Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Stability criteria for unsupervised temporal association networks

A biologically realizable, unsupervised learning rule is described for the online extraction of object features, suitable for solving a range of object recognition tasks. Alterations to the basic learning rule are proposed which allow the rule to better suit the parameters of a given input space. One negative consequence of such modifications is the potential for learning instability. The criteria for such instability are modeled using digital filtering techniques and predicted regions of stability and instability tested. The result is a family of learning rules which can be tailored to the specific environment, improving both convergence times and accuracy over the standard learning rule, while simultaneously insuring learning stability.

Heritability and nineteen-year stability of long and short EPQ-R Neuroticism scales

The heritability and stability over a 19 year period of long (23-item) and short (12-item) versions of Eysenck's Neuroticism scale were compared in a large Australian twin-family sample. Stability over 19 years of the 23-item Neuroticism scale was 0.62 and for the 12-item scale 0.59. Correlations between scores obtained by mailed questionnaire and telephone interview a few weeks apart were 0.87 for the long scale and 0.85 for the short scale; scores obtained by mail were slightly higher, particularly for females. The 12-item scale had slightly reduced power to discriminate both high and low scoring individuals on the full 23-item scale. Mean Neuroticism score for the 12-item scale was atypically low when compared to the distribution of the complete set of scores for all possible combinations (> 1 million) of 12-items drawn from the full 23-item EPQ-R. Mean heritabilities for the lowest and highest 300,000 of these combinations were 43.2% and 42.7%, respectively, somewhat higher than the 41.0% for the actual EPQ-R-S 12-item scale. Heritability for the 23-item scale was 46.5%. We conclude that there is little loss of either stability or heritability in using the short EPQ-R scale, but the choice of which 12-items could have been better. (c) 2005 Elsevier Ltd. All rights reserved.

Factors influencing the stability of cyclotides: Proteins with a circular backbone and cystine knot motif

Cyclotides are a large family of mini-proteins that have the distinguishing features of a head-to-tail cyclised backbone and a cystine knot formed by six conserved cysteine residues. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. The unique structural features of the cyclotides make them extremely resistant to chemical, thermal and proteolytic degradation. In this article we review recent Studies from our laboratory that dissect the role of the individual structural elements in defining the stability of cyclotides. The resistance of cyclotides to chemical and proteolytic degradation is in large part due to the cystine knot, whereas the thermal stability is I composite of several features including the cystine knot, the cyclic backbone and the hydrogen bonding network. A range of biological activities of cyclotides is critically dependent oil the presence of the cyclic backbone.

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template

The cyclotides are a recently discovered family of miniproteins that contain a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. They are approximately 30 amino acids in size and are present in high abundance in plants from the Violaceae, Rubiaceae, and Cucurbitaceae families, with individual plants containing a suite of up to 100 cyclotides. They have a diverse range of biological activities, including uterotonic, anti-HIV, antitumor, and antimicrobial activities, although their natural function is likely that of defending their host plants from pathogens and pests. This review focuses on the structural aspects of cyclotides, which may be thought of as a natural combinatorial peptide template in which a wide range of amino acids is displayed on a compact molecular core made up of the cyclic cystine knot structural motif. Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The struclural features that contribute to their remarkable stability are described ill this review. (c) 2006 Wiley Periodicals, Inc.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Effect of grape seed extract on oxidative, color and sensory stability of pre-cooked, frozen, re-heated beef sausage model system

