Biology subjects

Handa, A.

Publications and source records attributed to Handa, A..

10 recordsLinked to original sources

bioRxiv2024

3D super-resolution imaging of PSD95 reveals an abundance of diffuse protein supercomplexes in the mouse brain

PSD95 is an abundant scaffolding protein that assembles multiprotein complexes controlling synaptic physiology and behavior. Confocal microscopy has previously shown that PSD95 is enriched in the postsynaptic terminals of excitatory synapses and 2D super-resolution microscopy further revealed that it forms nanoclusters. In this study, we utilized 3D super-resolution microscopy to examine the nanoarchitecture of PSD95 in the mouse brain, characterizing over 8 million molecules. While we were able to identify structural subtypes previously reported, imaging in 3D allowed us to classify these with higher accuracy. Furthermore, 3D super-resolution microscopy enabled the quantification of protein levels, revealing an abundance of PSD95 molecules existed outside of synapses as a diffuse population of supercomplexes, containing multiple copies of PSD95. Further analysis of the supercomplexes containing two units identified two populations: one that had PSD95 molecules separated by 39 {+/-} 2 nm, and a second with a separation of 94 {+/-} 27 nm. These results suggest that PSD95 supercomplexes containing multiple protein copies assemble outside the synapse and then integrate into the synapse to form a supramolecular nanocluster architecture.

biophysics↗

bioRxiv2023

The NFκB Dif is required for behavioral and molecular correlates of sleep homeostasis in Drosophila

The nuclear factor binding the {kappa} light chain in B-cells (NF{kappa}B) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, efforts have been limited toward understanding how specific NF{kappa}B transcription factors function in sleep. Drosophila fruit flies carry three genes encoding NF{kappa}B transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the distribution of a Dif-associated GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was suppressed in Dif mutants and pan-neuronal over-expression of nur also suppressed the Dif mutant phenotype. Together, these findings indicate that Dif functions from brain to target nemuri and to promote sleep. Statement of SignificanceNF{kappa}B transcription factors drive inflammatory processes that underly a multitude of human diseases that are accompanied by sleep disturbance. However, genetic studies in mammals that investigate a function of NF{kappa}B in sleep have been limited. Using genetic approaches in the Drosophila fruit fly, we show that the Dif NF{kappa}B, homologous to the Rel family of NF{kappa}Bs in humans, functions from neuronal tissue to regulate daily sleep and to mediate responses to sleep loss. These findings enhance our understanding of the role of a specific NF{kappa}B gene in sleep regulation.

neuroscience↗

bioRxiv2023

Overexpression of MUC1 influences the anti-proliferative effect of STAT3-inhibitor Napabucasin in epithelial cancers.

MUC1 is a transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in epithelial cancers. The cytoplasmic tail of MUC1 (MUC1 CT) aids in tumorigenesis by upregulating the expression of multiple oncogenes. Signal transducer and activator of transcription 3 (STAT3) plays a crucial role in several cellular processes and is aberrantly activated in many cancers. In this study, we focus on recent evidence suggesting that STAT3 and MUC1 regulate each others expression in cancer cells in an auto-inductive loop and found that their interaction plays a prominent role in mediating epithelial-to-mesenchymal transition (EMT) and drug resistance. The STAT3 inhibitor Napabucasin was in clinical trials but was discontinued due to futility. We found that higher expression of MUC1 increased the sensitivity of cancer cells to Napabucasin. Therefore, high-MUC1 tumors may have a better outcome to Napabucasin therapy. We report how MUC1 regulates STAT3 activity and provide a new perspective on repurposing the STAT3-inhibitor Napabucasin to improve clinical outcome of epithelial cancer treatment.

cancer biology↗

bioRxiv2023

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 {+/-} 0.1%, 0.1 locs m-2) and sensitivity (>86.6 {+/-} 0.9%, 0.1 locs m-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs m-2) in dense cytosolic tubulin datasets.

