Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria.

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Acute, chronic and conditioned effects of intranasal oxytocin in the mu-opioid receptor knockout mouse model of autism: Social context matters.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behaviours. Multiple studies have highlighted the potential of oxytocin (OT) to ameliorate behavioural abnormalities in animal models and subjects with ASD. Clinical trials, however, yielded disappointing results. Our study aimed at assessing the behavioural effects of different regimens of OT administration in the Oprm1 null mouse model of ASD. We assessed the effects of intranasal OT injected once at different doses (0.15, 0.3, and 0.6 IU) and time points (5, 15, and 30 min) following administration, or chronically, on ASD-related behaviours (social interaction and preference, stereotypies, anxiety, nociception) in Oprm1+/+ and Oprm1-/- mice. We then tested whether pairing intranasal OT injection with social experience would influence its outcome on ASD-like symptoms, and measured gene expression in the reward/social circuit. Acute intranasal OT at 0.3 IU improved social behaviour in Oprm1-/- mice 5 min after administration, with limited effects on non-social behaviours. Chronic (8-17 days) OT maintained rescuing effects in Oprm1 null mice but was deleterious in wild-type mice. Finally, improvements in the social behaviour of Oprm1-/- mice were greater and longer lasting when OT was administered in a social context. Under these conditions, the expression of OT and vasopressin receptor genes, as well as marker genes of striatal projection neurons, was suppressed. We detected no sex difference in OT effects. Our results highlight the importance of considering dosage and social context when evaluating the effects of OT treatment in ASD.

The IMS Library: from IN-Stock to Virtual.

A chemical library is a key element in the early stages of pharmaceutical research. Its design encompasses various factors, such as diversity, size, ease of synthesis, aimed at increasing the likelihood of success in drug discovery. This article explores the collaborative efforts of computational and synthetic chemists in tailoring chemical libraries for cost-effective and resource-efficient use, particularly in the context of academic research projects. It proposes chemoinformatics methodologies that address two pivotal questions: first, crafting a diverse panel of under 1000 compounds from an existing pool through synthetic efforts, leveraging the expertise of organic chemists; and second, expanding pharmacophoric diversity within this panel by creating a highly accessible virtual chemical library. Chemoinformatics tools were developed to analyse initial panel of about 10,000 compounds into two tailored libraries: eIMS and vIMS. The eIMS Library comprises 578 diverse in-stock compounds ready for screening. Its virtual counterpart, vIMS, features novel compounds guided by chemists, ensuring synthetic accessibility. vIMS offers a broader array of binding motifs and improved drug-like characteristics achieved through the addition of diverse functional groups to eIMS scaffolds followed by filtering of reactive or unusual structures. The uniqueness of vIMS is emphasized through a comparison with commercial suppliers' virtual chemical space.

Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

Rational Design of Cyanine-Based Fluorogenic Dimers to Reduce Nonspecific Interactions with Albumin and Lipid Bilayers: Application to Highly Sensitive Imaging of GPCRs in Living Cells.

Fluorogenic dimers with polarity-sensitive folding are powerful probes for live-cell bioimaging. They switch on their fluorescence only after interacting with their targets, thus leading to a high signal-to-noise ratio in wash-free bioimaging. We previously reported the first near-infrared fluorogenic dimers derived from cyanine 5.5 dyes for the optical detection of G protein-coupled receptors. Owing to their hydrophobic character, these dimers are prone to form nonspecific interactions with proteins such as albumin and with the lipid bilayer of the cell membrane resulting in a residual background fluorescence in complex biological media. Herein, we report the rational design of new fluorogenic dimers derived from cyanine 5. By modulating the chemical structure of the cyanine units, we discovered that the two asymmetric cyanine 5.25 dyes were able to form intramolecular H-aggregates and self-quenched in aqueous media. Moreover, the resulting original dimeric probes enabled a significant reduction of the nonspecific interactions with bovine serum albumin and lipid bilayers compared with the first generation of cyanine 5.5 dimers. Finally, the optimized asymmetric fluorogenic dimer was grafted to carbetocin for the specific imaging of the oxytocin receptor under no-wash conditions directly in cell culture media, notably improving the signal-to-background ratio compared with the previous generation of cyanine 5.5 dimers.

