Biology subjects

Yu, Z.

Publications and source records attributed to Yu, Z..

At least 289 records · Page 16Linked to original sources

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2021

Thermal Fogging with Disinfectant Didecyl Dimethyl Ammonium Bromide Effectively Kills A Coronavirus, An Influenza Virus and Two Indicator Bacteria in Subzero Cold-Chain Environment

BackgroundThe origin of several local COVID-19 outbreaks in China in 2020 were confirmed to be frozen food or packages contaminated with SARS-CoV-2, revealing the lack of effective disinfection measures in the frozen food chain. ObjectiveTo evaluate the disinfection efficacy at -20{degrees}C of a recently marketed thermal fogging disinfection product, with disinfectant-antifreeze combination being didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG). MethodCarriers with porcine epidemic diarrhea virus (PEDV) YN1, a coronavirus, and a swine influenza virus H1N1 (SIV-H1N1), and three indicator bacteria, E. coli, S. aureus, and B. subtilis endospores, were respectively placed in a -20{degrees}C freezer warehouse with or without DDAB-PPG fogging and activities of the microorganisms were tested. ResultsAt -20{degrees}C, DDAB-PPG fogging, which fully settles in 3.5-4.5 hours, fully inactivated PEDV of 10-3.5 TCID50/0.1ml and SIV-H1N1 of 26 hemagglutination titer within 15-30 min, and inactivated S. aureus and E. coli vegetative cells (106/ml) within 15 or 60 min, respectively, but had little effect on B. subtilis spores. The bactericidal effect lasted at least 3 hours for bacteria on carrier plates and for 6 hours for airborne bacteria. ConclusionsA practical subzero temperature disinfection technology was confirmed its efficacy in killing enveloped viruses and vegetative bacteria. It would help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity. HighlightsO_LIFirst report of efficacy of disinfectant didecyl dimethyl ammonium bromide (DDAB) at subzero temperatures C_LIO_LIThe customized thermal fogging machine makes fine droplets of DDAB-propylene glycol (PPG) fog which can suspend for ~4 hours at -20{degrees}C C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C effectively inactivated a coronavirus and an influenza virus C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C for effectively killed S. aureus and E. coli C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C inactivated airborne microorganism for up to 6 h C_LI

molecular biology↗

bioRxiv2021

Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

molecular biology↗

bioRxiv2021

Comparative genomics reveals evolutionary drivers of sessile life and left-right shell asymmetry in bivalves

Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems. Across these ancient lineages, colony-founding larvae anchor themselves either by byssus production or by cemented attachment. The latter mode of sessile life is strongly molded by left-right shell asymmetry during larval development of Ostreoida oysters such as Crassostrea hongkongensis. Here, we sequenced the genome of C. hongkongensis in high resolution and compared it to reference bivalve genomes to unveil genomic determinants driving cemented attachment and shell asymmetry. Importantly, loss of the homeobox gene antennapedia (Antp) and broad expansion of lineage-specific extracellular gene families are implicated in a shift from byssal to cemented attachment in bivalves. Evidence from comparative transcriptomics shows that the left-right asymmetrical C. hongkongensis plausibly diverged from the symmetrical Pinctada fucata in expression profiles marked by elevated activities of orthologous transcription factors and lineage-specific shell-related gene families including tyrosinases, which may cooperatively govern asymmetrical shell formation in Ostreoida oysters.

genomics↗

bioRxiv2021

HDAC6 promote HCCLM3 cell migration and invasion by regulating TGF-β1pathway

Histone deacetylase 6 (HDAC6) have been demonstrated to play critical roles in the progression of tumor migration and invasion in recent years.However, little research has been done on hepatocellular carcinoma (HCC). This study investigated the role of HDAC6 in the migration and invasion of HCCLM3 cells and its potential mechanism. Western blot and immunohistochemistry were used to detect the expression of HDAC6 in normal hepatic cell line LO2 and HCC cell line HCCLM3. Transforming growth factor 1 (TGF-{beta}1) was proved as a direct target of HDAC6 by Western blot and qRT-PCR.The changes of migration and invasion ability of HCCLM3 cells were confirmed by wound healing and Transwell assays.Morphological changes of HCCLM3 cells were observed by microscope.Our study proved that drownregulated HDAC6 supress HCCLM3 cells metastasis and invasion. Meanwhile, our study verified that HDAC6 regulate EMT by targeting TGF-{beta}1.In conclusion, HDAC6 inhibits the EMT of HCCLM3 cells and further inhibits the migration and invasion of HCCLM3 cells by downregulating the expression of TGF-{beta}1.

