The impact of comparative genomic hybridization/single-nucleotide polymorphism microarray in risk stratification of pediatric acute lymphoblastic leukemia.

BACKGROUND: The objective of this study is to assess the concordance and added value of combined comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH/SNP) analyses in pediatric acute lymphoblastic leukemia (ALL) risk stratification compared to conventional cytogenetic methods. PROCEDURE: This is a retrospective study that included patients aged 1-18 years diagnosed with de novo ALL at Sainte-Justine Hospital between 2016 and 2021. Results from conventional cytogenetic and molecular analyses were collected and compared to those of CGH/SNP. RESULTS: A total of 135 ALL patients were included. Sample failures or non-diagnostic analyses occurred in 17.8% cases with G-banding karyotypes versus 1.5% cases with CGH/SNP. The mean turnaround time for results was significantly faster for CGH/SNP than karyotype with 5.8 versus 10.7 days, respectively. The comparison of ploidy assessment by CGH/SNP and G-banding karyotype showed strong concordance (r = .82, p < .001, r2 = .68). Furthermore, G-banding karyotype did not detect additional clinically relevant aberrations that were missed by the combined analysis of CGH/SNP and fluorescence in situ hybridization. The most common gene alterations detected by CGH/SNP were deletions involving CDKN2A (35.8%), ETV6 (31.3%), CDKN2B (28.4%), PAX5 (20.1%), IKZF1 (12.7%), and copy-neutral loss of heterozygosity (CN-LOH) of 9p (9.0%). Among these, only ETV6 deletion was found to have a significant prognostic impact with superior event-free survival in both univariate and multivariate analyses (adjusted hazard ratio 0.08, 95% confidence interval: 0.01-0.50, p = .02). CONCLUSION: CGH/SNP provided faster, reliable, and highly concordant results than those obtained by conventional cytogenetics. CGH/SNP identified recurrent gene deletions in pediatric ALL, of which ETV6 deletion conferred a favorable prognosis.

Evolutionary trajectory for nuclear functions of ciliary transport complex proteins.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4.

Aging-related cardiac fibrosis represents the principal pathological progression in cardiovascular aging. The Muscleblind-like splicing regulator 2 (MBNL2) has been unequivocally established as being associated with cardiovascular diseases. Nevertheless, its role in aging-related cardiac fibrosis remains unexplored. This investigation revealed an elevation of MBNL2 levels in the aged heart and senescent cardiac fibroblasts. Notably, the inhibition of MBNL2 demonstrated a capacity to mitigate H2O2-induced myofibroblast transformation and aging-related cardiac fibrosis. Further mechanistic exploration unveiled that aging heightened the expression of SENP1 and impeded the SUMO1 binding with KLF4, and SUMOylation of KLF4 effectively increased by the inhibition of MBNL2. Additionally, the inhibition of TGF-ß1/SMAD3 signaling attenuated the impact of over-expression of MBNL2 in inducing senescence and cardiac fibrosis. MBNL2, by orchestrating SUMOylation of KLF4, upregulating the TGF-ß1/SMAD3 signaling pathway, emerges as a significant promoter of aging-related cardiac fibrosis. This discovery identifies a novel regulatory target for managing aging-related cardiac fibrosis.

Sirtuin 2 regulates neutrophil functions through NAD+ synthesis pathway in virus infection.

Neutrophils play an important role in antiviral immunity, but the underlying mechanisms remain unclear. Here, we found that SIRT2 deficiency inhibited the infiltration of neutrophils, as well as the secretion of inflammatory cytokines and the formation of neutrophil extracellular traps (NETs), ameliorating disease symptoms during acute respiratory virus infection. Mechanistically, SIRT2 deficiency upregulates quinolinic acid (QA)-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) and leads to expression of quinolinate phosphoribosyltransferase (QPRT), which promotes the synthesis of QA for NAD+ and limits viral infection when de novo NAD+ synthesis is blocked. Tryptophan-2,3-oxygenase expressed in epithelial cells metabolizes tryptophan to produce kynurenine and 3-hydroxyaminobenzoic acid, which is a source of intracellular QA in neutrophils. Thus, our findings reveal a previously unrecognized QPRT-mediated switch in NAD+ metabolism by exploiting neutrophil-derived QA as an alternative source of replenishing intracellular NAD+ pools induced by SIRT2 to regulate neutrophil functions during virus infection, with implications for future immunotherapy approaches.

miRNA-mediated inhibition of an actomyosin network in hippocampal pyramidal neurons restricts sociability in adult male mice.

