Biology subjects

Martinez, E.

Publications and source records attributed to Martinez, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Linking anterior cingulate sulcal variability to attention-deficit hyperactivity disorder behavior

Background: The anterior cingulate cortex (ACC) is consistently implicated in attention-deficit/hyperactivity disorder (ADHD). ACC variability, including its most variable structure, the paracingulate sulcus (PCGS), has been known since the late 1800s, and since then, has been linked to cognitive control, impulsivity, and psychiatric conditions comorbid with ADHD, including autism spectrum disorder and schizophrenia. Yet, the role of the PCGS in ADHD remains unexamined at the individual level. Methods: We characterized PCGS incidence, asymmetry, and morphology in 247 children and adolescents (144 ADHD: 10.9 +/- 2.73 years old, 75% male; 103 neurotypical: 12.2 +/- 3.17 years old, 50% male) from the ADHD-200 dataset. The PCGS was manually identified in FreeSurfer cortical surface reconstructions. Morphometric measures, including sulcal length, depth, and cortical thickness, were extracted and normalized to hemisphere-wide variables. Between-group analyses tested whether PCGS presence, asymmetry, and quantitative morphology differed by diagnosis, while behavioral analyses examined associations with inattention, hyperactivity-impulsivity, and cognitive measures. Age and gender were included as covariates. Results: Analyses revealed that, in both NT and ADHD individuals, there was significant leftward asymmetry in PCGS presence. Between groups, the PCGS was less likely to be present in individuals with ADHD in the left hemisphere. Leftward PCGS absence was also associated with significantly higher ADHD index and hyperactivity-impulsivity scores. Conclusions: These findings advance understanding of how early neurodevelopmental variation in cortical folding may serve as a structural intermediary between genetic liability and the behavioral expression of ADHD.

neuroscience↗

bioRxiv2026

A quantitative interactome map reveals affinity tailored hijacking of human cellular functions by resident and pathogenic bacteria

Bacteria employ molecular mimicry to hijack host cellular functions for their own benefit. Despite the wealth of data mapping human-bacteria interactions, a comprehensive view of how these networks are fine-tuned remains elusive. In particular, there have been no large-scale screenings measuring human-bacterial protein-protein binding affinities to explore their role in functional and pathological outcomes. Here, we report the largest quantitative interactome to date between the family of human PDZ domains and C-terminal PDZ-binding motifs (PBMs) in effectors from pathogenic and resident gut bacteria. We show that bacteria tailor effector sequences to bind PDZ domains with similar or higher binding affinities than human PBMs to hijack their associated cellular functions. For a subset of the dataset, we validate PDZ-PBM interactions with the corresponding full-length proteins and show that for pathogenic E. coli effector NleA, its C-terminal PBM is necessary for interactions with proteins involved in intracellular transport and membrane organization, probably impacting the host glycocalyx to favor infection. Finally, we show that motifs overlapping with effector PBMs target interaction modules other than PDZ domains, adding an additional layer of complexity to the host-microbe molecular dialogue.

systems biology↗

bioRxiv2026

Proteolytic control of the SARS-CoV-2 furin cleavage site defines the phenotypic evolution from the pandemic to endemic state

The successive emergence of SARS-CoV-2 variants with altered tissue tropism has progressively decoupled transmissibility from lower respiratory tract pathogenicity. Omicron lineages transmit exceptionally well whilst limiting severe lung disease. Here, we demonstrate that this phenotype has evolved through two temporally distinct tissue-specific proteolytic controls at the Spike (S) furin cleavage site (FCS). At the virion level, cell-type-dependent furin-mediated FCS hyper-cleavage depletes S in lung but not nasal epithelial cells. At the infected cell membrane during cell-cell spread, TMPRSS2 mediates S FCS cleavage but is negatively regulated in the presence of ACE2. Tissue-specific solute carriers SLC6A19 and SLC6A20 sequester ACE2, thereby relieving inhibition of TMPRSS2 and enabling S FCS cleavage during cell-cell spread. Critically, whilst all SARS-CoV-2 lineages benefit from this latter pathway, Omicron variants have evolved exclusive dependence on it, a strategic consolidation that focuses S proteolytic activation to TMPRSS2 alone. The combined outcomes of S protein regulation across viral and cell membranes reveal how tissue-specific proteolytic optimisation drives Omicron's transmission fitness advantage in the upper respiratory tract but at the cost of heavy attenuation in the lower respiratory tract.

