Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Topical sodium thiosulfate for calcinosis cutis associated with autoimmune connective tissue diseases: the Mayo Clinic experience, 2012-2017.

In this case series, we retrospectively identified all patients treated with topical sodium thiosulfate (TST) for calcinosis cutis (CC) associated with underlying autoimmune connective tissue diseases at Mayo Clinic (Rochester, MN, USA) during the period 1 January 2012 to 27 June 2017. Of 28 patients identified (mean age 57.0 years; 96% female), 19 (68%) had clinical improvement of their CC with TST, 7 (25%) had no response and 2 (7%) had unknown response. There were adverse events in three patients: two had skin irritation and the third, who had a zinc allergy, experienced pain with application. Overall, our findings support those of previous case reports that TST appears to be a relatively well-tolerated adjuvant treatment for CC, although future studies with a control group are warranted to assess the true efficacy of TST for the indication of CC.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

A survey of veterinary antimicrobial prescribing practices, Washington State 2015.

Antimicrobial resistance is a growing global health issue. It is also a recognised problem in veterinary medicine. Between September and December 2015 the authors administered a cross-sectional survey to licensed veterinarians in Washington State to assess factors affecting antimicrobial prescribing practices among veterinarians in Washington State. Two hundred and three veterinarians completed the survey. The majority of respondents (166, 82 per cent) were engaged in small animal or exotic animal practice. 24 per cent of respondents reported not ordering culture and sensitivity (C/S) testing in practice. Of the 76 per cent of veterinarians who reported ordering C/S tests, 36 per cent reported ordering such testing 'often' or 'always' when treating presumptive bacterial infections. Most respondents (65 per cent) mentioned cost as the most common barrier to ordering a C/S test. Only 16 (10 per cent) respondents reported having access to or utilising a clinic-specific antibiogram. This survey demonstrated that while antimicrobials are commonly used in veterinary practice, and veterinarians are concerned about antimicrobial resistance, cost is a barrier to obtaining C/S tests to guide antimicrobial therapy. Summaries of antimicrobial resistance patterns are rarely available to the practising veterinarian. Efforts to promote antimicrobial stewardship in a 'One Health' manner should address barriers to the judicious use of antimicrobials in the veterinary practice setting.

Cardiovascular diseases in older patients with osteoporotic hip fracture: prevalence, disturbances in mineral and bone metabolism, and bidirectional links.

BACKGROUND: Considerable controversy exists regarding the contribution of mineral/bone metabolism abnormalities to the association between cardiovascular diseases (CVDs) and osteoporotic fractures. AIMS AND METHODS: To determine the relationships between mineral/bone metabolism biomarkers and CVD in 746 older patients with hip fracture, clinical data were recorded and serum concentrations of parathyroid hormone (PTH), 25-hydroxyvitamin D, calcium, phosphate, magnesium, troponin I, parameters of bone turnover, and renal, liver, and thyroid functions were measured. RESULTS: CVDs were diagnosed in 472 (63.3%) patients. Vitamin D deficiency was similarly prevalent in patients with (78.0%) and without (82.1%) CVD. The CVD group had significantly higher mean PTH concentrations (7.6 vs 6.0 pmol/L, P < 0.001), a higher prevalence of secondary hyperparathyroidism (SPTH) (PTH > 6.8 pmol/L, 43.0% vs 23.3%, P < 0.001), and excess bone resorption (urinary deoxypyridinoline corrected by creatinine [DPD/Cr] > 7.5 nmol/µmol, 87.9% vs 74.8%, P < 0.001). In multivariate regression analysis, SHPT (odds ratio [OR] 2.6, P = 0.007) and high DPD/Cr (OR 2.8, P = 0.016) were independent indictors of CVD. Compared to those with both PTH and DPD/Cr in the normal range, multivariate-adjusted ORs for the presence of CVD were 17.3 (P = 0.004) in subjects with SHPT and 9.7 (P < 0.001) in patients with high DPD/Cr. CVD was an independent predicator of SHPT (OR 2.8, P = 0.007) and excess DPD/Cr (OR 2.5, P = 0.031). CVD was predictive of postoperative myocardial injury, while SHPT was also an independent predictor of prolonged hospital stay and in-hospital death. CONCLUSION: SHPT and excess bone resorption are independent pathophysiological mediators underlying the bidirectional associations between CVD and hip fracture, and therefore are important diagnostic and therapeutic targets.

