Biology subjects

Lu, J.

Publications and source records attributed to Lu, J..

At least 361 records · Page 20Linked to original sources

bioRxiv2019

Moonlighting α-PheRS connects JAK/STAT with Notch signaling for intestinal homeostasis

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that do neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, -PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated -PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype resembles partially the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between -PheRS and Notch suggest that their activities neutralize each other and that elevated -PheRS levels attenuate Notch signaling whether Notch induces differentiation into enterocytes, neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of -PheRS which accumulates naturally in the intestine. This truncated version of -PheRS (-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that -S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in -PheRS and -S, but also essential for attenuating Notch signaling. Author SummaryAminoacyl tRNA synthetases charge tRNAs with their cognate amino acid to ensure proper decoding of the genetic code during translation. Independent of its aminoacylation function, the alpha subunit of Drosophila cytoplasmic Phenylalanyl tRNA synthetase (-PheRS, FARSA in humans) has an additional activity that promotes growth and proliferation (Ho et al., 2021). Here we describe that elevated -PheRS levels also induce cell fate changes and tumorous phenotypes in Drosophila midguts. Excessive proliferating cells with stem and progenitor cell characteristics accumulate and the composition of the terminally differentiated cells changes, too. This phenotype together with observed genetic interactions between -PheRS and Notch levels show that -PheRS counteracts Notch signaling in many different tissues and developmental stages. This novel activity of -PheRS maps to its N-terminal part, which is naturally produced. The fragment contains a DNA binding domain, translocates into nuclei, and displays essential similarities to a Notch domain that binds to the downstream transcription factor. This suggests that it might be competing with Notch for binding to a common target. Not only because Notch plays important roles in many tumors, but also because FARSA mRNA levels are considerably upregulated in many tumors, this novel activity deserves more attention for cancer research.

cell biology↗

bioRxiv2019

Effective Combination Immunotherapy using Oncolytic Viruses to Deliver CAR Targets to Solid Tumors

Chimeric antigen receptor (CAR)-engineered T cell therapy for solid tumors is limited by the lack of tumor-restricted and homogeneous expression of tumor antigens1,2. Therefore, we engineered an oncolytic virus to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-specific CAR T cells. Infecting tumor cells with a chimeric oncolytic vaccinia virus coding for CD19t (OV19t) produced de novo CD19 surface-antigen expression prior to virus-mediated tumor lysis. Co-cultured CD19-CAR T cells secreted cytokines and elicited potent cytolytic activity against infected tumors. Using multiple mouse tumor models, intratumoral delivery of OV19t induced tumor expression of CD19t and improved tumor control following CD19-CAR T cell administration. CAR T cell-mediated tumor killing also promoted release of virus from dying tumor cells, which propogated tumor expression of CD19t. These data demonstrate a novel immunotherapy approach utilizing oncolytic viruses to promote de novo CAR T cell targeting of solid tumors. One Sentence SummaryWe describe a novel and effective combination immunotherapy utilizing oncolytic viruses to deliver de novo cell surface expression of CD19 antigen promoting CD19-CAR T cell anti-tumor responses against solid tumors.

cancer biology↗

bioRxiv2019

BDKANN - Biological Domain Knowledge-based Artificial Neural Network for drug response prediction

MotivationOne of the main goals of precision oncology is to predict the response of a patient to a given cancer treatment based on their genomic profile. Although current models for drug response prediction are becoming more accurate, they are also black boxes and cannot explain their predictions, which is of particular importance in cancer treatment. Many models also do not leverage prior biological knowledge, such as the hierarchical information on how proteins form complexes and act together in pathways. ResultsIn this work, we use this prior biological knowledge to form the architecture of a deep neural network to predict cancer drug response from cell line gene expression data. We find that our approach not only has a low prediction error compared to baseline models but also allows meaningful interpretation of the network. These interpretations can both explain predictions made and discover novel connections in the biological knowledge that may lead to new hypotheses about mechanisms of drug action. AvailabilityCode at https://github.com/osnow/BDKANN Supplementary informationIncluded with submission

