T cells in SARS-CoV-2 infection and vaccination.

While antibodies garner the lion's share of attention in SARS-CoV-2 immunity, cellular immunity (T cells) may be equally, if not more important, in controlling infection. Both CD8+ and CD4+ T cells are elicited earlier and are associated with milder disease, than antibodies, and T-cell activation appears to be necessary for control of infection. Variants of concern (VOCs) such as Omicron have escaped the neutralizing antibody responses after two mRNA vaccine doses, but T-cell immunity is largely intact. The breadth and patient-specific nature of the latter offers a formidable line of defense that can limit the severity of illness, and are likely to be responsible for most of the protection from natural infection or vaccination against VOCs which have evaded the antibody response. Comprehensive searches for T-cell epitopes, T-cell activation from infection and vaccination of specific patient groups, and elicitation of cellular immunity by various alternative vaccine modalities are here reviewed. Development of vaccines that specifically target T cells is called for, to meet the needs of patient groups for whom cellular immunity is weaker, such as the elderly and the immunosuppressed. While VOCs have not yet fully escaped T-cell immunity elicited by natural infection and vaccines, some early reports of partial escape suggest that future VOCs may achieve the dreaded result, dislodging a substantial proportion of cellular immunity, enough to cause a grave public health burden. A proactive, rather than reactive, solution which identifies and targets immutable sequences in SARS-CoV-2, not just those which are conserved, may be the only recourse humankind has to disarm these future VOCs before they disarm us.

Predicted Coronary Occlusion and Impella Salvage During Valve-in-Valve Transcatheter Aortic Valve Replacement.

We describe an interesting case of a 71 years old fragile female, with progressive shortness of breath on exertion and ankle swelling, cardiac failure NYHA class III. She also had chest irradiation due to Hodgkin's disease many years before, previous surgical aortic valve replacement using bioprosthetic stent-less Freestyle #25 mm valve (Medtronic, Inc) in 2000 for severe aortic stenosis, history of cardiac arrest in 2012 and angioplasty to ostial RCA, PCI to ostial RCA in 2014, CABG (RA graft to RCA) in 2014 (RCA intra-stent restenosis with refractory ischemia), anemia requiring regular transfusions, bronchiectasis and chronic kidney disease. Because of the great comorbidities, STS 4.9% and worsening of the symptoms due to severe aortic valve regurgitation, heart team decided to perform "valve-in-valve" Transcatheter Aortic Valve Replacement (VIV-TAVR), but we already predicted coronary occlusion while performing this procedure because of the low left main coronary ostium and short aortic valve sinus. So regarding the probable left main coronary occlusion during the valve implantation, we decided to perform the placement of a not deployed stent inside the left main prior to the valve procedure, and to deploy it in case the predicted left main occlusion occurred. So just after the VIV-TAVR procedure, we observed left main coronary occlusion and the patient got ischemic cardiogenic shock and cardiac arrest, so we performed immediate PCI and deployed the bailout stent. After some minutes of chest compressions, an Impella mechanical circulatory support system (Abiomed, Danvers, MA) had to be installed. Patient recovered spontaneous circulation, and after hemodynamic stabilization, she was sent to the Intensive Coronary Unit, without further complications. She was discharged successfully without neurological or cardiac sequelae after 1 week.

Coupling high-throughput genetics with phylogenetic information reveals an epistatic interaction on the influenza A virus M segment.

BACKGROUND: Epistasis is one of the central themes in viral evolution due to its importance in drug resistance, immune escape, and interspecies transmission. However, there is a lack of experimental approach to systematically probe for epistatic residues. RESULTS: By utilizing the information from natural occurring sequences and high-throughput genetics, this study established a novel strategy to identify epistatic residues. The rationale is that a substitution that is deleterious in one strain may be prevalent in nature due to the presence of a naturally occurring compensatory substitution. Here, high-throughput genetics was applied to influenza A virus M segment to systematically identify deleterious substitutions. Comparison with natural sequence variation showed that a deleterious substitution M1 Q214H was prevalent in circulating strains. A coevolution analysis was then performed and indicated that M1 residues 121, 207, 209, and 214 naturally coevolved as a group. Subsequently, we experimentally validated that M1 A209T was a compensatory substitution for M1 Q214H. CONCLUSIONS: This work provided a proof-of-concept to identify epistatic residues by coupling high-throughput genetics with phylogenetic information. In particular, we were able to identify an epistatic interaction between M1 substitutions A209T and Q214H. This analytic strategy can potentially be adapted to study any protein of interest, provided that the information on natural sequence variants is available.

