Identification and characterization of intestinal lactobacilli strains capable of degrading immunotoxic peptides present in gluten.

AIM: Identify and characterize bacteria from the proximal gastrointestinal tract of pigs capable of degrading immunogenic gluten peptides. METHODS AND RESULTS: Bacteria were cultured from the small intestine of pigs fed a 20% gluten diet and from an enrichment media with the 18-mer peptide LQLQPFPQPQLPYPQPQL. Isolates were screened for the production of specialized proteolytic enzymes and the ability to degrade and remove metastable peptides from α-gliadin (16-mer and 33-mer) and ω-gliadin (17-mer), with established roles in the aetiology of coeliac disease. Degradation was determined by ELISA and mass spectrometry (UHPLC-MS/MS in MRM mode), and hydrolysis fragments were characterized by LC-MS/MS. Four strains from the species Lactobacillus ruminis, Lactobacillus johnsonii, Lactobacillus amylovorus and Lactobacillus salivarius showed the highest peptide-degrading activities. Strains displayed different degradation rates and cleavage patterns that resulted in reduction but not complete removal of immunotoxic epitopes. CONCLUSIONS: We employed a unique enrichment process to select for bacteria adapted to the conditions of the proximal gastrointestinal tract with the ability to partially detoxify well-characterized peptides involved in coeliac disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a basis for the selection of Lactobacillus strains for probiotic applications aimed to reduce epitope-containing gluten peptides before reaching the epithelium of the small intestine of patients with coeliac disease.

Performance of dairy cows fed silage and grain produced from second-generation insect-protected (Bacillus thuringiensis) corn (MON 89034), compared with parental line corn or reference corn.

Corn grain and corn silage are major feed components in lactating dairy cow rations. Bacillus thuringiensis (B.t.) is a naturally occurring soil bacterium that produces a protein that is toxic to lepidopteran insects that may damage plant tissues and reduce corn quality and yields. During each of the four 28-d periods, cows were offered 1 of 4 rations in which the corn grain and silage originated from different corn hybrids: a nontransgenic corn control (from hybrid DKC63-78; Monsanto Co., St. Louis, MO), a B.t. test substance corn (MON 89034 in hybrid DKC63-78; Monsanto Co.), and 2 commercial nontransgenic reference (Ref) hybrids: DKC61-42 (Ref 1) and DKC62-30 (Ref 2; Monsanto Co.). Sixteen multiparous Holstein cows averaging 110 ± 21 d in milk and weighing 684 ± 62.3 kg were blocked by days in milk and milk yield and randomly assigned to one of four 4 × 4 Latin squares. Diets were formulated to contain 36.4% corn silage and 16.3% corn grain. Dry matter intake was greater for cows consuming B.t. corn (26.6 ± 0.59 kg/d) compared with the control, Ref 1, and Ref 2 corn diets (25.4, 25.0, and 25.6 ± 0.59 kg/d, respectively). Milk yield, fat yield, and percentage of fat (36.8 ± 0.98 kg/d, 1.22 ± 0.05 kg/d, and 3.3 ± 0.10%), milk protein yield and percentage of protein (1.11 ± 0.03 kg/d and 3.01 ± 0.05%), milk urea nitrogen concentration (14.01 ± 0.49 mg/dL), and 3.5% fat-corrected milk yield (35.7 ± 1.07 kg/d) were not different across treatments. The results from this study show that lactating dairy cows that consume B.t. corn (MON 89034) do not differ from lactating dairy cows that consume nontransgenic corn in milk yield, 3.5% fat-corrected milk per unit of dry matter intake, or milk components.

Peripartum anesthetic management in patients with left ventricular noncompaction: a case series and review of the literature.