Social deficits are frequently observed in patients suffering from neurodevelopmental disorders, but the molecular mechanisms regulating sociability are still poorly understood. We recently reported that the loss of the microRNA (miRNA) cluster miR-379-410 leads to hypersocial behavior and anxiety in mice. Here, we show that ablating miR-379-410 in excitatory neurons of the postnatal mouse hippocampus recapitulates hypersociability, but not anxiety. At the cellular level, miR-379-410 loss in excitatory neurons leads to larger dendritic spines, increased excitatory synaptic transmission, and upregulation of an actomyosin gene network. Re-expression of three cluster miRNAs, as well as pharmacological inhibition of the actomyosin activator ROCK, is sufficient to reinstate normal sociability in miR-379-410 knockout mice. Several actomyosin genes and miR-379-410 family members are reciprocally dysregulated in isogenic human induced pluripotent stem cell (iPSC)-derived neurons harboring a deletion present in patients with Williams-Beuren syndrome, characterized by hypersocial behavior. Together, our results show an miRNA-actomyosin pathway involved in social behavior regulation.

Protocol for three-dimensional whole-mount imaging of the vascular network in the intestinal muscle.

Three-dimensional (3D) imaging of vascular networks is essential for the investigation of vascular patterning and organization. Here, we present a step-by-step protocol for the 3D visualization of the vasculature within whole-mount preparations of the mouse intestinal muscularis propria layer. We then detail the quantitative analysis of the resulting images for parameters such as vessel density, vessel diameter, the number of endothelial cells, and proliferation. The protocol can be easily extended to study cell-cell interactions such as neuro-vascular or immune-vascular interactions. For complete details on the use and execution of this protocol, please refer to Schrenk et al.1.

DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.

Colombian consensus for the molecular diagnosis of endometrial cancer / Consenso colombiano para el diagnóstico molecular del cáncer de endometrio.

Objective: The Cancer Genome Atlas research program (TCGA) developed the molecular classification for endometrial cancer with prognostic and therapeutic utility, which was replaced by the ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) classification by consensus and international guidelines due to its high cost. This article aims to present national recommendations from an expert consensus that allows unification and implementation of the molecular classification for women with endometrial cancer nationwide, with a rational use of resources and technology. Methods: Consensus of 36 experts in clinical oncology, oncological gynecology, pathology, and genetics, with clinical practice in the national territory. The leader group performed a literature review and structuring of questions rated 1 to 9 points. A modified nominal group technique was used. There was a face-to-face meeting with master presentations, deliberative dialogue, and Google Forms (Google LLC, Mountain View, CA, USA) questionnaire voting with analysis and discussion of responses. The non-consensual responses led to a second round of voting. The final manuscript was finally prepared and revised. Results: Seven recommendations were formulated integrating the panelist responses based on evidence, but adjusted to the Colombian context and reality. Recommendation 1. The molecular classification is recommended in all the endometrial cancers using the immunohistochemistry markers as subrogated results from the molecular profile initially proposed in the TCGA classification. Recommendation 2. The sequential test strategy is recommended, starting with the immunohistochemistry markers (p53, MLH1, MSH 2, MSH6, PMS2) simultaneously in all the patients, defining to request POLE (DNA polymerase epsilon) (if available) according to the risk classification based on the surgical piece. Recommendation 3. It is recommended, that the gynecologist oncologist should be the one to request the POLE (if available) according to the final pathology report. This test must be requested for all endometrial cancers stage I-II, except in low risk (stage IA low grade endometrioid histology without linfovascular invasion normal p53) and, stages III-IV without residual disease, without affecting the request of subrogated immunohistochemistry molecular markers upon histology. The consensus proposes that the POLE is requested after the immunohistochemistry and according to the categories in the risk classification established by the 2020 ESGO/ESTRO/ESP guidelines. Recommendation 4. It is recommended to perform immunohistochemistry for hormonal receptors for all women with endometrial cancer and the HER2 in patients with p53abn, simultaneously with the others immunohistochemistry markers. Recommendation 5. It is recommended to perform the immunohistochemistry markers (p53, MLH1, MSH2, MSH6 y PMS2) in an initial endometrial biopsy or curettage when the specimen is adequate and available. In case the initial immunohistochemistry is inconclusive, or there are histological discrepancies between the initial and definitive pathology, it is recommended to repeat the molecular profile in the surgical pathology. The immunohistochemistry markers must be reported in the pathology report according to the CAP (College of American Pathologists) recommendations, independently of the type of sample. Recommendation 6. It is recommended to perform MLH1 promoter methylation testing in patients who exhibit loss of expression of MLH1 in immunohistochemistry whether it is accompanied or not with loss of expression of PMS2. All the patients with deficient MMR (mismatch repair), should be sent for genetic counseling to rule out Lynch syndrome. Recommendation 7. It is recommended to consider the molecular classification in addition to the classical histopathological criteria when making adjuvant judgments, as incorporated by the classification of prognostic groups of the 2020 ESGO/ESTRO/ESP guidelines. Conclusions: It is necessary to implement the molecular classification of endometrial cancer in clinical practice in accordance to the Colombian context, due to its prognostic and probably predictive value. This will enable the characterization of the Colombian population in order to offer individualized guided treatments. This is an academic and nonregulatory document.

