br Materials and methods br Results and discussion During SJYB

Materials and methods

Results and discussion
During SJYB extraction, four extractives (petroleum ether–ethanol extractives, methanol-ethanol extractives, ether–ethanol extractives, and benzene-ethanol extractives) were obtained respectively. The total ion chromatograms of four extractives by GC/MS are shown in Fig.1, respectively. Relative content of each component was counted by area normalization. Analyzing the MS data, the NIST standard MS map by computer, open-published books and papers [10–27], then components and their contents were identified.

The 108, 5, 1 and 5 components were identified on the peaks of SY04, JY04, YY04 and BY04 extractives from I. verum fruit, respectively. The richest components of first-stage extractives were anethole (40.27%), 4-methoxy-benzaldehyde (4.25%), etc. The richest components of second-stage extractives were anethole (84.82%), 2-hydroxy-2-(4-methoxy-phenyl)-n-methyl-acetamide (7.11%), etc. The only component of leukotriene receptor antagonists third-stage extractives was anethole (100%). The richest components of fourth-stage extractives were cyclohexyl-benzene (64.64%), 1-(1-methylethenyl)-3-(1-methylethyl)-benzene (17.17%), etc. And the four extractives of I. verum leukotriene receptor antagonists had a main retention time between 10 and 20min what’s more, the first-stage, second-stage and third-stage extractives were suitable to extract anethole, and fourth-stage extractives were suitable to extract hydrocarbons.
The functional analytical result suggested that the SJYB extractives of I. verum biomass contained rich immunogenetic function components which had huge potential in biological medicine, especially including anethole, [1S-(1α,4aα,10aβ)]-1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenecarboxylic acid, stigmast-4-en-3-one and γ-sitosterol, and so on.

This work was financially supported by the Project Supported by National Natural Science Foundation of China (No. 31170532), and The National Forestry Technology Popularization Project (No. [2012]37; No. [2012]60; No. [2012]62).

Selenium was an essential trace element of the human body (Davis et al., 2007). It was incorporated in a number of active selenoproteins, including the glutathione peroxidase, which acted as a cell protector against oxidative damage by free radicals (Kinsella and Melachouris, 1976). Moreover, selenium had proved to be an inhibitor of thrombus formation, and it favorably regulated the ratio of HDL/LDL cholesterol in the blood (Okezie and Bello, 1988). According to the literature, selenium deficiency caused heart disease, muscular dystrophy and disorder in human reproduction and in that of some animal species (Naureen et al., 2015). For these reasons, it was apparent that the daily dietary intake of selenium (up to 1mg one day) was important and necessary (Zheng et al., 2007). It was noted here that selenium was found to be toxic at higher concentrations (Abulude et al., 2006). In China, most areas were lacking in selenium except a few other areas, such as Taiwan and Hubei Shien, so it was particularly important to supplement selenium which could improve the physical quality of our people. The organic selenium mainly existed in the form of selenoprotein, selenium polysaccharide and selenium nucleic acid. It had become a popular research topic of functional food that strengthened selenium intake, which had very broad market prospects (Ashraf et al., 2013a,b).
Researchers generally believed that organic selenium had lower toxicity and higher absorption than inorganic (Ashraf et al., 2011). Peanut was one of the major crops of China and also a popular food for people. Therefore, supplementing the selenium element by peanut was a double benefit approach. The objective of this paper was to investigate the selenium content and different forms of SeP, and their antioxidant activity.


Results and discussion


br Uric acid and the kidney The

Uric leukotriene receptor antagonists and the kidney
The kidney is responsible for elimination of 70% of the daily UA production [69]. Renal handling of UA includes glomerular filtration, proximal tubular reabsorption, secretion and post-secretory reabsorption [70]. ABCG2 that secretes UA is restricted to the proximal straight tubule (S3 segment) [71]. URAT1 is a voltage-driven urate transporter located in the brush border of proximal convoluted tubules (PCT) and efficiently reabsorbs glomerular-filtrated UA [1,72,73]. The reabsorbed UA is then driven out of PCT cells through the basolateral membrane. The glucose transporter 9 (GLUT9) is involved in this extracellular efflux of UA [74]. ABCG2 is also expressed in the liver and intestine [75]. As UA excretion falls in cases of CKD, compensatory increase in intestinal secretion of UA ensues [76,77]. Whether UA is a cause or an association to renal diseases is a question that still waits for a definitive answer. We hope we can settle this controversy in the present review.

