Biology subjects

Munoz, A.

Publications and source records attributed to Munoz, A..

At least 19 recordsLinked to original sources

bioRxiv2025

Passive Acoustic Monitoring within the Northwest Forest Plan Area: 2024 Annual Report

Passive acoustic monitoring (PAM) has become the primary framework for assessing the status, distribution, and habitat associations of forest-dependent wildlife under the Northwest Forest Plan (NWFP). In 2024, the NWFP PAM program continued to operate as a fully integrated, range-wide monitoring network, building on deployments initiated in 2018 and now conducting surveys across approximately 24 million acres of federal land. Using a randomized 2+20% sampling design, more than 4,000 autonomous recording units generated over 2 million hours of acoustic data analyzed with convolutional neural networks, enabling statistically rigorous inference at regional and range-wide scales. This report summarizes 2024 field efforts and analytical advancements in the context of recent peer-reviewed and in-progress studies. Updated analyses confirm persistent, low occupancy of northern spotted owls (Strix occidentalis caurina), with detections concentrated in the Klamath Mountains and southern Cascades, consistent with long-term demographic declines. Range-wide models of barred owl (Strix varia) landscape use continue to show high occupancy across much of the NWFP area, reinforcing competitive displacement as the dominant driver of spotted owl range contraction. The PAM network also advanced monitoring of marbled murrelets (Brachyramphus marmoratus), with 2024 results supporting PAM as a cost-efficient alternative to traditional audio-visual surveys for assessing inland nesting habitat use in coastal forests. Beyond focal species, the program increasingly supports multi-taxa biodiversity monitoring through bioacoustic bycatch and soundscape analyses, linking species occurrence, habitat condition, and disturbance dynamics. Collectively, 2024 results demonstrate that PAM has reached operational maturity as the cornerstone of Phase II NWFP effectiveness monitoring. Sustaining adequate sampling effort and field capacity remains a challenge but essential, as reduced deployment intensity would increase uncertainty and weaken inference. Continued investment in PAM infrastructure and expertise is critical for informing adaptive management, supporting sustainable timber harvest, and evaluating ecosystem resilience under changing disturbance regimes.

ecology↗

bioRxiv2025

Age-dependent chemotherapy response and vitamin D impact in paired patient-derived normal and tumor colorectal organoids

Early-onset colorectal cancer (EO-CRC, <50 years) incidence is rising worldwide. Using paired patient-derived colorectal normal and tumor organoids, we demonstrate the therapeutic window of three standard chemotherapies. Normal organoids were more resistant than tumor organoids to 5-fluorouracil (5-FU) and oxaliplatin in both EO-CRC and late-onset CRC (LO-CRC) patients. However, for SN38, this therapeutic window was observed in LO-CRC but not in EO-CRC patients, revealing age-dependent differences in drug resistance. We also evaluated the effect of calcitriol, the active vitamin D metabolite with anti-CRC activity, on drug sensitivity. Calcitriol reduced the cytotoxicity of 5-FU and SN38 in normal organoids regardless of patient age, while it selectively decreased their cytotoxicity in EO-CRC but not in LO-CRC tumor organoids. This protective effect correlated with calcitriol antiproliferative action and transcriptional effects on drug-metabolism pathways. These findings identify clinically relevant age-dependent differences in chemotherapy response and support vitamin D-based strategies to design age-tailored precision treatments.

cancer biology↗

bioRxiv2025

Targeting Tmem63b and Piezo2 in C-fiber low threshold mechanoreceptor: limitation of Vglut3-IRES-Cre