biochemistry↗

bioRxiv2021

Mendelian Randomization Analysis Using Multiple Biomarkers of an Underlying Common Exposure

SO_SCPLOWUMMARYC_SCPLOWMendelian Randomization (MR) analysis is increasingly popular for testing the causal effect of exposures on disease outcomes using data from genome-wide association studies. In some settings, the underlying exposure, such as systematic inflammation, may not be directly observable, but measurements can be available on multiple biomarkers or other types of traits that are coregulated by the exposure. We propose a method for MR analysis on latent exposures (MRLE), which tests the significance for, and the direction of, the effect of a latent exposure by leveraging information from multiple related traits. The method is developed by constructing a set of estimating functions based on the second-order moments of GWAS summary association statistics for the observable traits, under a structural equation model where genetic variants are assumed to have indirect effects through the latent exposure and potentially direct effects on the traits. Simulation studies show that MRLE has well-controlled type I error rates and enhanced power compared to single-trait MR tests under various types of pleiotropy. Applications of MRLE using genetic association statistics across five inflammatory biomarkers (CRP, IL-6, IL-8, TNF-, and MCP-1) provide evidence for potential causal effects of inflammation on increasing the risk of coronary artery disease, colorectal cancer, and rheumatoid arthritis, while standard MR analysis for individual biomarkers fails to detect consistent evidence for such effects.

genetics↗

bioRxiv2020

Gut microbiome couples gut and brain during calorie restriction in treating obesity

Calorie restriction (CR) has been widely recognized for its effect in reducing body weight and alleviating diabetes in humans, as well as prolonging life span in animal studies. Gut microbiome shifts contribute to part of the effects of CR, but little is known regarding their influences except on metabolism and immunity. Here we monitored gut microbiome using metagenomics and metatranscriptomics in obese individuals undergoing CR, and revealed microbial determinants that could contribute to successful weight loss. Microbiome changes are linked to changes in blood metabolome and hormones, which eventually correlate to brain functional changes as studied using functional magnetic resonance imaging (fMRI). Brain functional shifts indicate response of central neural system (CNS) to CR, and microbiome constitutes the keystone of gut-brain axis. Animal experiment further reaffirms the gut microbiome changes, metabolic and hormonal shifts of CR, while proteomic analysis of brain tissues suggest that epigenetic modifications of key proteins could explain responses of CNS to CR. Our study establishes linkage between CR, gut microbiome, metabolome/ hormones and CNS function, and demonstrates that CR has multi-facet, coordinated effects on the host, of which many could contribute to weight loss and other beneficial effects.

microbiology↗

bioRxiv2020

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Virus Infection as an Innate Antiviral Mechanism

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Redistribution of centrosomal proteins by centromeres and Polo kinase controls nuclear envelope breakdown

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB - yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high- resolution structured illumination microscopy (SIM), we show that the conserved SUN- domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.

cell biology↗

bioRxiv2020

Effect size, sample size and power of forced swim test assays in mice: Guidelines for investigators to optimize reproducibility

A recent flood of publications has documented serious problems in scientific reproducibility, power, and reporting of biomedical articles, yet scientists persist in their usual practices. Why? We examined a popular and important preclinical assay, the Forced Swim Test (FST) in mice used to test putative antidepressants. Whether the mice were assayed in a naive state vs. in a model of depression or stress, and whether the mice were given test agents vs. known antidepressants regarded as positive controls, the mean effect sizes seen in the experiments were indeed extremely large (1.5 - 2.5 in Cohens d units); most of the experiments utilized 7-10 animals per group which did have adequate power to reliably detect effects of this magnitude. We propose that this may at least partially explain why investigators using the FST do not perceive intuitively that their experimental designs fall short -- even though proper prospective design would require ~21-26 animals per group to detect, at a minimum, large effects (0.8 in Cohens d units) when the true effect of a test agent is unknown. Our data provide explicit parameters and guidance for investigators seeking to carry out prospective power estimation for the FST. More generally, altering the real-life behavior of scientists in planning their experiments may require developing educational tools that allow them to actively visualize the inter-relationships among effect size, sample size, statistical power, and replicability in a direct and intuitive manner.

scientific communication and education↗

bioRxiv2020

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

Integrated and quantitative observations of antibiotic resistance genes (ARGs) in urban water systems (UWSs) are lacking. We sampled three UWSs for clinically important extended spectrum {beta}-lactamase (ESBL) and carbapenemase (CP) genes, mobile genetic elements and microbial communities. Sewage - especially from hospitals - carried substantial loads of ESBL and CP genes (106 - 107 per person equivalent), but those loads progressively declined along the UWS, resulting in minimal emissions (101 - 104 copies per person equivalent). Removal was primarily during sewage conveyance (65% {+/-} 36%) rather than within sewage treatment (34% {+/-} 23%). The ARGs clustered in groups based on their persistence; less persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while more persistent groups appear horizontally transferred as they correlated with mobile genetic elements. This first documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based ARG surveillance.