The processing of meats at the factory level can trigger the onset of lipid oxidation, which can lead to meat quality deterioration. Warmed over flavor is an off-flavor, which is associated with oxidative deterioration in meat. To avoid or delay the auto-oxidation process in meat products, synthetic and natural antioxidants have been successfully used. Grape (Vitis Vinifera) is of special interest due to its high content of phenolic compounds. Grape seed extract sold commercially as a dietary supplement, has the potential to reduce lipid oxidation and WOF in cooked ground beef when added at 1%. The objective of study 1 was to compare the antioxidant activity of natural antioxidants including grape seed extract and some herbs belonging to the Lamiaciae family: rosemary (Rosmarinus Officinalis), sage (Salvia Officinalis) and oregano (Origanum Vulgare) with commercial synthetic antioxidants like BHT, BHA, propyl gallate and ascorbic acid using the ORAC assay. All sample solutions were prepared to contain 1.8 gm sample/10 ml solvent. The highest antioxidant activity was observed for the grape seed extract sample (359.75 µM TE), while the lowest was observed for BHA, propyl gallate and rosemary also showed higher antioxidant potential with ORAC values above 300 μmol TE/g. ORAC values obtained for ascorbic acid and Sage were between 250-300μ mol TE/g while lowest values were obtained for Butylated Hydroxytoluene (28.50 µM TE). Based on the high ORAC values obtained for grape seed extract, we can conclude that byproducts of the wine/grape industry have antioxidant potential comparable to or better than those present in synthetic counterparts. The objective of study 2 was to compare three levels of grape seed extract (GSE) to commonly used antioxidants in a pre-cooked, frozen, stored beef and pork sausage model system. Antioxidants added for comparison with control included grape seed extract (100, 300, 500 ppm), ascorbic acid (AA, 100 ppm of fat) and propyl gallate (PG, 100 ppm of fat). Product was formed into rolls, frozen, sliced into patties, cooked on a flat griddle to 70C, overwrapped in PVC, and then frozen at –18C for 4 months. GSE- and PG-containing samples retained their fresh cooked beef odor and flavor longer (p<0.05) than controls during storage. Rancid odor and flavor scores of GSE-containing samples were lower (p<0.05) than those of controls after 4 months of storage. The L* value of all samples increased (p<0.05) during storage. Thiobarbituric acid reactive substances (TBARS) of the control and AA-containing samples increased (p<0.05); those of GSE-containing samples did not change significantly (p>0.05) over the storage period.

Transcription of alternative oxidase (AOX) and plastid terminal oxidase (PTOX) during stress-regulated root tissue growth in Daucus carota L. - An approach to identify functional marker candidates for breeding on carrot yield stability

A presente tese explora a hipótese de utilização dos genes da oxidase alternativa (AOX) e da oxidase terminal da plastoquinona (PTOX) como genes-alvo para o desenvolvimento de marcadores funcionais (MF) para avaliar a performance do crescimento em cenoura, fator determinante da produtividade. Para avaliar se os referidos genes estão associados com o crescimento da cenoura procedeu—se ao seu isolamento e posterior análise dos seus perfis de transcrição em diversos sistemas biológicos. O sistema in vitro selecionado, denominado sistema de culturas primárias, permitiu avaliar alterações na quantidade de transcritos desses genes durante os processos de reprogramação celular e crescimento. Ao nível da planta foi também estudado o efeito do frio na expressão precoce dos genes AOX. Ambos os genes DcAOX1 e DcAOX2a revelaram uma resposta rápida e um padrão semelhante apos stresse (inoculação in vitro e resposta ao frio). Foi igualmente verificado um incremento na expressão do gene DcPTOX durante a fase inicial do processo de reprogramação celular. Estudos de expressão dos genes AOX durante o desenvolvimento da raiz da cenoura revelaram que o gene DcAOX2a será potencialmente o gene mais envolvido neste processo. De modo a avaliar a hipótese de envolvimento do gene DcPTOX no crescimento da raíz procederam—se a estudos de expressão ao nível do tecido meristemático. Todavia, para um mais completo entendimento da ligação entre DcPTOX e o crescimento secundário e/ou acumulação de carotenos, a expressão do gene DcPTOX foi também avaliada em raízes de cenoura durante o desenvolvimento, utilizando cultivares caracterizadas por distintos conteúdos de carotenos. Os resultados obtidos demonstraram a associação do gene DcPTOX a ambos os processos. O envolvimento da PTOX no crescimento adaptativo da raiz foi analisado com um ensaio que permitiu identificar, no tecido meristemático, uma resposta precoce do gene DcPTOX face a uma diminuição da temperatura. Adicionalmente, foi efetuada a seleção de genes de referência para uma analise precisa da expressão génica por RT-qPCR em diversos sistemas biológicos de cenoura, e a importância do seu estudo ao nível do sistema biológico foi realçada. Os resultados desta tese são encorajadores para prosseguir os estudos de utilização dos genes AOX e PTOX como MF no melhoramento da performance do crescimento adaptativo em cenoura, fator determinante para a produtividade; ABSTRACT: This thesis explores the hypothesis of using the alternative oxidase (AOX) and theplastid terminal oxidase (PTOX) as target genes for functional marker (FM) development for yield-determining growth performance in carrot. To understand if these genes are associated to growth, different AOX gene family members and the single PTOX gene were isolated, and their expression patterns evaluated in diverse carrot plant systems. An in-vitro primary culture system was selected to study AOX and PTOX transcript changes during cell reprogramming and growth performance. At plant level, a putative early response of AOX to chilling was also evaluated. In fact, both DcAOXl and DcAOXZa were early responsive and showed similar patterns under stress conditions (in vitro inoculation and chilling). A role for DcPTOX during earliest events of cell reprogramming was also suggested. Next, the expression profiles of AOX gene family members during carrot tap root development were investigated. DcAOXZa was identified as the most responsive gene to root development. In order to evaluate if DcPTOX is associated with carrot tap root growth performance, DcPTOX transcript levels were measured in the central root meristem. To further understand whether DcPTOX is associated with secondary growth and/or carotenoids accumulation, DcPTOX expression was also studied in deveIOping carrot tap roots in cultivars with different carotenoids contents. The results indicated that DcPTOX associates to both carotenoid biosynthesis and secondary growth during storage root development. To obtain further insights into the involvement of PTOX on adaptive growth, the early effects of temperature decrease were explored in the root meristem, where a short—term early response in DcPTOX was found, probably associated with adaptive growth. Furthermore, a selection of the most suitable reference genes for accurate RT—qPCR analysis in several carrot experimental systems was performed and discussed. The present research provides the necessary toolbox for continuing studies in carrot AOX and PTOX genes as promising resources for FM candidates in order to assist breeding on yield—determining adaptive growth performance.

