Biology subjects

Tomlinson, R.

Publications and source records attributed to Tomlinson, R..

7 recordsLinked to original sources

bioRxiv2025

ABCC6 and ANK regulate extracellular homeostasis of pyrophosphate and citrate and affect mineral deposition in bones and soft connective tissues

ABCC6 and ANK are integral membrane proteins involved in the efflux of specific organic anions. ABCC6, primarily expressed in the liver, regulates circulating levels of the mineralization inhibitor pyrophosphate (PPi). In contrast, ANK is widely expressed and plays a dual role, maintaining extracellular PPi homeostasis and, notably, mediating cellular citrate efflux. We studied how both proteins affected extracellular metabolite levels, ectopic calcification, and bone homeostasis. We observed that plasma PPi was reduced by [~]36% in Ankank/ankmice and [~]60% in Abcc6-/- mice. However, plasma citrate levels depended primarily on ANK, dropping [~]75% in Ankank/ank and double mutants, but remaining unchanged in Abcc6-/- mice. MicroCT revealed extreme ectopic calcification in double mutants, far exceeding either single knockout, affecting muzzle skin and ear cartilage. Oral citrate was bioavailable and, at high doses, prevented soft tissue calcification in Abcc6-/- mice, suggesting a systemic protective role. In bone, ANK was essential for incorporating both PPi and citrate, while ABCC6 mainly affected PPi. ANK deficiency led to reduced trabecular volume, cortical thickness, cortical area fraction, and mineral density, with more pronounced effects in males. Biomechanical testing showed decreased ultimate moment, bending rigidity, and energy in ANK-deficient femora, alongside increased post-yield displacement, indicating compensatory matrix changes. Collectively, our findings identify ANK as a dual regulator of PPi and citrate, with a previously unrecognized role in preventing soft tissue calcification. This study positions ANK as a potential therapeutic target for mineralization disorders like pseudoxanthoma elasticum (caused by ABCC6 deficiency) and conditions of low bone mineral density like osteoporosis.

developmental biology↗

bioRxiv2025

Discovery of a potent anti-Zika virus benzamide series targeting the viral protein NS4B

1.Zika virus (ZIKV), a member of the Flaviviridae family, causes significant public health concerns through congenital Zika syndrome and Guillain-Barre syndrome, yet no effective anti-ZIKV drugs or vaccines are available. To address this critical need, we conducted phenotypic, cytopathic effect-based, high-throughput screening followed by medicinal chemistry optimization and discovered novel benzamide anti-ZIKV leads. Current best compounds demonstrated superior potency (EC50 values 40-400 nM, CC50 > 50 {micro}M) compared to NITD-008, the most potent known anti-ZIKV agent. Time-of-addition assays, resistant virus selection studies, and biophysical binding experiments confirmed that NS4B interference constitutes the primary antiviral mechanism. Notably, resistance mutations mapped to the C-terminus of NS4B, distinct from other flavivirus NS4B inhibitors targeting dengue or yellow fever viruses, revealing novel insights into a critical function of the region. These findings establish NS4B as an Achilles heel for flaviviruses and support the development of pan-flavivirus therapeutics targeting this essential viral protein. 2. AUTHOR SUMMARYSince its pandemic spread in 2015-2016, Zika virus infection remains a significant public health threat worldwide. The virus can cause severe brain damage in developing babies and serious neurological complications like Guillain-Barre syndrome in adults. Despite these devastating consequences, we currently lack effective medicines or vaccines to prevent the virus from spreading through communities or from mothers to their unborn children. To address this critical gap, we conducted a large-scale screening of chemical compounds and discovered a promising new class of molecules that can effectively stop Zika virus from replication. Using medicinal chemistry techniques, we were able to make these compounds even more potent against the virus. In follow-up studies, we found that our compounds work by interfering with a specific viral protein called NS4B, which the virus needs to replicate its genome within the cell. Remarkably, other research teams studying related other flaviviruses (e.g., dengue and yellow fever virus) have independently discovered that this same protein is a vulnerable target. Our findings suggest that NS4B represents a universal weakness across the entire flavivirus family, making it an attractive target for developing broad-spectrum antiviral treatments.

