CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts.

The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.

Optical pH Sensor Based on a Long-Period Fiber Grating Coated with a Polymeric Layer-by-Layer Electrostatic Self-Assembled Nanofilm.

An optical fiber pH sensor based on a long-period fiber grating (LPFG) is reported. Two oppositely charged polymers, polyethylenimine (PEI) and polyacrylic acid (PAA), were alternately deposited on the sensing structure through a layer-by-layer (LbL) electrostatic self-assembly technique. Since the polymers are pH sensitive, their refractive index (RI) varies when the pH of the solution changes due to swelling/deswelling phenomena. The fabricated multilayer coating retained a similar property, enabling its use in pH-sensing applications. The pH of the PAA dipping solution was tuned so that a coated LPFG achieved a pH sensitivity of (6.3 ± 0.2) nm/pH in the 5.92-9.23 pH range. Only two bilayers of PEI/PAA were used as an overlay, which reduces the fabrication time and increases the reproducibility of the sensor, and its reversibility and repeatability were demonstrated by tracking the resonance band position throughout multiple cycles between different pH solutions. With simulation work and experimental results from a low-finesse Fabry-Perot (FP) cavity on a fiber tip, the coating properties were estimated. When saturated at low pH, it has a thickness of 200 nm and 1.53 ± 0.01 RI, expanding up to 310 nm with a 1.35 ± 0.01 RI at higher pH values, mostly due to the structural changes in the PAA.

Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

BACKGROUND: Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. METHODS: We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). RESULTS: Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. CONCLUSIONS: Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.

Evaluating priority locations and potential benefits for building a nation-wide fuel break network in Portugal.

Despite growing interest in developing extensive fuel treatment programs to prevent catastrophic wildfires in the Mediterranean region, there is little information on the projected effectiveness of fuel treatments in terms of avoided exposure and risk. In Portugal, a fuel management plan aiming to prevent loss of lives, reduce large fires (>500 ha), and reduce annual burned area is under implementation, with particular emphasis on the nation-wide fuel break network (FBN). In this study, we evaluated the effectiveness of the planned FBN in terms of meeting fire management objectives, costs, and benefits. We first estimated the overall effectiveness of the FBN at intersecting modeled large fires (>500 ha) and at reducing exposure to protected areas and residential buildings using wildfire simulation modeling. Then, the fuel break burn-over percentage, i.e. the percentage of fires that are not contained at the FBN, was modeled as a function of pre-defined flame length thresholds for individual FBN segments. For the planned FBN, the results suggested a potential reduction of up to 13% in the annual burned area due to large fires (ca. 13,000 ha), of up to 8% in the annual number of residential buildings exposed (ca. 100 residential buildings), and up to 14% in the annual burned area in protected areas (ca. 2400 ha). The expected burn-over percentage was highly variable among the segments in response to estimated fire intensity, and an average decrease of 40% of the total benefits was estimated. The most important fuel breaks typically showed a higher percentage of fire burn-over, and hence reduction in effectiveness. We also showed that the current implementation of FBN follows a random sequence, suboptimal for all objectives. Our results suggest that additional landscape-scale fuel reduction strategies are required to meet short-term national wildfire management targets.

MacroH2As regulate enhancer-promoter contacts affecting enhancer activity and sensitivity to inflammatory cytokines.

MacroH2A histone variants have a function in gene regulation that is poorly understood at the molecular level. We report that macroH2A1.2 and macroH2A2 modulate the transcriptional ground state of cancer cells and how they respond to inflammatory cytokines. Removal of macroH2A1.2 and macroH2A2 in hepatoblastoma cells affects the contact frequency of promoters and distal enhancers coinciding with changes in enhancer activity or preceding them in response to the cytokine tumor necrosis factor alpha. Although macroH2As regulate genes in both directions, they globally facilitate the nuclear factor κB (NF-κB)-mediated response. In contrast, macroH2As suppress the response to the pro-inflammatory cytokine interferon gamma. MacroH2A2 has a stronger contribution to gene repression than macroH2A1.2. Taken together, our results suggest that macroH2As have a role in regulating the response of cancer cells to inflammatory signals on the level of chromatin structure. This is likely relevant for the interaction of cancer cells with immune cells of their microenvironment.

Wild, tamed, and domesticated: Three fire macroregimes for global pyrogeography in the Anthropocene.

Climate and natural vegetation dynamics are key drivers of global vegetation fire, but anthropogenic burning now prevails over vast areas of the planet. Fire regime classification and mapping may contribute towards improved understanding of relationships between those fire drivers. We used 15 years of daily active fire data from the MODIS fire product (MCD14ML, collection 6) to create global maps of six fire descriptors (incidence, size inequality, season length, interannual variability, intensity, and fire season modality). Using multiple correspondence analysis (MCA) and hierarchical agglomerative clustering, we identified three fire macroregimes: Wild, Tamed, and Domesticated, each of which splitting into prototypical and transitional regimes. Interpretation of the six fire regimes in terms of their main drivers relied on the global maps of anthromes and Köppen climate types. The analysis yielded a two-dimensional space where the principal dimension of variability is primarily defined by interannual variability in fire activity and fire season length, and the secondary axis is based mainly on fire incidence. The Wild fire macroregime occurs mostly in cold wildlands, where burning is sporadic and fire seasons are short. Tamed fires predominate in seasonally dry tropical rangelands and croplands with high fire incidence. Domesticated fires are characteristic of humid, warm temperate and tropical croplands and villages with low fire incidence. The Tamed and Domesticated fire macroregimes, representing managed burning, account for 86% of all active fires in our dataset and for 70% of the global burnable area. Fourteen percent of active fires were found in the cold wildlands, and in the rangelands and forests of steppe and desert climates of the Wild macroregime. These results highlight the extent of human control over global pyrogeography in the Anthropocene.

Tuning the magnetic properties of NiPS3 through organic-ion intercalation.

Atomically thin van der Waals magnetic crystals are characterized by tunable magnetic properties related to their low dimensionality. While electrostatic gating has been used to tailor their magnetic response, chemical approaches like intercalation remain largely unexplored. Here, we demonstrate the manipulation of the magnetism in the van der Waals antiferromagnet NiPS3 through the intercalation of different organic cations, inserted using an engineered two-step process. First, the electrochemical intercalation of tetrabutylammonium cations (TBA+) results in a ferrimagnetic hybrid compound displaying a transition temperature of 78 K, and characterized by a hysteretic behavior with finite remanence and coercivity. Then, TBA+ cations are replaced by cobaltocenium via an ion-exchange process, yielding a ferrimagnetic phase with higher transition temperature (98 K) and higher remanent magnetization. Importantly, we demonstrate that the intercalation and cation exchange processes can be carried out in bulk crystals and few-layer flakes, opening the way to the integration of intercalated magnetic materials in devices.

Novel Antiretroviral Therapeutic Strategies for HIV.

When the first cases of HIV infection appeared in the 1980s, AIDS was a deadly disease without any therapeutic alternatives. Currently, there is still no cure for most cases mainly due to the multiple tissues that act as a reservoir for this virus besides the high viral mutagenesis that leads to an antiretroviral drug resistance. Throughout the years, multiple drugs with specific mechanisms of action on distinct targets have been approved. In this review, the most recent phase III clinical studies and other research therapies as advanced antiretroviral nanodelivery systems will be here discussed. Although the combined antiretroviral therapy is effective in reducing viral loading to undetectable levels, it also presents some disadvantages, such as usual side effects, high frequency of administration, and the possibility of drug resistance. Therefore, several new drugs, delivery systems, and vaccines have been tested in pre-clinical and clinical trials. Regarding drug delivery, an attempt to change the route of administration of some conventional antiretrovirals has proven to be successful and surpassed some issues related to patient compliance. Nanotechnology has brought a new approach to overcoming certain obstacles of formulation design including drug solubility and biodistribution. Overall, the encapsulation of antiretroviral drugs into nanosystems has shown improved drug release and pharmacokinetic profile.

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression.

Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes.

Putting fire on the map of Brazilian savanna ecoregions.

The Brazilian savanna (Cerrado) is considered the most floristically diverse savanna in the world, home to more than seven thousand species. The region is a mosaic of savannas, grasslands and forests whose unique biophysical and landscape attributes are on the basis of a recent ecoregional map, paving the way to improved region-based strategies for land management actions. However, as a fire-prone ecosystem, Cerrado owes much of its distribution and ecological properties to the fire regime and contributes to an important parcel of South America burned area. Accordingly, any attempt to use ecoregion geography as a guide for management strategies should take fire into account, as an essential variable. The main aim of this study is to complement the ecoregional map of the Cerrado with information related to the fire component. Using remotely sensed information, we identify patterns and trends of fire frequency, intensity, seasonality, extent and scar size, and combine this information for each ecoregion, relying on a simple classification that summarizes the main fire characteristics over the last two decades. Results show a marked north-south fire activity gradient, with increased contributions from MATOPIBA, the latest agricultural frontier. Five ecoregions alone account for two thirds of yearly burned area. More intense fires are found in the Arc of Deforestation and eastern ecoregions, while ecoregions in MATOPIBA display decreasing fire intensity. An innovative analysis of fire scars stratified by size class shows that infrequent large fires are responsible for the majority of burned area. These large fires display positive trends over many ecoregions, whereas smaller fires, albeit more frequent, have been decreasing in number. The final fire classification scheme shows well defined spatially-aggregated groups, where trends are found to be the key factor to evaluate fire within their regional contexts. Results presented here provide new insights to improve fire management strategies under a changing climate.

Topologically Associating Domains and Regulatory Landscapes in Development, Evolution and Disease.

Animal genomes are folded in topologically associating domains (TADs) that have been linked to the regulation of the genes they contain by constraining regulatory interactions between cis-regulatory elements and promoters. Therefore, TADs are proposed as structural scaffolds for the establishment of regulatory landscapes (RLs). In this review, we discuss recent advances in the connection between TADs and gene regulation, their relationship with gene RLs and their dynamics during development and differentiation. Moreover, we describe how restructuring TADs may lead to pathological conditions, which explains their high evolutionary conservation, but at the same time it provides a substrate for the emergence of evolutionary innovations that lay at the origin of vertebrates and other phylogenetic clades.

Assessing the conservation of Miombo timber species through an integrated index of anthropogenic and climatic threats.

AIM: Angolan Miombo woodlands, rich in timber species of the Leguminosae family, go through one of the highest rates of deforestation in sub-Saharan Africa. This study presents, on the basis of updated information of the distribution of Leguminosae timber species native to Angola, an integrated index framing the main threats for trees, which aims to support new conservation measures. LOCATION: Sub-Saharan Africa, Republic of Angola. METHODS: The current distribution areas of six Leguminosae timber species (i.e., Afzelia quanzensis, Brachystegia spiciformis, Guibourtia coleosperma, Isoberlinia angolensis, Julbernardia paniculata, and Pterocarpus angolensis) were predicted through ensemble modeling techniques. The level of threat to each species was analyzed, comparing the species potential distribution with a threat index map and with the protected areas. The threat index of anthropogenic and climatic factors encompasses the effects of population density, agriculture, proximity to roads, loss of tree cover, overexploitation, trends in wildfires, and predicted changes in temperature and precipitation. RESULTS: Our results revealed that about 0.5% of Angola's area is classified as of "Very high" threat, 23.9% as "High" threat, and 66.5% as "Moderate" threat. Three of the studied species require special conservation efforts, namely B. spiciformis and I. angolensis, which have a large fraction of predicted distribution in areas of high threat, and G. coleosperma since it has a restricted distribution area and is one of the most valuable species in international markets. The priority areas for the conservation of Leguminosae timber species were found in Benguela and Huíla. MAIN CONCLUSIONS: This study provides updated data that should be applied to inform policymakers, contributing to national conservation planning and protection of native flora in Angola. Moreover, it presents a methodological approach for the predictions of species distribution and for the creation of a threat index map that can be applied in other poorly surveyed tropical regions.

Step-by-step design of proteins for small molecule interaction: A review on recent milestones.

Protein design is the field of synthetic biology that aims at developing de novo custom-made proteins and peptides for specific applications. Despite exploring an ambitious goal, recent computational advances in both hardware and software technologies have paved the way to high-throughput screening and detailed design of novel folds and improved functionalities. Modern advances in the field of protein design for small molecule targeting are described in this review, organized in a step-by-step fashion: from the conception of a new or upgraded active binding site, to scaffold design, sequence optimization, and experimental expression of the custom protein. In each step, contemporary examples are described, and state-of-the-art software is briefly explored.

Genome-Wide Mapping of Histone H3 Lysine 4 Trimethylation (H3K4me3) and Its Involvement in Fatty Acid Biosynthesis in Sunflower Developing Seeds.