Provision of vascular surgery in England in 2012.

INTRODUCTION: In 2009 the Vascular Society of Great Britain and Ireland reported its recommendations for The Provision of Vascular Services for Patients with Vascular Disease. The objective is to halve the UK elective surgery mortality rate for Abdominal Aortic Aneurysm to 3.5% by 2013. From 16th March 2012, statutory approval has been given by Parliament to recognise Vascular Surgery as a Specialty in the UK. This study assesses the provision of vascular surgery in acute trusts across England. METHOD: From the Department of Health, 169 acute trusts were identified in England and each acute trust was emailed under the Freedom of Information Act. RESULTS: There was a 98.8% response rate. There are currently 80 trusts in England providing acute and elective arterial and aortic surgery, with 48 vascular hubs and 32 trusts which either provide a local on call network or are currently under review. Within the 48 vascular hubs there are a mean of 4.8 consultants and 3.75 middle grades. The on call rota was on average a 1 in 6. CONCLUSION: This study has shown that currently 80 trusts in England provide acute and elective arterial and aortic surgery with 48 centralised complex and arterial vascular services. An integrated vascular service will provide the best quality of care, develop the latest techniques and improve clinical standards.

Identification and evaluation of agents isolated from traditionally used herbs against Ophiophagus hannah venom.

The aim of this study was firstly to identify active molecules in herbs, that are traditionally used for the treatment of snake bite, such as Curcuma antinaia, Curcuma contravenenum, Andrographis paniculata, and Tanacetum parthenium; secondly to test similar structurally related molecules and finally to prepare and evaluate an efficient formulation against Ophiophagus hannah venom intoxification. Three labdane based compounds, including labdane dialdehyde, labdane lactone, and labdane trialdehyde and two lactones including 14-deoxy-11,12-didehydroandrographolide and parthenolide were isolated by column chromatography and characterised. Using the isolated rat phrenic nerve-hemidiaphragm preparation, the antagonistic effect of crude extracts, isolated compounds and prepared formulations were measured in vitro on the inhibition of the neuromuscular transmission. Inhibition on muscle contraction, produced by the 5 µg/mL venom, was reversed by test agents in organ bath preparations. A labdane trialdehyde, isolated from C. contravenenum, was identified as the best antagonising agent in the low micromolar range. Tests on formulations of the most potent C. contravenenum extract showed, that the suppository with witepsol H15 was an effective medicine against O. hannah venom. This study elucidated the active compounds, accounting for the antivenin activity of traditionally used herbs and suggested the most suitable formulation, which may help to develop potent medicines for the treatment of snake bite in the future.

Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype.

OBJECTIVES: Acetazolamide has been the most commonly used treatment for hypokalemic periodic paralysis since 1968. However, its mechanism of efficacy is not fully understood, and it is not known whether therapy response relates to genotype. We undertook a clinical and genetic study to evaluate the response rate of patients treated with acetazolamide and to investigate possible correlations between response and genotype. METHODS: We identified a total of 74 genotyped patients for this study. These included patients who were referred over a 15-year period to the only U.K. referral center or to a Chinese center and who underwent extensive clinical evaluation. For all genotyped patients, the response to acetazolamide therapy in terms of attack frequency and severity was documented. Direct DNA sequencing of CACNA1S and SCN4A was performed. RESULTS: Only 46% of the total patient cohort (34 of 74) reported benefit from acetazolamide. There was a greater chance of benefit in patients with mutations in CACNA1S (31 responded of 55 total) than in those with mutations in SCN4A (3 responded of 19 total). Patients with mutations that resulted in amino acids being substituted by glycine in either gene were the least likely to report benefit. CONCLUSIONS: This retrospective study indicates that only approximately 50% of genotyped patients with hypokalemic periodic paralysis respond to acetazolamide. We found evidence supporting a relationship between genotype and treatment response. Prospective randomized controlled trials are required to further evaluate this relationship. Development of alternative therapies is required.

Efficacy of omeprazole versus high-dose famotidine for prevention of exercise-induced gastritis in racing Alaskan sled dogs.

