Sugar ring alignment and dynamics underline cytarabine and gemcitabine inhibition on Pol η catalyzed DNA synthesis.

Nucleoside analogue drugs are pervasively used as antiviral and chemotherapy agents. Cytarabine and gemcitabine are anti-cancer nucleoside analogue drugs that contain C2' modifications on the sugar ring. Despite carrying all the required functional groups for DNA synthesis, these two compounds inhibit DNA extension once incorporated into DNA. It remains unclear how the C2' modifications on cytarabine and gemcitabine affect the polymerase active site during substrate binding and DNA extension. Using steady-state kinetics, static and time-resolved X-ray crystallography with DNA polymerase η (Pol η) as a model system, we showed that the sugar ring C2' chemical groups on cytarabine and gemcitabine snugly fit within the Pol η active site without occluding the steric gate. During DNA extension, Pol η can extend past gemcitabine but with much lower efficiency past cytarabine. The Pol η crystal structures show that the -OH modification in the ß direction on cytarabine locks the sugar ring in an unfavorable C2'-endo geometry for product formation. On the other hand, the addition of fluorine atoms on gemcitabine alters the proper conformational transition of the sugar ring for DNA synthesis. Our study illustrates mechanistic insights into chemotherapeutic drug inhibition and resistance and guides future optimization of nucleoside analogue drugs.

Primer terminal ribonucleotide alters the active site dynamics of DNA polymerase η and reduces DNA synthesis fidelity.

DNA polymerases catalyze DNA synthesis with high efficiency, which is essential for all life. Extensive kinetic and structural efforts have been executed in exploring mechanisms of DNA polymerases, surrounding their kinetic pathway, catalytic mechanisms, and factors that dictate polymerase fidelity. Recent time-resolved crystallography studies on DNA polymerase η (Pol η) and ß have revealed essential transient events during the DNA synthesis reaction, such as mechanisms of primer deprotonation, separated roles of the three metal ions, and conformational changes that disfavor incorporation of the incorrect substrate. DNA-embedded ribonucleotides (rNs) are the most common lesion on DNA and a major threat to genome integrity. While kinetics of rN incorporation has been explored and structural studies have revealed that DNA polymerases have a steric gate that destabilizes ribonucleotide triphosphate binding, the mechanism of extension upon rN addition remains poorly characterized. Using steady-state kinetics, static and time-resolved X-ray crystallography with Pol η as a model system, we showed that the extra hydroxyl group on the primer terminus does alter the dynamics of the polymerase active site as well as the catalysis and fidelity of DNA synthesis. During rN extension, Pol η error incorporation efficiency increases significantly across different sequence contexts. Finally, our systematic structural studies suggest that the rN at the primer end improves primer alignment and reduces barriers in C2'-endo to C3'-endo sugar conformational change. Overall, our work provides further mechanistic insights into the effects of rN incorporation on DNA synthesis.

Predicting lung function decline in cystic fibrosis: the impact of initiating ivacaftor therapy.

BACKGROUND: Modulator therapies that seek to correct the underlying defect in cystic fibrosis (CF) have revolutionized the clinical landscape. Given the heterogeneous nature of lung disease progression in the post-modulator era, there is a need to develop prediction models that are robust to modulator uptake. METHODS: We conducted a retrospective longitudinal cohort study of the CF Foundation Patient Registry (N = 867 patients carrying the G551D mutation who were treated with ivacaftor from 2003 to 2018). The primary outcome was lung function (percent predicted forced expiratory volume in 1 s or FEV1pp). To characterize the association between ivacaftor initiation and lung function, we developed a dynamic prediction model through covariate selection of demographic and clinical characteristics. The ability of the selected model to predict a decline in lung function, clinically known as an FEV1-indicated exacerbation signal (FIES), was evaluated both at the population level and individual level. RESULTS: Based on the final model, the estimated improvement in FEV1pp after ivacaftor initiation was 4.89% predicted (95% confidence interval [CI]: 3.90 to 5.89). The rate of decline was reduced with ivacaftor initiation by 0.14% predicted/year (95% CI: 0.01 to 0.27). More frequent outpatient visits prior to study entry and being male corresponded to a higher overall FEV1pp. Pancreatic insufficiency, older age at study entry, a history of more frequent pulmonary exacerbations, lung infections, CF-related diabetes, and use of Medicaid insurance corresponded to lower FEV1pp. The model had excellent predictive accuracy for FIES events with an area under the receiver operating characteristic curve of 0.83 (95% CI: 0.83 to 0.84) for the independent testing cohort and 0.90 (95% CI: 0.89 to 0.90) for 6-month forecasting with the masked cohort. The root-mean-square errors of the FEV1pp predictions for these cohorts were 7.31% and 6.78% predicted, respectively, with standard deviations of 0.29 and 0.20. The predictive accuracy was robust across different covariate specifications. CONCLUSIONS: The methods and applications of dynamic prediction models developed using data prior to modulator uptake have the potential to inform post-modulator projections of lung function and enhance clinical surveillance in the new era of CF care.

