Miracle-dz/UniFAR-FTUs huggingface dataset:
Deconstructing Complexity: A Computational Topology Approach to Trajectory Inference in the Human Thymus with tviblindi

Understanding complex, organ-level single-cell datasets represents a formidable interdisciplinary challenge. This study aims to describe developmental trajectories of thymocytes and mature T cells. We developed tviblindi, a trajectory inference algorithm that integrates several autonomous modules - pseudotime inference, random walk simulations, real-time topological classification using persistent homology, and autoencoder-based 2D visualization using the vaevictis algorithm. This integration facilitates interactive exploration of developmental trajectories, revealing not only the canonical CD4 and CD8 development but also offering insights into checkpoints such as TCRβ selection and positive/negative selection. Furthermore, it allows us to thoroughly characterize thymic regulatory T cells, tracing their development from the negative selection stage to mature thymic regulatory T cells with an extensive proliferation history and an immunophenotype of activated and recirculating cells. tviblindi is a versatile and generic approach suitable for any mass cytometry or single-cell RNA-seq dataset, equipping biologists with an effective tool for interpreting complex data.
On the variability of dynamic functional connectivity assessment methods

Dynamic functional connectivity (dFC) has become an important measure for understanding brain function and as a potential biomarker. However, various methodologies have been developed for assessing dFC, and it is unclear how the choice of method affects the results. In this work, we aimed to study the results variability of commonly-used dFC methods. We implemented seven dFC assessment methods in Python and used them to analyze fMRI data of 395 subjects from the Human Connectome Project. We measured the pairwise similarity of dFC results using several similarity metrics in terms of overall, temporal, spatial, and inter-subject similarity. Our results showed a range of weak to strong similarity between the results of different methods, indicating considerable overall variability. Surprisingly, the observed variability in dFC estimates was comparable to the expected natural variation over time, emphasizing the impact of methodological choices on the results. Our findings revealed three distinct groups of methods with significant inter-group variability, each exhibiting distinct assumptions and advantages. These findings highlight the need for multi-analysis approaches to capture the full range of dFC variation. They also emphasize the importance of distinguishing neural-driven dFC variations from physiological confounds, and developing validation frameworks under a known ground truth. To facilitate such investigations, we provide an open-source Python toolbox that enables multi-analysis dFC assessment. This study sheds light on the impact of dFC assessment analytical flexibility, emphasizing the need for careful method selection and validation, and promoting the use of multi-analysis approaches to enhance reliability and interpretability of dFC studies.
Voltage tunes mGlu5 receptor function, impacting synaptic transmission

Voltage sensitivity is a common feature of many membrane proteins, including some G-protein coupled receptors (GPCRs). However, the functional consequences of voltage sensitivity in GPCRs are not well understood. In this study, we investigated the voltage sensitivity of the post-synaptic metabotropic glutamate receptor mGlu5 and its impact on synaptic transmission. Using biosensors and electrophysiological recordings in non-excitable HEK293T cells or neurons, we found that mGlu5 receptor function is optimal at resting membrane potentials. We observed that membrane depolarization significantly reduced mGlu5 receptor activation, Gq-PLC/PKC stimulation, Ca2+ release, and mGlu5 receptor-gated currents through TRPC6 channels or NMDA receptors. Notably, we report a previously unknown activity of the NMDA receptor at the resting potential of neurons, enabled by mGlu5. Our findings suggest that mGlu5 receptor activity is directly regulated by membrane voltage which may have a significant impact on synaptic processes and pathophysiological functions.
Divergent evolution of head morphology between marine and freshwater sticklebacks

