An open-source, high-resolution, automated fluorescence microscope.

Fluorescence microscopy is a fundamental tool in the life sciences, but the availability of sophisticated equipment required to yield high-quality, quantitative data is a major bottleneck in data production in many laboratories worldwide. This problem has long been recognized and the abundancy of low-cost electronics and the simplification of fabrication through 3D-printing have led to the emergence of open-source scientific hardware as a research field. Cost effective fluorescence microscopes can be assembled from cheaply mass-produced components, but lag behind commercial solutions in image quality. On the other hand, blueprints of sophisticated microscopes such as light-sheet or super-resolution systems, custom-assembled from high quality parts, are available, but require a high level of expertise from the user. Here, we combine the UC2 microscopy toolbox with high-quality components and integrated electronics and software to assemble an automated high-resolution fluorescence microscope. Using this microscope, we demonstrate high resolution fluorescence imaging for fixed and live samples. When operated inside an incubator, long-term live-cell imaging over several days was possible. Our microscope reaches single molecule sensitivity, and we performed single particle tracking and SMLM super-resolution microscopy experiments in cells. Our setup costs a fraction of its commercially available counterparts but still provides a maximum of capabilities and image quality. We thus provide a proof of concept that high quality scientific data can be generated by lay users with a low-budget system and open-source software. Our system can be used for routine imaging in laboratories that do not have the means to acquire commercial systems and through its affordability can serve as teaching material to students.

A renal clearable fluorogenic probe for in vivo ß-galactosidase activity detection during aging and senolysis.

Accumulation of senescent cells with age leads to tissue dysfunction and related diseases. Their detection in vivo still constitutes a challenge in aging research. We describe the generation of a fluorogenic probe (sulfonic-Cy7Gal) based on a galactose derivative, to serve as substrate for ß-galactosidase, conjugated to a Cy7 fluorophore modified with sulfonic groups to enhance its ability to diffuse. When administered to male or female mice, ß-galactosidase cleaves the O-glycosidic bond, releasing the fluorophore that is ultimately excreted by the kidneys and can be measured in urine. The intensity of the recovered fluorophore reliably reflects an experimentally controlled load of cellular senescence and correlates with age-associated anxiety during aging and senolytic treatment. Interestingly, our findings with the probe indicate that the effects of senolysis are temporary if the treatment is discontinued. Our strategy may serve as a basis for developing fluorogenic platforms designed for easy longitudinal monitoring of enzymatic activities in biofluids.

Dopaminergic neuron loss in mice due to increased levels of wild-type human α-Synuclein only takes place under conditions of accelerated aging.

Understanding the intricate pathogenic mechanisms behind Parkinson's disease (PD) and its multifactorial nature presents a significant challenge in disease modeling. To address this, we explore genetic models that better capture the disease's complexity. Given that aging is the primary risk factor for PD, this study investigates the impact of aging in conjunction with overexpression of wild-type human α-synuclein (α-Syn) in the dopaminergic system. This is achieved by introducing a novel transgenic mouse strain overexpressing α-Syn under the TH-promoter within the senescence-accelerated SAMP8 (P8) genetic background. Behavioral assessments, conducted at both 10 and 16 months of age, unveil motor impairments exclusive to P8 α-SynTg mice, a phenomenon conspicuously absent in α-SynTg mice. These findings suggest a synergistic interplay between heightened α-Syn levels and the aging process, resulting in motor deficits. These motor disturbances correlate with reduced dopamine (DA) levels, increased DA turnover, synaptic terminal loss, and notably, the depletion of dopaminergic neurons in the substantia nigra and noradrenergic neurons in the locus coeruleus. Furthermore, P8 α-SynTg mice exhibit alterations in gut transit time, mirroring early PD symptoms. In summary, P8 α-SynTg mice effectively replicate parkinsonian phenotypes by combining α-Syn transgene expression with accelerated aging. This model offers valuable insights into the understanding of PD and serves as a valuable platform for further research.

Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

Laser Therapy for Vulvar Lichen Sclerosus, a Systematic Review.

Lichen sclerosus (LS) is a chronic inflammatory disease that mainly affects the anogenital area, with a higher incidence in post-menopausal women. In the long term, it can lead to loss of vulvar architecture or progress to squamous cell carcinoma. The evidence-based treatment involves high-potency topical corticosteroids in long regimens. However, second-line treatments are not well-established, including laser therapy. This current study aims to assess the level of evidence supporting this therapy. We conducted a search for primary-level studies published before April 2023 through MEDLINE/PubMed, Embase, Web of Science, Scopus, and CENTRAL, with no restrictions on the publication language or date. The methodological quality and risk of bias of the included studies were evaluated using the updated Cochrane Collaboration's tool for assessing risk of bias (RoB-2). Six studies (177 patients) met our eligibility criteria. Laser therapy was compared to topical corticosteroid treatment in five out of six studies. No significant histological differences were found, except for an increase in collagen production in the laser group. A greater reduction in itching, pain, and dyspareunia at 1 and 3 months of treatment in the laser group, as well as in the Skindex-29 at 6 months, was reported. Patient satisfaction was significantly higher among those who received laser therapy. Tolerability was excellent. No significant differences were observed in any of the previous aspects in the study compared to the placebo. In conclusion, there is not enough evidence to recommend laser therapy as a standalone treatment.

