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Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2–3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

Mitochondria are essential eukaryotic double-membrane organelles. The convoluted mitochondrial inner membrane forms highly organized invaginations, termed cristae, which are crucial for energy metabolism. Cristae formation requires MICOS, a conserved hetero-oligomeric inner membrane complex. The MICOS core subunit MIC10 is a small transmembrane protein that oligomerizes through highly conserved glycine-rich motifs to control cristae formation. Sequence alignments show thatD. melanogasterexhibits three MIC10-like proteins with different tissue-specific expression patterns. Here, we show that the ubiquitously expressed Dmel_CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Loss of DmMIC10b disturbs cristae architecture of mitochondria and reduces the life-span and fertility of flies. Moreover, using fluorescence nanoscopy and electron tomography, we demonstrate that despite its high similarity to the MIC10 proteins from yeast and humans, DmMIC10b exhibits the unique ability to polymerize into elongated filaments upon overexpression. DmMIC10b filaments form bundles which accumulate in the intermembrane space and alter the shape of mitochondrial cristae membranes. We show that the formation of the filaments relies on conserved glycine and cysteine residues and is suppressed by co-expression of other MICOS proteins. Thereby, our findings provide new insights into the regulation of MICOS in flies.

Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defining the molecular mechanism that underpins this metabolic programming will have direct significance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on different metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines (selected to match the biomarker phenotypes in tissue from PCa patients) revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with β3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly β3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which has significance for disease progression and how androgen-deprivation therapy may promote castration-resistant PCa.

BackgroundHaemorrhage is a principal contributory factor in trauma-related deaths in military (battlefield) and civilian settings. A significant proportion of severely injured casualties develop an early trauma- induced coagulopathy (TIC), which is an independent predictor of death. One aspect of TIC is an alteration in platelet function. In vivo trauma models are often essential to develop new treatments. The aim of this study was to evaluate whether an established model of trauma incorporates platelet dysfunction.MethodThe study was conducted in accordance with the Animals (Scientific Procedures) Act, 1986. Blood was collected from terminally anaesthetised pigs before (Baseline) and 30 minutes after the induction of trauma/haemorrhagic shock (S30), and again after 90 minutes of hypotensive resuscitation (R90) with either 0.9% saline, 1:1 packed red cells and plasma (PRBC:FFP) or whole blood (WB). Platelet function was assessed by aggregometry in response to ADP and TRAP (Multiplate®). Platelet count was obtained using a haematology analyser, and shock was quantified by measuring Actual Base Excess (ABE) of arterial blood.ResultsABE fell significantly from Baseline to S30 and remained negative (-7 ± 1 mM) until R90 in all groups (PConclusionsOur model of trauma results in an attenuation of platelet function that, in the early phase, is independent of resuscitation strategy.

Cancer cells that transit from primary tumours into the circulatory system are known as circulating tumour cells (CTCs). These cancer cells have unique phenotypic and genotypic characteristics which allow them to survive within the circulation, subsequently extravasate and metastasise. CTCs have emerged as a useful diagnostic tool using “liquid biopsies” to report on the metastatic potential of cancers. However, CTCs by their nature interact with components of the blood circulatory system on a constant basis, influencing both their physical and morphological characteristics as well as metastatic capabilities. These properties and the associated molecular profile may provide critical diagnostic and prognostic capabilities in the clinic. Platelets interact with CTCs within minutes of their dissemination and are crucial in the formation of the initial metastatic niche. Platelets and coagulation proteins also alter the fate of a CTC by influencing EMT, promoting pro-survival signalling and aiding in evading immune cell destruction. CTCs have the capacity to directly hijack immune cells and utilise them to aid in CTC metastatic seeding processes. The disruption of CTC clusters may also offer a strategy for the treatment of advance staged cancers. Therapeutic disruption of these heterotypical interactions as well as direct CTC targeting hold great promise, especially with the advent of new immunotherapies and personalised medicines. Understanding the molecular role that platelets, immune cells and the coagulation cascade play in CTC biology will allow us to identify and characterise the most clinically relevant CTCs from patients. This will subsequently advance the clinical utility of CTCs in cancer diagnosis/prognosis.

Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC-assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology. Refereed/Peer-reviewed

Infectious diseases like infective endocarditis (IE) may manifest or progress differently between sexes. This study sought to identify the differences in demographic and clinical characteristics among male and female patients with IE. Data were obtained from a newly developed registry comprising all adult patients with first IE admission at the four major tertiary cardiovascular centers in West Virginia, USA during 2014–2018. Patient characteristics were compared between males and females using Chi-square test, Fisher’s exact test, and Wilcoxon rank-sum test. A secondary analysis was restricted to IE patients with drug use only. Among 780 unique patients (390 males, 390 females), significantly more women (a) were younger than males (median age 34.9 vs. 41.4, p < 0.001); (b) reported drug use (77.7% vs. 64.1%, p < 0.001); (c) had tricuspid valve endocarditis (46.4% vs. 30.8%, p < 0.001); and (d) were discharged against medical advice (20% vs. 9.5%, p < 0.001). These differences persisted even within the subgroup of patients with drug use-associated IE. In a state with one of the highest incidences of drug use and overdose deaths, the significantly higher incident IE cases in younger women and higher proportion of women leaving treatment against medical advice are striking. Differential characteristics between male and female patients are important to inform strategies for specialized treatment and care.

Y-box-binding protein 1 (YB-1) is a DNA/RNA binding protein increasingly implicated in the regulation of cancer cell biology. Normally located in the cytoplasm, nuclear localisation in prostate cancer is associated with more aggressive, potentially treatment-resistant disease. This is attributed to the ability of YB-1 to act as a transcription factor for various target genes associated with androgen receptor signalling, survival, DNA repair, proliferation, invasion, differentiation, angiogenesis and hypoxia. This review aims to examine the clinical potential of YB-1 in the detection and therapeutic management of prostate cancer.

Objective\ud The therapeutic benefits of poly(ADP-ribose) polymerase inhibitors highlight the need to evaluate BRCA1/2 defects in tubal/ovarian cancer (OC). We sought to determine the pattern and disease characteristics associated with tumor BRCA1/2 mutations and BRCA1 methylation in women with OC.\ud Methods\ud We obtained 111 OC specimens from 2 university hospitals and assessed BRCA1/2 mutations and BRCA1 methylation in tumor DNA. The frequency and pattern of BRCA1/2 defects were examined. Associations between patient/disease characteristics and BRCA1/2 defects were ascertained (Fisher’s exact test). Platinum-free interval (PFI), progression-free survival (PFS), and overall survival (OS) based on the underlying BRCA1/2 defect were determined (Kaplan-Meier analysis [log-rank test]).\ud Results\ud We observed a BRCA1/2 dysfunction rate of 40% (28/70) in high-grade serous tubal/ovarian cancer (HGSC), including 14.3% BRCA1 methylation (n=10), 7.1% BRCA1 mutation (n=5), and 18.6% BRCA2 mutation (n=13). Defects in BRCA1/2 genes were associated with stage III/IV HGSC (BRCA1 methylation: P=0.005 [stage III/IV] and P=0.004 [HGSC]; BRCA1/2 mutation: P=0.03 [stage III/IV] and P

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 – 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

Introduction Life-threatening infections such as infective endocarditis (IE) are increasing simultaneously with the injection drug use epidemic in West Virginia (WV). We utilized a newly developed, statewide database to describe epidemiologic characteristics and healthcare utilization among patients with (DU-IE) and without (non-DU-IE) drug use-associated IE in WV over five years. Materials and methods This retrospective, observational study, incorporating manual review of electronic medical records, included all patients aged 18–90 years who had their first admission for IE in any of the four university-affiliated referral hospitals in WV during 2014–2018. IE was identified using ICD-10-CM codes and confirmed by chart review. Demographics, clinical characteristics, and healthcare utilization were compared between patients with DU-IE and non-DU-IE using Chi-square/Fisher’s exact test or Wilcoxon rank sum test. Multivariable logistic regression analysis was conducted with discharge against medical advice/in-hospital mortality vs. discharge alive as the outcome variable and drug use as the predictor variable. Results Overall 780 unique patients had confirmed first IE admission, with a six-fold increase during study period (p = .004). Most patients (70.9%) had used drugs before hospital admission, primarily by injection. Compared to patients with non-DU-IE, patients with DU-IE were significantly younger (median age: 33.9 vs. 64.1 years; p < .001); were hospitalized longer (median: 25.5 vs. 15 days; p < .001); had a higher proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates (42.7% vs. 29.9%; p < .001), psychiatric disorders (51.2% vs. 17.3%; p < .001), cardiac surgeries (42.9% vs. 26.6%; p < .001), and discharges against medical advice (19.9% vs. 1.4%; p < .001). Multivariable regression analysis showed drug use was an independent predictor of the combined outcome of discharge against medical advice/in-hospital mortality (OR: 2.99; 95% CI: 1.67–5.64). Discussion and conclusion This multisite study reveals a 681% increase in IE admissions in WV over five years primarily attributable to injection drug use, underscoring the urgent need for both prevention efforts and specialized strategies to improve outcomes.

