Synthetic protein protease sensor platform.

Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.

Biopsy bacterial signature can predict patient tissue malignancy.

Considerable recent research has indicated the presence of bacteria in a variety of human tumours and matched normal tissue. Rather than focusing on further identification of bacteria within tumour samples, we reversed the hypothesis to query if establishing the bacterial profile of a tissue biopsy could reveal its histology / malignancy status. The aim of the present study was therefore to differentiate between malignant and non-malignant fresh breast biopsy specimens, collected specifically for this purpose, based on bacterial sequence data alone. Fresh tissue biopsies were obtained from breast cancer patients and subjected to 16S rRNA gene sequencing. Progressive microbiological and bioinformatic contamination control practices were imparted at all points of specimen handling and bioinformatic manipulation. Differences in breast tumour and matched normal tissues were probed using a variety of statistical and machine-learning-based strategies. Breast tumour and matched normal tissue microbiome profiles proved sufficiently different to indicate that a classification strategy using bacterial biomarkers could be effective. Leave-one-out cross-validation of the predictive model confirmed the ability to identify malignant breast tissue from its bacterial signature with 84.78% accuracy, with a corresponding area under the receiver operating characteristic curve of 0.888. This study provides proof-of-concept data, from fit-for-purpose study material, on the potential to use the bacterial signature of tissue biopsies to identify their malignancy status.

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis.

The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3-V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1-V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1-V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.

A novel cell permeability assay for macromolecules.

BACKGROUND: Many cell permeabilisation methods to mediate internalisation of various molecules to mammalian or bacterial cells have been developed. However, no size-specific permeability assay suitable for both cell types exists. RESULTS: We report the use of intrinsically biotinylated cell components as the target for reporter molecules for assessing permeabilisation. Due to its well-described biotin binding activity, we developed an assay using Streptavidin (SAv) as a molecular weight marker for assessing eukaryotic and prokaryotic cell internalisation, using flow cytometry as a readout. This concept was tested here as part of the development of host DNA depletion strategies for microbiome analysis of formalin-fixed (FF) samples. Host depletion (HD) strategies require differential cell permeabilisation, where mammalian cells but not bacterial cells are permeabilised, and are subsequently treated with a nuclease. Here, the internalisation of a SAv-conjugate was used as a reference for nucleases of similar dimensions. With this assay, it was possible to demonstrate that formalin fixation does not generate pores which allow the introduction of 60 KDa molecules in mammalian or bacterial membranes/envelopes. Among surfactants tested, Saponin derived from Quillaja bark showed the best selectivity for mammalian cell permeabilisation, which, when coupled with Benzonase nuclease, provided the best results for host DNA depletion, representing a new HD strategy for formalin fixed samples. CONCLUSION: The assay presented provides researchers with a sensitive and accessible tool for discerning membrane/cell envelop permeability for different size macromolecules.

Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma.

BACKGROUND: The density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma. METHODS: Fifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression. RESULTS: CD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival. CONCLUSIONS: Thus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

Sequence-Based Characterization of Intratumoral Bacteria-A Guide to Best Practice.

Tumors are hospitable environments to bacteria and several recent studies on cancer patient samples have introduced the concept of an endogenous tumor microbiome. For a variety of reasons, this putative tumor microbiome is particularly challenging to investigate, and a failure to account for the various potential pitfalls will result in erroneous results and claims. Before this potentially extremely medically-significant habitat can be accurately characterized, a clear understanding of all potential confounding factors is required, and a best-practice approach should be developed and adopted. This review summarizes all of the potential issues confounding accurate bacterial DNA sequence analysis of the putative tumor microbiome, and offers solutions based on related research with the hope of assisting in the progression of research in this field.

Diagnostic Performance of Contrast-Enhanced MRI With Secretin-Stimulated MRCP for Non-Calcific Chronic Pancreatitis: A Comparison With Histopathology.

