Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Antioxidant and Anti-Inflammatory Activities of Stellera chamaejasme L. Roots and Aerial Parts Extracts.

Natural products, mainly plants, have a crucial role in folk medicine. Particularly, Stellera chamaejasme L. has been traditionally used in Mongolian medicine to treat various diseases, including chronic tracheitis, tuberculosis, and psoriasis. In this study, ethanol (EtOH) and dichloromethane (DCM) extracts of its roots (R) and aerial parts (AP) were evaluated for their antioxidant and anti-inflammatory activities. Thin-layer chromatography demonstrated the presence of flavonoids, namely kaempferol and quercetin-3-O-glucopyranoside, only in the EtOH-AP. Conversely, it showed that kaempferol, quercetin-3-O-glucopyranoside, coumarin, luteolin, rutin, morin, and riboflavin were not present in the other three extracts. The S. chamaejasme extracts exhibited strong antioxidant activity. In addition, the roots extracts presented the highest antioxidant activity against peroxyl radicals, with the EtOH-R being the most potent (IC50 = 0.90 ± 0.07 µg/mL). S. chamaejasme extracts also efficiently inhibited the production of one of the main pro-inflammatory cytokines, interleukin (IL)-6, in a dose-dependent manner by lipopolysaccharide-stimulated macrophages. Particularly, DCM-R was the strongest extract, reducing ≈ 91.5% of the IL-6 production. Since this extract was the most effective, gas chromatography-mass spectrometry (GC-MS) analyses were performed and demonstrated the presence of two fatty acids (palmitic acid and 9-octadecenoic acid), one fatty alcohol (1-hexadecanol), and one triterpenoid (squalene) that can contribute to the observed bioactivity. Herewith, S. chamaejasme extracts, mainly DCM-R, exhibit antioxidant and anti-inflammatory activities that could be applied as new and innovative natural formulations for the treatment of chronic inflammatory diseases.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Echinacea purpurea Fractions Represent Promising Plant-Based Anti-Inflammatory Formulations.

Echinacea purpurea is traditionally used in the treatment of inflammatory diseases. Therefore, we investigated the anti-inflammatory capacity of E. purpurea dichloromethanolic (DE) and ethanolic extracts obtained from flowers and roots (R). To identify the class of compounds responsible for the strongest bioactivity, the extracts were fractionated into phenol/carboxylic acid (F1) and alkylamide fraction (F2). The chemical fingerprint of bioactive compounds in the fractions was evaluated by LC-HRMS. E. purpurea extracts and fractions significantly reduced pro-inflammatory cytokines (interleukin 6 and/or tumor necrosis factor) and reactive oxygen and nitrogen species (ROS/RNS) production by lipopolysaccharide-stimulated primary human monocyte-derived macrophages. Dichloromethanolic extract obtained from roots (DE-R) demonstrated the strongest anti-inflammatory activity. Moreover, fractions exhibited greater anti-inflammatory activity than whole extract. Indeed, alkylamides must be the main compounds responsible for the anti-inflammatory activity of extracts; thus, the fractions presenting high content of these compounds presented greater bioactivity. It was demonstrated that alkylamides exert their anti-inflammatory activity through the downregulation of the phosphorylation of p38, ERK 1/2, STAT 3, and/or NF-κB signaling pathways, and/or downregulation of cyclooxygenase 2 expression. E. purpurea extracts and fractions, mainly DE-R-F2, are promising and powerful plant-based anti-inflammatory formulations that can be further used as a basis for the treatment of inflammatory diseases.

Base editing screens map mutations affecting interferon-γ signaling in cancer.

Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.

A spatially resolved atlas of the human lung characterizes a gland-associated immune niche.

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health.

On the Bioactivity of Echinacea purpurea Extracts to Modulate the Production of Inflammatory Mediators.