Intraspecific phenotypic differentiation is of common place occurrence, but the degree to which it reflects phenotypic plasticity or local adaptation remains often unclear. To be considered as adaptive, the differentiation must be genetically based and exceed what could be expected by neutral processes only. Using laboratory reared full-sib family data from replicate nine-spined stickleback (Pungitius pungitius) populations, we show that freshwater and marine fish display genetically based adaptive differentiation in head size and shape. Utilising identity-by-descent relationships among full-sibs as estimated with the aid of molecular markers, we further show that the studied traits are also highly heritable in all populations indicating and that they can respond to future episodes of natural selection. The head shape and size of pond fish suggests that observed adaptive differentiation has been driven by selection favoring limnetic feeding strategy among the pond fish. Analyses of gill-raker morphology were less conclusive: genetic differentiation was found in gill-raker length (pond > marine) and number, but the degree of divergence in these traits did not exceed neutral expectations. Yet, the direction of divergence in gill raker traits are suggestive of the limnetic feeding mode of pond fish, aligning with the inference from the head morphology analyses.
Interoceptive signals shape the earliest markers and neural pathway to awareness at the visual threshold

Conscious awareness for threshold-stimuli fluctuates stochastically with the pre-stimulus brain state and variations of interoceptive signals across the cardiac and respiratory cycle. It remains debated whether interoceptive signals are suppressed or not and at what stages of processing they affect awareness in different sensory modalities. Because cardiac and respiratory activity are coupled through respiratory sinus arrythmia (RSA), we investigated the influence of the cardiac (systole/diastole) and the respiratory (inhalation/exhalation) phase on awareness-related ERPs. Subjects discriminated visual threshold stimuli while their EEG, ECG and respiration were simultaneously recorded. We compared ERPs and their intracranial generators for stimuli identified correctly with and without awareness as a function of the cardiac and respiratory phase. Cyclic variations of interoceptive signals from the baroreceptors (BRs) modulated both the earliest electrophysiological markers and the trajectory of brain activity when subjects became aware of the stimuli: the P1 was the earliest marker of awareness for low (diastole/inhalation) and the VAN for high (systole/exhalation) BR activity, indicating that interoceptive signals interfere with sensory processing of the visual input. Likewise, activity spread from the anterior insula to prefrontal cortex during low and from the posterior insula to posterior parietal cortex during high BR activity, providing a new solution to the debate whether activity in prefrontal or posterior parietal cortex is crucial for awareness: these regions were differentially recruited as a function of cyclic variations of BR activity rather than task demands. Our results suggest that the cardiac and respiratory rhythms are important rhythms that shape awareness-related brain activity. Significance Statement The brain continuously processes stimuli from inside and outside the body, and interoceptive stimuli can modulate the perception of external stimuli. Cardiac and respiratory rhythms are important pacemakers of the organism and we show how they shape awareness-related brain activity for visual threshold stimuli in two ways. Variations of baroreceptor (BR) activity across the cardiac and respiratory cycle affect 1) the earliest electrophysiological marker (P1 for low (diastole/inhalation), VAN for high (systole/exhalation) BR activity) and 2) the brain areas activated (frontal cortex for low and parietal cortex for high BR activity) when subjects become aware of a stimulus. We propose to consider cardiac and respiratory activity as important modulators of brain activity rather than dismissing them as noise.
Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.
Impact of angiotensin-receptor blockers on intrarenal renin-angiotensin system activity in hypertension: A PK/PD modelling study

The renin-angiotensin system (RAS) is a primary regulator of volume homeostasis and blood pressure, whose over-activation is commonly associated with hypertension. Indeed, medications that target the RAS are generally effective in reducing blood pressure. However, more can be learned about how these medications influence the intrarenal RAS. Angiotensin-receptor blockers (ARBs) in particular have been shown to exert different effects on the intrarenal and systemic RASs in various experimental models of hypertension. In rats chronically infused with angiotensin II (Ang II), ARBs consistently prevent intrarenal, but not systemic Ang II levels from rising. The former effect is sufficient in preventing the development of hypertension. The regulation of intrarenal RAS, independently of the systemic RAS, by ARBs has been hypothesized to be mediated by the inhibition of all positive feedback loops inherent to the intrarenal RAS, also known as the “key point breakdown effect. ” To investigate the validity of this hypothesis, we developed a PK/PD model of the ARB Losartan that considers the kidney, and applied the model to study how this class of medication influences intrarenal RAS activity and consequently blood pressure regulation in male rats. Simulations indicate that ARBs more effectively inhibit the activation of the intrarenal RAS because, unlike in the plasma, this process relies on the accumulation of cell-associated Ang II. We hypothesize that it is by blocking this intracellular uptake pathway, and restricting Ang II to extracellular regions of the kidney where the peptide cannot initiate downstream signalling, that Losartan normalizes blood pressure. While the key point break down effect assists in this response, it alone is not sufficient. Our results highlight the intrarenal RAS as the key pharmacological target of ARB treatment and emphasize the importance of this local tissue RAS in the development of hypertension.
Activity of natural occurring entomopathogenic fungi on nymphal and adult stage of Philaenus spumarius

