Fragment-Based Discovery of a Series of Allosteric-Binding Site Modulators of ß-Glucocerebrosidase.

ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Early-stage lung cancer is driven by a transitional cell state dependent on a KRAS-ITGA3-SRC axis.

Glycine-12 mutations in the GTPase KRAS (KRASG12) are an initiating event for development of lung adenocarcinoma (LUAD). KRASG12 mutations promote cell-intrinsic rewiring of alveolar type-II progenitor (AT2) cells, but to what extent such changes interplay with lung homeostasis and cell fate pathways is unclear. Here, we generated single-cell RNA-seq (scRNA-seq) profiles from AT2-mesenchyme organoid co-cultures, mice, and stage-IA LUAD patients, identifying conserved regulators of AT2 transcriptional dynamics and defining the impact of KRASG12D mutation with temporal resolution. In AT2WT organoids, we found a transient injury/plasticity state preceding AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics, most notably retention of the injury/plasticity state. The injury state in AT2KRAS cells of patients, mice, and organoids was distinguishable from AT2WT states via altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors impaired AT2KRAS organoid growth. Together, our data show that an injury/plasticity state essential for lung repair is co-opted during AT2 self-renewal and LUAD initiation, suggesting that early-stage LUAD may be susceptible to interventions that target specifically the oncogenic nature of this cell state.

Evaluation of the feasibility for replacing sheep blood with human blood in culture media used in microbiological diagnostics.

The present study aimed to suggest the replacement of animal blood with human blood in culture media, involving alternative methods and ethical considerations, such as animal welfare, in addition to potential laboratory cost reduction. Characteristics of growth and hemolysis development were compared in different culture media, using both sheep blood and human blood. Blood types from the ABO blood group system were tested, and commercially acquired sheep blood agar was used for comparison. Bacteria of the genus Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were tested. It was observed that growth in media with type A and O positive blood showed closer similarities to those performed in agar with sheep blood. Depending on the bacterial species, the results were either more positive or not, with faster-growing and less demanding bacteria showing better results than, for example, S. pneumoniae, which demonstrated difficulty in the growth process and hemolysis generation in human blood agar. The research suggests that in some situations, sheep blood could be replaced, especially when the goal is growth and isolation, but may not be as suitable when the objective is to analyze hemolysis or when the studied species is demanding.

Soil greenhouse gas emissions from dead and natural mangrove forests in Southeastern Brazil.

Mangroves forests may be important sinks of carbon in coastal areas but upon their death, these forests may become net sources of carbon dioxide (CO2) and methane (CH4) to the atmosphere. Here we assessed the spatial and temporal variability in soil CO2 and CH4 fluxes from dead mangrove forests and paired intact sites in SE-Brazil. Our findings demonstrated that during warmer and drier conditions, CO2 soil flux was 183 % higher in live mangrove forests when compared to the dead mangrove forests. Soil CH4 emissions in live forests were > 1.4-fold higher than the global mangrove average. During the wet season, soil GHG emissions dropped significantly at all sites. During warmer conditions, mangroves were net sources of GHG, with a potential warming effect (GWP100) of 32.9 ± 10.2 (±SE) Mg CO2e ha-1 y-1. Overall, we found that dead mangroves did not release great amounts of GHG after three years of forest loss.

A human-specific enhancer fine-tunes radial glia potency and corticogenesis.

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications 1-3 . Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown 4,5 . HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development 6 . Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.

Airway injury induces alveolar epithelial and mesenchymal responses mediated by macrophages.

Acute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics and in vivo validation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation of Spp1 which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.

Circular economyeast: Saccharomyces cerevisiae as a sustainable source of glucans and its safety for skincare application.

