Benefits and risks of orthokeratology treatment: a systematic review and meta-analysis.

BACKGROUND: This meta-analysis reviews the evidence for the risks and benefits associated with orthokeratology (OK) treatment compared with other methods of myopia control in children and adults. METHODS: A systematic search of Cochrane Central Register of Controlled Trials, Pubmed, Embase and Ovid was conducted from database inception to 22nd August 2021. Studies that reported on risks, visual and ocular biometric effects of OK in patients > 5 years of age with myopia (- 0.75 to - 6.00D) were included. Main outcomes are change in axial length and any adverse event. RESULTS: Fourty-five papers were included in this systematic review and meta-analysis. The quality of data was variable and of moderate certainty, and selection bias likely skewed the results towards a relative benefit for OK. The rate of axial elongation in children was lower for OK treatment compared to other treatment modalities at one year (MD - 0.16 mm, 95% CI - 0.25 to - 0.07). Rate of change in axial length in children rebounded after OK discontinuation compared to participants who continued treatment (MD 0.10 mm, 95% CI 0.06 to 0.14). Adults and children wearing OK were up to 3.79 times more likely to experience an adverse event when compared with conventional contact lenses (OR 3.79, 95% CI 1.24 to ll.), though this evidence base is underdeveloped and requires additional well-designed studies for substantial conclusions to be drawn. CONCLUSIONS: OK arrests myopia progression while in use, however, there remain unanswered questions about the optimal duration of treatment, discontinuation effects and long-term risk for adverse events.

BACH2: The Future of Induced T-Regulatory Cell Therapies.

BACH2 (BTB Domain and CNC Homolog 2) is a transcription factor that serves as a central regulator of immune cell differentiation and function, particularly in T and B lymphocytes. A picture is emerging that BACH2 may function as a master regulator of cell fate that is exquisitely sensitive to cell activation status. In particular, BACH2 plays a key role in stabilizing the phenotype and suppressive function of transforming growth factor-beta (TGF-ß)-derived human forkhead box protein P3 (FOXP3)+ inducible regulatory T cells (iTregs), a cell type that holds great clinical potential as a cell therapeutic for diverse inflammatory conditions. As such, BACH2 potentially could be targeted to overcome the instability of the iTreg phenotype and suppressive function that has hampered their clinical application. In this review, we focus on the role of BACH2 in T cell fate and iTreg function and stability. We suggest approaches to modulate BACH2 function that may lead to more stable and efficacious Treg cell therapies.

Successful Treatment of Massive Pulmonary Embolism in Pregnancy with Catheter-Directed Embolectomy.

Introduction Pulmonary embolism (PE) is associated with approximately 10.5% of maternal deaths in the United States. Despite heightened awareness of its mortality potential, there islittle data available to guide its management in pregnancy. We present the case of a massive PE during gestation successfully treated with catheter-directed embolectomy. Case Presentation A 37-year-old G2P1001 presented with a syncopal episode preceded by dyspnea and chest pain. Upon presentation, she was hypotensive, tachycardiac, and hypoxic. Imaging showed an occlusive bilateral PE, right heart strain, and a possible intrauterine pregnancy. Beta-human chorionic gonadotropin was positive. She was taken emergently for catheter-directed embolectomy. Her condition immediately improved afterward. Postprocedure pelvic ultrasound confirmed a viable intrauterine pregnancy at 10 weeks gestation. She was discharged with therapeutic enoxaparin and gave birth to a healthy infant at 38 weeks gestation. Conclusion Despite being the gold standard for PE treatment in nonpregnant adults, systemic thrombolysis is relatively contraindicated in pregnancy due to concern for maternal or fetal hemorrhage. Surgical or catheter-based thrombectomies are rarely recommended. Limited alternative options force their consideration, particularly in a hemodynamically unstable patient. Catheter-directed embolectomy can possibly bypass such complications. Our case exemplifies the consideration of catheter-directed embolectomy as the initial treatment modality of a hemodynamically unstable gestational PE.

Impact of continuous testosterone exposure on reproductive physiology, activity, and pain-related behavior in young adult female rats.

