Miracle-dz/UniFAR-FTUs huggingface dataset:
Pathogen effector-associated nuclear-localization of RPW8.2 amplifies its expression to boost immunity in Arabidopsis

RESISTANCE TO POWDERY MILDEW 8 (RPW8) defines a unique N-terminal coiled-coil domain of nucleotide-binding and leucine-rich repeat immune receptors required for immune signaling in plants. Arabidopsis RPW8.2 is specifically induced by the powdery mildew (PM) fungus (Golovinomyces cichoracearum) in the infected epidermal cells to activate immunity. The mechanism of RPW8.2-induction is not well understood. Here, we identify a G. cichoracearum factor delivered to the nucleus of the host cell, named Gc-RPW8.2 interacting protein 1 (GcR8IP1). Ectopic expression of GcR8IP1 in Arabidopsis or Nicotiana benthamiana suppressed host immune responses and enhanced susceptibility to PM. Host-induced gene silencing of GcR8IP1 compromised PM infectivity in susceptible Arabidopsis plants. Co-expression of GcR8IP1 with RPW8.2 increased nuclear localization of RPW8.2, which in turn, promoted transcriptional amplification of RPW8.2. Thus, RPW8.2-dependent defense strengthening is due to altered partitioning of RPW8.2 by an effector of a PM fungus, which exemplifies an atypical form of effector-triggered immunity.
Perinatal correlated retinal activity is required for the wiring of visual axons in non-image forming nuclei

The formation of the visual system is a complex multistep process that includes proper assembly between retinal ganglion cell (RGC) axon terminals and their relay neurons in the different visual nuclei of the brain. RGC axons reach the main image-forming nuclei (IFN) —the superior colliculus and the lateral geniculate nucleus— at perinatal stages and extensively arborize to then refine throughout the first postnatal weeks. Spontaneous activity generated in the immature retina plays an essential role in the fine-tune refinement of exhuberant axonal arborizations but the molecular mechanisms underlying this activity-dependent remodeling process remain poorly characterized. RGC axons, in addition to innervate IFN, target non-image forming nuclei (NIFN), but the impact of spontaneous retinal activity in the development of these accessory nuclei has not yet been described. Here, by genetically altering spontaneous activity in the RGCs of mice we demonstrate that correlated retinal activity also shapes the connectivity of the non-image forming circuit and identify the transcriptional programs modulating this process. Together, our data contribute to a better understanding of the molecular mechanisms ruling activity-dependent axon refining in the building of visual circuits. with 25%-50%-80%-100% methanol/H2O for 20’ at RT, a bleaching in 3% H2O2 in methanol for 1h at RT, two washes in methanol, rehydration in 80%-50%-25% methanol/PBS 20’ each at RT and blocking in 5% BSA PBST (3% Tween) for 1 hour at RT. In situ hybridization for mRNA Syt13 was performed in P4 retinal cryosections and combined with anti- Pou4f2 antibodies (Chemicon).
Retromer oligomerization drives SNX‐BAR coat assembly and membrane constriction

The retromer coat mediates protein exit from endosomes and impacts many signaling pathways, lysosomal biogenesis, and diseases such as Parkinson’s, Alzheimer’s and COVID-19. Retromer complexes (CSC in yeast) form coats by interconnecting sorting nexins (SNX). The dynamics of this process is poorly explored. Here, we analyze the oligomerization of CSC/SNX-BAR retromer coats on oriented synthetic lipid tubules. SNX-BARs and CSC assemble a static tubular coat that does not exchange subunits. Coat formation proceeds bidirectionally, adding new subunits at both ends of the coat. High concentrations of SNX-BARs alone suffice to constrict membrane tubes to an invariant radius of 19 nm. At lower concentrations, CSC-complexes must drive constriction, which requires their oligomerization. CSCs populate the SNX-BAR layer at densities that increase with the starting radius of the membrane tube. We hence propose that retromer-mediated crosslinking of SNX-BARs at variable densities tunes the coat according to the energy required to deform the membrane. This model is supported by the effects of mutations interfering with retromer oligomerization, which impair retromer function in yeast and human cells.
ATM and MSH2 control blunt DNA end joining in immunoglobulin class switch recombination

Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain (Igh) gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene Msh2. Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele [Atmf] and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared to AtmD/Dand Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from AtmD/DMsh2-/-B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, AtmD/D, or Msh2-/- B cells. This data indicates that the absence of both ATM and MSH2 blocks non-homologous end joining (NHEJ), leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID. Summary Loss of the DNA repair genes Atm and Msh2 produces a novel synthetic lethality in mice. B cell specific deletion of Atm on an Msh2-/- background reduces Ig CSR and inhibits NHEJ.
Acinetobacter baumannii defends against oxidative stress through a Mn2+-dependent small RNA-mediated suppression of type VI secretion system

Acinetobacter baumannii employs a plethora of strategies during infection to compete with other pathogens and mitigate host-mediated oxidative stress. A. baumannii utilizes the type VI secretion system (T6SS) to induce contact-dependent killing off the competitor microbes. However, the role of T6SS during host-induced oxidative stress is not explored in A. baumannii. Here, we show that A. baumannii T6+ cells cannot cope with phagocytic cell-mediated oxidative stress due to inadequate uptake of Mn2+, which is crucial for bacterial physiology and reactive oxygen species (ROS) breakdown. Deleting the Mn2+-uptake system (MntH) causes a significant increase in the T6+ population, stipulating a deleterious effect on T6SS modulation in A. baumannii. Intriguingly, we identify a bonafide sRNA, AbsR28, that meditates the crosstalk between MntH and T6SS. This work elucidates a detailed mechanism of Mn2+-dependent AbsR28-mediated post-transcriptional repression of T6SS, exploited by A. baumannii to survive in the host and establish pathogenesis.
TROP2 Represents a Negative Prognostic Factor in Colorectal Adenocarcinoma and Its Expression Is Associated with Features of Epithelial–Mesenchymal Transition and Invasiveness

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions and its expression is maintained in most colorectal cancer (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli (Apc) tumor suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with transcription of genes involved in epithelial-mesenchymal transition, regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by β-catenin and additionally by Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/β-catenin signaling, YAP, and TROP2 expression. Simple Summary Colorectal cancer (CRC) is one of the most common cancers worldwide. While systemic treatment of CRC is based on chemotherapy, subsequent therapeutic options are far less effective. Trophoblast cell surface antigen 2 (TROP2) is highly expressed in many carcinomas, including CRC, where its expression correlates with poor prognosis. Anti-TROP2-targeted therapy was approved for the treatment of breast and urothelial carcinomas. We aimed to determine whether TROP2 is a suitable target for the treatment of CRC. We demonstrated that TROP2 expression in CRC correlates with lymph node metastasis and poor tumor differentiation. Analysis of mouse tumor models, patient-derived organoids, and tumor cells revealed that TROP2 expression is associated with features related to epithelial-mesenchymal transition and invasiveness. Our results suggest that TROP2 targeting may be a promising approach, especially in the early phase of treatment.
Improved Nanopore full-length cDNA sequencing by PCR-suppression

Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads. Contribution to the field Long-read RNA sequencing aims to sequence expressed transcripts in their entirety. However, this has remained a challenge, mainly due to inherent inefficiencies in cDNA library preparation. Herein, we provide a new Nanopore cDNA library preparation protocol, termed Panhandle, that improves the efficiency of cDNA PCR with yields 2 – 8 times the yields obtained with ordinary PCR. This is key, as this should in turn reflect in the possibility of lowering the number of PCR cycles needed to obtain ample sequencing material, which in turn could reduce PCR biases, PCR artifacts, turnaround time, reagents, and could increase general quality of the library. Further, transcripts generated using the Panhandle method show better gene body coverage and more accurate transcription start site mapping than regular methods. This represents an important step towards full-length cDNA sequencing by Nanopore.
Distance‐dependent distribution thresholding in probabilistic tractography