microbiology↗

bioRxiv2025

High-dose oral pyrophosphate inhibits connective tissue calcification in Abcc6 null mice but affects bone structure

Pseudoxanthoma elasticum is a rare inherited disorder marked by abnormal calcium phosphate deposition in soft connective tissues, particularly the skin, arteries, and eyes. It is caused by inactivating mutations in the ABCC6 gene, which encodes a hepatic efflux transporter. Loss of ABCC6 function leads to reduced plasma levels of pyrophosphate, a key inhibitor of calcification, thereby promoting ectopic mineralization. Oral pyrophosphate therapy has emerged as a potential treatment, but its effectiveness is uncertain. Most ingested pyrophosphate is hydrolyzed in the gut to inorganic phosphate, which may worsen calcification. Moreover, its impact on mineralized tissues remains largely unexplored. Abcc6-/- mice closely mimic human pseudoxanthoma elasticum and are widely used in preclinical studies. Although patients are most concerned about ocular complications, eye calcification is rarely assessed in translational studies using Abcc6-/- mice. Using microcomputed tomography we found that ectopic calcification at the ciliary margin is a reliable marker of ocular disease progression in these mice. Administering pyrophosphate in drinking water at concentrations up to 90 mM did not increase calcification in skin or eyes. However, only very high doses effectively prevented ectopic calcification - doses that would equate to an impractical 2.5 g/kg/day of disodium pyrophosphate in humans. These high doses also led to pyrophosphate accumulation in bone and negatively affected bone structure and strength. In summary, only supraphysiological doses of orally administered pyrophosphate inhibited ectopic calcification in Abcc6-/- mice, but these doses are not feasible for human use and may compromise bone function. These data are especially important considering the currently ongoing clinical trial evaluating the safety and efficacy of oral pyrophosphate administration as a treatment for pseudoxanthoma elasticum. LAY SUMMARYPseudoxanthoma elasticum (PXE) is a rare inherited mineralization disorder caused by the absence of functional ABCC6, a liver-expressed protein. This deficiency leads to reduced plasma levels of pyrophosphate, a key inhibitor of mineralization, resulting in abnormal calcium phosphate deposition in the skin, eyes, and blood vessels. Oral pyrophosphate has been proposed as a therapeutic strategy for PXE, and a clinical trial evaluating its efficacy recently began in France. In a PXE mouse model, we show that only very high oral doses of pyrophosphate are effective, but these doses impair bone quality and are not suitable for human use.

pathology↗

bioRxiv2025

Osteoblast-derived Nerve Growth Factor is Required for Skeletal Adaptation to Mechanical Load and the Osteoanabolic Effect of Gambogic Amide in Mice

In adult mice, new bone accrual following mechanical load is mediated by the neurotrophin nerve growth factor (NGF) that is expressed by osteoblasts on the bone surface. NGF can bind to its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), on peripheral sensory nerves resident in bone and support new bone formation. However, the osteoanabolic therapeutic potential of NGF-TrkA signaling to repair bone is limited due to the long-lasting thermal and mechanical hyperalgesia induced by administration of NGF in mice and humans. Here, we investigated whether 1) mature osteoblasts are the primary source of NGF required for bone accrual following loading, and 2) a small molecule TrkA receptor agonist - gambogic amide - can harness the downstream osteoanabolic potential of NGF-TrkA signaling in the absence of endogenous NGF. Loss of Ngf transcription in mature osteoblasts did not appear to affect bone structure or bone mass in adulthood. However, Ngf knockout mice significantly reduced periosteal bone accrual and osteogenic Wnt transcription in response to loading compared to wildtype mice. Intraperitoneal injection of gambogic amide prior to loading was unable to produce its osteoanabolic effects in Ngf knockout mice, suggesting that gambogic amide primarily functions in collaboration with endogenous NGF in bone. In total, our study reveals an important role for osteoblastic NGF in the skeletal adaptation of bone to mechanical forces.

bioengineering↗

bioRxiv2021

Abcc6 null mice a model for mineralization disorder PXE show vertebral osteopenia without enhanced intervertebral disc calcification with aging