Objetivos: el programa Cancer Genome Atlas Research (TCGA) desarrolló la clasificación molecular para cáncer endometrial con utilidad pronóstica y terapéutica, la cual ha sido reemplazada por consensos y guías internacionales por la clasificación ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) debido a su alto costo. El objetivo de este artículo es presentar recomendaciones a nivel nacional derivadas de un consenso de expertos que permitan unificar e implementar la clasificación molecular para mujeres con cáncer endometrial, mediante un uso racional de recursos y tecnología. Materiales y métodos: consenso de 36 expertos en oncología clínica, ginecología oncológica, patología y genética con práctica clínica en el territorio nacional. El grupo líder realizó una revisión de la literatura y estructuración de preguntas calificadas de 1 a 9 puntos. Se utilizó la técnica de grupo nominal modificada. Se efectuaron reuniones presenciales con presentaciones magistrales, diálogo deliberativo y votación de cuestionario Google Forms (Google LLC, Mountain View, CA, USA) con análisis y discusión de respuestas. Las respuestas no consensuadas se llevaron a una segunda ronda de votación. Finalmente, se elaboró y revisó el manuscrito final. Resultados: se formularon siete recomendaciones integrando las respuestas de las panelistas basadas en evidencia, pero ajustadas al contexto y a la realidad colombiana. Recomendación 1. Se recomienda realizar la clasificación molecular en todos los carcinomas endometriales utilizando los marcadores de inmunohistoquímica como resultados subrogados del perfil molecular inicialmente propuesto en la clasificación del TCGA. Recomendación 2. Se recomienda la estrategia secuencial de testeo iniciando por los marcadores de inmunohistoquímica (p53, MLH1, MSH 2, MSH6, PMS2) simultáneamente en todas las pacientes, y definir la solicitud del POLE (polimerasa épsilon del DNA) (si se encuentra disponible) de forma diferida de acuerdo con la clasificación de riesgo basado en la pieza quirúrgica. Recomendación 3. Se recomienda que sea el ginecólogo oncólogo quien solicite el POLE (si se encuentra disponible) de acuerdo con el reporte de patología definitivo. Esta prueba se debe solicitar a todos los cánceres endometriales de estadio I-II, excepto los de bajo riesgo (estadio IA endometrioide de bajo grado sin invasión linfovascular p53 normal) y estadio III-IV sin enfermedad residual, sin afectar la solicitud de los marcadores moleculares subrogados por inmunohistoquímica de acuerdo con la histología. El consenso propone que la solicitud del POLE se realice posterior a la inmunohistoquímica y de acuerdo con la clasificación del riesgo según las categorías establecidas por la guía ESGO/ESTRO/ESP del 2020. Recomendación 4. Se recomienda realizar simultáneamente con los otros marcadores de inmunohistoquímica la prueba para receptores hormonales en todas las pacientes con cáncer endometrial y el HER2 en pacientes con p53abn. Recomendación 5. Se recomienda que los marcadores de inmunohistoquímica (p53, MLH1, MSH2, MSH6 y PMS2) se realicen en la biopsia/legrado endometrial inicial cuando la muestra es adecuada y está disponible. En caso de inmunohistoquímica inicial no concluyente, o discrepancias histológicas entre la patología inicial y definitiva, se recomienda repetir el perfil molecular en la patología quirúrgica. Los marcadores de inmunohistoquímica deben reportarse en el informe de patología de acuerdo con las recomendaciones del CAP (College of American Pathologists), independientemente del tipo de muestra. Recomendación 6. Se recomienda realizar estudio de metilación de promotor de MLH1 en pacientes con pérdida de expresión de MLH1 en la inmunohistoquímica, acompañado o no de pérdida de expresión de PMS2. Todas las pacientes con déficit de MMR (mismatch repair), deben ser enviadas a genética para descartar síndrome de Lynch. Recomendación 7. Se recomienda tener en cuenta la clasificación molecular, además de los criterios histopatológicos clásicos para la toma de decisiones de adyuvancia, tal como los incorpora la clasificación de los grupos pronósticos de la guía ESGO/ ESTRO/ESP del 2020. Conclusiones: es necesario implementar la clasificación molecular de cáncer de endometrio en la práctica clínica acorde al contexto colombiano, dado su valor pronóstico y posiblemente predictivo. Esto permitirá la caracterización de la población colombiana para ofrecer tratamientos guiados de manera individualizada. Se trata de un documento académico y no regulatorio.

