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.

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).

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.

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.

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.

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.

Single cell derived mRNA signals across human kidney tumors.

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.

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.

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.

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.

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.

Robotic thoracic surgery for inflammatory and infectious lung disease: initial experience in Brazil / Cirurgia torácica robótica para doença pulmonar inflamatória e infecciosa: experiência inicial no Brasil

ABSTRACT 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.

RESUMO Objetivo: na América Latina, especialmente no Brasil, a adoção da plataforma robótica para cirurgia torácica está aumentando gradativamente nos últimos anos. No entanto, apesar da tuberculose e doenças pulmonares inflamatórias serem endêmicas em nosso país, faltam estudos que descrevam os resultados do tratamento cirúrgico robótico das bronquiectasias. Este estudo tem como objetivo avaliar os resultados cirúrgicos da cirurgia robótica para doenças inflamatórias e infecciosas, determinando a extensão da ressecção, complicações pós-operatórias, tempo operatório e tempo de internação hospitalar. Métodos: estudo retrospectivo a partir de um banco de dados envolvendo pacientes com diagnóstico de bronquiectasia e submetidos à cirurgia torácica robótica em três hospitais brasileiros entre janeiro de 2017 e janeiro de 2020. Resultados: foram incluídos 7 pacientes. A média de idade foi 47 + 18,3 anos (variação, 18-70 anos). A maioria dos pacientes apresentou bronquiectasia não fibrose cística (n=5), seguida de bronquiectasia tuberculosa (n=1) e abscesso pulmonar (n=1). As cirurgias realizadas foram lobectomia (n=3), segmentectomia anatômica (n=3) e bilobectomia (n=1). O tempo médio do console foi de 147 minutos (variação de 61-288 min.) e não houve necessidade de conversão para toracotomia. Complicação pós-operatória ocorreu em um paciente, tratando-se de obstipação com necessidade de lavagem intestinal. A mediana do tempo de drenagem torácica e internação hospitalar, em dias, foi de 1 (variação, 1-6 dias) e 5 (variação, 2-14 dias), respectivamente. Conclusões: a cirurgia torácica robótica para doenças inflamatórias e infecciosas é um procedimento viável e seguro, com baixo risco de complicações e morbidade.

One-Pot Synthesis of Indoles and Pyrazoles via Pd-Catalyzed Couplings/Cyclizations Enabled by Aqueous Micellar Catalysis.

An effective one-pot synthesis of either indoles or pyrazoles can be achieved via Pd-catalyzed aminations followed by subsequent cyclizations facilitated by aqueous micellar catalysis. This new technology includes efficient couplings with low loadings of palladium, a more stable source of the required hydrazine moiety, greater atom economy for the initial coupling, and reduced reaction temperatures, all leading to environmentally responsible processes.

Sustainable Palladium-Catalyzed Tsuji-Trost Reactions Enabled by Aqueous Micellar Catalysis.

Palladium-catalyzed allylic substitution, or "Tsuji-Trost" reactions, can be run under micellar catalysis conditions featuring not only chemistry in water but also numerous combinations of reaction partners that require low levels of palladium, typically on the order of 1000 ppm (0.1 mol %). These couplings are further characterized by especially mild conditions, leading to a number of cases not previously reported in an aqueous micellar medium. Inclusion of diverse nucleophiles, such as N-H heterocycles, alcohols, dicarbonyl compounds, and sulfonamides is described. Intramolecular cyclizations further illustrate the broad utility of this process. In addition to recycling studies, a multigram scale example is reported, indicative of the prospects for scale up.

Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.

Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) and are traditionally defined as being BDCA-2+CD123+. pDCs are not readily detectable in healthy human skin, but have been suggested to accumulate in wounds. Here, we describe a CD1a-bearing BDCA-2+CD123int DC subset that rapidly infiltrates human skin wounds and comprises a major DC population. Using single-cell RNA sequencing, we show that these cells are largely activated DCs acquiring features compatible with lymph node homing and antigen presentation, but unexpectedly express both BDCA-2 and CD123, potentially mimicking pDCs. Furthermore, a third BDCA-2-expressing population, Axl+Siglec-6+ DCs (ASDC), was also found to infiltrate human skin during wounding. These data demonstrate early skin infiltration of a previously unrecognized CD123intBDCA-2+CD1a+ DC subset during acute sterile inflammation, and prompt a re-evaluation of previously ascribed pDC involvement in skin disease.

Spatiotemporal immune zonation of the human kidney.

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly or by cross-talk with immune cells. We used single-cell RNA sequencing to resolve the spatiotemporal immune topology of the human kidney. We reveal anatomically defined expression patterns of immune genes within the epithelial compartment, with antimicrobial peptide transcripts evident in pelvic epithelium in the mature, but not fetal, kidney. A network of tissue-resident myeloid and lymphoid immune cells was evident in both fetal and mature kidney, with postnatal acquisition of transcriptional programs that promote infection-defense capabilities. Epithelial-immune cross-talk orchestrated localization of antibacterial macrophages and neutrophils to the regions of the kidney most susceptible to infection. Overall, our study provides a global overview of how the immune landscape of the human kidney is zonated to counter the dominant immunological challenge.

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.

A cellular census of human lungs identifies novel cell states in health and in asthma.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.