BACKGROUND: This retrospective review focuses on peripartum anesthetic management and outcome of a series of five pregnant women with left ventricular noncompaction (LVNC). METHODS: The Mayo Clinic Advanced Cohort Explorer medical database was utilized to identify women diagnosed with LVNC who had been admitted for delivery at the Mayo Clinic in Rochester, Minnesota, between January 2001 and September 2021. Echocardiograms were independently reviewed by two board-certified echocardiographers, and those determined by both to meet the Jenni criteria and/or having compatible findings on magnetic resonance imaging (MRI) were included. Electronic medical records were reviewed for information pertaining to cardiac function, labor, delivery, and postpartum management. RESULTS: We identified 44 patients whose medical record included the term "noncompaction" or "hypertrabeculation" and who had delivered at our institution during the study period. Upon detailed review of the medical records, 36 did not meet criteria for LVNC, and three additional patients did not receive the diagnosis until after delivery, leaving five patients with confirmed LVNC who had undergone six deliveries during the study interval. All five patients had a history of arrhythmias or had developed arrhythmias during pregnancy. One patient underwent emergency cesarean delivery due to sustained ventricular tachycardia requiring three intra-operative cardioversions. CONCLUSIONS: This case series adds new evidence to that already available about pregnancies among women with LVNC. Favorable obstetrical outcomes were achievable when multidisciplinary teams were prepared to manage the maternal and fetal consequences of intrapartum cardiac arrhythmias and hemodynamic instability.

Unusual kinematics of the Papatea fault (2016 Kaikoura earthquake) suggest anelastic rupture.

A key paradigm in seismology is that earthquakes release elastic strain energy accumulated during an interseismic period on approximately planar faults. Earthquake slip models may be further informed by empirical relations such as slip to length. Here, we use differential lidar to demonstrate that the Papatea fault-a key element within the 2016 Mw 7.8 Kaikoura earthquake rupture-has a distinctly nonplanar geometry, far exceeded typical coseismic slip-to-length ratios, and defied Andersonian mechanics by slipping vertically at steep angles. Additionally, its surface deformation is poorly reproduced by elastic dislocation models, suggesting the Papatea fault did not release stored strain energy as typically assumed, perhaps explaining its seismic quiescence in back-projections. Instead, it slipped in response to neighboring fault movements, creating a localized space problem, accounting for its anelastic deformation field. Thus, modeling complex, multiple-fault earthquakes as slip on planar faults embedded in an elastic medium may not always be appropriate.

Kinetics of Plasma Cell-Free DNA and Creatine Kinase in a Canine Model of Tissue Injury.

BACKGROUND: Cell-free DNA (cfDNA) comprises short, double-stranded circulating DNA sequences released from damaged cells. In people, cfDNA concentrations correlate well with disease severity and tissue damage. No reports are available regarding cfDNA kinetics in dogs. OBJECTIVES/HYPOTHESIS: Cell-free DNA will have a short biological half-life and would be able to stratify mild, moderate, and severe tissue injury. Our study aims were to determine the kinetics and biological half-life of cfDNA and to contrast them with those of creatine kinase (CK). ANIMALS: Three groups of 10 dogs undergoing open ovariohysterectomy, surgery for cranial cruciate ligament rupture (CCLR), or hemilaminectomy. METHODS: Plasma for cfDNA and CK analysis was collected at admission, at induction of anesthesia, postsurgery (time 0) and at 6, 12, 24, 36, 48, 60, and 72 hours after surgery. RESULTS: The biological half-life of plasma cfDNA and CK were 5.64 hours (95% confidence interval [CI 95], 4.36-7.98 hours) and 28.7 hours (CI95, 25.3-33.3 hours), respectively. In the hemilaminectomy group, cfDNA concentrations differed significantly from admission at 6-12 hours after surgery. Creatine kinase activity differed among the surgical groups and reached a peak 6 hours after surgery. In the ovariohysterectomy and CCLR groups, plasma CK activity 72 hours after surgery did not differ from admission activity of the ovariohysterectomy group. In contrast, in the hemilaminectomy group, plasma CK activity after 72 hours did not return to the ovariohysterectomy group admission activity. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma CK activity has a longer biological half-life than previously thought. In contrast to plasma CK activity, cfDNA has a short half-life and could be a useful marker for peracute severe tissue injury.

Protein kinase C delta involvement in mammary tumor cell metastasis.

Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKCdelta levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKCdelta is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKCdelta (RDdelta) under the control of a tetracycline-inducible promoter. RDdelta expression attenuated endogenous PKCdelta activity, as demonstrated by decreased phosphorylation of the PKCdelta substrate adducin in migrating cells. Thus, in MT cells, RDdelta appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RDdelta expression influenced metastatic potential in vivo, MTLn3/RDdelta cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RDdelta expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RDdelta significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKCdelta activity by expressing the inhibitory RDdelta fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.

The zebrafish genome editing toolkit.

Zebrafish (Danio rerio) is a unique model organism at the functional intersection between a high fecundity and conserved vertebrate physiology while being amenable to a multitude of genome editing techniques. The genome engineering field has experienced an unprecedented rate of growth in the recent years since the introduction of designer endonucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-Cas9 systems. With the ever-evolving toolset available to the scientific community, the important question one should ask is not simply how to make a mutant line, but rather how best to do so. For this purpose, understanding the toolset is just one end of the equation; understanding how DNA is repaired once double-strand breaks are induced by designer endonucleases, as well as understanding proper fish handling and line maintenance techniques, are also essential to rapidly edit the zebrafish genome. This chapter is outlined to provide a bird's-eye view on each of these three components. The goal of this chapter is to facilitate the adoption of the zebrafish as a model to study human genetic disease and to rapidly analyze the function of the vertebrate genome.

Increased protein kinase C delta in mammary tumor cells: relationship to transformtion and metastatic progression.

Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed alpha, delta, epsilon and zeta PKCs; however, PKC delta levels were relatively increased in the highly metastatic cells. To determine if increased PKC delta could contribute to promotion/progression, we overexpressed PKC delta in the low and moderately metastatic cell lines. PKC delta overexpression had no significant effect on growth of adherent cells, but significantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKC delta (RD delta), a putative PKC delta inhibitory fragment, inhibited anchorage-independent growth. The efficacy of RD delta as a PKC delta inhibitor was demonstrated by showing that RD delta selectively interfered with PKC delta subcellular location and significantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKC delta activity and phenotypic changes in cytoskeletal-dependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth effects of expressing PKC delta and RD delta as gain and loss of function constructs, respectively, provide strong evidence that PKC delta regulates processes important for anchorage-independent growth in these mammary tumor cells.

Phorbol esters modulate the turnover of both ether- and ester-linked phospholipids in cultured mammalian cells.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the metabolism of ester- and ether derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were studied in HeLa and HEL-37 cells. TPA stimulated the incorporation of [3H]choline into diacyl-, alkylacyl- and alkenylacy/PC in HeLa cells, but inhibited the incorporation of [3H]ethanolamine into the corresponding derivatives of PE. TPA also stimulated the incorporation of [3H]ethanolamine into lysoPE and the release of labelled ethanolamine and phosphoethanolamine from HeLa cells prelabelled with [3H]ethanolamine. All responses to TPA were abolished in HeLa cells preincubated with the phorbol ester and which were deficient in protein kinase C. In HEL-37 cells TPA stimulated label incorporation into both ester- and ether-forms of PE. The marked effects of TPA on ether-lipid metabolism raises the possibility that hydrolysis products of this class of lipid are important in transmembrane signalling pathways.

Microsatellite mutation of type II transforming growth factor-beta receptor is rare in atherosclerotic plaques.

A somatic mutation within a microsatellite polyA tract in the coding region of the type II transforming growth factor (TGF)-beta receptor gene was reported to occur in human atherosclerotic and restenotic lesions. This mutation occurs frequently in colorectal cancer with the replication error repair phenotype and results in loss of sensitivity to the growth inhibitory effects of TGF-beta in cells from the tumors. The mutation was proposed to account for the clonal expansion of vascular smooth muscle cells observed in atherosclerotic plaques, through loss of the growth inhibitory effect of TGF-beta. The frequency of the mutation and the extent of clonal expansion of the mutated cells have major implications for the mechanism of atherogenesis and therapeutic strategies. We analyzed a set of 22 coronary arterial and 9 aortic samples containing early to advanced atherosclerotic lesions for the mutation in the type II TGF-beta receptor polyA tract. Only 1 coronary arterial sample from an advanced lesion showed detectable amounts of the mutation, present at a low level (8% of the DNA sample). The data imply that the mutation occurs only at low frequency and is not a major mechanistic contributor to the development of atherosclerosis.