Glucans, a polysaccharide naturally present in the yeast cell wall that can be obtained from side streams generated during the fermentation process, have gained increasing attention for their potential as a skin ingredient. Therefore, this study focused on the extraction method to isolate and purify water-insoluble glucans from two different Saccharomyces cerevisiae strains: an engineered strain obtained from spent yeast in an industrial fermentation process and a wild strain produced through lab-scale fermentation. Two water-insoluble extracts with a high glucose content (> 90 %) were achieved and further subjected to a chemical modification using carboxymethylation to improve their water solubility. All the glucans' extracts, water-insoluble and carboxymethylated, were structurally and chemically characterized, showing almost no differences between both yeast-type strains. To ensure their safety for skin application, a broad safety assessment was undertaken, and no cytotoxic effect, immunomodulatory capacity (IL-6 and IL-8 regulation), genotoxicity, skin sensitization, and impact on the skin microbiota were observed. These findings highlight the potential of glucans derived from spent yeast as a sustainable and safe ingredient for cosmetic and skincare formulations, contributing to the sustainability and circular economy.

The Peptide Antibiotic Corramycin Adopts a ß-Hairpin-like Structure and Is Inactivated by the Kinase ComG.

The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.

Systematic Review of Neoadjuvant Immunotherapy for Mismatch Repair Deficient Locally Advanced Colon Cancer: An Emerging Strategy.

BACKGROUND: In April 2023, the National Comprehensive Cancer Network endorsed neoadjuvant immunotherapy for select patients with nonmetastatic mismatch repair deficient colon cancer. Approximately 15% of incident colon cancers are mismatch repair deficient, resulting in a distinct molecular subtype with high microsatellite instability that is responsive to immune checkpoint inhibition. OBJECTIVE: To describe the existing evidence supporting neoadjuvant immunotherapy for mismatch repair deficient, microsatellite unstable nonmetastatic colon cancer. DATA SOURCES: A medical librarian performed PubMed, Embase, and Web of Science searches most recently on April 24, 2023. The PubMed search was re-run on September 26, 2023, to identify any additional studies published between April 24 and September 26, 2023. STUDY SELECTION: Two authors screened titles and abstracts in the published studies. The inclusion criteria were 1) English language, 2) adults with primary cancer of the colon, 3) nonmetastatic disease, 4) neoadjuvant immunotherapy, and 5) reporting on 10 or more cases. INTERVENTION: Neoadjuvant immunotherapy. MAIN OUTCOME MEASURES: Safety (grade 3+ treatment-related adverse events) and efficacy (complete pathologic responses). RESULTS: From 7691 studies identified, 6370 were screened and 8 were included. Various agents, dosing regimens, and treatment durations were used, with durations of immunotherapy ranging from 1 to 16 cycles. Complete R0 resections were consistently achieved in 98% to 100% of resections. Of patients who received neoadjuvant immunotherapy and underwent resection, 50% to 91% had ypT0N0 pathology. The safety profiles were generally favorable, with grade 1 to 2 treatment-related adverse events (mostly immune-related) during immunotherapy reported in 22.2% to 70% of patients. Postoperative complications after neoadjuvant immunotherapy were reassuring, with no severe complications reported. LIMITATIONS: Small number of heterogeneous and uncontrolled studies precluding a meta-analysis. CONCLUSIONS: Neoadjuvant immune checkpoint inhibition is associated with high rates of pathologic complete responses in locally advanced colon cancer. The literature is limited, particularly for postoperative outcomes, and more studies are needed to understand the safety and positioning of these regimens in the neoadjuvant context.

The role of advanced endoscopy in appendiceal polyp management and outcomes.

BACKGROUND: Appendiceal orifice lesions are often managed operatively with limited or oncologic resections. The aim is to report the management of appendiceal orifice mucosal neoplasms using advanced endoscopic interventions. METHODS: Patients with appendiceal orifice mucosal neoplasms who underwent advanced endoscopic resections between 2011 and 2021 with either endoscopic mucosal resection (EMR), endoscopic mucosal dissection (ESD), hybrid ESD, or combined endoscopic laparoscopic surgery (CELS) were included from a prospectively collected dataset. Patient and lesion details and procedure outcomes are reported. RESULTS: Out of 1005 lesions resected with advanced endoscopic techniques, 41 patients (4%) underwent appendiceal orifice mucosal neoplasm resection, including 39% by hybrid ESD, 34% by ESD, 15% by EMR, and 12% by CELS. The median age was 65, and 54% were male. The median lesion size was 20 mm. The dissection was completed piecemeal in 49% of patients. Post-procedure, one patient had a complication within 30 days and was admitted with post-polypectomy abdominal pain treated with observation for 2 days with no intervention. Pathology revealed 49% sessile-serrated lesions, 24% tubular adenomas, and 15% tubulovillous adenomas. Patients were followed up for a median of 8 (0-48) months. One patient with a sessile-serrated lesion experienced a recurrence after EMR which was re-resected with EMR. CONCLUSION: Advanced endoscopic interventions for appendiceal orifice mucosal neoplasms can be performed with a low rate of complications and early recurrence. While conventionally lesions at the appendiceal orifice are often treated with surgical resection, advanced endoscopic interventions are an alternative approach with promising results which allow for cecal preservation.