Testosterone may reduce pain in cisgender women and transgender men. Rodents can provide a useful model for investigating physiological effects of hormone therapy. To this end, continuous-release testosterone or blank (placebo) capsules were implanted s.c. into young adult female rats, and three weeks later rats were either ovariectomized or sham-ovariectomized. Testosterone treatment that mimicked previously reported endogenous levels in males eliminated estrous cycling and decreased uterine weight. Testosterone also significantly increased body weight and suppressed the increases in daily wheel running observed in placebo controls over time. Subsequent ovariectomy or sham-ovariectomy decreased wheel running in all groups, but testosterone-treated rats recovered significantly more quickly than did placebo-treated rats. Neither testosterone nor ovariectomy significantly altered hindpaw mechanical threshold. Two weeks after sham/ovariectomy surgery, injection of Complete Freund Adjuvant (CFA) into one hindpaw reduced wheel running and mechanical threshold in all groups; running significantly decreased from the first to second day after CFA in testosterone- but not in placebo-treated rats. Morphine 1.0 but not 3.2 mg/kg increased CFA-suppressed wheel running similarly in all groups, whereas both doses of morphine increased CFA-suppressed mechanical threshold. These data suggest that weeks-long testosterone treatment with or without ovariectomy may provide a useful physiological model of testosterone therapy as used in human gender transition. Although testosterone administered at levels similar to those in gonadally intact males tended to hasten female rats' recovery from surgery, it did not decrease maximal pain-related behaviors after surgery or hindpaw inflammatory insult, nor did it alter opioid antinociception.

The mitophagy receptor NIX induces vIRF-1 oligomerization and interaction with GABARAPL1 for the promotion of HHV-8 reactivation-induced mitophagy.

Recently, viruses have been shown to regulate selective autophagy for productive infections. For instance, human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), activates selective autophagy of mitochondria, termed mitophagy, thereby inhibiting antiviral innate immune responses during lytic infection in host cells. We previously demonstrated that HHV-8 viral interferon regulatory factor 1 (vIRF-1) plays a crucial role in lytic replication-activated mitophagy by interacting with cellular mitophagic proteins, including NIX and TUFM. However, the precise molecular mechanisms by which these interactions lead to mitophagy activation remain to be determined. Here, we show that vIRF-1 binds directly to mammalian autophagy-related gene 8 (ATG8) proteins, preferentially GABARAPL1 in infected cells, in an LC3-interacting region (LIR)-independent manner. Accordingly, we identified key residues in vIRF-1 and GABARAPL1 required for mutual interaction and demonstrated that the interaction is essential for mitophagy activation and HHV-8 productive replication. Interestingly, the mitophagy receptor NIX promotes vIRF-1-GABARAPL1 interaction, and NIX/vIRF-1-induced mitophagy is significantly inhibited in GABARAPL1-deficient cells. Moreover, a vIRF-1 variant defective in GABARAPL1 binding substantially loses the ability to induce vIRF-1/NIX-induced mitophagy. These results suggest that NIX supports vIRF-1 activity as a mitophagy mediator. In addition, we found that NIX promotes vIRF-1 aggregation and stabilizes aggregated vIRF-1. Together, these findings indicate that vIRF-1 plays a role as a viral mitophagy mediator that can be activated by a cellular mitophagy receptor.

Multifaceted roles of TAX1BP1 in autophagy.

TAX1BP1 is a selective macroautophagy/autophagy receptor that plays a central role in host defense to pathogens and in regulating the innate immune system. TAX1BP1 facilitates the xenophagic clearance of pathogenic bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis and regulates TLR3 (toll-like receptor 3)-TLR4 and DDX58/RIG-I-like receptor (RLR) signaling by targeting TICAM1 and MAVS for autophagic degradation respectively. In addition to these canonical autophagy receptor functions, TAX1BP1 can also exert multiple accessory functions that influence the biogenesis and maturation of autophagosomes. In this review, we will discuss and integrate recent findings related to the autophagy function of TAX1BP1 and highlight outstanding questions regarding its functions in autophagy and regulation of innate immunity and host defense.Abbreviations: ATG: autophagy related; CALCOCO: calcium binding and coiled-coil domain; CC: coiled-coil; CHUK/IKKα: conserved helix-loop-helix ubiquitous kinase; CLIR: noncanonical LC3-interacting region; GABARAP: gamma-aminobutyric acid receptor associated protein; HTLV-1: human T-lymphotropic virus 1; IFN: interferon; IL1B/IL1ß: interleukin 1 beta; LIR: LC3-interacting region; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/JNK: mitogen-activated protein kinase; mATG8: mammalian Atg8 homolog; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MTB: Mycobacterium tuberculosis; MYD88: myeloid differentiation primary response gene 88; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; OPTN: optineurin; Poly(I:C): polyinosinic:polycytidylic acid; PTM: post-translational modification; RB1CC1: RB1-inducible coiled-coil 1; RIPK: receptor (TNFRSF)-interacting serine-threonine kinase; RLR: DDX58/RIG-I-like receptor; RSV: respiratory syncytia virus; SKICH: SKIP carboxyl homology; SLR: SQSTM1 like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TBK1: TANK-binding kinase 1; TICAM1: toll-like receptor adaptor molecule 1; TLR: toll-like receptor; TNF: tumor necrosis factor; TNFAIP3: TNF alpha induced protein 3; TNFR: tumor necrosis factor receptor; TOM1: target of myb1 trafficking protein; TRAF: TNF receptor-associated factor; TRIM32: tripartite motif-containing 32; UBD: ubiquitin binding domain; ZF: zinc finger.