Antiproliferative Potential of Olneya tesota PF2 Lectin in Human Acute Monocytic Leukemia Cells.

Acute monocytic leukemia is a type of myeloid leukemia that develops in monocytes. The current clinical therapies for leukemia are unsatisfactory due to their side effects and nonspecificity toward target cells. Some lectins display antitumor activity and may specifically recognize cancer cells by binding to carbohydrate structures on their surface. Therefore, this study evaluated the response of the human monocytic leukemia cell lines THP-1 to the Olneya tesota PF2 lectin. The induction of apoptosis and reactive oxygen species production in PF2-treated cells was evaluated by flow cytometry, and the lectin-THP-1 cell interaction and mitochondrial membrane potential were evaluated by confocal fluorescence microscopy. PF2 genotoxicity was evaluated by DNA fragmentation analysis via gel electrophoresis. The results showed that PF2 binds to THP-1 cells, triggers apoptosis and DNA degradation, changes the mitochondrial membrane potential, and increases reactive oxygen species levels in PF2-treated THP-1 cells. These results suggest the potential use of PF2 for developing alternative anticancer treatments with enhanced specificity.

Mathematics of neural stem cells: Linking data and processes.

Adult stem cells are described as a discrete population of cells that stand at the top of a hierarchy of progressively differentiating cells. Through their unique ability to self-renew and differentiate, they regulate the number of end-differentiated cells that contribute to tissue physiology. The question of how discrete, continuous, or reversible the transitions through these hierarchies are and the precise parameters that determine the ultimate performance of stem cells in adulthood are the subject of intense research. In this review, we explain how mathematical modelling has improved the mechanistic understanding of stem cell dynamics in the adult brain. We also discuss how single-cell sequencing has influenced the understanding of cell states or cell types. Finally, we discuss how the combination of single-cell sequencing technologies and mathematical modelling provides a unique opportunity to answer some burning questions in the field of stem cell biology.

Interferon regulates neural stem cell function at all ages by orchestrating mTOR and cell cycle.

Stem cells show intrinsic interferon signalling, which protects them from viral infections at all ages. In the ageing brain, interferon signalling also reduces the ability of stem cells to activate. Whether these functions are linked and at what time interferons start taking on a role in stem cell functioning is unknown. Additionally, the molecular link between interferons and activation in neural stem cells and how this relates to progenitor production is not well understood. Here we combine single-cell transcriptomics, RiboSeq and mathematical models of interferon to show that this pathway is important for proper stem cell function at all ages in mice. Interferon orchestrates cell cycle and mTOR activity to post-transcriptionally repress Sox2 and induces quiescence. The interferon response then decreases in the subsequent maturation states. Mathematical simulations indicate that this regulation is beneficial for the young and harmful for the old brain. Our study establishes molecular mechanisms of interferon in stem cells and interferons as genuine regulators of stem cell homeostasis and a potential therapeutic target to repair the ageing brain.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis.

Neural stem cells (NSCs) in the adult murine subependymal zone balance their self-renewal capacity and glial identity with the potential to generate neurons during the lifetime. Adult NSCs exhibit lineage priming via pro-neurogenic fate determinants. However, the protein levels of the neural fate determinants are not sufficient to drive direct differentiation of adult NSCs, which raises the question of how cells along the neurogenic lineage avoid different conflicting fate choices, such as self-renewal and differentiation. Here, we identify RNA-binding protein MEX3A as a post-transcriptional regulator of a set of stemness associated transcripts at critical transitions in the subependymal neurogenic lineage. MEX3A regulates a quiescence-related RNA signature in activated NSCs that is needed for their return to quiescence, playing a role in the long-term maintenance of the NSC pool. Furthermore, it is required for the repression of the same program at the onset of neuronal differentiation. Our data indicate that MEX3A is a pivotal regulator of adult murine neurogenesis acting as a translational remodeller.

In the pursuit of new social neurons. Neurogenesis and social behavior in mice: A systematic review.

Social behaviors have become more relevant to our understanding of the human nervous system because relationships with our peers may require and modulate adult neurogenesis. Here, we review the pieces of evidence we have to date for the divergence of social behaviors in mice by modulation of adult neurogenesis or if social behaviors and the social environment can drive a change in neurogenic processes. Social recognition and memory are deeply affected by antimitotic drugs and irradiation, while NSC transgenic mice may run with lower levels of social discrimination. Interestingly, social living conditions can create a big impact on neurogenesis. Social isolation and social defeat reduce the number of new neurons, while social dominance and enrichment of the social environment increase their number. These new "social neurons" trigger functional modifications with amazing transgenerational effects. All of these suggest that we are facing two bidirectional intertwined variables, and the great challenge now is to understand the cellular and genetic mechanisms that allow this relationship to be used therapeutically.

