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Biology subjects

Gomez, M.

Publications and source records attributed to Gomez, M..

At least 37 records · Page 2Linked to original sources

Failed reprogramming of transformed cells due to induction of apoptosis and senescence impairs tumor progression in lung cancer

Cell reprogramming to pluripotency applied to the study of cancer has identified transformation and pluripotency as two independent and incompatible cell fates. A detailed knowledge of the relationship between transformation and reprogramming could lead to the identification of new vulnerabilities and therapeutic targets in cancer. Here, we explore this interplay and find that OSKM expression limits tumor cell growth by inducing apoptosis and senescence. We identify Oct4 and Klf4 as the main individual reprogramming factors responsible for this effect. Mechanistically, the induction of cell cycle inhibitor p21 downstream of the reprogramming factors acts as mediator of cell death and senescence. Using a variety of in vivo systems, including allografts, orthotopic transplantation and KRAS-driven lung cancer mouse models, we demonstrate that OSKM expression impairs tumor growth and reduces tumor burden.

cell biology↗

Programmable delivery of fluoxetine via wearable bioelectronics for wound healing in vivo

The ability to deliver drugs with precise dosages at specific time points can significantly improve disease treatment while reducing side effects. Drug encapsulation for gradual delivery has opened up the doors for superior treatment regimen. To expand on this ability, programming bioelectronic devices to deliver small molecules enables ad-hoc personalized therapeutic profiles that are more complex than simple gradual release. Here, we introduce a wearable bioelectronic bandage with an integrated electrophoretic ion pump that affords on-demand drug delivery with precise dose control. Delivery of fluoxetine to wounds in mice resulted in a 27.2% decrease in the macrophage ratio (M1/M2) and a 39.9% increase in re-epithelialization, indicating a shorter inflammatory phase and faster overall healing. Programmable drug delivery using wearable bioelectronics in wounds introduces a broadly applicable strategy for the long-term delivery of a prescribed treatment regimen with minimal external intervention.

bioengineering↗

A Low-Cost Stage-Top Incubation Device For Human Cell Imaging Using Rapid Prototyping Methods

Live imaging of human or other mammalian cells at multi-hour time scales with minimal perturbation to their growth state requires that the specimens optimal growth conditions are met while fixed to a microscope stage. In general, ideal conditions include culturing in complete growth media, an ambient temperature of 36-37 C, and a humidity-controlled atmosphere comprising typically 5-7% CO2. Commercially available devices that achieve these conditions are not a financially viable option for many labs, with the price ranging anywhere from $12000 to $40000. The advent of 3D printing technology has allowed for low-cost rapid prototyping with precision comparable to traditional fabrication methods, opening the possibility for in-lab design and production of otherwise prohibitively expensive equipment such as stage-top incubation devices. The continued usefulness and widespread availability of single-board computers (SBC) such as Arduino and Raspberry Pi also simplify the process by which these devices can be controlled. Here we report the production of a do-it-yourself (DIY) device for stage-top incubation with temperature and atmospheric control with a cost reduction of approximately 100x.

cell biology↗

Increased LPS-Induced Fever and Sickness Behavior in Adult Male and Female Rats Perinatally Exposed to Morphine

As a result of the current opioid crisis, the rate of children born exposed to opioids has skyrocketed. Later in life, these children have an increased risk for hospitalization and infection, raising concerns about potential immunocompromise, as is common with chronic opioid use. Opioids can act directly on immune cells or indirectly via the central nervous system to decrease immune system activity, leading to increased susceptibility, morbidity, and mortality to infection. However, it is currently unknown how perinatal opioid exposure (POE) alters immune function. Using a clinically relevant and translatable model of POE, we have investigated how baseline immune function and the reaction to an immune stimulator, lipopolysaccharide, is influenced by in utero opioid exposure in adult male and female rats. We report here that POE potentiates the febrile and neuroinflammatory response to lipopolysaccharide, likely as a consequence of suppressed immune function at baseline (including reduced antibody production). This suggests that POE increases susceptibility to infection by manipulating immune system development, consistent with the clinical literature. Investigation of the mechanisms whereby POE increases susceptibility to pathogens is critical for the development of potential interventions for immunosuppressed children exposed to opioids in utero. HighlightsO_LIPerinatal exposure to morphine leads to increased fever and sickness post-LPS C_LIO_LIPerinatal exposure to morphine alters peripheral immunity C_LIO_LIPerinatal exposure to morphine increases microglial activation C_LI

neuroscience↗

Delivering biochemicals with precision using bioelectronic devices enhanced with feedback control