Multilevel joint model of longitudinal continuous and binary outcomes for hierarchically structured data.

Joint modeling has been a useful strategy for incorporating latent associations between different types of outcomes simultaneously, often focusing on a longitudinal continuous outcome characterized by an LME submodel and a terminal event subject to a Cox proportional hazard or parametric survival submodel. Applications to hierarchical longitudinal studies have been less frequent, particularly with respect to a binary process, which is commonly specified by a GLMM. Furthermore, many of the joint model developments have not allowed for investigations of nested effects, such as those arising from multicenter studies. To fill this gap, we propose a multilevel joint model that encompasses the LME submodel and GLMM through a Bayesian approach. Motivated by the need for timely detection of pulmonary exacerbation and characterization of irregularly observed lung function measurements in people living with cystic fibrosis (CF) receiving care across multiple centers, we apply the model to the data arising from US CF Foundation Patient Registry. In parallel, we examine the extent of bias induced by a non-hierarchical model. Our simulation study and application results show that incorporating the center effect along with individual stochastic variation over time within the LME submodel improves model estimation and prediction. Given that the center effect is evident in lung function observed in the CF population, accounting for center-specific power parameters by incorporating the symmetric power exponential power (spep) link function in the GLMM can facilitate more accurate conclusions in clinical studies.

Topical corticosteroids for the prevention of severe radiation dermatitis: a systematic review and meta-analysis.

PURPOSE: Radiation dermatitis (RD) is a frequently occurring adverse reaction during radiotherapy in cancer patients. While the use of topical corticosteroids (TCs) is common for the treatment of RD, its role in preventing severe reactions remains unclear. This systematic review and meta-analysis aim to evaluate the evidence on the use of TCs as prophylaxis of RD. METHODS: A systematic search was conducted using OVID MedLine, Embase, and Cochrane databases (between 1946 and 2023) to identify studies examining TC use in the prevention of severe RD. Statistical analysis was completed using RevMan 5.4 to calculate pooled effect sizes and 95% confidence intervals. Forest plots were then developed using a random effects model. RESULTS: Ten RCTs with a total of 1041 patients met the inclusion criteria. Six studies reported on mometasone furoate (MF) and four studies reported on betamethasone. Both TCs were associated with a significant improvement in the prevention of moist desquamation [OR = 0.34, 95% CI [0.25, 0.47], p < 0.00001], but betamethasone was found to be more effective than MF [OR = 0.29, 95% CI [0.18, 0.46], p < 0.00001 and OR = 0.39, 95% CI [0.25, 0.61], p < 0.0001, respectively]. A similar finding was seen in reducing the development of grade 2 or higher RD according to the Radiation Therapy Oncology Group scale. CONCLUSIONS: The current evidence supports the use of TCs in preventing severe reactions of RD. Both MF and betamethasone were found to be effective; however, betamethasone, a higher potency TC, is more effective despite MF being more commonly reported in literature.

IFNgamma-inducible CXCL10/CXCR3 axis alters the sensitivity of HEp-2 cells to ionizing radiation.

Radiotherapy is a conventional approach for anti-cancer treatment, killing tumor cells through damaging cellular DNA. While increasing studies have demonstrated that tumors generated the tolerance to radiation and tumor immune system was found to be correlated to radiotherapy resistance. Therefore, it is critical to identify potential immune factors associated with the efficacy of radiotherapy. Here in this study, we evaluated the sensitivities of different tumor cells to radiation and determined HEp-2 cells as the radio-resistant tumor cells for further investigation. IFNgamma as a key regulator of host immune response showed the potential to sensitize tumors to ionizing radiation (IR). Besides, IFNgamma-induced CXC chemokine ligand 10 (CXCL10) was found to be necessary for effective IR-induced killing of cultured HEp-2 cells. Increased clonogenic survival was observed in CXCL10-depleted HEp-2 cells and CXCL10-KO cells. Additionally, the loss of CXCL10 in HEp-2 cells showed less progression of the G0/G1 phase to G2/M when exposed to IR (8 Gy). Local IR (20 Gy) to nude mice bearing HEp-2 tumors significantly reduced tumor burden, while fewer effects on tumor burden in mice carrying CXCL10-KO tumors were observed. We furtherly evaluated the possible roles the chemokine receptor CXCR3 plays in mediating the sensitivity of cultured HEp-2 cells to IR. Altered expression of CXCR3 in HEp-2 cells affected IR-induced killing of HEp-2 cells. Our data suggest the IFNgamma-activated CXCL10/CXCR3 axis may contribute to the effective radiation-induced killing of HEp-2 cells in vitro.

hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells.

hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment.IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNß in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals.IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

GADD45γ Activated Early in the Course of Herpes Simplex Virus 1 Infection Suppresses the Activation of a Network of Innate Immunity Genes.

The stress response genes encoding GADD45γ, and to a lesser extent GADD45ß, are activated early in infection with herpes simplex virus 1 (HSV-1). Cells that had been depleted of GADD45γ by transfection of short hairpin RNA (shRNA) or in which the gene had been knocked out (ΔGADD45γ) yielded significantly less virus than untreated infected cells. Consistent with lower virus yields, the ΔGADD45γ cells (either uninfected or infected with HSV-1) exhibited significantly higher levels of transcripts of a cluster of innate immunity genes, including those encoding IFI16, IFIT1, MDA5, and RIG-I. Members of this cluster of genes were reported by this laboratory to be activated concurrently with significantly reduced virus yields in cells depleted of LGP2 or HDAC4. We conclude that innate immunity to HSV-1 is normally repressed in unstressed cells and repression appears to be determined by two mechanisms. The first, illustrated here, is through activation by HSV-1 infection of the gene encoding GADD45γ. The second mechanism requires constitutively active expression of LGP2 and HDAC4.IMPORTANCE Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.

Exosomes containing miRNAs targeting HER2 synthesis and engineered to adhere to HER2 on tumor cells surface exhibit enhanced antitumor activity.

BACKGROUND: Exosomes are small, cellular membrane-derived vesicles with a diameter of 50-150 nm. Exosomes are considered ideal drug delivery systems with a wide range of applications in various diseases, including cancer. However, nonspecific delivery of therapeutic agents by exosomes in vivo remains challenging. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor tyrosine kinase, and its overexpression is usually associated with cell survival and tumor progression in various cancers. In this study, we aim to develop novel exosomes with dual HER2-targeting ability as a nanoparticle delivery vehicle to enhance antitumor efficacy in vivo. RESULTS: Here, we report the generation of two kinds of exosomes carrying miRNAs designed to block HER2 synthesis, which consequently showed a distinct anti-tumor effect. The 293-miR-HER2 exosomes package and deliver miRNAs targeting HER2 to recipient cells to block HER2 synthesis. The anti-tumor effect of these exosomes on cancer cells dependent on HER2 for survival but do not affect cells that lack HER2 or that are engineered to express HER2 but are not dependent on it for survival. In contrast, 293-miR-XS-HER2 exosomes carry an additional peptide, which enables them to adhere to HER2 on the surface of cancer cells. Consequently, these exosomes preferentially enter these cells with surface expression of HER2 and further displayed a tumoricidal effect. The 293-miR-XS-HER2 exosomes are significantly more effective than the 293-miR-HER2 exosomes in shrinking HER2-positive tumors implanted in mice. CONCLUSIONS: Collectively, as novel antitumor drug delivery vehicles, HER2 dual-targeting exosomes exhibit increased target-specific delivery efficiency and can be further utilized to develop new nanoparticle-based targeted therapies.

PUM1 is a biphasic negative regulator of innate immunity genes by suppressing LGP2.