Harvesting the Power of Green Synthesis: Gold Nanoparticles Tailored for Prostate Cancer Therapy.

Biosynthetic gold nanoparticles (bAuNPs) present a promising avenue for enhancing bio-compatibility and offering an economically and environmentally responsible alternative to traditional production methods, achieved through a reduction in the use of hazardous chemicals. While the potential of bAuNPs as anticancer agents has been explored, there is a limited body of research focusing on the crucial physicochemical conditions influencing bAuNP production. In this study, we aim to identify the optimal growth phase of Pseudomonas aeruginosa cultures that maximizes the redox potential and coordinates the formation of bAuNPs with increased efficiency. The investigation employs 2,6-dichlorophenolindophenol (DCIP) as a redox indicator. Simultaneously, we explore the impact of temperature, pH, and incubation duration on the biosynthesis of bAuNPs, with a specific emphasis on their potential application as antitumor agents. Characterization of the resulting bAuNPs is conducted using ATR-FT-IR, TEM, and UV-Vis spectroscopy. To gain insights into the anticancer potential of bAuNPs, an experimental model is employed, utilizing both non-neoplastic (HPEpiC) and neoplastic (PC3) epithelial cell lines. Notably, P. aeruginosa cultures at 9 h/OD600 = 1, combined with biosynthesis at pH 9.0 for 24 h at 58 °C, produce bAuNPs that exhibit smaller, more spherical, and less aggregated characteristics. Crucially, these nanoparticles demonstrate negligible effects on HPEpiC cells while significantly impacting PC3 cells, resulting in reduced viability, migration, and lower IL-6 levels. This research lays the groundwork for the development of more specialized, economical, and ecologically friendly treatment modalities.

Epithelial stem and progenitor cells of the upper airway.

The upper airway acts as a conduit for the passage of air to the respiratory system and is implicated in several chronic diseases. Whilst the cell biology of the distal respiratory system has been described in great detail, less is known about the proximal upper airway. In this review, we describe the relevant anatomy of the upper airway and discuss the literature detailing the identification and roles of the progenitor cells of these regions.

Structure and properties of Quercus robur acorn starch extracted by pulsed electric field technology.

Pulsed electric field (PEF) technology was used to extract starch from Q. robur flours using low-intensity electric fields (0 and 0.1 kV/cm) and study the impact of PEF on the structure and properties of acorn starch concerning commercial starch. PEF technology is an advantageous method for starch extraction than the aqueous steeping from an industrial perspective since reduces extraction time and allows for continuous processing of larger suspension volumes. PEF technology preserved the amylose and amylopectin contents, hydrogen bonds, and diffraction patterns, as well as the starch native properties. Hence, PEF could be used to obtain native starches, but future studies should verify its economic viability. Acorn starches have lower damaged starch content, gelatinization temperatures, enthalpies, improved pseudoplastic behavior, reduced in-vitro digestibility, and lower resistance to deformation compared to commercial corn starch. The higher solubility and swelling power of acorn starches up to 80 °C make them a suitable food additive in fermented yogurt and milk products and thus help to value acorn and acorn starches. Hence, acorns can be used to obtain native starches, a food ingredient with a wide range of food and non-food usage, using PEF.

Vaginal prevention of Candida albicans: synergistic effect of lactobacilli and mannan oligosaccharides (MOS).