Fluorinated apelin-13 mediates neuroprotective effects in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease leading to demyelination and axonal loss. Current treatments are immunomodulatory or immunosuppressive drugs acting on the inflammatory component. However, these treatments do not adequately address the crucial aspect of neuroprotection. Recently, an association between an altered balance of adipokines and MS has been proposed as both a risk factor for developing MS and a chronic disease aggravating factor. Specifically, a decrease of apelin plasma levels in MS patients compared to controls correlates with the number of relapses and disease severity. Here we report a dramatic downregulation of apelin levels in the CNS of EAE mice which is also detected in MS patients brain samples compared to controls. Exploiting innovative design and synthesis techniques, we engineered a novel fluorinated apelin-13 peptide characterized by enhanced plasmatic stability compared to its native counterpart. With this peptide, we assessed the potential therapeutic benefits of apelin preventive supplementation in the EAE mouse model. We show that the fluorinated Apelin-13 peptide ameliorates EAE clinical score and preserves myelin content in the EAE MOG model recapitulating the progressive form of disease. These results combined with ex-vivo experiments in brain organotypic slices and in vitro studies in neurons and primary microglia and macrophages suggest that apelin has neuroprotective effects and influences the microglia/macrophages function.

Influence of donor-recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation.

Immunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years, reproducibility, as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here, we used various immunodeficient mouse models to characterize the effect of donor-recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex-specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly, human female donor cells display heightened sensitivity to the recipient mice's gender, influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics, shedding light on important considerations for future studies, particularly in the context of pre-clinical research.

Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Alared: Solvatochromic and Fluorogenic Red Amino Acid for Ratiometric Live-Cell Imaging of Bioactive Peptides.

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid-phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared-labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions. Due to its red-shifted absorption and emission spectra, Alared-labeled peptides could be followed in living cells with minimal interference from cellular autofluorescence. Using ratiometric fluorescence microscopy, we were able to track the fate of the Alared-labeled peptide agonists of the apelin G protein-coupled receptor upon receptor activation and internalization. Due to its color-shifting environmentally sensitive emission, Alared allowed for distinguishing the fractions of peptides that are specifically bound to the receptor or unspecifically bound to different cellular membranes.

Distinct ontogenetic lineages dictate cDC2 heterogeneity.

Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet+ cDC2As and T-bet- cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H+ pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H- CD8α+ pre-cDC2As in tissues, which differentiated into T-bet+ cDC2As. In contrast, a Siglec-H- fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet- cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment.

CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus.

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.

Loss of TET2 in human hematopoietic stem cells alters the development and function of neutrophils.

Somatic mutations commonly occur in hematopoietic stem cells (HSCs). Some mutant clones outgrow through clonal hematopoiesis (CH) and produce mutated immune progenies shaping host immunity. Individuals with CH are asymptomatic but have an increased risk of developing leukemia, cardiovascular and pulmonary inflammatory diseases, and severe infections. Using genetic engineering of human HSCs (hHSCs) and transplantation in immunodeficient mice, we describe how a commonly mutated gene in CH, TET2, affects human neutrophil development and function. TET2 loss in hHSCs produce a distinct neutrophil heterogeneity in bone marrow and peripheral tissues by increasing the repopulating capacity of neutrophil progenitors and giving rise to low-granule neutrophils. Human neutrophils that inherited TET2 mutations mount exacerbated inflammatory responses and have more condensed chromatin, which correlates with compact neutrophil extracellular trap (NET) production. We expose here physiological abnormalities that may inform future strategies to detect TET2-CH and prevent NET-mediated pathologies associated with CH.

Human CD34+ hematopoietic stem cell hierarchy: how far are we with its delineation at the most primitive level?