bioinformatics↗

bioRxiv2019

microRNA seedless sites attenuate strong-seed-site-mediated target repression

MicroRNAs (miRNAs) regulate protein-coding gene expression primarily through cognitive binding sites in the 3 untranslated regions (3' UTRs). Seed sites are sequences in messenger RNAs (mRNAs) that form perfect Watson-Crick base-paring with a miRNAs seed region, which can effectively reduce mRNA abundance and/or repress protein translation. Some seedless sites, which do no form perfect seed-pairing with a miRNA, can also lead to target repression, often with lower efficacy. Here we report the surprising finding that when seedless sites and seed sites are co-present in the same 3UTR, seedless sites attenuate strong-seed-site-mediated target suppression, independent of 3' UTR length. This attenuation effect is detectable in >70% of transcriptomic datasets examined, in which specific miRNAs are experimentally increased or decreased. The attenuation effect is confirmed by 3UTR reporter assays and mediated through base-pairing between miRNA and seedless sites. Furthermore, this seedless-site-based attenuation effect could affect seed sites of the same miRNA or another miRNA, thus partially explaining the variability in target suppression and miRNA-mediated gene upregulation. Our findings reveal an unexpected principle of miRNA-mediated gene regulation, and could impact the understanding of many miRNA-regulated biological processes.

bioinformatics↗

bioRxiv2019

Broad-Spectrum Antifungal Activities and Mechanism of Drimane Sesquiterpenoids

Eight drimane sesquiterpenoids including (-)-drimenol and (+)-albicanol were synthesized from (+)-sclareolide and evaluated for their antifungal activities. Three compounds, (-)-drimenol, (+)-albicanol, and (1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyl-decahydronaphthalene-1-carbaldehyde (4) showed strong activity against C. albicans. (-)-Drimenol, the strongest inhibitor of the three, (at concentrations of 8 - 64 g/ml, causing 100% death of fungi), acts not only against C. albicans as a fungicidal manner, but also inhibits other fungi such as Aspergillus, Cryptococcus, Pneumocystis, Blastomyces, Fusarium, Rhizopus, Saksenaea and FLU resistant strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. auris. These observations suggest drimenol is a broad-spectrum antifungal agent. At high concentration (100 g/ml), drimenol caused a rupture of the fungal cell wall/membrane. In a nematode model of C. albicans infection, drimenol rescued the worms from C. albicans-mediated death, indicating drimenol is tolerable and bioactive in a metazoan. Genome-wide fitness profiling assays of both S. cerevisiae (nonessential homozygous and essential heterozygous) and C. albicans (Tn-insertion mutants) collections revealed putative genes and pathways affected by drimenol. Using a C. albicans mutants spot assay, the Crk1 kinase associated gene products, Ret2, Cdc37, and novel putative targets orf19.759, orf19.1672, and orf19.4382 were revealed to be the potential targets of drimenol. Further, computational modeling results suggest possible modification of the structure of drimenol including the A ring for improving antifungal activity.

microbiology↗

bioRxiv2019

Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimers disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD metabolism and Tau homeostasis in aging and neurodegeneration.

biochemistry↗

bioRxiv2019

Selective constraints and pathogenicity of mitochondrial DNA variants inferred from a novel database of 196,554 unrelated individuals

High quality population allele frequencies of DNA variants can be used to discover new biology, and study rare disorders. Here, we created a public catalog of mitochondrial DNA variants based on a population of 195,983 individuals. We focused on 3 criteria: (i) the population is not enriched for mitochondrial disorders, or other clinical phenotypes, (ii) all genomes are sequenced and analyzed in the same clinical laboratory, and (iii) both homoplasmic and heteroplasmic variants are reported. We found that 47% of the mitochondrial genome was invariant in this population, including large stretches in the 2 rRNA genes. This information could be used to annotate the mitochondrial genome in future studies. We also showed how to use this resource for the interpretation of pathogenic variants for rare mitochondrial disorders. For example, 42% of variants previously reported to be pathogenic for Leber Hereditary Optic Neuropathy (LHON) should be reclassified.

genetics↗

bioRxiv2019

MicroRNA-210 Knockout Alters Dendritic Density and Behavioural Flexibility

MicroRNAs exert post-transcriptional control over eukaryotic genomes and are integral for regulating complex functions in neurodevelopment, neuroplasticity and cognitive processing. The microRNA-210 (miR-210) is a highly-conserved, hypoxia-regulated miRNA that has been shown to be dysregulated in Alzheimers Disease. We have recently also identified a significant enrichment of neurodegenerative diseases among miR-210 targets within the human transcriptome. To further elucidate the role of miR-210 in neuronal function and cognitive behaviour, we utilised conditional miR-210 knockout mice to characterise miR-210 regulation and function in primary hippocampal neurons and measured visual discrimination and reversal learning using rodent touchscreen assays. We found that miR-210 expression was induced by neuronal activation and loss of neuronal miR-210 increased oxidative metabolism and ROS production as well as dendritic density and branching in vitro. We also found that loss of miR-210 in knockout mice increased behavioural flexibility and reduced perseverative responding during reversal learning which required updating of information and feedback. These data support a conserved, activity-dependent role for miR-210 in cognitive function through modulation of neuronal metabolism.