Herpesvirus lytic infection-induced mitophagy via viral interferon regulatory factor 1.

Viral control of mitochondria via mitophagy has a dampening effect on mitochondrion-mediated innate immune responses. We previously found that human herpesvirus 8 (HHV-8) could activate mitophagy via its lytic gene product vIRF-1 (viral interferon regulatory factor 1). Mechanistically, we previously demonstrated that vIRF-1 interacts with the mitophagic proteins BNIP3L (BCL2 interacting protein 3 like) and TUFM (Tu translation elongation factor, mitochondrial). Despite these significant findings, however, the precise molecular mechanisms underlying vIRF-1-activated mitophagy, particularly with core components of the autophagy machinery, remained to be fully elucidated. We recently reported that vIRF-1 binds preferentially and directly to GABARAPL1 (GABA type A receptor associated protein like 1) in a noncanonical manner, and this interaction is essential for virus-productive replication. Furthermore, we found that BNIP3L is a crucial factor that promotes vIRF-1 oligomerization and associated mitophagy activation, including GABARAPL1 interaction with vIRF-1 and TUFM dimerization. Together, our findings deepen our understanding of lytic infection-induced mitophagy and provide the key protein-protein interactions involved in vIRF-1-mediated mitophagy.

Pasteurella Endocarditis in a Patient with Rheumatic Mitral Valve Disease.

BACKGROUND As a common member of the oral bacterial flora of cats and dogs, Pasteurella multocida can cause skin and soft tissue infection in humans after bites, licks, or scratches from animals. Uncommonly, infection due to Pasteurella can cause sepsis in humans. Even more rare is the development of infectious endocarditis from a Pasteurella infection. CASE REPORT A 76-year-old woman presented with malaise and symptoms of fluid overload. Blood cultures were positive for Pasteurella multocida, and an echocardiogram was significant for mitral valve vegetation and severe biatrial enlargement. A diagnosis of Pasteurella endocarditis was made. Surgical intervention was recommended, but owing to the risk involved, the patient elected for conservative management involving long-term treatment with intravenous antibiotics. CONCLUSIONS While exceedingly rare, Pasteurella multocida can cause infectious endocarditis in patients with predisposing factors. This patient had a known history of rheumatic heart disease, which is believed to have caused the significant findings on imaging. To the best of our knowledge, our case is the only one to depict Pasteurella endocarditis in a patient with rheumatic heart disease and severe biatrial enlargement. It is the authors' belief that the rheumatic heart disease and remodeling of the heart increased her susceptibility to severe infection from Pasteurella. The purpose of this case is to describe the pathogenicity of an otherwise low-attack bacterial infection in an elderly patient with underlying structural acquired heart damage.

Autophagy-competent mitochondrial translation elongation factor TUFM inhibits caspase-8-mediated apoptosis.

Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases.

Characteristics, Comorbidities, Complications, and Outcomes Among 802 Patients With Severe Acute Respiratory Syndrome Coronavirus 2 in a Community Hospital in Florida.