The ability to isolate and characterize different hematopoietic stem cell (HSC) or progenitor cell populations opens avenues to understand how hematopoiesis is regulated during development, homeostasis, and regeneration as well as in age-related conditions such as clonal hematopoiesis and leukemogenesis. Significant progress has been made in the past few decades in determining the composition of the cell types that exist in this system, but the most significant advances have come from mouse studies. However, recent breakthroughs have made significant strides that have enhanced the resolution of the human primitive hematopoietic compartment. Therefore, we aim to review this subject not only from a historical perspective but also to discuss the progress made in the characterization of the human postnatal CD34+ HSC-enriched populations. This approach will enable us to shed light on the potential future translational applicability of human HSCs.

The CKS1/CKS2 Proteostasis Axis Is Crucial to Maintain Hematopoietic Stem Cell Function.

Long-term hematopoietic stem cells are rare, highly quiescent stem cells of the hematopoietic system with life-long self-renewal potential and the ability to transplant and reconstitute the entire hematopoietic system of conditioned recipients. Most of our understanding of these rare cells has relied on cell surface identification, epigenetic, and transcriptomic analyses. Our knowledge of protein synthesis, folding, modification, and degradation-broadly termed protein homeostasis or "proteostasis"-in these cells is still in its infancy, with very little known about how the functional state of the proteome is maintained in hematopoietic stem cells. We investigated the requirement of the small phospho-binding adaptor proteins, the cyclin-dependent kinase subunits (CKS1 and CKS2), for maintaining ordered hematopoiesis and long-term hematopoietic stem cell reconstitution. CKS1 and CKS2 are best known for their roles in p27 degradation and cell cycle regulation, and by studying the transcriptome and proteome of Cks1 -/- and Cks2 -/- mice, we demonstrate regulation of key signaling pathways that govern hematopoietic stem cell biology including AKT, FOXO1, and NFκB, together balancing protein homeostasis and restraining reactive oxygen species to ensure healthy hematopoietic stem cell function.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

Rapid and Highly Selective Fluorescent Labeling of Peptides via a Thia-Diels-Alder Cycloaddition: Application to Apelin.

Herein, we describe a catalyst-free thia-Diels-Alder cycloaddition for the chemoselective labeling of fully deprotected phosphonodithioester-peptides in solution with fluorophores functionalized with an exocyclic diene. The reaction was optimized on the model tripeptide 1 containing a lysine residue, which enabled its rapid and straightforward labeling with three different fluorophores (fluorescein, lissamine rhodamine B, and squaraine) in very mild conditions (H2O/iPrOH, 37 °C, 1 h). The reaction was then successfully applied to the chemoselective labeling of fully deprotected apelin-13 with squaraine dye. The resulting fluorescent ligand 18 exhibited a high affinity (0.17 ± 0.03 nM) for apelinR. It enabled the development of time-resolved FRET-based competition assays for high-throughput screening and drug discovery. Thanks to its fluorogenic properties, ligand 18 was also successfully involved in the live-cell optical imaging of apelinR in no-wash conditions.

Hetero-Diels-Alder click reaction of dithioesters for a catalyst-free indirect 18F-radiolabelling of peptides.

The HDA reaction of dithioesters was developed as a new click-reaction compatible with the indirect 18F-labelling of peptides. It involves dithioester-peptides and a radiofluorinated diene as a novel prosthetic group. The method was applied to a PSMA-ligand for the in vivo detection of LNCap tumors in xenografted mice.

CKS1 inhibition depletes leukemic stem cells and protects healthy hematopoietic stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low 2-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs and toxicity of therapy toward healthy hematopoietic cells. We studied the role of cyclin-dependent kinase regulatory subunit 1 (CKS1)-dependent protein degradation in primary human AML and healthy hematopoiesis xenograft models in vivo. Using a small-molecule inhibitor (CKS1i), we demonstrate a dual role for CKS1-dependent protein degradation in reducing patient-derived AML blasts in vivo and, importantly, depleting LSCs, whereas inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Proteomic analysis of responses to CKS1i in our patient-derived xenograft mouse model demonstrate that inhibition of CKS1 in AML leads to hyperactivation of RAC1 and accumulation of lethal reactive oxygen species, whereas healthy hematopoietic cells enter quiescence in response to CKS1i, protecting hematopoietic stem cells. Together, these findings demonstrate that CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

Single cell analyses identify a highly regenerative and homogenous human CD34+ hematopoietic stem cell population.

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)- (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level.