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.

Biodiversity: the overlooked source of human health.

Biodiversity is the measure of the variation of lifeforms in a given ecological system. Biodiversity provides ecosystems with the robustness, stability, and resilience that sustains them. This is ultimately essential for our survival because we depend on the services that natural ecosystems provide (food, fresh water, air, climate, and medicine). Despite this, human activity is driving an unprecedented rate of biodiversity decline, which may jeopardize the life-support systems of the planet if no urgent action is taken. In this article we show why biodiversity is essential for human health. We raise our case and focus on the biomedicine services that are enabled by biodiversity, and we present known and novel approaches to promote biodiversity conservation.

Understanding and harnessing triple-negative breast cancer-related microbiota in oncology.

Bacterial inhabitants of the body have the potential to play a role in various stages of cancer initiation, progression, and treatment. These bacteria may be distal to the primary tumour, such as gut microbiota, or local to the tissue, before or after tumour growth. Breast cancer is well studied in this context. Amongst breast cancer types, Triple Negative Breast Cancer (TNBC) is more aggressive, has fewer treatment options than receptor-positive breast cancers, has an overall worse prognosis and higher rates of reoccurrence. Thus, an in-depth understanding of the bacterial influence on TNBC progression and treatment is of high value. In this regard, the Gut Microbiota (GM) can be involved in various stages of tumour progression. It may suppress or promote carcinogenesis through the release of carcinogenic metabolites, sustenance of proinflammatory environments and/or the promotion of epigenetic changes in our genome. It can also mediate metastasis and reoccurrence through interactions with the immune system and has been recently shown to influence chemo-, radio-, and immune-therapies. Furthermore, bacteria have also been found to reside in normal and malignant breast tissue. Several studies have now described the breast and breast tumour microbiome, with the tumour microbiota of TNBC having the least taxonomic diversity among all breast cancer types. Here, specific conditions of the tumour microenvironment (TME) - low O2, leaky vasculature and immune suppression - are supportive of tumour selective bacterial growth. This innate bacterial ability could enable their use as delivery agents for various therapeutics or as diagnostics. This review aims to examine the current knowledge on bacterial relevance to TNBC and potential uses while examining some of the remaining unanswered questions regarding mechanisms underpinning observed effects.

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.

Characterization of FFPE-induced bacterial DNA damage and development of a repair method.

Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterized. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterize this FFPE-induced bacterial DNA damage and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised - an optimized decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.

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.

Seeding sustainable education in developing countries: Teaching biotech in low-income areas.

Synthetic biology has the potential to seed research in impoverished countries. Teaching students and academics has to take into account reality - lessons from Honduras.

Protoblock - A biological standard for formalin fixed samples.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissue is the gold standard in pathology tissue storage, representing the largest collections of patient material. Their reliable use for DNA analyses could open a trove of potential samples for research and are currently being recognised as a viable source material for bacterial analysis. There are several key features which limit bacterial-related data generation from this material: (i) DNA damage inherent to the fixing process, (ii) low bacterial biomass that increases the vulnerability to contamination and exacerbates the host DNA effects and (iii) lack of suitable DNA extraction methods, leading to data bias. The development and systematic use of reliable standards is a key priority for microbiome research. More than perhaps any other sample type, FFPE material urgently requires the development of standards to ensure the validity of results and to promote reproducibility. RESULTS: To address these limitations and concerns, we have developed the Protoblock as a biological standard for FFPE tissue-based research and method optimisation. This is a novel system designed to generate bespoke mock FFPE 'blocks' with a cell content that is user-defined and which undergoes the same treatment conditions as clinical FFPE tissues. The 'Protoblock' features a mix of formalin-fixed cells, of known number, embedded in an agar matrix which is solidified to form a defined shape that is paraffin embedded. The contents of various Protoblocks populated with mammalian and bacterial cells were verified by microscopy. The quantity and condition of DNA purified from blocks was evaluated by qPCR, 16S rRNA gene amplicon sequencing and whole genome sequencing. These analyses validated the capability of the Protoblock system to determine the extent to which each of the three stated confounding features impacts on eventual analysis of cellular DNA present in FFPE samples. CONCLUSION: The Protoblock provides a representation of biological material after FFPE treatment. Use of this standard will greatly assist the stratification of biological variations detected into those legitimately resulting from experimental conditions, and those that are artefacts of the processed nature of the samples, thus enabling users to relate the outputs of laboratory analyses to reality. Video Abstract.

In situ biomolecule production by bacteria; a synthetic biology approach to medicine.

The ability to modify existing microbiota at different sites presents enormous potential for local or indirect management of various diseases. Because bacteria can be maintained for lengthy periods in various regions of the body, they represent a platform with enormous potential for targeted production of biomolecules, which offer tremendous promise for therapeutic and diagnostic approaches for various diseases. While biological medicines are currently limited in the clinic to patient administration of exogenously produced biomolecules from engineered cells, in situ production of biomolecules presents enormous scope in medicine and beyond. The slow pace and high expense of traditional research approaches has particularly hampered the development of biological medicines. It may be argued that bacterial-based medicine has been "waiting" for the advent of enabling technology. We propose that this technology is Synthetic Biology, and that the wait is over. Synthetic Biology facilitates a systematic approach to programming living entities and/or their products, using an approach to Research and Development (R&D) that facilitates rapid, cheap, accessible, yet sophisticated product development. Full engagement with the Synthetic Biology approach to R&D can unlock the potential for bacteria as medicines for cancer and other indications. In this review, we describe how by employing Synthetic Biology, designer bugs can be used as drugs, drug-production factories or diagnostic devices, using oncology as an exemplar for the concept of in situ biomolecule production in medicine.

Synthetic Biology in the Driving Seat of the Bioeconomy.

Synthetic biology is revolutionising the biotech industry and is increasingly applied in previously unthought-of markets. Here, we discuss the importance of this industry to the bioeconomy and two of its key factors: the synthetic biology approach to research and development (R&D), and the unique nature of the carefully designed, stakeholder-inclusive, community-directed evolution of the field.