Biology subjects

Diaz, J.

Publications and source records attributed to Diaz, J..

At least 19 recordsLinked to original sources

bioRxiv2026

The comparative strengths and limitations of Nile Red and 9-(dicyanovinyl)-julolidine (DCVJ) fluorescent dyes for detecting microplastics and nanoplastics

Nile Red is widely used for the detection of microplastics because its fluorescence emission is sensitive to local polarity and can distinguish hydrophobic plastics from hydrophilic ones. The fluorescence of the molecular rotor, 9-(dicyanovinyl)-julolidine (DCVJ) is less sensitive to polarity but more to viscosity. DCVJ is less widely used for microplastic analysis, although it has been used to detect polystyrene nanobeads. Here, we compared these dyes with standard samples from the Hawaii Pacific University Polymer Kit 1.0 and confirmed that Nile Red, in general, was better for the detection and identification of microplastics. Fluorescence emission was analyzed using photography, as well as spectroscopy. The color and peak emission wavelength of some stained environmental microplastics were affected by additives. Raman spectroscopy was used to confirm the chemical identity of such samples. Although DCVJ emits green fluorescence on binding to some microplastics, a peak at 620 nm has been reported with polystyrene nanobeads, attributed to dimer/excimer formation. We confirmed this property and directly observed diffraction-limited spots using fluorescence microscopy, attributed to single or just a few nanobeads. Nile Red also stains polystyrene nanobeads and gave stronger signals than with DCVJ, but Nile Red was prone to false positives due to dye aggregation in aqueous solutions.

biophysics↗

bioRxiv2026

Molecular basis for chikungunya virus recognition of a mosquito-specific receptor

Alphaviruses are arthropod-borne viruses that recognize cellular receptors in both vertebrate hosts and mosquito vectors to complete their transmission cycle, yet how they maintain recognition of receptors across evolutionarily divergent host species remains unresolved. Among alphaviruses, chikungunya virus (CHIKV), which is primarily vectored in urban settings by Aedes species mosquitoes, is the most widespread, and causes explosive outbreaks that can involve hundreds of thousands to millions of cases annually. The cell adhesion protein Lachesin is a mosquito-specific cellular receptor for CHIKV and multiple other arthritogenic alphaviruses. The envelope E2-E1 glycoproteins of these alphaviruses broadly recognize Lachesin orthologs from diverse mosquito species, but not other insects or arachnids. Lachesin genetic manipulation to prevent mosquito virus infection without interfering with endogenous receptor function could have a major impact for CHIKV control. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of alphaviruses bound to Aedes albopictus Lachesin. Comparative analysis of Lachesin-bound CHIKV, Semliki Forest virus (SFV), and Middelburg virus (MIDV) revealed that these three genetically divergent viruses use a similar surface to recognize Lachesin domain 1, but with reorganized E2-E1 glycoprotein contact residues. We show that a soluble Ae. albopictus Lachesin receptor decoy protein blocks the E2-E1-mediated entry of CHIKV and other arthritogenic alphaviruses into mammalian cells with greater breadth than a vertebrate receptor MXRA8 decoy and protects against lethal SFV challenge and CHIKV pathogenesis in murine models. Additionally, we identified a naturally occurring single residue Lachesin polymorphism that is found in some Anopheles (malaria vector) mosquitoes, and fully ablates CHIKV E2-E1 recognition, informing strategies for mosquito-targeted genetic interventions that could prevent mosquito vector infection and virus transmission. These findings define distinct determinants of receptor binding in mosquitoes and humans for arthritogenic alphaviruses, with implications for countermeasure development and outbreak preparedness.

microbiology↗

bioRxiv2026

Cell jamming transition is regulated by mitochondrial pyruvate transport and endocytosis

