Designing crossing and selection strategies to combine diagnostic markers and quantitative traits

Tese de doutoramento em Biociências, ramo de especialização em Toxicologia, apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraDoxorubicin (DOX) is one of the most potent antineoplastic drugs. Although possessing a superior anti-­‐‑cancer activity, a broader clinical use of DOX is limited by a dose-­‐‑dependent, constant and cumulative cardiomyopathy involving deterioration of mitochondrial function. Although acute effects of DOX treatment often disappear when treatment finishes, chronic effects often result in a persistent cardiotoxicity, including a progressive deterioration of mitochondrial metabolism, the development of cardiomyopathy and ultimately congestive heart failure. Important in the context of DOX-­‐‑induced cardiotoxicity, mitochondrial disruption has been observed in different models. This alteration of mitochondrial function is often sub-­‐‑clinical and is only manifested as cardiomyopathy when other factors are combined, including age or different types of cardiovascular stress. Also, metabolic alterations in the cardiac cell occur, which contribute to the ability of the heart to withstand increased workloads. Although mitochondrial disruption is an early and sensitive marker of DOX cardiotoxicity, how metabolic stress contributes to the development of cardiomyopathy remains to be determined. Because of this gap in knowledge, the objective of this work was to use of model of metabolic inhibition in perfused hearts from saline (SAL) and DOX-­‐‑ treated rats in order to identify metabolic alterations caused by an acute and sub-­‐‑ chronic treatment. Our assumption for this strategy is that a lower susceptibility to a determined inhibitor during perfusion, would be a sign of a more robust (i.e. more capacity) of the targeted pathway(s). XV For the acute treatment protocol, sixteen week-­‐‑old male Wistar rats were i.p. injected with 20mg/Kg DOX or 1mg/Kg 0.9%NaCl and sacrificed 24 hours later. For sub-­‐‑ chronic protocol, eight weeks-­‐‑old male Wistar rats received seven weekly s.c. injections with DOX(2mg/Kg) or equivalent SAL solution and sacrificed one week after the last injection. Following the protocol treatments, animals were sacrificed and hearts were removed and perfused using a Langendorff apparatus with distinct cardiac substrates (glucose, galactose plus glutamine -­‐‑ GG or octanoate plus malate – OM). Glycolytic (iodoacetate) and oxidative phosphorylation (rotenone-­‐‑ Rot or cyanide-­‐‑ KCN) inhibitors were separately added to the different metabolic perfusates, aiming to detect undercover mitochondrial defects in the DOX-­‐‑treated group. In non-­‐‑perfused hearts, or hearts perfused in the absence (time controls, TC) or presence of inhibitors, selected metabolic and mitochondrial proteins were semi-­‐‑ quantified by Western blotting, and mRNA levels were quantified by RT-­‐‑PCR. In the acute DOX treatment model, hearts perfused with glucose as substrate suffered a decline in the number of heart beat and rate pressure product (RPP) when iodoacetate was added, contrarily to Rot or KCN which had no effect. With GG, inhibitor titration decreased the heart rate, despite that the decrease in the RPP was more evident in SAL vs. DOX group with iodoacetate and KCN. Perfusion with OM resulted in decreased heart rate an RPP in the presence of the inhibitors, showing equal response between treatments. When glycolytic and mitochondrial proteins were semi-­‐‑quantified by Western blotting, alterations in proteins involved in mitochondrial biogenesis and autophagy were observed in DOX hearts perfused with inhibitors. The data from the acute protocol study, appears to suggest that hearts from DOX-­‐‑treated animals have improved function in the presence of XVI metabolic inhibitors, thus indicating that DOX triggers adaptations that allow the hearts to be less susceptible to mitochondrial and glycolytic inhibition. In the sub-­‐‑chronic model and using glucose as substrate, the DOX-­‐‑treated group showed again a better tolerability to inhibitors than SAL. With GG, titration with iodoacetate caused a decrease in heart beat and on RPP in DOX group, when rot or KCN was added the number of heart beat and RPP remains identical between the two groups. Glycolytic and mitochondrial proteins semi-­‐‑quantification suggested an impairment of autophagy in DOX perfused hearts perfused, more evident during GG perfusion. The presence of inhibitors in the perfusion also generally decreased the total amount of proteins detected by Western Blotting, although glycolytic proteins were increased when hearts were perfused with glucose, contrarily to GG perfusion. The results suggest that sub-­‐‑chronic DOX-­‐‑treated rats suffered a metabolic remodeling which is based on stronger glycolytic fluxes to maintain contractility, although no overt mitochondrial defect was uncovered. A surprising result is that regardless of the perfusion buffer used, no striking differences between SAL and DOX hearts in terms of hemodynamic parameters were found. The present work suggests that metabolic remodeling during DOX acute and sub-­‐‑ chronic treatment maintains cardiac function in the treated animals. This remodeling is apparently based in a stronger contribution of glycolysis to overall metabolism. Data from protein amount analyzed suggest that DOX treatment in both models affect important regulators of autophagy, mitochondrial biogenesis as well as the adenine nucleotide translocator. The results also suggest that a longer treatment XVII protocol or resting period should also be tested in order to uncover more profound differences.A doxorrubicina (DOX) é um dos fármacos antineoplásicos mais potentes. Apesar de possuir uma actividade anti-­‐‑cancro superior, uma mais ampla utilização