neuroscience↗

bioRxiv2019

Improved metagenomic analysis with Kraken 2

Although Krakens k-mer-based approach provides fast taxonomic classification of metagenomic sequence data, its large memory requirements can be limiting for some applications. Kraken 2 improves upon Kraken 1 by reducing memory usage by 85%, allowing greater amounts of reference genomic data to be used, while maintaining high accuracy and increasing speed five-fold. Kraken 2 also introduces a translated search mode, providing increased sensitivity in viral metagenomics analysis.

bioinformatics↗

bioRxiv2019

γ-Secretase controls the specification of astrocytes from oligodendrocyte precursor cells via Stat3

Oligodendrocytes (OLs) and astrocytes play critical roles in a variety of brain functions. OL precursor cells (OPCs) are known to give rise to OLs as well as astrocytes. However, little is known about the mechanism by which OPCs determine their specification choice for OLs versus astrocytes in the central nervous system (CNS). Here we show that genetic inhibition of {gamma}-secretase in OPCs reduces OL differentiation but enhances astrocyte specification. Mechanistic analysis reveals that inhibition of {gamma}-secretase results in decreased levels of Hes1, and that Hes1 down-regulates the expression of signal transducer and activator of transcription3 (Stat3) via binding to specific regions of its promoter. We demonstrate that conditional inactivation of Stat3 in OL lineages restores the number of astrocytes in {gamma}-secretase mutant mice. In summary, this study identifies a key mechanism which controls OPCs specification choice for OL versus astrocyte during postnatal development. This {gamma}-secretase-dependent machinery may be essential for the CNS to maintain the population balance between OLs and astrocytes.

neuroscience↗

bioRxiv2019

The PRMT7-dependent methylation of shank2 modulates invasion-proliferation switching during breast cancer metastasis

Invasiveness of cancer cells is associated with proliferation inhibition in multiple types of cancers. Here, we identified the pivotal roles of Arginine methyltransferase PRMT7 in promoting invasion and attenuating proliferation of breast cancer cells. PRMT7 exerted its functions through binding to the scaffold protein shank2 to induce the di-methylation of shank2 at R240. Shank2 R240 methylation exposed ANK domain by disrupting its SPN-ANK domain blockade. Moreover, shank2 R240 methylation rendered recruitment of FAK that elicited the FAK auto-phosphorylation, which consequently augmented the shank2-dependent migration and invasion of breast cancer cells. On the other hand, the shank2 R240 methylation impeded proliferation of breast cancer cells by antagonizing the Ras-Raf binding via tethering the mono-ubiquitinated H-Ras. These findings characterize the PRMT7-dependent shank2 methylation as a key player in mediating reciprocal switching between invasion and proliferation, also point to the value of shank2 R240 methylation as a target for tumour metastasis treatment strategies.

cancer biology↗

bioRxiv2019

Non-antibiotic pharmaceuticals can enhance the spread of antibiotic resistance via conjugation

Antibiotic resistance is a global threat for public health. It is widely acknowledged that antibiotics at sub-inhibitory concentrations are important in disseminating antibiotic resistance via horizontal gene transfer. While there is high use of non-antibiotic human-targeted pharmaceuticals in our societies, the potential contribution of these on the spread of antibiotic resistance has been overlooked so far. Here, we report that commonly consumed non-antibiotic pharmaceuticals, including nonsteroidal anti-inflammatories (ibuprofen, naproxen, diclofenac), a lipid-lowering drug (gemfibrozil), and a {beta}-blocker (propanolol), at clinically and environmentally relevant concentrations, significantly accelerated the conjugation of plasmid-borne antibiotic resistance genes. We looked at the response to these drugs by the bacteria involved in the gene transfer through various analyses that included monitoring reactive oxygen species (ROS) and cell membrane permeability by flow cytometry, cell arrangement, and whole-genome RNA and protein sequencing. We found the enhanced conjugation correlated well with increased production of ROS and cell membrane permeability. We also detected closer cell-to-cell contact and upregulated conjugal genes. Additionally, these non-antibiotic pharmaceuticals caused the bacteria to have responses similar to those detected when exposed to antibiotics, such as inducing the SOS response, and enhancing efflux pumps. The findings advance our understanding of the bacterial transfer of antibiotic resistance genes, and importantly emphasize concerns of non-antibiotic human-targeted pharmaceuticals for enhancing the spread of antibiotic resistance.