Peripheral somatosensory neurons in the dorsal root ganglia (DRG) transduce mechanical force in the skin and other organs into electrical signals using specialized mechanically activated (MA) ion channels that initiate neuronal activation in response to force. Increasing evidence highlights PIEZO2 as the primary transducer of low-threshold mechanical force in DRG neurons. However, in the absence of Piezo2, mice and humans still respond to noxious painful stimuli like pinch, suggesting that additional MA channel(s) likely exist in DRG neurons. Strategies to identify Cre lines and DRG subpopulations that select for non-PIEZO2 expressing neurons is therefore an ongoing effort in the field to discover unknown mechanosensors. Here, we investigated a Vglut3 labeled mouse line as a candidate to identify non-PIEZO2 MA channels in a subtype of DRG neurons called C-fiber low threshold mechanoreceptors (C-LTMRs). Our study carefully demonstrates that the Vglut3-IRES-Cre mouse line specifically and efficiently labels C-LTMR neurons of the DRG. Electrophysiological recordings using two different in vitro mechanical stimulation assays show that the genetically labelled Vglut3 neurons have robust indentation- and stretch-activated MA currents that are exclusively slowly or ultra-slowly adapting. To determine whether the Vglut3-IRES-Cre mouse line can be used to delete genes of interest and identify the underlying MA ion channels in C-LTMRs we attempted to generate a Tmem63b conditional knockout using this Cre-line but detected incomplete loss of Tmem63b transcript and lack of TMEM63B- dependent effect on C-LTMR MA currents. Together, our results emphasize that although the Vglut3-IRES-Cre line is robust in driving expression of a conditional reporter gene, it is inefficient in deleting genes like Tmem63b as well as Piezo2. Statement of SignificanceIn this study, we identify a previously uncharacterized Vglut3-IRES-Cre as a tool to uncover unknown mechanosensors in C-LTMRs of the DRG. We methodically demonstrate that the Vglut3-IRES-Cre line labels C-LTMRs and express two mechanosensors, Tmem63b and Piezo2. However, we were unable to discern which gene contributes to the MA currents due to poor knockout efficiency induced by the Vglut3-IRES-Cre. Our studies highlight the nuances and challenges associated with Cre lines and alert future investigations to exercise caution while using Cre lines to discover the contribution of specific genes to mechanosensation.

neuroscience↗

bioRxiv2025

Sarcomeric Remodelling in Human Heart Failure unraveled by single molecule long read sequencing

Dysregulation of alternative splicing - mediated by factors such as RBM20 or SLM2 - can affect proper gene isoform control disrupts gene isoform homeostasis and underpins severe cardiomyopathy in both animal models and patients. Although innovative therapies target various sarcomeric components, the impact of isoform switching in cardiac disease remains poorly understood. Here, we applied nanopore long-read sequencing to map the full-length transcriptome of left ventricular tissue from thirteen nonfailing controls, ten patients with dilated cardiomyopathy (DCM), and ten with ischemic cardiomyopathy (ICM). Our analysis identified 78,520 transcripts, 31% of which represent novel isoforms of known genes. Notably, the transcriptomes of DCM and ICM were largely indistinguishable, indicating that end-stage heart failure is characterized by a convergent isoform landscape, irrespective of disease etiology. Among 11 prototypical sarcomere genes, 10 displayed highly significant isoform shifts (p= 5.23x10-45 - 2.89x10-200). Focusing on tropomyosin, we observed that while the predominant cardiac gene TPM1 showed moderate up-regulation of its transcript isoforms, transcripts derived from TPM3--typically expressed at lower levels in the healthy heart--were markedly increased in heart failure.

genetics↗

bioRxiv2024

TMEM63A, associated with hypomyelinating leukodystrophies, is an evolutionarily conserved regulator of myelination

Infantile hypomyelinating leukodystrophy 19 (HLD19) is a rare genetic disorder where patients exhibit reduced myelin in central nervous system (CNS) white matter tracts and present with varied neurological symptoms. The causative gene TMEM63A encodes a mechanosensitive ion channel whose role in myelination has not been explored. Our study shows that TMEM63A is a major regulator of OL-driven myelination in the CNS. In mouse and zebrafish, Tmem63a inactivation led to early deficits in myelination, recapitulating the HLD19 phenotype. OL-specific conditional mouse knockouts of Tmem63a exhibited transient reductions in myelin, indicating that TMEM63A regulates myelination cell-autonomously. We show that TMEM63A is present at plasma membrane and on lysosomes and modulates myelin/myelin-associated protein production. Intriguingly, HLD19-associated TMEM63A variants from patients blocked trafficking to cell membrane. Together, our results reveal an ancient role for TMEM63A in fundamental aspects of myelination in vivo and highlight two exciting new models for the development of treatments for devastating hypomyelinating leukodystrophies.