OBJECTIVES: Coronavirus disease 2019 continues to increase throughout the United States. Despite the rapid progression of the disease, there is limited information of the factors associated with mortality in Florida. This study aims to review the demographics, characteristics, comorbidities, complications, and outcomes of hospitalized patients, and their association with mortality. DESIGN: Cohort study. SETTING: A community-based tertiary-care hospital of Orlando Health, Orlando Regional Medical Center. PATIENTS/SUBJECTS: Data of hospitalized patients who tested positive for severe acute respiratory syndrome coronavirus 2 between March 1, 2020, and August 31, 2020, at the Orlando Regional Medical Center. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Main data assessed included patient demographics, clinical characteristics, comorbidities, complications, outcomes, and inhospital mortality. The median age for hospitalized patients was 61 years; among them, 56% were males. Most were of African American (n = 288, 35.9%), Hispanic (n = 237, 29.6%), and Caucasian (n = 217, 27.1%) descent. More patients presented with symptoms developing at home (n = 589, 75.9%) than from skilled nursing and long-term acute care facilities. The most common comorbidities were diabetes mellitus (42.8%), obesity (39.2%), lung disease (23.3%), coronary artery disease (20.2%), and congestive heart failure (18.3%). Complications with higher odds of mortality were mechanical ventilation (odds ratio, 148.00, p < 0.001), coinfections (odds ratio, 56.42, p < 0.001), acute kidney injury (odds ratio, 84.01, p < 0.001), atrial fibrillation (odds ratio, 28.30, p < 0.001), acute myocardial infarction (odds ratio, 23.29, p < 0.001), and acute venous thromboembolism (odds ratio, 26.43, p < 0.001). CONCLUSIONS: We identified an increase of severity of coronavirus disease 2019 within older patients of African American and Hispanic descent with comorbidities such as diabetes, coronary artery disease, congestive heart failure, chronic kidney disease, cancer, liver disease, or cerebrovascular disease. Noninvasive positive-pressure ventilation and high-flow nasal cannula oxygen may have helped avert mechanical ventilation, and this may have improved patient outcomes over the course of the study period.

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

Herpesvirus Regulation of Selective Autophagy.

Selective autophagy has emerged as a key mechanism of quality and quantity control responsible for the autophagic degradation of specific subcellular organelles and materials. In addition, a specific type of selective autophagy (xenophagy) is also activated as a line of defense against invading intracellular pathogens, such as viruses. However, viruses have evolved strategies to counteract the host's antiviral defense and even to activate some proviral types of selective autophagy, such as mitophagy, for their successful infection and replication. This review discusses the current knowledge on the regulation of selective autophagy by human herpesviruses.

Pneumothorax and Pneumomediastinum Secondary to COVID-19 Disease Unrelated to Mechanical Ventilation.

In the recent worldwide coronavirus 2019 pandemic, a notable rise in pneumomediastinum and pneumothorax complications has been witnessed in numerous mechanically ventilated patients infected with severe acute respiratory syndrome coronavirus 2. Most cases have reported these complications as barotrauma from mechanical ventilation with COVID-19 disease. We aim to report three polymerase chain reaction-confirmed COVID-19 patients who developed pneumomediastinum and pneumothorax unrelated to mechanical ventilation. We originally analyzed 800 patients with COVID-19 disease at Orlando Regional Medical Center from March 1, 2020, to July 31, 2020, of which 12 patients developed pneumomediastinum and pneumothorax in their hospital course. Interestingly, three patients developed pneumomediastinum on chest imaging prior to intubation. We present these three patients, one female and two males, ages of 42, 64, and 65, respectively, who were diagnosed with COVID-19 disease through nasopharyngeal sampling tests with acute respiratory distress syndrome. Spontaneous pneumomediastinum and pneumothorax are potential complications of COVID-19 disease in the lungs unrelated to mechanical ventilation. This is similar to previous outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) diseases. Further investigation is needed to define the causality of pneumomediastinum in nonintubated COVID-19 patients to define the incidence of disease.

Acetylcysteine for the Treatment of Suspected Remdesivir-Associated Acute Liver Failure in COVID-19: A Case Series.

Remdesivir is a direct-acting nucleoside RNA polymerase inhibitor with activity against the novel severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus used in the treatment of coronavirus disease 2019 (COVID-19) pneumonia. Here, we present two cases of suspected remdesivir-associated acute liver failure (ALF) in which the liver failure improved after continuous infusion acetylcysteine and withdrawal of remdesivir. Both patients had significant increases in transaminases between day 3 and day 10 of remdesivir therapy accompanied by coagulopathy and encephalopathy. After initiation of continuous infusion acetylcysteine, the transaminases of both patients rapidly improved. Ultimately, one patient fully recovered while the other died of suspected septic shock. Due to its novel nature and only recent widespread use, there are very little data on the risk of ALF from remdesivir. Additionally, the data for the use of acetylcysteine to manage non-acetaminophen-induced ALF are limited. It is important to consider the risk of remdesivir-associated ALF when weighing the risk versus benefits of use, and acetylcysteine may have a role in its management.

Extensive catastrophic thromboses from elevation of factor VIII.

