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Rodriguez, K.

Publications and source records attributed to Rodriguez, K..

10 recordsLinked to original sources

Single-cell learning in Stentor coeruleus is governed by a fractional-order low-pass filter

Single cells display a range of complex behaviors normally associated with a nervous system, including basic forms of learning like habituation. The giant ciliate Stentor coeruleus habituates to mechanical stimuli and shows many of the hallmark features characteristic of habituation in animal cells. When Stentor cells are mechanically stimulated by a predator or other stimuli, an action potential fires and leads to calcium-dependent contraction. When the same cell is repeatedly stimulated, it becomes less likely to respond, thus showing habituation. While the molecular basis of habituation in Stentor is not yet known, it has been shown to involve CaMKII, which also plays a key role in learning in neurons. The presence of an action potential, the role of calcium in the response, and the involvement of CaMKII in habituation, all suggest a potential deep conservation of learning mechanisms between single-celled protists and the neurons of animals. A number of different models have been proposed to explain habituation in a single cell but existing data in Stentor are unable to clearly rule out any of these models or favor others. Here we report a frequency domain analysis of habituation in which we measure the response probability of Stentor cells to pulsatile stimuli delivered at a range of frequencies. We find that the Bode plot of the frequency response resembles a classic low pass filter, with a flat passband at low frequencies, a clear corner frequency, and a linear roll-off. However, unlike standard low pass filter, the roll-off occurs with a slope of -30dB/decade, thus showing a fractional-order behavior. None of the existing models for habituation in Stentor, at least in their current form, predict this form of the frequency response, leading us to look for other explanations. We tested, and ruled out, a model based on a refractory period associated with the re-extension of cells following contraction. Inspired by methods used in analog circuit design to approximate fractional order systems using conventional lumped devices, we developed a model in which a series of distinct molecular species, such as different multimeric complexes of CaMKII, acting in parallel to inhibit the response, produce a fractional-order effect. The fractional order behavior of habituation in Stentor resembles the fractional-order behavior of adaptation in neurons, further supporting the idea that neurons may employ similar mechanisms for learning as were already present in unicellular eukaryotes prior to the evolution of metazoa.

cell biology↗

Intermittent exposure to high ambient heat during the second half of gestation in mice causes mild alterations of reproductive endpoints in male embryos

Periods of elevated ambient temperature challenge the bodys ability to maintain internal homeostasis, and heat stress poses particular risks during pregnancy. Epidemiological studies associate gestational heat exposure with higher rates of congenital anomalies such as hypospadias, yet the direct link between gestational heat exposure and reproductive anomalies remains unknown. In this study, we examined the effects of intermittent heat exposure on reproductive development in male mouse offspring. Pregnant dams either remained at constant temperature of 22{degrees}C (control) or were exposed to 38{degrees}C for 2 hours daily (experimental) from embryonic day (E)10 to E18, modeling intermittent heat exposure during mid-to-late gestation. Embryos were collected at E18 for analysis. While heat exposure did not affect pregnancy outcomes, including placental development, litter size, sex ratio, or fetal growth, male embryos exhibited significantly reduced anogenital distance and increased hypospadias scores, which are both markers of disrupted androgen signaling. Despite these phenotypic changes, expression of genes involved in androgen synthesis in the fetal testis, as well as gene expression in external genitalia, remained unchanged. Instead, transcriptomic analysis revealed significant alterations in testicular pathways related to RNA splicing and mRNA processing. Together, these findings reveal that maternal heat stress disrupts reproductive development of male offspring, with altered gene regulatory processes being a potential driver.

developmental biology↗

Targeting the COP9 signalosome overcomes platinum resistance in ovarian cancer through two distinct genome stability mechanisms