microbiology↗

bioRxiv2019

Enhancing single-cell cellular state inference by incorporating molecular network features

In biological systems, genes function in conjunction rather than in isolation. However, traditional single-cell RNA-seq (scRNA-seq) analyses heavily rely on the transcriptional similarity of individual genes, ignoring the inherent gene-gene interactions. Here, we present SCORE, a network-based method, which incorporates the validated molecular network features to infer cellular states. Using real scRNA-seq datasets, SCORE outperforms existing methods in accuracy, robustness, scalability, data integration and removal of batch effect. When applying SCORE to a newly generated human ileal scRNA-seq dataset, we identified several novel stem/progenitor clusters, including a Cripto-1+ cluster. Moreover, two distinct groups of goblet cells were identified and only one of them tended to secrete mucus. Besides, we found that the recently identified BEST4+OTOP2+ microfold cells also highly expressed CFTR, which is different from their colonic counterparts. In summary, SCORE enhances cellular state inference by simulating the dynamic changes of molecular networks, providing more biological insights beyond statistical interpretations.

bioinformatics↗

bioRxiv2019

Molecular mechanism of mitochondrial phosphatidate transfer by Ups1/Mdm35

Cardiolipin plays many important roles for mitochondrial physiological function and is synthesized from phosphatidic acid (PA) at inner mitochondrial membrane (IMM). PA synthesized from endoplasmic reticulum needs to transfer to IMM via outer mitochondrial membrane (OMM). The transfer of PA between IMM and OMM is mediated by Ups1/Mdm35 protein family. Although there are many structures of this family available, the detailed molecular mechanism of how PA is transferred between membranes is yet unknown. Here, we report another crystal structures of Ups1/Mdm35 in the authentic monomeric apo state and the DHPA bound state. By combining subsequent all-atom molecular dynamics simulations, extensive structural comparisons and biophysical assays, we discovered the conformational changes of Ups1/Mdm35, identified key structural elements and residues during membrane binding and PA entry. We found the monomeric Ups1 on membrane is an important transit for the success of PA transfer, and the equilibrium between monomeric Ups1 and Ups1/Mdm35 complex on membrane affects the PA transfer rate and can be regulated by many factors including environmental pH.

biochemistry↗

bioRxiv2019

New genotype invasion of dengue virus serotype 1 drove massive outbreak in Guangzhou, China

BackgroundDengue fever is a mosquito-borne infectious disease that has caused major health problems. Variations in dengue virus (DENV) genes are important features of epidemic outbreaks. However, the associations of DENV genes with epidemic scale have not been extensively examined. Here, we assessed new genotype invasion of DENV-1 isolated from Guangzhou in China to evaluate associations with epidemic outbreaks.\n\nMethodology/Principal FindingsWe used DENV-1 strains isolated from sera of dengue cases from 2002 to 2016 in Guangzhou for complete genome sequencing. A neighbor-joining phylogenetic tree was constructed to elucidate the genotype characteristics and determine if new genotype invasion correlated with major outbreaks. In our study, a new genotype invasion event was observed during each significant outbreak period in 2002-2003, 2006-2007 and 2013-2014. Genotype II was the main epidemic genotype in 2003 and before. Invasion of genotype I in 2006 caused an unusual outbreak with 765 cases (relative risk (RR)=16.24, 95% confidence interval (CI) =12.41-21.25). At the middle and late stages of the 2013 outbreak, genotype III was introduced to Guangzhou as a new genotype invasion responsible for 37340 cases with RR 541.73 (95%CI=417.78-702.45), after which genotypes I and III began co-circulating. Base mutations occurred after new genotype invasion, and the gene sequence of NS3 protein had the lowest average similarity ratio (99.82%), followed by the gene sequence of E protein (99.86%), as compared to the 2013 strain.\n\nConclusions/SignificanceGenotype replacement and co-circulation of multiple DENV-1 genotypes were observed. New genotype invasion was highly correlated with local unusual outbreaks. In addition to DENV-1 genotype I in the unprecedented outbreak in 2014, new genotype invasion by DENV-1 genotype III occurred in Guangzhou.\n\nAuthor SummaryNew genotype invasion of dengue virus highly correlates with the massive outbreaks. In this study, we examined the association of the genotype of dengue virus serorype 1 (DENV-1) from human cases through complete genome sequencing with outbreak scale during 2002 and 2016 in Guangzhou, China. It was observed that genotype replacement and co-circulation of multiple genotypes occurred. Most importantly, it indicated that new genotype invasion was highly related with local unusual outbreaks in major outbreak periods in 2002-2003, 2006-2007 and 2013-2014. DENV-1 genotype II was the main epidemic genotype in 2003 and before. Invasion of genotype I in 2006 caused an unusual outbreak with 765 cases reported. In addition to genotype I circulation, new genotype invasion by genotype III was the key determinant for the 2014 massive outbreak reaching the highest number of cases with 37340. Furthermore, base mutations appeared after genotype III invasion, and the gene sequence of NS3 protein had the lowest average similarity ratio, followed by the gene sequence of E protein, as compared to the 2013 strain.

