Biology subjects

Kalluri, R.

Publications and source records attributed to Kalluri, R..

25 records · Page 2Linked to original sources

bioRxiv2022

Identification of unique α4 chain structure and conserved anti-angiogenic activity of α3NC1 type IV collagen in zebrafish

Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding 1 and 2 chains, in contrast with the six genes in humans, encoding 1 to 6 chains. The chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. We report on the molecular evolution of type IV collagen genes. The zebrafish 4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter 4 chain interactions with other chains, as supported by temporal and anatomic expression patterns of collagen IV chains during zebrafish development. Despite the divergence between zebrafish and human 3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish 3 NC1 domain exhibits conserved anti-angiogenic activity in human endothelial cells. Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the 4 chain.

developmental biology↗

bioRxiv2022

The onset of hearing triggers nuclear translocation in inner hair cells

Nuclear position is precisely orchestrated during cell division, migration, and maturation of cells and tissues. Here we report a previously unrecognized, programmed movement of the nucleus in rat cochlear inner hair cells. This cell-type specific nuclear movement among hair cells of the inner ear offers a new glimpse into the cellular mechanisms involved in the functional maturation of inner hair cells. In early post-natal days, the nucleus sits at the very basal pole of the hair cell, far from the apically located mechano-transducing stereocilia, but close to where synapses with primary afferent and efferent neurons are forming. By maturity, the nucleus moves to occupy a new position half-way along the length of the cell. Remarkably, nuclear translocation happens quickly over the course of 1-2 post-natal days, coinciding with the onset of hearing. The movement of the nucleus likely signals the closing of a critical period correlated with the forming and refining of synapses at the onset of hearing. Significance StatementThe misplacement of a cells nucleus has been implicated in hearing, vision, and muscular pathologies. In this study, we report a remarkably abrupt change in nuclear position in cochlear inner hair cells that coincides with the onset of hearing in rats, when hair cells switch from producing sensory-independent action potentials to sensory-dependent graded potentials. This work suggests that post-mitotic nuclear migration may be critical to the final phase of post-natal development in mammalian hearing.

neuroscience↗

bioRxiv2022

Quantification of phosphonate drugs by 1H-31P HSQC shows that rats are better models of primate drug exposure than mice

The phosphonate group is a key pharmacophore in many anti-viral, anti-microbial, and anti-neoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods is challenging and requires derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton-phosphorous (1H-31P) heteronuclear single quantum correlation (HSQC) NMR allows for quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissue at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 hr), the plasma half-life of HEX was more than 1hr in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1H-31P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.

pharmacology and toxicology↗

bioRxiv2022

Muscarinic acetylcholine receptors modulate HCN channel properties in vestibular ganglion neurons

Vestibular efferent neurons play an important role in shaping vestibular afferent excitability and, accordingly, on the information encoded by their spike patterns. Efferent- modulation is linked to muscarinic signaling cascades that affect ion channel conductances, most notably low-voltage gated potassium channels such as KCNQ. Here we tested and found that muscarinic signaling cascades also modulate hyperpolarization- activated cyclic-nucleotide gated channels (HCN). HCN channels play a key role in controlling spike-timing regularity and a non-chemical form of transmission between type I hair cells and vestibular afferents. The impact of cholinergic efferent input on HCN channels was assessed using voltage-clamp methods, which measure currents in the disassociated cell bodies of vestibular ganglion neurons (VGN). Membrane properties in VGN were characterized before and after administration of the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M). We found that Oxo-M shifted the voltage-activation range of HCN channels in the positive direction by 3.2 {+/-} 0.7 mV, which more than doubled the available current when held near rest at -70 mV (a 139 {+/-} 43% increase, n=12). This effect was not blocked by pre-treating the cells with a KCNQ channel blocker, linopirdine, which suggests that this effect is not dependent on KCNQ currents. We also found that HCN channel properties in the baseline condition and sensitivity to mAChR activation depended on cell size and firing patterns. Large-bodied neurons with onset firing patterns had the most depolarized activation range and least sensitivity to mAChR activation. Together, our results highlight the complex and dynamic regulation of HCN channels in VGN. Significance StatementVestibular afferents express a diverse complement of ion channels. This diversity is key to shaping their various response properties, allowing them to encode both fast and slow head movements. In vitro studies that characterized afferent diversity identified low-voltage activated potassium channels and hyperpolarization-activated cyclic- nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Afferent excitability is known to be controlled by a network of acetylcholine-releasing efferent neurons that close a type of low-voltage activated potassium channel found on the afferent neuron. Here, we show that the same efferent signaling cascade that shuts these potassium channels also enhances the activation of HCN channels by depolarizing their voltage-activation range. The size of this effect varies from cell to cell depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system robust and flexible control over the excitability and timing properties vestibular afferent activity.

