Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Retrotransposon LINE-1 bodies in the cytoplasm of piRNA-deficient mouse spermatocytes: Ribonucleoproteins overcoming the integrated stress response.

Transposable elements (TE) are mobile DNA sequences whose excessive proliferation endangers the host. Although animals have evolved robust TE-targeting defenses, including Piwi-interacting (pi)RNAs, retrotransposon LINE-1 (L1) still thrives in humans and mice. To gain insights into L1 endurance, we characterized L1 Bodies (LBs) and ORF1p complexes in germ cells of piRNA-deficient Maelstrom null mice. We report that ORF1p interacts with TE RNAs, genic mRNAs, and stress granule proteins, consistent with earlier studies. We also show that ORF1p associates with the CCR4-NOT deadenylation complex and PRKRA, a Protein Kinase R factor. Despite ORF1p interactions with these negative regulators of RNA expression, the stability and translation of LB-localized mRNAs remain unchanged. To scrutinize these findings, we studied the effects of PRKRA on L1 in cultured cells and showed that it elevates ORF1p levels and L1 retrotransposition. These results suggest that ORF1p-driven condensates promote L1 propagation, without affecting the metabolism of endogenous RNAs.

Genomic Characterization of Prostatic Basal Cell Carcinoma.

Basal cell carcinoma (BCC) of the prostate is a rare tumor. Compared with the more common acinar adenocarcinoma (AAC) of the prostate, BCCs show features of basal cell differentiation and are thought to be biologically distinct from AAC. The spectrum of molecular alterations of BCC has not been comprehensively described, and genomic studies are lacking. Herein, whole genome sequencing was performed on archival formalin-fixed, paraffin-embedded specimens of two cases with BCC. Prostatic BCCs were characterized by an overall low copy number and mutational burden. Recurrent copy number loss of chromosome 16 was observed. In addition, putative driver gene alterations in KIT, DENND3, PTPRU, MGA, and CYLD were identified. Mechanistically, depletion of the CYLD protein resulted in increased proliferation of prostatic basal cells in vitro. Collectively, these studies show that prostatic BCC displays distinct genomic alterations from AAC and highlight a potential role for loss of chromosome 16 in the pathogenesis of this rare tumor type.

Comparison of traditional growth rods and magnetically controlled growing rods in early-onset scoliosis: a case-matched mid term follow-up study.

PURPOSE: Early-onset scoliosis (EOS) has always been a challenging situation for spine surgeons. The aim of treatment is to control the direction of curve progression to allow for the complete development of lungs. Among all the growth constructs available, traditional growth rods (TGR) and magnetically controlled growth rods (MCGR) are most widely used. The MCGR has been introduced a few years back and there is a dearth of long-term follow-up studies. The purpose of this study is to compare the effectiveness of TGR and MCGR for the treatment of EOS. METHODS: All patients of EOS managed with either TGR or MCGR were included in the study. The patients managed with other methods or having follow-up < 2-years were excluded from the study. A total of 20 patients were recruited in the MCGR group and 28 patients were recruited in the TGR group. Both groups were matched by etiology, gender, pre-operative radiological parameters, and complications including unplanned surgeries. RESULTS: The mean age in our study was 7.90 years in the MCGR group and 7.46 years in the TGR group. The mean duration of follow-up in the MCGR group was 50.89 months and in the TGR group 94.2 months. Pre-operative cobb's angle in the coronal plane and T1-S1 were comparable in both groups with a mean cobb's angle of 65.4 in MCGR and 70.5 in TGR. The mean T1-S1 length in the MCGR group was 36.1cms and in the TGR group was 35.2 cms (p = 0.18). The average increase in T1-S1 length was 1.3 cm/year in the TGR group and 1.1 cm/year in the MCGR group (p > 0.05). The TGR patients underwent 186 open lengthening surgeries and 11 unplanned surgeries for various complications. The MCGR group has 180 non-invasive lengthening with only 4 unplanned returns to OT for various causes. CONCLUSION: The curve correction was similar in both TGR and MCGR groups. The average T1-S1 length achieved on final follow-up was similar in both groups. The MCGR patients have attained similar correction with fewer invasive procedures and lesser complications compared to the TGR group.