neuroscience↗

bioRxiv2024

Methemoglobin aggregation is modulated by the anti-sickling drug voxelotor

Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T) state. The drug voxelotor was recently approved to treat sickle cell disease by preventing HbSS fiber formation. Voxelotor acts as an allosteric inhibitor of polymerization by maintaining the HbSS protein in the relaxed (R) conformation, limiting polymerization of T-state fibers. Normal blood cells contain small amounts of natural Hb fibers and a few percent of the Fe3+ ferric form, metHb, incapable of binding oxygen. Although the drug Voxelotor is now in use, the effect of the drug on the oxidized metHb state has not been reported. Here we assessed the influence of voxelotor on normal human metHb. We compared the aggregation of metHb at two pH values (5.5 and 7.1). MetHb is known to form organized fiber structures at or below pH 5.5. We find that voxelotor significantly enhances fiber formation of metHb R-state at pH 5.5, consistent with the mode of action for this drug in maintaining the Hb R conformation. The opposite effect is observed at physiological pH values. Voxelotor significantly decreases the rate of metHb aggregate formation at pH 7.1 but did not affect protein stability. Notably, drug binding drives metHb into novel spherical particles with a morphology never seen before for Hb. The formation of these particles should be considered in patients being treated for sickle cell disease with voxelotor. WHY IT MATTERSVoxelotor is an FDA-approved drug for sickle cell anemia, known to prevent hemoglobin fiber formation. Here, we investigate its effect on methemoglobin, the form of hemoglobin in which iron takes on the ferric Fe3+ state. Our study examines voxelotors impact on methemoglobin aggregation and stability. At pH 7.1, we found voxelotor to have an effect on methemoglobin solubility as evidenced by the formation of novel methemoglobin spherical structures. We observe that voxelotor significantly increases methemoglobin fiber formation at pH 5.5 but, notably, reduces methemoglobin aggregation at physiological pH levels. Minimal impact on methemoglobin thermodynamic stability is noted. These findings suggest voxelotors potential therapeutic efficacy for various hemoglobinopathies, including conditions characterized by Heinz body formation.

biophysics↗

bioRxiv2024

The Genotype and Phenotypes in Families (GPF) platform manages the large and complex data at SFARI

The exploration of genotypic variants impacting phenotypes is a cornerstone in genetics research. The emergence of vast collections containing deeply genotyped and phenotyped families has made it possible to pursue the search for variants associated with complex diseases. However, managing these large-scale datasets requires specialized computational tools tailored to organize and analyze the extensive data. GPF (Genotypes and Phenotypes in Families) is an open-source platform (https://github.com/iossifovlab/gpf) that manages genotypes and phenotypes derived from collections of families. The GPF interface allows interactive exploration of genetic variants, enrichment analysis for de novo mutations, and phenotype/genotype association tools. In addition, GPF allows researchers to share their data securely with the broader scientific community. GPF is used to disseminate two large-scale family collection datasets (SSC, SPARK) for the study of autism funded by the SFARI foundation. However, GPF is versatile and can manage genotypic data from other small or large family collections. Our GPF-SFARI GPF instance (https://gpf.sfari.org/) provides protected access to comprehensive genotypic and phenotypic data for the SSC and SPARK. In addition, GPF-SFARI provides public access to an extensive collection of de novo mutations identified in individuals with autism and related disorders and to gene-level statistics of the protected datasets characterizing the genes roles in autism. Here, we highlight the primary features of GPF within the context of GPF-SFARI.

bioinformatics↗

bioRxiv2024

SIRT1 mediates the antagonism of Wnt/β-catenin pathway by vitamin D in colon carcinoma cells.