PUM1 is an RNA binding protein shown to regulate the stability and function of mRNAs bearing a specific sequence. We report the following: (i) A key function of PUM1 is that of a repressor of key innate immunity genes by repressing the expression of LGP2. Thus, between 12 and 48 hours after transfection of human cells with siPUM1 RNA there was an initial (phase 1) upsurge of transcripts encoding LGP2, CXCL10, IL6, and PKR. This was followed 24 hours later (phase 2) by a significant accumulation of mRNAs encoding RIG-I, SP100, MDA5, IFIT1, PML, STING, and IFNß. The genes that were not activated encoded HDAC4 and NF-κB1. (ii) Simultaneous depletion of PUM1 and LGP2, CXCL10, or IL6 revealed that up-regulation of phase 1 and phase 2 genes was the consequence of up-regulation of LGP2. (iii) IFNß produced 48-72 hours after transfection of siPUM1 was effective in up-regulating LGP2 and phase 2 genes and reducing the replication of HSV-1 in untreated cells. (iv) Because only half of genes up-regulated in phase 1 and 2 encode mRNAs containing PUM1 binding sites, the upsurge in gene expression could not be attributed solely to stabilization of mRNAs in the absence of PUM1. (v) Lastly, depletion of PUM2 does not result in up-regulation of phase 1 or phase 2 genes. The results of the studies presented here indicate that PUM1 is a negative regulator of LGP2, a master regulator of innate immunity genes expressed in a cascade fashion.

miRNAs Targeting ICP4 and Delivered to Susceptible Cells in Exosomes Block HSV-1 Replication in a Dose-Dependent Manner.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.

Role of activating transcription factor 3 in the synthesis of latency-associated transcript and maintenance of herpes simplex virus 1 in latent state in ganglia.

A key property of herpes simplex viruses (HSVs) is their ability to establish latent infection in sensory or autonomic ganglia and to reactivate on physical, hormonal, or emotional stress. In latently infected ganglia, HSVs express a long noncoding RNA, a latency-associated transcript (LAT), which plays a key role in maintaining latently infected neurons, but not viral proteins. To investigate the events leading to reactivation, we examined the use of ganglionic organ cultures that enable rapid reactivation in medium containing antibody to nerve growth factor (NGF) or delayed reactivation in medium containing NGF and epidermal growth factor (EGF). Here we report the discovery that activating transcription factor 3 (ATF3), a stress response protein, profoundly affects the interaction of HSV with its host. Specifically, (i) ATF3 is induced by stress, such as inhibition of protein synthesis or infection; (ii) in infected cells, ATF3 enhances the accumulation of LAT by acting on the response elements in the promoter of the LAT precursor RNA; (iii) ATF3 is induced nearly 100-fold in ganglionic organ cultures; and (iv) ATF3 plays a key role in the maintenance of the latent state, inasmuch as expression of ATF3 bereft of the C-terminal activation domain acts as a dominant negative factor, inducing HSV gene expression in ganglionic organ cultures harboring latent virus and incubated in medium containing NGF and EGF. Thus, ATF3 is a component of a cluster of cellular proteins that together with LAT maintain the integrity of the neurons harboring latent virus.

Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection, latency, and on reactivation.

The key events in herpes simplex virus (HSV) infections are (i) replication at a portal of entry into the body modeled by infection of cultured cells; (ii) establishment of a latent state characterized by a sole latency-associated transcript and microRNAs (miRNAs) modeled in murine peripheral ganglia 30 d after inoculation; and (iii) reactivation from the latent state modeled by excision and incubation of ganglia in medium containing anti-NGF antibody for a timespan of a single viral replicative cycle. In this report, we examine the pattern of synthesis and accumulation of 18 HSV-1 miRNAs in the three models. We report the following: (i) H2-3P, H3-3P, H4-3P, H5-3P, H6-3P, and H7-5P accumulated in ganglia harboring latent virus. All but H4-3P were readily detected in productively infected cells, and most likely they originate from three transcriptional units. (ii) H8-5P, H15, H17, H18, H26, and H27 accumulated during reactivation. Of this group, only H26 and H27 could be detected in productively infected cells. (iii) Of the 18 we have examined, only 10 miRNAs were found to accumulate above background levels in productively infected cells. The disparity in the accumulation of miRNAs in cell culture and during reactivation may reflect differences in the patterns of regulation of viral gene expression during productive infection and during reactivation from the latent state.

Observing one-divalent-metal-ion dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo.

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The superfamily of His-Me nucleases is renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how this single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases. Teaser: Soaking crystals of the HNH Cas9 family I-PpoI nuclease reveals how one metal ion and a histidine residue are sufficient for cleaving DNA.

Understanding the perspective of community co-educators on community-based service learning: a qualitative analysis.

