The HLA-II immunopeptidome of SARS-CoV-2.

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4+ T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize vaccine effectiveness.

Minimalistic approach to enhanced recovery after pediatric scoliosis surgery.

PURPOSE: Prior studies of enhanced recovery protocols (ERP) have been conducted at large institutions with abundant resources. These results may not apply at institutions with less resources directed to quality improvement efforts. The purpose of this study was to assess the value of a minimalistic enhanced recovery protocol in reducing length of stay (LOS) following PSF for adolescent idiopathic scoliosis. We hypothesized that accelerated transition to oral pain medications and mobilization alone could shorten hospital length of stay in the absence of a formal multimodal pain regimen. METHODS: AIS patients aged 10-18 who underwent PSF at a tertiary pediatric hospital between January 1, 2014 and December 31, 2017 were reviewed. The study population was further narrowed to consecutive patients from a single surgeon's practice that piloted the modified ERP. Reservation from key stakeholders regarding the feasibility of implementing widespread protocol change led to the minimal alterations made to the postoperative protocol following PSF. Patients were divided into either the Standard Recovery Protocol (SRP) or Enhanced Recovery Protocol (ERP). Primary variables analyzed were hospital LOS, complications, readmissions, and total narcotic requirement. RESULTS: A total of 92 patients met inclusion criteria. SRP and ERP groups consisted of 44 (47.8%) and 48 (52.2%) patients. There was no difference between the two groups with regard to age, sex, and ASA score (p > 0.05). Fusion levels and EBL did not differ between treatment groups (p > 0.05). PCA pumps were discontinued later in the SRP group (39.5 ± 4.3 h) compared to the ERP group (17.4 ± 4.1 h, p < 0.0001). Narcotic requirement was similar between groups (p = 0.94) Patients in the SRP group had longer hospital stays than patients in the ERP group (p < 0.0001). 83% of the ERP group had LOS ≤ 3 days compared to 0% in the SRP group, whose mean LOS was 4.2 days. There was no difference in complications between the groups (2.2% vs 6.0%, p = 0.62). Readmission to the hospital within 30 days of surgery was rare in either group (2 SRP patients: 1 superior mesenteric artery syndrome, 1 bowel obstruction vs 0 ERP patients, p = 0.23). CONCLUSION: In this cohort, minor changes to the postoperative protocol following surgery for AIS led to a significant decrease in hospital length of stay. This minimalistic approach may ease implementation of an ERP in the setting of stakeholder apprehension.

Radiographic considerations for pediatric supracondylar humerus fractures.

Although supracondylar humerus fractures are common pediatric injuries, guidelines for postoperative imaging remain unclear. This study's purpose was to evaluate decision-making at various points in the postoperative period. The secondary objective was to compare the use of mini C arm fluoroscopy and flat plate X-rays at the first postoperative visit. A retrospective, cohort study was performed at one level I trauma center. Patients ages 1 to 14 with extension Gartland type II-IV supracondylar fractures sustained between January 2013 and May 2020 and treated with closed or open reduction and percutaneous fixation were included. Data collected included demographics, fracture characteristics, and imaging information. Of 553 patients who underwent surgery, 375 (67.8%) received intraoperative images after casting; none resulted in an intraoperative intervention. Of 463 patients with imaging at first follow-up, nine (1.9%) had a management modification, including seven for loss of reduction, all determined by the original operating surgeon. The method of imaging, did not differ significantly with respect to revision surgery. Twenty-six (4.0%) of 532 patients with imaging at pin removal received additional casting after pin removal, but no patients had their pins retained. This retrospective study examined the efficacy of imaging in pediatric supracondylar fractures. Intraoperative, postcasting images did not change management and should be discontinued. Imaging at first follow-up can be useful in identifying patients with loss of reduction and mini C arm serves as a viable alternative to standard X-rays. Finally, imaging at pin removal resulted in additional casting only in type III fractures. Level of evidence: Level III-retrospective, cohort study.

Is Electroacupuncture Contraindicated for Preventing and Treating Chemotherapy-Induced Peripheral Neuropathy?