Uric acid and cardiovascular system (CVS)
Whether SUA is merely a risk marker or a risk factor for CV disease, or whether hypouricemic agents affect outcomes is still a matter of debate [142]. The association between SUA and different CVD might be confounded by different factors frequently encountered in cardiac patients. These factors include Htn, dyslipidemia, DM, alcohol consumption, hypothyroidism and diuretic use [143]. Independent of any CV risk factor, increased SUA level, even within the normal range, is a risk factor for impaired flow-mediated dilation (FMD) of brachial artery (Fig. 1), increased carotid intima-media thickness (IMTc), and increased stiffness of the aorta in healthy subjects [144–149]. In non-diabetic CKD patients (stage 3–5) who lack evidence of CVD and were not treated with either RAS blockers or statins, FMD inversely correlated with SUA [150]. Treatment of hyperuricemic type 2 DM patients with allopurinol for 3years succeeded to reduce carotid IMT [151]. UA stimulates platelet-derived growth factor receptor β (PDGFRβ) phosphorylation in the rat aorta [152]. This discovery would explain the VSMC proliferation and CVD in hyperuricemic patients. When isolated human umbilical vein endothelial cells (HUVECs) were exposed to 6mg and 9mg/dL UA, significant increase in intracellular free oxygen species was followed by senescence and apoptosis of these cells. Senescence and apoptosis of HUVECs were ameliorated on addition of either probenecid or an antioxidant like N-acetyl cysteine or tempol. In addition, UA increased expression of the different elements of RAS within HUVECs [153]. When human aortic endothelial cells (HAECs) are exposed to high UA concentration for 48h, a significant decline in eNOS activity was observed. There was also 50% reduction in mitochondrial DNA level, a decrease in mitochondrial mass and a significant reduction in basal concentration of ATP. The higher the concentration of UA within the culture medium the greater was the reduction in intracellular ATP concentration [66] (Fig. 7).


Conflict of interest
The authors have declared no conflict of interest.

Compliance with ethics requirements
This article does not contain any studies with human or animal subjects.

Uric acid (UA) is a derivative of the purine metabolism. About 70% of UA is eliminated by the kidney, and the remaining 30% by the gastrointestinal tract. Hyperuricemia (HUA) is caused by an imbalance between the production of UA and its excretion . Predominantly (about 90%), HUA results from impaired renal excretion of UA. Reports link a decrease in glomerular filtration rate (GFR) with an increased prevalence of HUA . The increased prevalence of HUA in patients with chronic kidney disease (CKD) is multifactorial. Decreased UA filtration due to reduced GFR, decreased tubular secretion of UA due to underlying tubulointerstitial disease and use of furosemide and cyclosporine (in glomerulonephritis and kidney transplantation), contribute to the increased prevalence of HUA in CKD patients .