A case of intestinal schwannoma initially suspected by transvaginal ultrasound.

Schwannomas are peripheral nerve sheath tumors. Due to their low incidence, few cases of colorectal schwannomas have been published, which increases the diagnostic challenge. The aim of this case report is to discuss the role of transvaginal ultrasound in different areas than the gynecological disorders, when on hands of properly trained professionals that perform systematized procedures. A 56-year-old woman consulted for postmenopausal genital bleeding. During transvaginal ultrasound, a colonic solid, hypervascularized mass of 23 × 26 mm was visualized. As a result of this incidental finding, the patient underwent a sigmoidectomy, with a final diagnosis of intestinal schwannoma. Transvaginal ultrasound is today one of the most useful and accurate diagnostic tools in the assessment of gynecological disorders. However, the proximity of other pelvic structures makes it possible to evaluate the presence of nongynecological conditions. This fact should encourage gynecologists to systematize the transvaginal ultrasound procedure.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition.

Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.

High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids.

Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth. For complete details on the use and execution of this protocol, please refer to Clark (2019), Clark et al. (2018), and Clark et al., 2018, Hagemann-Jensen et al., 2020a, Hagemann-Jensen et al., 2020b.

Single versus tandem autologous stem-cell transplantation in patients with newly diagnosed multiple myeloma and high-risk cytogenetics. A retrospective, open-label study of the PETHEMA/Spanish Myeloma Group (GEM).

Tandem ASCT has been suggested as a valid approach to improve the prognosis of patients with MM and HR cytogenetic. In this observational, retrospective study, 213 patients with newly diagnosed MM and HR cytogenetic in 35 hospitals from the Spanish Myeloma Group underwent single or tandem ASCT between January 2015 and December 2019 after induction with VTD/VRD. HR cytogenetic was defined as having ≥1 of the following: del17p, t(4;14), t(14;16) or gain 1q21. More patients in the tandem group had R-ISS 3 and >1 cytogenetic abnormality at diagnosis. With a median follow-up of 31 months (range, 10-82), PFS after single ASCT was 41 months versus 48 months with tandem ASCT (p = 0.33). PFS in patients with del17p undergoing single ASCT was 41 months, while 52% of patients undergoing tandem ASCT were alive and disease free at 48 months. In conclusion, tandem ASCT partly overcomes the bad prognosis of HR cytogenetic.

Dermatological adverse reactions after vaccination with BNT162b2 in a cohort of healthcare workers.

BACKGROUND: The description of the skin reactions produced by the different vaccines against SARS-CoV-2 has focused on the symptoms reported by the general population. There are few studies with very different measurement methods focused on healthcare workers. METHODS: A longitudinal observational study was conducted on all the healthcare workers from the Hospital Universitario San Cecilio that received vaccination against COVID-19 with BNT162b2. The recruitment period was from December 2020 to September 2021. The recommended regimen was double, with a minimum interval of 21 days between doses. All dermatological reactions reported as adverse effects of the vaccine were evaluated by the Staff of the Dermatology Unit of our center. RESULTS: A total of 3969 healthcare workers of our center were followed. Only 0.7% of them reported dermatological adverse reactions. The most frequently reported reactions were morbilliform rash and COVID arm. In the multivariate analysis, the vaccination regimen (one dose) and the history of COVID-19 infection remained the main factors associated with the report of dermatological adverse reactions. CONCLUSION: The rate of dermatological adverse reactions after vaccination with BNT162b2 (Pfizer-BioNTech) is extraordinarily low. No patient required hospitalization, which supports the safety of this vaccination in a population of healthcare workers.

Centrosome linker protein C-Nap1 maintains stem cells in mouse testes.

The centrosome linker component C-Nap1 (encoded by CEP250) anchors filaments to centrioles that provide centrosome cohesion by connecting the two centrosomes of an interphase cell into a single microtubule organizing unit. The role of the centrosome linker during development of an animal remains enigmatic. Here, we show that male CEP250-/- mice are sterile because sperm production is abolished. Premature centrosome separation means that germ stem cells in CEP250-/- mice fail to establish an E-cadherin polarity mark and are unable to maintain the older mother centrosome on the basal site of the seminiferous tubules. This failure prompts premature stem cell differentiation in expense of germ stem cell expansion. The concomitant induction of apoptosis triggers the complete depletion of germ stem cells and consequently infertility. Our study reveals a role for centrosome cohesion in asymmetric cell division, stem cell maintenance, and fertility.

Secondary loss of miR-3607 reduced cortical progenitor amplification during rodent evolution.

The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/ß-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the ß-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.