Metabolic reprogramming and dysregulated IL-17 production impairs CD4 T cell function post sepsis.

Sepsis survivors are at high risk for infection-related rehospitalization and mortality for years following the resolution of the acute septic event. These infection-causing microorganisms generally do not cause disease in immunocompetent hosts, suggesting that the post-septic immune response is compromised. Given the importance of CD4 T cells in the development of long-lasting protective immunity, we analyzed their post-septic function. Here we showed that sepsis induced chronic increased and non-specific production of IL-17 by CD4 T cells, resulting in the inability to mount an effective immune response to a secondary pneumonia challenge. Altered cell function was associated with metabolic reprogramming, characterized by mitochondrial dysfunction and increased glycolysis. This metabolic reprogramming began during the acute septic event and persisted long after sepsis had resolved. Our findings reveal cell metabolism as a potential therapeutic target. Given the critical role of cell metabolism in the physiological and pathophysiological processes of immune cells, these findings reveal a potential new therapeutic target to help mitigate sepsis survivors' susceptibility to secondary infections.

Supernumerary proteins of the human mitochondrial ribosomal small subunit are integral for assembly and translation.

Mitochondrial ribosomes (mitoribosomes) have undergone substantial evolutionary structural remodeling accompanied by loss of ribosomal RNA, while acquiring unique protein subunits located on the periphery. We generated CRISPR-mediated knockouts of all 14 unique (mitochondria-specific/supernumerary) human mitoribosomal proteins (snMRPs) in the small subunit to study the effect on mitoribosome assembly and protein synthesis, each leading to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Surprisingly, the stability of mS37 was reduced in all our snMRP knockouts of the small and large ribosomal subunits and patient-derived lines with mitoribosome assembly defects. A redox-regulated CX9C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.

BTLA and PD-1 signals attenuate TCR-mediated transcriptomic changes.

T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

CHK1 inhibitor SRA737 is active in PARP inhibitor resistant and CCNE1 amplified ovarian cancer.