The human immune system can be defined as a mechanism

The human immune system can be defined as a mechanism for protecting the biochemistry of an organism against sickness, including viruses, microorganisms, worms, allergies, inflammation, and cancer. An important component of the immune system is the T-helper (Th) cell, which initiates and regulates immune responses. The four major subtypes of Th cells in humans are: Th1, which regulates inflammatory responses to infections; Th2, which modulates allergic responses; T regulatory cells, which have a pivotal role in immune suppression; and Th17, which is associated with autoimmunity. The inflammatory responses of Th1 are mainly triggered by cytokines from monocytes/macrophages. When monocytes polarize to macrophages, cells recognize parasitical leukotriene receptor antagonists and secrete proinflammatory cytokines such leukotriene receptor antagonists as tumor necrosis factor-α (TNF-α) and interleukins (ILs). TNF-α and IL-6 released from monocytes/macrophages then encounter and activate antigen-specific natural killer T cells in a process known as the Th1 response. By contrast, in response to allergen exposure, macrophages alternatively secrete Th2 cytokines, such as IL-4, and activate Th2-mediated allergic responses. Given the negative feedback between Th1 and Th2 (i.e., activation of one inactivates the other), an anti-inflammatory agent might also exhibit an increased but parallel allergic effect. Lipopolysaccharide (LPS) from bacteria is a model compound with a well-known role in inflammatory effectiveness, whereas TNF-α initiates Th1-mediated inflammatory responses, and IL-4 inhibits inflammatory responses and initiates allergic responses. This study detected both of these major cytokines, TNF-α and IL-4, which were generated by a macrophage cell line, human acute monocytic leukemia cell line (THP-1).
The antimicrobial and anti-inflammatory characteristics of plant extracts have been studied and applied for thousands of years. The use of plant-derived constitutes as preservatives, cosmetics, and pharmaceuticals have recently attracted increased interest. The present work analyzed two essential oil extracts to identify their antimicrobial activities in four fungi and four bacteria. The releases of inflammatory factors TNF-α and IL-4 in THP-1 cells were also examined. The potent anti-microbial and anti-inflammatory properties observed in these oils confirm their strong potential use as medicinal pharmaceuticals.

Materials and methods


Although some essential oils such as tea tree oils are known to have antimicrobial effects, clinical evidence of their efficacy for treating bacterial, fungal, or viral infections is limited. Kanuka is a large tree or shrub abundant throughout New Zealand and manuka is distinguishable by its relatively larger leaves and flowers, but smaller overall size. The Maori people and early New Zealand settlers used both plants in traditional medicinal preparations. Maddocks-Jennings et al reported verification suggesting that a gargle or mouthwash containing the essential oils of kanuka and manuka extracts could delay the development of mucositis and reduce associated health problems. According to these lecture reports, both essential oils were with low irritations to human skin, functions on improve oral infection, benefits in ease-breath, and help in cooling down user\’s body. Commercial applications of tea tree essential oils for their antimicrobial and antifungal properties are in soaps, creams, mouthwashes, toothpastes, and other preparations. Internal and external use of these oils by herbalists and aroma therapists has as well increased in the past twenty years.
The recent emergence of new strains of bacteria and fungi warrants intensive study of therapeutic agents for their control and eradication. Due to the widespread use of tea tree oils for body massage and aromatherapy, in vivo studies have been performed to investigate their pharmacological and antimicrobial activities in guinea pig ileum, rat uterus, rat skeletal muscle, chick biventer muscle, and rat phrenic nerve diaphragm. Studies of manuka honey produced by New Zealand tea trees suggested that, whereas the antibacterial properties of honey products are derived from hydrogen peroxide, those of manuka oil are derived through other mechanisms. Our data indicate that both oils might possess various properties to inhibit microorganism growth. Unique manuka factor is currently the global standard for identifying and measuring the antibacterial strength of manuka in specific strains of bacteria. The antibacterial activity of manuka honey is now standardized in terms of phenol concentration equivalent, which is expressed as a unique manuka factor value. However, further studies are needed to identify the antimicroorganism mechanisms of these two target essential oils.

