Biology subjects

Hernandez, J. A.

Publications and source records attributed to Hernandez, J. A..

9 recordsLinked to original sources

bioRxiv2026

Asymmetric condensin loop extrusion is regulated by RPA-coated single-stranded DNA in quiescent cells

SMC complexes influence virtually all DNA-dependent processes by organizing the genome via the process of loop extrusion. Although transcription has been implicated in regulating SMC complex function, the underlying mechanisms remain unclear. Further, the directionality of loop extrusion observed in biochemical experiments has been difficult to reconcile with the chromatin condensation observed in cells. Here, we use a quiescent yeast model to uncover the relationship between condensin loop extrusion and transcription. Condensin gradually relocates to transcribed gene promoters during quiescence entry, allowing us to dissect condensin targeting mechanisms temporally. Through targeted degradation experiments, we discover that topological stress generated by transcription leads to single-stranded DNA accumulation at promoters, and that these RPA-bound regions are loading sites and extrusion barriers for condensin. We further use a condensin mutant to determine that condensin extrudes loops asymmetrically in cells. We propose that antagonism by RPA universally regulates SMC complex function.

molecular biology↗

bioRxiv2025

The structure, redox chemistry and motor neuron toxicity of heterodimeric zinc-deficient SOD1-Implications for the toxic gain of function observed in ALS.

A subset of familial cases of amyotrophic lateral sclerosis (fALS) are caused by mutations to copper, zinc superoxide dismutase (Cu, Zn SOD1). There are over 200 mutations to SOD1 that have been associated with fALS and the majority of these mutations are dominantly inherited. Thus, individuals are heterozygous and express both wild-type SOD1 and the mutant form of the protein. Paradoxically, when rodent models are produced that mimic the co-expression of wild-type SOD1 with mutant fALS SOD1 the motor neuron disease accelerates. Previously, we have shown that the loss of zinc from an SOD1 kills cultured motor neurons due to a gained, redox activity catalyzed by the active-site copper. Furthermore, motor neuron toxicity of zinc-deficient SOD1 is enhanced by wild-type Cu, Zn SOD1. Because SOD1 exists as a non-covalent dimer, the enhanced toxicity might result from stabilization of the heterodimeric interface between zinc-deficient SOD1 and Cu, Zn-SOD1. However, experimentation with the heterodimer is difficult because SOD1 subunits exchange in minutes. To better characterize the role of dimer stabilization on the enhanced toxicity of fALS mutant SOD1 by wild type SOD1, we genetically tethered a zinc-deficient SOD1 subunit with a Cu, Zn SOD1 subunit with a 16-residue linker. The x-ray structure of the tethered heterodimer shows that zinc-deficient subunit adopts a wild-type-like conformation and is not misfolded. The heterodimer intermediate also produced peroxynitrite from nitric oxide, and the tethered SOD1 was strikingly toxic to primary cultures of motor neurons. This work supports the concept that zinc-deficient SOD1 is a likely toxic intermediate in ALS. Furthermore, the wild-type allele in human familial-SOD1 ALS patients may physically contribute to the dominant inheritance of SOD1 mutations through heterodimer formation.

biochemistry↗

bioRxiv2025

Mechanical conditions preventing live cell extrusion during primary neurulation in amniotes

During primary neurulation in amniote embryos, the neural plate gives rise to the neural tube in a process requiring the coordination of forces at different scales throughout a geometrically complex tissue. The ways in which this process fails inform us of the complex mechanical conditions required for its correct completion. Previous results showed that the functional disruption of MARCKS, a protein which simultaneously interacts with the plasma membrane and actin filaments, resulted in neural tube closure defects with apical cell extrusion. Here, we demonstrate that this is an example of "live cell extrusion", wherein extruded cells are not undergoing apoptosis. This suggests that extrusion in this case might be due to a mechanical instability in the neural plate. Using an expanded energy-based vertex model of pseudostratified epithelia we then show that extrusion may be elicited by a reduction in the relative surface tension of apical and basal interfaces with respect to cell-cell interfaces. Finally, by considering a continuum description of a simplified epithelium we derive an approximate quantitative threshold for single-layered epithelial stability in the form of a power law relating cell density to the relative value of interfacial surface tensions. Our work serves to explain an example of how alterations in polarization and forces at the single-cell level can produce tissue-scale instabilities which not only greatly alter its morphology but can also ultimately lead to severe developmental defects. HighlightsO_LIPMA treatment induces live apical cell extrusion in the chick embryo neural plate C_LIO_LIOur energy-based vertex model mechanically simulates a pseudostratified epithelium C_LIO_LIHigher apical/basal compared to lateral surface tension leads to epithelial stability C_LIO_LILower apical/basal surface tension leads to epithelial instability and cell extrusion C_LIO_LIA power law between cell density and surface tension defines the stability threshold C_LI

