Porphyromonas gingivalis impairs glucose uptake in skeletal muscle associated with altering gut microbiota.

Skeletal muscles have a high metabolic capacity, which play key roles in glucose metabolism. Although periodontal disease increases the risk of metabolic syndrome, the relationship between periodontal bacterial infection and skeletal muscle metabolic dysfunction is unclear. We found that anti-Porphyromonas gingivalis (Pg) antibody titers positively correlated with intramuscular adipose tissue content (IMAC), fasting blood glucose, and HOMA-IR in metabolic syndrome patients. In C57BL/6J mice fed a high-fat diet, recipients of oral Pg (HFPg) had impaired glucose tolerance, insulin resistance, and higher IMAC compared to recipients of saline (HFco). The soleus muscle in HFPg mice exhibited fat infiltration and lower glucose uptake with higher Tnfa expression and lower insulin signaling than in HFco mice. Gene set enrichment analysis showed that TNFα signaling via NFκB gene set was enriched in the soleus muscle of HFPg mice. Moreover, TNF-α also decreased glucose uptake in C2C12 myoblast cells in vitro. Based on 16S rRNA sequencing, Pg administration altered the gut microbiome, particularly by decreasing the abundance of genus Turicibacter. Microbial network of the gut microbiome was dramatically changed by Pg administration. Our findings suggest that infection with Pg is a risk factor for metabolic syndrome and skeletal muscle metabolic dysfunction via gut microbiome alteration.

Epidemiological survey, general blood biochemistry, and histological examination of slaughtered heavy horse breeds with hemorrhage in the adipose tissue in the crest of the neck.

Fifty-four slaughtered horses were classified into groups having adipose tissue in the crest of the neck with or without hemorrhage (AH and NH groups, respectively). Blood biochemical tests (Alb, TP, T-bil, GOT, GPT, LDH, T-cho, and BUN) and an epidemiological survey (age, gender, weight, origin, breed, BCS, CNS, and hoof disease) were performed. T-bil tended to be high, while the other parameters were normal. Weight, BCS, and CNS were higher in the AH group (P<0.05). GOT was lower in the AH group (P<0.05). It was suspected that the horses in the AH group had lipomatosis. It was assumed that the adipose tissue of the horses in the AH group contained damaged capillaries, and inflammation was confirmed based on evidence of macrophages and lymphocytes.

Effect of a novel rice fermented extract on gastric ulcers in horses.

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.

Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions.

We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.

Photoactivatable Hydrogel Interfaces for Resolving the Interplay of Chemical, Mechanical, and Geometrical Regulation of Collective Cell Migration.

Collective migration is the mechanobiological interplay within migrating cell clusters and against extracellular matrixes (ECMs) underneath, mediating various physiological and pathological processes. Therefore, it is crucial to develop a robust platform on which collective migration can be studied under standardized conditions to understand how cells migrate differently between normal and disease states. We herein demonstrated phtotoactivatable hydrogel interfaces as suitable candidates for such applications. The substrate was composed of a poly(acrylamide) (PAAm) hydrogel whose surface was sequentially functionalized with poly-d-lysine (PDL) and photocleavable poly(ethylene glycol) (PEG). On the surface of the gel substrates, cell clusters with any given geometries can be prepared by controlling the irradiation patterns (geometrical cue), and their collective migration can be induced by the subsequent irradiation of the surrounding regions. Moreover, the substrate mechanical properties can be controlled by changing the composition of the PAAm hydrogel (mechanical cue), and the chemical properties were controlled by changing the amount of immobilized PDL, thereby altering the adsorbed amount of ECM proteins (chemical cue). The photoactivatable gel substrates were characterized by fluorescence microscopy, ζ-potential measurements, and the protein adsorption test. Through the study of the interplay of chemical, mechanical, and geometrical cues in the regulation of collective characteristics, we found additive effects of chemical and mechanical cues on the suppression of circular expansion by up-regulating the epithelial morphology. Also, the impact of geometrical cues became more significant by decreasing the chemical cue. We believe the present platform will be a useful research tool for the comprehensive mechanobiological analysis of collective cell migration.

Influence of Porphyromonas gingivalis in gut microbiota of streptozotocin-induced diabetic mice.