Epithelial tissues undergo dynamic transitions between fluid-like collective motion and mechanically jammed states during development, injury repair, and disease progression. However, the cellular programs that drive these transitions and regulate collective behavior remain unclear. Using a controlled crowding model integrated with live-cell imaging and time-resolved multi-omics, we demonstrate that epithelial crowding triggers early metabolic changes characterized by increased mitochondrial pyruvate anaplerosis that precedes the jamming transition. Functional inhibition of mitochondrial pyruvate import is sufficient to sustain collective cell motility, impeding jamming transition in crowded cells. This unjammed state is driven by enhanced cytoskeletal remodeling and requires RhoA-myosin II activity. Mechanistically, we show that elevated cytoskeletal signaling promotes macropinocytic uptake, which serves as a required feedback loop to maintain motility. These findings identify mitochondrial pyruvate utilization as a key regulator that links metabolic remodeling to the endocytic control of epithelial fluidity.

cell biology↗

bioRxiv2026

Mechanistic Language Modeling and Oxygenated 3D Screening Reveal Berberine and Enzalutamide Synergy in Resistant Prostate Cancer

Resistance to androgen receptor inhibitors remains a primary challenge in prostate cancer treatment, yet identifying synergis-tic co-therapies is hindered by immense combinatorial search spaces and the limited interpretability of predictive computation models. Here, we developed an integrated discovery-validation axis coupling knowledge-augmented large language models with oxygen-supplemented 3D spheroid assays. By leveraging inherent model stochasticity, our framework measures the degree of consensus across independent predictions to establish a formal metric for predictive accuracy. This principle enables high-throughput assessment of complex signaling crosstalk, yielding mechanistic rationales for all predictions and defining a high-confidence zone that minimizes experimental attrition. Utilizing this approach to screen 3,592 natural products, we identified a previously unrecognized synergy between berberine and enzalutamide that re-sensitizes resistant cells. Validation confirms that berberine perturbs the PI3K/AKT/mTOR and AMPK axes, a finding consistent with the mechanistic rationales computationally derived by the framework. Integrating interpretable AI with physiologically relevant 3D screening provides a scalable methodology for the rational discovery of synergistic therapies. SignificanceIntegrating mechanistic AI with oxygenated 3D screening, we identify a novel berberine-enzalutamide synergy. This framework resolves complex signaling dependencies, providing a scalable, transparent methodology for the rational discovery of effective combination therapies.

bioengineering↗

bioRxiv2025

Envelope analysis as a tool for identifying epileptic EEG patterns during the sleep-wake cycle in rats

BackgroundThis research aimed to study electroencephalographic abnormalities in an animal model of temporal lobe epilepsy induced by pilocarpine. By using the coefficient of variation of the envelope (CVE) of electroencephalography (EEG), we characterize and quantify morphological alterations in sleep-wake cycle (SWC) stages. MethodsEpilepsy was induced in 13 rats using a single dose of intraperitoneal pilocarpine injection. A second group of 13 rats served as the control. All the rats underwent polysomnography for at least two days. To do so, three channels were created using four electrodes for EEG and one more for EMG. Channels were built using contralateral lead. ResultsEnvelope analysis revealed global alterations in EEG morphology. Significant differences were observed in the delta and theta bands between the epileptic and control groups. Epileptic animals showed a near-total suppression of theta rhythm activity during REM sleep. Additionally, we identified a novel pattern of synchronized slow waves (sDelta), distinct from the physiological delta waves observed in non-REM sleep. ConclusionIdentifying both subtle and overt morphological abnormalities in EEG is challenging for human experts and computational methods. By applying CVE analysis to the well-known pilocarpine model, we reveal abnormal EEG dynamics with exceptional summarizing capabilities. For example, a 24-hour EEG can be synthesized into a single, easily interpretable visualization of EEG morphology. This technique and its underlying framework may serve as valuable tools for epilepsy research and potential diagnostic applications.

neuroscience↗

bioRxiv2025

Basal Ganglia Dopamine Availability and Immune Activation Interact and Relate to Anhedonia Severity among Youth with Depression

