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.

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.

The effects of 808-nm near-infrared laser light irradiation on actin cytoskeleton reorganization in bone marrow mesenchymal stem cells.

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms.

Use of nutraceuticals in the stallion: Effects on semen quality and preservation.

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.

Management of otitis externa with an led-illuminated gel: a randomized controlled clinical trial in dogs.

BACKGROUND: Canine otitis externa is a painful condition which can be challenging to treat due to difficulties in the administration of otic medication. This can be due to lack of owner compliance in the application of ear drops or due to the resentment that some dogs demonstrate when attempts are made to administer topical medication into a sensitive ear canal. The aim of the study was to assess the efficacy of a topical LED-illuminated gel (LIG) in canine otitis externa in comparison to standard of care therapy. Dogs with spontaneous otitis externa were randomly allocated in three groups: groups QW received LIG once weekly; BW received LIG twice weekly; group C received enrofloxacin and silver sulfadiazine twice daily. LIG consists of a topical application of a gel containing chromophores that, when illuminated by a LED lamp, re-emit fluorescent light which can stimulate physiological responses, promoting healing and controlling bacteria. The evaluation protocol (T0 to T5) considered clinical assessment (OTIS-3-index-scoring-system; pruritus-severity-scale; pain-severity-score; aural temperature), cytological scoring system, quali-quantitative bacteriologic assessment. RESULTS: All groups (QW, n = 21; BW, n = 23; C, n = 20) showed improvement during the study (QW: P < 0.02 for cytological and pain scores, P < 0.003 for bacteriologic assessment, P < 10- 4 for pruritus, total OTIS-3 and temperature assessments; BW: P < 10- 4 for all clinical, cytological and bacteriologic assessments; C: P < 0.02 for all clinical and cytological assessments, P < 10- 4 for bacteriologic assessment). The highest clinical score reduction occurred in Group BW (P < 0.014 in T3; P < 0.001 in T4 and P < 10- 4 in T5). BW reached the clinically relevant effect level at T3 (- 3.26 ± 1.21 levels), QW reached it at T4 (- 3.24 ± 0.99), C did not reach it. No differences between groups were seen in the reduction of CFU/mL (T0-T5). CONCLUSIONS: All treatment groups showed a positive clinical effect. LIG administered twice-a-week was the most favourable protocol of the study. LIG may be considered beneficial in the management of canine otitis externa; it seems to be effective in controlling the clinical condition, including the signs of inflammation and local pain, the bacterial growth, and it may help increasing treatment compliance.

Management of canine perianal fistula with fluorescence light energy: preliminary findings.

BACKGROUND: Canine perianal fistula (CPF) is a severe, painful, debilitating skin condition affecting the perianal skin. It often interferes with the quality of life of both dog and owner. Conventional medical treatment involves the use of immunosuppressive therapy; however, the successful resolution of lesions can be limited by poor owner compliance, adverse drug effects and dependence on costly therapies. HYPOTHESIS/OBJECTIVES: The present study aimed to assess the potential benefits of fluorescent light energy (FLE) on cases of CPF. ANIMALS: Four dogs with active perianal fistulas METHODS AND MATERIALS: FLE was applied as sole management therapy once a week with two consecutive applications in the same session for each dog until clinical signs had significantly improved, with weekly assessments for a six week period. Dogs were assessed by measuring the size of lesions at the start of the study and then weekly for six weeks, using planimetry software. Owners recorded vocalization and straining frequency scores during their pet's defaecation, and perianal licking frequency on a 0-5 point scale to assess the response to therapy. RESULTS: All dogs improved with FLE, achieving a significant reduction in vocalization, straining and licking after two weeks (P = 0.002). After five weeks of FLE therapy, lesional areas had significantly decreased (P = 0.04). Only one dog required more than seven applications. No adverse events were reported. CONCLUSION AND CLINICAL IMPORTANCE: FLE may be a promising alternative therapy for CPF.

