Biology subjects

Marchetti, M.

Publications and source records attributed to Marchetti, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Dysregulated neutrophil extracellular trap formation is a novel driver of clonogenicity and therapeutic vulnerability in CMML

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

cancer biology↗

bioRxiv2026

IsdH binding to hemoglobin:haptoglobin is decoupled from iron acquisition in Staphylococcus aureus

Staphylococcus aureus requires iron for proliferation during infection and acquires it mainly from host hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The hemophores IsdB and IsdH mediate the initial steps of Hb recognition. Notably, IsdH also recognizes the hemoglobin:haptoglobin (HbHp) complex, although the molecular determinants and physiological relevance of this interaction remain unclear. Here, combining cryo-electron microscopy and biochemical and cellular assays, we define the basis of HbHp recognition by IsdH. The structure reveals how IsdH engages HbHp and captures the intrinsic conformational flexibility of the HbHp assembly. Functional analyses demonstrate that, despite retaining the ability to extract heme from HbHp, IsdH does not support S. aureus growth under iron-restricted conditions when HbHp is the sole iron source. These findings suggest that HbHp recognition by IsdH may serve functions beyond nutrient iron acquisition, contributing to modulation of host-pathogen interactions.

biochemistry↗

bioRxiv2026

Longitudinal Changes in Nasal and Oral Microbiome and Antimicrobial Resistance Gene Profiles in Response to Human Fecal Microbiota Transplantation

The gut-lung axis describes interactions between intestinal and respiratory mucosal systems through microbial, metabolic, and immune pathways, but the systemic impact of gut-targeted therapies on upper respiratory tract (URT) communities remains underexplored. We conducted a longitudinal study in adult patients undergoing fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (CDI) alongside healthy controls. Fecal, nasal, and oral samples were collected at baseline (Day 0) and on Days 14 and 56 following FMT. Shotgun metagenomic sequencing was performed to quantify microbial diversity, taxonomic composition, and the abundance of antimicrobial resistance genes (ARGs). FMT was associated with increased gut diversity and decreased levels of key intestinal taxa commonly considered pathobionts, including Klebsiella spp., Escherichia spp., Shigella spp., and Klebsiella pneumoniae. At the phylum level, fecal Bacteroidota increased, while Mucoromycota decreased following treatment. Post-FMT nasal microbiome changes included reduced richness and diversity, expansion of Moraxella, and decreases in taxa linked with respiratory colonization, including Staphylococcus aureus and Streptococcus pneumoniae. By Day 56, nasal communities partially recovered toward healthy profiles. Baseline nasal ARG abundance decreased following FMT, particularly among {beta}-lactam, aminoglycoside, and fluoroquinolone resistance genes, and remained comparable to healthy controls by Day 56. In contrast, the oral microbiome and oral resistome remained largely stable, with only minor fluctuations, and no consistent increases in respiratory pathobiont-associated taxa. In summary, FMT was associated with broader effects beyond the gut, including changes in the URT microbial ecology and antimicrobial resistance profiles. Together, these findings are consistent evidence of gut-lung microbial interactions, linking intestinal dynamics with respiratory microbial composition and antimicrobial resistance patterns.

microbiology↗

bioRxiv2026

Engineering a PD-L1-sensing synthetic receptor for programmable macrophage-mediated phagocytosis

Macrophages are abundant immune cells within the tumor microenvironment with intrinsic phagocytic capabilities, yet their antitumor functions are frequently suppressed by inhibitory signals. Synthetic biology enables the rational design of ligand-responsive genetic circuits to reprogram immune cell behavior. Here, we report the engineering of a synthetic Notch-based receptor that detects PD-L1, an immune-checkpoint broadly expressed by cancer cells. Upon PD-L1 engagement, the circuit triggers programmable outputs, including expression of a fluorescent reporter or CV1-Fc, that locally blocks the CD47 "dont eat me" signal. We show that circuit activation scales with PD-L1 levels, partially attenuates PD-1/PD-L1 signaling, and that conditional CV1-Fc expression enhances engulfment of SKOV-3 ovarian cancer cells by THP-1-derived macrophages in vitro. Collectively, this work reframes PD-L1 from an end-point therapeutic target to a primary input signal for synthetic circuit activation and establishes a modular framework for engineering macrophage behaviour through spatially confined, ligand-responsive control. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/700810v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1cbb391org.highwire.dtl.DTLVardef@7a0900org.highwire.dtl.DTLVardef@1e4f6d6org.highwire.dtl.DTLVardef@109838d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. A novel -PDL1 SNIPR receptor to program macrophage outputs. PD-L1 expressed on cancer cells is detected by -PD-L1 SNIPR-engineered macrophages, triggering release of the GAL4-VP64 transcription factor and activation of a programmable actuator. Circuit activation leads to customized outputs, including biosensing and enhanced macrophage phagocytosis via CD47 blockade, as well as immunomodulatory effects through interference with the PD-1/PD-L1 checkpoint axis. This sensor-actuator framework enables spatially confined and ligand-dependent reprogramming of macrophage function. C_FIG

synthetic biology↗

bioRxiv2025

Natural variation in an NLR pair confers thermostable resistance to a devastating bacterial pathogen