Physicochemical characterization of a new family of small alkyl phosphonium imide ionic liquids

Despite their promising properties, phosphonium based ionic liquids have attracted little attention as compared to their nitrogen-based cation counterparts. This study focuses on the properties of a family of small phosphonium imide ionic liquids, as well as the effect of lithium salt addition to these. The 6 ionic liquids were either alkyl, cyclic or nitrile functionalised phoshonium cations with bis(trifluoromethanesulfonyl)imide, NTf2, or bis(fluorosulfonyl)imide (FSI) as anion. Amongst the properties investigated were ionic conductivity, viscosity, thermal behaviour, electrochemical stability and the reversibility of electrochemical lithium cycling. All ionic liquids showed very promising properties e.g. having low transition temperatures, high electrochemical stabilities, low viscosities and high conductivities. Particularly the trimethyl phosphonium ionic liquids showed some of the highest conductivities reported amongst phosphonium ionic liquids generally. The combination of electrochemical stability, high conductivity and reversible lithium cycling makes them promising systems for energy storage devices such as lithium batteries.

Persistence and subtype stability of ADHD among substance use disorder treatment seekers

OBJECTIVE: To examine ADHD symptom persistence and subtype stability among substance use disorder (SUD) treatment seekers. METHOD: In all, 1,276 adult SUD treatment seekers were assessed for childhood and adult ADHD using Conners' Adult ADHD Diagnostic Interview for Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV; CAADID). A total of 290 (22.7%) participants met CAADID criteria for childhood ADHD and comprise the current study sample. RESULTS: Childhood ADHD persisted into adulthood in 72.8% (n = 211) of cases. ADHD persistence was significantly associated with a family history of ADHD, and the presence of conduct disorder and antisocial personality disorder. The combined subtype was the most stable into adulthood (78.6%) and this stability was significantly associated with conduct disorder and past treatment of ADHD. CONCLUSION: ADHD is highly prevalent and persistent among SUD treatment seekers and is associated with the more severe phenotype that is also less likely to remit. Routine screening and follow-up assessment for ADHD is indicated to enhance treatment management and outcomes.

Financial Characteristics of Family Businesses and Financial Aspects of Succession

Family businesses are special in many respects. By examining their financial characteristics one can come to unique conclusions/results. This paper explores the general characteristics of the financial behaviour of family businesses, presents the main findings of the INSIST project’s company case studies concerning financing issues and strategies, and intends to identify the financial characteristics of company succession. The whole existence of family businesses is characterized by a duality of the family and business dimensions and this remains the case in their financial affairs. The financial decisions in family businesses (especially SMEs) are affected by aspects involving a duality of goals rather than exclusively profitability, the simultaneous presence of family and business financial needs, and the preferential handling of family needs at the expense of business needs (although it has to be said that there is evidence of family investments being postponed for the sake of business, too. Family businesses, beyond their actual effectiveness, are guided by individual goals like securing living standards, ensuring workplaces for family members, stability of operation, preservation of the company’s good reputation, and keeping the company’s size at a level that the immediate family can control and manage. The INSIST project’s company case studies revealed some interesting traits of family business finances like the importance of financial support from the founder’s family during the establishment of the company, the use of bootstrapping techniques, the financial characteristics of succession, and the role of family members in financial management.

