Biology subjects

King, G. F.

Publications and source records attributed to King, G. F..

9 recordsLinked to original sources

bioRxiv2025

A Minimally Invasive, Scalable and Reproducible Neonatal Rat Model of Severe Focal Brain Injury

BackgroundNeonatal brain injuries such as stroke cause focal ischemic lesions that often result in lifelong neurological disabilities, as treatment options are limited. To speed up the discovery of potential therapies, early-phase screening with models that reliably reproduce brain injury, with scalable injury volume and minimal confounders, such as varying anaesthesia duration and painful procedures, is essential. MethodsPostnatal day 10 Sprague-Dawley rats of both sexes, with four litters per group and timepoint, were randomly allocated to delivery of intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and 10 minutes of light-emitting diode illumination through the intact scalp and skull. Infarct progression and reproducibility were assessed at 24 hours, 7 days, and 14 days post-injury. Outcomes included infarct volume and sensorimotor function, and cleaved caspase-3, glial fibrillary acidic protein (GFAP), and ionised calcium-binding adaptor molecule 1 (Iba1) immunoreactivity, with analysis of sex differences. Data were analysed using one-way or two-way ANOVA with Sidaks post-hoc tests. ResultsThere was no mortality due to the infarct, and procedure time was approximately 19 minutes across all groups; the lesion was consistent and supported scalability. The 25 mg/kg dose produced a reproducible cortical infarct (3.74 {+/-} 0.58 mm3; CV = 31%). Lesion size increased with dose and decreased over time (11.15 {+/-} 0.63 mm3 at 60 mg/kg versus 0.05 {+/-} 0.007 mm3 at 14 days; p < 0.0001). Cleaved caspase-3 and glial activation persisted for 14 days, indicating ongoing apoptosis and gliosis. No sex-dependent effects were observed in lesion volume, behaviour, or gliosis. ConclusionsThis refined neonatal photothrombotic ischaemia model is reproducible, scalable, and ethically improved, requiring no skin incision. Its minimal surgical burden, absence of mortality, consistent histopathology, and measurable functional outcomes make it an ideal platform for preclinical screening of neuroprotective and reparative interventions in the developing brain.

developmental biology↗

bioRxiv2023

High-content fluorescence bioassay to investigate pore formation, ion channel modulation and cell membrane lysis induced by venoms

Venoms comprise highly evolved bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membrane. This array of targets and bioactivity requires high-content bioassays to aid the development of novel envenomation treatments and biotechnological and pharmacological agents. To address this gap in venoms research, we developed a fluorescence-based high-throughput and high-content cellular assay to simultaneously identify common cellular activities produced by venoms: membrane lysis, pore-formation, and ion channel modulation. By combining intracellular calcium with extracellular nucleic acid measurements, we distinguished these venom mechanisms using one cellular assay. We applied our high-content bioassay in three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond the distinct profiles produced by these three types of action mechanisms, we found that the pore-forming latrotoxin -Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings demonstrate that our high-content bioassay distinguish clades and interspecific traits and capture the clinical observations at venom level and is capable of differentiate ion pore-forming from membrane lysis and ion channel modulation. We hope our research will accelerate the understanding of venom biology and the diversity of toxins inducing cytotoxic, cardiotoxic and neurotoxic effects and assist in identifying venom components whose properties could benefit humankind.

pharmacology and toxicology↗

bioRxiv2022

Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

ShK from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. We identified ten SA8 genes in two clusters and six SA8 genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

genetics↗

bioRxiv2022

Holistic profiling of the venom from the lethal spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics

Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. In this study, we explored the neuroactivity and molecular diversity P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggests that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

pharmacology and toxicology↗

bioRxiv2022

The voltage-gated sodium channel NaV1.7 underlies endometriosis-associated chronic pelvic pain

Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera have shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar therapies have not been explored for endometriosis-associated CPP. Here we examined the role of the voltage-gated sodium (NaV) channel NaV1.7 in the sensitivity of vagina-innervating sensory afferents and investigated whether NaV1.7 inhibition reduces nociceptive signals from the vagina and ameliorates endometriosis-associated CPP. The mechanical responsiveness of vagina-innervating sensory afferents was assessed with ex vivo single unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV1.7 with OD1 sensitised vagina-innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitized vagina-innervating pelvic afferents to mechanical stimuli. The highly selective NaV1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV1.7-dependent manner. Moreover, in vivo intra-vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD that is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity or in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV1.7 plays a crucial role in endometriosis-induced vaginal hyperalgesia. Importantly, NaV1.7 inhibition selectively alleviated endometriosis-associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV1.7 could improve the quality of life of women with endometriosis.

cell biology↗

bioRxiv2021

Non-invasive Imaging of Sense of Smell by Tracking the Voltage-Gated Sodium Channel NaV1.7

The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (NaV1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of NaV1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets NaV1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.

neuroscience↗

bioRxiv2020

Heterodimeric insecticidal peptide provides new insights into the molecular and functional diversity of ant venoms

Ants use venom for predation, defence and communication, however, the molecular diversity, function and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modelling, stability studies, and a series of in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide {Delta}-pseudomyrmecitoxin-Pp1a ({Delta}-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain crosslinked by two disulfide bonds in an antiparallel orientation. We chemically synthesised {Delta}-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that {Delta}-PSDTX-Pp1a had the most potent insecticidal effects in blow fly assays (LD50 = 3 nM). Molecular modelling and circular dichroism studies revealed strong alpha-helical features, indicating its cytotoxic effects could derive from membrane disruption, which was further supported by insect cell calcium assays. The native heterodimer was also substantially more stable against proteolytic degradation (t1/2 =13 h) than its homodimers or monomers (t1/2 <20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterisation of {Delta}-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom, and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability; a concept that is also highly relevant for the design and development of peptide therapeutics, molecular probes and bioinsecticides.

pharmacology and toxicology↗

bioRxiv2018

Structural venomics: evolution of a complex chemical arsenal by massive duplication and neofunctionalization of a single ancestral fold

Spiders are the most successful venomous animals on the planet, with more than 47,000 extant species. Most spider venoms are dominated by disulfide-rich peptides (DRPs) with a diverse range of pharmacological activities. Although some venoms contain thousands of unique peptides, little is known about the mechanisms used to generate such complex chemical arsenals. We used a combined transcriptomic, proteomic and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins, more than described for any other arachnid. We show that 15 of the 26 DRP superfamilies form an ultra-stable inhibitor cystine knot motif, and that these DRPs are the major contributor to the diversity of the venom peptidome. NMR data reveal that most of these DRPs are structurally related and range in complexity from simple to highly elaborated knottin domains that likely evolved from a single ancestral fold.

biochemistry↗

bioRxiv2018

Zinc-binding to the cytoplasmic PAS domain regulates the essential WalK histidine kinase of Staphylococcus aureus

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone appears not to explain its essentiality. To understand WalKR function we investigated a suppressor mutant that arose when WalKR activity was impaired; a single histidine to tryptophan substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introduction of the WalKH271Y mutation into wild-type S. aureus activated the WalKR regulon. Structural analyses of the WalK PASCYT domain revealed a hitherto unknown metal binding site, in which a zinc ion (Zn2+) was tetrahedrally-coordinated by four amino acid residues including H271. The WallkH271Y mutation abrogated metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR activity. Identification of a metal ligand sensed by the WalKR system substantially expands our understanding of this critical S. aureus regulon.

microbiology↗