Vulvovaginal candidiasis (VVC) affects approximately 30-50% of women at least once during their lifetime, causing uncomfortable symptoms and limitations in their daily quality of life. Antifungal therapy is not very effective, does not prevent recurrencies and usually causes side effects. Therefore, alternative therapies are urgently needed. The goal of this work was to investigate the potential benefits of using mannan oligosaccharides (MOS) extracts together with a Lactobacillus sp. pool, composed by the most significant species present in the vaginal environment, to prevent infections by Candida albicans. Microbial growth of isolated strains of the main vaginal lactobacilli and Candida strains was assessed in the presence of MOS, to screen their impact upon growth. A pool of the lactobacilli was then tested against C. albicans in competition and prophylaxis studies; bacterial and yeast cell numbers were quantified in specific time points, and the above-mentioned studies were assessed in simulated vaginal fluid (SVF). Finally, adhesion to vaginal epithelial cells (HeLa) was also evaluated, once again resorting to simultaneous exposure (competition) or prophylaxis assays, aiming to measure the effect of MOS presence in pathogen adherence. Results demonstrated that MOS extracts have potential to prevent vaginal candidiasis in synergy with vaginal lactobacilli, with improved results than those obtained when using lactobacilli alone. KEY POINTS: Potential benefits of MOS extracts with vaginal lactobacilli to prevent C. albicans infections. MOS impacts on growth of vaginal lactobacilli pool and C. albicans in SVF. MOS extracts in synergy with L. crispatus inhibit C. albicans adhesion in HeLa cells.

Practice Patterns for Organ Preservation in US Patients With Rectal Cancer, 2006-2020.

Importance: In March 2023, the National Comprehensive Cancer Network endorsed watch and wait for those with complete clinical response to total neoadjuvant therapy. Neoadjuvant therapy is highly efficacious, so this recommendation may have broad implications, but the current trends in organ preservation in the US are unknown. Objective: To describe organ preservation trends among patients with rectal cancer in the US from 2006 to 2020. Design, Setting, and Participants: This retrospective, observational case series included adults (aged ≥18 years) with rectal adenocarcinoma managed with curative intent from 2006 to 2020 in the National Cancer Database. Exposure: The year of treatment was the primary exposure. The type of therapy was chemotherapy, radiation, or surgery (proctectomy, transanal local excision, no tumor resection). The timing of therapy was classified as neoadjuvant or adjuvant. Main Outcomes and Measures: The primary outcome was the absolute annual proportion of organ preservation after radical treatment, defined as chemotherapy and/or radiation without tumor resection, proctectomy, or transanal local excision. A secondary analysis examined complete pathologic responses among eligible patients. Results: Of the 175 545 patients included, the mean (SD) age was 63 (13) years, 39.7% were female, 17.4% had clinical stage I disease, 24.7% had stage IIA to IIC disease, 32.1% had stage IIIA to IIIC disease, and 25.7% had unknown stage. The absolute annual proportion of organ preservation increased by 9.8 percentage points (from 18.4% in 2006 to 28.2% in 2020; P < .001). From 2006 to 2020, the absolute rate of organ preservation increased by 13.0 percentage points for patients with stage IIA to IIC disease (19.5% to 32.5%), 12.9 percentage points for patients with stage IIIA to IIC disease (16.2% to 29.1%), and 10.1 percentage points for unknown stages (16.5% to 26.6%; all P < .001). Conversely, patients with stage I disease experienced a 6.1-percentage point absolute decline in organ preservation (from 26.4% in 2006 to 20.3% in 2020; P < .001). The annual rate of transanal local excisions decreased for all stages. In the subgroup of 80 607 eligible patients, the proportion of complete pathologic responses increased from 6.5% in 2006 to 18.8% in 2020 (P < .001). Conclusions and Relevance: This case series shows that rectal cancer is increasingly being managed medically, especially among patients whose treatment historically relied on proctectomy. Given the National Comprehensive Cancer Network endorsement of watch and wait, the increasing trends in organ preservation, and the nearly 3-fold increase in complete pathologic responses, international professional societies should urgently develop multidisciplinary core outcome sets and care quality indicators to ensure high-quality rectal cancer research and care delivery accounting for organ preservation.