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Preprocedure CT Findings of Right Heart Failure as a Predictor of Mortality After Transcatheter Aortic Valve Replacement.

OBJECTIVE: The purpose of this study is to determine whether imaging features of right heart failure seen on CT performed before transcatheter aorta valve replacement (TAVR) predict poor outcomes after the procedure. MATERIALS AND METHODS: We retrospectively evaluated findings on CT performed before TAVR for 505 consecutive patients seen from 2014 to 2018. Of these patients, 300 underwent TAVR. Patient demographic characteristics and clinical and procedural data were recorded. Imaging features, including signs of right heart failure, left heart failure, lung disease, coronary artery disease, and concomitant mitral valve and apparatus calcifications were evaluated. The primary outcome was all-cause mortality at 1 year after TAVR. Patients were divided into two groups: those who were alive (group 1) and those who had died (group 2) by 1 year after TAVR. These groups were compared using the Mann-Whitney U test and the Pearson chi-square and Fisher exact tests when applicable. Multivariate logistic regression with a backward stepwise approach was performed. Results were correlated with echo-cardiography findings. RESULTS: A total of 31 patients (10.3%) died within 1 year of TAVR. The presence and size of pericardial effusions were strongly associated with mortality within 1 year after TAVR (p = 0.002). Pericardial effusion was noted in 25 patients in group 1 (9.3%) and eight patients in group 2 (25.8%). Increased size of the main pulmonary artery was associated with death (p = 0.024), with a median main pulmonary artery size of 2.9 cm (interquartile range, 2.6-3.3 cm) in group 1 and 3.2 cm (interquartile range, 2.9-3.5 cm) in group 2. In multivariate analysis, pericardial effusion size and pulmonary artery size, both of which are indicative of right heart failure, were predictors of death, independent of the routinely used clinical Society of Thoracic Surgeons score (AUC, 0.758; 95% CI, 0.671-0.845). Depressed right ventricular ejection fraction, as identified on echocardiography, was associated with mortality within 1 year after TAVR (p = 0.034), further corroborating the CT findings. CONCLUSION: Features related to right heart failure on pre-TAVR CT were associated with increased all-cause mortality within the first year after TAVR, even after adjustment for the Society of Thoracic Surgeons score. Such imaging findings can help in further risk stratification of patients before TAVR.

Observer-Based Deconvolution of Deterministic Input in Coprime Multichannel Systems With Its Application to Noninvasive Central Blood Pressure Monitoring.

Estimating central aortic blood pressure (BP) is important for cardiovascular (CV) health and risk prediction purposes. CV system is a multichannel dynamical system that yields multiple BPs at various body sites in response to central aortic BP. This paper concerns the development and analysis of an observer-based approach to deconvolution of unknown input in a class of coprime multichannel systems applicable to noninvasive estimation of central aortic BP. A multichannel system yields multiple outputs in response to a common input. Hence, the relationship between any pair of two outputs constitutes a hypothetical input-output system with unknown input embedded as a state. The central idea underlying our approach is to derive the unknown input by designing an observer for the hypothetical input-output system. In this paper, we developed an unknown input observer (UIO) for input deconvolution in coprime multichannel systems. We provided a universal design algorithm as well as meaningful physical insights and inherent performance limitations associated with the algorithm. The validity and potential of our approach were illustrated using a case study of estimating central aortic BP waveform from two noninvasively acquired peripheral arterial pulse waveforms. The UIO could reduce the root-mean-squared error (RMSE) associated with the central aortic BP by up to 27.5% and 28.8% against conventional inverse filtering (IF) and peripheral arterial pulse scaling techniques.

Direct Targeting of the mTOR (Mammalian Target of Rapamycin) Kinase Improves Endothelial Permeability in Drug-Eluting Stents-Brief Report.