biophysics↗

bioRxiv2021

PROTECTIVE ROLE FOR SMOOTH MUSCLE CELL HEPCIDIN IN ABDOMINAL AORTIC ANEURYSM

RationaleHepcidin (HAMP) is a hormone produced primarily in the liver. It controls systemic iron homeostasis by inhibiting the iron exporter ferroportin (FPN) in the gut and spleen, respective sites of iron absorption and recycling. HAMP and FPN are also found ectopically in tissues not involved in systemic iron homeostasis. The physiological functions of ectopic HAMP and FPN are only just beginning to be uncovered. We observed that HAMP expression is markedly increased in smooth muscle cells (SMCs) of abdominal aortic aneurysms (AAA), both in patients and in an experimental mouse model of AAA. ObjectiveTo understand the role of SMC-derived HAMP in the pathophysiology of AAA. Methods and ResultsWe generated mice harbouring an inducible, SMC-specific deletion of the hamp gene. We then applied the experimental model of AAA and simultaneously induced deletion of hamp in SMCs. We found that these mice developed large aneurysms and had greater incidences of rupture and of fatal dissection than mice with intact hamp in SMCs. A similar phenotype was observed in mice harbouring an inducible SMC-specific knock-in of HAMP-resistant FPNC326Y. Additionally, we observed that expression of Lipocalin-2 (LCN2), a protein known to promote AAA, was suppressed in AAA tissue from patients and from mice with intact hamp in SMCs, but not in mice lacking hamp in SMCs. Treatment of these mice with a LCN2-neutralising antibody protected them from the otherwise detrimental effects of loss of hamp in SMCs. ConclusionsThe present study demonstrates that the rise in SMC-derived HAMP within the aneurysm tissue is protective in the setting of AAA, and that such protection involves the cell-autonomous action of HAMP, and suppression of local LCN2. These findings are the first example of a protective role for ectopic HAMP in disease. They expand understanding of the multifaceted functions of HAMP outside the liver.

physiology↗

bioRxiv2020

Development of a Novel Index to Characterise Arterial Dynamics Using Ultrasound Imaging

Abdominal aortic aneurysms (AAA) are associated with systemic inflammation and endothelial dysfunction. We previously reported flow mediated dilatation (FMD) of the brachial artery as a predictor of AAA growth. We hence hypothesised that other physical characteristics of the brachial artery correlate with AAA growth. Using a prospectively cohort of AAA patients, we devised a brachial artery relaxation index (BARI) and examined its role as a biomarker for AAA growth. However, no correlation between BARI and future aneurysm growth was observed (p=0.5). Therefore, our investigations did not substantiate the hypothesis that other physical characteristics of the brachial artery predicts AAA growth.

bioengineering↗

bioRxiv2020

Genome-wide association analysis and replication in 810,625 individuals identifies novel therapeutic targets for varicose veins

BackgroundVaricose veins (VVs) affect one-third of Western society, with a significant subset of patients developing venous ulceration, and ongoing management of venous leg ulcers costing around $14.9 billion annually in the USA. There is no current medical management for VVs, with approaches limited to compression stockings, ablation techniques, or open surgery for more advanced disease. A significant proportion of patients report a positive family history, and heritability is ~17%, suggesting a strong genetic component. We aimed to identify novel therapeutic targets by improving our understanding of the aetiopathology and genetic architecture of VVs. MethodsWe performed the largest two-stage genome-wide association study of VVs in 401,656 subjects from UK Biobank, and replication in 408,969 subjects from 23andMe (total 135,514 varicose veins cases and 675,111 controls). We constructed a genetic risk score for VVs to investigate its use as a prognostic tool. Genes and pathways were prioritised using a suite of bioinformatic tools, and therapeutic targets identified using the Open Targets Platform. ResultsWe discovered 49 signals at 46 susceptibility loci associated with VVs, including 29 previously unreported genetic associations (28 susceptibility loci). We demonstrated that patients with VVs requiring surgery have a higher genetic risk score than those managed non-surgically. We map 237 genes to these loci, many of which are biologically relevant and tractable to therapeutic targeting or repurposing (notably VEGFA, COL27A1, EFEMP1, PPP3R1 and NFATC2). Tissue enrichment analyses implicated vascular tissue, and several genes were enriched in biological pathways relating to extracellular matrix biology, inflammation, angiogenesis, lymphangiogenesis, vascular smooth muscle cell migration, and apoptosis. ConclusionsGenes and pathways identified represent biologically plausible contributors to the pathobiology of VVs, identifying promising candidates for further investigation of venous biology and potential therapeutic targets. We have provided the proof-of-principle that genetic risk score correlates with disease severity, which represents a first step in personalised medicine approaches to varicose veins.