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a common side-effect of several drugs used to combat cancer. Thus, researchers have sought better treatments for and prevention of CIPN, such as electroacupuncture (EA). Some trials show EA worsens or prolongs CIPN pain and recommend against further studies on this. This narrative review explores EA for preventing or treating CIPN, comparing positive and negative outcomes. Methods: PubMed, ScienceDirect, and Google Scholar were searched for electroacupuncture, CIPN, and peripheral neuropathy. A snowballing method was used to find systematic reviews and studies in systematic reviews. Results: Seven English-language trials were found on using EA for preventing or treating CIPN. In 3 prevention studies, 1 had significant benefits, 1 had modest benefits, and 1 had worse pain in an EA group at follow-up, compared to sham controls. In 4 treatment studies, 2 had significant benefits, 1 had no difference from 3 controls, and 1 had sham control was superior to verum EA. Conclusions: Most of the studies were limited by small sample sizes, and some studies used EA protocols and treatment doses (frequency and total number of sessions) that were potentially suboptimal. The quantity and quality of the studies are insufficient to draw firm conclusions on effectiveness and safety. More studies must test optimal EA protocols and treatment dosages. It is inappropriate to say that EA is not recommended for CIPN prevention or treatment, because there is no robust evidence to justify this. Generally, research has found benefits and no harms.

Beyond identity: Understanding the contribution of the 5' nucleotide of the antisense strand to RNAi activity.

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene's mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5' nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5' nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5' nt identities and different pairing states between the 5' antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5' nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5' thermodynamic rules governing topical siRNA efficacy across plants and animals.

Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species.

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

Elav-Mediated Exon Skipping and Alternative Polyadenylation of the Dscam1 Gene Are Required for Axon Outgrowth.

Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms because of alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon outgrowth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing.

Effect of Academic Grade Level on Return to Athletic Competition After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: After anterior cruciate ligament (ACL) reconstruction, 63% to 87% of high school athletes return to competition. Although physical and psychological factors are known contributors for failure to return to play, little attention has been paid to effect of academic grade level. Our purpose was to determine the influence of effect of academic grade level on return to competitive play. The primary hypothesis is that high school seniors who undergo ACL reconstruction or knee arthroscopy will be less likely to return to competitive play at 1 year than those in grades 9 to 11. METHODS: We retrospectively reviewed high school athletes who injured their knee during competitive athletic activity and underwent arthroscopic knee surgery, including ACL reconstruction. We included those 14 to 18 years old at time of surgery and analyzed records for grade level, sporting activity, surgery details, and date of return to play. The definition of return to competitive play was return to same preinjury sport within 1 year of surgery and the sport had to be organized. RESULTS: Our study group included 225 patients that underwent an ACL reconstruction and 74 had knee arthroscopy. Athletes undergoing ACL reconstructions were less likely to return to preinjury sport within 1 year than those undergoing knee arthroscopy (P=0.0163). Seniors were significantly less likely to return to play at 1 year than athletes in grades 9 to 11 after both ACL reconstruction (P<0.0001) and knee arthroscopy (P=0.0335). Although return to competitive play rates remained fairly constant within grades 9 to 11, a precipitous decline by 28.9% and 29.4% in return to play rates occurred in the ACL reconstruction and knee arthroscopy groups, respectively, between the junior and senior years of high school. DISCUSSION: Although return to competition rates were lower for high school athletes undergoing ACL reconstruction than those undergoing knee arthroscopy, both had declines in return when the surgery occurs during their senior season. These data are useful when interpreting return to play rates. Future studies would benefit from further defining this relationship, or at least, noting the number of "senior" athletes studied. LEVEL OF EVIDENCE: Therapeutic study-Level III.

Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

Cost-comparison of two trabecular micro-bypass stents versus selective laser trabeculoplasty or medications only for intraocular pressure control for patients with open-angle glaucoma.