Further development of hydrogen bond functions for use in determining energetically favorable binding sites on molecules of known structure. 1. Ligand probe groups with the ability to form two hydrogen bonds.

The directional properties of hydrogen bonds play a major role in determining the specificity of intermolecular interactions. An energy function which takes explicit account of these properties has been developed for use in the determination of energetically favorable ligand binding sites on molecules of known structure by the GRID method (Goodford, P.J.J. Med. Chem. 1985, 28, 849. Boobbyer, D.N.A.; Goodford, P.J.; McWhinnie, P.M.; Wade, R.C.J. Med. Chem. 1989, 32, 1083). In this method, the interaction energy between a target molecule and a small chemical group (a probe), which may be part of a larger ligand, was calculated using an energy function consisting of Lennard-Jones, electrostatic, and hydrogen bond terms. The latter term was a function of the length of the hydrogen bond, its orientation at the hydrogen-bonding atoms, and their chemical nature. We now describe hydrogen bond energy functions which take account of the spatial distribution of the hydrogen bonds made by probes with the ability to form two hydrogen bonds. These functions were designed so as to model the experimentally observed angular dependence of the hydrogen bonds. We also describe the procedure to locate the position and orientation of the probe at which the interaction energy is optimized. The use of this procedure is demonstrated by examples of biological and pharmacological interest which show that it can produce results that are consistent with other theoretical approaches and with experimental observations.

In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus.

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

An anatomy-based coordinate system for the description of the kinematic displacements in the human knee.

The effects and interaction of the anatomical displacements in the human knee are a prerequisite to an accurate assessment and communication of the kinematic data. For the kinematic information to be used to improve diagnosis and treatment, and for better prosthetic design and installation, there must be clear, concise, and universal definitions of the displacements. In general, the displacements are defined as three translations and three rotations. In this paper, anatomic landmarks on the femur and on the tibia are used to define the locations and orientations of the six displacement axes; i.e. three translational and three rotational displacement axes. The most commonly accepted kinematic representation of the knee joint, in the literature, is a special geometry three-cylindric open chain in which the axes of the cylindric joints are defined according to the rotational displacement axes. The sequentially adjacent joint axes are assumed to not only intersect but to intersect at right-angles. The open chain permits a total of six degrees of freedom between a Cartesian reference frame attached to the femur and a Cartesian reference frame attached to the tibia. In this paper, the three rotational axes are shown to be skewed and off-set from each other, therefore, a three-cylindric open chain with skewed joint axes is proposed to measure the six displacements between the two reference frames. The authors believe that the proposed open chain is the most general to date and provides a more realistic representation of the displacements in the knee. To illustrate the significance of the reference frames on the interpretation of measured data, the anterior/posterior drawer is plotted against per cent gait cycle for three existing open chains and the proposed open chain.

An unusual group-A rotavirus associated with an epidemic of diarrhea among three-month-old calves.

During the 1992 and 1993 breeding seasons, epidemics of diarrhea among calves approximately 3 months old in a cow/calf operation were reported. Rotavirus was determined to be the probable cause, but because rotavirus typically affects younger calves, further investigations were conducted to determine the characteristics of the virus. Virus isolated from the feces of 1 affected calf was found to be antigenically distinct from the vaccine strain used. The primary water source was a slough, and rapid spread of infection may have been a result of fecal contamination of the slough. In both years, the epidemic began shortly after migrating cattle egrets arrived in the district.

A transgenic zebrafish model for monitoring glucocorticoid receptor activity.