genetics↗

bioRxiv2020

Integrated Plasma and Tissue Proteomics Reveals Attractin Release by Intraluminal Thrombus of Abdominal Aortic Aneurysms and Improves Aneurysm Growth Prediction in Humans

Background: Abdominal aortic aneurysms (AAA) are pathological dilatations of the aorta which can result in rupture and mortality. Novel methods of predicting AAA growth is a recognised priority in AAA research. Patient with AAAs have increased risk of cardiovascular morbidity. We have previously observed accelerated systemic endothelial dysfunction (measured by brachial artery FMD) in AAA patients and FMD correlates with future AAA growth. Further, systemic endothelial dysfunction is reversed by AAA repair. AAAs contain intra-luminal thrombus (ILT). Since ILT is either removed or excluded from circulation after successful repair of AAAs, we hypothesise that ILT to be the source of mediators that contribute to AAA growth. Methods: Patients were prospectively recruited to the Study (Ethics Ref SC/13/0250). Plasma samples were collected at baseline and at 1 year from each patient. Plasma samples were also collected before and at 10-12 weeks after surgery from each patient (n=29). Paired aneurysm wall, ILT, omental biopsies were collected intra-operatively during open surgical repair (n=3). In addition to analyses of the tissue, supernatant was obtained from ex vivo culture of these paired tissue samples. Samples were subjected to non-targeted LC-MSMS workflow after trypsin digest, using the Universal method to discover novel proteins. LC-MSMS data was analysed using the Progenesis QI pipeline. Results: The median AAA size at baseline was 48 mm. 59 patients were prospectively followed for 12 months. The median growth rate of AAA was 3.8%/year (IQR 1.9% to 6.8%). Comparison between patients with the fastest vs the slowest (n=10 each) showed 116 proteins to be differentially expressed in their plasma. Among these proteins, 35 also changed significantly before and after AAA repair, suggesting their origin to from the AAA complex. Comparison of the proteomics profile of aneurysm tissue, ILT, and omental artery show 128 proteins to be uniquely present in ILT. Analyses of the tissue culture supernatant further revealed 3 proteins that are: (i) uniquely present in ILT; (ii) released by ILT; (iii) systemic levels reduced after AAA surgery; (iv) differs between fast and slow growth AAAs. One of these proteins is attractin. To validate the LC-MSMS data, attractin level in individual patient was measured by ELISA. Consistent with the LC-MSMS data, plasma attractin level is higher in patients with fast AAA growth. Plasma attractin level correlates significantly with future AAA growth rate (Spearman r=0.35, P<;0.005). Using attractin and AAA diameter as input variables, the AUROC for predicting no growth of AAA at 12 months is 85% (P<0.001). Conclusion: We show that ILT of AAAs releases mediators (such as attractin) during the natural history of AAA growth. These are novel biomarkers for AAA growth prediction in humans.

systems biology↗

bioRxiv2020

vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching3. While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced volume) a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.

biophysics↗

bioRxiv2019

Thymosin β4 mediates vascular protection via regulation of Low Density Lipoprotein Related Protein 1 (LRP1)

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic switch which promotes atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin {beta}4 (T{beta}4) may additionally function to maintain healthy vasculature and protect against disease throughout postnatal life. This was supported by identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. T{beta}4-null mice display aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. We confirmed predisposition to disease in models of atherosclerosis and AAA. Diseased vessels and plaques were characterised by accelerated contractile-synthetic VSMC switching and augmented PDGFR{beta} signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of T{beta}4, coincided with dysregulated endocytosis, leading to increased recycling of LRP1-PDGFR{beta} and reduced lysosomal targeting. Our study identifies T{beta}4 as a key regulator of LRP1 for maintaining vascular health, providing insight which may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression.

cell biology↗