bioRxiv Science⌕ Search

Biology subjects

Schubert, R.

Publications and source records attributed to Schubert, R..

16 recordsLinked to original sources

Aerosol delivery-based serial femtosecond crystallography

Serial femtosecond crystallography (SFX) has revolutionised structural biology by enabling direct visualization of conformational dynamics of biomacromolecules at near-physiological temperatures. However, conventional SFX sample delivery at X-ray free-electron laser (XFEL) facilities introduces a significant X-ray scattering background, which may limit the data quality and resolution. Here, we introduce an aerosol delivery-based method that drastically reduces the back-ground scattering by minimising the liquid environment surrounding the nanocrystals. We validate our method by solving the structure of Cydia pomonella granulovirus nanocrystals at 1.9 [A] resolution, achieving orders-of-magnitude lower background scattering compared to a liquid jet-based method. Structural comparison with the liquid jet-based model revealed similar overall structure, suggesting that the native structure is largely preserved despite dehydration during aerosolisation. Our method enables efficient SFX studies, particularly pump-probe time-resolved SFX on protein nanocrystals with enhanced signal-to-noise ratios, as well as high-throughput small molecule SFX (smSFX) applications for pharmaceuticals and functional materials.

biophysics↗

Benchmarking long-read RNA-seq across modalities, methods, and sequencing depth in iNeurons

Long-read RNA sequencing (lrRNA-seq) provides advantages for transcript discovery and quantification through the sequencing of full-length transcripts. Although recent benchmarks have evaluated long-read technologies and quantification tools, to the best of our knowledge, no study to date has jointly compared sequencing technology, quantification choice, and depth across both bulk and single-cell platforms. Here, we generate a matched dataset using NGN2-induced neurons derived from Fragile X syndrome and isogenic rescue lines, profiled with bulk and single-cell Illumina, Oxford Nanopore Technologies (ONT), and Pacific Biosciences (PB) Kinnex technologies. All platforms and technologies capture the expected FMR1 reactivation signal. We find that PB bulk under-detects and under-quantifies short transcripts (less than 1.25 kb), ONT bulk under-detects and under-quantifies long transcripts (greater than 5 kb), and single-cell long-read technologies a large number of single-cell specific transcripts associated with truncations. Across six bulk and four single-cell long-read quantification tools, Isosceles, Miniquant, and Oarfish provide the best compromise between computational efficiency and performance in terms of quantification accuracy as measured by spike-ins, comparisons to Illumina, and on consensus based down-stream tasks such as differential transcript expression (DTE). Depth-equivalency analyses reveal that PB single-cell sequencing requires approximately three- to four-fold greater depth than bulk to reach comparable power for transcript discovery and differential transcript expression. Our work aims to offer practical guidance for study design, including the choice of technology, sequencing depth, and quantification method. In addition, we hope our data may serve a reference dataset to evaluate emerging long-read transcriptomic protocols and methods as well as more closely investigate FMR1 biology.

bioinformatics↗

The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5

The autosomal recessive disease ataxia-telangiectasia (A-T) presents with cerebellar degeneration, immunodeficiency, radiosensitivity, capillary dilatations, and pulmonary infections. Most symptoms outside the nervous system can be explained by failures of the disease protein ATM as Ser/Thr-kinase to coordinate DNA damage repair. However, ATM in adult neurons has cytoplasmic localization and vesicle association, where its roles remain unclear. Here, we defined novel ATM protein targets in human neuroblastoma cells and filtered initial pathogenesis events in ATM-null mouse cerebellum. Profiles of global proteome and phosphorylome - both direct ATM/ATR-phosphopeptides and overall phosphorylation changes - confirmed previous findings on NBN, MRE11, MDC1, CHEK1, EIF4EBP1, AP3B2, PPP2R5C, SYN1 and SLC2A1. Even stronger downregulation of ATM/ATR-phosphopeptides after ATM-depletion was documented for CHGA, EXPH5, NBEAL2 and CHMP6 as key factors of protein secretion and endosome dynamics, as well as for CRMP5, DISP2, PHACTR1, PLXNC1, INA and TPX2 as neurite extension factors. Prominent affection of semaphorin-CRMP5-microtubule signals and ATM association with CRMP5 were validated. As a functional consequence, microtubules were stabilized, and neurite retraction ensued. The ATM impact on secretory granules confirms previous ATM-null cerebellar transcriptome findings. Our study provides the first link of A-T neural atrophy to growth cone collapse and aberrant microtubule dynamics.

cell biology↗

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. SynopsisStructures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

biophysics↗

Abatacept treatment in LRBA deficient patients results in an increase in circulating FoxP3+ Helios + natural Treg