Managing Succession and Knowledge Transfer in Family Businesses: Lessons from a Comparative Research

The most natural mode of family firm succession is the intergenerational ownership transfer. Statistical evidence, however, suggests that in most cases the succession process fails. There can be several reasons as a lot of personal, emotional and structural factors can act as an inhibitor to succession. The effectiveness of the implementation of any succession strategy is strongly dependent on the efficiency of intergenerational knowledge transfer, which is related to the parties’ absorptive capacity and willingness to learn. The paper is based on the experiences learned from the INSIST project. In the framework of the project different aspects of family business succession have been investigated in three participating countries (Hungary, Poland and the United Kingdom). The aim of the paper is to identify the patterns of management, succession, knowledge transfer and learning in family businesses. Issues will be examined in detail such as the succession strategies of companies investigated and the efforts family businesses and their managers make in order to harmonize family goals (such as emotional stability, harmony, and reputation) with business- related objectives (e.g. survival, growth or profitability).

The actinin family of actin crosslinking proteins: natural functions and potential applications in synthetic biology

Actinin and spectrin proteins are members of the Spectrin Family of Actin Crosslinking Proteins. The importance of these proteins in the cytoskeleton is demonstrated by the fact that they are common targets for disease causing mutations. In their most prominent roles, actinin and spectrin are responsible for stabilising and maintaining the muscle architecture during contraction, and providing shape and elasticity to the red blood cell in circulation, respectively. To carry out such roles, actinin and spectrin must possess important mechanical and physical properties. These attributes are desirable when choosing a building block for protein-based nanoconstruction. In this study, I assess the contribution of several disease-associated mutations in the actinin-1 actin binding domain that have recently been linked to a rare platelet disorder, congenital macrothrombocytopenia. I investigate the suitability of both actinin and spectrin proteins as potential building blocks for nanoscale structures, and I evaluate a fusion-based assembly strategy to bring about self-assembly of protein nanostructures. I report that the actinin-1 mutant proteins display increased actin binding compared to WT actinin-1 proteins. I find that both actinin and spectrin proteins exhibit enormous potential as nano-building blocks in terms of their stability and ability to self-assemble, and I successfully design and create homodimeric and heterodimeric bivalent building blocks using the fusion-based assembly strategy. Overall, this study has gathered helpful information that will contribute to furthering the advancement of actinin and spectrin knowledge in terms of their natural functions, and potential unnatural functions in protein nanotechnology.

