A Prospective, Blinded, Open-Label Clinical Trial to Assess the Ability of Fluorescent Light Energy to Enhance Wound Healing after Mastectomy in Female Dogs.

Mammary gland tumors represent the most frequently diagnosed malignant neoplasm in intact female dogs, and surgical removal represents the current gold standard treatment. To promote wound healing and prevent possible bacterial contamination, perioperative antimicrobials are commonly used in clinical practice, even though there are no publications establishing guidelines for the use of such drugs in canine mastectomy. The aim of the present study was to evaluate the ameliorative effect of fluorescent light energy on the quality of the healing process after mastectomy surgery in female dogs, in the absence of perioperative antimicrobial administration. Nine female dogs received a multiple-gland mastectomy due to gland tumors and received FLE application immediately after surgery and then five days after. The surgical incisions were evaluated by a blind investigator over time using the Modified Hollander Cosmesis and Modified Draize Wound Healing Score systems. Statistical analysis revealed a significant ameliorative effect of FLE in the control of step-off borders, contour irregularities, and excessive distortion. In addition, erythema, edema, and serous discharge were lower for those wounds managed with FLE. These results underscore the advantageous impact of FLE on the healing of post-mastectomy wounds in female dogs, offering the dual benefits of reducing potential infection risks and lessening the home care burden for pet owners.

Synovial Fluid Metabolome Can Differentiate between Healthy Joints and Joints Affected by Osteoarthritis in Horses.

Osteoarthritis (OA) is a common cause of lameness in sport horses with a significant economic impact. The prevention of OA is crucial since no effective treatment is available. This study aimed to apply untargeted metabolomic analysis to investigate the differences in synovial fluid (SF) composition between healthy and OA-affected joints in horses. SF collected from healthy (n.8) and OA (n.11) horses was analyzed using H-NMR analysis. Metabolomic analysis allowed 55 different metabolites to be identified and quantified in SF samples. Nineteen metabolites were found to be differently concentrated in OA compared to control horses. Synovial fluids from the OC group were found to be higher in 1,3-dihydroxyacetone but lower in tryptophan, phenylalanine, tyrosine, uridine, creatinine, creatine, glycine, choline, asparagine, glutamine, arginine, 3-hydroxybutyrate, valine, 2-hydroxyisovalerate, α-ketoisovaleric acid, 3-methyl-2-oxovalerate, 3-hydroxyisobutyrate, isoleucine, and methionine compared to the controls. A variety of SF metabolites significantly changed following joint disease, demonstrating the complex mechanism underlying osteoarthritis in horses and highlighting the value of applying the metabolomic approach in clinical research.

Fecal Proteome Profile in Dogs Suffering from Different Hepatobiliary Disorders and Comparison with Controls.

In the present study, the fecal proteomes of clinically healthy dogs (HD = n. 10), of dogs showing clinical, ultrasonographic, and/or laboratory evidence of different hepatobiliary dysfunction (DHD = n. 10), and of dogs suffering from chronic hepatitis (CHD = n. 10) were investigated with an Ultimate 3000 nanoUPLC system, coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Fifty-two different proteins of canine origin were identified qualitatively in the three study groups, and quantitative differences were found in 55 proteins when comparing groups. Quantitatively, a total of 41 and 36 proteins were found differentially abundant in the DHD and CHD groups compared to the control HD, and 38 proteins resulted dysregulated in the CHD group as compared to the DHD group. Among the various proteins, differently abundant fecal fibronectin and haptoglobin were more present in the feces of healthy and DHD dogs than in chronic ones, leading us to hypothesize its possible diagnostic/monitoring role in canine chronic hepatitis. On the other hand, the trefoil factor 2 was increased in DHD dogs. Our results show that the analysis of the fecal proteome is a very promising field of study, and in the case of dogs suffering from different hepatobiliary disorders, it was able to highlight both qualitative and quantitative differences among the three groups included. Results need to be confirmed with western blotting and in further studies.

Exploring fluorescent light energy as management option for canine superficial bacterial folliculitis.

Superficial bacterial folliculitis (SBF) represents a common dermatological diagnosis in dogs that can be successfully managed with either topical and/or systemic treatments. In the present study we evaluated the efficacy of a fluorescent light energy (FLE) device as sole management for SBF. The same FLE device has been shown, as adjunct therapy to systemic antibiotic or alone, to effectively control clinical manifestation of interdigital furunculosis. Twenty dogs were randomized to receive FLE once (six dogs) or twice (six dogs) weekly in comparison with oral anti-biotic (eight dogs) until complete healing. FLE regimen was able to significantly reduce the time needed to clinical resolution for oral antibiotic, supporting owners' compliance and welfare of dogs.