clínica da DOX é limitada por uma dose-­‐‑dependente, constante e cumulativa cardiomiopatia que envolve a deterioração da função mitocondrial. Embora os efeitos agudos do tratamento DOX normalmente desaparece quando se conclui o tratamento, os efeitos crónicos resultam muitas vezes numa cardiotoxicidade persistente, incluindo a deterioração progressiva do metabolismo mitocondrial, o desenvolvimento de cardiomiopatia e por fim insuficiência cardíaca congestiva. Importante no contexto da cardiotoxicidade induzida pela DOX, perturbação mitocondrial foi observada em diferentes modelos. Esta alteração da função mitocondrial é muitas vezes sub-­‐‑clínica e só se manifesta como cardiomiopatia quando outros factores são combinados, incluindo a idade ou diferentes tipos de estresse cardiovascular. Além disso, ocorrem alterações metabólicas na célula cardíaca, o que contribui para a capacidade do coração resistir a maior demanda. Embora a perturbação mitocondrial seja um marcador precoce e sensível de DOX cardiotoxicidade, como o stress metabólico contribui para o desenvolvimento de cardiomiopatia permanece por determinar. Devido a essa lacuna no conhecimento, o objetivo deste trabalho foi a utilização de um modelo de inibição metabólica em corações perfundidos de ratos tratados com uma solução salina (SAL) e ratos tratados com DOX, a fim de identificar alterações metabólicas causadas por um tratamento agudo e sub-­‐‑crônico. XIX O nosso pressuposto para esta estratégia é que uma menor susceptibilidade a um determinado inibidor durante a perfusão, seria um sinal de uma forma mais robusta (ou seja, mais de capacidade) da via-­‐‑alvo (s). Para o protocolo de tratamento agudo, ratos machos Wistar de 16 semanas de idade foram injectados i.p. com 20 mg / kg de DOX ou de 1 mg / kg a 0,9% de NaCl e sacrificados 24 horas mais tarde. Para o protocolo sub-­‐‑crônico, ratos machos Wistar de oito semanas de idade, receberam sete injecções s.c. semanais com DOX (2 mg / kg) ou uma equivalente da solução SAL sendo sacrificados uma semana após a última injecção. Após o protocolo de tratamentos, os animais foram sacrificados e os corações foram perfundidos com um aparelho de Langendorff com substratos cardíacos distintos (glucose, galactose mais glutamina -­‐‑ GG ou octanoato mais malato -­‐‑ OM). Inibidores glicolíticos (iodoacetato) e inibidores da fosforilação oxidativa (Rot-­‐‑ rotenona ou KCN-­‐‑ cianeto) foram adicionados separadamente nos diferentes substratos metabólicos, com o objetivo de detectar defeitos mitocondriais no grupo tratado com DOX. Em corações não perfundidos, ou corações perfundidos na ausência (controlos de tempo, TC) ou na presença de inibidores, algumas proteínas metabólicas e proteínas mitocondriais foram semi-­‐‑quantificadas por Western blotting, e os níveis de mRNA foram quantificados por RT-­‐‑PCR. No modelo de tratamento agudo DOX, corações perfundidos com glucose como substrato sofreram um declínio no número de batimentos cardíacos e produto da taxa de pressão (RPP), quando iodoacetato foi adicionado, ao contrário da Rot ou KCN, que não teve nenhum efeito. Com GG, a titulação com o inibidor diminuiu a frequência cardíaca, apesar de que a diminuição da RPP foi mais evidente no grupo SAL vs. DOX com iodoacetato e KCN. Perfusão com OM resultou em diminuição da XX frequência cardíaca e do RPP na presença dos inibidores, mostrando uma resposta igual entre os tratamentos. Quando proteínas glicolíticas e mitocondriais foram semi-­‐‑ quantificadas por Western blotting, alterações de proteínas envolvidas na biogênese mitocondrial e autofagia foram observados em corações DOX perfundidos com inibidores. Os dados do protocolo de estudo agudo, parecem sugerir que os corações provenientes de animais tratados com DOX na presença de inibidores, têm a função metabólica melhorada, indicando assim que a DOX desencadeia adaptações que permitem que os corações sejam menos susceptíveis à inibição mitocondrial e glicolítica. No modelo sub-­‐‑crónica e utilizando glucose como substrato, o grupo tratado com DOX mostrou novamente um melhor tolerabilidade para inibidores que SAL. Com GG, a titulação com iodoacetato causou uma diminuição no batimento cardíaco e no RPP em grupo DOX, quando rot ou KCN foi adicionado o número de batimentos cardíacos e RPP permaneceram idênticos entre os dois grupos. A semi-­‐‑quantificação de proteínas glicolíticas e mitocondriais sugerem uma deficiência da autofagia em corações DOX perfundidos, mais evidente durante a perfusão com GG. A presença de inibidores da perfusão geralmente também reduziu a quantidade total de proteínas detectadas através de Western Blot, embora proteínas glicolíticas aumentaram quando os corações foram perfundidos com glucose, ao contrário da perfusão com GG. Os resultados sugerem que ratos tratados sub-­‐‑cronicamente com DOX sofreram uma remodelação metabólica, que é baseado em fluxos glicolíticos mais fortes para manter a contratilidade, embora nenhum defeito mitocondrial ostensivo foi descoberto. XXI Um resultado surpreendente é que, independentemente do tampão de perfusão utilizado, não foram encontradas diferenças marcantes entre SAL e DOX corações em termos de parâmetros hemodinâmicos. O presente trabalho sugere que o remodelação metabólica durante o tratamento agudo e sub-­‐‑crônico com DOX, mantém a função cardíaca nos animais tratados. Esta remodelação é, aparentemente, baseado numa contribuição mais forte da glicólise ao metabolismo geral. Os resultados de quantidade de proteína analisados sugerem que o tratamento com DOX em ambos os modelos afectam importantes reguladores da autofagia e biogénese mitocondrial, bem como o translocador de nucleótidos de adenina. Os resultados também sugerem que um protocolo de tratamento mais longo ou com um período de repouso também devem ser testados a fim de descobrir diferenças mais profundas

Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in biliary brush cytology

Background: Biliary brush cytology is the standard method of evaluating biliary strictures, but is insensitive at detecting malignancy. In pancreaticobiliary cancer minichromosome maintenance replication proteins (MCM 2–7) are dysregulated in the biliary epithelium and MCM5 levels are elevated in bile samples. This study aimed to validate an immunocolorimetric ELISA assay for MCM5 as a pancreaticobiliary cancer biomarker in biliary brush samples. methods: Biliary brush specimens were collected prospectively at ERCP from patients with a biliary stricture. Collected samples were frozen at −80 °C. The supernatant was washed and lysed cells incubated with HRP-labelled anti-MCM5 mouse monoclonal antibody. Test positivity was determined by optical density absorbance. Patients underwent biliary brush cytology or additional investigations as per clinical routine. results: Ninety-seven patients were included in the study; 50 had malignant strictures. Median age was 65 years (range 21–94) and 51 were male. Compared with final diagnosis the MCM5 assay had a sensitivity for malignancy of 65.4% compared with 25.0% for cytology. In the 72 patients with paired MCM5 assay and biliary brush cytology, MCM5 demonstrated an improved sensitivity (55.6% vs 25.0%; P=0.0002) for the detection of malignancy. conclusions: Minichromosome maintenance replication protein5 is a more sensitive indicator of pancreaticobiliary malignancy than standard biliary brush cytology

The relationship between reductions in knee loading and immediate pain response whilst wearing lateral wedged insoles in knee osteoarthritis

Studies of lateral wedge insoles (LWIs) in medial knee osteoarthritis (OA) have shown reductions in the average external knee adduction moment (EKAM) but no lessening of knee pain. Some treated patients actually experience increases in the EKAM which could explain the overall absence of pain response. We examined whether, in patients with painful medial OA, reductions in the EKAM were associated with lessening of knee pain. Each patient underwent gait analysis whilst walking in a control shoe and two LWI's. We evaluated the relationship between change in EKAM and change in knee pain using Spearman Rank Correlation coefficients and tested whether dichotomizing patients into biomechanical responders (decreased EKAM) and non-responders (increased EKAM) would identify those with reductions in knee pain. In 70 patients studied, the EKAM was reduced in both LWIs versus control shoe (−5.21% and −6.29% for typical and supported wedges, respectively). The change in EKAM using LWIs was not significantly associated with the direction of knee pain change. Further, 54% were biomechanical responders, but these persons did not have more knee pain reduction than non-responders. Whilst LWIs reduce EKAM, there is no clearcut relationship between change in medial load when wearing LWIs and corresponding change in knee pain

