Systematic benchmarking of single-cell ATAC-sequencing protocols.

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

Hydrop enables droplet-based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.

Single-cell RNA-seq and single-cell assay for transposase-accessible chromatin (ATAC-seq) technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost and pave the way for more specialized multiome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyze a small population of fluorescence-activated cell sorted neurons from the Drosophila brain, confirming the protocol's applicability to low input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.

Scientists are now able to determine the order of chemical blocks, or nucleic acids, that make up the genetic code. These sequencing tools can be used to identify which genes are active within a biological sample. They do this by extracting and analysing open chromatin (regions of DNA that are accessible to the cell's machinery), or sequences of RNA (the molecular templates cells use to translate genes into working proteins). Initially, most sequencing tools could only provide an 'averaged-out' profile of the genes activated in bulk pieces of tissue which contain multiple types of cell. However, advances in technology have led to new methods that can extract and analyse open chromatin or RNA from individual cells. First, the cells are separated, via a technique called microfluidics, into tiny droplets of water along with a single bead that carries a unique barcode. The cell is then broken apart inside the droplet and the barcode within the bead gets released and attaches itself to the genetic material extracted from the cell. All the genetic material inside the droplets is then pooled together and sequenced. Researchers then use the barcode tags to identify which bits of RNA or DNA belong to each cell. Single-cell sequencing has many advantages, including being able to pinpoint precise genetic differences between healthy and abnormal cells, and to create cell atlases of whole organisms, tissues and microbial communities. But existing methods for extracting chromatin are very expensive, and there were no openly available tools for processing thousands of cells at speed. Furthermore, while several single-cell RNA sequencing tools are already freely available, they are not very sensitive or practical to use. Here, De Rop et al. have developed a new open-source platform called HyDrop that overcomes these barriers. The method entails a new type of barcoded bead and optimised elements of existing microfluidics protocols using open-source reagents. These changes created a more user-friendly workflow and increased sensitivity of sequencing at no additional cost. De Rop et al. used their new platform to screen the RNA and open chromatin of thousands of individuals cells from the brains of mice and flies. HyDrop outperformed other open-source methods when working in RNA-sequencing mode. It also provides the first open-source tool for sequencing open chromatin in single cells. Further improvements are expected as researchers tweak the platform, which for now provides an affordable alternative to existing methods.

Decoding gene regulation in the fly brain.

The Drosophila brain is a frequently used model in neuroscience. Single-cell transcriptome analysis1-6, three-dimensional morphological classification7 and electron microscopy mapping of the connectome8,9 have revealed an immense diversity of neuronal and glial cell types that underlie an array of functional and behavioural traits in the fly. The identities of these cell types are controlled by gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. Here, to characterize GRNs at the cell-type level in the fly brain, we profiled the chromatin accessibility of 240,919 single cells spanning 9 developmental timepoints and integrated these data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which 70,000 are linked to developmental trajectories involving neurogenesis, reprogramming and maturation. For 40 cell types, uniquely accessible regions were associated with their expressed transcription factors and downstream target genes through a combination of motif discovery, network inference and deep learning, creating enhancer GRNs. The enhancer architectures revealed by DeepFlyBrain lead to a better understanding of neuronal regulatory diversity and can be used to design genetic driver lines for cell types at specific timepoints, facilitating their characterization and manipulation.

Nanoporous Block Copolymer Membranes with Enhanced Solvent Resistance Via UV-Mediated Cross-Linking Strategies.

In this work, a block copolymer (BCP) consisting of poly((butyl methacrylate-co-benzophenone methacrylate-co-methyl methacrylate)-block-(2-hydroxyethyl methacrylate)) (P(BMA-co-BPMA-co-MMA)-b-P(HEMA)) is prepared by a two-step atom-transfer radical polymerization (ATRP) procedure. BCP membranes are fabricated applying the self-assembly and nonsolvent induced phase separation (SNIPS) process from a ternary solvent mixture of tetrahydrofuran (THF), 1,4-dioxane, and dimethylformamide (DMF). The presence of a porous top layer of the integral asymmetric membrane featuring pores of about 30 nm is confirmed via scanning electron microscopy (SEM). UV-mediated cross-linking protocols for the nanoporous membrane are adjusted to maintain the open and isoporous top layer. The swelling capability of the noncross-linked and cross-linked BCP membranes is investigated in water, water/ethanol mixture (1:1), and pure ethanol using atomic force microscopy, proving a stabilizing effect of the UV cross-linking on the porous structures. Finally, the influence of the herein described cross-linking protocols on water-flux measurements for the obtained membranes is explored. As a result, an increased swelling resistance for all tested solvents is found, leading to an increased water flux compared to the pristine membrane. The herein established UV-mediated cross-linking protocol is expected to pave the way to a new generation of porous and stabilized membranes within the fields of separation technologies.