BackgroundMutations in the lipopolysaccharide-responsive beige-like anchor (LRBA) gene were identified to cause autoimmunity and immunodeficiency with a broad spectrum of clinical manifestations. Increased lysosomal degradation of the CTLA-4 due to the lack of recylcing by LRBA protein has been described resulting in a reduced suppressive capacity of regulatory T cells (Treg). >ObjectiveWe sought to explore the extended Treg profile of patients with LRBA deficiency (N=6) compared to reference values for Treg subpopulations from an age-matched healthy cohort (N=39) prior and during abatacept treatment as well as after allogeneic haematopoietic stem cell transplantation. In parallel we assessed the feasibility and robustness of the CHAI and the IDDA2.1 scores for this cohort. MethodsUsing a flow cytometric approach with a pre-formulated antibody panel in peripheral blood samples, we analyzed Treg subsets including CD4+CD25hiFoxP3+ Treg, Helios+ natural Treg, Helios- induced Treg, CD39+ Treg, CD62L+CD45RA+ naive Treg, CD62L+CD45RA- memory Treg, and FoxP3hiCD45RA- effector Treg as well as CD4+CD25hiCD127low Treg. Longitudinal data were collected while patients were receiving abatacept and in three patients after alloHSCT. CHAI and IDDA2.1 scores were performed. ResultsThe healthy cohort including individuals from the ages of 2 to 40 years showed a steady total CD25hiFoxP3+ Treg population around 5%, while there was a significant age-dependent increase in Helios+ natural Treg (P=0.003), Helios- induced Treg (P=0.046), CD62L+CD45RA- memory Treg (P=0.020) with a continuous decrease in CD62L+CD45RA+ naive Treg (P=0.015) and CD4+CD25hiCD127low Treg (P=0.024) over time. LRBA deficient patients showed a significant lack in FoxP3+Helios+ natural Treg (P=0.003), CD62L+CD45RA+ naive Treg (P<0.001), FoxP3hi CD45RA+ effector Treg (P=0.001) and CD4+CD25hiCD127low Treg (P=0.005), while their CD62L+CD45RA+ memory Treg (P=0.016) were significantly elevated. Abatacept treatment led to a significant increase in natural Treg (P=0.003) in patients without having a measurable effect on the other subpopulations. This was accompanied by a decrease in sIL2R levels. The IDDA2.1 score was feasible for this mainly pediatric cohort and correlated with patients clinical course of the disease. ConclusionWithin the Treg subsets in peripheral blood of LRBA deficient patients, there is a significant lack of natural, naive, effector and CD4+CD25hiCD127low Treg, while memory Treg are elevated. During abatacept treatment we observe a significant increase in circulating Helios+ natural Treg levels. IDDA2.1 scoring system is feasible for pediatric patients to assess the severity of their disease and the response to the treatment.

immunology↗

Light-induced Trpin/Metout switching during BLUF domain activation in ATP-bound photoactivatable adenylate cyclase OaPAC

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in a non-productive conformation. However, FTIR spectroscopic experiments confirm that the non-productive conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the initial events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Initial changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180{degrees} rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via -helix 3 and 4 in the linker region between sensor and effector domain is discussed.

biochemistry↗

Transcriptional dysregulation and impaired neuronal activity in FMR1 knock-out and Fragile X patients iPSC-derived models.

The lack of fragile X mental retardation protein (FMRP) protein, due to a repression of the FMR1 gene, causes Fragile X syndrome (FXS), one of the most prevalent forms of syndromic autisms. The FMR1 gene codes for an RNA binding protein involved in the regulation of gene expression through RNA processing, control of local translation, and protein-protein interactions; processes that are crucial for proper brain development. Taking advantage of induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 genome editing technologies, we generated iPSC-derived cortical neural progenitors and cortical neurons from an FMR1 knock-out and patient cell line with the aim of identifying common phenotypes between the two cellular models. Using RNA sequencing, quantitative PCR and multielectrode array approaches, we assessed how the absence of the functional FMR1 gene affects the transcriptional profiles and the activities of iPSC-derived cortical neuronal progenitor cells (NPCs) and neurons with both models. We observed that FMR1 KO and FXS patient cells have a decrease in their mean firing rate; a cellular activity that can also be blocked by tetrodotoxin (TTX) application in wild-type active neurons. Relative to wild-type neurons, in FMR1 KO neurons, increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and Autism Related Genes found amongst downregulated genes. This implies that an absence of functional FMRP affects transcriptional profiles at the NPC stage, resulting in impaired activity and differentiation of the progenitors into mature neurons over time. These findings from the FMR1 KO lines were also shared with FXS patients iPSC-derived cells that also present with an impairment in activity and neuronal differentiation, illustrating the critical role of FMRP protein in neuronal development.

