Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3ß. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

Gene expression variability across cells and species shapes innate immunity.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

The Thiol-Modifier Effects of Organoselenium Compounds and Their Cytoprotective Actions in Neuronal Cells.

Most pharmacological studies concerning the beneficial effects of organoselenium compounds have focused on their ability to mimic glutathione peroxidase (GPx). However, mechanisms other than GPx-like activity might be involved on their biological effects. This study was aimed to investigate and compare the protective effects of two well known [(PhSe)2 and PhSeZnCl] and two newly developed (MRK Picolyl and MRK Ester) organoselenium compounds against oxidative challenge in cultured neuronal HT22 cells. The thiol peroxidase and oxidase activities were performed using the glutathione reductase (GR)-coupled assay. In order to evaluate protective effects of the organoselenium compounds against oxidative challenge in neuronal HT22 cells, experiments based on glutamate-induced oxytosis and SIN-1-mediated peroxynitrite generation were performed. The thiol peroxidase activities of the studied organoselenium compounds were smaller than bovine erythrocytes GPx enzyme. Besides, (PhSe)2 and PhSeZnCl showed higher thiol peroxidase and lower thiol oxidase activities compared to the new compounds. MRK Picolyl and MRK Ester, which showed lower thiol peroxidase activity, showed higher thiol oxidase activity. Both pre- or co-treatment with (PhSe)2, PhSeZnCl, MRK Picolyl and MRK Ester protected HT22 cells against glutamate-induced cytotoxicity. (PhSe)2 and MRK Picolyl significantly prevented peroxinitrite-induced dihydrorhodamine oxidation, but this effect was observed only when HT22 were pre-treated with these compounds. The treatment with (PhSe)2 increased the protein expression of antioxidant defences (Prx3, CAT and GCLC) in HT22 cells. Taking together, our results suggest that the biological effects elicited by these compounds are not directly related to their GPx-mimetic and thiol oxidase activities, but might be linked to the up-regulation of endogenous antioxidant defences trough their thiol-modifier effects.

The Human Lung Cell Atlas: A High-Resolution Reference Map of the Human Lung in Health and Disease.

Lung disease accounts for every sixth death globally. Profiling the molecular state of all lung cell types in health and disease is currently revolutionizing the identification of disease mechanisms and will aid the design of novel diagnostic and personalized therapeutic regimens. Recent progress in high-throughput techniques for single-cell genomic and transcriptomic analyses has opened up new possibilities to study individual cells within a tissue, classify these into cell types, and characterize variations in their molecular profiles as a function of genetics, environment, cell-cell interactions, developmental processes, aging, or disease. Integration of these cell state definitions with spatial information allows the in-depth molecular description of cellular neighborhoods and tissue microenvironments, including the tissue resident structural and immune cells, the tissue matrix, and the microbiome. The Human Cell Atlas consortium aims to characterize all cells in the healthy human body and has prioritized lung tissue as one of the flagship projects. Here, we present the rationale, the approach, and the expected impact of a Human Lung Cell Atlas.

Blimp-1 induces and Hobit maintains the cytotoxic mediator granzyme B in CD8 T cells.

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA+ effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm.

Building a Large Robotic Thoracic Surgery Program in an Emerging Country: Experience in Brazil.

BACKGROUND: In the last decade, robotic video-assisted thoracic surgery (R-VATS) has grown significantly and consolidated as an alternative to video-assisted thoracic surgery. The objective of this study is to present the implementation as well as the experience with R-VATS accumulated by 2 Brazilian groups. We also compared the outcomes of procedures performed during the learning curve and after a more mature experience. METHODS: Retrospective cohort study included all R-VATS procedures performed since April 2015 until April 2018. We describe the process of implantation of robotic surgery, highlighting the peculiarities and difficulties found in a developing country. Moreover, we reported our descriptive results and compared the first 60 patients to the subsequent cases. RESULTS: Two hundred and five patients included 101 females/104 males. Mean age was 61.7 years. There were hundred and sixty-four pulmonary resections, 39 resections of mediastinal lesions, 1 diaphragmatic plication, and 1 resection of a hilar tumor. Median operative times were 205 min for lung resections and 129 min for mediastinal. There was no conversion to VATS or thoracotomy or major intraoperative complications. Median length of stay was 3 days for pulmonary resections and 1 day for mediastinal. Postoperative complications occurred in 35 cases (17.0%)-prolonged air leak was the most common (17 cases). One fatality occurred in an elderly patient with pneumonia and sepsis (0.4%). Comparison of the first 60 patients (learning curve) with subsequent 145 patients (consolidated experience) showed significant differences in surgical and ICU time, both favoring consolidated experience. CONCLUSIONS: Our results were comparable to the literature. Robotic thoracic surgery can be safely and successfully implemented in tertiary hospitals in emerging countries provided that all stakeholders are involved and compromised with the implementation process.