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia and Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P. spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius cadavers collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on the adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P. spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave the way for testing EPF isolates against P. spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.
Neurotrophin-3 from the dentate gyrus supports postsynaptic sites of mossy fiber-CA3 synapses and contextual memory

At the center of the hippocampal tri-synaptic loop are synapses formed between mossy fiber (MF) terminals from granule cells in the dentate gyrus (DG) and proximal dendrites of CA3 pyramidal neurons. However, the molecular mechanism regulating the development and function of these synapses is poorly understood. In this study, we showed that neurotrophin-3 (NT3) was expressed in nearly all mature granule cells but not CA3 cells. We selectively deleted the NT3-encoding Ntf3 gene in the DG during the 1st two postnatal weeks to generate a Ntf3 conditional knockout (Ntf3-cKO). Ntf3-cKO mice had normal hippocampal cytoarchitecture but displayed elevated anxiety level and impairments in contextual memory, spatial reference memory and nest building. As MF-CA3 synapses are essential for encoding of contextual memory, we examined synaptic transmission at these synapses using ex vivo electrophysiological recordings. We found that Ntf3-cKO mice showed impaired basal synaptic transmission due to deficits in excitatory postsynaptic currents mediated by AMPA receptors but normal presynaptic function and intrinsic excitability of CA3 pyramidal neurons. Consistent with this selective postsynaptic deficit, Ntf3-cKO mice had fewer and smaller thorny excrescences on proximal apical dendrites of CA3 neurons and lower GluR1 levels in the stratum lucidum area where MF-CA3 synapses reside but normal MF terminals, compared with control mice. Thus, our study indicates that NT3 expressed in the dentate gyrus is crucial for the postsynaptic structure and function of MF-CA3 synapses and hippocampal-dependent memory.
Rapid identification of key genes for the rod-shaped morphology in bacteria using multi-species genomes

Accurate identification of key genes is pivotal in biological research. Here, we introduce machine learning to the field of functional gene identification, enabling precise prediction of bacterial shape based on genomic information. Our machine learning model successfully predicts bacterial shape, and we determine the influence of various protein domains on shape using the model. This approach facilitates the identification of candidate genes involved in regulating bacterial shape. Through targeted knockout experiments on eight potential key regulatory genes (pal, yicC, mreB, tolQ, ftsX, amiC, yddB, and rpoZ) in Escherichia coli, we observe significant alterations in rod-shaped morphology upon individual knockout of pal and mreB genes. E. coli transitions from rod-shaped to spherical or cell wall-deficient protoplasmic states. Experimental validations validate the robustness of our newly developed method. This study establishes an innovative avenue for exploring functional genes, harnessing large-scale genomic information to promptly uncover key genes governing shared traits across species.
Distinct senescence mechanisms restrain progression of dysplastic nevi

Abstract Telomerase reverse transcriptase (TERT) promoter mutations (TPMs) are frequently found in different cancer types, including ∼70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma-sequencing datasets, where melanomas with BRAF V600E mutations were more likely to have a CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.
Tandemly duplicated MYB genes specifically in the Phaseoleae lineage are functionally diverged in the regulation of anthocyanin biosynthesis