microbiology↗

bioRxiv2026

Rescue of ribosomal protein bL27 in Streptococcus pneumoniae TIGR4 by an alternate protease

Streptococcus pneumoniae is a major human respiratory pathogen. The bacterial 70S ribosome is a target of many clinically important antibiotics. The N-terminus of ribosomal protein bL27 extends into the peptidyl transferase center and contributes to the translation process. In Firmicutes, full length bL27 contains an 8-12 amino acid N-terminal extension that is absent from Gram-negative bacteria. This extension is cleaved by the protease Prp, which is absent from organisms lacking the extension. Prp-mediated cleavage of bL27 is essential in Staphylococcus aureus, and Prp has been proposed as a potential antibiotic target. Here, we show that in S. pneumoniae strain TIGR4, a {Delta}prp mutant remained viable, and produced ribosomes containing cleaved bL27, whereas deletion of prp was not tolerated in strain D39. These results suggested the presence of an alternate bL27-processing protease in TIGR4 that was absent from D39. Using a combination of genomics, proteomics and biochemical analyses, we identified this enzyme as the product of previously uncharacterized gene SP_1145, encoding a protease that we named Ribosome rescue protease (Rrp). SP_1145 is carried on a mobile genetic element that is present in strain TIGR4, but absent from D39. Our findings shed light on an alternative mechanism for bL27 maturation, and indicate that some strains of S. pneumoniae harbor horizontally acquired redundant pathways for this essential ribosome processing step.

microbiology↗

bioRxiv2026

A Bioactive Phospholipid Promotes Rapid Progenitor Lung Progenitor Activation via AP-1

Upon injury to the distal lung, alveolar type 2 cells (AT2s) must make a discrete switch from surfactant factories to stem cells capable of regeneration, which involves both proliferation and differentiation into oxygen-exchanging alveolar type 1 (AT1) cells. However, the discrete signals and molecular pathways facilitating this fundamental switch in AT2 functionality are uncertain. Here we demonstrate that the bioactive lipid lysophosphatidic acid (LPA), typically associated with driving fibrosis, is an extremely efficient inducer of this state change in comparison to previously implicated signals IL-1{beta} and p53 stabilization. We observed endogenous production and accumulation of LPA in influenza-injured murine lungs, creating a microenvironment that facilitates AT2 progenitor switching. Multiple transcriptomic approaches reveal elevation of Fosl1 and Jun, core members of the Activator Protein 1 (AP-1) transcription factor family, in response to LPA. Using novel genetic models combined with influenza injury, we demonstrate that AP-1 activity in AT2s is necessary for effective alveolar regeneration at both the cellular and physiologic levels. These findings unveil a critical relationship between paracrine LPA and cell-intrinsic AP-1 in facilitating effective lung alveolar regeneration. HighlightsO_LILPA facilitates progenitor switching in lung regeneration via initiation of a discrete transcriptomic state C_LIO_LILPA promotes AT2 state switching via Jun (AP-1) C_LIO_LIImpaired AP-1 signaling significantly restricts recovery from influenza infection C_LI

cell biology↗

bioRxiv2026

SARS-CoV-2 nucleocapsid protein engages with viral RNA and ERGIC lipids to drive viral core assembly

Severe acute respiratory syndrome coronavirus 2 assembles at the ER-Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA- N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.

biophysics↗

bioRxiv2026

Influenza-induced tuft cell expansion alters ILC-mediated inflammation

Tuft cells act as sentinels that amplify type 2 inflammation primarily by activating type 2 innate lymphoid cells (ILC2s). Although normally absent from the distal lung, ectopic tuft cells form after severe lung injury including influenza infection in mice. Here, we investigated the function of these ectopic tuft cells in shaping innate immunity following influenza injury. We observed that IFN{gamma} restrains tuft cell differentiation, whereas ILC2s drive tuft cell expansion, establishing a reciprocal regulatory axis. Tuft cell-deficient mice exhibited reduced eosinophilic inflammation and expansion of ILC1s and ILC3s after influenza injury resolution. Single-cell RNA-seq of influenza infected whole lung revealed transcriptional signatures consistent with type 1 pathway activation, type 2 suppression and oxidative stress. Following influenza injury and subsequent Alternaria alternata challenge, tuft cell-deficient mice also showed neutrophilic and ILC3 expansion. Together, these data identify a distal-airway tuft-cell-ILC2 circuit that helps maintain a balanced inflammatory environment in response to viral injury and aeroallergens.