Precision medicine tailors treatment in a way that accounts for variations in patient response. Treatment strategies can be determined based on factors such as genetic mutations, age, and diet. Another way of implementing precision medicine in a dynamic fashion is through bioelectronics equipped with real-time sensing and intelligent actuation. Bioelectronic devices such as ion pumps can be utilized to deliver therapeutic drugs. To be able to perform precision medicine, medical devices need to be able to deliver drugs with high precision. For this, closed-loop control is required to be able to change the treatment strategy as new information about the response and progression of the biological system is received. To this end, a sliding mode controller is utilized given its ability to perform satisfactory control actions when there is model uncertainty. The controller is used in an experiment with the goal of delivering a pre-determined dosage of fluoxetine throughout a period of time.

systems biology↗

Quantifying innervation facilitated by deep learning in wound healing

The peripheral nerves (PNs) innervate the dermis and epidermis, which have been suggested to play an important role in wound healing. Several methods to quantify skin innervation during wound healing have been reported. Those usually require multiple observers, are complex and labor-intensive, and noise/background associated with the Immunohistochemistry (IHC) images could cause quantification errors/user bias. In this study, we employed the state-of-the-art deep neural network, DnCNN, to perform pre-processing and effectively reduce the noise in the IHC images. Additionally, we utilized an automated image analysis tool, assisted by Matlab, to accurately determine the extent of skin innervation during various stages of wound healing. The 8mm wound is generated using a circular biopsy punch in the wild-type mouse. Skin samples were collected on days 3,7,10 and 15, and sections from paraffin-embedded tissues were stained against pan-neuronal marker- protein-gene-product 9.5 (PGP 9.5) antibody. On day 3 and day 7, negligible nerve fibers were present throughout the wound with few only on the lateral boundaries of the wound. On day 10, a slight increase in nerve fiber density appeared, which significantly increased on day 15. Importantly we found a positive correlation (R-2 = 0.933) between nerve fiber density and re-epithelization, suggesting an association between re-innervation and re-epithelization. These results established a quantitative time course of re-innervation in wound healing, and the automated image analysis method offers a novel and useful tool to facilitate the quantification of innervation in the skin and other tissues.

molecular biology↗

Sex-dependent contributions of ventrolateral orbitofrontal cortex and basolateral amygdala to learning under uncertainty

Reversal learning measures the ability to form flexible associations between choice outcomes with stimuli and actions that precede them. This type of learning is thought to rely on several cortical and subcortical areas, including highly interconnected orbitofrontal cortex (OFC) and basolateral amygdala (BLA), and is often impaired in various neuropsychiatric and substance use disorders. However, unique contributions of these regions to stimulus- and action-based reversal learning have not been systematically compared using a chemogenetic approach and particularly before and after the first reversal that introduces new uncertainty. Here, we examined the roles of ventrolateral OFC (vlOFC) and BLA during reversal learning. Male and female rats were prepared with inhibitory DREADDs targeting projection neurons in these regions and tested on a series of deterministic and probabilistic reversals during which they learned about stimulus identity or side (left or right) associated with different reward probabilities. Using a counterbalanced within-subject design, we inhibited these regions prior to reversal sessions. We assessed initial and pre-post reversal changes in performance to measure learning and adjustments to reversals, respectively. We found that inhibition of vlOFC, but not BLA, eliminated adjustments to stimulus-based reversals. Inhibition of BLA, but not vlOFC, selectively impaired action-based probabilistic reversal learning, leaving deterministic reversal learning intact. vlOFC exhibited a sex-dependent role in early adjustment to action-based reversals, but not in overall learning. These results reveal dissociable roles for BLA and vlOFC in flexible learning and highlight a more crucial role for BLA in learning meaningful changes in the reward environment. Significance StatementInflexible learning is a feature of several neuropsychiatric disorders. We investigated how the ventrolateral orbitofrontal cortex (vlOFC) and basolateral amygdala (BLA) are involved in learning of stimuli or actions under reinforcement uncertainty. Following chemogenetic inhibition of these regions in both male and females, we measured learning and adjustments to deterministic and probabilistic reversals. For action learning, BLA, but not vlOFC, is needed for probabilistic reversal learning. However, BLA is not necessary for initial probabilistic learning or retention, indicating a critical role for learning of unexpected changes. For stimulus learning, vlOFC, but not BLA, is required for adjustments to reversals, particularly in females. These findings provide insight into the complementary cortico-amygdalar substrates of learning under different forms of uncertainty.

neuroscience↗

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. One sentence summaryThe human origin recognition complex subunit 1 ORC1, binds to RNAs transcribed from genes with origins of replication at the TSS, which is required for optimal origin activation.

molecular biology↗

Establishing cell motility patterns as predictors of macrophage subtypes and their relation to cell morphology

The motility of macrophages in response to microenvironment stimuli is a hallmark of innate immunity, where macrophages play pro-inflammatory or pro-reparatory roles depending on their activation status during wound healing. Cell size and shape have been informative in defining macrophage subtypes. Studies show pro and anti-inflammatory macrophages exhibit distinct migratory behaviors, in vitro, in 3D and in vivo but this link has not been rigorously studied. We apply both morphology and motility-based image processing approaches to analyze live cell images consisting of macrophage phenotypes. Macrophage subtypes are differentiated from primary murine bone marrow derived macrophages using a potent lipopolysaccharide (LPS) or cytokine interleukin-4 (IL-4). We show that morphology is tightly linked to motility, which leads to our hypothesis that motility analysis could be used alone or in conjunction with morphological features for improved prediction of macrophage subtypes. We train a support vector machine (SVM) classifier to predict macrophage subtypes based on morphology alone, motility alone, and both morphology and motility combined. We show that motility has comparable predictive capabilities as morphology. However, using both measures can enhance predictive capabilities. While motility and morphological features can be individually ambiguous identifiers, together they provide significantly improved prediction accuracies (75%) from a training dataset of 1000 cells tracked over time using only phase contrast time-lapse microscopy. Thus, the approach combining cell motility and cell morphology information can lead to methods that accurately assess functionally diverse macrophage phenotypes quickly and efficiently. This can support the development of cost efficient and high through-put methods for screening biochemicals targeting macrophage polarization. Author summaryPrevious work has shown that macrophage phenotypes can be distinguished by their morphological characteristics. We extend this work to show that distinct motility patterns are linked to macrophage morphology. Thus, motility patterns can be used to differentiate phenotypes. This can enable high-throughput classification of cell phenotypes without regard for the high-resolution images needed to quantify morphological characteristics. Furthermore, combining motility-based features with morphological information improves prediction of macrophage subtypes by a machine learning based classification model.

cell biology↗

Neuregulin1 regulates cardiomyocyte dynamics, proliferation, and maturation during ventricular chamber morphogenesis

BACKGROUNDCardiac ventricles are essential for providing the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for fundamental biology and regenerative medicine. Trabeculation is dependent on the signaling protein Neuregulin-1 (Nrg1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODSIn this study we investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with conditional cardiac-specific inactivation or overexpression of Nrg1. RESULTSAnalysis of cardiac-specific-Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 signaling and pErk activation. Early endothelial loss of Nrg1 and below normal pErk activation diminished cardiomyocyte Pard3 and Crumbs2 protein, and altered cytoskeletal gene expression and organization. These changes were associated with aberrant expression of genes involved in mitotic spindle organization and a directional shift from perpendicular to parallel/obliquely-oriented cardiomyocyte division. Further analysis indicated that Nrg1 is required for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchyme transition (EMT)-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, maintained high EMT-like gene expression and prolonged trabeculation, blocking compact myocardium maturation. Likewise, alterations of myocardial trabecular patterning resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with disorganization of the sarcomere actin cytoskeleton. The Nrg1 loss- and gain-of-function transcriptomes were enriched for yes-associated protein-1 (Yap1) gene signatures, identifying Yap1 as a potential downstream effector. Biochemical and imaging data showed that pErk activation and nuclear-cytoplasmic distribution of Yap1 during trabeculation are dependent on Nrg1. CONCLUSIONSThese data establish the Nrg1-ErbB2/4-pErk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development. Moreover, our data identify a Nrg1-dependent signaling cascade that could be leveraged for future cardiac regenerative therapies. Novelty and Significance WHAT IS KNOWN?O_LIMyocardial trabeculae play important roles in ventricular chamber growth, development of the conduction system, and formation of the coronary arteries. C_LIO_LITrabeculae are formed through oriented cell division (OCD), and their growth is driven by directional migration. C_LIO_LIThe membrane glycoprotein Neuregulin-1 (Nrg1) mediates cell-cell signaling and is essential for trabecular development. C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?O_LINrg1 signaling is essential for the expression of cardiomyocyte polarity genes and the organization of the cytoskeleton during the oriented cell division process underlying trabeculation. C_LIO_LINrg1 is required for the formation of the inner ventricular wall but not the coronaries. C_LIO_LINrg1 regulates motility and cell-cycle progression during ventricular wall growth. C_LIO_LIEctopic expression of Nrg1 leads to excessive trabeculation of the myocardium and disrupts compaction. C_LIO_LINrg1 regulates ventricular patterning mediated by cytoskeletal dynamics and modulates pErk-dependent Yap1 S274 phosphorylation during trabeculation. C_LIO_LINrg1 is not required for ventricular compaction. C_LI

developmental biology↗

Male harm suppresses female fitness to affect the dynamics of adaptation and evolutionary rescue

One of the most pressing questions we face as biologists is to understand how climate change will affect the evolutionary dynamics of natural populations and how these dynamics will in turn affect population recovery. Increasing evidence shows that sexual selection favours population viability and local adaptation. However, sexual selection can also foster sexual conflict and drive the evolution of male harm to females. Male harm is extraordinarily widespread and has the potential to suppress female fitness and compromise population growth, yet we currently ignore its net effects across taxa, or its effects on local adaptation and evolutionary rescue. We conducted a comparative meta-analysis to quantify the impact of male harm on female fitness and found an overall negative effect of male harm on female fitness. Negative effects seem to depend on proxies of sexual selection, increasing in species with larger sexual size dimorphism and strong sperm competition. We then developed theoretical models to explore how male harm affects adaptation and evolutionary rescue. We show that, when sexual conflict depends on local adaptation, population decline is reduced, but at the cost of slowing down genetic adaptation. This trade-off suggests that eco-evolutionary feedbacks on sexual conflict can act like a double-edge sword, reducing extinction risk by buffering the demographic costs of climate change, but delaying genetic adaptation. However, variation in the mating system and male harm type can mitigate this trade-off. Our work shows that male harm has widespread negative effects on female fitness and productivity, identifies potential mechanistic factors underlying variability in such costs across taxa, and underscores the importance of male harm on the demographic and evolutionary processes that impact how species adapt to environmental change. Impact summaryFor species to persist in the face of climate change, adaptation needs to be fast enough to prevent extinction. If population decline is too abrupt, adaptation will be less likely to promote recovery, leading to extinction. Therefore, numerous studies have sought to determine how species can adapt and escape extinction. Sexual selection can promote genetic adaptation, but often has a by-product, sexual conflict, that promotes adaptations beneficial for one sex and detrimental to the other. Such is the case of male adaptations that increase male reproduction by harming females (male harm). Male harm is widespread and has been shown to decrease female and population productivity in some species, facilitating extinction. Furthermore, there is increasing evidence that the degree of male harm to females depends on environmental changes and how well males are adapted to them. However, we ignore how strong the effects of sexual conflict across taxa are, or how ecological feedback on sexual conflict may affect the rate of adaptation and population recovery. Here, we first conducted a meta-analysis to quantify the effect of male harm on female fitness and show, across taxa, that there is an overall negative effect that seems to be dependent on proxies of sexual selection. Then, we used a series of theoretical models to show that, although eco-evolutionary feedback on sexual conflict can limit population decline, this comes at the cost of slowing down the rate of adaptation and population recovery. Our study suggests that understanding how quick environmental changes affect sexual conflict can increase our understanding of how populations adapt and recover in the face of climate change.

evolutionary biology↗

The structure of insulin granule core determines secretory capacity being reduced in type-2 diabetes

Exocytosis in excitable cells is essential for their physiological functions. Although the exocytotic machinery controlling cellular secretion has been well investigated, the function of the vesicular cargo, i.e. secretory granular content remains obscure. Here we combine dSTORM imaging and single-domain insulin antibody, to dissect the in situ structure of insulin granule cores (IGCs) at nano level. We demonstrate that the size and shape of the IGCs can be regulated by the juxta-granular molecules Nucleobindin-2 and Enolase-1, that further contribute to the stimulated insulin secretion. IGCs located at the plasma membrane are larger than those in the cytosol. The IGCs size is decreased by [~]20% after glucose stimulation due to the release of the peripheral part of IGCs through incomplete granule fusion. Importantly, the reduction of the IGCs size is also observed in non-stimulatory pancreatic {beta}-cells from diabetic db/db mice, Akita (Ins2+/-) mice and human Type-2 diabetic donors, in accordance with impaired secretion. These findings overall highlight the structure of exocytotic insulin cores as a novel modality amenable to targeting in the stimulated exocytosis in {beta}-cells with impaired insulin secretion.

physiology↗

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we have identified a new function of histone H1 in chromatin structure and transcription using a range of genomic approaches. We show that histone H1-depleted cells accumulate nascent non-coding RNAs on chromatin, suggesting that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accumulated non-coding transcripts have reduced levels of m6A modification and cause replication-transcription conflicts. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism and DNA replication to maintain genome performance.

molecular biology↗

Effective spatio-temporal regimes for wound treatment by way of macrophage polarization: A mathematical model

Wound healing consists of a sequence of biological processes often grouped into different stages. Interventions applied to accelerate normal wound healing must take into consideration timing with respect to wound healing stages in order to maximize treatment effectiveness. Macrophage polarization from M1 to M2 represents a transition from the inflammatory to proliferation stage of wound healing. Accelerating this transition may be an effective way to accelerate wound healing; however, it must be induced at the appropriate time. We search for an optimal spatio-temporal regime to apply wound healing treatment in a mathematical model of wound healing. In this work we show that to maximize effectiveness, treatment must not be applied too early or too late. We also show that effective spatial distribution of treatment depends on the heterogenity of the wound surface. In conclusion, this research provides a possible optimal regime of therapy that focuses on macrophage activity and a hypothesis of treatment outcome to be tested experimentally in future. Finding best regimes for treatment application is a first step towards development of intelligent algorithms of wound treatment that minimize healing time.

systems biology↗

Automatic wound detection and size estimation using deep learning algorithms

Evaluating and tracking wound size is a fundamental metric for the wound assessment process. Good location and size estimates can enable proper diagnosis and effective treatment. Traditionally, laboratory wound healing studies include a collection of images at uniform time intervals exhibiting the wounded area and the healing process in the test animal, often a mouse. These images are then manually observed to determine key metrics --such as wound size progress- relevant to the study. However, this task is a time-consuming and laborious process. In addition, defining the wound edge could be subjective and can vary from one individual to another even among experts. Furthermore, as our understanding of the healing process grows, so does our need to efficiently and accurately track these key factors for high throughput (e.g., over large-scale and long-term experiments). Thus, in this study, we develop a deep learning-based image analysis pipeline that aims to intake non-uniform wound images and extract relevant information such as the location of interest, wound only image crops, and wound periphery size over-time metrics. In particular, our work focuses on images of wounded laboratory mice that are used widely for translationally relevant wound studies and leverages a commonly used ring-shaped splint present in most images to predict wound size. We apply the method to a dataset that was never meant to be quantified and, thus, presents many visual challenges. Additionally, the data set was not meant for training deep learning models and so is relatively small in size with only 256 images. We compare results to that of expert measurements and demonstrate preservation of information relevant to predicting wound closure despite variability from machine-to-expert and even expert-to-expert. The proposed system resulted in high fidelity results on unseen data with minimal human intervention. Furthermore, the pipeline estimates acceptable wound sizes when less than 50% of the images are missing reference objects. Author summaryKnowledge of the wound size changes over-time allows us to observe important insights such as rate of closure, time to closure, and expansion events, which are key indicators for predicting healing status. To better perform wound measurements it is essential to utilize a technique that returns accurate and consistent results every time. Over the last years, collecting wound images is becoming easier and more popular as digital cameras and smartphones are more accessible. Commonly, scientists/clinicians trace the wound in these images manually to observe changes in the wound, which is normally a slow and labor-intensive process and also requires a trained eye. The clinical goal is to more efficiently and effectively treat wounds by employing easy to use and precise wound measurement techniques. Therefore, the objective should be devising automatic and precise wound measurement tools to be used for wound assessment. To this end, we leveraged a combination of various state-of-the-art computer vision and machine learning-based methods for developing a versatile and automatic wound assessment tool. We applied this tool to analyze the images of wound inflicted lab mice and showed that our developed tool automated the overall wound measurement process, therefore, resulting in high fidelity results without significant human intervention. Furthermore, we compared results to two expert measurements. We found variability in measurement even across experts further validating the need for a consistent approach. However, qualitative behavior, which is most important for predicting wound closure, is preserved.

bioinformatics↗

Screening of Cultivars for Tissue Culture Response and Establishment of Genetic Transformation in a High-yielding and Disease-resistant Cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning the initiation of floral material to the regeneration of plantlets into soil has been tested and improved in cacao. Fourteen cultivars were screened for their tissue culture response and transfer DNA (T-DNA) delivery efficiency via Agrobacterium. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from immature somatic embryos ranging in size between 4-10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in secondary callus growth medium increased the percentage of explants producing greater than ten embryos by 504% and 443%, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant level. Grafting transgenic scions with undeveloped roots to wild-type seedlings with strong, healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The present methods can be applied to improve tissue culture response and transformation frequency in other cacao cultivars. Key messageTissue culture and genetic transformation methods for a high-yielding, disease-resistant cultivar of Theobroma cacao were established while factors affecting T-DNA delivery and somatic embryogenesis were identified.

plant biology↗

Peripheral Natural Killer cells from chronic hepatitis B patients display molecular hallmarks of T cell exhaustion

A significant proportion of individuals infected by HBV develops chronic infection. Antiviral effectors such as Natural Killer (NK) cells have impaired functions in these patients, but the molecular mechanism responsible for this dysfunction remains poorly characterized. Here, we show that peripheral NK cells from chronic hepatitis B (CHB) patients have a defective capacity to produce IFN-{gamma}, MIP1-{beta} and TNF- but retain an intact killing capacity. This functional phenotype was associated with a decrease in the expression of NKp30 and CD16, combined with defects in IL-15 stimulation of the mTOR pathway. Transcriptome analysis of NK cells in CHB patients further revealed a strong enrichment for transcripts typically expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion rely on common molecular mechanisms. In particular, the transcription factor thymocyte selection-associated HMG box protein (TOX) and several of its targets, including immune checkpoints, were over-expressed in NK cells of CHB patients. This T cell exhaustion signature was predicted to be dependent on the calcium (Ca2+)-associated transcription factor NFAT. In line with this, when stimulating the Ca2+-dependent pathway in isolation, we recapitulated the dysfunctional phenotype. Thus, deregulated Ca2+ signalling could be a central event in both T cell exhaustion and NK cell dysfunction that occur during chronic infections.

immunology↗

Three-dimensional connectivity and chromatin environment mediate the activation efficiency of mammalian DNA replication origins

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled and bidirectional DNA synthesis is established. The slowdown of DNA polymerases at endogenous or exogenous obstacles triggers the activation of additional dormant origins whose genomic positions and regulation are not well understood. Here we report a comparative study of origin activity in mouse embryonic stem cells growing in control conditions or in the presence of mild replication stress. While stress-responsive origins can be identified, we find that the majority of them are also active, albeit with lower frequency, in the control population. To gain insights into the molecular and structural determinants of origin efficiency, we have analyzed the genetic and epigenetic features of origins stratified according to their frequency of activation. We have also integrated the linear origin maps into three-dimensional (3D) chromatin interaction networks, revealing a hierarchical organization in which clusters of connected origins are brought together by longer-range chromatin contacts. Origin efficiency is proportional to the number of connections established with other origin-containing fragments. Interacting origins tend to be activated with similar efficiency and share their timing of replication even when located in different topologically associated domains. Our results are consistent with a model in which clusters of origins are arranged in 3D in replication factories. Within each factory, main and dormant origins are functionally defined by a combination of chromatin environment and 3D connectivity.

genomics↗