Biology subjects

Hurtado, R.

Publications and source records attributed to Hurtado, R..

6 recordsLinked to original sources

bioRxiv2025

Restoration of Spermatogenesis is Dependent on Activation of a SPRY4-ERK Checkpoint Following Germline Stem Cell Damage

Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury in adult mice, germline-specific Spry4 gene deletion (Spry4G-KO) reduces stem cell regeneration with an enhanced genotoxic stress response and differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs). Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor Rapamycin, was sufficient to promote spermatogonial proliferation in Spry4G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, reducing spermatogonial genome integrity, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.

cell biology↗

bioRxiv2024

Atacama Clear for Complex 3D Imaging of Organs

3-dimensional (3D) imaging is a powerful tool for interrogation of intact tissues, but can suffer from poor resolution due to impediments such as high tissue autofluorescence that remains a significant challenge in imaging cleared samples, including human clinical specimens. We developed Atacama Clear (ATC), a 3D imaging technology that increases signal-to-noise ratios (SNRs) while simultaneously augmenting the capacity of tissue to be cleared. ATC exhibited SNRs that are up to 200% of widely used 3D imaging methods, potentiated all tested optical clearing solutions by up to 600%, decreased the time of optical clearing by up to a factor of 8, and enabled detection of poorly recognized antigens with a remarkable 4-fold increase in signal detection while using up to 10-fold lower antibody concentrations. Strikingly, ATC produced up to a 5x increase in transgenic fluorescent reporter protein signal detection, which is instead often diminished with currently used 3D imaging methods. This increased imaging efficacy enabled multiplex interrogation of tough fibrous tissue and specimens that naturally exhibit high levels of background noise, including the heart, kidney, and human biopsies. Indeed, ATC facilitated the use of AI based auto-segmentation with simple low tech stereo fluorescence microscopy, visualization of previously undocumented adjacent nephron segments that exhibit notoriously high autofluorescence, elements of the cardiac conduction system, and distinct human glomerular tissue layers, with cellular resolution. Taken together, these studies establish ATC as a platform for complex 3D imaging studies of basic and clinical specimens with superior resolution.

molecular biology↗

bioRxiv2023

Three-dimensional Isotropic Imaging of Live Suspension Cells Enabled by Droplet Microvortices

Three-dimensional (3D) imaging of non-adherent cells in suspension media is challenging due to their propensity to drift when not fixed to a substrate, as required by optical sectioning technologies. Resolution differences in the lateral versus depth directions typically present in those systems further complicates single-cell morphometry of cellular features indicative of effector functions, such as cytosol and organelle volumetric distribution, and cell membrane topography. Here, we present a method for 3D fluorescent isotropic imaging of live, non-adherent single cells encapsulated in picoliter droplets using Optical Projection Tomography (OPT) enabled by droplet microvortices. Our microfluidic platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intra-droplet microvortices, which in turn are modulated to rotate single-cells on their axis to enable OPT-based imaging. This strategy allows observation of cells encapsulated inside non-toxic isotonic buffer droplets and facilitates scalable OPT acquisition by the simultaneous spinning of hundreds of cells. Specifically, we demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells--small cells prone to movement in their suspended phenotype. In addition, morphometry of primary T cells under different immunological activation states allowed us to identify six distinct nuclear content distributions, which differ from the conventional 2D images depicting spheroid and bean-like nuclear shapes commonly associated with lymphocytes. This Arrayed-Droplet Optical Projection Tomography (ADOPT) technology is capable of isotropic, single live-cell 3D imaging and has the potential to perform large-scale morphometry of immune cell effector function states, while providing compatibility with microfluidic droplet operations.