Gene duplications have long been recognized as a driving force in the evolution of genes, giving rise to novel functions. The soybean genome is characterized by a large extent of duplicated genes. However, the extent and mechanisms of functional divergence among these duplicated genes in soybean remain poorly understood. In this study, we revealed that tandem duplication of MYB genes, which occurred specifically in the Phaseoleae lineage, exhibited a stronger purifying selection in soybean compared to common bean. To gain insights into the diverse functions of these MYB genes in anthocyanin biosynthesis, we examined the expression, transcriptional activity, metabolite, and evolutionary history of four MYB genes (GmMYBA5, GmMYBA2, GmMYBA1 and Glyma.09g235000), which were presumably generated by tandem duplication in soybean. Our data revealed that Glyma.09g235000 had become a pseudogene, while the remaining three MYB genes exhibited strong transcriptional activation activity and promoted anthocyanin biosynthesis in different soybean tissues. Furthermore, GmMYBA5 produced distinct compounds in Nicotiana benthamiana leaves compared to GmMYBA2 and GmMYBA1 due to variations in their DNA binding domains. The lower expression of anthocyanin related genes in GmMYBA5 resulted in lower levels of anthocyanins compared to GmMYBA2 and GmMYBA1. Metabolomics analysis further demonstrated the diverse and differential downstream metabolites, suggesting their functional divergence in metabolites following gene duplication. Together, our data provided evidence of functional divergence within the MYB gene cluster following tandem duplication, which shed light on the potential evolutionary direction of gene duplications during legume evolution.
Cohesin forms fountains at active enhancers in C. elegans

Transcriptional enhancers must find their target genes both efficiently and specifically. Chromatin conformation capture revealed the critical function of three-dimensional chromosome segmentation by topologically associated domains (TADs) to limit the search space of enhancers for promoters in mammals. In nematodes, although more than 30’000 sequences with characteristic enhancer chromatin features have been identified, the autosomal genome is not segmented by TADs, raising the question of the mechanism directing enhancer-promoter specificity. Using high-resolution HiC, we show that enhancer loci correlate with 3D hairpin-like structures extending 10-50 kb from the enhancers, hereafter designated as fountains. Fountains are specific to active enhancers, accumulate the major somatic cohesin and disappear when the latter is cleaved in vivo. Fountains accumulate topological constraints and are enriched for topoisomerases and the negatively-supercoiled DNA binder psoralen. Short-term topoisomerase depletion leads to small-scale structural changes at the fountain tip. Functionally, fountain disappearance correlates with enhancer-proximal gene activation, suggesting fountains play a similar role as TADs and direct enhancer-promoter interactions, in particular for genes expressed in neurons. We directly observe this cell-type specific upregulation for the skn-1/Nrf gene in a pair of head neurons. Phenotypically, cohesin cleavage has a major impact on nematode movement and foraging attitudes, demonstrating that changes in neuronal gene expression impact nervous system function, reminiscent of pathologies caused by cohesin mutations in humans. Together, this study highlights a clear link between 3D genome organization at enhancers by cohesin, transcriptional gene regulation and animal behavior.
Leaf susceptibility of Macaronesian laurel forest species to Phytophthora ramorum

Phytophthora ramorum (Pr) is an invasive oomycete in Europe and North America and the causal agent of sudden oak death (SOD), which occurs along the coastal fog belt of California and southwestern Oregon, and it also causes sudden larch death in the UK. The Macaronesian laurel forest (MLF), a relict subtropical evergreen forest of the North Atlantic islands, shares climatic and some taxonomic affinities with those areas affected by SOD. To assess the disease risk, we tested the foliage susceptibility of MLF species and their capacity to sustain Pr sporulation and compared the climatic suitability with other areas where the pathogen is established. Detached leaves of 15 species were inoculated with zoospores and mycelium (through wounding) with five Pr isolates belonging to the EU1 and NA1 clonal lineages. MLF species showed diverse responses to Pr, ranging from extensive necroses on Viburnum tinus to asymptomatic sporulation on Picconia excelsa. Eleven species developed necrotic lesions to different degrees through zoospore inoculation while this increased to 13 species through wound treatment. Overall, small necrotic lesions (i.e. tolerance) were predominant, but Pr was rather aggressive to V. tinus, Arbutus canariensis and Ilex canariensis. Although the mean sporangial production was generally low (25-201 sporangia) in all species, the number of sporangia per leaf in five MLF species was similar to those reported for Umbellularia californica, a key host driving the SOD epidemics in California. Climatic suitability indexes in MLF areas were similar to those where SOD is found in California. Our results indicate a moderate to high risk of Pr establishment if the pathogen is introduced in the MLF.