Tubo-ovarian high-grade serous carcinoma (HGSC) is a leading cause of gynecologic cancer mortality, largely due to the emergence of platinum resistance, which serves as the mainstay of chemotherapy. Here, we identify COPS5 as a therapeutic target and use an available small molecule inhibitor to overcome platinum resistance. A genetic screen for platinum-induced DNA damage in a platinum resistant ovarian cancer model identified COPS5 and COPS6, two components of the COP9 signalosome. Consistently, high COPS5 expression correlated with poor clinical outcomes in patients with HGSC. In both in vitro and in vivo experiments, COPS5 depletion sensitized ovarian cancer cells to carboplatin. A small molecule COPS5 inhibitor, CSN5i-3, synergized with carboplatin in homologous recombination-deficient and -proficient cells. This combination was also effective in xenografts and in a syngeneic mouse model of carboplatin-resistant HGSC. Importantly, we demonstrate that CSN5i-3 is selective for cancer cells, with patient-derived HGSC cells exhibiting up to 50-fold greater sensitivity to CSN5i-3 than benign cells. Finally, we show that genetic or small molecule inhibition of COPS5 impaired both nucleotide excision repair (NER) and interstrand crosslink (ICL) repair, leading to increased DNA platinum adducts. Mechanistically, this was due to increased ubiquitination and degradation of DNA-specific DNA binding protein 1 (DDB1) and other key NER and ICL repair proteins, consistent with the role of COPS5 in the regulation of these factors. Our findings highlight the importance of NER and ICL regulation in chemotherapy response and indicate that targeting COPS5 can enhance the efficacy of platinum-based chemotherapy in HGSC. One Sentence SummaryCOPS5 depletion or inhibition using a small molecule COPS5 inhibitor CSN5i-3 sensitizes high-grade serous carcinoma to platinum chemotherapy through downregulation of nucleotide excision repair and interstrand crosslink repair.

cancer biology↗

Single-nucleus multiomics of murine gonadal cells reveals transcriptional regulatory network underlying supporting lineage differentiation

Sex determination of mammalian gonads hinges upon sex-specific differentiation of gonadal supporting cells: Sertoli cells in the testis and granulosa cells in the ovary. To gain insights into how supporting cells acquire their identities, we performed joint single-nucleus transcriptomics and chromatin accessibility assays on murine gonadal cells across sex determination. By contrasting sex-specific gene expression and corresponding chromatin accessibility among progenitor and differentiated cells, we discovered that sex-specific chromatin regions in supporting cells are established shortly after sex determination, accompanied by the acquisition of active histone marks. The presence of potential transcription factor binding motifs in the open chromatin regions revealed regulatory networks underlying ovary-enriched factors LEF1 and MSX1, which promote granulosa fate by inducing granulosa-specific genes such as Foxl2 and Fst. Our results not only identify the gene regulatory framework underlying supporting cell sex differentiation, but also provide invaluable resources for the field. TeaserSex-specific changes in chromatin and gene expression underly the divergent developmental fates of the bipotential gonadal primordium.

developmental biology↗

Replication stress marker phospho-RPA2 predicts response to platinum and PARP inhibitors in homologous recombination-proficient ovarian cancer

BackgroundOvarian cancer treatment includes cytoreductive surgery, platinum-based chemotherapy, and often poly (ADP-ribose) polymerase (PARP) inhibitors. Homologous recombination (HR)-deficiency is a well-established predictor of therapy sensitivity. However, over 50% of HR-proficient tumors also exhibit sensitivity to standard-of-care treatments. Currently, there are no biomarkers to identify which HR-proficient tumors will be sensitive to standard-of-care therapy. Replication stress may serve as a key determinant of response. MethodsWe evaluated phospho-RPA2-T21 (pRPA2) foci via immunofluorescence as a potential biomarker of replication stress in formalin-fixed, paraffin-embedded tumor samples collected at diagnosis from patients treated with platinum chemotherapy (discovery cohort: n = 31, validation cohort: n = 244) or PARP inhibitors (n = 87). Recurrent tumors (n = 37) were also analyzed. pRPA2 scores were calculated using automated imaging analysis. Samples were defined as pRPA2-High if > 16% of cells had [≥] 2 pRPA2 foci. ResultsIn the discovery cohort, HR-proficient, pRPA2-High tumors demonstrated significantly higher rates of pathologic complete response to platinum chemotherapy than HR-proficient, pRPA2-Low tumors. In the validation cohort, patients with HR-proficient, pRPA2-High tumors had significantly longer survival after platinum treatment than those with HR-proficient, pRPA2-Low tumors. Additionally, the pRPA2 assay effectively predicted survival outcomes in patients treated with PARP inhibitors and in recurrent tumor samples. ConclusionOur study underscores the importance of considering replication stress markers alongside HR status in therapeutic planning. Our work suggest that this assay could be used throughout a patients treatment course to expand the number of patients receiving effective therapy while reducing unnecessary toxicity.