An inductive force sensor for in-shoe plantar normal and shear load measurement

Diabetic foot ulcers (DFUs) are a severe global public health issue. Plantar normal and shear load are believed to play an important role in the development of foot ulcers and could be a valuable indicator to improve assessment of DFUs. However, despite their promise, plantar load measurements currently have limited clinical application, primarily due to the lack of reliable measurement techniques particularly for shear load measurements. In this paper we report on the design and evaluation of a novel tri-axis force sensor to measure both normal and shear load on the foot’s plantar surface simultaneously. The sensor consists of a group of inductive sensing coils above which a conductive target is placed on a hyperelastic elastomer. Movement of the target under load affects the coil inductances which are measured and digitized by an embedded system. Using a computational finite element model, we investigated the influence of sensing coil form and configuration on sensor performance. A sensor configured with four-square coils and maximal turns provided the best performance for plantar load measurements. A prototype was fabricated and calibrated using a neural network to map the non-linear relationship between the sensor output and the applied tri-axis load. Experimental evaluation indicates that the tri-axis sensor can effectively detect shear load of �16 N and normal load up to 105 N (RMS errors: 1.05 N and 1.73 N respectively) with a high performance. Overall, this sensor provides a promising basis for plantar normal and shear load measurement which are crucial for improved assessment of DFU

A Review of Wearable Sensor Systems to Monitor Plantar Loading in the Assessment of Diabetic Foot Ulcers

Diabetes is highly prevalent throughout the world and imposes a high economic cost on countries at all income levels. Foot ulceration is one devastating consequence of diabetes, which can lead to amputation and mortality. Clinical assessment of diabetic foot ulcer (DFU) is currently subjective and limited, impeding effective diagnosis, treatment and prevention. Studies have shown that pressure and shear stress at the plantar surface of the foot plays an important role in the development of DFUs. Quantification of these could provide an improved means of assessment of the risk of developing DFUs. However, commercially-available sensing technology can only measure plantar pressures, neglecting shear stresses and thus limiting their clinical utility. Research into new sensor systems which can measure both plantar pressure and shear stresses are thus critical. Our aim in this paper is to provide the reader with an overview of recent advances in plantar pressure and stress sensing and offer insights into future needs in this critical area of healthcare. Firstly, we use current clinical understanding as the basis to define requirements for wearable sensor systems capable of assessing DFU. Secondly, we review the fundamental sensing technologies employed in this field and investigate the capabilities of the resultant wearable systems, including both commercial and research-grade equipment. Finally, we discuss research trends, ongoing challenges and future opportunities for improved sensing technologies to monitor plantar loading in the diabetic foot

Phylogenetic diversity analysis of shotgun metagenomic reads describes gut microbiome development and treatment effects in the post-weaned pig.

Intensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. Although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. Shotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 post-weaning piglets and 42 sows were analysed in a phylogenetic framework. Differences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. The discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors

Identification of potential serum peptide biomarkers of biliary tract cancer using MALDI MS profiling.

The aim of this discovery study was the identification of peptide serum biomarkers for detecting biliary tract cancer (BTC) using samples from healthy volunteers and benign cases of biliary disease as control groups. This work was based on the hypothesis that cancer-specific exopeptidase activities in serum can generate cancer-predictive peptide fragments from circulating proteins during coagulation

A large-scale metagenomic survey dataset of the post-weaning piglet gut lumen

BackgroundEarly weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows.ResultsHere we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning.ConclusionsThis study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host

Pancreatoscopy-Directed Electrohydraulic Lithotripsy for Pancreatic Ductal Stones in Painful Chronic Pancreatitis Using SpyGlass.

OBJECTIVE: Painful chronic pancreatitis is often associated with main duct obstruction due to stones. Approaches to management are challenging, including surgery, extracorporeal shock wave lithotripsy, or endoscopic approaches. Here, we report our experience of pancreatoscopy + electrohydraulic lithotripsy (EHL) for pancreatic duct (PD) stones using SpyGlass. METHODS: We retrospectively audited the use of SpyGlass (Legacy and DS) + EHL. Indication, procedural details, and clinical outcomes were assessed. RESULTS: A total of 118 SpyGlass + EHL procedures for stones were performed, of which 8 (7%) for pancreatic stones, in 6 patients (3 female; mean [standard deviation] age, 45 [7] years). All patients had painful chronic pancreatitis, with radiological evidence of a dilated PD, and main duct stone disease. Surgical options had been considered in all cases. Stone fragmentation and PD decompression were achieved in 83% (n = 5) without complications. Two patients required 2 EHL procedures to achieve clearance. In 1 patient with failed clearance, pancreatoscopy revealed a stone in the adjacent parenchyma and not in PD. All patients with successful EHL had pain relief/marked improvement at clinical review (mean [standard deviation] follow-up, 2.7 [1.1] years). CONCLUSIONS: Pancreatoscopy + EHL may have a valuable role in treating obstructing PD stones, possibly avoiding the need for surgery in some patients