Anhedonia emerges in adolescence, has putative substrates in neural reward and dopamine systems, and is a hallmark of poor course in depression. Psychoneuroimmunology models suggest altered immune and reward systems may jointly underlie its development. When dopamine availability (rather than function) is considered, immune activation may be understood as a moderator amplifying how lower dopamine levels translate into motivational deficits. This cross-sectional study analyzed data from 55 youth with current depression (Mage=21.4 years; Female=75%). Plasma was assayed for relative quantification of 92 immune proteins, followed by dimensionality reduction via principal component (PC) analysis, forming five PC scores. Dopamine availability was indexed by basal ganglia tissue iron, quantified by MRI R2, a measure detectable in youth. Anhedonia was assessed using the Snaith-Hamilton Pleasure Scale (SHAPS), thought to capture overall anhedonia, and Positive Valence Systems Scale (PVSS), which provides overall, anticipatory, and consummatory anhedonia scores. PC1 scores--whose loadings indicated broadly distributed immune protein elevations consistent with general immune activation--moderated the relationship between basal ganglia dopamine availability and anhedonia: At higher PC1 levels, lower dopamine availability was linked to greater overall anhedonia (as measured by SHAPS) and anticipatory anhedonia, but not to consummatory anhedonia. Lower PC2 scores were associated with greater PVSS overall and consummatory anhedonia. Analyses examining individual basal ganglia regions suggest potential differences in dopaminergic-immune interactions across these regions, with effects most consistently observed in the putamen. Findings highlight nuanced neuroimmune pathways underlying anhedonia components in youth and support inflammation-based models of depression emphasizing dopaminergic-immune interplay.

neuroscience↗

bioRxiv2025

Maximum water stress is decoupled from climate, traits and growth in a xeric oak

O_LIWidespread drought-induced forest mortality highlights the ecological consequences of climate change, yet our ability to explain, let alone predict, the spatial patterns of forest mortality remains limited. C_LIO_LIWe conducted a range-wide survey of drought stress, growth, and allocation traits in a widespread oak species (Quercus douglasii, blue oak) to test the predictability of stress across populations and explore how water availability and allocation mediate spatial variation in tree growth. C_LIO_LIAcross 15 sites, we found little relationship between end-of-season water availability (predawn leaf water potentials) or maximum water stress (midday water potentials) and climate or soils. Instead, water availability (within and among sites) was predicted by access to deep water resources inferred from stem water stable isotopes. C_LIO_LIWe also found a remarkable three-way decoupling of water stress, growth, and allocation to leaf tissue, which challenged a data-parameterized mechanistic plant model. C_LIO_LIOur results reveal that deeply rooted trees can be hydrologically decouple from above-ground climate, and that the seasonality of growth, trait development and hydraulic risk are phenologically disconnected. The complicated relationship between carbon gain and hydraulic risk in seasonal environments and limited data on critical zone hydrology are key challenges to predicting drought vulnerability. C_LI Plain Language SummaryBelow-ground structure complicates our understanding of plant water stress. We show that below-ground complexity and highly seasonal environments can both disconnect trees from their above-ground climate and disconnect drought mortality risk from growth and trait development.

ecology↗

bioRxiv2025

Metabolic Adaptations of Prostate Cancer Cells Under Chronic Fructose Stimulation

BackgroundDietary sugars, particularly fructose, are increasingly implicated in cancer progression through their impact on tumor metabolism. However, the specific metabolic adaptations driven by fructose in prostate cancer (PCa) remain unexplored. MethodsWe investigated the metabolic consequences of chronic fructose exposure in androgen-sensitive (LNCaP) and androgen-independent (PC-3) PCa cell lines. We evaluated the expression, localization, and function of the fructose transporter Glut-5 and assessed metabolic fluxes, enzyme expression, lipid accumulation, and global metabolite profiles using molecular, imaging, and metabolomic approaches. ResultsGlut-5 was primarily localized to early endosomes under basal and fructose-stimulated conditions, suggesting a non-canonical role potentially consistent with transceptor function. Chronic fructose exposure significantly upregulated Glut-5 expression and enhanced fructose uptake, but did not alter substantially its subcellular localization. Functionally, fructose reduced lactate production and mitochondrial ATP output, indicating a metabolic shift away from glycolysis and oxidative phosphorylation. In LNCaP cells, fructose induced robust activation of de novo lipogenesis (DNL), evidenced by upregulation of FASN and G6PD, increased lipid droplet accumulation, and enhanced levels of key fatty acid metabolites (e.g., TG, EPA, DHA). In contrast, PC-3 cells exhibited a distinct metabolic response, characterized by increased ceramide and amino acid metabolites. Notably, pharmacological inhibition of lipid metabolism using etomoxir abrogated proliferation in both cell lines under fructose stimulation. ConclusionsThese findings reveal that fructose promotes a metabolic reprogramming in PCa cells that is cell type- and AR-dependent, enhancing lipogenesis and potentially contributing to tumor progression. Our study identifies Glut-5-mediated fructolysis and lipid metabolic pathways as key vulnerabilities in PCa, offering potential avenues for metabolic intervention.