Background Parachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer-term RBC survival is undocumented. This study aimed to determine post-drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage. Study design and methods Twenty-two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6-8 days post-donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4-h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters. Results Blood units were maintained at 2-6°C and recovered intact after recorded ground impacts of 341-1038 m s-2 . All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB. Discussion Appropriately packaged aerial delivery by parachute can be successfully used for blood supply.

Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data., Adaptation of current algorithms to 3D SMLM data is currently problematic. Here the authors report a method that increases the signal-to-noise ratio and resolution of 3D single particle analysis in localization microscopy and enables determination of the symmetry groups of macromolecular complexes.

Background ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. Methods In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). Results ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. Conclusion FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

Highlights • Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype. • Platelet cancer cell interactions appear to be mediated by 5 key genes which have established roles in metastasis. • Targeting these mediators of metastasis could improve outcomes for cancer patients., Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

Oesophageal adenocarcinoma (OAC) is a disease with an incredibly poor survival rate and a complex makeup. The growth and spread of OAC tumours are profoundly influenced by their surrounding microenvironment and the properties of the tumour itself. Constant crosstalk between the tumour and its microenvironment is key to the survival of the tumour and ultimately the death of the patient. The tumour microenvironment (TME) is composed of a complex milieu of cell types including cancer associated fibroblasts (CAFs) which make up the tumour stroma, endothelial cells which line blood and lymphatic vessels and infiltrating immune cell populations. These various cell types and the tumour constantly communicate through environmental cues including fluctuations in pH, hypoxia and the release of mitogens such as cytokines, chemokines and growth factors, many of which help promote malignant progression. Eventually clusters of tumour cells such as tumour buds break away and spread through the lymphatic system to nearby lymph nodes or enter the circulation forming secondary metastasis. Collectively, these factors need to be considered when assessing and treating patients clinically. This review aims to summarise the ways in which these various factors are currently assessed and how they relate to patient treatment and outcome at an individual level.

Pleiner, Bates, and Görlich introduce anti–mouse and anti–rabbit IgG nanobodies that can be produced in E. coli and fused to reporters or labeled fluorescently to create bright and specific detection reagents with unique advantages over conventional polyclonal secondary antibodies., Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.

A modular assembly of water-soluble diarylethenes (DAEs), applicable as biomarkers for optical nanoscopy, is reported. Reversibly photoswitchable 1,2-bis(2-alkyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes possessing a fluorescent "closed" form were decorated with one or two methoxy group(s) attached to the para-position(s) of phenyl ring(s) and two, four, or eight carboxylic acid groups. Antibody conjugates of these DAEs feature low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and photophysical properties (high emission efficiencies and low cycloreversion quantum yields) enabling their application in superresolution microscopy. Images of tubulin, vimentin, and nuclear pore complexes are presented. The superresolution images can also be acquired by using solely 488 nm light without additional photoactivation with UV light. These DAEs exhibit reversible photoswitching without requiring any additives to the imaging media and open new paths toward the modular design of fluorescent dyes for bioimaging with optical superresolution.

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance. Refereed/Peer-reviewed