Gene regulation resulting from glucocorticoid receptor and glucocorticoid response element interactions is a hallmark feature of stress response signaling. Imbalanced glucocorticoid production and glucocorticoid receptor activity have been linked to socioeconomically crippling neuropsychiatric disorders, and accordingly there is a need to develop in vivo models to help understand disease progression and management. Therefore, we developed the transgenic SR4G zebrafish reporter line with six glucocorticoid response elements used to promote expression of a short half-life green fluorescent protein following glucocorticoid receptor activation. Herein, we document the ability of this reporter line to respond to both chronic and acute exogenous glucocorticoid treatment. The green fluorescent protein expression in response to transgene activation was high in a variety of tissues including the brain, and provided single-cell resolution in the effected regions. The specificity of these responses is demonstrated using the partial agonist mifepristone and mutation of the glucocorticoid receptor. Importantly, the reporter line also modeled the temporal dynamics of endogenous stress response signaling, including the increased production of the glucocorticoid cortisol following hyperosmotic stress and the fluctuations of basal cortisol concentrations with the circadian rhythm. Taken together, these results characterize our newly developed reporter line for elucidating environmental or genetic modifiers of stress response signaling, which may provide insights to the neuronal mechanisms underlying neuropsychiatric disorders such as major depressive disorder.

Surgical management of suspected congenital luxation of the radial head in three dogs.

CASE HISTORIES: Three dogs, aged between 11 and 20 weeks, were presented with unilateral forelimb lameness, with an associated bony prominence on the lateral elbow. CLINICAL FINDINGS AND DIAGNOSIS: Radiographs revealed a caudolateral luxation of the radial head in all cases, consistent with a diagnosis of suspected congenital luxation of the radial head. Surgical reduction and stabilisation involved open reduction of the radial head, and in two cases the use of a trans articular pin. CLINICAL RELEVANCE: Congenital luxation of the radial head is an uncommon condition that has conflicting reports in the literature regarding its cause, heritability, breed predisposition, treatment and prognosis. The three cases here occurred in young dogs of English Bull Terrier, Jack Russell Terrier, and Staffordshire Bull Terrier breeds. Surgical reduction and stabilisation provided successful outcomes, in all cases. Only six cases of congenital luxation of the radial head managed surgically have previously been reported in the literature.

Evidence that the bradykinin-induced activation of phospholipase D and of the mitogen-activated protein kinase cascade involve different protein kinase C isoforms.

The effect of alkylglycerol supplementation on protein kinase C (PKC)-mediated signaling events has been studied in fibroblasts from Zellweger patients (SF 3271 cells). Western blotting analysis established that Zellweger fibroblasts express PKC alpha, epsilon, and zeta. Incubation with bradykinin induced a rapid transient translocation of PKC alpha and a more sustained translocation of PKC epsilon to the particulate fraction; translocation of PKC zeta was unaffected. Bradykinin-induced translocation and activation of PKC alpha, but not translocation of PKC epsilon, was blocked in SF 3271 cells which had been incubated with 1-O-hexadecylglycerol (1-O-HDG; 20 micrograms/ml) for 24 h and then incubated in the absence of 1-O-HDG and serum for a further 24 h. Supplementation with 1-O-HDG increased the mass of ether-linked phospholipid. Bradykinin initiated a transient increase in cytosolic Ca2+ concentration in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplementation. Bradykinin also caused a rapid activation of phospholipase D (PLD), measured by phosphatidylbutanol accumulation, and mitogen-activated protein kinase (MAPK) determined by myelin basic protein phosphorylation of Mono Q fractions. Both events were blocked by preincubation of the cells with 12-O-tetradecanoylphorbol-13-acetate for 24 h to deplete PKC protein. 1-O-HDG supplementation prevented the bradykinin-induced activation of PLD, but had no effect on the stimulation of MAPK activity. These results establish that modulation of the ether lipid composition of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC alpha, most likely PKC epsilon, is involved in MAPK activation.

Carbon dioxide-induced hypoxia causes selective loss of rat brain protein kinase C-gamma.

Protein kinase C purified from the brains of rats killed by decapitation contained isozymes -alpha, beta and -gamma, whereas brain from animals killed by CO2-anoxia contained only the -alpha and -beta forms. Immunoblotting experiments established that exposure to CO2 resulted in a selective loss of protein kinase C-gamma protein.