BACKGROUND: Omeprazole and famotidine both reduce severity of exercise-induced gastritis, but administering famotidine is easier than administering omeprazole during racing competition. HYPOTHESIS: Famotidine is more efficacious than no treatment in reducing severity of exercise-induced gastritis; and high-dose famotidine is more efficacious than omeprazole in reducing severity of exercise-induced gastritis. ANIMALS: Experiment 1: Randomized placebo-controlled study, 36 sled dogs (3-8 years); Experiment 2: Randomized positive-control study, 52 sled dogs (2-8 years). METHODS: Experiment 1: Equal numbers of dogs randomly assigned to famotidine (20 mg q24h) or no treatment groups. Gastroscopy was performed 24 hours after the dogs ran 330 miles. Mucosal appearance was blindly scored by previously described scoring system. Experiment 2: Equal numbers of dogs randomly assigned to omeprazole (20 mg q24h) or high-dose famotidine (40 mg q12h) groups. Gastroscopy was performed 48 hours before and 24 hours after the dogs ran 300 miles. Mucosal appearance was blindly scored by previously described scoring system. RESULTS: Famotidine reduced the prevalence of clinically relevant, exercise-induced gastric lesions compared with no treatment (7/16 versus 11/16, P = .031). Compared with high-dose famotidine, omeprazole significantly decreased the severity (0.4 versus 1.2, P = .0002) and prevalence (2/23 versus 7/21, P = .049) of gastric lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, omeprazole is superior to famotidine in preventing gastritis in dogs running 300 miles. Routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing Alaskan sled dogs.

Towards the routine use of brain imaging to aid the clinical diagnosis of disorders of consciousness.

Clinical audits have highlighted the many challenges and dilemmas faced by clinicians assessing persons with disorders of consciousness (vegetative state and minimally conscious state). The diagnostic decision-making process is highly subjective, dependent upon the skills of the examiner and invariably dictated by the patients' ability to move or speak. Whilst a considerable amount has been learnt since Jennett and Plum coined the term 'vegetative state', the assessment process remains largely unchanged; conducted at the bedside, using behavioural assessment tools, which are susceptible to environmental and physiological factors. This has created a situation where the rate of misdiagnosis is unacceptably high (up to 43%). In order to address these problems, various functional brain imaging paradigms, which do not rely upon the patient's ability to move or speak, have been proposed as a source of additional information to inform the diagnostic decision making process. Although accumulated evidence from brain imaging, particularly functional magnetic resonance imaging (fMRI), has been encouraging, the empirical evidence is still based on relatively small numbers of patients. It remains unclear whether brain imaging is capable of informing the diagnosis beyond the behavioural assessment and whether brain imaging has any prognostic utility. In this study, we describe the functional brain imaging findings from a group of 41 patients with disorders of consciousness, who undertook a hierarchical speech processing task. We found, contrary to the clinical impression of a specialist team using behavioural assessment tools, that two patients referred to the study with a diagnosis of vegetative state did in fact demonstrate neural correlates of speech comprehension when assessed using functional brain imaging. These fMRI findings were found to have no association with the patient's behavioural presentation at the time of investigation and thus provided additional diagnostic information beyond the traditional clinical assessment. Notably, the utility of brain imaging was further underlined by the finding that the level of auditory processing revealed by functional brain imaging, correlated strongly (rs = 0.81, P < 0.001) with the patient's subsequent behavioural recovery, 6 months after the scan, suggesting that brain imaging may also provide valuable prognostic information. Although further evidence is required before consensus statements can be made regarding the use of brain imaging in clinical decision making for disorders of consciousness, the results from this study clearly highlight the potential of imaging to inform the diagnostic decision-making process for persons with disorders of consciousness.

Continuous expression of corticotropin-releasing factor in the central nucleus of the amygdala emulates the dysregulation of the stress and reproductive axes.