Climate change reshapes host-pathogen interactions by increasing pathogen aggressiveness and weakening plant immune responses, particularly under heat stress. Nucleotide-binding leucine-rich repeat (NLR) immune receptors, key players in pathogen recognition, are altered under elevated temperatures in both plants and mammals, posing a major challenge to disease resistance. In Arabidopsis thaliana, the well-described RPS4/RRS1 NLR pair confers resistance to the worldwide devastating phytopathogenic Ralstonia pseudosolanacearum. Here, by combining the exploration of natural genetic variation with genetic mapping, polymorphism analysis, structural modeling, and functional complementation, we demonstrated that the paucity of full thermostable resistance is primarily associated with a unique RPS4/RRS1 haplotype carrying key substitutions in leucine-rich repeat domains. These findings reveal natural genetic diversity as a source of thermostable resistance, highlighting promising opportunities to engineer climate-resilient plants.

plant biology↗

bioRxiv2025

Extracellular matrix and cytoskeletal reverse remodeling pathways are key drivers of myocardial recovery following left ventricular assist device therapy

Transcriptomic changes in heart failure (HF) patients prior to and following left ventricular assist device (LVAD) support have been extensively studied. Recent studies focused on understanding DNA methylation changes in patients with cardiovascular diseases (CVD) and the role of circulating markers of DNA methylation as clinical predictors of the risk of CVD related morbidity and mortality. In this study, we used paired (pre- and post-LVAD) myocardial samples to examine changes in DNA methylation alongside RNA and protein expression. Our data suggests that patients with no improvement in cardiac function after LVAD therapy, despite showing an improvement in energy production (increased {beta}-oxidation of fatty acid) exhibited persistent activation of profibrotic signaling, increased collagen deposition and cytoskeletal disarray evident from abnormal increase in sarcomeric distance following LVAD support. Contrarily, patients with improvement in cardiac function after LVAD therapy showed activation of pro-inflammatory signaling, collagen degradation and myogenesis. Both RNA sequencing and western blot data showed increased COL1A1 and decreased TPPP3 in post-NR thereby suggesting increased fibrosis and disrupted cytoskeletal signaling as potential barriers to myocardial recovery. Additionally, responders to LVAD therapy showed a significant reversal in myocardial interstitial fibrosis with a preserved sarcomeric architecture. Mice model of HF and recovery also confirmed our human findings, with reduced fibrotic signaling and improved cytoskeletal remodeling signaling observed in mice that showed improvement in cardiac function compared to mice with HF. Overall, our data suggests that altering extracellular matrix regulation and cytoskeletal signaling pathways may contribute to myocardial recovery. Further studies targeting these pathways are required to identify new HF therapeutic targets.

genomics↗

bioRxiv2025

A comprehensive genetic toolkit for Zymomonas mobilis; allowing rapid, high-resolution, combinatorial engineering of metabolic pathways.

The limited availability of reliable genetic tools has constrained the precise engineering of Zymomonas mobilis, a promising bacterium for the sustainable bioproduction of fuels and commodity chemicals. Here, we present a comprehensive, modular genetic toolkit that enables high-resolution, combinatorial control of gene expression in Z. mobilis. We constructed and characterised a synthetic promoter library spanning the broadest dynamic range reported for this organism, alongside a suite of strong transcriptional terminators that effectively insulate genetic cassettes and prevent transcriptional readthrough. To achieve predictable translation across diverse genetic contexts, we further implemented bicistronic design (BCD) elements, allowing context-independent control of expression. Promoter activities were shown to be stable under both aerobic and anaerobic conditions, highlighting the robustness of the regulatory parts across physiologically relevant environments. The toolkit was integrated into the Start-Stop Assembly system, facilitating high-throughput construction of multi-gene pathways. We demonstrate its utility through the rapid assembly and testing of the possible expression space of genes encoding a 2,3-butanediol biosynthetic pathway. Collectively, this genetic toolkit now allows the high-resolution engineering of multi-enzyme pathways in Z. mobilis for industrial biotechnology applications.