neuroscience↗

USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING

Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFN (IFN-{lambda}) exhibit cell-type specific and long-lasting functions in autoinflammation, tumorigenesis and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-{lambda} secretion and SARS-CoV-2 infections in native human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-{lambda} and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 23-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-{lambda} expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.

cell biology↗

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that - in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 [A] resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae from Anopheles stephensi, Aedes albopictus and Culex tarsalis - substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained using Culex cell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components. Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 [A]. Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity. We expand the known target range to include three more mosquito species: Anopheles stephensi, Aedes albopictus and Culex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

biochemistry↗

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

Protein prediction for trait mapping in diverse populations

Genetically regulated gene expression has helped elucidate the biological mechanisms underlying complex traits. Improved high-throughput technology allows similar interrogation of the genetically regulated proteome for understanding complex trait mechanisms. Here, we used the Trans-omics for Precision Medicine (TOPMed) Multi-omics pilot study, which comprises data from Multi-Ethnic Study of Atherosclerosis (MESA), to optimize genetic predictors of the plasma proteome for genetically regulated proteome-wide association studies (PWAS) in diverse populations. We built predictive models for protein abundances using data collected in TOPMed MESA, for which we have measured 1,305 proteins by a SOMAscan assay. We compared predictive models built via elastic net regression to models integrating posterior inclusion probabilities estimated by fine-mapping SNPs prior to elastic net. In order to investigate the transferability of predictive models across ancestries, we built protein prediction models in all four of the TOPMed MESA populations, African American (n=183), Chinese (n=71), European (n=416), and Hispanic/Latino (n=301), as well as in all populations combined. As expected, fine-mapping produced more significant protein prediction models, especially in African ancestries populations, potentially increasing opportunity for discovery. When we tested our TOPMed MESA models in the independent European INTERVAL study, fine-mapping improved cross-ancestries prediction for some proteins. Using GWAS summary statistics from the Population Architecture using Genomics and Epidemiology (PAGE) study, which comprises ~50,000 Hispanic/Latinos, African Americans, Asians, Native Hawaiians, and Native Americans, we applied S-PrediXcan to perform PWAS for 28 complex traits. The most protein-trait associations were discovered, colocalized, and replicated in large independent GWAS using proteome prediction model training populations with similar ancestries to PAGE. At current training population sample sizes, performance between baseline and fine-mapped protein prediction models in PWAS was similar, highlighting the utility of elastic net. Our predictive models in diverse populations are publicly available for use in proteome mapping methods at https://doi.org/10.5281/zenodo.4837328. Author summaryGene regulation is a critical mechanism underlying complex traits. Transcriptome-wide association studies (TWAS) have helped elucidate potential mechanisms because each association connects a gene rather than a variant to the complex trait. Like genome-wide association studies (GWAS), most TWAS are still conducted exclusively in populations of European ancestry, which misses the opportunity to test the full spectrum of human genetic variation for associations with complex traits. Here, move beyond the transcriptome and because protein measurement assays are growing to allow interrogation of the proteome, we use data from TOPMed MESA to develop genetic predictors of protein abundance in diverse ancestry populations. We compare model-building strategies with the goal of providing the best resource for protein association discovery with available data. We demonstrate how these prediction models can be used to perform proteome-wide association studies (PWAS) in diverse populations. We show the most protein-trait associations were discovered, colocalized, and replicated in independent cohorts using proteome prediction model training populations with similar ancestries to individuals in the GWAS. We shared our protein prediction models and performance statistics publicly to facilitate future proteome mapping studies in diverse populations.