OBJECTIVES: Increasing evidence suggests that periodontitis can exacerbate diabetes, and gut bacterial dysbiosis appears to be linked with the diabetic condition. The present study examined the effects of oral administration of the periodontopathic bacterium, Porphyromonas gingivalis, on the gut microbiota and systemic conditions in streptozotocin-induced diabetic mice. MATERIALS AND METHODS: Diabetes was induced by streptozotocin injection in C57BL/6J male mice (STZ). STZ and wild-type (WT) mice were orally administered P. gingivalis (STZPg, WTPg) or saline (STZco, WTco). Feces were collected, and the gut microbiome was examined by 16S rRNA gene sequencing. The expression of genes related to inflammation, epithelial tight junctions, and glucose/fatty acid metabolism in the ileum or liver were examined by quantitative PCR. RESULTS: The relative abundance of several genera, including Brevibacterium, Corynebacterium, and Facklamia, was significantly increased in STZco mice compared to WTco mice. The relative abundances of Staphylococcus and Turicibacter in the gut microbiome were altered by oral administration of P. gingivalis in STZ mice. STZPg mice showed higher concentrations of fasting blood glucose and inflammatory genes levels in the ileum, compared to STZco mice. CONCLUSIONS: Oral administration of P. gingivalis altered the gut microbiota and aggravated glycemic control in streptozotocin-induced diabetic mice.

Label-Free Rapid Separation and Enrichment of Bone Marrow-Derived Mesenchymal Stem Cells from a Heterogeneous Cell Mixture Using a Dielectrophoresis Device.

Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.

Effect of Porphyromonas gingivalis infection in the placenta and umbilical cord in pregnant mice with low birth weight.

OBJECTIVE: Growing evidence indicates an association between periodontitis and delivery outcome; however, the mechanism is unclear. This study aimed to investigate the influence of Porphyromonas gingivalis (Pg) infection on delivery outcome in mice. MATERIALS AND METHODS: Bacteremia was induced in pregnant Slc:ICR mice (8 weeks old) by intravenous injection of Pg. Mice were randomly divided into a control group (CO), and those receiving Pg injection at gestational day 1 (GD1), gestational day 15 (GD15) or every day (ED). Delivery outcome, Pg infection, and gene expression in the placenta and umbilical cord were evaluated. RESULTS: Birth weight was lower in the ED and GD15 groups than in the CO group. A remarkable increase in anti-Pg IgG antibody was observed in the ED and GD1 groups, although Pg was not detected in the placenta or umbilical cord. mRNA expression of Tnfα and Il6 in the placenta, and Hif1α in the umbilical cord, was significantly increased in the ED group. Microarray analysis of the umbilical cord revealed increased expression of several genes including Orm1, Mgl2, Rps6ka3 and Trim15 in the ED group. CONCLUSIONS: Pg infection during the third trimester caused low birth weight and inflammation in the placenta and umbilical cord.

Displacement of the large colon in a horse with enterolithiasis due to changed positions observed by computed tomography.

Computed tomography (CT) was performed for an 18-year-old female pony with enterolithiasis in the prone and supine positions. CT images from the prone position revealed displacement of the large dorsal colon, which contained an enterolith to the ventral side of the abdomen, and those from the supine position revealed displacement to the dorsal side. A high-density material suggestive of a metallic foreign body was also observed in the enterolith core. An enterolith (422 g, 104 mm) was surgically removed from the large dorsal colon. This caused no complications after surgery and increased the horse's weight. Changing positions during CT helps identify the exact location of enterolith and intestinal displacement due to enterolith weight, as well as size and number.

Molecular crowding improves bead-based padlock rolling circle amplification.

Bead-based padlock rolling circle amplification (RCA), an ultrasensitive and accurate DNA detection technique, was conducted in a molecular crowding environment created by poly(ethylene glycol) (PEG). The number of RCA products generated increased and exhibited a bell-shaped dependence on PEG concentration. Experiments using magnetic beads suggested that facilitation of DNA ligation and hybridization is the main reason for the observed increase. Selectivity of the technique was retained in the presence of PEG. This technique is simple and can be utilized to detect target DNA with high accuracy and sensitivity in a variety of areas such as medical diagnosis and food analysis.

Characteristic findings of magnetic resonance imaging (MRI) and computed tomography (CT) for severe chronic laminitis in a Thoroughbred horse.