Inflammatory diseases are the focus of several clinical studies, due to limitations and serious side effects of available therapies. Plant-based drugs (e.g., salicylic acid, morphine) have become landmarks in the pharmaceutical field. Therefore, we investigated the immunomodulatory effects of flowers, leaves, and roots from Echinacea purpurea. Ethanolic (EE) and dichloromethanolic extracts (DE) were obtained using the Accelerated Solvent Extractor and aqueous extracts (AE) were prepared under stirring. Their chemical fingerprint was evaluated by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The pro- and anti-inflammatory effects, as well as the reduction in intracellular reactive oxygen and nitrogen species (ROS/RNS), of the different extracts were evaluated using non-stimulated and lipopolysaccharide-stimulated macrophages. Interestingly, AE were able to stimulate macrophages to produce pro-inflammatory cytokines (tumor necrosis factor -TNF-α, interleukin -IL-1ß, and IL-6), and to generate ROS/RNS. Conversely, under an inflammatory scenario, all extracts reduced the amount of pro-inflammatory mediators. DE, alkylamides-enriched extracts, showed the strongest anti-inflammatory activity. Moreover, E. purpurea extracts demonstrated generally a more robust anti-inflammatory activity than clinically used anti-inflammatory drugs (dexamethasone, diclofenac, salicylic acid, and celecoxib). Therefore, E. purpurea extracts may be used to develop new effective therapeutic formulations for disorders in which the immune system is either overactive or impaired.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Fishroesomes as carriers with antioxidant and anti-inflammatory bioactivities.

The great diversity of marine habitats and organisms renders them a high-value source to find/develop novel drugs and formulations. Therefore, herein, sardine (Sardina pilchardus) roe was used as a lipidic source to produce liposomes. This fish product presents high nutritional value, being its lipidic content associated with important health benefits. Consequently, it can be advantageously used to produce therapeutically active delivery devices. Roe lipids were extracted using the Matyash method. After lipid film hydration and extrusion, sardine roe-derived large unilamellar liposomes (LUVs), designated as fishroesomes, presented a size of ≈330 nm and a significant negative surface charge (≈-27 mV). Radical scavenging assays demonstrated that fishroesomes efficiently neutralized peroxyl, hydroxyl and nitric oxide radicals. Moreover, fishroesomes significantly reduced the expression of pro-inflammatory cytokines and chemokines by LPS-stimulated macrophages at non-toxic concentrations for L929 and THP-1 cells. Consequently, the developed liposomes exhibit unique properties as bioactive drug carriers for inflammatory diseases treatment.

Werner Helicase Is a Synthetic-Lethal Vulnerability in Mismatch Repair-Deficient Colorectal Cancer Refractory to Targeted Therapies, Chemotherapy, and Immunotherapy.

Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer. The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of patients receiving immunotherapy have disease that is refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR colorectal cancer preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in patients with dMMR/MSI-H colorectal cancer and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies. SIGNIFICANCE: We found that a large, diverse set of dMMR/MSI-H colorectal cancer preclinical models, including models of treatment-refractory disease, are WRN-dependent. Our results support WRN as a promising synthetic-lethal target in dMMR/MSI-H colorectal cancer tumors as a monotherapy or in combination with targeted agents, chemotherapy, or immunotherapy.This article is highlighted in the In This Issue feature, p. 1861.

Antioxidant and Anti-Inflammatory Activities of Cytocompatible Salvia officinalis Extracts: A Comparison between Traditional and Soxhlet Extraction.

Chronic inflammation is characterized by an overproduction of several inflammatory mediators (e.g., reactive species and interleukins -IL) that play a central role in numerous diseases. The available therapies are often associated with serious side effects and, consequently, the need for safer drugs is of utmost importance. A plant traditionally used in the treatment of inflammatory conditions is Salvia officinalis. Therefore, conventional maceration and infusion of its leaves were performed to obtain hydroethanolic (HE-T) and aqueous extracts (AE-T), respectively. Their efficacy was compared to soxhlet extracts, namely aqueous (AE-S), hydroethanolic (HE-S), and ethanolic extracts (EE-S). Thin-layer chromatography demonstrated the presence of rosmarinic acid, carnosol, and/or carnosic acid in the different extracts. Generally, soxhlet provided extracts with higher antioxidant activities than traditional extraction. Moreover, under an inflammatory scenario, EE-S were the most effective, followed by HE-S, HE-T, AE-T, and AE-S, in the reduction of IL-6 and TNF-α production. Interestingly, the extracts presented higher or similar anti-inflammatory activity than diclofenac, salicylic acid, and celecoxib. In conclusion, the extraction method and the solvents of extraction influenced the antioxidant activity, but mainly the anti-inflammatory activity of the extracts. Therefore, this natural resource can enable the development of effective treatments for oxidative stress and inflammatory diseases.

Dissolvable Microcarriers Allow Scalable Expansion And Harvesting Of Human Induced Pluripotent Stem Cells Under Xeno-Free Conditions.

The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno-free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner-flasks. A maximum 4.0 ± 0.8-fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno-free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration-based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier-based hiPSC expansion, leading to a more cost-effective and efficient bioprocess.