cell biology↗

bioRxiv2025

OGDHL regulates nucleotide metabolism, tumor growth, and neuroendocrine marker expression in prostate cancer

As cancer cells evade therapeutic pressure and adopt alternate lineage identities not commonly observed in the tissue of origin, they likely adopt alternate metabolic programs to support their evolving demands. Targeting these alternative metabolic programs in distinct molecular subtypes of aggressive prostate cancer may lead to new therapeutic approaches to combat treatment-resistance. We identify the poorly studied metabolic enzyme Oxoglutarate Dehydrogenase-Like (OGDHL), named for its structural similarity to the tricarboxylic acid (TCA) cycle enzyme Oxoglutarate Dehydrogenase (OGDH), as an unexpected regulator of tumor growth, treatment-induced lineage plasticity, and DNA Damage in prostate cancer. While OGDHL has been described as a tumor-suppressor in various cancers, we find that its loss impairs prostate cancer cell proliferation and tumor formation. Loss of OGDHL profoundly alters Androgen Receptor inhibition-induced plasticity, including suppressing the neuroendocrine markers DLL3 and HES6, induces accumulation of the DNA damage response marker {gamma}H2AX, and reduces nucleotide synthesis. Our data suggest that OGDHL has minimal impact on TCA cycle activity, and that mitochondrial localization is not required for its regulation of prostate cancer plasticity and nucleotide metabolism. Finally, we demonstrate that OGDHL expression is tightly correlated with neuroendocrine differentiation in clinical prostate cancer. These findings underscore the importance of investigating poorly characterized metabolic genes as potential regulators of distinct molecular subtypes of aggressive cancer.

cancer biology↗

bioRxiv2025

Epistasis at the cell surface: what is the role of Erg3 loss-of-function in acquired echinocandin resistance?

Echinocandins, which target the fungal {beta}-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus (Candida glabrata) in which combined mutations in ERG3 and FKS2, but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude ERG3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic crosstalk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes.

molecular biology↗

bioRxiv2025

LARP6 regulates the mRNA translation of fibrogenic genes in liver fibrosis

Metabolic syndrome and excessive alcohol consumption result in liver injury and fibrosis, which is characterized by increased collagen production by activated Hepatic Stellate Cells (HSCs). LARP6, an RNA-binding protein, was shown to facilitate collagen production. However, LARP6 expression and functionality as a regulator of fibrosis development in a disease relevant model remains elusive. By using snRNA-sequencing, we show that LARP6 is upregulated mainly in HSCs of liver fibrosis patients. Moreover, LARP6 knockdown in human HSCs suppresses fibrogenic gene expression. By integrating eCLIP analysis and ribosome profiling in HSCs, we show that LARP6 interacts with mature mRNAs comprising over 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. Furthermore, LARP6 knockdown in HSC attenuates fibrosis development in human liver spheroids. Altogether, our results suggest that targeting LARP6 in human HSCs may provide new strategies for anti-fibrotic therapy. HighlightsO_LILARP6 is upregulated in liver fibrosis, mainly in HSCs. C_LIO_LILARP6 knockdown in human HSCs reduces liver fibrosis development. C_LIO_LIOf the hundreds of gene targets, LARP6 interacts most with collagen mRNAs. C_LIO_LILARP6 regulates mRNA translation via interaction with 5UTRs. C_LI

cell biology↗

bioRxiv2024

Biosynth Pipeline: An integrated platform for merging the computational design of chimeric type I polyketide synthases with enzymatic pathways for chemical biosynthesis