Genetics and immunity in the era of single-cell genomics.

Recent developments in the field of single-cell genomics (SCG) are changing our understanding of how functional phenotypes of cell populations emerge from the behaviour of individual cells. Some of the applications of SCG include the discovery of new gene networks and novel cell subpopulations, fine mapping of transcription kinetics, and the relationships between cell clonality and their functional phenotypes. Immunology is one of the fields that is benefiting the most from such advancements, providing us with completely new insights into mammalian immunity. In this review, we start by covering new immunological insights originating from the use of single-cell genomic tools, specifically single-cell RNA-sequencing. Furthermore, we discuss how new genetic study designs are starting to explain inter-individual variation in the immune response. We conclude with a perspective on new multi-omics technologies capable of integrating several readouts from the same single cell and how such techniques might push our biological understanding of mammalian immunity to a new level.

Molecular characterization of HCMV-specific immune responses: Parallels between CD8(+) T cells, CD4(+) T cells, and NK cells.

CD8(+) T cells are important for immunity against human cytomegalovirus (HCMV). The HCMV-specific CD8(+) T-cell response is characterized by the accumulation of terminally differentiated effector cells that have downregulated the costimulatory molecules CD27 and CD28. These HCMV-specific CD8(+) T cells maintain high levels of cytotoxic molecules such as granzyme B and rapidly produce the inflammatory cytokine IFN-γ upon activation. Remarkably, HCMV-specific CD8(+) T cells are able to persist long term as fully functional effector cells, suggesting a unique differentiation pathway that is distinct from the formation of memory CD8(+) T cells after infection with acute viruses. In this review, we aim to highlight the most recent developments in HCMV-specific CD8(+) T-cell differentiation, maintenance, tissue distribution, metabolism and function. HCMV also induces the differentiation of effector CD4(+) T cells and NK cells, which share characteristics with HCMV-specific CD8(+) T cells. We propose that the overlap in differentiation of NK cells, CD4(+) and CD8(+) T cells after HCMV infection may be regulated by a shared transcriptional machinery. A better understanding of the molecular framework of HCMV-specific CD8(+) T-cell responses may benefit vaccine design, as these cells uniquely combine the capacity to rapidly respond to infection with long-term survival.

Blimp-1 homolog Hobit identifies effector-type lymphocytes in humans.

Human cytomegalovirus (CMV) induces the formation of effector CD8(+) T cells that are maintained for decades during the latent stage of infection. Effector CD8(+) T cells appear quiescent, but maintain constitutive cytolytic capacity and can immediately produce inflammatory cytokines such as IFN-γ after stimulation. It is unclear how effector CD8(+) T cells can be constitutively maintained in a terminal stage of effector differentiation in the absence of overt viral replication. We have recently described the zinc finger protein Homolog of Blimp-1 in T cells (Hobit) in murine NKT cells. Here, we show that human Hobit was uniformly expressed in effector-type CD8(+) T cells, but not in naive or in most memory CD8(+) T cells. Human CMV-specific but not influenza-specific CD8(+) T cells expressed high levels of Hobit. Consistent with the high homology between the DNA-binding Zinc Finger domains of Hobit and Blimp-1, Hobit displayed transcriptional activity at Blimp-1 target sites. Expression of Hobit strongly correlated with T-bet and IFN-γ expression within the CD8(+) T-cell population. Furthermore, Hobit was both necessary and sufficient for the production of IFN-γ. These data implicate Hobit as a novel transcriptional regulator in quiescent human effector-type CD8(+) T cells that regulates their immediate effector functions.

