A Poxvirus Decapping Enzyme Colocalizes with Mitochondria To Regulate RNA Metabolism and Translation and Promote Viral Replication.

Decapping enzymes remove the 5' cap of eukaryotic mRNA, leading to accelerated RNA decay. They are critical in regulating RNA homeostasis and play essential roles in many cellular and life processes. They are encoded in many organisms and viruses, including vaccinia virus, which was used as the vaccine to eradicate smallpox. Vaccinia virus encodes two decapping enzymes, D9 and D10, that are necessary for efficient viral replication and pathogenesis. However, the underlying molecular mechanisms regulating vaccinia decapping enzymes' functions are still largely elusive. Here, we demonstrated that vaccinia D10 almost exclusively colocalized with mitochondria. As mitochondria are highly mobile cellular organelles, colocalization of D10 with mitochondria can concentrate D10 locally and mobilize it to efficiently decap mRNAs. Mitochondria were barely observed in "viral factories," where viral transcripts are produced, suggesting that mitochondrial colocalization provides a spatial mechanism to preferentially decap cellular mRNAs over viral mRNAs. We identified three amino acids at the N terminus of D10 that are required for D10's mitochondrial colocalization. Loss of mitochondrial colocalization significantly impaired viral replication, reduced D10's ability to remove the RNA 5' cap during infection, and diminished D10's gene expression shutoff and mRNA translation promotion abilities. IMPORTANCE Decapping enzymes comprise many members from various organisms, ranging from plants, animals, and viruses. The mechanisms regulating their functions vary and are still largely unknown. Our study provides evidence that a vaccinia virus-encoded decapping enzyme, D10, colocalizes with mitochondria. Loss of mitochondrial colocalization significantly impairs viral replication, D10's gene expression shutoff, and mRNA translation promotion ability. Overall, our results suggest that mitochondrial colocalization is a spatial mechanism to concentrate D10 locally and mobilize it to efficiently and preferentially target cellular mRNAs for decapping and promote viral mRNA translation. Our results have broad impacts for understanding the functions and regulatory mechanisms of decapping enzymes.

Interventional pain training using phantom model during COVID-19 pandemic.

BACKGROUND: Fluoroscopic-guided lumbar procedures have increased in daily pain practice because the lumbar spine is one of the most common sources of pain. Interventional pain fellows must develop a minimum number of skills during their training in order to achieve the competences without neglecting radiological safety. However, medical training in fluoroscopic-guided interventions is being affected by the current coronavirus disease 2019 (COVID-19) situation. METHODS: The objective of this study was to evaluate the use of a phantom model for lumbar injection as a training strategy during the COVID-19 pandemic in fellows of interventional pain. The study was divided into theoretical and practical modules. The hands-on practice was performed in a lumbar model phantom where fellows were evaluated in four fluoroscopically guided approaches: intra-articular facet block (IAFB), medial branch block (MBB), transforaminal block (TFB), and interlaminar block (ILB) divided in 5 sessions. The aim was to make as many punctures as possible in every session. We measured total procedural performance (TPP), total needle hand time (TNH), and total radiation dose generated by the fluoroscopic machine (TRD) during each procedure. Additionally, a survey was applied to evaluate confidence and satisfaction before and after training. RESULTS: A total of 320 lumbar punctures were completed. The results were statistically significant in all approaches attempted (p < 0.01). The fellow's survey for satisfaction and confidence demonstrated a significant difference between pre and post-test (p < 0.01). CONCLUSIONS: The results of this study highlight the importance of adaptations and adoption of new educational models. The use of the phantom model for simulation could be a strategy for other emerging situations, like the COVID-19 pandemic. Including this practice in the interventional pain programs could lead to better results for the patient and operator radiology safety.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs.

Cellular decapping enzymes negatively regulate gene expression by removing the methylguanosine cap at the 5' end of eukaryotic mRNA, rendering mRNA susceptible to degradation and repressing mRNA translation. Vaccinia virus (VACV), the prototype poxvirus, encodes two decapping enzymes, D9 and D10, that induce the degradation of both cellular and viral mRNAs. Using a genome-wide survey of translation efficiency, we analyzed vaccinia virus mRNAs in cells infected with wild type VACV and mutant VACVs with inactivated decapping enzymes. We found that VACV decapping enzymes are required for selective translation of viral post-replicative mRNAs (transcribed after viral DNA replication) independent of PKR- and RNase L-mediated translation repression. Further molecular characterization demonstrated that VACV decapping enzymes are necessary for efficient translation of mRNA with a 5'-poly(A) leader, which are present in all viral post-replicative mRNAs. Inactivation of D10 alone in VACV significantly impairs poly(A)-leader-mediated translation. Remarkably, D10 stimulates mRNA translation in the absence of VACV infection with a preference for RNA containing a 5'-poly(A) leader. We further revealed that VACV decapping enzymes are needed for 5'-poly(A) leader-mediated cap-independent translation enhancement during infection. Our findings identified a mechanism by which VACV mRNAs are selectively translated through subverting viral decapping enzymes to stimulate 5'-poly(A) leader-mediated translation.