Tractography is widely used in human studies of connectivity with respect to every brain region, function, and is explored developmentally, in adulthood, aging, and in disease. However, the core issue of how to systematically threshold, taking into account the inherent differences in connectivity values for different track lengths, and to do this in a comparable way across studies has not been solved. The study adopted Monte Carlo derived distance-dependent distributions (DDDs) to generate distance-dependent thresholds with various levels of alpha for connections of varying lengths. As a test case, we applied the DDD approach to generate a language connectome. The resulting connectome showed expected short- and long-distance structural connectivity in the close and distant regions within the language network. The finding demonstrates that the DDD approach can be used for both individual and group thresholding. Critically, it offers a standard method that can be applied to various probabilistic tracking datasets.
Infection- or vaccine mediated immunity reduces SARS-CoV-2 transmission, but increases competitiveness of Omicron in hamsters

Omicron has demonstrated a competitive advantage over Delta in vaccinated people. To understand this, we designed a transmission chain experiment using naïve, intranasally (IN) or intramuscularly (IM) vaccinated, and previously infected (PI) hamsters. Vaccination and previous infection protected animals from disease and virus replication after Delta and Omicron dual challenge. A gradient in transmission blockage was observed: IM vaccination displayed moderate transmission blockage potential over three airborne chains (approx. 70%), whereas, IN vaccination and PI blocked airborne transmission in >90%. In naïve hamsters, Delta completely outcompeted Omicron within and between hosts after dual infection in onward transmission. Although Delta also outcompeted Omicron in the vaccinated and PI transmission chains, an increase in Omicron competitiveness was observed in these groups. This correlated with the increase in the strength of the humoral response against Delta, with the strongest response seen in PI animals. These data highlight the continuous need to assess the emergence and spread of novel variants in populations with pre-existing immunity and address the additional evolutionary pressure this may exert on the virus.
Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone

Colony stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and thus an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 stimulating osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Single cell RNA- sequencing indicated specific expression of Csf1 in MALPs, which is further increased during aging. To investigate its role, we constructed Csf1 CKO mice using Adipoq-Cre. These mice showed increased femoral trabecular bone over time, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prx1-Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs are a critical player in controlling bone remodeling and hematopoiesis.
Comprehensive analysis of the transcription factor REST regulatory networks in IDH-mutant and IDH-wild type glioma cells and gliomas

REST is a widely expressed, dual role transcription factor that acts either as a transcriptional repressor or transcriptional activator depending on the genomic and cellular context. REST is an important oncogenic factor, a key player in brain cell differentiation and has a role in establishing DNA methylation status in proximity of its binding sites. Mutations in IDH cause significant changes to the epigenome contributing to blocking cell differentiation and are considered an oncogenic driver in glioma. We aimed at defining the REST role in the IDH mutation-related phenotype in gliomas accounting for its role in gene activation and repression. We studied the effects of REST knockdown, REST binding sites, and REST motifs methylation in context of IDH mutation, and found that both REST binding patterns and TF motif composition proximal to REST binding sites differed in IDH wild-type and mutant glioma. Among such REST targets were genes involved in glial cell differentiation and ECM organization. REST knockdown differently impacted glioma cell invasion depending on the IDH phenotype. DNA methylation of REST activated gene promoters showed positive correlation with gene expression. The canonical REST-repressed gene targets correlated with NPC-like cellular state properties in IDH-MUT grade 2/3 gliomas. The identified REST targets, gene regulatory networks and putative REST cooperativity with other TFs point to differential control of REST target gene expression in IDH-WT and IDH-MUT gliomas. We conclude that REST could be considered as a key factor in the design of targeted glioma therapies.
Unbiased identification of unknown cellular and environmental factors that mediate eQTLs using principal interaction component analysis