molecular biology↗

bioRxiv2026

A mobile ESX type VII secretion system enhances intracellular persistence in globally distributed Mycobacterium abscessus

Mycobacterium abscessus are non-tuberculous mycobacteria that are widespread in the environment and of increasing global clinical significance. Accumulating evidence shows that M. abscessus has emerged as an important pathogen, driven by highly drug-resistant lineages, enhanced transmissibility and the acquisition of specific virulence factors. In this study, we describe a previously uncharacterised ESX secretion system encoded on a 123-kbp plasmid identified in a clinical isolate of M. abscessus. This ESX system, termed ESX-pMA07, is distinct from ESX systems previously reported in M. abscessus in both sequence composition and locus organisation, characterised by a unique arrangement of core ESX components and low sequence identity to ESX-3, ESX-4 and plasmid-borne ESX-P systems. ESX-pMA07 was detected in geographically diverse clinical isolates but was restricted to particular genotypes within the global M. abscessus phylogeny. Transcriptional profiling revealed expression of ESX-pMA07 components in artificial cystic fibrosis media and during intracellular growth in macrophage cell lines. Using CRISPR interference, we show that inducible silencing of eccC, encoding the ATPase component of ESX-pMA07, significantly reduced intracellular survival of M. abscessus within macrophages. To our knowledge, this is the first characterisation of a functional, plasmid-borne ESX secretion system in M. abscessus, demonstrating that mobile genetic elements contribute to the pathogens intracellular persistence and may influence its evolving virulence. Author SummaryMycobacterium abscessus is a rapidly emerging, highly drug-resistant bacterium that causes chronic infections, particularly in people with underlying lung disease such as cystic fibrosis. The factors that enable certain M. abscessus strains to persist inside host cells are not fully understood. In this study, we identified a previously unrecognised type VII secretion system (ESX) encoded on a large plasmid in a clinical isolate of M. abscessus. This plasmid-borne ESX system, which we termed ESX-pMA07, is genetically distinct from the ESX systems normally found on the chromosome and was detected in geographically diverse clinical isolates, but restricted to specific lineages within the global M. abscessus population. We show that ESX-pMA07 genes are expressed under conditions relevant to lung infection and during intracellular growth in macrophages. Using inducible CRISPR interference to silence the ESX ATPase gene eccC, we demonstrate that ESX-pMA07 contributes to intracellular survival of M. abscessus in macrophages. These findings reveal that mobile genetic elements can encode functional secretion systems that enhance intracellular persistence, providing a mechanism for the emergence and spread of virulence traits in this important pathogen.

microbiology↗

bioRxiv2026

Engineering a soft tumor microenvironment: fibrin-enriched hydrogel promotes cancer cell invasion in a 3D bioprinted colorectal cancer model

Accurately modeling the tumor microenvironment is crucial for advancing our understanding of colorectal cancer (CRC) progression and therapeutic response. Three-dimensional (3D) hydrogel-based models that mimic the mechanical properties of native tissue serve as a valuable tool for studying tumor-stromal interactions and tumor invasion in vitro. In this work, we developed a 3D bioprinted CRC model by embedding spheroids and human intestinal fibroblasts (HIFs) within the GelMA-PEGDA and GelMA-PEGDA-Fibrin hydrogels to assess the effect of matrix composition and stiffness on spheroid invasiveness. The addition of fibrin resulted in a softer, more viscoelastic hydrogel that promoted fibroblast migration, elongation, and alignment, facilitating more dynamic tumor-stromal interactions. Moreover, both epithelial-like HT29 and mesenchymal-like SW480 spheroids showed more invasive behavior in GelMA-PEGDA-Fibrin hydrogel. This was reflected in distinct phenotypic responses. HT29 spheroids demonstrated greater growth, irregular morphology, and more interaction with elongated fibroblasts, whereas SW480 spheroids exhibited partial dissociation and disruption with a higher number of dispersed cells in GelMA-PEGDA-Fibrin hydrogel. These findings demonstrate the role of matrix softness in promoting the invasiveness of colorectal cancer. Overall, our results highlight the potential of fibrin-enriched soft hydrogel to mimic key features of the tumor microenvironment, offering a powerful tool for studying CRC invasion dynamics and supporting future applications in drug screening and personalized medicine.

bioengineering↗

bioRxiv2025

Graft protection by myeloid progenitor cells using lethal or non-lethal preconditioning.