developmental biology↗

bioRxiv2025

Integrating microfluidic automation into thermoplastic devices for analysis of small volumes of blood

Microfluidic devices hold considerable promise for miniaturizing and automating laboratory scale workflows involving sample preparation and biomarker analysis. By-and-large microfluidic automation has been implemented using polydimethyl siloxane (PDMS) - a material that presents challenges for scalable manufacturing. In this paper, we fabricated an automated microfluidic device composed of thermoplastic material, poly (methyl methacrylate) (PMMA), and demonstrated a sophisticated workflow consisting of plasma extraction, sample aliquoting and sample-reagent mixing steps performed in the device. Automation was accomplished using multiple on-board microvalves consisting of a pressure sensitive adhesive (PSA) membrane sandwiched between PMMA flow and control layers. In addition to novel microvalves, our device included features designed to address challenges arising from the gas impermeable nature of PMMA. These features included a bubble trap and air micro-tanks to supply O2 for enzymatic reactions. We rigorously characterized the performance of the microfluidic device and demonstrated that the quality of plasma extracted in the device is comparable to a standard plasma isolation method involving centrifugation. We also showed close agreement between glucose levels measured in the device and by standard assay. As a final demonstration of utility, we loaded 5 {micro}L of blood, isolated plasma and detected three analytes simultaneously - glucose, lactate and H2O2. In addition to minimizing the volume of blood and eliminating standard laboratory equipment (e.g., centrifuge), our device enabled a rapid response (<10 min). The use of thermoplastic materials may pave the way for inexpensive, mass-produced automated microfluidic devices for rapid detection of blood biomarkers at the point of care.

bioengineering↗

bioRxiv2025

Pleiotropic functions of an antenna-specific odorant binding protein linking xenobiotic adaptation and olfaction in the Colorado potato beetle, Leptinotarsa decemlineata

The Colorado potato beetle, Leptinotarsa decemlineata, is the primary defoliator of potatoes and is notorious for its robust capability to develop resistance to various insecticides used to control it. As the initial interface between the environment and the insect olfactory system, odorant binding proteins (OBPs) solubilize and transport hydrophobic odorant molecules from the sensillar lymph to olfactory receptors. Recently, evidence has suggested that OBPs may also sequester excess harmful molecules such as insecticides in the perireceptor space, preventing them from reaching vulnerable olfactory sensory neuronal dendrites. In this study, we identified an antenna-specific OBP (LdecOBP33) that is 2.5-fold higher expressed in a neonicotinoid resistant strain than the susceptible one. Competitive displacement fluorescence binding assays revealed that LdecOBP33 protein binds to a wide range of compounds including various plant volatiles and insecticides. Next, we used RNA interference to knock down LdecOBP33 and combined with toxicological bioassays, behavioral, and electrophysiological assays to investigate functions of LdecOBP33 in insecticide resistance and host location. We found that LdecOBP33 silencing increased male beetles susceptibility to imidacloprid, suggesting its role in insecticide resistance. Additionally, LdecOBP33 aids in host location through enhancing the detection of stress-induced potato plant volatiles, increasing the efficacy of CPB adult foraging. Taken together, our study is the first to functionally characterize an OBP in CPB linked to insecticide resistance and host location. Our findings provide insight into a key molecular factor involved in CPBs response to environmental challenges, suggesting a potential link between insect adaptation to xenobiotics and olfactory processing.

molecular biology↗

bioRxiv2020

Silencing NADPH-Cytochrome P450 reductase affects imidacloprid susceptibility, fecundity, and embryonic development in Leptinotarsa decemlineata