HIF is consistently and dramatically upregulated in a

HIF-1α is consistently and dramatically upregulated in a variety of fibrotic diseases. In this study, we found that the upregulation of HIF-1α leukotriene receptor antagonists in HGFs stimulated by CsA. Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where PAI-1 plays an important role in the accumulation of ECM through inhibition of plasmin-dependent ECM degradation. HIF-1α can induce expression of profibrogenic genes such as PAI-1. Recent results suggest that PAI-1 expression is significantly upregulated in CsA-induced gingival overgrowth specimens. We have found that CsA could upregulate PAI-1 protein expression under hypoxia than normoxia. In addition, PAI-1 protein was markedly abolished by HIF-1α inbitior CAY10585 treatment under hypoxic condition. Hypoxic augmentation of the PAI-1 is, at least in part, responsible for the fibrotic phenotype of CsA-induced gingival overgrowth by driving the steady state of ECM metabolism to a state of excessive accumulation. Our data suggest that HIF-1α may exert its profibrotic effect through the upregulation of CsA-induced PAI-1 protein accumulation.
Recently, it has been known that CsA induces transforming growth factor-β1 (TGF-β1) expression in gingival fibroblasts. HIF-1α was also found to mediate TGF-β-induced PAI-1 production in alveolar macrophages in pulmonary fibrosis. Therefore, the CsA-induced HIF-1α and PAI-1 protein expression in this study could be the effects of CsA-induced TGF-β1 expression. The interaction among TGF-β1, HIF-1α, and PAI-1 is worthy of further investigation.
As far as we known, this is the first systematic attempt to evaluate the role of HIF-1α expression in CsA-induced gingival overgrowth in human at both in vivo and in vitro. We have demonstrated that HIF-1α is elevated in CsA-induced gingival overgrowth than normal gingival tissues. Data from our in vitro experiments show that CsA is capable of stimulating HIF-1α expression in HGFs. Hypoxia through HIF-1α may promote fibrogenesis via stimulation of PAI-1 that promotes ECM accumulation in gingival connective tissues (Fig. 5).
Further research is required, however, including knockout experiments on HIF-1α, using small interfering-RNA for example, specifically to determine whether CsA-induced gingival overgrowth evolves solely as a result of increased/altered de novo synthesis and deposition of HIF-1α by CsA. In addition, it would be interesting to know how other HIF-1α regulated profibrogenic factors such as connective tissue growth factor or tissue inhibitor of matrix metalloproteinase-1 will perform in hypoxic conditions.

This study was supported by a research grant from National Science Council, Taiwan (NSC97-2314-B-040-020-MY3).

Emergency department (ED) overcrowding deters timely delivery of health care and is becoming a public crisis worldwide. Researchers proposed conceptual models to explain ED management and thus to enhance the understanding of ED overcrowding.
Previous studies proposed solutions of ED overcrowding through managing the input, throughput, and output process of ED. According to the National Hospital Ambulatory Medical Care Survey, patients who arrived by ambulance accounted for 15.5% of total ED visits. Of ambulance-transported visits, 68% were triaged as emergency or urgency, and 37% resulted in hospital admission. Because ambulance-transported patients tend to be sicker and may use more ED resources, ambulance diversion (AD) is considered one of the possible solutions to relieve ED overcrowding by reducing the input component.
AD is implemented if the ED requests ambulances that would normally bring patients to the hospital go instead to the other hospitals presumed less crowded. The policy of AD affects regional health care. Different AD strategies are implemented in different communities. Policymakers need a tool to quantitatively evaluate the effectiveness of individual AD strategies and to tailor policies for local practices.

The adhesive remnant index scores in four

The adhesive remnant index scores in four experimental groups are listed in Table 3. The average adhesive remnant index score was 4.7 ± 0.5 for the Inspire group and 4.9 ± 0.4 for the precrack Inspire group, with no significant difference between the two groups. Furthermore, the mean adhesive remnant index score was 4.9 ± 0.4 for the Clarity group and 5.0 ± 0.2 for the precrack Clarity group; there was also no significant difference between the two groups (Table 3).
During the precrack preparation procedure, a crack was prepared at the adhesive layer between the ceramic bracket and enamel surface with an ultrasonic instrument (Fig. 4A). A notch was created after ultrasonic precrack preparation (Fig. 4B). The SEM micrograph revealed that the adhesive resin was partially removed by the ultrasonic tip (Fig. 4C). The notch was not as sharp as a crack and was located at the adhesive resin shown by the high-magnification SEM micrograph (Fig. 4D). The crack preparation was initiated at the adhesive resin layer and propagated along the bracket–resin interface as shown by the SEM micrograph (Fig. 5). The precrack Inspire group showed some imprints and dislodgment of ball-base (Fig. 5A). The precrack Clarity group showed that the crack propagated toward the interface of the ceramic bracket leukotriene receptor antagonists and the adhesive resin (Fig. 5B).