Donkey Colostrum and Milk: How Dietary Probiotics Can Affect Metabolomic Profile, Alkaline Sphingomyelinase and Alkaline Phosphatase Activity.

Positive results on animal health, feed efficiency, and milk's nutritional content have been obtained after oral administration of probiotics. The aim of the present study was therefore to evaluate the effect of dietary supplementation with high numbers of multispecies probiotic formulations on the milk metabolomic profiles of alkaline sphingomyelinase (alk-SMase) and alkaline phosphatase (ALP) in donkeys. Twenty animals were randomly allocated to receive either a normal diet (group B) or a supplemented diet (group A). Colostrum and milk samples were obtained within 48 h, at 15 days (supplementation start), and at 45 days after parturition. Different metabolomic profiles were observed between colostrum and milk, as were the concentrations of 12 metabolites that changed following 30 days of probiotic supplementation. Alk-SMase activity was found to be higher in donkey colostrum (vs. milk at 15 days); this enzyme, together with ALP, increased in milk after 30 days of probiotic supplementation. The results of the present study provide new insight into the complex changes in donkey colostrum and milk composition in the first 45 days of lactation and how the milk metabolome can be modulated by probiotic supplementation.

Sensitivity and specificity of in vivo COVID-19 screening by detection dogs: Results of the C19-Screendog multicenter study.

Trained dogs can recognize the volatile organic compounds contained in biological samples of patients with COVID-19 infection. We assessed the sensitivity and specificity of in vivo SARS-CoV-2 screening by trained dogs. We recruited five dog-handler dyads. In the operant conditioning phase, the dogs were taught to distinguish between positive and negative sweat samples collected from volunteers' underarms in polymeric tubes. The conditioning was validated by tests involving 16 positive and 48 negative samples held or worn in such a way that the samples were invisible to the dog and handler. In the screening phase the dogs were led by their handlers to a drive-through facility for in vivo screening of volunteers who had just received a nasopharyngeal swab from nursing staff. Each volunteer who had already swabbed was subsequently tested by two dogs, whose responses were recorded as positive, negative, or inconclusive. The dogs' behavior was constantly monitored for attentiveness and wellbeing. All the dogs passed the conditioning phase, their responses showing a sensitivity of 83-100% and a specificity of 94-100%. The in vivo screening phase involved 1251 subjects, of whom 205 had a COVID-19 positive swab and two dogs per each subject to be screened. Screening sensitivity and specificity were respectively 91.6-97.6% and 96.3-100% when only one dog was involved, whereas combined screening by two dogs provided a higher sensitivity. Dog wellbeing was also analyzed: monitoring of stress and fatigue suggested that the screening activity did not adversely impact the dogs' wellbeing. This work, by screening a large number of subjects, strengthen recent findings that trained dogs can discriminate between COVID-19 infected and healthy human subjects and introduce two novel research aspects: i) assessement of signs of fatigue and stress in dogs during training and testing, and ii) combining screening by two dogs to improve detection sensitivity and specificity. Using some precautions to reduce the risk of infection and spillover, in vivo COVID-19 screening by a dog-handler dyad can be suitable to quickly screen large numbers of people: it is rapid, non-invasive and economical, since it does not involve actual sampling, lab resources or waste management, and is suitable to screen large numbers of people.

Fecal Protein Profile in Eight Dogs Suffering from Acute Uncomplicated Diarrhea before and after Treatment.

Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present findings.

p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats.

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.

Fluorescent Light Energy in the Management of Multi Drug Resistant Canine Pyoderma: A Prospective Exploratory Study.