Myeloid progenitor cell (MPC) therapy can protect against infection and can be used as an irradiation countermeasure, but can also prevent rejection of matched grafts. Here we report on the matching requirements between MPCs and skin grafts, and on the development of a non-lethal preconditioning model that supports MPC-induced protection of skin grafts. Mouse MPCs (mMPC) were obtained from HSC following 10-day ex vivo expansion. Skin grafts were tested in host mice that either received a lethal dose of irradiation and reconstitution with allogeneic mMPC and third party HSCs or were given injections with depleting antibodies and chemotherapeutics followed by transplantation of mMPC without HSC. mMPC-matched grafts were protected and no significant difference was observed between mice receiving irradiation and fully matched grafts (major and minor transplantation antigens), partially matched grafts (major only), and fully matched grafts in mice that received sublethal irradiation. Haploidentical grafts (half of the MHC alleles not matched at all) are not protected. We conclude that closely matched mMPCs are sufficient to prevent rejection. We also show that mMPCs are effective in a trachea transplant model. Graft protection does not require either lethal preconditioning or an accompanying HSC transplantation, and affects both T- and B-cell responses.

immunology↗

bioRxiv2025

Replication of SARS-CoV-2 Omicron lineages is defined by TMPRSS2 use in environments where ACE2 is complexed with solute carriers SLC6A19 and SLC6A20.

The Omicron variant of SARS-CoV-2 emerged in late 2021 and since then Omicron subvariants have continued to evolve and dominate globally. The viral S protein evolved towards highly efficient antibody evasion and replicative capacity in the upper respiratory tract resulting in high transmissibility. At the same time, the mutations acquired in the S protein diminish infection of the lung epithelium and pathogenic potential. The changing entry requirements for Omicron sub-lineages that lead to this shift in tropism remain poorly understood. We resolve the changing replication requirements of SARS-CoV-2 to be related to two distinct pools of ACE2. The first pool relates to ACE2s role in the renin angiotensin system (RAS) and this pool can complex with TMPRSS2 (RAS-ACE2). The second pool relates to ACE2s role as a protein solute carrier chaperone than cannot complex with TMPRSS2 (Chaperone ACE2). Here, we demonstrate that pre-Omicron lineages replicate in a TMPRSS2 dependent manner across both ACE2 pools, whilst Omicron lineages can only spread and replicate using chaperone ACE2. This provides a mechanistic basis for the evolving infectivity requirements of SARS-CoV-2 and furthermore provides approaches to track and monitor ACE2 utilizing coronaviruses. Graphical AbstractMechanistic basis for shift in SARS-CoV-2 tropism with the arrival of Omicron. A. Chaperone ACE2 is defined structurally as a heterodimer of dimers with a solute carrier protein-SLC6A19 or SLC6A20. Here this ACE2 structure can exist uncomplexed from TMPRSS2 and enables TMPRSS2 use by both pre-Omicron and Omicron lineages. B. Renin Angiotensin ACE2 is defined by ACE2 with an exposed collectrin-like domain (CLD), which enables binding of TMPRSS2 or ADAM-17. Here ACE2 can form a complex with TMPRSS2 in a manner that allows pre-Omicron but not Omicron lineages to utilize TMPRSS2 to facilitate infection. Here Omicron lineages are heavily attenuated as they cannot use TMPRSS2 to spread. C. to E. Based on single cell profiles, ACE2 can exist as a chaperone with SLC6A20 in C. the Nasal Cavity or D. primarily as RAS-ACE2 in the lung to respond to acute lung injury. E. The largest pool of ACE2 in our body resides within the small intestine on enterocytes and this further facilitates replication in this tissue by pre-Omicron and Omicron lineages. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/663433v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@172aa47org.highwire.dtl.DTLVardef@109285eorg.highwire.dtl.DTLVardef@394f8corg.highwire.dtl.DTLVardef@958e31_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

CHD7 binds distinct regions in the Sox11 locus to regulate neuronal differentiation