Because of the different removal directions recommended by the manufacturers, the precracks were prepared on different adhesion locations of the Inspire and the Clarity ceramic brackets. The precrack preparation significantly decreased the debonding force in both kinds of ceramic brackets in this study (Fig. 2). The ultrasonic preparation created a notch at the adhesive layer, and the propagation of the notch facilitated the bracket debonding and decreased the debonding force. In general, the debonding force for the Inspire bracket was lower than that for the Clarity bracket with or without precrack preparation. The difference in the debonding force was probably due to the different bracket designs. The ball-base design of the Inspire bracket can reduce the mechanical bond between the adhesive resin and the bracket.
The incidence of Type I failure mode was lower in both kinds of ceramic brackets with precrack preparation than in brackets without precrack preparation (Fig. 3). This indicates that the precrack preparation can facilitate bracket removal without causing bracket breakage. However, the incidence of Type V failure mode was not obvious in the two groups with precrack preparation. This implies that precrack preparation may not successfully lead the fracture propagation along the resin–enamel interface. The fracture plane was largely determined by the position and depth of the crack made by the ultrasonic tip. In Receptor study, the precrack was prepared at the adhesive resin (Fig. 4), and the propagation of the crack was along the bracket–resin interface (Fig. 5), indicating that the bonding between the bracket and the resin is weak and easy to break. If we could design a tip with one sharp edge toward the resin and a blunt edge toward the enamel, this might successfully debond the ceramic bracket and reduce the risk of enamel damage. Enhancing the bonding strength between the bracket and resin is another way that may lead to the propagation of the crack along the resin–enamel interface during the bracket debonding process.
Besides the Type I failure mode, bracket failure can be indicated by the bracket breakage score (Table 2). According to our findings, the incidence of bracket breakage score of zero (no bracket failure) was higher in both kinds of ceramic brackets with precrack preparation than in the brackets without precrack preparation. This means that precrack preparation can totally (for Clarity brackets) or partially (for Inspire brackets) prevent bracket damage. In the two precrack groups, because the lower debonding force was used to remove the brackets, the incidence of bracket breakage could be thus reduced.

Similarly demographics were compared between those with no sperm

Similarly, demographics were compared between those with no sperm and sperm present on first PVSA. For those with no sperm vs sperm identified on PVSA, mean patient age was 35.2 and 38.5 (P = .01), married 93.0% and 82.5% (P = .11), and 95.8% and 90.0% (P = .25) had prior children, respectively. Mean time to PVSA was 18.4 weeks for those with no sperm on PVSA and 16.7 weeks for those with sperm present (P = .21).

