Film practice as interdisciplinary research: a case study

Referring primarily to my own doctoral practice-as-research project as a case study, this chapter explores cinematic practice as a mode of interdisciplinary research. The project traces the journey of the Kladovo transport, a large group of Jewish refugees from central Europe, who attempted to flee Nazi persecutions in 1939 via the river Danube. The majority of the passengers never got further than Serbia, where their journey fatally ended in 1941/1942. This failed escape attempt is charged with striking relationships to time, like the long periods of stasis that the Kladovo transport spent on the Danube waters. While drawing from large an interdisciplinary field, including history, Holocaust geographies and archaeology, I explore this journey as a multi-temporal event, with the camera as my main research tool. In this chapter, I will take a closer look at some of the elements of the interdisciplinary encounters as they appeared in my study

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni

Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. This thesis aims to address this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression and thereby investigate gene function. To achieve this, a “dead” cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. The tool was validated through successful repression of the arylsulphatase gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was futher validated against the hippicurase gene hipO, and trialled with an Anhydrotetracycline (ATc) inducible promoter, which demonstrated leaky expression in the absence of ATc. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). Finally, CRISPRi-based repression of the flagella genes fliE and fliQ was attempted to trial targeting of essential genes in C. jejuni. To date, this is the first report of a CRISPR-based interference system demonstrated in C. jejuni.Wellcome Trus

Development of a CRISPR interference system in Campylobacter jejuni

Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuniresponsible for 80% of these infections. There is no current vaccine and antibiotic resistance is emerging. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which impacts studies into the function of essential and conditionally essential genes. My project aims to address this by applying a CRISPR-based interference system known as CRISPRi in C. jejuni as a means to control gene expression and thereby investigate gene function. To validate the CRISPRi system in C. jejuni, I have paired the dCas9 and sgRNA backbone from the Streptococcus pyogenesCRISPRi system with several C. jejuni-derived promoters to develop a series of CRISPRi constructs targeting several genes. Through rigorous sgRNA target design I have successfully targeted and repressed expression of the endogenous arylsulphatase (AstA) enzyme, as well as achieving partial repression of expression of the regulatory flagellar protein FlgR in two clinically relevant C. jejuni strains. This is the first report of a CRISPRi system for Campylobacter.</jats:p

Substrate Utilisation and Energy Metabolism in Non-Growing Campylobacter jejuni M1cam.

Campylobacter jejuni, the major cause of bacterial foodborne illness, is also a fastidious organism that requires strict growth requirements in the laboratory. Our aim was to study substrate utilisation and energy metabolism in non-growing C. jejuni to investigate the ability of these bacteria to survive so effectively in the food chain. We integrated phenotypic microarrays and genome-scale metabolic modelling (GSM) to investigate the survival of C. jejuni on 95 substrates. We further investigated the underlying metabolic re-adjustment associated with varying energy demands on each substrate. We identified amino acids, organic acids and H2, as single substrates supporting survival without growth. We identified several different mechanisms, which were used alone or in combination, for ATP production: substrate-level phosphorylation via acetate kinase, the TCA cycle, and oxidative phosphorylation via the electron transport chain that utilised alternative electron donors and acceptors. The benefit of ATP production through each of these mechanisms was associated with the cost of enzyme investment, nutrient availability and/or O2 utilisation. C. jejuni can utilise a wide range of substrates as energy sources, including organic acids commonly used for marination or preservation of ingredients, which might contribute to the success of their survival in changing environments

Human Leukocytes Kill <i>Brugia malayi</i> Microfilariae Independently of DNA-Based Extracellular Trap Release

<div><p>Background</p><p><i>Wuchereria bancrofti</i>, <i>Brugia malayi</i> and <i>Brugia timori</i> infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases.</p><p>Principal findings</p><p><i>In vitro</i>, PMNs expel large amounts of NETs that capture but do not kill <i>B</i>. <i>malayi</i> Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill <i>B</i>. <i>malayi</i> Mf <i>in vitro</i> and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf <i>in vitro</i>, which may reflect the pathological heterogeneity associated with filarial parasitic infections.</p><p>Conclusions/Significance</p><p>Human innate immune cells are able to recognize, attach to and kill <i>B</i>. <i>malayi</i> microfilariae in an <i>in vitro</i> system. This suggests that, <i>in vivo</i>, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.</p></div

Extracellular DNA release from human PMNs.