Background: Community-based service learning (CBSL) is a core component of the Canadian medical education system. However, the unique role of community partner organizations (CPOs) in supporting CBSL remains unclear. This qualitative study evaluates the perspective of CPOs as co-educators in the undergraduate medical curriculum. Methods: We conducted eight semi-structured, one-on-one interviews with CPOs at a medical school in Toronto, Ontario between 2020-2021. Interviews were conducted following a pre-determined interview guide and then recorded, de-identified, and transcribed. Three reviewers independently performed an inductive thematic analysis of codes followed by a group review of discrepancies. Results: Five main findings were identified: 1) CPOs share a common interest in serving as co-educators; 2) considerable heterogeneity in the understanding of co-education exists; 3) there is an opportunity for increased partnerships between CPOs and faculty; 4) the role of co-educators is limited by curriculum structure; and 5) co-educators facilitate unique teachings of social determinants of health otherwise not available through traditional didactic teaching. Conclusions: There is an emerging, unique role for community co-educators in the undergraduate medical curriculum, supported by interest from CPOs. Its emphasis may contribute to future cohorts of medical students capable of understanding and addressing the needs of the populations they serve.

Contexte: L'apprentissage par le service communautaire (APSC) est une composante essentielle du système d'éducation médicale canadien. Cependant, le rôle unique des organismes communautaires partenaires (OCP) dans le soutien de l'APSC n'est toujours pas clair. Cette étude qualitative évalue le point de vue des OCP en tant que co-éducateurs dans le programme d'études médicales de premier cycle. Méthodes: Nous avons mené huit entrevues individuelles semi-structurées avec des OCP d'une faculté de médecine de Toronto, en Ontario, entre 2020 et 2021. Les entrevues ont été menées en suivant un guide d'entrevue prédéterminé, puis enregistrées, dépersonnalisées et transcrites. Trois examinateurs ont effectué indépendamment une analyse thématique inductive des codes, suivie d'un examen collectif des divergences. Résultats: Cinq conclusions principales ont été identifiées : 1) les OCP ont un intérêt commun à agir en tant que co-éducateurs; 2) il existe une grande hétérogénéité dans la compréhension de la co-éducation; 3) il est possible d'accroître les partenariats entre les OCP et le corps professoral; 4) le rôle des co-éducateurs est limité par la structure du curriculum médical et 5) les co-éducateurs facilitent des opportunités d'apprentissage uniques sur les déterminants sociaux de la santé qui ne sont pas disponibles dans le cadre de l'enseignement didactique traditionnel. Conclusions: Les co-éducateurs communautaires jouent un rôle novateur et unique dans le programme d'études médicales de premier cycle, soutenu par l'intérêt des OCP. L'accent mis sur ce rôle peut contribuer à former de futures cohortes d'étudiants en médecine capables de comprendre et de répondre aux besoins des populations qu'ils servent.

Enhanced therapeutic efficacy for glioblastoma immunotherapy with an oncolytic herpes simplex virus armed with anti-PD-1 antibody and IL-12.

Glioblastoma is the most common and aggressive malignant brain tumor and has limited treatment options. Hence, innovative approaches are urgently needed. Oncolytic virus therapy is emerging as a promising modality for cancer treatment due to its tumor-specific targeting and immune-stimulatory properties. In this study, we developed a new generation of oncolytic herpes simplex virus C5252 by deletion of a 15-kb internal repeat region and both copies of γ34.5 genes. Additionally, C5252 was armed with anti-programmed cell death protein 1 antibody and interleukin-12 to enhance its therapeutic efficacy for glioblastoma immune-virotherapy. In vitro and in vivo experiments demonstrate that C5252 has a remarkable safety profile and potent anti-tumor activity against glioblastoma. Mechanistic studies demonstrated that C5252 specifically induces cell apoptosis by caspase-3/7 activation via downregulating ciliary neurotrophic factor receptor α. Furthermore, the enhanced anti-tumor therapeutic efficacy of C5252 in a subcutaneous glioblastoma model and an orthotopic glioblastoma model was confirmed. Moreover, syngeneic mouse models showed that the murine surrogate of C5252 has superior anti-tumor activity compared to the unarmed backbone virus, with enhanced immune activation. Taken together, our findings support C5252 as a promising therapeutic option for glioblastoma treatment, positioning it as a highly promising candidate for clinical translation.

Utility of Methicillin-Resistant Staphylococcus aureus Nasal PCR Testing in Pediatric Patients With Suspected Respiratory Infections.

Observational studies in adults suggest nasal methicillin-resistant Staphylococcus aureus (MRSA) swabs have a high negative predictive value (NPV) for ruling out MRSA pneumonia, however, pediatric data are limited. This retrospective study of 505 pediatric patients found a 99.8% NPV among children with suspected respiratory infections.