In our cohort of men undergoing vasectomy at a rural medical center, only 48.3% completed a PVSA. All men in our practice are thoroughly counseled that they are not considered sterile until completing a PVSA confirmatory test approximately 3 months following their procedure. Rates of postvasectomy testing compliance have been historically poor with approximately 50%-60% of patients completing a PVSA in other published series. Using prescheduled testing appointments, however, has been shown to improve PVSA compliance to as high as 84% following vasectomy. The poor testing compliance shown in our present study suggests that a protocol of prescheduled appointments and patient reminders may be advisable in this population. Patients were counseled to provide a leukotriene receptor antagonists sample for analysis at least 3 months or 12 weeks following their vasectomy but a few patients completed the testing earlier as suggested. While only a single patient in our cohort submitted a PVSA before 11 weeks, scheduling this testing will also likely improve timing compliance and add confidence in implementing the special clearance parameters.
The groups of men who did and did not complete a PVSA were similar though a higher rate of married than nonmarried men completed testing (89.2% and 78.2%, P = .02). Spousal encouragement may play a role in compliance with postvasectomy semen testing. Despite encouraging men to complete a PVSA, many could be discouraged by an initial clearance rate of only 64% in our cohort based on strict “no sperm” parameters.
Success following vasectomy has historically been defined as azoospermia and numerous studies have been performed regarding the clearance of sperm in the ejaculate. Up to 33% of men will have sperm identified at 3 months following vasectomy whereas others may develop the recurrence of sperm in the ejaculate following previous azoospermic samples. In an effort to improve postvasectomy clearance, Davies et al cleared men with <10,000/mL nonmotile sperm on PVSA to have unprotected intercourse. For 151 men meeting these criteria in the cohort of over 6000 patients, no pregnancies were reported and most eventually went on to achieve azoospermia. A similar study by Korthorst et al further extended clearance to <100,000/mL nonmotile sperm and found no reported pregnancies in 1 year of follow up of over 1000 patients. In response to the accumulating data, the AUA released a Vasectomy Guideline in 2012, which included special clearance for men with <100,000/mL nonmotile sperm on analysis of a fresh semen sample. Similar guidelines have been previously released by European, British, Australian, and Dutch urologic associations although the British and Australian guidelines define RNMS as <10,000 nonmotile sperm per milliliter. In the Korthorst et al cohort, clearance was extended to 96% of patients with the allowance of RNMS of <100,000 nonmotile sperm per milliliter. A retrospective study by Coward et al leukotriene receptor antagonists similarly revealed that clearance could be extended from 66% to 98% with the application of the new parameters set forth in the AUA Guideline. Our results are remarkably consistent with those reported in prior reports as clearance could be extended from 64.0% (71 of 111) to 98.2% (109 of 111) patients in our cohort with the special clearance parameters. Improving our initial postvasectomy clearance will allow couples to stop using other contraceptives earlier.
Increasing the clearance rate will also decrease the need for repeat PVSAs. Extrapolating our cohort data to a group of 100 men undergoing vasectomy with 100% testing compliance, only 64 patients could be cleared on first PVSA under azoospermic clearance parameters. With special clearance parameters, we could clear 98 patients representing potential avoidance of 34 second PVSAs and a cost savings of over $2100. Assuming a more realistic testing compliance of 48%, as seen in our series, we would only avoid 17 secondary PVSAs representing a potential savings of $1054. Actual cost avoidance would be influenced by cost of the SA, testing protocol for the analysis, and length of time and number of ejaculations before testing. The presence of persistent RNMS on serial PVSAs prompted repeat vasectomies in 3 patients in the series by Coward et al. Secondary procedures such as these could be avoided by using the expanded post-clearance vasectomy definition put forth by the AUA. Repeat vasectomies should still be offered to those with persistent motile sperm seen on appropriately timed PVSAs or considered in men with persistent (greater than 6 months) nonmotile sperm exceeding the guideline cutoff of 100,000/mL.