High-throughput identification of loss-of-function mutations for anti-interferon activity in the influenza A virus NS segment.

UNLABELLED: Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE: Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.

High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.

Assessment of presurgical psychological screening in patients undergoing spine surgery: use and clinical impact.

STUDY DESIGN: Prospective survey. OBJECTIVE: To determine the prevalence of use of presurgical psychological screening (PPS) among spine surgeons in the United States, identify factors associated with PPS use, evaluate surgeons' opinions of PPS, and investigate how PPS is applied in clinical practice. SUMMARY OF BACKGROUND DATA: The United States Preventive Services Task Force recommends PPS for patients undergoing back surgery. The prevalence of PPS is unknown. Thus, it may be difficult to improve preoperative care for such patients with psychological conditions. METHODS: An online survey invitation was emailed to 340 spine surgeons. Questions addressed surgeon characteristics (eg, number of years in practice), practice characteristics (eg, practice type), inclusion of integrated rehabilitation and psychological services, and use of PPS. The impact of psychological factors on rehabilitation and recovery was assessed using an 11-point Likert scale (0, no impact; 10, highest impact). We analyzed the 110 (32%) responses with a χ(2) test (significance, P<0.05). RESULTS: PPS was used by 37% (41) to screen for depression (100%) or anxiety (85%). PPS use was highest among surgeons with more experience, higher annual volume, and no university affiliation. Among those screening for fear avoidance, use was highest among surgeons more recently entering the field. Surgeons reported a strong belief regarding the impact of psychological factors on pain relief, adherence to therapy, and return to work (mean impact rating, >7.0); however, impact on return for follow-up was only moderate (mean rating, 5.8). CONCLUSIONS: A minority of surgeons reported using PPS. Surgeons were less likely to use PPS if they had completed residency or begun practice within 14 years, had fewer than 200 cases annually, or were university affiliated. This study highlights the need to advocate for the use of North American Spine Society guidelines regarding the use of PPS.

Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.

Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors.

Pax3 is an essential myogenic regulator of fetal and embryonic development, but its role in postnatal myogenesis remains a topic of debate. We show that constitutive expression of Pax3 in postnatal, juvenile mouse skeletal muscle stem cells, a subset of the heterogeneous satellite cell pool highly enriched for myogenic activity, potently induces differentiation. This differentiation-promoting activity stands in contrast to the differentiation-inhibiting effects of Pax3 in the commonly used mouse myoblast cell line C2C12. Pax3 mRNA levels in distinct muscles correlate with the rate of myogenic differentiation of their muscle stem cells. Although Pax3 controls embryonic myogenesis through regulation of the canonical myogenic regulatory factors (MRFs) Myf-5, MyoD, myogenin and Mrf4, we find that in postnatal muscle stem cells, ectopic Pax3 expression fails to induce expression of any of these factors. Unexpectedly, overexpression of neither Myf-5 nor myogenin is sufficient to induce differentiation of juvenile stem cells; and knockdown of Myf-5, rather than inhibiting differentiation, promotes it. Taken together, our results suggest that there are distinct myogenic regulatory pathways that control the embryonic development, juvenile myogenesis and adult regeneration of skeletal myofibers.

VHL loss actuates a HIF-independent senescence programme mediated by Rb and p400.

Germline von Hippel-Lindau tumour suppressor gene (VHL) mutations cause renal cell carcinomas, haemangioblastomas and phaeochromocytomas in humans. Mutations in VHL also occur in sporadic renal cell carcinomas. The protein encoded by VHL, VHL, is part of the ubiquitin ligase that downregulates the heterodimeric transcription factor Hif under well-oxygenated conditions. Here we show that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo. This phenotype was independent of p53 and Hif but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a Hif-independent fashion. Our results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.

Differential regulation of apoptotic genes by Rb in human versus mouse cells.

The retinoblastoma protein (Rb) controls cellular proliferation and suppresses tumor formation through its effects upon E2F transcriptional regulation of the cell cycle. Unexpectedly, however, in proliferating human cells, Rb was present at the promoters of eight of eight E2F-regulated apoptotic genes tested, but zero of six E2F-regulated cell cycle genes tested. Binding of apoptotic gene promoters by Rb was constitutive, and inhibition of Rb in human cells by E2Fdb or E1A expression resulted in induction of these apoptotic genes and efficient cell death. E1A induced apoptosis much more efficiently in human fibroblasts than in mouse fibroblasts, suggesting a difference in susceptibility to loss of Rb function between human cells and mouse cells. Abrogation of Rb function in mouse cells did not induce expression of these apoptotic genes. Underlying this species difference in susceptibility to apoptosis following loss of Rb function was the absence of Rb on apoptotic gene promoters in mouse cells. Rb protein levels were 20-35-fold higher in primary human cells than in primary mouse cells. The constitutive repression of a multitude of apoptotic genes by Rb in human cells but not in mouse cells may provide a partial explanation for the well-known difference between human and mouse cells in transformation and tumorigenic potential.