A national riparian restoration programme in New Zealand: Is it value for money?

National scale initiatives are being attempted in New Zealand (NZ) to meet important environmental goals following land-use intensification over recent decades. Riparian restoration to filter agricultural spillover effects is currently the most widely practised mitigation measure but few studies have investigated the cumulative value of these practices at a national level. We use an applied economic land use model the benefits (GHG emissions, N leaching, P loss, sedimentation and biodiversity gain) and relevant costs (fencing, alternative stock water supplies, restoration planting and opportunity costs) of restoring riparian margins (5-50 m) on all streams in NZ flowing through current primary sector land. Extensive sensitivity analysis reveals that depending on margin width and cost assumptions, riparian margin restoration generates net benefits of between NZ$1.7 billion - $5.2 billion/yr and benefit-cost ratios ranging between 1.4 and 22.4. This suggests that even when not monetising the increase in biodiversity or components of stream ecosystem health and other benefits from planting riparian strips, the benefits to climate and freshwater are significantly greater than the implementation costs of riparian restoration.

Non-Market Values in a Cost-Benefit World: Evidence from a Choice Experiment.

In support of natural resource and ecosystem service policy, monetary value estimates are often presented to decision makers along with other types of information. There is some evidence that, presented with such 'mixed' information, people prioritise monetary over non-monetary information. We conduct a discrete choice experiment among New Zealand decision makers in which we manipulate the information presented to participants. We find that providing explicit monetary information strengthens the pursuit of economic benefits as well as the avoidance of environmental damage. Cultural impacts, of which we provided only qualitative descriptions, did not affect respondents' choices. Our study provides further evidence that concerns regarding the use of monetary information in decisions with complex, multi-value impacts are valid. Further research is needed to validate our results and find ways to reduce any bias in monetary and non-market information.

Derivation of injury-responsive dendritic cells for acute brain targeting and therapeutic protein delivery in the stroke-injured rat.

Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs), we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.

Magnetic field interaction with guided light for detection of an active gaseous medium within an optical fiber.

We report a novel fiber-optic sensing architecture for the detection of paramagnetic gases. By interacting a modulated magnetic field with guided light within a microstructured optical fiber, it is possible to exploit Faraday Rotation Spectroscopy (FRS) within unprecedentedly small sample volumes. This approach, which utilizes magnetic circular birefringence and magnetic circular dichroism effects, is applied to a photonic bandgap fiber to detect molecular oxygen and operates at a wavelength of 762.309 nm. The optical fiber sensor has a 4.2 nL detection volume and 14.8 cm long sensing region. The observed FRS spectra are compared with a theoretical model that provides a first understanding of guided-mode FRS signals. This FRS guided-wave sensor offers the prospect of new compact sensing schemes.

[Occurrence and control of the Clostridium perfringens type A associated diarrhea of the suckling pigs with special consideration of the immunoprophylaxis]. / Vorkommen und Bekämpfung des Clostridium-perfringens -Typ-A-assoziierten Durchfalls der Saugferkel unter besonderer Berücksichtigung der Immunprophylaxe.

OBJECTIVE: Clostridium (C.) perfringens type A is often associated with diarrhoea in suckling piglets. The impact of the alpha (α) and beta (ß)2 toxins on the pathogenesis of the disease has not been conclusively answered. Previously, autologous vaccines were used to control this disease. The aim of our investigation was the classification of C. perfringens strains and the quantitative determination of the α and ß2 toxins during the production of autologous vaccines. The efficacy of a commercial vaccine against the C. perfringens type A associated diarrhoea was tested using an animal intoxication model. MATERIAL AND METHODS: A total of 1,434 C. perfringens strains from piglets with diarrhoea were classified using a multiplex PCR in the first part of the examination. In parallel, the α and ß2 toxin content was determined quantitatively using ELISA tests. In the second part of the study 18 gilts were vaccinated twice in the last third of gestation to evaluate the α and ß2 toxoid containing C.perfringens type A vaccine (Clostriporc A, IDT Biologika GmbH). RESULTS: Of the C. perfringens strains which were found, 87.9% were type A ( cpa , cpb2 ), 6.3% type A ( cpa ), and 5.8% type C ( cpa , cpb , cpb2 ). Low to middle a toxin-producing strains often strongly expressed the ß2 toxin. The vaccination led to the production of antibodies against the α and ß2 toxins, which were transferred to the offspring by colostrum. Piglets were significantly protected (p<0.05) by the antibodies after having been challenged with an α and ß2-containing supernatant of a heterologous C. perfringens type A strain. CONCLUSION AND CLINICAL RELEVANCE: These results underline the importance of α and ß2 toxin producing C.perfringens type A strains in diarrhoea in suckling piglets. Using the intoxication model, this vaccine showed significant protective effects against the α and ß2 toxins.