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the ABCC6-/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. ABCC6-/- mice exhibited diminished trabecular bone quality parameters at 7-months which remained significantly lower than the wild-type mice at 18 months-of-age. ABCC6-/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling. Author SummaryInherited mutations in the ABCC6 transporter gene results in mineralization, often in the form of hydroxyapatite, of connective tissues throughout the body, predominantly affecting the skin, eyes, and blood vessels. Functional loss of ABCC6 causes reduced levels of the potent mineralization inhibitor pyrophosphate (PPi) in blood resulting in these pathologies. Pathological mineralization is also a prominent feature of intervertebral disc degeneration, but the role of ABCC6 and systemic PPi levels and its correlation to disc mineralization and vertebral bone health has remained unexplored. In this study, we show for the first time that loss of ABCC6 in mice results in significant decline in vertebral bone quality and mild age-related disc degeneration without increased incidence of abnormal mineralization. Importantly, treatment of ABCC6 deficient mice with K3Citrate resulted in restoration of early cellular changes which drive bone loss and mechanical function of the vertebrae. In summary, our data reveal that ABCC6 is dispensable for mineralization prevention in the intervertebral disc. Unexpectedly, we found that vertebral bone quality and bone cell activities are linked to ABCC6 function.

cell biology↗

bioRxiv2021

Enhancing Precision in Bioprinting Utilizing Fuzzy Systems

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for a variety of applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of substrate materials (bioinks) that can be accommodated, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that limit bioprinting precision, particularly when generating constructs from different bioinks. Here, we aimed to achieve high precision (i.e. repeatability) across samples by generating bioink-specific printing parameters using a systematic approach. We hypothesized that a fuzzy system could be used as a "black box" method to tackle the inherent vagueness and imprecision in 3D bioprinting data and uncover the optimal printing parameters for a specific bioink that would result in high accuracy and precision. Our fuzzy model was used to approximate and quantify the precision and ease of printability for two common bioinks - type I collagen and Pluronic F127, with or without dilution in MEM culture media. The model consisted of three inputs (pressure, speed, and dilution percent of bioink) and a single output (layer width). Using this system, we introduce the Bioink Precision Index (BPI), a metric that can be used to quantify and compare the precision of any bioink. Here, we show that printing with parameters optimized using BPI increases the precision for collagen (+15%) and Pluronic F127 (+29%) as compared to the manufacturers recommended printing parameters.

bioinformatics↗

bioRxiv2018

Naproxen impairs load-induced bone formation, reduces bone toughness, and delays stress fracture repair in mice

Debilitating stress fractures are surprisingly common in physically active individuals, including athletes, military recruits, and dancers. These individuals are overrepresented in the 30 million daily users of non-steroidal anti-inflammatory drugs (NSAIDs). We hypothesized that regular use of NSAIDs would predispose habitually loaded bones to stress fracture and delay the repair of these injuries. To test this hypothesis, adult mice were subjected to six bouts of axial forelimb compression over two weeks. Aspirin, naproxen, or vehicle was administered 24 hours before loading. Naproxen-treated mice had diminished load-induced bone formation as well as a significant loss in toughness in non-loaded bone, which were not observed in aspirin-treated mice. Furthermore, there were no differences in RANKL/OPG ratio or cortical bone parameters. Picrosirius red staining and second harmonic generation imaging revealed that alterations in bone collagen fibril size and organization were driving the loss of toughness in naproxen-treated mice. Separately, adult mice were subjected to an ulnar stress fracture generated by a single bout of fatigue loading, with NSAIDs provided 24 hours before injury. Both aspirin-treated and naproxen-treated mice had normal forelimb use in the week after injury, whereas control mice favored the injured forelimb until day 7. However, woven bone volume was only significantly impaired by naproxen. Both NSAIDs were found to significantly inhibit Cox2 and Ngf expression following stress fracture, but only naproxen significantly affected serum PGE2 concentration. Overall, our results suggest that naproxen, but not aspirin, may increase the risk of stress fracture and extend the healing time of these injuries, warranting further clinical evaluation for patients at risk for fatigue injuries.

bioengineering↗