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

Aims To evaluate systemic inflammation in TBI by exploration of the inflammasome pathway, a component of the innate immune system that regulates and induces inflammation. We examine the pathway at baseline in TBI compared with healthy control children, and in vitro in response to both LPS stimulation and melatonin therapy. Melatonin has protective effects against NLRP3 inflammasome activation and has therapeutic implications. Methods Whole blood was sampled from children with TBI (n=10) within 24 hours of injury and compared to healthy age-matched controls (n=8) at baseline, following stimulation with bacterial endotoxin (LPS) (10ng/ml) and melatonin treatment (10–3M). Granulocytes were delineated as CD66b+ and FSC, SSC-A. Measurements of mean channel fluorescence (MCF) of CD11b and TLR4 expression on FACS Canto II were recorded and analysed with FloJo software v10. Gene Expression of NLRP3 via rtPCR was recorded in 10 patients and 10 controls at baseline and following LPS and melatonin treatment. Results Granulocyte CD11b expression was lower in children with TBI compared to controls (p=0.04) Both upregulated CD11b with LPS stimulation. Melatonin significantly decreased this LPS upregulation. There was no significant difference in baseline TLR4 expression between TBI and controls, but LPS upregulation of TLR4 was decreased by melatonin in the TBI cohort. Inflammasome was upregulated via NLRP3 expression in children with TBI compared to controls (p= 0.02). Melatonin significantly decreased LPS-induced upregulation of NLRP3 only in controls. Conclusion Inflammation is altered in TBI compared to controls with altered responsiveness to melatonin treatment following LPS stimulation. The inflammasome is downregulated in children immediately following TBI. Selective inhibition of systemic inflammation targeting the inflammasome may have a future immunomodulatory role as a target in treating TBI.

Aims To evaluate components of the innate immune system, the inflammsome, in mild Traumatic Brain Injury (TBI) and the correlation with symptom burden 2 weeks from injury. We examine activation of the pathway at presentation, and also at 2 weeks from injury compared with healthy control children. Methods Whole blood was sampled from children with mild TBI within 24 hours of injury and at two weeks from injury and compared to healthy paediatric controls at baseline. RNA was isolated and cDNA was synthesized. Gene Expression of NLRP3 and IL1 β via rtPCR was recorded in 22 patients and 5 controls at baseline and 15 patients at 2 weeks. The Post Concussive Symptom Inventory was administered at 2 weeks. A change from pre-injury baseline was recorded. Results Inflammasome was upregulated via NLRP3 expression in children with TBI compared to controls across groups however this did not correlate with symptoms at 2 weeks. Higher IL-1β transcription levels at presentation were positively correlated by Pearson correlation (p = 0.029) with higher symptom scores at 2 weeks. Conclusion Inflammation is altered in TBI compared to controls The NLRP3 component of inflammasome while elevated does not correlate with symptom burden. IL=1 β gene transcription does. IL-1 β holds promise in predicting symptom burden following mTBI. Selective inhibition of systemic inflammation targeting the inflammasome may have a future immunomodulatory role as a target in treating mTBI.

Objective To investigate human papillomavirus (HPV) DNA testing and p16/Ki-67 staining for detecting cervical intraepithelial grade 2 or worse (CIN2+) and CIN3 in women referred to colposcopy with minor abnormal cervical cytology low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of undermined significance (ASC-US). The clinical performance of both tests was evaluated as stand-alone tests and combined, for detection CIN2+ and CIN3 over 2 years. Methods ThinPrep® liquid-based cytology (LBC) specimens were collected from 1349 women with repeat LSIL or ASC-US. HPV DNA was performed using Hybrid Capture. Where adequate material remained (n = 471), p16/Ki-67 overexpression was assessed. Clinical performance for detection of histologically diagnosed CIN2+ and CIN3 was calculated. Results Approximately 62.2% of the population were positive for HPV DNA, and 30.4% were positive for p16/Ki-67. p16/Ki-67 showed no significant difference in positivity between LSIL and ASC-US referrals (34.3% versus 28.6%; P = 0.189). Women under 30 years had a higher rate of p16/Ki-67 compared to those over 30 years (36.0% versus 26.6%; P = 0.029). Overall HPV DNA testing produced a high sensitivity for detection of CIN3 of 95.8% compared to 79.2% for p16/Ki-67. In contrast, p16/Ki-67 expression offered a higher specificity, 75.2% versus 40.4% for detection of CIN3. Combining p16/Ki-67 with HPV DNA improved the accuracy in distinguishing between CIN3 and