The increase in prevalence of staphylococcal antimicrobial resistance has been also associated with pyoderma in dogs, and prolonged antibiotic treatment, as often needed in severe cases of pyoderma, has been related to influencing possible development of multidrug resistance (MDR). Fluorescent light energy (FLE) has been indicated to improve pyoderma lesions as adjunct therapy to systemic antibiotics. In the present study, we evaluated the effect of FLE on clinical signs of MDR canine deep pyoderma (CDP) and interdigital furunculosis (CIF) when administered as solely management. Sixteen client-owned dogs affected by CIF (five dogs) and CDP (eleven dogs) were scored using a dedicated scoring system and received a single FLE applications twice weekly, until clinical resolution was achieved. Mean time to achieve complete resolution was 5.20 ± 3.56 weeks (median 3 weeks) for CIF cases and 4.18 ± 1.47 weeks (median 4 weeks) for CDP ones. FLE shows promise as an aid to managing clinical signs while reducing reliance on antibiotics for MDR CDP and CIF. In this study, FLE was responsible for the decrease in lesion scores and resolution of MDR pyoderma infection without any adjunct therapy, having a potential useful role to play in antibiotic stewardship programs, efficiently promoting complete clinical resolution of MDR lesions while optimizing the use of antibiotics.

Dog-human translational genomics: state of the art and genomic resources.

Innovative models for medical research are strongly required nowadays. Convincing evidence supports dog as the most suitable spontaneous model for several human genetic diseases. Decades of studies on dog genome allowed the identification of hundreds of mutations causing genetic disorders, many of which are proposed as counterparts responsible for human diseases. Traditionally, the murine model is the most extensively used in human translational research. However, this species shows large physiological differences from humans, and it is kept under a controlled artificial environment. Conversely, canine genetic disorders often show pathophysiological and clinical features highly resembling the human counterpart. In addition, dogs share the same environment with humans; therefore, they are naturally exposed to many risk factors. Thus, different branches of translational medicine aim to study spontaneously occurring diseases in dogs to provide a more reliable model for human disorders. This review offers a comprehensive overview of the knowledge and resources available today for all the researchers involved in the field of dog-human translational medicine. Some of the main successful examples from dog-human translational genomics are reported, such as the canine association studies which helped to identify the causal mutation in the human counterpart. We also illustrated the ongoing projects aiming to create public canine big datasets. Finally, specific online databases are discussed along with several information resources that can speed up clinical translational research.

Evaluation of the Fecal Proteome in Healthy and Diseased Cheetahs (Acinonyx jubatus) Suffering from Gastrointestinal Disorders.

Fecal proteomics allows for the identification of proteins and peptides present in stools and is useful in finding possible new biomarkers for diagnosing and/or monitoring gastrointestinal (GI) disorders. In the present study, we investigated the fecal proteome in healthy and diseased cheetahs (Acinonyx jubatus). Captive individuals of this species frequently show gastrointestinal disorders characterized by recurrent episodes of diarrhea, rare episodes of vomiting and weight loss, associated with Helicobacter spp. infection. Fecal proteomic evaluation has been performed by two-dimensional electrophoresis followed by liquid chromatography-tandem mass spectrometry. In healthy cheetahs, the results showed the presence of the following proteins: collagen alpha-1 (II) chain, transthyretin, IgG Fc-binding protein, titin, dystonin, isopentenyl-diphosphate Delta-isomerase 1, sodium/potassium-transporting ATPase subunit alpha-1 and protein disulfide-isomerase A6. The presence of albumin isoforms was found only in diseased cheetahs. The present paper reports the study of the fecal proteome in the cheetah, evidences some differences between healthy and diseased patients and confirms, once again, the potential of fecal proteomics for the study of the GI environment, with promising developments regarding the identification of new diagnostic/monitoring markers.

Fluorescence Biomodulation for Canine Interdigital Furunculosis: Updates for Once-Weekly Schedule.

Interdigital furunculosis is a common multifactorial, inflammatory disease of the canine interdigital skin in which lesions commonly become secondarily infected. Fluorescence biomodulation (FBM) administered twice weekly has shown to effectively control clinical manifestation as adjunct therapy to systemic antibiotic. Since twice weekly regimen could be unaffordable for some pet owners, the aim of this study was to evaluate the effect of once weekly application of FBM in combination with systemic antibiotic on clinical manifestations of canine interdigital pyoderma, comparing the results to those present in literature. Twelve dogs diagnosed with interdigital pyoderma received antibiotic plus once weekly FBM application. Dogs were scored until complete healing based on global lesion score and neutrophil engulfing bacterial score. The results obtained demonstrated that once weekly application of FBM exerts the same beneficial effect on interdigital furunculosis healing as per twice weekly, indicating that once weekly regimen is well tolerated and is yielding similar results to twice weekly applications.