Expression quantitative trait loci (eQTL) can reveal the regulatory mechanisms of trait associated variants. eQTLs are highly cell-type and context-specific, but often these contexts are unknown or not measured. Here, we introduce PICALO (Principal Interaction Component Analysis through Likelihood Optimization), an unbiased method to identify known and hidden contexts that influence eQTLs. PICALO uses expectation maximization to identify latent components, referred to as Principal Interaction Components (PIC), that interact with genotypes to maximize explained eQTL effect-sizes. We applied PICALO to bulk RNA-seq eQTL datasets in blood (n=2,932) and brain (n=2,440). We identify 31 PICs in blood, interacting with 4,169 (32%) unique cis-eQTLs (BH-FDR≤0.05). In brain, we identified 21 PICs, interacting with 4,058 (39%) unique cis-eQTLs (BH-FDR≤0.05). These PICs are associated with RNA quality, cell type composition or environmental influences. Furthermore, PICs clearly disentangle distinct eQTL contexts, for example technical from non-technical factors. Combined, 3,065 unique genes showed a cis-eQTL effect that is dependent on a cell type or other non-technical context, emphasizing the value of methods like PICALO. PICALO is robust, works well with heterogeneous datasets, yields reproducible interaction components, and identifies eQTL interactions and contexts that would have been missed when using cell counts or expression based principal components. Since PICALO allows for the identification of many context-dependent eQTLs without any prior knowledge of such contexts, this method can help to reveal and quantify the influence of previously unknown environmental factors that play a role in common diseases.
Genetic diversity, population structure and parentage analysis of Brazilian grapevine hybrids after half a century of genetic breeding

In the 1940s, the Agronomic Institute of Campinas (IAC) started a grapevine breeding program to develop new cultivars adapted to the tropical and subtropical regions of Brazil. More than 2,000 crosses were carried out over 50 years, using 850 varieties as parents. However, among the thousands of hybrids developed by the program, only 130 are still maintained in the IAC grapevine germplasm collection. Little is known about their genetic makeup and usefulness for current breeding programs. In this study, we genotyped 130 Brazilian grape hybrids at 21 polymorphic microsatellite markers to evaluate the genetic diversity and population structure of the hybrids and verified their disclosed pedigrees. The results showed that the hybrid collection is highly diverse, with an expected heterozygosity (HE) of 0.80 and an observed heterozygosity (Ho) of 0.78. Strong structure in three subgroups based mainly on the usage and combination of parental groups was revealed by STRUCTURE software and confirmed by discriminant analysis of principal components (DAPC). Through molecular profiling analysis, fourteen synonyms, one homonym and one duplicate were identified. Parentage analysis confirmed 24 full parentages, as well as 33 half-kinships. In addition, 18 pedigrees were invalidated, and seven mislabeling events were identified. No compatible parent was identified for 33% of the IAC hybrids, highlighting severe genetic erosion in the IAC germplasm. The molecular characterization of the breeding hybrid bank collection contributes to our understanding of the genetic basis of the varieties, guiding the efficient utilization of available genetic diversity. Together, our results could be applied to other breeding programs and assist in the selection of parents, management of the breeding collection, and conservation of grapevine genetic resources.
The nervous system leverages the non-linear properties of the Achilles tendon to regulate ankle impedance during postural control

Regulating ankle impedance is essential for controlled interactions with the environment and rejecting unexpected disturbances. Ankle impedance in the sagittal plane depends strongly on the triceps surae and Achilles tendon, but their relative contributions remain unknown. It is commonly assumed that ankle impedance is controlled by changing muscle activation and, thereby, muscle impedance, but this ignores the fact that tendon impedance also changes with loading caused by increases in muscle activation. Thus, we sought to determine the relative contributions from the triceps surae and Achilles tendon during conditions relevant to postural control. We used a novel technique that combines B-mode ultrasound imaging with joint-level perturbations to quantify ankle, muscle, and tendon impedance simultaneously across activation levels from 0 – 30% of maximum voluntary contraction. We found that muscle and tendon stiffness, the static component of impedance, increased with voluntary plantarflexion contractions, but that muscle stiffness exceeded tendon stiffness at very low loads (21 ± 7 N). Above these loads, corresponding to 1.3% of maximal strength for an average participant in our study, ankle stiffness was determined predominately by Achilles tendon stiffness. Hence, the nervous system leverages the non-linear properties of the Achilles tendon to increase ankle stiffness during postural conditions.