synthetic biology↗

bioRxiv2025

Microcephaly-Associated Genes asp and Sas4 Control Chromatin Organization and Nuclear Lamina Structure in Drosophila melanogaster

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and non-progressive intellectual disability. Mutations in over 30 genes have been linked to MCPH. Nearly a half of the genes identified by these mutations encode proteins involved in centrosome biogenesis or microtubule (MT) dynamics, suggesting a central role for mitotic spindle organization and division plane orientation in disease aetiology. However, it has been suggested that disruptions in spindle positioning alone are not sufficient to lead to microcephaly. Here, we investigate the contribution of the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) to nuclear architecture, chromatin organization, and genome stability. We show that loss of either Sas4 or Asp leads to aberrant microtubule architecture, mislocalization of the LINC complex, and deformation of the nuclear lamina. These defects are accompanied by reduced levels of both lamin and HP1 and impaired centromere clustering in interphase cells. Sas4 and asp mutants also exhibit a global reduction in heterochromatin-associated histone marks (H3K9me2/3 and H3K27me3) and increased levels of the euchromatin-associated mark H3K4me3. Remarkably, treatment with Methylstat, a demethylase inhibitor, reduced nuclear invaginations by partially restoring H3K9me3 levels. Additionally, Sas4 or Asp depletion leads to DNA damage, increased sensitivity to genotoxic stress, and delayed DNA repair. Together, these findings reveal a previously underappreciated role for Asp and Sas4 in preserving nuclear architecture and chromatin integrity, offering new insight into the pathogenesis of MCPH. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/666102v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@bfdfbborg.highwire.dtl.DTLVardef@f983eaorg.highwire.dtl.DTLVardef@143857borg.highwire.dtl.DTLVardef@1f0895b_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Two birds with one stone: a novel potential antibiotic blocking IsdB-mediated heme extraction by Staphylococcus aureus with serendipitous hemoglobin left-shifting activity

Infections caused by Staphylococcus aureus are closely linked to its ability to secure essential nutrients, including iron, which is extracted from the heme of human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[[2-[[5- (1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetyl]amino]benzoate (C35) was recently identified as a new potential antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by specifically targeting the hemophore-driven iron acquisition system. Our findings confirm both the potential of C35 as a first-in-class protein-protein interaction inhibitor with antimicrobial activity, and the effectiveness of targeting hemophores as a strategy to inhibit S. aureus growth. To gain information for drug discovery purposes, the X-ray structure of Hb in the presence of the compound was solved. Unexpectedly, we discovered that, rather than the predicted binding pose, the molecule binds to tetrameric Hb in a cleft between the alpha subunits, stabilizing an R2 relaxed Hb conformation. This triggered further investigation of the effect of C35 on Hb functional properties, which showed a pronounced left-shift activity on oxygen binding curve (i.e., it strongly increases the Hb oxygen affinity). These results highlight C35 as a promising dual-acting compound with both antimicrobial activity and the ability to modulate Hb function through non-covalent stabilization of a high-affinity state. Author SummaryStaphylococcus aureus is a dangerous bacterium that can cause severe infections in humans. To grow and survive it needs iron, which it steals from our red blood cells by taking it from hemoglobin, the protein that carries oxygen in the blood. In this study, we focused on a small molecule, called C35, that blocks the interaction between hemoglobin and a key bacterial protein involved in heme acquisition. We found that C35 strongly inhibits the growth of S. aureus when hemoglobin is the only available source of iron, showing a potential new method to starve the pathogen and consequently fight the infection. Surprisingly, we also found that C35 increases the affinity of hemoglobin for oxygen. This dual action makes C35 a unique molecule for future therapeutic development, with potential applications both as a new antimicrobial agent and in the treatment of diseases related to hemoglobin function.

microbiology↗

bioRxiv2025

Schistosoma mansoni antigen induced innate immune memory features mitochondrial biogenesis and can be inhibited by ovarian produced hormones

We have previously identified that S. mansoni infection induces a unique form of myeloid training that protects male but not female mice from high fat diet induced disease. Here we demonstrate that ovarian derived hormones account for this sex specific difference. Ovariectomy of females prior to infection permits metabolic reprogramming of the myeloid lineage, with BMDM exhibiting carbon source flexibility for cellular respiration, and mice protected from systemic metabolic disease. The innate training phenotype of infection can be replicated by in vivo injection of SEA, and by exposure of bone marrow to SEA in culture prior to macrophage differentiation (Day 0). This protective phenotype is linked to increased chromatin accessibility of lipid and mitochondrial pathways in BMDM including Nrf1 and Tfam, as well as mitochondrial biogenesis. This work provides evidence that S. mansoni antigens induce a unique form of innate training inhibited by ovarian-derived hormones in females.