epidemiology↗

bioRxiv2019

Genome-wide rare variant analysis for thousands of phenotypes in 54,000 exomes

Defining the effects that rare variants can have on human phenotypes is essential to advancing our understanding of human health and disease. Large-scale human genetic analyses have thus far focused on common variants, but the development of large cohorts of deeply phenotyped individuals with exome sequence data has now made comprehensive analyses of rare variants possible. We analyzed the effects of rare (MAF<0.1%) variants on 3,166 phenotypes in 40,468 exome-sequenced individuals from the UK Biobank and performed replication as well as meta-analyses with 1,067 phenotypes in 13,470 members of the Healthy Nevada Project (HNP) cohort who underwent Exome+ sequencing at Helix. Our analyses of non-benign coding and loss of function (LoF) variants identified 78 gene-based associations that passed our statistical significance threshold (p<5x10-9). These are associations in which carrying any rare coding or LoF variant in the gene is associated with an enrichment for a specific phenotype, as opposed to GWAS-based associations of strictly single variants. Importantly, our results do not suffer from the test statistic inflation that is often seen with rare variant analyses of biobank-scale data because of our rare variant-tailored methodology, which includes a step that optimizes the carrier frequency threshold for each phenotype based on prevalence. Of the 47 discovery associations whose phenotypes were represented in the replication cohort, 98% showed effects in the expected direction, and 45% attained formal replication significance (p<0.001). Six additional significant associations were identified in our meta-analysis of both cohorts. Among the results, we confirm known associations of PCSK9 and APOB variation with LDL levels; we extend knowledge of variation in the TYRP1 gene, previously associated with blonde hair color only in Solomon Islanders to blonde hair color in individuals of European ancestry; we show that PAPPA, a gene in which common variants had previously associated with height via GWAS, contains rare variants that decrease height; and we make the novel discovery that STAB1 variation is associated with blood flow in the brain. Our results are available for download and interactive browsing in an app (https://ukb.research.helix.com). This comprehensive analysis of the effects of rare variants on human phenotypes marks one of the first steps in the next big phase of human genetics, where large, deeply phenotyped cohorts with next generation sequence data will elucidate the effects of rare variants.

genetics↗

bioRxiv2019

Germline-encoded TCR-MHC contacts promote TCR V gene bias in umbilical cord blood T cell repertoire

T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the \"net\" genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR {beta} chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.

immunology↗

bioRxiv2019

Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

We have discovered that basement membrane and its major components can induce rapid, strikingly robust fibronectin organization. In this new matrix assembly mechanism, 5{beta}1 integrin-based focal adhesions slide actively on the underlying matrix towards the ventral cell center through the dynamic shortening of myosin IIA-associated actin stress fibers to drive rapid fibronectin fibrillogenesis distal to the adhesion. This mechanism contrasts with classical fibronectin assembly based on stable/fixed-position focal adhesions containing V{beta}3 integrins plus 5{beta}1 integrin translocation into proximal fibrillar adhesions. On basement membrane components, these sliding focal adhesions contain standard focal adhesion constituents but completely lack classical V{beta}3 integrins. Instead, peripheral 3{beta}1 or 2{beta}1 adhesions mediate initial cell attachment, but over time are switched to 5{beta}1 integrin-based sliding focal adhesions to assemble fibronectin matrix. This basement membrane-triggered mechanism produces rapid fibronectin fibrillogenesis, providing a mechanistic explanation for the well-known widespread accumulation of fibronectin at many organ basement membranes.

cell biology↗