Cancer initiation and progression result from both genetic alterations and epigenetic reprograming caused by environmental or endogenous factors which can lead to aberrant cell signalling. Most colorectal cancers (CRC) are linked to the abnormal activation of the Wnt/ {beta}-catenin pathway, whose key feature is the accumulation of acetylated {beta}-catenin protein within the nucleus of colon epithelial cells. Nuclear {beta}- catenin acts as a transcriptional co-activator that alters the expression of many target genes involved in cell proliferation and invasion. The most active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol) can antagonize the over-activated Wnt/ {beta}-catenin pathway via binding to its high affinity receptor VDR. Here, we show that the activation of the SIRT1 deacetylase by 1,25(OH)2D3-bound VDR promotes deacetylation and nuclear exclusion of {beta}-catenin and, consequently, the downregulation of its pro-tumorigenic target genes and the inhibition of human colon carcinoma cell proliferation. Notably, orthogonal SIRT1 activation systematically drives nuclear exclusion of {beta}-catenin, highlighting the key role of SIRT1 in CRC. Since nuclear localization of {beta}-catenin is a critical driver of CRC initiation and progression that requires its acetylation, our results provide a mechanistic basis for the epidemiological evidence linking vitamin D deficiency and increased CRC risk and mortality.

cancer biology↗

bioRxiv2023

Human outperform mouse Purkinje cells in dendritic complexity and computational capacity

Purkinje cells (PC) of the cerebellum are amongst the largest neurons of the brain and have been extensively investigated in rodents. However, their morphological and physiological properties in humans are still poorly understood. Here, we have taken advantage of high-resolution morphological reconstructions and of unique electrophysiological recordings of human PCs ex vivo to generate computational models and estimate computational capacity. An inter-species comparison showed that human PCs had similar fractal structure but were bigger than mouse PCs. Consequently, given a similar spine density (2/m), human PCs hosted about 5 times more dendritic spines. Moreover, human had higher dendritic complexity than mouse PCs and usually emitted 2-3 main dendritic trunks instead than 1. Intrinsic electroresponsiveness was similar in the two species but model simulations revealed that the dendrites generated ~6.5 times (n=51 vs. n=8) more combinations of independent input patterns in human than mouse PCs leading to an exponential 2n increase in Shannon information. Thus, while during evolution human PCs maintained similar patterns of spike discharge as in rodents, they developed more complex dendrites enhancing computational capacity up to the limit of 10 billion times.

neuroscience↗

bioRxiv2023

Vitamin D induces SIRT1 activation through K610 deacetylation in colon cancer

Posttranslational modifications of epigenetic modifiers provide a flexible and timely mechanism for rapid adaptations to the dynamic environment of cancer cells. SIRT1 is an NAD+-dependent epigenetic modifier whose activity is classically associated with healthy aging and longevity, but its function in cancer is not well understood. Here, we reveal that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), the active metabolite of vitamin D (VD), promotes SIRT1 activation through auto-deacetylation in human colon carcinoma cells, and identify lysine 610 as an essential driver of SIRT1 activity. Remarkably, our data show that the post-translational control of SIRT1 activity mediates the antiproliferative action of 1,25(OH)2D3. This effect is reproduced by the SIRT1 activator SRT1720, suggesting that SIRT1 activators may offer new therapeutic possibilities for colon cancer patients who are VD deficient or unresponsive. Moreover, this might be extrapolated to inflammation and other VD deficiency-associated and highly prevalent diseases in which SIRT1 plays a prominent role.

cancer biology↗

bioRxiv2022

Neuromodulatory organization in the developing rat somatosensory cortex

The vast majority of cortical synapses are found in the neuropil which is implicated in multiple and diverse functions underlying brain computation. Unraveling the organizing principles of the cortical neuropil requires an intricate characterization of synaptic connections established by excitatory and inhibitory axon terminals, of intrinsic and extrinsic origin and from ascending projections that govern the function of cortical microcircuits through the release of neuromodulators either through point-to-point chemical synapses or diffuse volume transmission (VT). Even though neuromodulatory release has been studied for almost a century it is still not clear if one modality prevails upon the other. The hindlimb representation of the somatosensory cortex (HLS1) of two-week old Wistar rats has served as a model system to dissect the microcircuitry of neurons and their synaptic connections. In the present study, we quantified the fiber length per cortical volume and the density of varicosities for cholinergic, catecholaminergic and serotonergic neuromodulatory systems in the cortical neuropil using immunocytochemical staining and stereological techniques. Acquired data were integrated into a novel computational framework to reconcile the specific modalities and predict the effects of neuromodulatory release in shaping neocortical network activity. We predict that acetylcholine (ACh), dopamine (DA), serotonin (5-HT) release desynchronizes cortical activity by inhibiting slow oscillations (delta range), and that 5-HT triggers faster oscillations (theta). Moreover, we found that high levels (>40%) of neuromodulatory VT are sufficient to induce network desynchronization, but also that combining volume release with synaptic inputs leads to more robust and stable effects, meaning that lower levels of VT are needed to achieve the same outcome (10%).

