Informing plasmid compatibility with bacterial hosts using protein-protein interaction data.

The compatibility of plasmids with new host cells is significant given their role in spreading antimicrobial resistance (AMR) and virulence factor genes. Evaluating this using in vitro screening is laborious and can be informed by computational analyses of plasmid-host compatibility through rates of protein-protein interactions (PPIs) between plasmid and host cell proteins. We identified large excesses of such PPIs in eight important plasmids, including pOXA-48, using most known bacteria (n = 4363). 23 species had high rates of interactions with four blaOXA-48-positive plasmids. We also identified 48 species with high interaction rates with plasmids common in Escherichia coli. We found a strong association between one plasmid and the fimbrial adhesin operon pil, which could enhance host cell adhesion in aqueous environments. An excess rate of PPIs could be a sign of host-plasmid compatibility, which is important for AMR control given that plasmids like pOXA-48 move between species with ease.

Paternally Expressed Imprinted Genes under Positive Darwinian Selection in Arabidopsis thaliana.

Genomic imprinting is an epigenetic phenomenon where autosomal genes display uniparental expression depending on whether they are maternally or paternally inherited. Genomic imprinting can arise from parental conflicts over resource allocation to the offspring, which could drive imprinted loci to evolve by positive selection. We investigate whether positive selection is associated with genomic imprinting in the inbreeding species Arabidopsis thaliana. Our analysis of 140 genes regulated by genomic imprinting in the A. thaliana seed endosperm demonstrates they are evolving more rapidly than expected. To investigate whether positive selection drives this evolutionary acceleration, we identified orthologs of each imprinted gene across 34 plant species and elucidated their evolutionary trajectories. Increased positive selection was sought by comparing its incidence among imprinted genes with nonimprinted controls. Strikingly, we find a statistically significant enrichment of imprinted paternally expressed genes (iPEGs) evolving under positive selection, 50.6% of the total, but no such enrichment for positive selection among imprinted maternally expressed genes (iMEGs). This suggests that maternally- and paternally expressed imprinted genes are subject to different selective pressures. Almost all positively selected amino acids were fixed across 80 sequenced A. thaliana accessions, suggestive of selective sweeps in the A. thaliana lineage. The imprinted genes under positive selection are involved in processes important for seed development including auxin biosynthesis and epigenetic regulation. Our findings support a genomic imprinting model for plants where positive selection can affect paternally expressed genes due to continued conflict with maternal sporophyte tissues, even when parental conflict is reduced in predominantly inbreeding species.

Evaluating amino acids as protectants against ß-N-methylamino-l-alanine-induced developmental neurotoxicity in a rat model.

With accumulating evidence that supports the role of ß-N-methylamino-l-alanine (BMAA) in neurodegeneration, it is necessary to elucidate the mechanisms and modes of BMAA toxicity so as to facilitate the search for potential preventative/therapeutic strategies. Daily supplementation with l-serine was suggested as a possible therapy to treat BMAA-induced neurotoxicity, based on the hypothesized mechanism of BMAA misincorporation into proteins for l-serine. As an alternative to misincorporation, it was hypothesized that BMAA toxicity may, in part, be due to its high affinity for associating with hydroxyl group-containing amino acids, and that a dietary excess of the hydroxyl-containing l-serine might offer protection by binding to BMAA and reducing its toxicity. Additionally, l-serine can also reduce the uptake of BMAA into human cells by competitive uptake at ASCT2, and l-phenylalanine, by competitive uptake at LAT1, and l-alanine, by competitive uptake at SNAT2, can also reduce BMAA uptake into human cells. The aim of this study was therefore to determine the protective value of l-serine, l-phenylalanine and l-alanine in reducing the effects of neonatal exposure to BMAA in a Sprague Dawley rat model. Pre-treatment with l-phenylalanine reduced the observed behavioral abnormalities and neuropathologies by 60-70% in most cases. l-serine was also effective in reducing some of the behavioral abnormalities and neuropathologies, most markedly spinal cord neuronal loss. However, the protective effect of l-serine was obfuscated by neuropathies that were observed in l-serine-treated control male rats. l-alanine had no effect in protecting against BMAA-induced neurotoxicity, suggesting that competitive amino acid uptake plays a minor role in protecting against BMAA-induced neurotoxicity.

ß- N-methylamino-L-alanine Inhibits Human Catalase Activity: Possible Implications for Neurodegenerative Disease Development.