The role of cancer related inflammation, Src family kinases and matrix metalloproteinase 9 in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the UK with 41,000 new cases diagnosed in 2011. Despite undergoing potentially curative resection, a significant amount of patients develop recurrence. Biomarkers that aid prognostication or identify patients who are suitable for adjuvant treatments are needed. The TNM staging system does a reasonably good job at offering prognostic information to the treating clinician, but it could be better and identifying methods of improving its accuracy are needed. Tumour progression is based on a complex relationship between tumour behaviour and the hosts’ inflammatory responses. Sustained tumour cell proliferation, evading growth suppressors, resisting apoptosis, replicative immortality, sustained angiogenesis, invasion & metastasis, avoiding immune destruction, deregulated cellular energetics, tumour promoting inflammation and genomic instability & mutation have been identified as hallmarks. These hallmarks are malignant behaviors are what makes the cell cancerous and the more extreme the behaviour the more aggressive the cancer the more likely the risk of a poor outcome. There are two primary genomic instability pathways: Microsatellite Instability (MSI) and Chromosomal Instability (CI) also referred to as Microsatellite Stability (MSS). Tumours arising by these pathways have a predilection for specific anatomical, histological and molecular biological features. It is possible that aberrant molecular expression of genes/proteins that promote malignant behaviors may also act as prognostic and predictive biomarkers, which may offer superior prognostic information to classical prognostic features. Cancer related inflammation has been described as a 7th hallmark of cancer. Despite the systemic inflammatory response (SIR) being associated with more aggressive malignant disease, infiltration by immune cells, particularly CD8+ lymphocytes, at the advancing edge of the tumour have been associated with improved outcome and tumour MSI. It remains unknown if the SIR is associated with tumour MSI and this requires further study. The mechanisms by which colorectal cancer cells locally invade through the bowel remain uncertain, but connective tissue degradation by matrix metalloproteinases (MMPs) such as MMP-9 have been implicated. MMP-9 has been found in the cancer cells, stromal cells and patient circulation. Although tumoural MMP-9 has been associated with poor survival, reports are conflicting and contain relatively small sample sizes. Furthermore, the influence of high serum MMP-9 on survival remains unknown. Src family kinases (SFKs) have been implicated in many adverse cancer cell behaviors. SFKs comprise 9 family members BLK, C-SRC, FGR, FYN, HCK, LCK, LYN, YES, YRK. C-SRC has been the most investigated of all SFKs, but the role of other SFKs in cellular behaviors and their prognostic value remains largely unknown. The development of Src inhibitors, such as Dasatinib, has identified SFKs as a potential therapeutic target for patients at higher risk of poor survival. Unfortunately, clinical trials so far have not been promising but this may reflect inadequate patient selection and SFKs may act as useful prognostic and predictive biomarkers. In chapter 3, the association between cancer related inflammation, tumour MSI, clinicopathological factors and survival was tested in two independent cohorts. A training cohort consisting of n=182 patients and a validation cohort of n=677 patients. MSI tumours were associated with a raised CRP (p=0.003). Hypoalbuminaemia was independently associated with poor overall survival in TNM stage II cancer (HR 3.04 (95% CI 1.44 – 6.43);p=0.004), poor recurrence free survival in TNM stage III cancer (HR 1.86 (95% 1.03 – 3.36);p=0.040) and poor overall survival in CI colorectal cancer (HR 1.49 (95% CI 1.06 – 2.10);p=0.022). Interestingly, MSI tumours were associated with poor overall survival in TNM stage III cancer (HR 2.20 (95% CI 1.10 – 4.37);p=0.025). In chapter 4, the role of MMP-9 in colorectal cancer progression and survival was examined. MMP-9 in the tissue was assessed using IHC and serum expression quantified using ELISA. Serum MMP-9 was associated with cancer cell expression (Spearman’s Correlation Coefficient (SCC) 0.393, p<0.001)) and stromal expression (SCC 0.319, p=0.002). Serum MMP-9 was associated with poor recurrence-free (HR 3.37 (95% CI 1.20 – 9.48);p=0.021) and overall survival (HR 3.16 (95% CI 1.22 – 8.15);p=0.018), but tumour MMP-9 was not survival or MSI status. In chapter 5, the role of SFK expression and activation in colorectal cancer progression and survival was studied. On PCR analysis, although LYN, C-SRC and YES were the most highly expressed, FGR and HCK had higher expression profiles as tumours progressed. Using IHC, raised cytoplasmic FAK (tyr 861) was independently associated with poor recurrence free survival in all cancers (HR 1.48 (95% CI 1.02 – 2.16);p=0.040) and CI cancers (HR 1.50 (95% CI 1.02 – 2.21);p=0.040). However, raised cytoplasmic HCK (HR 2.04 (95% CI 1.11 – 3.76);p=0.022) was independently associated with poor recurrence-free survival in TNM stage II cancers. T84 and HT29 cell lines were used to examine the cellular effects of Dasatinib. Cell viability was assessed using WST-1 assay and apoptosis assessed using an ELISA cell death detection assay. Dasatinib increased T84 tumour cell apoptosis in a dose dependent manner and resulted in reduced expression of nuclear (p=0.008) and cytoplasmic (p=0.016) FAK (tyr 861) expression and increased nuclear FGR expression (p=0.004). The results of this thesis confirm that colorectal cancer is a complex disease that represents several subtypes of cancer based on molecular biological behaviors. This thesis concentrated on features of the disease related to inflammation in terms of genetic and molecular characterisation. MSI cancers are closely associated with systemic inflammation but despite this observation, they retain their relatively improved survival. MMP-9 is a feature of tissue remodeling during inflammation and is also associated with degradation of connective tissue, advanced T-stage and poor outcome when measured in the serum. The lack of stromal quantification due to TMA use rather than full sections makes the value of tumoural MMP-9 immunoreactivity in the prognostication and its association with MSI unknown and requires further study. Finally, SFK activation was also associated with SIR, however, only cytoplasmic HCK was independently associated with poor survival in patients with TNM stage II disease, the group of patients where identifying a novel biomarker is most needed. There is still some way to go before these biomarkers are translated into clinical practice and future work needs to focus on obtaining a reliable and robust scientific technique with validation in an adequately powered independent cohort.