genomics↗

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

Multi-ethnic transcriptome-wide association study of prostate cancer

The genetic risk for prostate cancer has been governed by a few rare variants with high penetrance and over 150 commonly occurring variants with lower impact on risk; however, most of these variants have been identified in studies containing exclusively European individuals. People of non-European ancestries make up less than 15% of prostate cancer GWAS subjects. Across the globe, incidence of prostate cancer varies with population due to environmental and genetic factors. The discrepancy between disease incidence and representation in genetics highlights the need for more studies of the genetic risk for prostate cancer across diverse populations. To better understand the genetic risk for prostate cancer across diverse populations, we performed PrediXcan and GWAS in a cohort of 4,769 self-identified African American (2,463 cases and 2,306 controls), 2,199 Japanese American (1,106 cases and 1,093 controls), and 2,147 Latin American (1,081 cases and 1,066 controls) individuals from the Multiethnic Genome-wide Scan of Prostate Cancer. We used prediction models from 46 tissues in GTEx version 8 and five models from monocyte transcriptomes in the Multi-Ethnic Study of Artherosclerosis. Across the three populations, we predicted 19 gene-tissue pairs, including five unique genes, to be significantly (lfsr < 0.05) associated with prostate cancer. One of these genes, NKX3-1, replicated in a larger European cohort. At the SNP level, 110 SNPs met genome-wide significance in the African American cohort while 123 SNPs met significance in the Japanese American cohort. Fine mapping revealed three significant independent loci in the African American cohort and two significant independent loci in the Japanese American cohort. These identified loci confirm findings from previous GWAS of prostate cancer in diverse cohorts while PrediXcan-identified genes suggest potential new directions for prostate cancer research in populations across the globe.View Full Text

genetics↗

Preparation and characterization of phospholipid stabilized nanoemulsions in small-scale

Phospholipids have been used to prepare liposomes. The use of phospholipids to stabilize nanoemulsions may cause spontaneous formation of liposomes. The main objective of this study is to develop a method to prepare phospholipid stabilized nanoemulsions in small scale (< 1 mL) and to minimize the formation of liposomes.\n\nA combination of hand extrusion and detergent removal methods was used in this study. Extrusion through polycarbonate membranes was performed in two steps, firstly using membranes of 400 nm followed by 200 nm membranes as the second step. Sodium cholate was used as a detergent to solubilize the formed liposomes which was later removed via dialysis. Nanoemulsions were characterized by measuring their particle size, polydispersity index and zeta-potential using Photon Correlation Spectroscopy and Cryo-TEM pictures. The stability of nanoemulsion stored under refrigeration was also studied.\n\nFifty-one extrusion cycles through polycarbonate membrane of 400 nm pore size followed by one-hundred fifty-three cycles through polycarbonate membrane of 200 nm produced nanoemulsions having particle size below 200 nm (diameter). The nanoemulsions were found to be homogenous as depicted by polydispersity index (PDI) value below 0.1. Similarly, the zeta-potential was measured to be above -30 mV which is sufficient to keep nanoemulsions stable for as long as 7 months when stored under refrigeration. The Cryo-TEM pictures revealed 30 mM to be an optimum concentration of sodium cholate to prepare homogenous nanoemulsions with negligible proportion of liposomes.\n\nIt was concluded that this method could be established as a small scale method of preparing nanoemulsions which will not only reduce the cost of preparation but also the disposal cost of toxic chemicals used for functionalizing nanoemulsions for scientific research.

biophysics↗

Ataxia Telangiectasia triggers deficits in Reelin pathway

Autosomal recessive Ataxia Telangiectasia (A-T) is characterized by radiosensitivity, immunodeficiency and cerebellar neurodegeneration. A-T is caused by inactivating mutations in the Ataxia-Telangiectasia-Mutated (ATM) gene, a serine-threonine protein kinase involved in DNA-damage response and excitatory neurotransmission. The selective vulnerability of cerebellar Purkinje neurons (PN) to A-T is not well understood.\n\nEmploying global proteomic profiling of cerebrospinal fluid from patients at ages around 15 years we detected reduced Calbindin, Reelin, Cerebellin-1, Cerebellin-3, Protocadherin Fat 2, Sempahorin 7A and increased Apolipoprotein -B, -H, -J peptides. Bioinformatic enrichment was observed for pathways of chemical response, locomotion, calcium binding and complement immunity. This seemed important, since secretion of Reelin from glutamatergic afferent axons is crucial for PN radial migration and spine homeostasis. Reelin expression is downregulated by irradiation and its deficiency is a known cause of ataxia. Validation efforts in 2-month-old Atm-/- mice before onset of motor deficits confirmed transcript reductions for Reelin receptors Apoer2/Vldlr with increases for their ligands Apoe/Apoh and cholesterol 24-hydroxylase Cyp46a1. Concomitant dysregulations were found for Vglut2/Sema7a as climbing fiber markers, glutamate receptors like Grin2b and calcium homeostasis factors (Atp2b2, Calb1, Itpr1), while factors involved in DNA damage, oxidative stress, neuroinflammation and cell adhesion were normal at this stage.\n\nThese findings show that deficient levels of Reelin signaling factors reflect the neurodegeneration in A-T in a sensitive and specific way. As an extracellular factor, Reelin may be accessible for neuroprotective interventions.

neuroscience↗