immunology↗

bioRxiv2024

Organ injury accelerates stem cell differentiation by modulating a fate-transducing lateral inhibition circuit

To rebuild tissue form and function, injured organs accelerate the differentiation of replacement stem cell progeny. Here we demonstrate that injury-induced factors open the throttle on faster differentiation by streamlining the archetypal signaling circuit that patterns cell fates. During normal turnover of the adult Drosophila intestine, fates are patterned by a conserved lateral inhibition circuit: In stem cell pairs, mutual activation of Notch receptor by Delta ligand feeds back to create opposing states of high Notch/low Delta and low Notch/high Delta; cells terminally differentiate once their Notch activity exceeds a fate-deciding threshold. After feeding flies a gut-damaging toxin, we perform in vivo imaging of real-time intestinal repair and trace Notch reporter dynamics in single cells. We find that tissue damage causes the speed of Notch signal activation to accelerate dramatically; faster activation expedites terminal differentiation by propelling cells past the critical Notch threshold more quickly. Combining single-cell analyses with dynamical modeling, we show that faster activation results from aberrant elevation of Delta ligand due to loss of time-delaying circuit feedback. Injury abolishes feedback via a cytokine-JAK-STAT relay from damaged cells to stem cells, causing stem cells to deactivate the Notch co-repressor that normally turns off Delta. Thus, organ injury unmasks latent plasticity in Notch-Delta lateral inhibition to propel the differentiation of new replacement cells. By unifying temporal and spatial fate control in a single, adaptable signaling circuit, organs tune stem cell dynamics to meet environmental challenges.

developmental biology↗

bioRxiv2024

Bwa, an ortholog of alkaline ceramidase-ACER2, promotes intestinal stem cell proliferation through pro-inflammatory cytokine signaling in Drosophila melanogaster

Sphingolipids, including ceramides, are an important component of high-fat diets. These molecules can regulate fatty acid oxidation and intestinal stem cell proliferation, predisposing the gut to tumorigenesis. However, the molecular mechanisms involved in ceramide metabolism-mediated intestinal stem cell (ISC) proliferation and tumorigenesis are poorly understood. To understand how changes in sphingolipid metabolite flux affect intestinal stem cells, we manipulated the activities of each of the enzymes of the ceramide synthetic pathway using cell type-specific over-expression or depletion of the corresponding mRNAs in each intestinal cell type of the Drosophila midgut. We documented cell-autonomous and non-cell-autonomous effects, including alterations in cell size, number, differentiation, and proliferation. In our screen, the altered expression of several ceramide metabolism enzymes led to changes in ISC proliferation, cell sizes, and overall cellularity. Among other genes, over-expression of ceramidase homolog, Brain washing (bwa) in gut enteroblasts (EB) increased EB cell size and caused a non-cell-autonomous, 7-8-fold increase in ISC proliferation. Our analysis confirmed previous reports that bwa does not have ceramidase activity, and lipidomic studies indicated that bwa increases the saturation status of sphingolipids, free fatty acids, and other lipids. The pro-proliferative effects of bwa could be counter-acted by depleting a serine palmitoyltransferase, Lace, or a sphingosine acyltransferase, Schlank, which are needed for ceramide synthesis, or by co-expressing a ceramide desaturase enzyme, ifc, indicating that increased saturated ceramides were causal for ISC proliferation and the disruption of gut homeostasis. Accumulating saturated sphingolipids and fatty acids induced inflammatory signaling in the gut, and activated ISC proliferation through the pro-inflammatory cytokines, Upd3 and Upd2. We propose that saturated sphingolipids promote ISC proliferation through pro-inflammatory pathways.

cell biology↗

bioRxiv2024

Mating and ecdysone signaling modify growth, metabolism, and digestive efficiency in the female Drosophila gut

Adaptive changes in organ size and physiology occur in most adult animals, but how these changes are regulated is not well understood. Previous research found that mating in Drosophila females drives not only increases in gut size and stem cell proliferation but also alters feeding behavior, intestinal gene expression, and whole-body lipid storage, suggesting altered gut metabolism. Here, we show that mating dramatically alters female gut metabolism and digestive function. In addition to promoting a preference for a high-protein diet, mating also altered levels of TCA cycle intermediates and fatty acids in the gut, increased total gut lipids and protein, reduced relative carbohydrate levels, and enhanced the efficiency of protein digestion relative to carbohydrate digestion. The expression of genes that mediate each of these metabolic processes was similarly altered. In addition, we noted the mating-dependent downregulation of oxidative stress response and autophagy genes. Mating-dependent increases in ecdysone signaling played an important role in re-programming many, but not all, of these changes in the female gut. This study contributes to our understanding of how steroid signaling alters gut physiology to adapt to the demands of reproduction.

physiology↗

bioRxiv2024

Neuronal progenitors suffer genotoxic stress in the Drosophila clock mutant per0.