Catastrophic thrombotic syndrome, otherwise known as thrombotic storm (TS) is an extreme prothrombotic clinical syndrome that presents as rapid onset of multiple thromboembolic events affecting a large variety of vasculature. In recent studies, there has been a correlation of high plasma levels of factor VIII with thrombotic events. We present the case of a young man who exhibited multi-organ failure due to thrombotic storm. A 38-year-old male presented to the emergency department for progressive dyspnea and was diagnosed to have pulmonary embolism. The patient developed respiratory distress requiring intubation and was diagnosed with both an ST-elevation myocardial infarction and right cerebral infarction during the hospital course. The patient expired and autopsy revealed the cause of death to be myocardial, cerebral and renal infarction from widespread vascular thrombosis. Autopsy revealed cause of death to be elevated factor VIII associated thrombotic coagulopathy. Factor VIII level upon autopsy was 375% (55-200%). Although TS is rare, it can be lifethreatening if not recognized early. Survival depends on the prompt initiation and duration of anticoagulation.

Hypercalcemia Resulting from Necrotizing Leiomyoma in a Pregnant Female.

BACKGROUND Hypercalcemic crisis is defined as a serum calcium level >14 mg/dL in a symptomatic patient. While severe hypercalcemia during pregnancy is rare, it poses a life-threatening risk to both mother and fetus. Hypercalcemia in association with a benign tumor such as a leiomyoma is exceedingly rare. CASE REPORT A 38-year-old primagravida at 31.2 week's gestation conceived by in vitro fertilization presented to the emergency department for complaints of nausea, vomiting, and epigastric abdominal pain. Her fetal monitor strip was reassuring. A complete metabolic panel on admission was significant for severely elevated calcium of 15.9 mg/dL (8.6-10.3 mg/dL) and an elevated lipase of 1457U/L (11-82 U/L). She was started on aggressive intravenous fluid resuscitation, but became confused and lethargic, unarousable to verbal stimuli, as a result of hypercalcemia. Computed tomography (CT) scan of the abdomen and pelvis revealed a heterogeneously enhancing, placental-appearing soft tissue mass extending posteriorly and to the right that measured 2414 cm. The patient subsequently underwent planned low transverse cesarean delivery and exploratory laparotomy for myomectomy with removal of a 2834-g benign leiomyoma measuring 19.018.514.0 cm. Her serum parathyroid hormone-related protein (PTHrP) was elevated to 9.6 pmol/L (<4.2 pmol/L). The patient's calcium normalized to 9.8 mg/dL (8.6-10.3mg/dL) immediately following surgery. CONCLUSIONS Leiomyoma as a cause of hypercalcemia should be included in the differential diagnosis because surgical removal of leiomyoma is curative. Particularly in pregnant patients, for whom medical therapies for hypercalcemia are limited and those available can result in complications, early identification and surgical resection can be life saving.

Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells.

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Sleep Parameters and Overnight Urinary Melatonin Production in Children With Persistent Post-concussion Symptoms.

BACKGROUND: Sleep disturbance is common after a mild traumatic brain injury (mTBI) in children, yet its biology is poorly understood. We aimed to explore sleep-related problems (SRPs), sleep-activity patterns, and endogenous melatonin production in children with different recovery trajectories following mTBI. We hypothesized that children with delayed recovery would have more SRPs and abnormal sleep-activity patterns, which would correlate with lower overnight melatonin production. METHODS: In this prospective controlled cohort study, we enrolled 83 children with persistent symptoms, 26 children who had clinically recovered following mTBI, and 25 healthy controls. SRPs were evaluated using the sleep subscale of the Post-Concussion Symptom Inventory. Sleep actigraphy was performed for five to seven days at 37 (S.D. 7) days post-injury. Health-related quality of life and mood disturbance was assessed using the Child Health Questionnaire and the Behavior Assessment System for Children, respectively. Endogenous melatonin production was assessed using overnight urine collection. RESULTS: The groups were similar in age (13.9 [S.D. 2.6] years) and sex (52% female). Regression analysis demonstrated increased SRP in the symptomatic group (9.0; 95% confidence interval: 7.6, 11.1) compared with the recovered group (1.6; 95% confidence interval: 1.0, 2.4) and controls (2.0; 95% confidence intervals: 1.2, 3.2). Actigraphy parameters and urinary melatonin levels were not significantly different between groups. Neither SRPs nor actigraphy parameters correlated with anxiety and depression scores. CONCLUSIONS: Although children with persistent post-concussion symptoms reported more SRPs, this was not related to actigraphy sleep parameters or melatonin production. Further research is warranted to understand the pathophysiology of post-traumatic sleep disturbance.

Tristetraprolin posttranscriptionally downregulates PFKFB3 in cancer cells.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.

DRG2 supports the growth of primary tumors and metastases of melanoma by enhancing VEGF-A expression.

Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1α to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases.