Miracle-dz/UniFAR-FTUs huggingface dataset:
S-Acylation of plant immune receptors mediates immune signaling in plasma membrane nanodomains

S-Acylation is a reversible protein post-translational modification mediated by Protein S-acyltransferases (PATs). Here, we demonstrate that three plant immune receptors P2K1 (DORN1), FLS2 and CERK1, representing three distinct families of receptor like-kinases, are S-acylated by Arabidopsis PAT5 and PAT9 on the plasma membrane (PM). Mutations in Atpat5 and Atpat9 resulted in an elevated plant immune response, increased phosphorylation and decreased degradation of FLS2, P2K1 and CERK1 during immune signaling. Mutation of key cysteine residues S-acylated in these receptors resulted in a similar phenotype as exhibited by Atpat5/Atpat9 mutant plants. The data indicate that S-acylation also controls localization of the receptors in distinct PM nanodomains and, thereby, controls the formation of specific protein-protein interactions. Our study reveals that S-acylation plays a critical role in mediating spatiotemporal dynamics of plant receptors within PM nanodomains, suggesting a key role for this modification in regulating the ability of plants in respond to external stimuli.
Gross, histological and histochemical observations on the gland of third eyelid in buffalo calf (Bubalus bubalis).

The present study was conducted on 10 male buffalo calves of non-descript breed having 1-2 year of age. The gland of third eyelid was located at medial canthus of eye. It was consisted of two parts viz: superficial or cartilaginous part or nictitans gland and deep or non cartilaginous part or harderian gland. The gland was compound tubuloalveolar type and mixed in nature. The capsule of the gland was rich in collagen and reticular fibres. The reticular fibres supported the basement membrane of epithelium of all acini. Myoepithelial cells were observed between basement membrane and nucleus of acinar cells. The acinar cell cytoplasm, basement membrane and ductular epithelium showed strong periodic acid schiff reaction. Mucous acini showed strong reaction to acid mucopolysaccharide while serous acini were poorly stained with acid mucopolysaccharide. Bound lipids were observed in the basement membrane of acinar epithelium. In cartilaginous part the hyaline cartilage had lacunae filled with one or two chondrocytes; some chondrocytes were in dividing stage. The collagen fibres were observed in perichondrium of cartilage.
Morphogenesis is transcriptionally coupled to neurogenesis during olfactory placode development

Morphogenesis of sense organs occurs concomitantly with the differentiation of sensory cells and neurons necessary for their function. While our understanding of the mechanisms controlling morphogenesis and neurogenesis has grown, how they are coordinated remains relatively unknown. The earliest wave of neurogenesis in the zebrafish olfactory placode requires the bHLH proneural transcription factor Neurogenin1 (Neurog1). To address whether Neurog1 couples neurogenesis and morphogenesis in this system, we analyzed the morphogenetic behavior of early olfactory neural progenitors. Our results indicate that the oriented movements of these progenitors are disrupted in neurog1 mutants. Morphogenesis is similarly affected by mutations in the chemokine receptor, cxcr4b, making it a potential Neurog1 target gene. We find that Neurog1 directly regulates cxcr4b through an E-boxes cluster located just upstream of the cxcr4b transcription start site. Our results suggest that proneural transcription factors, such as Neurog1, directly couple distinct aspects of nervous system development. One Sentence Summary Neurog1 controls olfactory placode morphogenesis via cxcr4b
Correction de mutations causant l’épidermolyse bulleuse simplex par recombinaison homologue avec la technologie CRISPR/Cas9

L’epidermolyse bulleuse simplex (EBS) fait partie d’un regroupement de maladies hereditaires dont le tissu cible est la peau. Cette maladie dermatologique est causee par une mutation dans les genes KRT5 ou KRT14, laquelle entraine un probleme au niveau de la formation des filaments intermediaires de la keratine qui composent la peau. Un mesappariement au niveau des keratines 5 et 14 est responsable de l’apparition des symptomes de la maladie incluant la formation de cloques suite au trauma mecanique que subit la peau. Ces cloques entrainent d’importantes douleurs chez le patient, et de l’inflammation et de l’irritation. Actuellement, aucun traitement curatif n’est disponible pour soulager les patients atteints de la maladie. Depuis quelques annees, les chercheurs utilisent des technologies pour modifier directement l’ADN des patients afin de reparer la mutation responsable de l’apparition des symptomes. Des technologies d’edition du genome telles que CRISPR/Cas9 constituent donc une avenue prometteuse pour le traitement de maladies hereditaires monogeniques comme l’EBS. Cette technologie permet de cibler un endroit precis dans le genome par la combinaison d’un ARN guide simple brin et d’une proteine Cas9. La reparation de l’ADN se fait par l’induction d’une coupure double brin de l’ADN par la proteine Cas9 et sa reparation par un fragment d’ADN donneur double ou simple brin. Ce projet a pour objectif principal de corriger les mutations causant l’EBS chez deux patients possedant des mutations heterozygotes. Plus specifiquement, ce projet de maitrise a pour objectif d’optimiser la methode permettant l’utilisation de CRISPR/Cas9 dans un contexte d’EBS. La technologie CRISPR/Cas9 a ete utilisee sur des cellules HEK293T, permettant de demontrer que la Cas9 est en mesure d’induire une coupure double brin de l’ADN au site cible dans l’ADN. Ensuite, il a ete demontre que la coupure de la Cas9 jumelee a l’utilisation d’un ADN double ou simple brin permet de reparer la coupure en realisant de la recombinaison homologue. Le sequencage a permis de confirmer ces resultats. A ce jour, les resultats dans les cellules HEK293T se sont montres concluants et ont permis d’initier les travaux dans les fibroblastes de patients.
Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we developed methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlighted one of three copies of histone H3 genes for which transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation caused defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial ‘wake-up’ trigger at dormancy breaking.
Cooling and freezing effects on development of flies of forensic importance

Abstract. Cooling and freezing experiments were carried out at Oswaldo Cruz Foundation (Fiocruz / RJ). Flies colonies were established from specimens collected at the campus using a baited trap, as described for the species: Chrysomya putoria (Wiedemann), Chrysomya megacephala (Fabricius), Lucilia cuprina (Wiedemann), Musca domestica Linnaeus, Peckia ruficornis (Fabricius) and Peckia chrysostoma (Wiedemann). Prolonged exposures at lethal low temperatures can produce deleterious effects (including death) even if the insect does not freeze; during long-term exposure to low temperatures, the organisms may finally die from the exhaustion of energy reserves.Efeitos do resfriamento e congelamento no desenvolvimento de moscas de importância forenseResumo. Experimentos de resfriamento e congelamento foram realizados na Fundação Oswaldo Cruz (Fiocruz / RJ). Colônias de moscas foram estabelecidas a partir de espécimes coletados no campus usando uma armadilha com isca, conforme descrito para as espécies: Chrysomya putoria (Wiedemann), Chrysomya megacephala (Fabricius), Lucilia cuprina (Wiedemann), Musca domestica Linnaeus, Peckia ruficornis (Fabricius) e Peckia chrysostoma (Wiedemann). Exposições prolongadas em baixas temperaturas podem produzir efeitos deletérios (incluindo morte) mesmo se o inseto não congelar; durante a exposição prolongada a baixas temperaturas, os organismos podem morrer pelo esgotamento das reservas de energia.
Genomic sequencing and recombinant expression of proinsulin isolated from cow and buffalo in Pakistan

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector. There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo. Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies. Key words: Bubalus bubalis, Bos taurus, cDNA, T/A cloning vector, BL21 Codon plus (DE3)-RIL cells.
MIXTURE: an improved algorithm for immune tumor microenvironment estimation based on gene expression data

RNA sequencing has proved to be an efficient high-throughput technique to robustly characterize the presence and quantity of RNA in tumor biopsies at a given time. Importantly, it can be used to computationally estimate the composition of the tumor immune infiltrate and to infer the immunological phenotypes of those cells. Given the significant impact of anti-cancer immunotherapies and the role of the associated immune tumor microenvironment (ITME) on its prognosis and therapy response, the estimation of the immune cell-type content in the tumor is crucial for designing effective strategies to understand and treat cancer. Current digital estimation of the ITME cell mixture content can be performed using different analytical tools. However, current methods tend to over-estimate the number of cell-types present in the sample, thus under-estimating true proportions, biasing the results. We developed MIXTURE, a noise-constrained recursive feature selection for support vector regression that overcomes such limitations. MIXTURE deconvolutes cell-type proportions of bulk tumor samples for both RNA microarray or RNA-Seq platforms from a leukocyte validated gene signature. We evaluated MIXTURE over simulated and benchmark data sets. It overcomes competitive methods in terms of accuracy on the true number of present cell-types and proportions estimates with increased robustness to estimation bias. It also shows superior robustness to collinearity problems. Finally, we investigated the human immune microenvironment of breast cancer, head and neck squamous cell carcinoma, and melanoma biopsies before and after anti-PD-1 immunotherapy treatment revealing associations to response to therapy which have not seen by previous methods.
Promiscuous Chemokine Antagonist (BKT130) Suppresses Laser-Induced Choroidal Neovascularization by Inhibition of Monocyte Recruitment

Background Age-related macular degeneration (AMD), the most common cause of blindness in the developed world, usually affects individuals older than 60 years of age. The majority of visual loss in this disease is attributable to the development of choroidal neovascularization (CNV). Mononuclear phagocytes, including monocytes and their tissue descendants, macrophages, have long been implicated in the pathogenesis of neovascular AMD (nvAMD). Current therapies for nvAMD are based on targeting vascular endothelial growth factor (VEGF). This study is aimed at assessing if perturbation of chemokine signaling and mononuclear cell recruitment may serve as novel complementary therapeutic targets for nvAMD. Methods A promiscuous chemokine antagonist (BKT130), aflibercept treatment, or combined BKT130+aflibercept treatment was tested in an in vivo laser-induced model of choroidal neovascularization (LI-CNV) and in an ex vivo choroidal sprouting assay (CSA). Quantification of CD11b+ cell in the CNV area was performed, and mRNA levels of genes implicated in CNV growth were measured in the retina and RPE-choroid. Results BKT130 reduced the CNV area and recruitment of CD11b+ cells by 30-35%. No effect of BKT130 on macrophages' proangiogenic phenotype was demonstrated ex vivo, but a lower VEGFA and CCR2 expression was found in the RPE-choroid and a lower expression of TNFα and NOS1 was found in both RPE-choroid and retinal tissues in the LI-CNV model under treatment with BKT130. Conclusions Targeting monocyte recruitment via perturbation of chemokine signaling can reduce the size of experimental CNV and should be evaluated as a potential novel therapeutic modality for nvAMD.
Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

Kaposi’s sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production. IMPORTANCE KSHV ORF66, a late gene product, and vPIC are thought to contribute significantly to late gene expression during the lytic replication. However, the physiological importance of ORF66 in terms of viral replication and vPIC formation remains poorly understood. Therfore, we generated a ORF66-deficient BAC clone and evaluated its viral replication. Results showed that ORF66 played a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing ORF66 function in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacted with the transcription start site of K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among β- and γ-herpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.
Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications

Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cell’s morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work. Statement of significance Actively patterned illumination in fluorescence microscopy can reduce bleaching and phototoxicity as well as actively manipulate proteins in optogenetic applications. Matching the coordinate system of the camera and the light patterning device such as digital mirror arrays (DMA) remains a challenge. We developed a fast and easy calibration procedure that determines and corrects for the transformation between the camera and DMA coordinate system with ~50 nm precision. Using this approach, we develop spatial feedback photoactivation for Single Molecule Localization Microscopy (SMLM) to photoswitch only intracellular regions containing photoswitchable fluorophores. Our results show a 44% improvement in the possible data acquisition time before phototoxic effects become detectable and a 28% increase in detected localizations. Spatial feedback photoactivation thus significantly improves SMLM experiments.
Impact of Colletotrichum acutatum Pathogen on Olive Phenylpropanoid Metabolism