High-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) are initially responsive to poly (ADP-ribose) polymerase inhibitors (PARPi), but resistance ultimately emerges. HGSOC with CCNE1 amplification (CCNE1 amp) are associated with resistance to PARPi and platinum treatments. High replication stress in HRD and CCNE1 amp HGSOC leads to increased reliance on checkpoint kinase 1 (CHK1), a key regulator of cell cycle progression and the replication stress response. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable CHK1 inhibitor (CHK1i), SRA737, in both acquired PARPi-resistant BRCA1/2 mutant and CCNE1 amp HGSOC models. We demonstrated that SRA737 increased replication stress and induced subsequent cell death in vitro. SRA737 monotherapy in vivo prolonged survival in CCNE1 amp models, suggesting a potential biomarker for CHK1i therapy. Combination SRA737 and PARPi therapy increased tumor regression in both PARPi-resistant and CCNE1 amp patient-derived xenograft models, warranting further study in these HGSOC subgroups.

Osteopontin regulation of MerTK+ macrophages promotes Crohn's disease intestinal fibrosis.

The pathogenesis of intestinal fibrosis in Crohn's disease (CD) remains unclear. Mer receptor tyrosine kinase (MerTK) is an immunosuppressive protein specifically expressed in macrophages. Osteopontin (OPN), also known as secreted phosphoprotein 1, contributes to inflammation and wound repair. This study investigates the potential profibrotic pathway in MerTK+ macrophages in order to provide a possible therapeutic target for intestinal fibrosis. MerTK expression in the inflamed and stenotic bowels was evaluated. The MerTK/ERK/TGF-ß1 pathway was overactivated in the fibrotic intestinal tissues of patients with CD. This pathway was induced by epithelial cell apoptosis, resulting in activated fibroblasts with increased TGF-ß1 secretion. OPN upregulated TGF production by altering ERK1/2 phosphorylation, as evidenced by OPN or MerTK knockdown and OPN overexpression in vitro. MerTK inhibitor UNC2025 alleviated intestinal fibrosis in mouse colitis models, suggesting a potential therapeutic target for intestinal fibrosis in patients with CD.

Msc1 is a nuclear envelope protein that reinforces DNA repair in late mitosis.

Precise double-strand break (DSB) repair is a paramount for genome stability. Homologous recombination (HR) repairs DSBs when cyclin-dependent kinase (CDK) activity is high, which correlates with the availability of the sister chromatid as a template. However, anaphase and telophase are paradoxical scenarios since high CDK favors HR despite sister chromatids being no longer aligned. To identify factors specifically involved in DSB repair in late mitosis, we have undertaken comparative proteomics in Saccharomyces cerevisiae and found that meiotic sister chromatid 1 (Msc1), a poorly characterized nuclear envelope protein, is significantly enriched upon both random and guided DSBs. We further show that Δmsc1 is more sensitive to DSBs in late mitosis, and has a delayed repair of DBSs, as indicated by increased Rad53 hyperphosphorylation, a higher presence of RPA foci, fewer Rad52 repair factories, and slower HR completion. We propose that Msc1 favors the later stages of HR and the timely completion of DSB repair before cytokinesis.

Insulin receptor isoform B is required for efficient proinsulin processing in pancreatic ß cells.

The insulin receptor (INSR, IR) has two isoforms, IRA and IRB, through alternative splicing. However, their distinct functions in vivo remain unclear. Here we generated ß cell-specific IRB knockout (KO) mice (ßIRBKO). The KO mice displayed worsened hyperinsulinemia and hyperproinsulinemia in diet-induced obesity due to impaired proinsulin processing in ß cells. Mechanistically, loss of IRB suppresses eukaryotic translation initiation factor 4G1 (eIF4G1) by stabilizing the transcriptional receptor sterol-regulatory element binding protein 1 (SREBP1). Moreover, excessive autocrine proinsulin in ßIRBKO mice enhances the activity of extracellular signal-regulated kinase (ERK) through the remaining IRA to further stabilize nuclear SREBP1, forming a feedback loop. Collectively, our study paves the way to dissecting the isoform-specific function of IR in vivo and highlights the important roles of IRB in insulin processing and protecting ß cells from lipotoxicity in obesity.

RHOF activation of AKT/ß-catenin signaling pathway drives acute myeloid leukemia progression and chemotherapy resistance.