Effect of the topical Klox fluorescence biomodulation system on the healing of canine surgical wounds.

OBJECTIVE: To determine the effect of the Klox fluorescence biomodulation system (Phovia) on the healing of surgical wounds. STUDY DESIGN: Prospective, blinded, controlled clinical trial. SAMPLE POPULATION: Healthy dogs undergoing orthopedic surgery (n = 10). METHODS: Half of the length of each surgical wound was treated with Phovia, and the remaining 50% was treated with saline solution on the first day after surgery and every 3 days until day 13. Wound healing of treated and control areas within each wound was evaluated via macroscopic assessment and histological and immunohistochemical analysis of treated and control wounds. RESULTS: The areas treated with Phovia achieved lower histology scores (P = .001), consistent with complete re-epithelialization, less inflammation of the dermal layer, and greater and more regular deposition of collagen. According to immunohistochemistry, expression of factor VIII, epidural growth factor, decorin, collagen III, and Ki67 was increased in treated compared with untreated tissues. CONCLUSION: Phovia therapy improved re-epithelialization, decreased dermal inflammation, and improved matrix formation in uncomplicated cutaneous incisional wounds by regulating the expression of key biological mediators. CLINICAL SIGNIFICANCE: Phovia may be a beneficial adjunct to promote the healing of incisional wounds.

P62 deficiency shifts mesenchymal/stromal stem cell commitment toward adipogenesis and disrupts bone marrow homeostasis in aged mice.

With advancing age have been observed bone and bone marrow phenotypic alterations due to the impaired bone tissue homeostatic features, involving bone remodeling, and bone marrow niche ontogeny. The complex "inflamm-aging" pathological scenario that culminates with osteopenia and mesenchymal/stromal and hematopoietic stem cell commitment breakdown, is controlled by cellular and molecular intramural components comprising adapter proteins such as the sequestosome 1 (p62/SQSTM1). p62, a "multiway function" protein, has been reported as an effective anti-inflammatory, bone-building factor. In this view, we considered for the first time the involvement of p62 in aging bone and bone marrow of 1 year and 2 years p62-/- mice. Interestingly, p62 deficiency provoked accelerated osteopenia and impaired niche operational activities within the bone marrow. The above findings unearthed the importance of p62 in mesenchymal stem cell maintenance/differentiation schedule in old animals and provide, at least in part, a mechanistic scenario of p62 action.

Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis.

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross-linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero-order release kinetics, and the release rate depended on cross-link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox-9 and Runx-2. Furthermore, the downregulation of proinflammatory mediators, such as TNF-α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA.

Preoperative topical liposomal ozone dispersion to reduce bacterial colonization in conjunctival sac and periocular skin: Preliminary study in dogs.

Prophylaxis represents a keystone to reduce periocular skin and ocular conjunctiva bacterial load before surgical procedures. Despite many prophylactic agents are available the preferred perioperative ocular surface antimicrobial is still unknown. The purpose of this study was to assess the effectiveness of preoperative liposomal ozone dispersion in reducing bacterial colonization from the conjunctival sac and periocular skin in dogs, in comparison with povidone-iodine and fluoroquinolone. Twenty-two owned dogs consisting with 44 eyes in total scheduled for ophthalmic surgical procedure were enrolled for the study and divided in four groups receiving either ozone dispersion or povidone iodine in eyelid and conjunctiva, fluoroquinolone or placebo. A swab was taken before and after the antisepsis protocol evaluating total microbial count, coagulase positive and negative staphylococci. Statistical analysis revealed a significant decrease in colony forming units (CFU) for total microbial count, coagulase positive and negative staphylococci both for liposomal ozone dispersion and povidone iodine. No statistical differences were detected in median CFU for both one-day placebo and fluoroquinolone preoperative prophylactic topical therapy. The results of this preliminary study demonstrate that liposomal ozone-dispersion is as effective as povidone iodine to reduce preoperative bacterial load in ocular surface.