Olive anthracnose caused by the hemibiotrophic fungal pathogen Colletotrichum acutatum is a serious threat to the olive sector. Olive oil and fruit production is severely constrained by Colletotrichum spp. infection, being C. acutatum the most distributed pathogen in Portuguese olive orchards. To understand the impact of C. acutatum on phenylpropanoids biosynthesis, the enzyme activity, phenolic compounds, ortho-diphenols, and flavonoids content were determined and correlated with the expression of gene encoding key enzymes within phenylpropanoids metabolism in susceptible and tolerant olive fruits, during maturation and when infected with C. acutatum. Differences between cultivars was observed, the tolerant olive cv. Picual presented a higher basal value and a stable phenolic content throughout the infection process, supporting its high C. acutatum tolerance, whereas in the susceptible olive cv. Galega these secondary metabolites were significantly increased only after the elicitation with C. acutatum.
Hyperglycemia Enhances Cancer Immune Evasion by Inducing Alternative Macrophage Polarization through Increased O-GlcNAcylation

Hyperglycemia increases O-GlcNAc in tumor-associated macrophages, shifting them to a protumor phenotype (M2-like) and contributing to cancer progression. Inhibition of O-GlcNAcylation reprograms intratumoral macrophages to an antitumoral phenotype, slowing tumor growth. Diabetes mellitus (DM) significantly increases the risk for cancer and cancer progression. Hyperglycemia is the defining characteristic of DM and tightly correlates with a poor prognosis in patients with cancer. The hexosamine biosynthetic pathway (HBP) is emerging as a pivotal cascade linking high glucose, tumor progression, and impaired immune function. Here we show that enhanced glucose flow through the HBP drives cancer progression and immune evasion by increasing O-GlcNAcylation in tumor-associated macrophages (TAM). Increased O-GlcNAc skewed macrophage polarization to a M2-like phenotype supporting tumor progression. Finally, we found an upregulation of M2 markers on TAMs in DM2 patients with colorectal cancer compared with nondiabetic normoglycemic patients. Our results provide evidence for a new and targetable mechanism of cancer immune evasion in patients with hyperglycemia, advocating for strict control of hyperglycemia in patients with cancer.
Complete mitochondrial genome of tiger cowrie Cypraea tigris (Linnaeus, 1758)

Abstract The complete mitochondrial genome of Cypraea tigris from the South China Sea has been determined, which was the first report of complete mitogenome in the superfamily Cypraeoidea. It is 16,177 bp long and consists of 21 tRNA genes, 2 rRNA genes, 13 protein-coding genes (PCGs), and 1 control region. The base composition of C. tigris mitogenome is biased (A, G, T, and C were 28.8, 17.9, 37.1, and 16.3%, respectively) with A + T contents of 58.5%. All of the PCGs use a typical start codon (ATN) and a complete stop codon (TAA or TAG) as normal. The maximum-likelihood tree demonstrated that the Cypraeoidea was closer to the superfamily Tonnoidea, and further clarified the phylogenetic relationships of each superfamily in Littorinimorpha.
Stochasticity-induced stabilization in ecology and evolution

The ability of random environmental variation to stabilize competitor coexistence was pointed out long ago and, in recent years, has received considerable attention. Here we suggest a novel and generic synthesis of stochasticity-induced stabilization (SIS) phenomena. The storage effect in the lottery model, together with other well-known examples drawn from population genetics, microbiology and ecology, are placed together, reviewed, and explained within a clear, coherent and transparent theoretical framework. Implementing the diffusion approximation we show that in all these systems (including discrete and continuous dynamics, with overlapping and non-overlapping generations) the ratio between the expected growth and its variance governs both qualitative and quantitative features of persistence and invasibility. We further clarify the relationships between bet-hedging strategies, generation time and SIS, study the dynamics of extinction when SIS fails and the explain effects of species richness and asymmetric competition on the stabilizing mechanism.