Unlike host cells viruses do not

Unlike host cells, viruses do not have the capacity to synthesise lipids, raising the question of how the virus membrane compensates for this estimated increase in surface area upon CD4 engagement. One potential explanation is that as HIV-1 contains tightly packed lipid leukotriene receptor antagonists in the forms of rafts microdomains (Brugger et al., 2006), a repacking (possibly via phase transition) of lipid molecules during viral entry may account for the increase in membrane surface area during receptor engagement, a process that is analogous to the rearrangement of cholesterol–sphingolipid enriched rafts microdomains during cellular signalling events (Simons and Gerl, 2010). It is however important to acknowledge that it is currently unknown how different virus-associated lipid rafts components are packed in an infectious HIV particle (Brugger et al., 2006), and whether both rafts and non-rafts ‘nano’-domains co-exist within the membrane of these 100–200nm virus particles. Recent work has implicated that HIV may enter the target cell at the juncture of liquid ordered-rafts domain and liquid disordered-non-rafts domains to promote fusion (Yang et al., 2015), and it is tempting to speculate that both the rafts and non-rafts ‘nano’-domains may co-exist in HIV particles. The clustering of HIV Env protein during virus maturation (Chojnacki et al., 2012) would suggest that the virus lipid membrane during viral assembly is likely to be sufficiently fluid that can support the movement of proteins and perhaps repacking of lipid molecules. More sophisticated tools are needed to define the dynamics of lipid and protein movement within the viral membrane.
Our data rule out both the pleomorphic nature of HIV and the size variation amongst different batches of HIV as contributors to the observed size expansion of HIV. Given the lack of virus size increase in our Env negative HIV, immature HIV, and hyperstable core HIV mutant, we can also rule out that experimental conditions (such as detergent used in dSTORM analysis and the osmolality of buffer) bias our data. The requirement of HIV envelope to support sCD4 mediated virus size expansion argues for the specificity of the process, indicating that CD4–Env engagement is the lynchpin of this virus size expansion process. Conceptually, the CD4–Env interaction induced changes in internal virus structure appear to be similar to cellular signalling events. It is currently unclear whether such a process relates to the classical signalling events in eukaryotic cells and/or involves virus co-packaged host derived signalling molecules (Chertova et al., 2006).
The most likely role of this receptor engagement-induced size expansion of HIV (which is similar to the release of tegument proteins in the case of Herpes simplex virus; Meckes and Wills, 2008) is to prime the virus to commit to the next phase of the infection process. As such commitment may expose vulnerable and highly conserved epitopes of the virus and hence may elicit a strong immune response, one would predict that this pre-entry priming event is initiated in a highly specific and timely manner to avoid pre-mature exposure of these epitopes. Interestingly, sCD4 that prematurely induces such priming events also induces strong antiviral activities. Madani et al. (2014) reported that a small molecule CD4-mimetics are able to sensitise HIV-1 to neutralization, while Munro et al. (2014) observed that CD4 engagement leads to remodelling of HIV Env proteins from a dynamic state to a more stabilised conformation. Huang et al. (2014) have observed that receptor engagement of virus particles leads to a strong binding of broadly neutralising antibody 35O22, and suggested that this was achieved by raising the Env spike 15Å away from the viral membrane after CD4 engagement (Huang et al., 2014). Although these studies focus primarily on the rearrangement of Env on the membrane surface upon CD4 engagement and structural rearrangement of the Env protein itself is sufficient to expose the Achille’s heel of HIV for immune attack, our data however provide direct physical evidence that these pre-entry priming structural rearrangements also extend to the internal virus architecture. These data are consistent with each other that the pre-entry priming of HIV particles via the unique properties of CD4–Env engagement can lead to remodelling across many viral compartments of HIV, highlighting the existence of a previously un-appreciated process in HIV entry.

MSCs have been isolated a range of tissues including bone

MSCs have been isolated a range of tissues, including bone marrow, adipose tissue, foetal tissue, placenta, umbilical cord and peripheral blood (PB). Although bone marrow and adipose tissue represent excellent sources of MSCs, the tissue harvesting process is invasive and requires anaesthesia in some species. Foetal tissues, placenta and umbilical cord are potentially attractive sources of MSCs, since they contain abundant MSCs and can be collected without the requirement for invasive methods, but are not always available when needed. MSCs have been isolated from PB (PB-MSCs) of human beings, mice, sheep, horses and dogs (Roufosse et al., 2004; Lyahyai et al., 2012; Spaas et al., 2013). Since collection of blood samples is minimally invasive, PB may be a source of progenitor cells in clinical situations.
Feline MSCs have been isolated from bone marrow (Martin et al., 2002; Munoz et al., 2012), adipose tissue (Webb et al., 2012; Kono et al., 2014; Zhang et al., 2014) and foetal tissues (Jin et al., 2008; Iacono et al., 2012; Vidane et al., 2014). MSCs derived from feline bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) have been applied in clinical settings, including chronic kidney disease (Quimby et al., 2011, 2013), chronic allergic leukotriene receptor antagonists (Trzil et al., 2014) and chronic enteropathy (Webb and Webb, 2015). Despite this trend, basic information regarding feline MSCs is still limited, and there are no reports on feline PB-MSCs (fPB-MSCs). The aim of the present study was to establish a procedure for isolating MSCs from feline PB, to characterise the markers of fPB-MSCs and to demonstrate the capacity of these cells to differentiate.