<p>A-C: Images of SYTOX Orange-labelled extracellular DNA present within Mf-treated (A), zero Mf (negative control) (B) and PMA-treated (positive control) (C) wells. The images were taken 7 hours post-assay set up. These wells did not contain serum. D-G: Changes in mean SYTOX Orange intensity were used to monitor the release of extracellular DNA from PMNs incubated for 7 hours <i>in vitro</i>. In each panel, the dotted lines indicate the Standard Error of the mean fluorescence intensity. D: 25nM phorbol myristate acetate (PMA)- and Mf-induced DNA release in the absence of serum was measured as described in Materials and Methods (n = 7). E: 10μM diphenyleneiodonium (DPI) and 30μg/ml DNAse I inhibited Mf-induced DNA release in the absence of serum (n≥4). F: Extracellular DNA release in Mf- and PMA-treated wells that contained 5% autologous serum (n = 6). G: 5% autologous serum and 5% autologous heat treated serum (HTS) inhibited Mf induced DNA release (n≥6).</p

Mf survival in the presence of PMNs and PBMCs.

<p>The percentage of Mf that survived to days 1 (A), 2 (B) and 5 (C) post-experimental set up (n = 5). White, blue, yellow, and red bars represent zero cell, 1500 PMN/Mf, 1500 PBMC/Mf and PMN plus PBMC treated wells respectively. All wells contained either 25% autologous serum or 25% heat treated serum (HTS). DNase I denotes the addition of 30μg/ml DNase I. Error bars represent standard error of the mean; *P<0.05, **P<0.01, ***P<0.001. D. Scatter plot comparing leukocyte attachment and Mf survival in the presence of PMNs and PBMCs. The percentage of Mf that had at least one PMN/PBMC adhered to their surface or indirectly fastened by extracellular DNA at 1 hour post-experimental set up plotted against the percentage of Mf that survived to day 5 (n = 15). Each point represents an individual biological replicate derived from distinct human blood donors. All wells contained 25% autologous serum, ~100 Mf, ~150,000 PMNs and ~150,000 PBMCs.</p

The attachment of PMNs to <i>B</i>. <i>malayi</i> Mf.

<p>A: The percentage of Mf that had at least one PMN adhered to their surface or indirectly fastened by extracellular DNA at 24 hours post-experimental set up (n≥6). White, yellow, red, blue, and black bars represent no serum, 5% autologous serum, 5% autologous heat-treated (55°C, 30 mins) serum, 10μM diphenyleneiodonium (DPI) and 30μg/ml DNase I treated wells respectively. Error bars represent standard error of the mean; *P<0.05, **P<0.01, ***P<0.001. B: Image of PMNs adhered to a Mf. Image taken after 24 hours of incubation in 5% autologous serum at 37°C and 5% CO₂. The preparation was stained with modified Wrights’ stain.</p

<i>B</i>. <i>malayi</i> Mf entangled within neutrophil extracellular traps.

<p>Fluorescence imaging of a worm-NET aggregate labelled with DAPI (blue) and antibodies to myeloperoxidase (MPO; purple), citrullinated histone H4 (histone; green) and human neutrophil elastase (HNE; red). The co-localization of markers with Mf is shown in the merged image with transmitted light (top left panel) and the overlap of MPO, histone and HNE with DNA shown in the merged image (bottom right panel). Live Mf were incubated in the presence of human neutrophils for 18 hours at 37°C and 5% CO₂. Scale bar = 10μm.</p