Photoinduced electron transfer based ion sensing within an optical fiber.

We combine suspended-core microstructured optical fibers with the photoinduced electron transfer (PET) effect to demonstrate a new type of fluorescent optical fiber-dip sensing platform for small volume ion detection. A sensor design based on a simple model PET-fluoroionophore system and small core microstructured optical fiber capable of detecting sodium ions is demonstrated. The performance of the dip sensor operating in a high sodium concentration regime (925 ppm Na(+)) and for lower sodium concentration environments (18.4 ppm Na(+)) is explored and future approaches to improving the sensor's signal stability, sensitivity and selectivity are discussed.

Simultaneous multi-laser, multi-species trace-level sensing of gas mixtures by rapidly swept continuous-wave cavity-ringdown spectroscopy.

The greenhouse-gas molecules CO(2), CH(4), and H(2)O are detected in air within a few ms by a novel cavity-ringdown laser-absorption spectroscopy technique using a rapidly swept optical cavity and multi-wavelength coherent radiation from a set of pre-tuned near-infrared diode lasers. The performance of various types of tunable diode laser, on which this technique depends, is evaluated. Our instrument is both sensitive and compact, as needed for reliable environmental monitoring with high absolute accuracy to detect trace concentrations of greenhouse gases in outdoor air.

[Psoas abscess as a chicken pox complication]. / Absceso de psoas como complicación de varicela.

Chicken pox is the most frequent exantematic illness; usually its course is self-limited and benign. Several bacterial complications are described due to the disruption of the skin as a defensive barrier because of the characteristics of the injuries and the associated inmunodepression. Psoas abscess is a rare illness and it's difficult to diagnose, with a general unspecified clinical presentation. We present the case of a 5-year-old girl, on her fifth day of chicken pox, who consults about a febrile convulsion, from which she recovers without any neurological symptoms, referring to functional impotence of her inferior left limb and pain in the lumbar and gluteal zone, which irradiates to the homolateral hip, making deambulation impossible. The definitive diagnosis was made with a CAT at hospital admission. The germ isolated was community-acquired methricillin-resistant Staphilococcus aureus. Treatment consisted in surgical drainage and endovenous antibiotics.

Absceso de psoas como complicación de varicela / Psoas abscess as a chicken pox complication

La varicela es la enfermedad exantemática más frecuente, sucurso suele ser autolimitado y benigno. Están descriptas variadascomplicaciones bacterianas debido a la disrupción de la piel como barrera defensiva por las características de suslesiones y a la inmunodepresión asociada.El absceso de psoas es una enfermedad poco frecuente y de difícil diagnóstico, con un cuadro clínico generalmente inespecífico.Presentamos el caso de una niña de 5 años, que, cursando el 5º día de varicela, consulta por convulsión febril de la cual se recupera sin síntomas neurológicos. Presenta impotencia funcional de miembro inferior izquierdo y dolor en región lumbar y glútea, que irradia a la cadera de dicho miembro con imposibilidad de deambular.El diagnóstico definitivo se realizó con tomografía axial computada (TAC) el día de ingreso.El germen aislado fue Staphylococcus meticilino-resistente (SAMR) de la comunidad.El tratamiento consistió en el drenaje quirúrgico y antibióticos endovenosos.

Absceso de psoas como complicación de varicela / Psoas abscess as a chicken pox complication

La varicela es la enfermedad exantemática más frecuente, sucurso suele ser autolimitado y benigno. Están descriptas variadascomplicaciones bacterianas debido a la disrupción de la piel como barrera defensiva por las características de suslesiones y a la inmunodepresión asociada.El absceso de psoas es una enfermedad poco frecuente y de difícil diagnóstico, con un cuadro clínico generalmente inespecífico.Presentamos el caso de una niña de 5 años, que, cursando el 5º día de varicela, consulta por convulsión febril de la cual se recupera sin síntomas neurológicos. Presenta impotencia funcional de miembro inferior izquierdo y dolor en región lumbar y glútea, que irradia a la cadera de dicho miembro con imposibilidad de

Neurogenesis and alterations of neural stem cells in mouse models of cerebral amyloidosis.