neuroscience↗

bioRxiv2022

X chromosome dosage drives statin-induced dysglycemia and mitochondrial dysfunction

Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects, particularly new-onset diabetes (NOD) and muscle weakness. We determined that female mice are more susceptible than males to glucose intolerance, fasting hyperglycemia, and muscle weakness after short-term statin treatment. Lipidomic, transcriptomic, and biochemical analyses identified reduced docosahexaenoic acid (DHA) levels, and impaired redox tone and mitochondrial respiration specifically in statin-treated female mice. Statin adverse effects could be prevented in females by complementation with a source of DHA. Statin adverse effects segregated with XX chromosome complement, and specifically dosage of the Kdm5c gene, which regulates fatty acid gene expression and has differential expression levels in females and males. In humans, we found that women experience more severe reductions than men in DHA levels after short-term statin administration, and that DHA reduction was correlated with increases in fasting glucose levels. Furthermore, induced pluripotent stem cells derived from women, but not men, who developed NOD exhibited impaired mitochondrial function when treated with statin. Overall, our studies identify biochemical mechanisms, biomarkers, and a genetic risk factor for susceptibility to statin adverse effects, and point to DHA supplementation as a preventive co-therapy.

physiology↗

bioRxiv2022

Dexamethasone Enhances CAR T Cell Persistence and Function by Upregulating Interleukin 7 Receptor

Dexamethasone (dex) is a glucocorticoid that is a mainstay for treatment of inflammatory pathologies, including immunotherapy-associated toxicities. Dex suppresses the endogenous immune response and is also believed to suppress the function of chimeric antigen receptor (CAR) T cells. However, recent reports observed higher CAR T cell numbers in patients treated with dex, highlighting the rationale for interrogating the specific effects of dex on CAR T cells. Here, we found that dex did not inhibit CAR T cell expansion or function. A single dose of dex during the manufacturing process upregulated the pro-persistence interleukin 7 receptor (IL7R) on CAR T cells and induced expression of genes involved in activation, migration, and persistence. The ex vivo upregulation of IL7R induced by dex significantly enhanced CAR T cell persistence and anti-tumor efficacy in vivo when combined with exogenous IL-7. Moreover, the combination of dex and IL-7 resulted in increased persistence of CAR T cells and led to complete remission of mice. Overall, our studies in both in vitro and in vivo treatment support a positive role of dex on CAR T cell potency and provide insight into the application of glucocorticoids in cellular anti-cancer therapy.

immunology↗

bioRxiv2022

Waning and boosting of functional humoral immunity to SARS-CoV-2

Since the emergence of the SARS-CoV-2 virus, we have witnessed a revolution in vaccine development with the rapid emergence and deployment of both traditional and novel vaccine platforms. The inactivated CoronaVac vaccine and the mRNA-based Pfizer/BNT162b2 vaccine are among the most widely distributed vaccines, both demonstrating high, albeit variable, vaccine effectiveness against severe COVID-19 over time. Beyond the ability of the vaccines to generate neutralizing antibodies, antibodies can attenuate disease via their ability to recruit the cytotoxic and opsinophagocytic functions of the immune response. However, whether Fc-effector functions are induced differentially, wane with different kinetics, and are boostable, remains unknown. Here, using systems serology, we profiled the Fc-effector profiles induced by the CoronaVac and BNT162b2 vaccines, over time. Despite the significantly higher antibody functional responses induced by the BNT162b2 vaccine, CoronaVac responses waned more slowly, albeit still found at levels below those present in the systemic circulation of BNT162b2 immunized individuals. However, mRNA boosting of the CoronaVac vaccine responses resulted in the induction of significantly higher peak antibody functional responses with increased humoral breadth, including to Omicron. Collectively, the data presented here point to striking differences in vaccine platform-induced functional humoral immune responses, that wane with different kinetics, and can be functionally rescued and expanded with boosting.