Differences in stability of repressor complexes at promoters underlie distinct roles for Rb family members.

Oncogenic transformation of cells can induce the cyclin-dependent kinase inhibitor, p16, which leads to hypophosphorylation and activation of retinoblastoma (Rb). Rb is capable of causing permanent growth arrest, which may underlie its role as a tumor suppressor. We show that repression by Rb at E2F target gene promoters involves the establishment of a stable repressor complex that is not displaced by the overexpression of E2F-1. Rather than displacing Rb, excess E2F-1 instead recruits more Rb, leading to direct transcriptional repression. In contrast, the Rb family members, p130 and p107, which have not been demonstrated to be tumor suppressors, bind preferentially to target promoters in the absence of growth factors and in proliferating cells, respectively, and these repressor complexes are displaceable by E2F-1. Heterochromatin protein 1 (HP1), which interacts with Rb, is associated with these distinct repressor complexes and follows a similar pattern of stability/displaceability. Efficient growth arrest by p16/Rb is dependent on histone H3 lysine 9 methylation, which provides a binding site for HP1. We propose that these differences in the stability of repressor complexes at promoters may, in part, underlie the different roles of Rb vs p130 and p107 in cell cycle regulation and tumor suppression.

Ras protects Rb family null fibroblasts from cell death: a role for AP-1.

The retinoblastoma protein (Rb) controls cell proliferation, differentiation, and senescence and provides an essential tumor suppressive function that cells must eliminate to attain unlimited proliferative potential. Elimination of the Rb pathway also results in apoptosis, however, thereby providing an efficient surveillance mechanism to sense the loss of Rb. To become tumorigenic cells must thus overcome not only Rb function but also the apoptotic response caused by the loss of Rb function. We show that oncogenic Ras (RasV12) potently blocks cell death in Rb family member knockout mouse embryo fibroblasts (TKO cells). Activation of phosphatidylinositol 3-kinase and Raf by oncogenic Ras mediated this protection, implying that multiple Ras effector pathways are required, in concert, for this pro-survival signal. Although activation of Raf by selective Ras mutants protected TKO cells from cell death, pharmacologic inhibition of MEK had little effect on RasV12 protection, suggesting that a Raf-dependent, MEK-independent pathway was important for this effect. We show that this Raf-dependent protection occurred through activation of c-Jun and thus AP-1 activation. These observations could account for the dependence of Ras transformation on c-Jun activity and for the roles of AP-1 in oncogenesis. Our results support the concept of two oncogenic events cooperating to achieve a balance between immortalization and survival.

Mechanisms of transcriptional regulation by Rb-E2F segregate by biological pathway.

The E2F family of transcription factors are critical regulators of the cell cycle and have also been implicated in apoptosis, development, DNA damage checkpoints, and differentiation. Retinoblastoma (Rb) proteins interact with E2F to regulate transcription, and several mechanisms have been proposed for Rb-E2F transcriptional regulation. We designed microarray-based experiments to characterize the relative contributions of each mechanism, and unexpectedly, we found that distinct functional gene groups show preference for one mechanism over the others. We propose that such a distribution may provide signaling specificity to enable regulatory proteins to turn on or off entire pathways that determine cell fate.

Auto-activation of the rta gene of human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus.

Rta, mainly encoded by open reading frame 50 (ORF50), is the product of an immediate-early gene of human herpesvirus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus. Rta is a transcriptional activator that is both necessary and sufficient to disrupt viral latency and activate the expression of downstream viral lytic genes. We report that ectopically expressed Rta protein could also activate the rta promoter on a reporter plasmid up to 144-fold, both in latently infected B cells and in uninfected epithelial cells, and that this activation was dose-dependent. Furthermore, by analysing the 5' untranslated region using ribonuclease protection assays, we demonstrated that transfection of an Rta expression plasmid into latently infected cells activated the expression of rta transcripts from endogenous viral genomes. We propose that auto-activation of the immediate-early gene, rta, is an important strategy for HHV-8 to effectively respond to environmental stimuli and maximally activate the virus lytic cycle.