Objective- Drug-eluting stents eluting canonical mTOR (mammalian target of rapamycin) inhibitors are widely used to treat coronary artery disease but accelerate the development of atherosclerosis within the stent (neoatherosclerosis)-a leading cause of late stent failure. We recently showed that canonical mTOR inhibitors bind FKBP12.6 (12.6-kDa FK506-binding protein 12), displace it from calcium release channels, resulting in activation of PKCα (protein kinase Cα) and dissociation of p-120-catenin (p120) from VE-CAD (vascular endothelial cadherin; promoting endothelial barrier dysfunction [EBD]). However, the relevance of these findings to drug-eluting stents remains unknown. Newer generation direct mTOR kinase inhibitors do not bind FKBP12.6 and offer the potential of improving endothelial barrier function while maintaining antirestenotic efficacy, but their actual effects are unknown. We examined the effects of 2 different pharmacological targeting strategies-canonical mTOR inhibitor everolimus and mTOR kinase inhibitors Torin-2-on EBD after stenting. Approach and Results- Using the rabbit model of stenting and a combination of Evans blue dye, confocal and scanning electron microscopy studies, everolimus-eluting stents resulted in long-term EBD compared with bare metal stents. EBD was mitigated by using stents that eluted mTOR kinase inhibitors (Torin-2-eluting stent). At 60 days after stent placement, everolimus-eluting stents demonstrated large areas of Evans blue dye staining and evidence of p120 VE-CAD dissociation consistent with EBD. These findings were absent in bare metal stents and significantly attenuated in Torin-2-eluting stent. As proof of concept of the role of EBD in neoatherosclerosis, 100 days after stenting, animals were fed an enriched cholesterol diet for an additional 30 days. Everolimus-eluting stents demonstrated significantly more macrophage infiltration (consistent with neoatherosclerosis) compared with both bare metal stents and Torin-2-eluting stent. Conclusions- Our results pinpoint interactions between FKBP12.6 and canonical mTOR inhibitors as a major cause of vascular permeability and neoatherosclerosis, which can be overcome by using mTOR kinase inhibitors. Our study suggests further refinement of molecular targeting of the mTOR complex may be a promising strategy (Graphic Abstract).

Endovascular Reconstruction of the Hepatic Arterial System for the Management of Mycotic Pseudoaneurysm in a Liver Transplant Patient.

BACKGROUND: Hepatic artery pseudoaneurysm is a rare but very morbid complication after liver transplant. Treatment options include ligation or endovascular embolization, followed by revascularization. We describe a new endovascular approach by stent exclusion in a high-risk patient. RESULTS: A 62-year-old male who received a second liver transplant after failed allograft presented with hemobilia and was diagnosed with a hepatic artery pseudoaneurysm in the setting of infection. Given his hostile abdomen, an endovascular approach was sought. We excluded the mycotic pseudoaneurysm with multiple covered stent grafts extending from the common hepatic artery to the right and left hepatic arteries. He was discharged with long-term antibiotics. On his 6-month follow-up visit, his stent was patent and hepatic function was stable. CONCLUSIONS: Endovascular stent-graft placement for management of hepatic artery pseudoaneurysm after liver transplant should be considered as a lower morbidity alternative to surgical repair, even in the setting of infection.

ETS2 is a prostate basal cell marker and is highly expressed in prostate cancers aberrantly expressing p63.