immunology↗

bioRxiv2021

Multiscale dynamics underlying neocortical slow oscillations

Slow oscillations in the sleeping and anesthetized brain invariantly emerge as an alternation between Up (high firing) and Down (almost quiescent) states. In cortex, they occur simultaneously in cell assemblies in different layers and propagate as traveling waves, a concerted activity at multiple scales whose interplay and role is still under debate. Slow oscillations have been reported to start in deep layers, more specifically in layer 5. Here, we studied the laminar organization of slow oscillations in the anesthetized rat cortex and we found that the activity leading to Up states actually initiates in layer 6, then spreads towards upper layers. Layer 5 cell assemblies have a threshold-like activation that can persist after layer 6 inactivation, giving rise to hysteresis loops like in "flip-flop" computational units. We found that such hysteresis is finely tuned by the columnar circuitry depending on the recent history of the local ongoing activity. Furthermore, thalamic inactivation reduced infragranular excitability without affecting the columnar activation pattern. We propose a role for layer 6 acting as a hub unraveling a hierarchy of cortical loops.

neuroscience↗

bioRxiv2021

Chemotherapy induces a YAP1-dependent fetal conversion to human Colorectal Cancer cells that is predictive of poor patient outcome

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription. Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

cancer biology↗

bioRxiv2020

Stellate cell computational modelling predicts signal filtering in the molecular layer circuit of cerebellum

The functional properties of cerebellar stellate cells and the way they regulate molecular layer activity are still unclear. We have measured stellate cells electroresponsiveness and their activation by parallel fiber bursts. Stellate cells showed intrinsic pacemaking, along with characteristic responses to depolarization and hyperpolarization, and showed a marked short-term facilitation during repetitive parallel fiber transmission. Spikes were emitted after a lag and only at high frequency, making stellate cells to operate as delay-high-pass filters. A detailed computational model summarizing these physiological properties allowed to explore different functional configurations of the parallel fiber - stellate cell - Purkinje cell circuit. Simulations showed that, following parallel fiber stimulation, Purkinje cells almost linearly increased their response with input frequency but such an increase was inhibited by stellate cells, which leveled the Purkinje cell gain curve to its 4 Hz value. When reciprocal inhibitory connections between stellate cells were activated, the control of stellate cells over Purkinje cell discharge was maintained only at very high frequencies. These simulations thus predict a new role for stellate cells, which could endow the molecular layer with low-pass and band-pass filtering properties regulating Purkinje cell gain and, along with this, also burst delay and the burst-pause responses pattern.

neuroscience↗

bioRxiv2020

Calcium homeostasis plays important roles in the internalisation and activities of the small synthetic antifungal peptide PAF26

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca2+ and the presence of fully functioning fungal Ca2+ homeostatic/signalling machinery. In a screen of mutants with deletions in Ca2+-signalling machinery, we identified three mutants more tolerant to PAF26. The Ca2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the{Delta} yvc-1 mutant. It is therefore apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca2+ and components of the Ca2+-regulatory machinery. AUTHOR SUMMARYLife threatening diseases can be caused when fungi invade human tissues. These invasions often occur when a persons immune defences are down, often due to treatments for cancer or transplantation. These infections are commonly buried deep within the body and as such are difficult to access and treat. Current medications are often highly toxic to the patient. There is also a worrying rise in drug resistance seen in fungi sampled from patients, with infections effectively untreatable - a death sentence. Antifungal peptides such as PAF26 provide a possible solution by offering a cheap and rapidly produced alternative to conventional drugs. However, unlike antibacterial peptides, little is known about how these small molecules mostly exert their effects and cause death. Using live-cell imaging and deletion mutants, this study provides an analysis of the important roles that Ca2+-homeostasis and Ca2+-signalling, and possible accompanying vacuolar fusion, play during the dynamic internalization and interaction with and within the fungal cell following PAF26 treatment.

molecular biology↗