The naturally produced, nonprotein amino acid ß- N-methylamino-l-alanine (BMAA) has been proposed as a significant contributor to sporadic neurodegenerative disease development worldwide. However, the existing hypothesized mechanisms of toxicity do not adequately explain the role of BMAA in neurodegenerative disease development. There is evidence for BMAA-induced enzyme inhibition, but the effect of BMAA on human stress response enzymes has received little attention, despite the well-described role of oxidative stress in neurodegenerative disease development. The aim of this study was therefore to investigate the effect of BMAA on human catalase activity and compare it to the known inhibitor 3-amino-1,2,4-triazole. BMAA inhibited human erythrocyte catalase in a cell-free exposure to the same extent as the known inhibitor. Based on enzyme kinetics, the inhibition appears to be noncompetitive, possibly as a result of BMAA binding in the nicotinamide adenine dinucleotide phosphate (NADPH) binding site. BMAA-induced catalase inhibition was also observed in a human cell line culture. We therefore propose that BMAA-induced enzyme inhibition, specifically catalase inhibition, is a mechanism of toxicity that may contribute to the neurotoxicity of BMAA, further supporting the role of BMAA in neurodegenerative disease development.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis.

A novel pan-Leishmania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 18S ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.

Redeploying ß-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that ß-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.

Tandem Amplification of the Staphylococcal Cassette Chromosome mec Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus.

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) ß-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 µg/ml to 130 µg/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ∼10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 µg/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.

Genomic confirmation of hybridisation and recent inbreeding in a vector-isolated Leishmania population.

Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.

The triploid East African Highland Banana (EAHB) genepool is genetically uniform arising from a single ancestral clone that underwent population expansion by vegetative propagation.

KEY MESSAGE: All East African Highland Banana varieties are genetically uniform having arisen from a single clone introduced to Africa. East African Highland bananas (EAHBs) are a subgroup of triploid (AAA genome) bananas of importance to food security in the Great Lakes region of Africa. Little is known about their genetic variation, population structure and evolutionary history. Ninety phenotypically diverse EAHB cultivars were genotyped at 100 SSR microsatellite markers to investigate population genetic diversity, the correlation of genetic variability with morphological classes, and evolutionary origins since introduction to Africa. Population-level statistics were compared to those for plantain (AAB) and dessert (AAA) cultivars representing other M. acuminata subgroups. EAHBs displayed minimal genetic variation and are largely genetically uniform, irrespective of whether they were derived from the distinct Ugandan or Kenyan germplasm collections. No association was observed between EAHB genetic diversity and currently employed morphological taxonomic systems for EAHB germplasm. Population size dynamics indicated that triploid EAHBs arose as a single hybridization event, which generated a genetic bottleneck during foundation of the EAHB genepool. As EAHB triploids are sterile, subsequent asexual vegetative propagation of EAHBs allowed a recent rapid expansion in population size. This provided a basis for emergence of genetically near-isogenic somatic mutants selected across farmers and environments in East Africa over the past 2000 years since EAHBs were first introduced to the African continent.

Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance.

Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that whole-genome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface- and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.

Uptake of ß-N-methylamino-L-alanine (BMAA) into glutamate-specific synaptic vesicles: Exploring the validity of the excitotoxicity mechanism of BMAA.

The first mechanism of toxicity proposed for the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) was excitotoxicity, and this was supported by numerous in vitro studies in which overactivation of both ionotropic and metabotropic glutamate receptors was reported. However, the excitotoxicity of BMAA is weak in comparison with other known excitotoxins and on par with that of glutamate, implying that to achieve sufficient synaptic concentrations of BMAA to cause classical in vivo excitotoxicity, BMAA must either accumulate in synapses to allow persistent glutamate receptor activation or it must be released in sufficiently high concentrations into synapses to cause the overexcitation. Since it has been shown that BMAA can be readily removed from synapses, release of high concentrations of BMAA into synapses must be shown to confirm its role as an excitotoxin in in vivo systems. This study therefore sought to evaluate the uptake of BMAA into synaptic vesicles and to determine if BMAA affects the uptake of glutamate into synaptic vesicles. There was no evidence to support uptake of BMAA into glutamate-specific synaptic vesicles but there was some indication that BMAA may affect the uptake of glutamate into synaptic vesicles. The uptake of BMAA into synaptic vesicles isolated from areas other than the cerebral cortex should be investigated before definite conclusions can be drawn about the role of BMAA as an excitotoxin.