biophysics↗

bioRxiv2022

An evolutionarily conserved pacemaker role for HCN ion channels in smooth muscle

Although HCN ion channels are well established to underlie cardiac pacemaker activity, their role in smooth muscle organs remains controversial. HCN expressing cells are localized to renal pelvic smooth muscle (RPSM) pacemaker tissues of the murine upper urinary tract and HCN channel conductance is required for peristalsis. To date, however, the Ih pacemaker current conducted by HCN channels has never been detected in these cells, raising questions on the identity of RPSM pacemakers. Indeed, the RPSM pacemaker mechanisms of the unique multicalyceal upper urinary tract exhibited by humans remains unknown. Here, we developed immunopanning purification protocols and demonstrate that 96% of isolated HCN+ cells exhibit Ih. Single molecule STORM to whole-tissue imaging showed HCN+ cells express single HCN channels on their plasma membrane and integrate into the muscular syncytium. By contrast, PDGFR-+ cells exhibiting the morphology of ICC gut pacemakers were shown to be vascular mural cells. Translational studies in the homologous human and porcine multicalyceal upper urinary tracts showed that contractions and pacemaker depolarizations originate in proximal calyceal RPSM. Critically, HCN+ cells were shown to integrate into calyceal RPSM pacemaker tissues, and HCN channel block abolished electrical pacemaker activity and peristalsis of the multicalyceal upper urinary tract. Cumulatively, these studies demonstrate that HCN ion channels play a broad, evolutionarily conserved pacemaker role in both cardiac and smooth muscle organs and have implications for channelopathies as putative etiologies of smooth muscle disorders.

physiology↗

bioRxiv2020

Genomic diversity, population structure and accessory genome analysis of Pasteurella multocida: New Insights into host adaptation and disease specialization

Pasteurella multocida is a multi-host pathogen that infects a wide spectrum of domestic and wild animals including humans. Despite its impact on health and economics, P. multocida is considered an enigmatic pathogen and the genetic basis of its pathogenicity and host adaptation still remains unclear. Here we present a detailed genomic framework based on 336 whole-genome sequences of P. multocida isolates from different animal species and countries. Our data provide genomic support of the existence of two very divergent phylogroups (PmI and PmII), which present a barrier to homologous recombination suggesting genetic isolation. Additionally, a torCAD operon, which reduces TMAO (trimethylamine N-oxide) to produce energy during bacterial anaerobic respiration, is present only in PmI and can act as a hypothetical driver of niche segregation between phylogroups. The PmI phylogroup harbors strains that infect a wider range of hosts than PmII, and shows a highly diverse phylogeny and accessory genome. We identified nine clonal lineages for PmI, seven of which are associated with specific hosts or diseases and contain distinct accessory gene pools that can confer ecologically relevant phenotypes. We found differential presence of a trehalose metabolism operon in the bovine lineage associated with pneumonic pasteurellosis; while, citrate, L-arabinose, L-fucose, and D-allose operons are only present in avian lineages. These findings suggest that alternative metabolic pathways may facilitate the establishment of P. multocida during host colonization in the early stages of infection promoting the adaptation of P. multocida lineages to certain hosts.

microbiology↗

bioRxiv2020

Taxonomic classification of strain PO100/5 shows a broader geographic distribution and genetic markers of the recently described Corynebacterium silvaticum

The bacterial strain PO100/5 was isolated from a skin abscess of a pig (Sus scrofa domesticus) in the Alentejo region of southern Portugal. It was identified as Corynebacterium pseudotuberculosis using biochemical tests, multiplex PCR and Pulsed Field Gel Electrophoresis. After genome sequencing and rpoB phylogeny, the strain was classified as C. ulcerans. To better understand the taxonomy of this strain and improve identification methods, we compared strain PO100/5 to other publicly available genomes from the C. diphtheriae group. Taxonomic analysis reclassified it and three others strains as belonging to the recently described C. silvaticum, which have been isolated from wild boar and roe deer in Germany and Austria. The results showed that PO100/5 is the first sequenced genome of a C. silvaticum strain from a domestic animal and a different geographical region, is a putative producer of the diphtheriae toxin, and has a unique sequence type. Genomic analysis of PO100/5 showed four prophages and eight conserved genomic islands when compared to C. ulcerans. Pangenome analysis of 38 C. silvaticum and 76 C. ulcerans samples suggest that C. silvaticum is a clonal species, with 73.6% of conserved genes and a pangenome near to being closed ( > 0.952). 172 conserved genes are unique to C. silvaticum when compared to C. ulcerans, with most related to nutrient uptake and metabolism, prophages or immune evasion. These unique genes could be used as genetic markers for species identification. This information can be useful for identification and surveillance of this pathogen, especially in regard to the possibility of zoonotic transmission.

genomics↗