BACKGROUND: Rare prostate carcinomas aberrantly express p63 and have an immunophenotype intermediate between basal and luminal cells. Here, we performed gene expression profiling on p63-expressing prostatic carcinomas and compared them to usual-type adenocarcinoma. We identify ETS2 as highly expressed in p63-expressing prostatic carcinomas and benign prostate basal cells, with lower expression in luminal cells and primary usual-type adenocarcinomas. METHODS: A total of 8 p63-expressing prostate carcinomas at radical prostatectomy were compared to 358 usual-type adenocarcinomas by gene expression profiling performed on formalin fixed paraffin embedded tumor tissue using Affymetrix 1.0 ST microarrays. Correlation between differentially expressed genes and TP63 expression was performed in 5239 prostate adenocarcinomas available in the Decipher GRID. For validation, ETS2 in situ hybridization was performed on 19 p63-expressing prostate carcinomas and 30 usual-type adenocarcinomas arrayed on tissue microarrays (TMA). RESULTS: By gene expression, p63-expressing prostate carcinomas showed low cell cycle activity and low Decipher prognostic scores, but were predicted to have high Gleason grade compared to usual-type adenocarcinomas by gene expression signatures and morphology. Among the genes over-expressed in p63-expressing carcinoma relative to usual-type adenocarcinoma were known p63-regulated genes, along with ETS2, an ETS family member previously implicated as a prostate cancer tumor suppressor gene. Across several cohorts of prostate samples, ETS2 gene expression was correlated with TP63 expression and was significantly higher in benign prostate compared to usual-type adenocarcinoma. By in situ hybridization, ETS2 gene expression was high in benign basal cells, and low to undetectable in benign luminal cells or usual-type adenocarcinoma. In contrast, ETS2 was highly expressed in 95% (18/19) of p63-expressing prostate carcinomas. CONCLUSIONS: ETS2 is a predominantly basally-expressed gene in the prostate, with low expression in usual-type adenocarcinoma and high expression in p63-expressing carcinomas. Given this pattern, the significance of ETS2 loss by deletion or mutation in usual-type adenocarcinomas is uncertain.

Metabolic modeling helps interpret transcriptomic changes during malaria.

Disease represents a specific case of malfunctioning within a complex system. Whereas it is often feasible to observe and possibly treat the symptoms of a disease, it is much more challenging to identify and characterize its molecular root causes. Even in infectious diseases that are caused by a known parasite, it is often impossible to pinpoint exactly which molecular profiles of components or processes are directly or indirectly altered. However, a deep understanding of such profiles is a prerequisite for rational, efficacious treatments. Modern omics methodologies are permitting large-scale scans of some molecular profiles, but these scans often yield results that are not intuitive and difficult to interpret. For instance, the comparison of healthy and diseased transcriptome profiles may point to certain sets of involved genes, but a host of post-transcriptional processes and regulatory mechanisms renders predictions regarding metabolic or physiological consequences of the observed changes in gene expression unreliable. Here we present proof of concept that dynamic models of metabolic pathway systems may offer a tool for interpreting transcriptomic profiles measured during disease. We illustrate this strategy with the interpretation of expression data of genes coding for enzymes associated with purine metabolism. These data were obtained during infections of rhesus macaques (Macaca mulatta) with the malaria parasite Plasmodium cynomolgi or P. coatneyi. The model-based interpretation reveals clear patterns of flux redistribution within the purine pathway that are consistent between the two malaria pathogens and are even reflected in data from humans infected with P. falciparum. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang.

stringMLST: a fast k-mer based tool for multilocus sequence typing.

Rapid and accurate identification of the sequence type (ST) of bacterial pathogens is critical for epidemiological surveillance and outbreak control. Cheaper and faster next-generation sequencing (NGS) technologies have taken preference over the traditional method of amplicon sequencing for multilocus sequence typing (MLST). But data generated by NGS platforms necessitate quality control, genome assembly and sequence similarity searching before an isolate's ST can be determined. These are computationally intensive and time consuming steps, which are not ideally suited for real-time molecular epidemiology. Here, we present stringMLST, an assembly- and alignment-free, lightweight, platform-independent program capable of rapidly typing bacterial isolates directly from raw sequence reads. The program implements a simple hash table data structure to find exact matches between short sequence strings (k-mers) and an MLST allele library. We show that stringMLST is more accurate, and order of magnitude faster, than its contemporary genome-based ST detection tools. AVAILABILITY AND IMPLEMENTATION: The source code and documentations are available at http://jordan.biology.gatech.edu/page/software/stringMLST CONTACT: lavanya.rishishwar@gatech.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Reconciling discordant myocardial perfusion imaging and coronary angiography.