Acute myeloid leukemia (AML) is a clonal malignancy originating from leukemia stem cells, characterized by a poor prognosis, underscoring the necessity for novel therapeutic targets and treatment methodologies. This study focuses on Ras homolog family member F, filopodia associated (RHOF), a Rho guanosine triphosphatase (GTPase) family member. We found that RHOF is overexpressed in AML, correlating with an adverse prognosis. Our gain- and loss-of-function experiments revealed that RHOF overexpression enhances proliferation and impedes apoptosis in AML cells in vitro. Conversely, genetic suppression of RHOF markedly reduced the leukemia burden in a human AML xenograft mouse model. Furthermore, we investigated the synergistic effect of RHOF downregulation and chemotherapy, demonstrating significant therapeutic efficacy in vivo. Mechanistically, RHOF activates the AKT/ß-catenin signaling pathway, thereby accelerating the progression of AML. Our findings elucidate the pivotal role of RHOF in AML pathogenesis and propose RHOF inhibition as a promising therapeutic approach for AML management.

Structural insights into ligand recognition, selectivity, and activation of bombesin receptor subtype-3.

Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence.

The development of antifungal drugs requires novel molecular targets due to limited treatment options and drug resistance. Through chemical screening and establishment of a novel genetic technique to repress gene expression in Trichophyton rubrum, the primary causal fungus of dermatophytosis, we demonstrated that fungal Cdc42 and Rac GTPases are promising antifungal drug targets. Chemical inhibitors of these GTPases impair hyphal formation, which is crucial for growth and virulence in T. rubrum. Conditional repression of Cdc24, a guanine nucleotide exchange factor for Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. EHop-016 inhibited the promotion of the guanine nucleotide exchange reaction in Cdc42 and Rac by Cdc24 as well as germination and growth on the nail fragments of T. rubrum and improved animal survival in an invertebrate infection model of T. rubrum. Our results provide a novel antifungal therapeutic target and a potential lead compound.

Investigating cerebral neurovascular responses to hyperglycemia in a rat model of type 2 diabetes using multimodal assessment techniques.

To study neurovascular function in type 2 diabetes mellitus (T2DM), we established a high-fat diet/streptozotocin (HFD/STZ) rat model. Electrocorticography-laser speckle contrast imaging (ECoG-LSCI) revealed that the somatosensory-evoked potential (SSEP) amplitude and blood perfusion volume were significantly lower in the HFD/STZ group. Cortical spreading depression (CSD) velocity was used as a measure of neurovascular function, and the results showed that the blood flow velocity and the number of CSD events were significantly lower in the HFD/STZ group. In addition, to compare changes during acute hyperglycemia and hyperglycemia, we used intraperitoneal injection (IPI) of glucose to induce transient hyperglycemia. The results showed that CSD velocity and blood flow were significantly reduced in the IPI group. The significant neurovascular changes observed in the brains of rats in the HFD/STZ group suggest that changes in neuronal apoptosis may play a role in altered glucose homeostasis in T2DM.

Fusobacterium nucleatum subsp. nucleatum RadD binds Siglec-7 and inhibits NK cell-mediated cancer cell killing.

Fusobacterium nucleatum is an oral commensal bacterium that can colonize extraoral tumor entities, such as colorectal cancer and breast cancer. Recent studies revealed its ability to modulate the immune response in the tumor microenvironment (TME), promoting cancer progression and metastasis. Importantly, F. nucleatum subsp. animalis was shown to bind to Siglec-7 via lipopolysaccharides, leading to a pro-inflammatory profile in human monocyte-derived dendritic cells. In this study, we show that F. nucleatum subsp. nucleatum RadD binds to Siglec-7 on NK cells, thereby inhibiting NK cell-mediated cancer cell killing. We demonstrate that this binding is dependent on arginine residue R124 in Siglec-7. Finally, we determine that this binding is independent of the known interaction of RadD with IgA. Taken together, our findings elucidate the targeting of Siglec-7 by F. nucleatum subsp. nucleatum RadD as a means to modulate the NK cell response and potentially promoting immune evasion and tumor progression.