A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer related to asbestos exposure. Early diagnosis is challenging due to generic symptoms and a lack of biomarkers. We previously demonstrated that mesothelial precursor cells (MPC) characterized by mesothelin (MSLN)+CD90+CD34+ could be implicated in the development of mesothelioma after asbestos exposure. Here, we aimed to determine the clinical significance of detecting MPC in blood for early-stage diagnosis and prognosis of mesothelioma. METHODS: Due to the rarity of MPC in blood, it is challenging to identify this cell population using conventional techniques. Hence, we have developed a microfluidic liquid biopsy platform called MesoFind that utilizes an immunomagnetic, mesothelin capture strategy coupled with immunofluorescence to identify rare populations of cells at high sensitivity and precision. To validate our technique, we compared this approach to flow cytometry for the detection of MPC in murine blood and lavage samples. Upon successful validation of the murine samples, we then proceeded to examine circulating MPC in 23 patients with MPM, 23 asbestos-exposed individuals (ASB), and 10 healthy donors (HD) to evaluate their prognostic and diagnostic value. FINDING: MPC were successfully detected in the blood of murine samples using MesoFind but were undetectable with flow cytometry. Circulating MPC were significantly higher in patients with epithelioid MPM compared to HD and ASB. The MPC subpopulation, MSLN+ and CD90+, were upregulated in ASB compared to HD suggesting an early role in pleural damage from asbestos. The MPC subpopulation, MSLN+ and CD34+, in contrast, were detected in advanced MPM and associated with markers of poor prognosis, suggesting a predominant role during cancer progression. INTERPRETATION: The identification of circulating MPC presents an attractive solution for screening and early diagnosis of epithelioid mesothelioma. The presence of different subtypes of MPC have a prognostic value that could be of assistance with clinical decisions in patients with MPM. FUNDING: Princess Margaret Hospital Foundation Mesothelioma Research Fund, Toronto General & Western Hospital Foundation.

Vaccinia Virus as a Master of Host Shutoff Induction: Targeting Processes of the Central Dogma and Beyond.

The synthesis of host cell proteins is adversely inhibited in many virus infections, whereas viral proteins are efficiently synthesized. This phenomenon leads to the accumulation of viral proteins concurrently with a profound decline in global host protein synthesis, a phenomenon often termed "host shutoff." To induce host shutoff, a virus may target various steps of gene expression, as well as pre- and post-gene expression processes. During infection, vaccinia virus (VACV), the prototype poxvirus, targets all major processes of the central dogma of genetics, as well as pre-transcription and post-translation steps to hinder host cell protein production. In this article, we review the strategies used by VACV to induce host shutoff in the context of strategies employed by other viruses. We elaborate on how VACV induces host shutoff by targeting host cell DNA synthesis, RNA production and processing, mRNA translation, and protein degradation. We emphasize the topics on VACV's approaches toward modulating mRNA processing, stability, and translation during infection. Finally, we propose avenues for future investigations, which will facilitate our understanding of poxvirus biology, as well as fundamental cellular gene expression and regulation mechanisms.

[The annual cost of medical care for patients with moderate to severe atopic dermatitis in Mexico. A multicenter study]. / Costo anual de la atención médica de pacientes con dermatitis atópica moderada a grave en México. Estudio multicéntrico.

BACKGROUND: In Mexico, the economic burden of medical care for patients with atopic dermatitis is unknown. OBJECTIVE: To determine the annual direct medical costs of the treatment for patients with moderate to severe atopic dermatitis who receive medical attention at "Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado" (Institute for Social Security and Social Services for State Workers, better known as ISSSTE), as well as the main variables associated with it. METHODS: A multicenter, retrospective cohort study. Clinical records of patients with moderate to severe atopic dermatitis were reviewed and a multivariate analysis was designed by using a generalized linear model. RESULTS: 65 patients were included; 41 of them (63.07 %) had moderate atopic dermatitis, and 24 (36.92 %) had severe AD; 39 (60 %) of them were female patients. In groups with severe atopic dermatitis, statistically significant differences were observed in matters of the duration of the evolution of the disease, comorbidities, intense pruritus, and depression. The average annual cost of medical care for patients with moderate atopic dermatitis was 1527 ± 623 USD, and for patients with severe atopic dermatitis the cost was 9487 ± 8990 USD. Significant differences were observed in dermatology consultations, referrals, laboratory and diagnostic studies, and the number of drugs prescribed by physicians. With the multivariate analysis, it was identified that the highest cost was presented by severe patients (p = 0.0001) who were younger and had comorbidities, along with diagnosis of depression. CONCLUSIONS: The severity of atopic dermatitis, the age average, the presence of comorbidities, and the diagnosis of depression are the variables with the highest association and impact on the direct cost of medical care.