Histone modifications are of paramount importance during plant development. Investigating chromatin remodeling in developing oilseeds sheds light on the molecular mechanisms controlling fatty acid metabolism and facilitates the identification of new functional regions in oil crop genomes. The present study characterizes the epigenetic modifications H3K4me3 in relationship with the expression of fatty acid-related genes and transcription factors in developing sunflower seeds. Two master transcriptional regulators identified in this analysis, VIV1 (homologous to Arabidopsis ABI3) and FUS3, cooperate in the regulation of WRINKLED 1, a transcriptional factor regulating glycolysis, and fatty acid synthesis in developing oilseeds.

Functional Conservation of Divergent p63-Bound cis-Regulatory Elements.

The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence.

Pioneer and repressive functions of p63 during zebrafish embryonic ectoderm specification.

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.

The Nup84 complex coordinates the DNA damage response to warrant genome integrity.

DNA lesions interfere with cellular processes such as transcription and replication and need to be adequately resolved to warrant genome integrity. Beyond their primary role in molecule transport, nuclear pore complexes (NPCs) function in other processes such as transcription, nuclear organization and DNA double strand break (DSB) repair. Here we found that the removal of UV-induced DNA lesions by nucleotide excision repair (NER) is compromised in the absence of the Nup84 nuclear pore component. Importantly, nup84Δ cells show an exacerbated sensitivity to UV in early S phase and delayed replication fork progression, suggesting that unrepaired spontaneous DNA lesions persist during S phase. In addition, nup84Δ cells are defective in the repair of replication-born DSBs by sister chromatid recombination (SCR) and rely on post-replicative repair functions for normal proliferation, indicating dysfunctions in the cellular pathways that enable replication on damaged DNA templates. Altogether, our data reveal a central role of the NPC in the DNA damage response to facilitate replication progression through damaged DNA templates by promoting efficient NER and SCR and preventing chromosomal rearrangements.

Biomarkers of fungal lung infection.

PURPOSE OF REVIEW: The incidence of lung fungal infections, namely invasive pulmonary aspergillosis (IPA) and mucormycosis, is increasing in neutropenic and nonneutropenic patients. As they are a major cause of death, early diagnosis and antifungal therapy are crucial for outcome. The role of biomarkers in the management of this infections is the scope of this review. RECENT FINDINGS: Galactomannan in bronchoalveolar lavage shows the best discriminatory power for IPA diagnosis. At baseline, serum galactomannan may be useful to predict outcome and its kinetics may be informative to assess response to antifungal therapy. Recent standardization of PCR technology brought some improvements in IPA and mucormycosis diagnosis. Several new biomarkers are currently under investigation, but none showed a better performance than current available biomarkers. To improve diagnostic accuracy, a combination of biomarkers, including galactomannan, has been proposed. SUMMARY: Biomarkers may play an important role in the early diagnosis of fungal lung infections and in prognostic assessment and response monitoring, but more research is needed to determine the best strategy for their clinical use.

Donor-Recipient Identification in Para- and Poly-phyletic Trees Under Alternative HIV-1 Transmission Hypotheses Using Approximate Bayesian Computation.

Diversity of the founding population of Human Immunodeficiency Virus Type 1 (HIV-1) transmissions raises many important biological, clinical, and epidemiological issues. In up to 40% of sexual infections, there is clear evidence for multiple founding variants, which can influence the efficacy of putative prevention methods, and the reconstruction of epidemiologic histories. To infer who-infected-whom, and to compute the probability of alternative transmission scenarios while explicitly taking phylogenetic uncertainty into account, we created an approximate Bayesian computation (ABC) method based on a set of statistics measuring phylogenetic topology, branch lengths, and genetic diversity. We applied our method to a suspected heterosexual transmission case involving three individuals, showing a complex monophyletic-paraphyletic-polyphyletic phylogenetic topology. We detected that seven phylogenetic lineages had been transmitted between two of the individuals based on the available samples, implying that many more unsampled lineages had also been transmitted. Testing whether the lineages had been transmitted at one time or over some length of time suggested that an ongoing superinfection process over several years was most likely. While one individual was found unlinked to the other two, surprisingly, when evaluating two competing epidemiological priors, the donor of the two that did infect each other was not identified by the host root-label, and was also not the primary suspect in that transmission. This highlights that it is important to take epidemiological information into account when analyzing support for one transmission hypothesis over another, as results may be nonintuitive and sensitive to details about sampling dates relative to possible infection dates. Our study provides a formal inference framework to include information on infection and sampling times, and to investigate ancestral node-label states, transmission direction, transmitted genetic diversity, and frequency of transmission.