Case Report: Oral Fecal Microbiota Transplantation in a Dog Suffering From Relapsing Chronic Diarrhea-Clinical Outcome and Follow-Up.

The present case report describes the effects of orally administered fecal microbiota transplantation (FMT) (frozen capsules) in a dog suffering from relapsing chronic diarrhea, needing a continuous low prednisolone dose to maintain the condition under acceptable control. Through FMT, we aimed at evaluating the possibility of improving the clinical score and/or reducing/suspending steroid administration. During a first period of strict monitoring (21 days), the canine inflammatory bowel disease activity index (CIBDAI) score passed from mild to clinically insignificant disease. Furthermore, two additional gastrointestinal signs that had been reported, bloating and episodes of painful defecation, rapidly improved (bloating) or even resolved (painful defecation). The patient was then followed for 18 months (to the authors' knowledge, the longest follow-up time ever reported in a dog), during which no serious relapses occurred and no increase in prednisolone dose was necessary. No adverse clinical effects were ever reported during monitoring. The present description provides a further experience increasing those already present in the veterinary literature, in which an agreement on how to use FMT has not yet been achieved although strongly needed and recommended.

Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone.

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions.

Clostridioides difficile toxin B alone and with pro-inflammatory cytokines induces apoptosis in enteric glial cells by activating three different signalling pathways mediated by caspases, calpains and cathepsin B.

Clostridioides difficile infection (CDI) causes nosocomial/antibiotic-associated gastrointestinal diseases with dramatically increasing global incidence and mortality rates. The main C. difficile virulence factors, toxins A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We demonstrated that TcdB induces caspase-dependent, mitochondria-independent enteric glial cell (EGC) apoptosis that is enhanced by the pro-inflammatory cytokines TNF-α and IFN-γ (CKs) by increasing caspase-3/7/9 and PARP activation. Because this cytotoxic synergism is important for CDI pathogenesis, we investigated the apoptotic pathways involved in TcdB- and TcdB + CK-induced apoptosis indepth. EGCs were pre-treated with the inhibitors BAF or Q-VD-OPh (pan-caspase), Z-DEVD-fmk (caspase-3/7), Z-IETD-fmk (caspase-8), PD150606 (calpains), and CA-074Me (cathepsin B) 1 h before TcdB exposure, while CKs were given 1.5 h after TcdB exposure, and assays were performed at 24 h. TcdB and TcdB + CKs induced apoptosis through three signalling pathways activated by calpains, caspases and cathepsins, which all are involved both in induction and execution apoptotic signalling under both conditions but to different degrees in TcdB and TcdB + CKs especially as regards to signal transduction mediated by these proteases towards downstream effects (apoptosis). Calpain activation by Ca2+ influx is the first pro-apoptotic event in TcdB- and TcdB + CK-induced EGC apoptosis and causes caspase-3, caspase-7 and PARP activation. PARP is also directly activated by calpains which are responsible of about 75% of apoptosis in TcdB and 62% in TcdB + CK which is both effector caspase-dependent and -independent. Initiator caspase-8 activation mediated by TcdB contributes to caspase-3/caspase-7 and PARP activation and is responsible of about 28% of apoptosis in both conditions. Caspase-3/caspase-7 activation is weakly responsible of apoptosis, indeed we found that it mediates 27% of apoptosis only in TcdB. Cathepsin B contributes to triggering pro-apoptotic signal and is responsible in both conditions of about 35% of apoptosis by a caspase-independent manner, and seems to regulate the caspase-3 and caspase-7 cleaved fragment levels, highlighting the complex interaction between these cysteine protease families activated during TcdB-induced apoptosis. Further a relevant difference between TcdB- and TcdB + CK-induced apoptosis is that TcdB-induced apoptosis increased slowly reaching at 72 h the value of 18.7%, while TcdB + CK-induced apoptosis increased strongly reaching at 72 h the value of 60.6%. Apoptotic signalling activation by TcdB + CKs is enriched by TNF-α-induced NF-κB signalling, inhibition of JNK activation and activation of AKT. In conclusion, the ability of C. difficile to activate three apoptotic pathways represents an important strategy to overcome resistance against its cytotoxic activity.

Chronic Stress-Related Gastroenteric Pathology in Cheetah: Relation between Intrinsic and Extrinsic Factors.