Fecal Proteomic Analysis in Healthy Dogs and in Dogs Suffering from Food Responsive Diarrhea.

Different laboratory markers are routinely used in the diagnosis and management of gastrointestinal (GI) disease in dogs. In the present study, starting from feces from both healthy dogs and dogs suffering from food responsive diarrhea (FRD), we tried to find proteins differently expressed in the two groups of dogs, by using a proteomic approach. Interestingly, we found that the immunoglobulin J-chain isoform 1 (species: Canis lupus familiaris) was identified only in diseased dogs (not in healthy). J-chain combines especially IgA monomers to IgA dimers and plays a crucial role for their secretions into mucosal interface. Being the first study of that kind in the dog, it is only possible to hypothesize that their presence could be likely due to an increased activation of the immune system or to a mucosal damage or both in FRD patients. Similarly, it is still impossible to assess whether this protein could be used as diagnostic/prognostic marker of GI disease; however, this study represents a promising first step toward fecal proteomics in canine GI disorders.

Fluorescence biomodulation in the management of canine interdigital pyoderma cases: a prospective, single-blinded, randomized and controlled clinical study.

BACKGROUND: Interdigital pyoderma is a common multifactorial, inflammatory disease of the canine interdigital skin. Lesions commonly become infected secondarily. In addition to management of the underlying cause, management of the chronic inflammatory changes in the interdigital skin created by secondary infection and by the release of keratin into deep tissues is required. Fluorescence biomodulation appears to modulate the inflammatory process in dermatological disorders and has shown promise in preliminary studies evaluating its use in superficial and deep pyoderma in dogs. HYPOTHESIS/OBJECTIVES: To evaluate the effect of a fluorescence biomodulation (FB) system used in conjunction with systemic antibiotic on clinical manifestations of canine interdigital pyoderma (CIP), compared to dogs treated with antibiotic alone. ANIMALS: Thirty-six dogs diagnosed with CIP. METHODS AND MATERIALS: Dogs were randomly allocated to treatment groups of either antibiotic alone (Group A) or antibiotic plus twice-weekly FB application (Group B). Dogs were scored over a 12 week period on the basis of two measured parameters: a global lesion score composed of four different lesions types and neutrophil engulfing bacterial scores. RESULTS: A statistically significant decrease was seen by Week 3 in both measured parameters for Group B compared to Group A. The mean time-to-resolution of lesions was 4.3 weeks in Group B and 10.4 weeks in Group A. CONCLUSION AND CLINICAL IMPORTANCE: The FB system shows promise as an adjunct therapy to systemic antibiotic use in the management of CIP.

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria.

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.

Nuclear Magnetic Resonance (NMR) Metabolomics: Current Applications in Equine Health Assessment.

Metabolomics can allow for the comprehensive identification of metabolites within biological systems, at given time points, in physiological and pathological conditions. In the last few years, metabolomic analysis has gained popularity both in human and in veterinary medicine, showing great potential for novel applications in clinical activity. The aim of applying metabolomics in clinical practice is understanding the mechanisms underlying pathological conditions and the influence of certain stimuli (i.e., drugs, nutrition, exercise) on body systems, in the attempt of identifying biomarkers that can help in the diagnosis of diseases. Proton Nuclear Magnetic Resonance spectroscopy (1H-NMR) is well tailored to be used as an analytical platform for metabolites' detection at the base of metabolomics studies, due to minimal sample preparation and high reproducibility. In this mini-review article, the scientific production of NMR metabolomic applications to equine medicine is examined. The research works are very different in methodology and difficult to compare. Studies are mainly focused on exercise, reproduction, and nutrition, other than respiratory and musculoskeletal diseases. The available information on this topic is still scant, but a greater collection of data could allow researchers to define new reliable markers to be used in clinical practice for diagnostic and therapeutical purposes.

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.

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.

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.

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.

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.

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.