Background: Circulating tumour cells (CTCs) are silent precursors of metastatic disease that utilise various mechanisms to survive in circulation and metastasise to distal sites. Classical CTC detection relies on EpCam affinity-based technologies; however, CTCs are highly heterogeneous and often undergo EMT. Recent research highlights the ability of platelets and neutrophils to ‘cloak' CTCs and crosstalk to aid in their proliferation and survival in circulation. Aims: Assess the role of platelets and neutrophils in the characterisation of CTCs. Methods: Cell lines: Breast cancer (MCF-7) and ovarian cancer (SKOV-3, OVCAR3, OAW42) cells were exposed to healthy donor isolated platelets and neutrophils for 24h. EMT and immune evasion gene assays, flow cytometry analysis and cell proliferation assays were performed following co-culture. Cell line-blood spike-in experiments were performed for gating strategy optimisation.Patient samples: Blood specimens were prospectively collected from breast and ovarian cancer patients (central venous and peripheral blood). CTCs and immune cells were then isolated using a ClearCell FX microfluidic device. FX isolated cells were immunophenotyped by either immunofluorescence (IF) or flow cytometry. Single cells were index sorted using BD FACS Melody for subsequent scRNAseq. Results: Platelet cloaking altered EpCam, PLEK2, CCL2 and TWIST1 mRNA expression in MCF-7 cells. Platelet and neutrophil co-culture altered EpCam, PAI-1 and PD-L1 expression in ovarian cancer cell lines. Healthy neutrophils co-cultured with ovarian cancer cells increased cell viability, while induction of NETosis increased cancer cell proliferation. CTCs isolated from peripheral blood in breast and ovarian patients were EpCam/panCK+/CD45- by IF. Immunophenotyped CTCs were identified as EpCam+/E-Cadherin+/CD45-, N-Cadherin+/CD45- and as PD-L1+/EpCam+/E-Cadherin+/CD45- cells by flow cytometry. We also identified EpCam+/CD45+/CD66b+ cells in ovarian central blood. Isolated classical and non-classical CTCs were sorted based on their different immunophenotypes for downstream scRNAseq using BD Precise Whole Transcriptome Assays. Conclusion: Platelets and neutrophils alter the expression of markers used in the identification of CTCs. Neutrophils were found to increase the proliferation of cancer cells as well as increase PD-L1 expression. We identified a heterogeneous population of CTCs across different patients and found that CTCs from central venous blood have increased numbers of non-classical CTCs (CD66b+/EpCam+ cells). Molecular scRNAseq signatures of CTC subsets form the basis for identifying the most clinically relevant CTCs in circulation. Our approach may improve diagnostic accuracy, allow for immunophenotyping of cloaked CTCs, and provide prognostic information. Citation Format: Mark P. Ward, Bashir M. Mohamed, Laura Kane, Mark Bates, Janina Berghoff, Cathy L. Spillane, Tanya Kelly, John Kennedy, Feras A. Saadeh, Karsten Hokamp, Noreen Gleeson, Orla Sheils, Cara Martin, Michael Gallagher, Sean Hannify, Eric P. Dixon, Sharon A. O'Toole, John J. O'Leary. Influence of platelets and neutrophils on circulating tumour cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 764.

The exact biological mechanism governing the radioresistant phenotype of prostate tumours at a high risk of recurrence despite the delivery of advanced radiotherapy protocols remains unclear. This study analysed the protein expression profiles of a previously generated isogenic 22Rv1 prostate cancer model of radioresistance using DigiWest multiplex protein profiling for a selection of 90 signalling proteins. Comparative analysis of the profiles identified a substantial change in the expression of 43 proteins. Differential PARP-1, AR, p53, Notch-3 and YB-1 protein levels were independently validated using Western Blotting. Pharmacological targeting of these proteins was associated with a mild but significant radiosensitisation effect at 4Gy. This study supports the clinical relevance of isogenic in vitro models of radioresistance and clarifies the molecular radiation response of prostate cancer cells.

Polyclonal anti-IgG secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in E. coli and could thus make secondary antibody-production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore-coupling. We demonstrate their superior performance in Western Blotting, both in peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and super-resolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols and even allow multi-target localization with primary IgGs from the same species and of the same class.

We present a general purpose, open-source software library for estimation of non-linear parameters by the Levenberg-Marquardt algorithm. The software, Gpufit, runs on a Graphics Processing Unit (GPU) and executes computations in parallel, resulting in a significant gain in performance. We measured a speed increase of up to 42 times when comparing Gpufit with an identical CPU-based algorithm, with no loss of precision or accuracy. Gpufit is designed such that it is easily incorporated into existing applications or adapted for new ones. Multiple software interfaces, including to C, Python, and Matlab, ensure that Gpufit is accessible from most programming environments. The full source code is published as an open source software repository, making its function transparent to the user and facilitating future improvements and extensions. As a demonstration, we used Gpufit to accelerate an existing scientific image analysis package, yielding significantly improved processing times for super-resolution fluorescence microscopy datasets.