An increase in corticotropin-releasing factor (CRF) is a putative factor in the pathophysiology of stress-related disorders. As CRF expression in the central nucleus of the amygdala (CeA) is important in adaptation to chronic stress, we hypothesized that unrestrained synthesis of CRF in CeA would mimic the consequences of chronic stress exposure and cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, increase emotionality and disrupt reproduction. To test this hypothesis, we used a lentiviral vector to increase CRF-expression site specifically in CeA of female rats. Increased synthesis of CRF in CeA amplified CRF and arginine vasopressin peptide concentration in the paraventricular nucleus of the hypothalamus, and decreased glucocorticoid negative feedback, both markers associated with the pathophysiology of depression. In addition, continuous expression of CRF in CeA also increased the acoustic startle response and depressive-like behavior in the forced swim test. Protein levels of gonadotropin-releasing hormone in the medial preoptic area were significantly reduced by continuous expression of CRF in CeA and this was associated with a lengthening of estrous cycles. Finally, sexual motivation but not sexual receptivity was significantly attenuated by continuous CRF synthesis in ovariectomized estradiol-progesterone-primed females. These data indicate that unrestrained CRF synthesis in CeA produces a dysregulation of the HPA axis, as well as many of the behavioral, physiological and reproductive consequences associated with stress-related disorders.Molecular Psychiatry (2009) 14, 37-50; doi:10.1038/mp.2008.91; published online 12 August 2008.

Effect of a Bacillus-based direct-fed microbial feed supplement on growth performance and pen cleaning characteristics of growing-finishing pigs.

A direct-fed microbial (DFM) based on a combination of Bacillus organisms specifically selected to increase the manure decomposition process was evaluated to determine its efficacy for improving growth performance and manure dissolution time. Three experiments involving 336 crossbred barrows and gilts were conducted to determine the effect of the Bacillus-based direct-fed microbial on growth performance and pen cleaning time. In each experiment, 2 dietary treatments (0 and 0.05% DFM) were fed during the growing-finishing period throughout the experiment, such that the DFM provided 1.47 x 10(8) cfu of Bacillus organisms per gram of supplement. Data from the 3 experiments were combined for analysis, such that there were 28 pens representing each of the 2 treatments. Pigs were weighed and feed intake was determined at the initiation and termination of each phase (starter, grower, and finisher). At the end of Exp. 1 and 3, pen cleaning time was determined by measuring the time required for each pen to be scraped and washed with a high-pressure sprayer. Additionally, 2 solid manure mat samples weighing approximately 4 g each were collected from solid manure buildup in each pen (16 pens/treatment), and the time required to completely disperse each manure mat sample was determined. Gain:feed improved (P < 0.05) in pigs fed Bacillus compared with the control diet during the finisher phase and throughout the combined growing-finishing period. The time required to dissolve the manure mat was reduced (P < 0.01) in samples collected from pens containing pigs fed Bacillus compared with samples from control pens. An additional evaluation was conducted in a commercial swine production facility using statistical process control analysis. Statistical process control analysis determined that supplementation with Bacillus increased the expected mean for ADG and decreased the expected mean for death loss percentage. Supplementation with a DFM composed of specifically selected Bacillus organisms improved G:F and decreased the time required to disperse a swine manure mat sample in a controlled study conducted at swine research facilities. Furthermore, when evaluated in a commercial swine production facility, the Bacillus-based DFM improved ADG and reduced mortality of pigs during the growing-finishing period.

Interactive effect of ractopamine and dietary fat source on quality characteristics of fresh pork bellies.

Crossbred pigs (n = 216) were used to test the interaction, if any, of ractopamine (RAC) and dietary fat source on the characteristics of fresh pork bellies. Pigs were blocked by BW (77.6 +/- 6.5 kg) and allotted randomly to pens (6 pigs/pen). After receiving a common diet devoid of RAC for 2 wk, pens within blocks were assigned randomly to 1 of 4 treatments arranged in a 2 x 2 factorial design, with 5% fat (beef tallow vs. soybean oil) and RAC (0 vs. 10 mg/kg). At the conclusion of the 35-d feeding period, pigs were slaughtered at a commercial pork packing plant (average BW of 108.8 +/- 0.6 kg), and fresh bellies were captured during carcass fabrication. Neither RAC (P = 0.362) nor fat source (P = 0.247) affected belly thickness. Subjective (bar-suspension) or objective (compression test) measures of belly firmness were not (P > or = 0.148) affected by the inclusion of RAC in the diet; however, bellies from pigs fed soybean oil (SBO) were softer than those from pigs fed beef tallow (BT), as indicated by perpendicular (P < or = 0.005) and parallel (P < 0.001) suspensions. Moreover, bellies from BT-fed pigs required more (P = 0.096) force to compress 50% of their thickness than bellies from SBO-fed pigs (52.29 vs. 43.51 kg). Color (L*, a*, and b* values) of the belly lean and fat was not (P > or = 0.131) affected by RAC, and lean color was similar (P > or = 0.262) between fat sources; however, belly fat from BT-fed pigs was lighter (P = 0.030) and redder (P = 0.013) in color than belly fat from SBO-fed pigs. Bellies of SBO-fed pigs had greater (P < 0.001) proportions of PUFA and lower (P < 0.001) proportions of SFA and MUFA than belly fat from pigs fed BT. Regardless of the RAC inclusion level, PUFA:SFA and iodine values were lower in belly fat from pigs fed BT than SBO; however, within SBO-fed pigs, PUFA:SFA and iodine values were further increased by feeding RAC (RAC x fat source, P < 0.001). As expected, dietary fat source altered the fatty acid composition of fresh pork bellies, which subsequently impacted fresh belly firmness. Interestingly, including RAC in swine finishing diets exacerbated the effect of feeding SBO on pork fat polyunsaturation.