ecology↗

bioRxiv2020

High-throughput phenotyping analysis of maize at the seedling stage using end-to-end segmentation network

Image processing technologies are available for high-throughput acquisition and analysis of phenotypes for crop populations, which is of great significance for crop growth monitoring, evaluation of seedling condition, and cultivation management. However, existing methods rely on empirical segmentation thresholds, thus can have insufficient accuracy of extracted phenotypes. Taking maize as an example crop, we propose a phenotype extraction approach from top-view images at the seedling stage. An end-to-end segmentation network, named PlantU-net, which uses a small amount of training data, was explored to realize automatic segmentation of top-view images of a maize population at the seedling stage. Morphological and color related phenotypes were automatic extracted, including maize shoot coverage, circumscribed radius, aspect ratio, and plant azimuth plane angle. The results show that the approach can segment the shoots at the seedling stage from top-view images, obtained either from the UAV or ground high-throughput phenotyping platform. The average segmentation accuracy, recall rate, and F1 score are 0.96, 0.98, and 0.97, respectively. The extracted phenotypes, including maize shoot coverage, circumscribed radius, aspect ratio, and plant azimuth plane angle, are highly correlated with manual measurements (R2=0.96-0.99). This approach requires less training data and thus has better expansibility. It provides practical means for high-throughput phenotyping analysis of early growth stage crop populations.

plant biology↗

bioRxiv2020

Polygenic Risk Scores for Kidney Function to the Circulating Proteome, and Incident Kidney Diseases: the Atherosclerosis Risk in Communities Study

Genome-wide association studies (GWAS) have revealed numerous loci for kidney function (estimated glomerular filtration rate, eGFR). The relationship of polygenic predictors of eGFR, risk of incident adverse kidney outcomes, and the plasma proteome is not known. We developed a genome-wide polygenic risk score (PRS) using a weighted average of 1.2 million SNPs for eGFR using the LDpred algorithm, summary statistics generated by a European-ancestry (EA) meta-analysis of the CKDGen Consortium (N=558,423) and UK Biobank GWAS for eGFR (90% of the cohort; N=289,432), followed by best parameter selection using data from the remaining 10% of the UK Biobank (N=32,159). We then tested the association of the PRS among 8,886 EA participants in the Atherosclerosis Risk in Communities (ARIC) study (mean age: 54{+/-}6 years, 53% female) with incident chronic kidney disease (CKD), end stage kidney disease (ESKD), kidney failure (KF), and acute kidney injury (AKI). We also examined 4,877 plasma proteins measured at two time points (visit 3 (1993-95) and visit 5 (2011-13)) in relation to the PRS and compared associations between the proteome and eGFR itself. All models were adjusted for age, sex, center, and the first 10 principal components of ancestry. The developed PRS had an R2 for eGFR of 0.07 in ARIC. Over 30 years of follow up, the number of incident CKD, ESKD, KF, and AKI were 2,959, 137, 470, and 1,723, respectively. The PRS showed significant associations with all outcomes: hazard ratios (95% CI) per 1 SD lower PRS were 1.33 (1.28, 1.39), 1.20 (1.00, 1.42), 1.17 (1.06, 1.28), and 1.07 (1.02, 1.12) for incident CKD, ESKD, KF, and AKI respectively. The PRS was significantly associated (Bonferroni threshold P<1.02 x 10-5) with 108 proteins at both time points. The strongest associations were with cystatin-C (a marker of kidney function used in clinical practice), collagen alpha-1 (XV) chain, and desmocollin-2. All significant correlations with the PRS were negative, except those of testican-2 and angiostatin. Correlations of proteins with eGFR were much stronger than those with the PRS. Overall, we demonstrated that the PRS for eGFR is now sufficiently strong to capture risk for a spectrum of incident kidney diseases as well as broadly influence the plasma proteome.

genetics↗

bioRxiv2020

Robust Population Single Neuronal Calcium Signal Extraction Using SCOUT Allows for Longitudinal Analysis of Behavior-associated Neural Ensemble Dynamics