Synthetic biology offers the promise of manufacturing chemicals more sustainably than petrochemistry. Yet, both the rate at which biomanufacturing can synthesize these molecules and the net chemical accessible space are limited by existing pathway discovery methods which can often rely on arduous literature searches. Here, we present an automated retrobiosynthesis tool, BioPKS Pipeline, that simultaneously tackles both problems by integrating multifunctional type I polyketide synthases (PKSs) with monofunctional enzymes to propose the synthesis of desired target chemicals via two new tools: DORAnet and RetroTide. While monofunctional enzymes are valuable for carefully decorating a substrates carbon backbone, they typically cannot expand the backbone itself. PKSs can, instead, predictably do this through their unique ability to catalyze carbon-carbon bond formation reactions iteratively. We have evaluated the performance of BioPKS Pipeline against a previously published set of 155 molecules of interest for biomanufacturing, and report that BioPKS Pipeline could produce exact designs for 93 of them, as well as pipelines to a chemically similar product for most of the remaining molecules. Furthermore, BioPKS Pipeline successfully proposes biosynthetic routes for complex therapeutic natural products (cryptofolione and basidalin) for which no known biosynthetic pathway currently exists.

bioinformatics↗

bioRxiv2024

Dogs density drives the reproductive effort of American Oystercatchers (Haematopus palliatus) in disturbed habitats of the Maule Region, central Chile

The American Oystercatcher (Haematopus palliatus) is a shorebird specialized in coastal environments across the Americas. Thus, anthropic extensive use of the shoreline including the introduction of dogs and vehicles to beaches could directly impact fitness, and ultimately the persistence of the species populations. In this study, we aimed to assess the impact of pedestrians, vehicles and dogs on the nest density, productivity and hatching success of the American Oystercatcher in the Maule Region, central Chile. To this end, we sampled sandy beaches, quantifying the number of nests, eggs, chicks, but also pedestrians, vehicles and dogs during the breeding seasons of 2023 and 2024. We assessed for the influence of these threats on parameters of reproductive output by using Generalized Linear Mixed Models. We found that the number of dogs is the only variable that negatively impacts the number of nests, eggs and chicks of the American Oystercatcher in the sampled areas. Reducing the impact of dogs is a ubiquitous challenge in coastal environments of central Chile, however current regulations do not allow management relying on removal of dogs from important areas for biodiversity.

ecology↗

bioRxiv2024

iSCORE-PD: an isogenic stem cell collection to research Parkinson Disease

Parkinsons disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer a unique experimental platform to advance our understanding of PD etiology by enabling the generation of disease-relevant cell types carrying patient mutations along with isogenic control cells. To facilitate this approach, we generated a collection of 65 human stem cell lines genetically engineered to harbor high risk or causal variants in genes associated with PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G/FS, SYNJ1 R258Q/FS, VPS13C A444P/FS, VPS13C W395C/FS, GBA1 IVS2+1/FS). All mutations were introduced into a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using different genome editing techniques. To ensure the genetic integrity of these cell lines, we implemented rigorous quality controls, including whole-genome sequencing of each line. Our analysis of the genetic variation in this cell line collection revealed that while genome editing, particularly using CRISPR/Cas9, can introduce rare off-target mutations, the predominant source of genetic variants arises from routine cell culture and are fixed in cell lines during clonal isolation. The observed genetic variation was minimal compared to that typically found in patient-derived iPSC experiments and predominantly affected non-coding regions of the genome. Importantly, our analysis outlines strategies for effectively managing genetic variation through stringent quality control measures and careful experimental design. This systematic approach ensures the high quality of our stem cell collection, highlights advantages of prime editing over conventional CRISPR/Cas9 methods and provides a roadmap for the generation of gene-edited hPSC collections at scale in an academic setting. Our iSCORE-PD collection represents an easily accessible and valuable platform to study PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

neuroscience↗

bioRxiv2023

Plasticity drives geographic variation and trait coordination in blue oak drought physiology