The hippocampus in Alzheimer's disease is burdened with amyloid plaques and is one of the few locations where neurogenesis continues throughout adult life. To evaluate the impact of amyloid-beta deposition on neural stem cells, hippocampal neurogenesis was assessed using bromodeoxyuridine incorporation and doublecortin staining in two amyloid precursor protein (APP) transgenic mouse models. In 5-month-old APP23 mice prior to amyloid deposition, neurogenesis showed no robust difference relative to wild-type control mice, but 25-month-old amyloid-depositing APP23 mice showed significant increases in neurogenesis compared to controls. In contrast, 8-month-old amyloid-depositing APPPS1 mice revealed decreases in neurogenesis compared to controls. To study whether alterations in neurogenesis are the result of amyloid-induced changes at the level of neural stem cells, APPPS1 mice were crossed with mice expressing green fluorescence protein (GFP) under a central nervous system-specific nestin promoter. Eight-month-old nestin-GFP x APPPS1 mice exhibited decreases in quiescent nestin-positive astrocyte-like stem cells, while transient amplifying progenitor cells did not change in number. Strikingly, both astrocyte-like and transient-amplifying progenitor cells revealed an aberrant morphologic reaction toward congophilic amyloid-deposits. A similar reaction toward the amyloid was no longer observed in doublecortin-positive immature neurons. Results provide evidence for a disruption of neural stem cell biology in an amyloidogenic environment and support findings that neurogenesis is differently affected among various transgenic mouse models of Alzheimer's disease.

An existentialist view on mortality salience effects: personal hardiness, death-thought accessibility, and cultural worldview defence.

Two studies examined the possible moderating role of hardiness on reactions to mortality salience inductions. A sample of 240 Israeli undergraduate students completed a hardiness scale, were exposed to a mortality salience or control induction, and then either rated the severity and punishment of 10 social transgressions (Study 1, N = 120) or performed a word-stem completion task, which tapped the accessibility of death-related thoughts (Study 2, N = 120). Results indicated that a mortality salience induction led to more severe judgments of social transgressions as well as to more severe punishments than a control induction only among participants scoring low in the hardiness scale. However, a mortality salience induction led to a higher cognitive accessibility of death-related thoughts than a control condition regardless of participants' hardiness scores. The discussion emphasizes the importance of considering inner resources when examining reactions to mortality reminders.

Mental health and marital adaptation among mothers of children with cerebral palsy.

Employing a theoretical model that emphasizes risk-resistance factors for maternal adaptation, this study compared 80 mothers of children with cerebral palsy to 80 mothers of children without physical disabilities. Results revealed differences between the two groups, and indicated that, among mothers of children with cerebral palsy, self-esteem, self-mastery, and family network size were the main variables that contributed to mothers' psychological and marital adaptation.

A new member of the sorting nexin family interacts with the C-terminus of P-selectin.

P-selectin is a cell adhesion molecule found in platelets and endothelial cells mediating binding of leukocytes. It is stored in secretory granules and expressed at the plasma membrane after cell activation. After rapid internalisation P-selectin recycles or is degraded. The 35 amino acid cytoplasmic domain of P-selectin contains signals for sorting into secretory granules, for endocytosis and for delivery to lysosomes. To investigate protein-protein interactions, we performed two-hybrid screening using the cytoplasmic domain of P-selectin as bait. KIAA0064 was identified as a putative intracellular P-selectin binding protein. Because the protein contains a phox homology (PX) domain in the N-terminus which is a characteristic feature of the sorting nexin (SNX) family, it was named SNX17. The PX domain is not required for binding of P-selectin in the two-hybrid system. Expression of a fusion protein between SNX17 and green fluorescent protein demonstrated localisation of SNX17 in the cytosol and to membranes.

Exploring individual differences in reactions to mortality salience: does attachment style regulate terror management mechanisms?

Five studies examined the contribution of attachment style to mortality salience effects. In Study 1, mortality salience led to more severe judgments of transgressions only among anxious-ambivalent and avoidant persons but not among secure persons. In addition, whereas anxious-ambivalent persons showed immediate and delayed increases in severity judgments, avoidant persons showed this response only after a delay period. In Study 2, anxious-ambivalent persons showed immediate and delayed increases in death-thought accessibility after death reminders. Avoidant and secure persons showed this effect only after a delay period. Study 3 revealed that worldview defense in response to mortality salience reduced death-thought accessibility only among avoidant persons. Studies 4-5 revealed that mortality salience led to an increase in the sense of symbolic immortality as well as in the desire of intimacy only among secure persons, but not among avoidant and anxious-ambivalent persons.

Does a threat appeal moderate reckless driving? A terror management theory perspective.

A series of two studies examined the effects of threat appeals on reckless driving from a terror management theory perspective. In both studies, all the participants (N = 109) reported on the relevance of driving to their self-esteem, and, then, half of them were exposed to a road trauma film and the remaining to a neutral film. In Study 1, the dependent variable was the self-report of intentions to drive recklessly in hypothetical scenarios. In Study 2, the dependent variable was actual behavior (driving speed) in a driving simulator. Findings indicated that a road trauma film led to less reported intentions of reckless driving, but to higher driving speed than a neutral film. These effects were only found among participants who perceived driving as relevant to their self-esteem. The discussion emphasized the self-enhancing mechanisms proposed by the terror management theory.