Continuous flow reactions in the preparation of active pharmaceutical ingredients and fine chemicals.

Herein, we present an overview of continuous flow chemistry, including photoflow and electroflow technologies in the preparation of active pharmaceutical ingredients (APIs) and fine chemical intermediates. Examples highlighting the benefits and challenges associated with continuous flow processes, mainly involving continuous thermal, photo- and electrochemical transformations, are drawn from the relevant literature, especially our experience and collaborations in this area, with emphasis on the synthesis and prospective scale-up.

Batch and Continuous Flow Total Synthesis of Cannabidiol.

Herein, we present a comprehensive total synthesis of cannabidiol integrating both batch and continuous flow conditions. Our approach is planned to streamline the synthesis of olivetolic acid derivatives and utilize an enantiomerically pure monoterpene moiety obtained from naturally occurring (R)-(+)-limonene by photocatalysis. Key reactions, including the synthesis of olivetolic ester and a Friedel-Crafts alkylation, are successfully adapted to continuous flow, resulting in improved yields and selectivities. This study not only offers a scalable and efficient route for cannabidiol synthesis but also contributes to the synthetic approaches to access cannabinoids (diversity synthesis), with potential applications in medicinal and industrial contexts.

Synthesis of Fentanyl under Continuous Photoflow Conditions.

An advantageous and original synthesis of fentanyl is described. This new approach includes two efficient continuous flow reductive aminations achieved via photoredox catalysis and a final batch acylation. A telescoped protocol for the two photocatalyzed steps is also presented, and overall, this protocol provides improved sustainability, significant efficiency, reduced temperatures and reaction times, and is functional for scaling up this relevant active pharmaceutical ingredient (API).

Technical Report: Percutaneous Reductions on Gartland Type III Supracondylar Fractures.

Various surgical treatments for supracondylar fracture in children remain the motive of research and study. A common factor in each technique is anatomic reduction, which is critical for the excellent recovery of the fracture. Anatomic reduction is preferably realized through closed and percutaneous approaches. This study aimed to present a closed percutaneous technique to treat Gartland III supracondylar fractures to aggregate a facilitating factor in the surgical approach to this condition. Our technique was applied to surgical patients in orthopedic and traumatology emergency care and illustrated by a synthetic elbow model, including soft tissue and intraoperative images.

Transcriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

Molecular characterization of colorectal cancer related peritoneal metastatic disease.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Robotic thoracic surgery for inflammatory and infectious lung disease: initial experience in Brazil.

OBJECTIVE: in Latin America, especially Brazil, the use of a robotic platform for thoracic surgery is gradually increasing in recent years. However, despite tuberculosis and inflammatory pulmonary diseases are endemic in our country, there is a lack of studies describing the results of robotic surgical treatment of bronchiectasis. This study aims to evaluate the surgical outcomes of robotic surgery for inflammatory and infective diseases by determining the extent of resection, postoperative complications, operative time, and length of hospital stay. METHODS: retrospective study from a database involving patients diagnosed with bronchiectasis and undergoing robotic thoracic surgery at three hospitals in Brazil between January of 2017 and January of 2020. RESULTS: a total of 7 patients were included. The mean age was 47 + 18.3 years (range, 18-70 years). Most patients had non-cystic fibrosis bronchiectasis (n=5), followed by tuberculosis bronchiectasis (n=1) and lung abscess (n=1). The performed surgeries were lobectomy (n=3), anatomic segmentectomy (n=3), and bilobectomy (n=1). The median console time was 147 minutes (range 61-288 min.) and there was no need for conversion to open thoracotomy. There were no major complications. Postoperative complications occurred in one patient and it was a case of constipation with the need for an intestinal lavage. The median for chest tube time and hospital stay, in days, was 1 (range, 1-6 days) and 5 (range, 2-14 days) respectively. CONCLUSIONS: robotic thoracic surgery for inflammatory and infective diseases is a feasible and safe procedure, with a low risk of complications and morbidity.

Integrated single-cell analysis unveils diverging immune features of COVID-19, influenza, and other community-acquired pneumonia.

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns-including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups-and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.

Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas.

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.