-of-reporting index (Detsky score), a modified Cochrane risk-of-bias tool, and abstraction of relevant data identifying predictors of quality, were conducted. RESULTS: A total of 5,780 publications were identified in JBJS from 2001 to 2013, with 285 RCTs (4.9%), representing an increase from the prior 13-year period. The overall mean transformed Detsky score (and standard error) increased significantly (p < 0.001) from 68.1% ± 1.67% to 76.24% ± 0.72%. The percentage of multicenter RCTs decreased from 67% to 31%. The percentage of positive trials also decreased from 80% to 50.5%, as did the mean sample size (212 to 166). Regression analysis indicated that trials with an epidemiologist as the first author and nonsurgical trials were significantly associated (p = 0.001) with a higher overall trial quality score. The categories of the lowest mean methodology scores were randomization and concealment, eligibility criteria, and reasons for patient exclusion, as identified with the Detsky score, and patient and assessor blinding, as identified with the risk-of-bias assessment. CONCLUSIONS: The quantity and quality of published RCTs in JBJS have increased in the 2001 to 2013 time period compared with the previous time period. Although these improvements are encouraging, trends to smaller, single-center trials were also observed. To efficiently determine the efficacy of orthopaedic treatments and limit bias, high-quality randomized trials of appropriate sample size and rigorous design are needed.

Rupture of ectopic pregnancy with minimally detectable beta-human chorionic gonadotropin levels: a report of 2 cases.

BACKGROUND: Several studies have demonstrated that 25-77% of ectopic pregnancies spontaneously resolve with expectant management. However, expectant management is controversial and should be considered only for patients with small, unruptured gestational sacs, low beta-human chorionic gonadotropin (beta-hCG) levels and absence of symptoms. There is no consensus on how long to follow such patients. CASES: Two patients with beta-hCG levels < 10 mIU/mL presented with ruptured ectopic pregnancy and hemoperitoneum. CONCLUSION: While expectant management of a suspected ectopic pregnancy may allow spontaneous resolution of such an ectopic pregnancy, rupture may occur at any time and even with extremely low beta-hCG levels. Patients need to be counseled about the risks of rupture and symptoms, immediate action should be taken if symptoms develop, and serum beta-hCG levels should be followed to zero.