AIM: Patients with open-angle glaucoma (OAG) whose intraocular pressure is not adequately controlled by one medication have several treatment options in the US. This analysis evaluated direct costs of unilateral eye treatment with two trabecular micro-bypass stents (two iStents) compared to selective laser trabeculoplasty (SLT) or medications only. MATERIALS AND METHODS: A population-based, annual state-transition, probabilistic, cost-of-care model was used to assess OAG-related costs over 5 years. Patients were modeled to initiate treatment in year zero with two iStents, SLT, or medications only. In years 1-5, patients could remain on initial treatment or move to another treatment option(s), or filtration surgery. Treatment strategy change probabilities were identified by a clinician panel. Direct costs were included for drugs, procedures, and complications. RESULTS: The projected average cumulative cost at 5 years was lower in the two-stent treatment arm ($4,420) compared to the SLT arm ($4,730) or medications-only arm ($6,217). Initial year-zero costs were higher with two iStents ($2,810) than with SLT ($842) or medications only ($996). Average marginal annual costs in years 1-5 were $322 for two iStents, $777 for SLT, and $1,044 for medications only. The cumulative cost differences between two iStents vs SLT or medications only decreased over time, with breakeven by 5 or 3 years post-initiation, respectively. By year 5, cumulative savings with two iStents over SLT or medications only was $309 or $1,797, respectively. LIMITATIONS: This analysis relies on clinical expert panel opinion and would benefit from real-world evidence on use of multiple procedures and treatment switching after two-stent treatment, SLT, or polypharmaceutical initial approaches. CONCLUSIONS: Despite higher costs in year zero, annual costs thereafter were lowest in the two-stent treatment arm. Two-stent treatment may reduce OAG-related health resource use, leading to direct savings, especially over medications only or at longer time horizons.

Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers.

Tumor genetics guides patient selection for many new therapies, and cell culture studies have demonstrated that specific mutations can promote metabolic phenotypes. However, whether tissue context defines cancer dependence on specific metabolic pathways is unknown. Kras activation and Trp53 deletion in the pancreas or the lung result in pancreatic ductal adenocarinoma (PDAC) or non-small cell lung carcinoma (NSCLC), respectively, but despite the same initiating events, these tumors use branched-chain amino acids (BCAAs) differently. NSCLC tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source, whereas PDAC tumors have decreased BCAA uptake. These differences are reflected in expression levels of BCAA catabolic enzymes in both mice and humans. Loss of Bcat1 and Bcat2, the enzymes responsible for BCAA use, impairs NSCLC tumor formation, but these enzymes are not required for PDAC tumor formation, arguing that tissue of origin is an important determinant of how cancers satisfy their metabolic requirements.

Circadian Rhythm Disruption Promotes Lung Tumorigenesis.

Circadian rhythms are 24-hr oscillations that control a variety of biological processes in living systems, including two hallmarks of cancer, cell division and metabolism. Circadian rhythm disruption by shift work is associated with greater risk for cancer development and poor prognosis, suggesting a putative tumor-suppressive role for circadian rhythm homeostasis. Using a genetically engineered mouse model of lung adenocarcinoma, we have characterized the effects of circadian rhythm disruption on lung tumorigenesis. We demonstrate that both physiologic perturbation (jet lag) and genetic mutation of the central circadian clock components decreased survival and promoted lung tumor growth and progression. The core circadian genes Per2 and Bmal1 were shown to have cell-autonomous tumor-suppressive roles in transformation and lung tumor progression. Loss of the central clock components led to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation. Our findings demonstrate that both systemic and somatic disruption of circadian rhythms contribute to cancer progression.

Environment Impacts the Metabolic Dependencies of Ras-Driven Non-Small Cell Lung Cancer.

Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells.

Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.

Organization of endoreduplicated chromosomes in the endosperm of Zea mays L.

The chromosomes of the maize endosperm proceed through an endoreduplication phase in later stages of development. Endoreduplication is a process in which the cell cycle continues DNA synthesis but does not proceed through cytokinesis. When this occurs, the normally triploid endosperm cell can reach ploidy levels greater than 200x in some lines of maize. In this work, we examined the structure of the endoreduplicated chromosomes. Previous cytological work has indicated that, although the DNA content per cell increases, the number of nucleoli and knobs remains the same. Using fluorescence in situ hybridization and slot blot techniques, we show that the highly repetitive heterochromatic areas both on the A and B chromosomes, as well as several actively transcribed genes, are endoreduplicated. This result suggests that the entire genome follows that same trend. Further evidence shows that the various chromatin strands stay associated throughout the length of the chromosomes after they have been replicated, and that the DNA at the centromeric and knob regions is more tightly associated than the other regions of the chromosomes. Interploidy crosses between diploid and tetraploid derivatives of the same inbred exhibit changes in the chromatin organization of centromeres and heterochromatic knobs.