The physiological role and the molecular architecture of the circadian clock in fully developed organisms are well established. Yet, we have a limited understanding about the function of the clock during ontogenesis. We have used a null mutant (per0) of the clock gene period (per) in Drosophila melanogaster to ask whether PER may play a role during normal brain development. In 3rd instar larvae, we have observed that absence of functional per results in increased genotoxic stress compared to wild type controls. We have detected increased double strand DNA breaks in the central nervous system and chromosome aberrations in dividing neuronal precursor cells. We have demonstrated that reactive oxygen species (ROS) are causal to the genotoxic effect and that expression of PER in glia is necessary and sufficient to suppress such a phenotype. Finally, preliminary evidence indicates that absence of PER may result in less condensed chromatin, which may contribute to DNA damage.

genetics↗

bioRxiv2024

Two-photon all-optical electrophysiology for the dissection of larval zebrafish brain functional connectivity

One of the most audacious goals of modern neuroscience is unraveling the complex web of causal relations underlying the activity of neuronal populations on a whole-brain scale. This endeavor, prohibitive just a couple of decades ago, has recently become within reach owing to the advancements in optical methods and the advent of genetically encoded indicators/actuators. These techniques, applied to the translucent larval zebrafish have enabled recording and manipulation of the activity of extensive neuronal populations spanning the entire vertebrate brain. Here, we present the conception of a custom two-photon optical system, coupling light-sheet imaging and 3D excitation with acousto-optic deflectors for simultaneous high-speed volumetric recording and optogenetic stimulation. By employing a zebrafish line with pan-neuronal expression of both the calcium reporter GCaMP6s and the red-shifted opsin ReaChR, we implemented a crosstalk-free, noninvasive all-optical approach and applied it to the reconstruction of the functional connectivity of the left habenula.

neuroscience↗

bioRxiv2023

Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms

Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome (e.g., calreticulin (CALR), Fc gamma receptor (Fc{gamma}RIIA) and galectin-1 (LGALS1) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease. HighlightsMPN patient platelet proteome identifies key pathobiological mediators of thrombosis and proteostasis. The MPN platelet proteomic profile validates our prior findings from the platelet transcriptome. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/563619v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb6290org.highwire.dtl.DTLVardef@1b3ec3eorg.highwire.dtl.DTLVardef@15b0c8eorg.highwire.dtl.DTLVardef@9575b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2023

Time-resolved X-ray solution scattering unveils the sequence of events leading to human Hb heme capture by Staphylococcus aureus IsdB

Infections caused by Staphylococcus aureus depend on its ability to acquire nutrients. One essential nutrient is iron, which is obtained from the heme of the human host hemoglobin (Hb) through a protein machinery called Iron-regulated Surface Determinant (Isd). IsdB is the protein in charge of heme extraction from Hb, which is the first step of the chain of events leading to iron transfer to the bacterium cell interior. In order to elucidate the molecular events leading from the formation of the initial IsdB:Hb complex to heme extraction, we have performed a time-resolved X-ray solution scattering (TR-XSS) investigation combined with a rapid mixing triggering approach. We succeeded in defining the stoichiometry of IsdB:Hb binding and in describing the kinetics of the subsequent structural changes. The presented approach is potentially applicable to unveil the complex kinetic pathways generated by protein-protein interaction in different biological systems.

biochemistry↗

bioRxiv2021

An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine

In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, current organ culture techniques are limited to imaging sessions of [≤]16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. Therefore, we developed a new organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na+/K+ ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelium damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation, we demonstrate that intestinal stem lineages can be traced through multiple cell divisions using live imaging. Both asymmetric and symmetric divisions can be identified in the reconstructed lineages. We find that daughter cells of asymmetric divisions remain in close proximity of each other, while the progeny of symmetric divisions actively move apart, with implications for cell differentiation and tissue organization. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 72 hours. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.

cell biology↗