The Colorado potato beetle (CPB) is a prominent insect pest of potatoes, tomatoes and eggplants all over the world, however, the management of CPB remains a challenging task for more than one hundred years. We have successfully developed bacteria-expressed dsRNA-mediated feeding RNA interference (RNAi) approach in our previous study. A critical step towards field management of CPB via feeding RNAi is to identify effective and environmentally safe target genes. NADPH-Cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The full length Leptinotarsa decemlineata CPR (LdCPR) cDNA was isolated from an imidacloprid resistant population. The LdCPR gene was ubiquitously expressed in all stages tested but showed an increase in expression during the early stage of embryonic development. The bacteria-expressed dsRNA-mediated feeding RNAi of LdCPR in adults caused systemic knock down expression of the gene coding for LdCPR in both adults and their eggs. Suppression of LdCPR expression increased susceptibility of imidacloprid in resistant beetles, as well as a significant decrease of fecundity in female beetles (29% less eggs/day) and the hatching rate (47%) of their eggs. These data suggest that LdCPR plays important roles in insecticide detoxification and biosynthetic pathways of endogenous compounds and may serve as an essential target to control CPB. HIGHLIGHTSO_LIHigh expression of LdCPR was observed in the egg stage. C_LIO_LISilencing of LdCPR reduced the CPR enzymatic activities. C_LIO_LILdCPR knockdown increased imidacloprid susceptibility. C_LIO_LILdCPR knockdown decreased the fecundity and enhanced embryonic lethality. C_LI

pharmacology and toxicology↗

bioRxiv2018

Mutations in the zebrafish hmgcs1 gene reveal a novel function for isoprenoids during red blood cell development.

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is one potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wildtype siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal two novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.\n\nKey PointsO_LIThe products of the cholesterol synthesis pathway regulate red blood cell development during primitive erythropoiesis.\nC_LIO_LIIsoprenoids regulate erythropoiesis by modulating the expression of the GATA1 transcription factor.\nC_LI

developmental biology↗

bioRxiv2017

The effect of abiotic and biotic stress on the salicylic acid biosynthetic pathway from mandelonitrile in peach

HighlightWe show that the recently suggested third pathway for SA biosynthesis from mandelonitrile in peach is also functional under both abiotic and biotic stress conditions.\n\nAbstractSalicylic acid (SA) plays a central role in plant responses to environmental stresses via the SA-mediated regulation of many metabolic and molecular processes. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, we show that this new SA biosynthetic pathway is also functional under abiotic (salt) and biotic (Plum pox virus infection) stress conditions, although the contribution of this pathway to the SA pool does not seem to be important under such conditions. Treating peach plants with MD not only affected the SA content, but it also had a pleiotropic effect on abscisic acid and jasmonic acid levels, two well-known stress related hormones, as well as on the H2O2-related antioxidant activities. Furthermore, MD improved plant performance under the stressful conditions, probably via the activation of different signaling pathways. We have thus proven that SA is not limited to biotic stress responses, but that it also plays a role in the response to abiotic stress in peach, although the physiological functions of this new SA biosynthetic pathway from MD remain to be elucidated.\n\nAbbreviations

plant biology↗

bioRxiv2017

Metabolomics and biochemical approaches link salicylic acid biosynthesis to cyanogenesis in peach plants

HighlightMandelonitrile, and hence cyanogenic glycosides turnover, is involved in salicylic acid (SA) biosynthesis in peach plants under control and stress conditions. A third pathway for SA synthesis in peach is proposed.\n\nAbstractDespite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathway has not been fully characterized. The proposed SA synthesis originates from chorismate by two distinct pathways: isochorismate and penhylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between both pathways. In this work, by metabolomics and biochemical approaches (including [13C]-labelled compounds), we provide evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions.\n\nThe main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from Phe, controlling their turnover. In peach plants MD is at the hub of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating both the amygdalin and SA biosynthesis. MD-treated peach plants displayed increased SA levels via benzoic acid (SA precursor). In addition, MD also provides partial protection against Plum pox virus infection in peach seedlings. Thus, we proposed a third pathway, alternative to the PAL pathway, for SA synthesis in peach plants.

physiology↗