Protection against ß-N-methylamino-l-alanineꟷinduced vesicular monoamine transporter 2 inhibition by hydroxyl-containing proteinogenic amino acids.

ß-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupꟷcontaining amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupꟷcontaining amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

Genetic markers for SSG resistance in Leishmania donovani and SSG treatment failure in visceral leishmaniasis patients of the Indian subcontinent.

The current standard to assess pentavalent antimonial (SSG) susceptibility of Leishmania is a laborious in vitro assay of which the result has little clinical value because SSG-resistant parasites are also found in SSG-cured patients. Candidate genetic markers for clinically relevant SSG-resistant parasites identified by full genome sequencing were here validated on a larger set of clinical strains. We show that 3 genomic locations suffice to specifically detect the SSG-resistant parasites found only in patients experiencing SSG treatment failure. This finding allows the development of rapid assays to monitor the emergence and spread of clinically relevant SSG-resistant Leishmania parasites.

A primer on correlation-based dimension reduction methods for multi-omics analysis.

The continuing advances of omic technologies mean that it is now more tangible to measure the numerous features collectively reflecting the molecular properties of a sample. When multiple omic methods are used, statistical and computational approaches can exploit these large, connected profiles. Multi-omics is the integration of different omic data sources from the same biological sample. In this review, we focus on correlation-based dimension reduction approaches for single omic datasets, followed by methods for pairs of omics datasets, before detailing further techniques for three or more omic datasets. We also briefly detail network methods when three or more omic datasets are available and which complement correlation-oriented tools. To aid readers new to this area, these are all linked to relevant R packages that can implement these procedures. Finally, we discuss scenarios of experimental design and present road maps that simplify the selection of appropriate analysis methods. This review will help researchers navigate emerging methods for multi-omics and integrating diverse omic datasets appropriately. This raises the opportunity of implementing population multi-omics with large sample sizes as omics technologies and our understanding improve.

ß-N-methylamino-l-alanine is a non-competitive inhibitor of vesicular monoamine transporter 2.

The neurotoxic, non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) has been implicated in the development of neurodegenerative diseases; however, the mechanism(s) and mode(s) of toxicity remain unclear. Similarities in the neuropathology and behavioural deficits of neonatal rats exposed to either BMAA or reserpine, a known vesicular monoamine transporter 2 (VMAT2) inhibitor, suggest a similar mode of action. The aims of this study were therefore to determine if BMAA could prevent the uptake of serotonin into dense granules via inhibition of VMAT2, and, if so, the type of inhibition caused by BMAA. Exposing platelet dense granules to BMAA resulted in a concentration-dependent reduction in serotonin uptake. The inhibition of VMAT2 was non-competitive. The findings from this study support previous reports that BMAA-associated neuropathologies in a neonatal rat model may be due to VMAT2 inhibition during critical periods of neurogenesis.

The differential evolutionary dynamics of avian cytokine and TLR gene classes.

The potential for investigating immune gene diversity has been greatly enhanced by recent advances in sequencing power. In this study, variation at two categories of avian immune genes with differing functional roles, pathogen detection and mediation of immune mechanisms, was examined using high-throughput sequencing. TLRs identify and alert the immune system by detecting molecular motifs that are conserved among pathogenic microorganisms, whereas cytokines act as mediators of resulting inflammation and immunity. Nine genes from each class were resequenced in a panel of domestic chickens and wild jungle fowl (JF). Tests on population-wide genetic variation between the gene classes indicated that allele frequency spectra at each group were distinctive. TLRs showed evidence pointing toward directional selection, whereas cytokines had signals more suggestive of frequency-dependent selection. This difference persisted between the distributions considering only coding sites, suggesting functional relevance. The unique patterns of variation at each gene class may be constrained by their different functional roles in the immune response. TLRs identify a relatively limited number of exogeneous pathogenic-related patterns and would be required to adapt quickly in response to evolving novel microbes encountered in new environmental niches. In contrast, cytokines interact with many molecules in mediating the power of immune mechanisms, and accordingly respond to the selective stimuli of many infectious diseases. Analyses also indicated that a general pattern of high variability has been enhanced by widespread genetic exchange between chicken and red JF, and possibly between chicken and gray JF at TLR1LA and TLR2A.