neuroscience↗

bioRxiv2021

Mutations in the spike RBD of SARS-CoV-2 omicron variant may increase infectivity without dramatically altering the efficacy of current multi-dosage vaccinations

With the continuous evolution of SARS-CoV-2, variants of concern (VOCs) and their mutations are a focus of rapid assessment. Vital mutations in the VOC are found in spike protein, particularly in the receptor binding domain (RBD), which directly interacts with ACE2 on the host cell membrane, a key determinant of the binding affinity and cell entry. With the reporting of the most recent VOC, omicron, we performed amino acid sequence alignment of the omicron spike protein with that of the wild type and other VOCs. Although it shares several conserved mutations with other variants, we found that omicron has a large number of unique mutations. We applied the Hopp-Woods scale to calculate the hydrophilicity scores of the amino acid stretches of the RBD and the entire spike protein, and found 3 new hydrophilic regions in the RBD of omicron, implying exposure to water, with the potential to bind proteins such as ACE2 increasing transmissibility and infectivity. However, careful analysis reveals that most of the exposed domains of spike protein can serve as antigenic epitopes for generating B cell and T cell-mediated immune responses. This suggests that in the collection of polyclonal antibodies to various epitopes generated after multiple doses of vaccination, some can likely still bind to the omicron spike protein and the RBD to prevent severe clinical disease. In summary, while the omicron variant might result in more infectivity, it can still bind to a reasonable repertoire of antibodies generated by multiple doses of current vaccines likely preventing severe disease. Effective vaccines may not universally prevent opportunistic infections but can prevent the sequelae of severe disease, as observed for the delta variant. This might still be the case with the omicron variant, albeit, with increased frequency of infection.

biochemistry↗

bioRxiv2017

A subgroup of mitochondrial extracellular vesicles discovered in human melanoma tissues are detectable in patient blood

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations are only beginning to be explored. Since EVs have been implicated in tumor microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Specifically, EVs positive for a combination of the two mitochondrial inner membrane proteins MT-CO2 (mitochondrial genome) and COX6c (nuclear genome) were detected in the plasma of melanoma patients, and in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs, contains active mitochondrial enzymes. Our findings show that tumor tissues are enriched in EVs with mitochondrial proteins and enzymatic activity, and these EVs can be detected in blood.

cancer biology↗

bioRxiv2017

Multiple antibodies identify Glypican-1 on serum exosomes from patients with pancreatic cancer

Exosomes are man-sized vesicles shed by all cells, including cancer cells. Exosomes can serve as novel liquid biopsies for diagnosis of cancer with potential prognostic value. The exact mechanism/s associated with sorting or enrichment of cellular components into exosomes are still largely unknown. We reported Glypican-1 (GPC1) on the surface of cancer exosomes and provided evidence for the enrichment of GPC1 in exosomes from patients with pancreatic cancer1. Several different laboratories have validated this novel conceptual advance and reproduced the original experiments using multiple antibodies from different sources. These include anti-GPC1 antibodies from ThermoFisher (PA5-28055 and PA-5-24972)1,2, Sigma (SAB270028), Abnova (MAB8351, monoclonal antibodies clone E9E)3, EMD Millipore (MAB2600-monoclonal antibodies)4, SantaCruz5, and R&D Systems (BAF4519)2. This report complements such independent findings and report on the specific detection of Glypican-1 on the exosomes derived from the serum of pancreas cancer patients using multiple antibodies. Additionally, the specificity of the antibodies to GPC1 was determined by western blot and Protein Simple analyses of pancreatic cancer cells and their exosomes. Interestingly, our results highlight a specific enrichment of high molecular weight GPC1 on exosomes, potentially contributed by heparan sulfate and other glycosylation modifications.

cancer biology↗