Antecedentes: En México se desconoce el impacto económico de la atención médica de los pacientes con dermatitis atópica. Objetivo: Determinar los costos médicos directos anuales del tratamiento de pacientes con dermatitis atópica moderada y grave que se atienden en el Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, y las principales variables asociadas. Métodos: Estudio multicéntrico de cohorte retrospectiva. Se revisaron los expedientes clínicos de pacientes con dermatitis atópica clasificada como moderada o grave y se diseñó un modelo de análisis multivariado mediante un modelo lineal generalizado. Resultados: Se incluyeron 65 pacientes, 41 (63.07 %) tuvieron dermatitis atópica moderada y 24 (36.92 %), grave; 39 (60 %) fueron del sexo femenino. En los grupos con dermatitis atópica grave se observaron diferencias estadísticamente significativas en años de evolución de la enfermedad, comorbilidades, prurito intenso y depresión. El costo promedio anual de la atención médica para dermatitis atópica moderada fue de 1527 ± 623 USD y para dermatitis atópica grave, de 9487± 8990 USD. Se obtuvieron diferencias estadísticamente significativas en consultas de dermatología, interconsultas, estudios de laboratorio y gabinete y número de medicamentos prescritos. Con el análisis multivariado se identificó que el costo mayor lo presentaban los pacientes graves (p = 0.0001), más jóvenes, con comorbilidades y diagnóstico de depresión. Conclusiones: La gravedad de la dermatitis atópica, la edad, presentar comorbilidades y contar con el diagnóstico de depresión son las variables con mayor asociación e impacto en el costo directo de la atención médica.

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. To dissect the role of 5'-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5'-UTR and 3'-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

Combining Desmopressin and Docetaxel for the Treatment of Castration-Resistant Prostate Cancer in an Orthotopic Model.

BACKGROUND/AIM: Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in breast cancer models. Docetaxel is a chemotherapy agent for castrate-resistant prostate cancer (CRPC). In this study, the ability of CRPC cells to grow and develop in vivo tumors in an animal model was evaluated, in order to investigate the anti-tumor effect of desmopressin in combination with docetaxel. MATERIALS AND METHODS: The CRPC cell line PC3 was used for orthotopic inoculation in male athymic nude mice. The mice were randomly assigned to one of the four treatment groups: Control, docetaxel, desmopressin or combination therapy. Following the last treatment, tumors were excised and measured. Blood samples were processed for CTC analysis. RESULTS: Docetaxel treatment resulted in a significant reduction in tumor volume compared to control. The combination therapy resulted in even more significant reduction (31.2%) in tumor volume. There was a complete absence of CTCs in the combination group. CONCLUSION: Our pilot study demonstrated an enhanced efficacy of docetaxel-based therapy in combination with desmopressin.

Dynamic CTC phenotypes in metastatic prostate cancer models visualized using magnetic ranking cytometry.

Tumors can shed thousands of cells into the circulation daily. These circulating tumor cells (CTCs) are heterogeneous, and their phenotypes change dynamically. Real-time monitoring of CTC phenotypes is crucial to elucidate the role of CTCs in the metastatic cascade. Here, we monitor phenotypic changes in CTCs in mice xenografted with tumors with varying aggressiveness during cancer progression and a course of chemotherapy to study the metastatic potential of CTCs and changes in the properties of these cells in response to treatment. A new device that enables magnetic ranking cytometry (MagRC) is employed to profile the phenotypic properties of CTCs. Overall, CTCs from metastatic xenografts in mice display dynamic and heterogeneous profiles while non-metastatic models had static profiles. Decreased heterogeneity followed by a reduction in metastasis incidence was observed after a course of chemotherapy administered to highly metastatic xenografts. Phenotypic profiling of CTCs could be employed to monitor disease progression and predict therapeutic responses.

Hemodynamic response to caudal epidural clonidine in a pediatric cardiac patient.

IMPLICATIONS: Postoperative management of sedation and analgesia in pediatric cardiac patients presents many challenges. This case report describes a child who experienced dramatic clinical improvement with the postoperative use of caudal morphine and clonidine after conventional therapy had failed.

Cutis verticis gyrata asociado a síndrome carcinoide pulmonar metastásico / Cutis verticis gyrata associated to the metastatic carcinoma of the lung syndrome

El cutis gyrata (CVG), es una enfermedad rara de la piel, que se ha asociado a muchas enfermedades, incluyendo neoplasias. Se presenta el caso de un paciente con asociación de carcinoide pulmonar metastásico al hígado. En la literatura mundial, existen pocos informes de esta asociación, la cual debe de considerarse importante en vista de que el CVG es un marcador cutáneo de malignidad interna