In vivo calcium imaging enables simultaneous recording of large neuronal ensembles while engaged in operations such as learning and memory. However, such in vivo optical recordings are typically subject to motion artifact and background contamination from neurons and blood vessels. Further, population cell tracking across multiple recordings is complicated by non-rigid transformation induced by cell movements and field shifts. We introduce the novel method SCOUT for Single-Cell SpatiOtemporal LongitUdinal Tracking, consisting of two crucial parts: (1) imposition of spatial constraints on neuronal footprints extracted from individual optical recordings to improve ROI selection and eliminate false discoveries, and (2) application of a predictor-corrector, using spatiotemporal correlation of extracted neurons across sessions, for population cell tracking across multiple sessions. SCOUT empirically outperforms current methods for cell extraction and tracking in long-term multi-session imaging experiments across multiple brain regions. Application of this method allows for robust longitudinal analysis of contextual discrimination associated neural ensemble dynamics in the hippocampus up to 60 days.

neuroscience↗

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

molecular biology↗

bioRxiv2020

Enhancing SNR and generating contrast for cryo-EM images with convolutional neural networks

In cryogenic electron microscopy (cryo-EM) of radiation-sensitive biological samples, both the signal-to-noise ratio (SNR) and the contrast of images are critically important in the image processing pipeline. Classic methods improve low-frequency image contrast experimentally, by imaging with high defocus, or computationally, by applying various types of low-pass filter. These contrast improvements typically come at the expense of high-frequency SNR, which is suppressed by high-defocus imaging and removed by low pass filtration. Here, we demonstrate that a convolutional neural network (CNN) denoising algorithm can be used to significantly enhance SNR and generate contrast in cryo-EM images. We provide a quantitative evaluation of bias introduced by the denoising procedure and its influences on image processing and three-dimensional reconstructions. Our study suggests that besides enhancing the visual contrast of cryo-EM images, the enhanced SNR of denoised images may facilitate better outcomes in the other parts of the image processing pipeline, such as classification and 3D alignment. Overall, our results provide a ground of using denoising CNNs in the cryo-EM image processing pipeline.

biophysics↗

bioRxiv2020

SWR1-independent association of H2A.Z to the LINC complex promotes meiotic chromosome motion

ABSTRACTThe H2A.Z histone variant is deposited into chromatin by the SWR1 complex affecting multiple aspects of meiosis. Here we describe a SWR1-independent localization of H2A.Z at meiotic telomeres and the centrosome. We demonstrate that H2A.Z colocalizes and interacts with Mps3, the SUN component of the LINC complex that spans the nuclear envelope and links meiotic telomeres to the cytoskeleton promoting meiotic chromosome movement. H2A.Z also interacts with the meiosis-specific Ndj1 protein that anchors telomeres to the nuclear periphery via Mps3. Telomeric localization of H2A.Z depends on Ndj1 and the N-terminal domain of Mps3. Although telomeric attachment to the nuclear envelope is maintained in the absence of H2A.Z, the distribution of Mps3 is altered. The velocity of chromosome movement during meiotic prophase I is reduced in the htz1Δ mutant lacking H2A.Z, but it is unaffected in swr1Δ cells. We reveal that H2A.Z is an additional LINC-associated factor that contributes to promote telomere-driven chromosome motion critical for error-free gametogenesis.Competing Interest StatementThe authors have declared no competing interest.Non-standard abbreviationsBiFCBimolecular Fluorescence ComplementationDICDifferential Interference ContrastKAcPotassium AcetateNENuclear EnvelopeLINCLinker of the Nucleoskeleton and CytoskeletonrDNARibosomal DNASCSynaptonemal ComplexSIMStructured Illumination MicroscopySPBSpindle Pole BodyVCC-terminal moiety of the Venus fluorescent proteinVNN-terminal moiety of the Venus fluorescent proteinWCEWhole cell extractsView Full Text

cell biology↗

bioRxiv2020

In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution

In Gram-negative bacteria, tripartite efflux pump AcrAB-TolC plays a prominent role in antibiotic resistance. We have used high resolution cryo-ET to visualize the structure of Escherichia coli AcrAB-TolC at a 7 [A] resolution in intact cells. The resulting structures show the detailed architecture of the assembled complex embedded into cell envelope. Interactions with the inner membrane enable crosstalk between AcrB and TolC through AcrA, suggesting that assembly in the native cellular environment is critical for the pump activation mechanism, where the allosteric activating signal is triggered by the alternate binding of AcrA to the lipid membrane and AcrB porter domain. We establish a platform for high resolution in situ structural studies of bacteria efflux pump, which can yield critical information in understanding complex assemblies function. One sentence summaryA 7 [A] in situ structure of the AcrAB-TolC pump from bacteria by cryo-ET reveals mechanisms for active efflux.

biophysics↗