The chromodomain helicase DNA binding protein 7 (CHD7) is a nucleosome repositioner implicated in multiple cellular processes, including neuronal differentiation. We identified CHD7 genome-wide binding sites that regulate neuronal differentiation in an otic stem cell line. We identified CHD7 enrichment at the Sox11 promoter and 3 untranslated region (UTR). Sox11 is a transcription factor essential for neuronal differentiation. CRISPRi of Sox11 promoter or 3UTR displayed decreased neurite lengths and reduced neuronal marker expression TUBB3 expression. We showed that the Sox11 locus resides at TAD boundaries, and CTCF marks the 3UTR. We propose that CHD7 modulates chromatin accessibility of the Sox11 promoter and CTCF-marked insulators in the 3UTR to facilitate neuronal differentiation. CRISPRi of the insulator site alters 3D chromatin organization, affects gene expression and ultimately perturbs cellular processes. Our results implicate a general mechanism of CHD7 in facilitating neuronal differentiation and provide insight into CHD7 dysfunction in CHARGE syndrome, a congenital disorder associated with hearing loss.

molecular biology↗

bioRxiv2025

CHD7 binds to insulators during neuronal differentiation

Spiral ganglion neurons (SGNs) are crucial for hearing, and the loss of SGNs causes hearing loss. Stem cell-based therapies offer a promising approach for SGN regeneration and require understanding the mechanisms governing SGN differentiation. We investigated the chromatin remodeler CHD7 in neuronal differentiation using immortalized multipotent otic progenitor (iMOP) cells. We demonstrated that CHD7 knockdown impaired neuronal differentiation. Genome-wide analysis revealed CHD7 binding at diverse cis-regulatory elements, with notable enrichment at sites marked by the insulator-binding protein CTCF between topologically associating domains (TADs). Insulators marked by the enrichment of CHD7 and CTCF resided near genes critical for neuronal differentiation, including Mir9-2. Targeting these regulatory regions in iMOPs with CRISPR interference (CRISPRi) and activation (CRISPRa) increased miR-9 transcription, irrespective of the method. Blocking the CHD7 and CTCF marked sites suggested that the elements function as insulators to regulate gene expression. The study highlights CHD7 activity at insulators and underscores an unreported mechanism for promoting neuronal differentiation.

molecular biology↗

bioRxiv2025

Short- and long-term effects of uterine disease on oocyte developmental capacity in postpartum dairy cows

The hypothesis was that early postpartum uterine disease would reduce the developmental capacity of oocytes thus contributing to the reduced fertility of dairy cows with uterine disease. Dairy cows were diagnosed healthy or with metritis at 7 to 10 d postpartum. The reproductive tract was collected at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of cumulus-oocyte complexes (COC). The COC were matured, co- incubated with sperm for fertilization, and cultured to the blastocyst stage (8 d) in vitro. For Exp.1, the disease diagnosis (healthy or metritis) did not affect the number of collected COC or the subsequent embryo development to the blastocyst stage. The presence of purulent material in the uterine lumen (endometritis) at time of oocyte collection, however, was associated with a reduced cleavage rate evaluated 3 d following fertilization. For Exp. 2, there was no effect of disease diagnosis (healthy or metritis) on the number of COC or their subsequent development. Reduced cleavage rates were observed in COC retrieved from cows slaughtered at 80 d postpartum, but not at 165 d postpartum, and this reduction was associated with a vaginal microbiome indicative of uterine disease at 4 to 5 wk postpartum. Regression analyses that included plasma haptoglobin or energy metabolite concentrations or uterine bacterial genera abundance did not explain a large percentage of the variation in oocyte development in vitro. We conclude that there is an effect of uterine disease at one month postpartum on the oocyte and its capacity for development (Exp. 1) and this effect may be present at 80 d postpartum (Exp. 2). In later postpartum cows (165 d postpartum; Exp. 2) there was no effect of uterine disease on in vitro oocyte development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/636469v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@7ab5d7org.highwire.dtl.DTLVardef@3845e3org.highwire.dtl.DTLVardef@743f52org.highwire.dtl.DTLVardef@1e95c38_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIOocytes collected at 1 month postpartum from dairy cows with endometritis (purulent uterine lumen) had a lower cleavage rate following in vitro fertilization when compared with oocytes collected from healthy cows (Exp. 1). C_LIO_LIOocytes collected at approximately 80 d postpartum from dairy cows with evidence of uterine disease at 4 to 5 wk postpartum had a lower cleavage rate following in vitro fertilization but this disease-associated difference was not observed when oocytes were collected from cows later postpartum (approximately 165 d postpartum; Exp. 2). C_LIO_LIRegardless of the study (Exp. 1 or 2), uterine disease primarily affected the percentage of oocytes that cleaved after fertilization. C_LIO_LIStatistical associations between circulating metabolites or relative abundance of uterine bacteria were either not significant or explained only a small percentage of the variation in the in vitro embryo development in either experiment. C_LI