Bacterial plasmid-associated and chromosomal proteins have fundamentally different properties in protein interaction networks.

Plasmids facilitate horizontal gene transfer, which enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-encoded genes can cooperate well with chromosomal genes. We hypothesise that such mobile genes are functionally different to chromosomal ones due to this ability to encode proteins performing non-essential functions like antimicrobial resistance and traverse distinct host cells. The effect of plasmid-driven gene gain on protein-protein interaction network topology is an important question in this area. Moreover, the extent to which these chromosomally- and plasmid-encoded proteins interact with proteins from their own groups compared to the levels with the other group remains unclear. Here, we examined the incidence and protein-protein interactions of all known plasmid-encoded proteins across representative specimens from most bacteria using all available plasmids. We found that plasmid-encoded genes constitute ~ 0.65% of the total number of genes per bacterial sample, and that plasmid genes are preferentially associated with different species but had limited taxonomical power beyond this. Surprisingly, plasmid-encoded proteins had both more protein-protein interactions compared to chromosomal proteins, countering the hypothesis that genes with higher mobility rates should have fewer protein-level interactions. Nonetheless, topological analysis and investigation of the protein-protein interaction networks' connectivity and change in the number of independent components demonstrated that the plasmid-encoded proteins had limited overall impact in > 96% of samples. This paper assembled extensive data on plasmid-encoded proteins, their interactions and associations with diverse bacterial specimens that is available for the community to investigate in more detail.

Aberrant calcium signalling downstream of mutations in TP53 and the PI3K/AKT pathway genes promotes disease progression and therapy resistance in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC) associated with poor patient outcomes. For the majority of patients, there is a lack of approved targeted therapies. Therefore, chemotherapy remains a key treatment option for these patients, but significant issues around acquired resistance limit its efficacy. Thus, TNBC has an unmet need for new targeted personalized medicine approaches. Calcium (Ca2+) is a ubiquitous second messenger that is known to control a range of key cellular processes by mediating signalling transduction and gene transcription. Changes in Ca2+ through altered calcium channel expression or activity are known to promote tumorigenesis and treatment resistance in a range of cancers including BC. Emerging evidence shows that this is mediated by Ca2+ modulation, supporting the function of tumour suppressor genes (TSGs) and oncogenes. This review provides insight into the underlying alterations in calcium signalling and how it plays a key role in promoting disease progression and therapy resistance in TNBC which harbours mutations in tumour protein p53 (TP53) and the PI3K/AKT pathway.

Data pertaining to aberrant intracellular calcium handling during androgen deprivation therapy in prostate cancer.

The data generated here in relates to the research article "CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer". A model of prostate cancer (PCa) progression to castration resistance was employed, with untreated androgen sensitive LNCaP cell line alongside two androgen deprived (bicalutamide) sublines, either 10 days (LNCaP-ADT) or 2 years (LNCaP-ABL) treatment, in addition to androgen insensitive PC3. With this PCa model, qPCR was used to examined fold change in markers linked to androgen resistance, androgen receptor (AR) and neuron specific enolase (NSE), observing an increase under androgen deprivation. In addition, the gene expression of a range of calcium channels was measured, with only the L-type Voltage gated calcium channel, CACNA1D, demonstrating an increase during androgen deprivation. With CACNA1D knockdown the channel was found not to influence the gene expression of calcium channels, ORAI1 and STIM1. The calcium channel blocker (CCB), nifedipine, was employed to determine the impact of CaV1.3 on the observed store release and calcium entry measured via Fura-2AM ratiometric dye in our outlined PCa model. In both the presence and absence of androgen deprivation, nifedipine was found to have no impact on store release induced by thapsigargin (Tg) in 0mM Ca2+ nor store operated calcium entry (SOCE) following the addition of 2mM Ca2+. However, CACNA1D siRNA knockdown was able to reduce SOCE in PC3 cells. The effect of nifedipine on CaV1.3 in PCa biology was measured through cell proliferation assay, with no observed change in the presence of CCB. While siCACNA1D reduced PC3 cell proliferation. This data can be reused to inform new studies investigating altered calcium handling in androgen resistant prostate cancer. It provides insight into the mechanism of CaV1.3 and its functional properties in altered calcium in cancer, which can be of use to researchers investigating this channel in disease. Furthermore, it could be helpful in interpreting studies investigating CCB's as a therapeutic and in the development of future drugs targeting CaV1.3.