cancer biology↗

Age-maintained human neurons demonstrate a developmental loss of intrinsic neurite growth ability

Injury to adult mammalian central nervous system (CNS) axons results in limited regeneration. Rodent studies have revealed a developmental switch in CNS axon regenerative ability, yet whether this is conserved in humans is unknown. Using human fibroblasts from 8 gestational-weeks to 72 years-old, we performed direct reprogramming to transdifferentiate fibroblasts into induced neurons (Fib-iNs), avoiding pluripotency which restores cells to an embryonic state. We found that early gestational Fib-iNs grew longer neurites than all other ages, mirroring the developmental switch in regenerative ability in rodents. RNA-sequencing and screening revealed ARID1A as a developmentally-regulated modifier of neurite growth in human neurons. These data suggest that age-specific epigenetic changes may drive the intrinsic loss of neurite growth ability in human CNS neurons during development. One-Sentence Summary: Directly-reprogrammed human neurons demonstrate a developmental decrease in neurite growth ability.

neuroscience↗

Glutathione supports lipid abundance in vivo

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the livers balance of redox buffering and triglyceride production.

biochemistry↗

YAP promotes cell-autonomous immune responses to tackle intracellular Staphylococcus aureus in vitro

Transcriptional cofactors YAP/TAZ have recently been found to support autophagy and inflammation, which are part of cell autonomous immunity and are critical in antibacterial defense. Here, we studied the role of YAP against Staphylococcus aureus using CRISPR/Cas9-mutated HEK293 cells and a primary cell-based organoid model. We found that S. aureus infection increases YAP transcriptional activity, which is required to reduce intracellular S. aureus replication. A 770-gene targeted transcriptomic analysis revealed that YAP upregulates genes involved in autophagy/lysosome and inflammation pathways in both infected and uninfected conditions. The YAP/TEAD transcriptional activity promotes autophagic flux and lysosomal acidification, which are important for defense against intracellular S. aureus. Furthermore, the staphylococcal toxin C3 exoenzyme EDIN-B was found effective in preventing YAP-mediated cell-autonomous immune response. This study provides new insights on the anti-S. aureus activity of YAP, which could be conserved for defense against other intracellular bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/492111v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@6158f1org.highwire.dtl.DTLVardef@1164aedorg.highwire.dtl.DTLVardef@911558org.highwire.dtl.DTLVardef@11044b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

NMR spectroscopy of a single mammalian embryo

The resolving power, chemical sensitivity and non-invasive nature of NMR has made it an established technique for in vivo studies of large organisms both for research and clinical applications. These features would clearly be beneficial at the nanoliter scale (nL), typical of early development of mammalian embryos, microtissues and organoids, the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 micrometers) and sensitivity issues have prevented the widespread adoption of NMR. Recently we have shown how these limitations can be overcome with ultra-compact single-chip probes. In this article we show that such probes have sufficient sensitivity to detect and resolve NMR signals from individual bovine pre-implantation embryos. In less than 1 hour these spherical samples of just 130-190 micrometers produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectral features, namely the peak intensities, vary from one sample to another despite their optical and morphological similarities.

biochemistry↗

Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening

ContextSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SRAS-CoV-2. MethodsVero E6 cells were treated with copper gluconate 18 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were maintained in fresh medium containing copper gluconate for an additional 48-hour period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT assay. ResultsThe viability of Vero E6 cells treated with copper gluconate up to 200 M was found to be similar to that of untreated cells, but it dropped below 40% with 400 M of this agent. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3%,5.2% in cells treated with 0, 2, 10, 25, 50 and 100 M of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 M of copper gluconate respectively (p < 0.05). ConclusionCopper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells. Furthers studies are needed to determine whether copper homeostasis could play a role in SARS-CoV-2 infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/422548v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f99949org.highwire.dtl.DTLVardef@1bebae6org.highwire.dtl.DTLVardef@e05e37org.highwire.dtl.DTLVardef@49a3b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