A common clinical conundrum presents itself in the discordance between nuclear stress testing and invasive coronary angiography (ICA) in the patient presenting with angina. A patient with an abnormal perfusion scan and "normal coronary angiography" may result in the patient's symptoms being dismissed as "non-cardiac." Alternatively, a patient with a "normal perfusion study," who nonetheless undergoes ICA and is found to have significant coronary artery disease may confound efforts to risk stratify and potentially treat patients with angina. This paper will review the current evidence to explain these apparent paradoxical scenarios.

Reasons and implications of agreements and disagreements between coronary flow reserve, fractional flow reserve, and myocardial perfusion imaging.

Information on coronary physiology and myocardial blood flow (MBF) in patients with suspected angina is increasingly important to inform treatment decisions. A number of different techniques including myocardial perfusion imaging (MPI), noninvasive estimation of MBF, and coronary flow reserve (CFR), as well as invasive methods for CFR and fractional flow reserve (FFR) are now readily available. However, despite their incorporation into contemporary guidelines, these techniques are still poorly understood and their interpretation to guide revascularization decisions is often inconsistent. In particular, these inconsistencies arise when there are discrepancies between the various techniques. The purpose of this article is therefore to review the basic principles, techniques, and clinical value of MPI, FFR, and CFR-with particular focus on interpreting their agreements and disagreements.

Vertical Transmission of Chikungunya Manifesting as Foetal Pericardial Effusion.

Chikungunya fever is believed to be a self-limiting illness, which can result in significant disability, but with a very low mortality. Chikungunya is uncommonly believed to be associated with serious manifestations. The present case is of a pregnant lady who had transient fever for two days in the third trimester, following which she developed foetal pericardial effusion and intrauterine growth restriction. After two weeks of the fever, in view of non-reactive non-stress test, breech and foetal pericardial effusion, patient was taken up for caesarean section. The neonate was positive for Chikungunya, detected by RT-PCR, while the mother tested positive for Chikungunya IgM antibodies. A diagnosis of Chikungunya pericardial effusion was made in the neonate, presumably acquired vertically secondary to the maternal Chikungunya infection occurring in the third trimester, which was also contributory to intrauterine growth restriction. No case of vertical transmission of Chikungunya has been reported in India, and foetal pericardial effusion has not been reported in world literature.

HES6 promotes prostate cancer aggressiveness independently of Notch signalling.

Notch signalling is implicated in the pathogenesis of a variety of cancers, but its role in prostate cancer is poorly understood. However, selected Notch pathway members are overrepresented in high-grade prostate cancers. We comprehensively profiled Notch pathway components in prostate cells and found prostate cancer-specific up-regulation of NOTCH3 and HES6. Their expression was particularly high in androgen responsive lines. Up- and down-regulating Notch in these cells modulated expression of canonical Notch targets, HES1 and HEY1, which could also be induced by androgen. Surprisingly, androgen treatment also suppressed Notch receptor expression, suggesting that androgens can activate Notch target genes in a receptor-independent manner. Using a Notch-sensitive Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) reporter assay, we found that basal levels of Notch signalling were significantly lower in prostate cancer cells compared to benign cells. Accordingly pharmacological Notch pathway blockade did not inhibit cancer cell growth or viability. In contrast to canonical Notch targets, HES6, a HES family member known to antagonize Notch signalling, was not regulated by Notch signalling, but relied instead on androgen levels, both in cultured cells and in human cancer tissues. When engineered into prostate cancer cells, reduced levels of HES6 resulted in reduced cancer cell invasion and clonogenic growth. By molecular profiling, we identified potential roles for HES6 in regulating hedgehog signalling, apoptosis and cell migration. Our results did not reveal any cell-autonomous roles for canonical Notch signalling in prostate cancer. However, the results do implicate HES6 as a promoter of prostate cancer progression.