Our ability to predict drought stress across the landscape remains limited. This uncertainty stems in part from an incomplete understanding of within-species variation in hydraulic physiology, particularly coordinated variation across multiple traits. This variation reflects genetic differentiation among populations (ecotypic variation) and phenotypic plasticity. We examined among-population differentiation in morphological and hydraulic traits in California blue oak (Quercus douglasii) using a 30 year-old common garden. We compared trait differentiation and trait-trait coordination in the garden to wild phenotypes from the original source populations. We found remarkably limited among-population differentiation in all traits in the common garden but considerable site-to-site variation in the field that could rarely be explained with site climate variables. Trait-trait relationships were also stronger in the field than in the garden, particularly links between leaf morphology, leaf hydraulic efficiency and stem hydraulic efficiency. Only four trait-trait relationship were present in both the wild and garden, but 12 of 45 relationships showed significant wild phenotypic correlations, with strong coordination among leaf and stem hydraulic efficiency apparently mediated by leaf size. Ultimately, we found limited evidence for ecotypic variation but considerable geographic in phenotypic integration in the wild, suggesting considerable acclimation potential in the face of climate change.

ecology↗

bioRxiv2023

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl4) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

cell biology↗

bioRxiv2023

Mycobacterium tuberculosis-Macrophage Interactome: Molecular Network Structure and Resilience against Antibiotics

Resistance to several antibiotics against Mycobacterium tuberculosis is a serious problem to be solved worldwide. In the present work, we made the statistical analysis of the gene regulatory network of Mycobacterium tuberculosis and of the Mycobacterium tuberculosis-macrophage interactome to find the probable cause of this resistance. The results from this analysis show that both the gene regulatory network and the interactoma have a hierarchical free scale modular structure that assures a high degree of resilience of these networks against external perturbations. In particular, the interactome is a complex hybrid network that results from the formation of novel links between the Mycobacterium tuberculosis and macrophage proteins and from the modification of the previously existing links between the native macrophage proteins, which give rise to novel negative and positive feedback loops that modify the dynamical behavior of the interactome and protect the mycobacterium against the attack with antibiotics by taking control of the macrophage immune response and apoptosis. The statistical analysis of the interactome shows that the highly connected mycobacterium proteins inhA, ahpC, kasA, katG and rpsL exert this control by creating new links with the host proteins FAS and NF-{kappa}B. These new hybrid circuits embedded in the hierarchical scale-free modular molecular structure of the interactome produce its high resistance to external perturbations like antibiotics. As consequence, the present work proposes the hypothesis that Mycobacterium tuberculosis antibiotic resistance in vivo during chronic tuberculosis is only a particular case of a more complex problem that is the interactome resilience against antibiotics. Thus, new strategies of drug design are necessary to shatter the complex structure of the Mycobacterium tuberculosis-macrophage interactome.

systems biology↗

bioRxiv2022

Identifying cell culturing parameters that improve endocytic uptake of the HIV-TAT cell penetrating peptide

Delivery tools, including cell-penetrating peptides (CPPs) are often inefficient due to a combination of poor endocytosis and endosomal escape. Herein, the impact of cell culturing techniques on the endocytic uptake of a typical CPP, the TAT peptide (derived from HIV1-TAT), was quantified. Parameters previously found to generally modulate endocytosis such as cell density, washing steps, and cell aging did not affect TAT endocytosis. In contrast, cell dissociation methods, media, temperature, serum starvation, and media composition all contributed to changes in uptake. The combination of these parameters in worst versus best-uptake protocols, led to changes in uptake of more than 13-fold and indicated that small variations in cell culturing techniques have a cumulative effect on CPP uptake. More specifically, modulating cell culture protocols does not result in an increased amount of peptide inside endosomes, rather the number of TMR-TAT containing endosomes increases. Taken together this study highlights how technical aspects of cell culture protocols can be used to improve experimental reproducibility, as well as parameters that can be potentially exploited to improve CPP accumulation in endosomes, and hence increase the possibility of endosomal escape and cytosolic access.

biochemistry↗