The rapid decline of cheetah (Acinonyx jubatus) throughout their range and long-term studies of captive breeding has increased conservation action for this species including the study of chronic diseases. Gastritis is one of the captive diseases that leads to high mortality presented with symptoms including vomiting, diarrhea, anorexia, and weight loss. The disease presents different histological lesions in the gastrointestinal tract that are characterized by inconstant and different clinical appearance in captive and free-range cheetahs. The aim of this review is to summarize the causes of chronic gastritis in the cheetah. Factors including diet, living conditions, infections with gastric Helicobacter-like organisms (GHLOs), the lack of genetic polymorphism and the cheetah's specific-immunocompetence are analyzed. All studies on gastroenteric cheetah pathologies, conducted between 1991 (to the best of our knowledge, the first report on online databases) and 2021, are included in this review, highlighting the possible correlation between stress-related captive conditions and chronic gastric pathology.

P62/SQSTM1 beyond Autophagy: Physiological Role and Therapeutic Applications in Laboratory and Domestic Animals.

Inflammation is the preceding condition for the development of mild and severe pathological conditions, including various forms of osteopenia, cancer, metabolic syndromes, neurological disorders, atherosclerosis, cardiovascular, lung diseases, etc., in human and animals. The inflammatory status is induced by multifarious intracellular signaling cascades, where cytokines, chemokines, arachidonic acid metabolites, adhesion molecules, immune cells and other components foster a "slow burn" at a local or systemic level. Assuming that countering inflammation limits the development of inflammation-based diseases, a series of new side-effects-free therapies was assessed in experimental and domestic animals. Within the targets of the drug candidates for quenching inflammation, an archetypal autophagic gear, the p62/sqstm1 protein, has currently earned attention from researchers. Intracellular p62 has been recently coined as a multi-task tool associated with autophagy, bone remodeling, bone marrow integrity, cancer progression, and the maintenance of systemic homeostasis. Accordingly, p62 can act as an effective suppressor of inflamm-aging, reducing oxidative stress and proinflammatory signals. Such an operational schedule renders this protein an effective watchdog for degenerative diseases and cancer development in laboratory and pet animals. This review summarizes the current findings concerning p62 activities as a molecular hub for cell and tissues metabolism and in a variety of inflammatory diseases and other pathological conditions. It also specifically addresses the applications of exogenous p62 (DNA plasmid) as an anti-inflammatory and homeostatic regulator in the treatment of osteoporosis, metabolic syndrome, age-related macular degeneration and cancer in animals, and the possible application of p62 plasmid in other inflammation-associated diseases.

Serum Amyloid A as a Promising Biomarker in Domestic Animals' Reproduction: Current Knowledge and Future Perspective.

The investigation of acute phase proteins in veterinary medicine has opened the doors towards the identification and use of new markers for a timely assessment of health status in both companion and food-producing animals. The aim of this paper is to review the literature available on the use of serum amyloid A (SAA), an acute phase protein, for the diagnosis and monitoring of reproductive disorders in animals. This review critically appraises the usefulness of such marker in clinical practice and summarizes the current state of knowledge. Recent advances in the diagnosis and monitoring of reproductive diseases are presented, highlighting where SAA evaluation may enhance early diagnostic tools for dogs, cats, cattle, and equines.

Clinicopathological and Fecal Proteome Evaluations in 16 Dogs Presenting Chronic Diarrhea Associated with Lymphangiectasia.

Canine intestinal lymphangiectasia (IL) is a condition characterized by variably severe gastrointestinal signs, frequently associated with laboratory abnormalities; the research for markers allowing a better understanding of the severity degree and/or obtaining an early diagnosis and/or monitoring is continuously progressing. In the present study, we investigated possible new diagnostic/follow-up markers in IL dogs, namely, serum C-reactive protein, serum bacterial lipopolysaccharide, serum cleaved cytokeratin 18, serum citrulline, and zonulin (in both serum and feces). A fecal proteomic study looking for possible confirmation and/or new marker candidates was also performed. All markers in both substrates, with the exception of serum citrulline, significantly differed between diseased and control dogs. Fecal proteomics allowed the retrieval of three proteins in IL dogs (Fc fragment of IgG-binding protein; transthyretin; proproteinase E) that were not previously found in clinically healthy subjects. Although further studies are needed, C-reactive protein, bacterial lipopolysaccharide, cleaved cytokeratin 18, and zonulin (in both serum and feces) resulted as promising markers for canine IL; similarly, fecal proteomics represents a road worthy of being pursued in the search for candidate biomarkers.