physiology↗

bioRxiv2024

Modeling the effect of daytime duration on the biosynthesis of terpenoid precursors

Terpenoids are valued chemicals in the pharmaceutical, biotechnological, cosmetic, and biomedical industries. Biosynthesis of these chemicals relies on polymerization of Isopentenyl di-phosphate (IPP) and/or dimethylallyl diphosphate (DMAPP) monomers, which plants synthesize using a cytosolic mevalonic acid (MVA) pathway and a plastidic methyleritritol-4-phosphate (MEP) pathway. Circadian regulation affects MVA and MEP pathway activity at three levels: substrate availability, gene expression of pathway enzymes, and utilization of IPP and DMAPP for synthesizing complex terpenoids. There is a gap in understanding the interplay between the circadian rhythm and the dynamics and regulation of the two pathways. In this paper we create a mathematical model of the MVA and MEP pathways in plants that incorporates the effects of circadian rhythms. We then used the model to investigate how annual and latitudinal variations in circadian rhythm affect IPP and DMAPP biosynthesis. We found that, despite significant fluctuations in daylight hours, the amplitude of oscillations in IPP and DMAPP concentrations remains stable, highlighting the robustness of the system. We also examined the impact of removing circadian regulation from different parts of the model on its dynamic behavior. We found that regulation of pathway substrate availability alone results in higher sensitivity to daylight changes, while gene expression regulation alone leads to less robust IPP/DMAPP concentration oscillations. Our results suggest that the combined circadian regulation of substrate availability, gene expression, and product utilization, along with MVA- and MEP-specific regulatory loops, create an optimal operating regime. This regime maintains pathway flux closely coupled to demand and stable across a wide range of daylight hours, balancing the dynamic behavior of the pathways and ensuring robustness in response to cellular demand for IPP/DMAPP.

systems biology↗

bioRxiv2024

3D micropatterned traction force microscopy: a technique to control three-dimensional cell shape while measuring cell-substrate force transmission.

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2-dimensional (2D) substrates, but not for cells in more physiological three-dimensional (3D) settings. Here, we demonstrate a technique called 3D micropatterned traction force microscopy (3D-TFM) to confine cells in three-dimensional wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-TFM, we show that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. We further show that cells switch from a global contractile to extensile behaviour as their volume is reduced. Our technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

bioengineering↗

bioRxiv2024

In a nutshell: pistachio genome and kernel development

Pistachio is a sustainable nut crop with exceptional climate resilience and nutritional value. To advance pistachios as a future food source and a model system for hard-shelled fruits, we generated a chromosome-scale reference genome of the most widely grown pistachio cultivar (Pistacia vera Kerman) and a spatiotemporal developmental study of the hull, shell, and kernel. Our study defined four distinct stages of pistachio growth and maturation by integrating tissue-level physiological and molecular data from thousands of nuts across twenty-four time points over three growing seasons. Transcriptional and metabolic changes in the kernel elucidate molecular pathways governing nutritional quality, such as the accumulation of unsaturated fatty acids, which are vital for shelf-life and dietary value. This work yields new knowledge and resources that will inform other woody crops and facilitate further improvement of pistachio as a globally significant, sustainable, and nutritious crop.

plant biology↗

bioRxiv2024

Streamlining Marker-less Allelic Replacement in Streptococcus pneumoniae Through a Single Transformation Step Strategy: easyJanus

The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic-exchange predominantly using the kanR-rpsL cassette known as "Janus". The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target genes flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our approach involves the integration of an additional gene downstream region upstream of the Janus cassette, resulting in a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. IMPORTANCEWe introduce a groundbreaking strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings, while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.

microbiology↗