OBJECTIVES: Diagnosis of non-calcific chronic pancreatitis (NCCP) in patients presenting with chronic abdominal pain is challenging and controversial. Contrast-enhanced magnetic resonance imaging (MRI) with secretin-stimulated MRCP (sMRCP) offers a safe and noninvasive modality to diagnose mild CP, but its findings have not been correlated with histopathology. We aimed to assess the correlation of a spectrum of MRI/sMRCP findings with surgical histopathology in a cohort of NCCP patients undergoing total pancreatectomy with islet autotransplantation (TPIAT). METHODS: Adult patients undergoing TPIAT for NCCP between 2008 and 2013 were identified from our institution's surgery database and were included if they had MRI/sMRCP within a year before surgery. Histology was obtained from resected pancreas at the time of TPIAT by wedge biopsy of head, body, and tail, and was graded by a gastrointestinal pathologist who was blinded to the imaging features. A fibrosis score (FS) of 2 or more was considered as abnormal, with FS ≥6 as severe fibrosis. A multivariate regression analysis was performed for MRI features predicting fibrosis, after taking age, sex, smoking, alcohol, and body mass index (BMI) into consideration. A quantitative receiver operating characteristic (ROC) curve analysis was performed and Spearman rank correlation coefficient (r) was calculated. RESULTS: Fifty-seven patients (females=49, males=8) with NCCP and MRI/sMRCP were identified. ROC curve analysis showed that two or more MRI/sMRCP features provided the best balance of sensitivity (65%), specificity (89%), and accuracy (68%) to differentiate abnormal (FS≥2) from normal pancreatic tissue. Two or more features provided the best cutoff (sensitivity 88%, specificity 78%) for predicting severe fibrosis (FS≥6). There was a significant correlation between the number of features and severity of fibrosis (r=0.6, P<0.0001). A linear regression after taking age, smoking, and BMI into consideration showed that main pancreatic duct irregularity, T1-weighted signal intensity ratio between pancreas and paraspinal muscle, and duodenal filling after secretin injection to be significant independent predictors of fibrosis. CONCLUSIONS: A strong correlation exists between MRI/sMRCP findings and histopathology of NCCP.

Central presynaptic terminals are enriched in ATP but the majority lack mitochondria.

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.

Neoadjuvant chemoradiotherapy for locally advanced pancreas cancer rarely leads to radiological evidence of tumour regression.

BACKGROUND: Neo-adjuvant chemo-radiotherapy has been proposed to improve resectability of locally-advanced pancreatic cancer (LAPC). However, the ability of neo-adjuvant therapy to induce radiological tumour regression has not been reported. METHODS: Pre- and post-treatment computed tomography (CT) scans of patients undergoing neo-adjuvant chemo-radiotherapy for LAPC were reviewed. LAPC was sub-classified into borderline resectable disease [≤ 180° involvement of the superior mesenteric artery (SMA); short-segment encasement/abutment of the common hepatic artery; or tumour-associated deformity, abutment or short-segment occlusion of the superior mesenteric vein (SMV)/ portal vein (PV) that was amenable to vascular resection and reconstruction] and locally advanced un-resectable pancreatic cancer (vascular involvement more than that described for borderline resectable pancreatic cancer). The radiological response and surgical resection rates were assessed. RESULTS: Sixteen patients received neo-adjuvant therapy for LAPC during 2005-2008. Regression of major vascular involvement, i.e. un-encasement or regression of abutment of any involved vessels was not observed in any patient. Pre- and post-treatment tumour densities were not statistically different. Fifty per cent of patients with borderline resectable disease and none of the patients with locally advanced un-resectable pancreatic cancer eventually underwent surgical resection. CONCLUSION: Neo-adjuvant treatment does not induce radiological tumour regression of LAPC with major vascular involvement. Patient selection for neo-adjuvant trial enrollment should remain focused on borderline disease which may have a potential for surgical resection.