A Thoroughbred horse with severe chronic laminitis of both forelimbs was evaluated on the same day with magnetic resonance imaging (MRI) and computed tomography (CT). Both MRI and CT revealed loss of the dorsal aspect of the cortical bone of the 3rd phalanx and sclerosis. CT reflected the status of the horny layer and bone of the affected feet, while MRI depicted inflammation of the laminar corium, together with tendon edema. On the 3-dimensional CT venogram, vessels were visualized in both the right and left forelimbs, although there was a difference in the vasculature of the coronary plexus and circumflex vessels between the right and left forelimbs. A combination of both MRI and CT provides detailed information regarding pathological conditions.

Computed tomography and magnetic resonance imaging findings for the initial stage of equine temporohyoid osteoarthropathy in a Thoroughbred foal.

Temporohyoid osteoarthropathy (THO) is characterized by progressive osseous proliferation of the stylohyoid and petrous temporal bones. Generally, diagnosis is confirmed by guttural pouch endoscopy and skull radiography. In the present case, computed tomography (CT) and magnetic resonance imaging (MRI) were performed in a 6-month-old Thoroughbred foal showing signs of head tilt and unilateral ear droop, consistent with the presence of a vestibular disease and unilateral facial paralysis. CT revealed bony fusion and proliferation of the right temporohyoid joint, while MRI revealed that otitis media was responsible for THO. In conclusion, this report suggests that CT and MRI provide a more concrete diagnosis and better understandings of the mechanism of THO etiology.

Four cases of equine motor neuron disease in Japan.

In this study, fasciculation of the limbs and tongue was observed in four horses kept by a riding club. Neurogenic muscle atrophy was also observed in biopsy of pathological tissues. In addition, in two cases that subjected to autopsy, Bunina-like bodies of inclusion in the cell bodies of neurons in the spinal cord ventral horn were confirmed, leading to a diagnosis of equine motor neuron disease (EMND). Serum vitamin E concentrations varied between 0.3 and 0.4µg/ml, which is significantly lower than the levels in normal horses. Although lack of vitamin E is speculated to be a contributory factor for development of EMND, no significant improvement was observed following administration of vitamin E.

Oxidative stress markers in Thoroughbred horses after castration surgery under inhalation anesthesia.

Oxidative stress has been reported to occur during surgery. It is important to reduce intraoperative oxidative stress to improve the postoperative prognosis. However, there are no reports regarding oxidative stress related to surgery in horses. In the present study, we measured pre and postsurgical diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP); the oxidative stress index (OSI) was then calculated (OSI=d-ROMs/BAP × 100). d-ROMs were not significantly different between the pre and postsurgical periods. However, BAP significantly decreased after surgery (P=0.02), and OSI significantly increased after surgery (P=0.02). Based on these results, it suggested that castration surgery under inhalation anesthesia decreases the antioxidant potential and causes oxidative stress in horses.

Autopsy imaging for cardiac tamponade in a Thoroughbred foal.

Autopsy imaging (Ai), postmortem imaging before necropsy, is used in human forensic medicine. Ai was performed using computed tomography (CT) for a 1-month-old Thoroughbred foal cadaver found in a pasture. CT revealed pericardial effusion, collapse of the aorta, bleeding in the lung lobe, gas in the ventricles and liver parenchyma, and distension of the digestive tract. Rupture in the left auricle was confirmed by necropsy; however, it was not depicted on CT. Therefore, Ai and conventional necropsy are considered to complement each other. The cause of death was determined to be traumatic cardiac tamponade. In conclusion, Ai is an additional option for determining cause of death.

Quantitative evaluation of cervical cord compression by computed tomographic myelography in Thoroughbred foals.

Five Thoroughbred foals (age, 8-33 weeks; median age, 31 weeks; weight, 122-270 kg; median weight, 249 kg) exhibiting ataxia with suspected cervical myelopathy (n=4) and limb malformation (n=1) were subjected to computed tomographic (CT) myelography. The areas of the subarachnoid space and cervical cord were measured on transverse CT images. The area of the cervical cord was divided by the area of subarachnoid space, and stenosis ratios were quantitatively evaluated and compared on the basis of histopathological examination. The sites with a ratio above 52.8% could have been primary lesion sites in the histopathological examination, although one site with a ratio of 54.1% was not a primary lesion site. Therefore, in this study, a ratio between 52.8-54.1% was suggested to be borderline for physical compression that damages the cervical cord. All the cervical vertebrae could not be scanned in three of the five cases. Therefore, CT myelography is not a suitable method for locating the site of compression, but it should be used for quantitative evaluation of cervical stenosis diagnosed by conventional myelography. In conclusion, the stenosis ratios determined using CT myelography could be applicable for detecting primary lesion sites in the cervical cord.