When thoughts become action: an fMRI paradigm to study volitional brain activity in non-communicative brain injured patients.

The assessment of voluntary behavior in non-communicative brain injured patients is often challenging due to the existence of profound motor impairment. In the absence of a full understanding of the neural correlates of consciousness, even a normal activation in response to passive sensory stimulation cannot be considered as proof of the presence of awareness in these patients. In contrast, predicted activation in response to the instruction to perform a mental imagery task would provide evidence of voluntary task-dependent brain activity, and hence of consciousness, in non-communicative patients. However, no data yet exist to indicate which imagery instructions would yield reliable single subject activation. The aim of the present study was to establish such a paradigm in healthy volunteers. Two exploratory experiments evaluated the reproducibility of individual brain activation elicited by four distinct mental imagery tasks. The two most robust mental imagery tasks were found to be spatial navigation and motor imagery. In a third experiment, where these two tasks were directly compared, differentiation of each task from one another and from rest periods was assessed blindly using a priori criteria and was correct for every volunteer. The spatial navigation and motor imagery tasks described here permit the identification of volitional brain activation at the single subject level, without a motor response. Volunteer as well as patient data [Owen, A.M., Coleman, M.R., Boly, M., Davis, M.H., Laureys, S., Pickard J.D., 2006. Detecting awareness in the vegetative state. Science 313, 1402] strongly suggest that this paradigm may provide a method for assessing the presence of volitional brain activity, and thus of consciousness, in non-communicative brain-injured patients.

The overall status in rheumatoid arthritis (OSRA) measure--further evidence to support its use in clinical practice.

OBJECTIVES: The overall status in rheumatoid arthritis (OSRA) instrument is a simple summary of health status, including disease activity (OSRA-A) and damage (OSRA-D) scores. Despite evidence of the validity of the OSRA, uptake has been low. This study aimed to assess the responsiveness and re-examine the validity of the OSRA using the measures from the British Rheumatoid Outcome Study Group (BROSG) randomized controlled trial of aggressive vs symptomatic treatment of rheumatoid arthritis (RA) patients. METHODS: 466 patients were recruited. Outcome measures included the OSRA, the OMERACT core set and the DAS28, and were collected at baseline and annually for the 3 yrs of the trial. X-rays of the hands and feet were taken at baseline and 3 yrs. Patients were assigned a Townsend score (a measure of social deprivation) according to area of residence. Construct validity was assessed by correlating the OSRA with a range of outcome measures, and testing for the known inequality in RA outcome between patients classified by social deprivation. Responsiveness to change was assessed against self-reported change over the first year of the trial. RESULTS: The OSRA-A and OSRA-D measures demonstrated construct validity, performing as hypothesized. The OSRA-A was the most responsive measure in the BROSG trial in detecting patient reported improvement and deterioration. The OSRA-D demonstrated similar responsiveness to alternative measures. CONCLUSIONS: Our results demonstrate the validity and responsiveness of the OSRA, and its potential for inclusion in clinical trials. More important, as the OSRA is quick and easily calculated, uses routinely collected information, and provides useful quantitative information about a patient's status and progress it is suitable for use in the routine clinic.