Caspase 3 silencing inhibits biomechanical overload-induced intervertebral disk degeneration.

Intervertebral disk (IVD) degeneration causes debilitating low back pain in much of the worldwide population. No efficient treatment exists because of an unclear pathogenesis. One characteristic event early in such degeneration is the apoptosis of nucleus pulposus (NP) cells embedded in IVDs. Excessive biomechanical loading may also be a major etiology of IVD degeneration. The present study used in vitro and in vivo models of compressive loading to elucidate the underlying mechanism of IVD degeneration. In addition, we investigated whether the inhibition of apoptosis is a potential clinical therapeutic strategy for the treatment of IVD degeneration induced by biomechanical stress. A TUNEL assay showed that NP cell-agarose three-dimensional composite cultures subjected to uniaxial, unconfined, static, compressive loading exhibited a time-dependent increase in apoptosis. Western blot analysis revealed the up-regulation of several extracellular matrix-degrading enzymes and down-regulation of tissue inhibitor of metalloproteinase 1. These responses to compressive loading were all significantly inhibited by caspase 3 siRNA. In the in vivo model of compressive loading-induced IVD degeneration, a single local injection of caspase 3 siRNA significantly inhibited IVD degeneration by magnetic resonance imaging, histological findings, IHC, and TUNEL assay. The present study suggests that caspase 3 siRNA attenuates overload-induced IVD degeneration by inhibiting NP cell apoptosis and the expression of matrix-degrading enzymes.

Alternating current cloud point extraction on a microchip: the effect of electrode geometry.

We report on the effect of electrode geometry on alternating current cloud point extraction (ACPE). ACPE is a technique utilized to extract membrane-associated biomolecules in an electrode-integrated microfluidic channel. In this study, we investigated the effect of gap size (4∼22 µm) between microband electrodes on ACPE. A decrease in gap size resulted in efficient and rapid concentration of fluorescent-labeled phospholipids, a model of membrane-associated biomolecules. We also investigated the effect of applied voltage amplitude on ACPE using devices with decreased electrode gap size. When the gap was small, ACPE was achieved with low applied voltages. ACPE of membrane proteins extracted from HeLa cells was also studied to demonstrate the applicability of the ACPE to real samples. The results provide a guideline to improve the performance of ACPE and facilitate application of the ACPE technique as part of an overall analytical process.

Experimental investigation of bone mineral density in Thoroughbreds using quantitative computed tomography.

Bone mineral density (BMD) is one of the indications of the strength and health. BMD measured by quantitative computed tomography (QCT) was compared with that measured by dual energy X-ray absorptiometry (DXA) and radiographic bone aluminum equivalence (RBAE). Limbs were removed from horses that had been euthanized for reasons not associated with this study. Sixteen limbs (left and right metacarpals and metatarsals) from 4 horses were used to compare BMD as measured by QCT with those measured by DXA and RBAE. There was a strong correlation between BMD values measured by QCT and those measured by DXA (R(2)=0.85); correlation was also observed between values obtained by QCT and those obtained by RBAE (R(2)=0.61). To investigate changes in BMD with age, 37 right metacarpal bones, including 7 from horses euthanized because of fracture were examined by QCT. The BMD value of samples from horses dramatically increased until 2 years of age and then plateaued, a pattern similar to the growth curve. The BMD values of bone samples from horses euthanized because of fracture were within the population range, and samples of morbid fracture were not included. The relationship between BMD and age provides a reference for further quantitative studies of bone development and remodeling. Quantitative measurement of BMD using QCT may have great potential for the evaluation of bone biology for breeding and rearing management.

Magnetic resonance imaging of a microvascular-interstitium model on a microfluidic device.

We report a microvascular-interstitium model on microfluidic devices to study leakage of drugs from blood vessels under in vivo-like flow conditions. We employed magnetic resonance imaging to demonstrate the compatibility of the model for experimental animals and humans. We observed transport of two types of different molecular-weight contrast agents into the model interstitium. The ratio of the transport rates of agents agreed with the ratio calculated from diffusion coefficients of the agents. We expect that the model will be useful for the estimation and evaluation of leakage of many kinds of agents in vivo.