Materials and methods


Feline MSCs were first isolated by Martin et al. (2002) from bone marrow and subsequently feline MSCs have been derived from adipose (Webb et al., 2012; Kono et al., 2014; Zhang et al., 2014) and foetal (Iacono et al., 2012; Vidane et al., 2014) tissue. In these studies, feline MSCs were characterised by morphological features, cell-surface antigen profiles and their capacity for differentiation. Although the existence of MSCs in PB has been demonstrated in many species, our study is the first to demonstrate the feasibility of isolating fPB-MSCs exhibiting appropriate morphology, mesenchymal surface markers and the capacity to differentiate into osteoblasts, chondroblasts and adipocytes.
Canine PB-derived fibroblast-like cells have been isolated in the presence of interleukin 6 (Huss et al., 2000). Tondreau et al. (2005) isolated MSCs from human PB using GM-CSF and culture medium containing conditioned medium from human BM-MSCs. Standard isolation methods used for BM-MSCs can be employed to isolate PB-derived precursor cells from several mammals, such as sheep and horses (Koerner et al., 2006; Lyahyai et al., 2012). Although we tried to isolate fPB-MSCs using previously described culture methods for isolation of feline BM-MSCs (Martin et al., 2002; Munoz et al., 2012; Webb et al., 2012), the seeded PBMCs diminished over time. Additionally, cell proliferation was not observed, despite the use of several different types of culture media, for example media containing feline GM-CSF or fBM-MSCs-CM. Culture medium containing both FBS and AP resulted in the enhanced adherence and propagation of fPB-MSCs. Our results suggest that methods used to isolate PB-MSCs differ between animal species.
In several species, MSCs have been successfully cultured and grown in autologous serum or platelet-rich plasma instead of medium supplemented with FBS (Stute et al., 2004; Edamura et al., 2012; Fukuda et al., 2015). To the best of our knowledge, no studies have used a culture medium supplemented with both AP and FBS to isolate MSCs. Culture medium supplemented with only FBS or AP did not facilitate the isolation of fPB-MSCs. It is unknown which components of feline plasma are responsible for the ability to isolate and maintain proliferation of fPB-MSCs. Growth factors, such as TGF-β and fibroblast growth factors, affect proliferation, morphogenesis and survival of MSCs (Rodrigues et al., 2010). We are investigating which factors are required during the growth stage of fPB-MSCs. Our preliminary findings suggest that the propagation of passaged fPB-MSCs might not require AP (data not shown).

Dorsal wedging of the TTB was

Dorsal wedging of the TTB was not significantly associated with increasing severity of OA for grades 0–2 in the DIT and TMT joints, which is in leukotriene receptor antagonists to previous studies in foals, in which narrowing of the dorsal aspect of the third tarsal bone was associated with the development of OA (Fretz, 1980; Auer et al., 1982; Ruohoniemi et al., 1995; Dutton et al., 1998; Butler et al., 2000). This might be due to the multifactorial nature of OA affecting those joints; such factors have more time to contribute to the development of this disease as the horse becomes older.
The most severe OA cases showed plantar wedging and were wider on the dorsal aspect; this finding was most evident medially. There are two possible explanations for this finding. Collapse may not occur at the most dorsal aspect, but at the level of the more plantar measurement, this has been seen in radiographs in some cases of tarsal bone collapse (Morgan, 1967; Sedrish and Moore, 1997). It could also be explained by extensive new bone formation and loss of joint space, resulting in an error in measurement; this correlates with reports that increased new bone formation is found predominantly on the dorsomedial aspect of the tarsus (Shelley and Dyson, 1984; Kidd et al., 2001).


Conflict of interest statement


Oral medication is the preferred method for extended post-operative pain control for ‘day-case’ procedures such as ovariohysterectomy (OHE) as the drug can be administered by owners. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for this purpose, although these can lead to a variety of side effects including gastrointestinal, renal, and haemostatic disorders (Curry et al., 2005; Luna et al., 2007; Monteiro-Steagall et al., 2013). Carprofen has been studied extensively in dogs in the peri-operative (Nolan and Reid, 1993; Fox and Johnston, 1997; Lascelles et al., 1998; Laredo et al., 2004; Shih et al., 2008) and also in the extended post-operative period (Leece et al., 2005). An advantage of carprofen is that it is generally administered only once daily, thereby improving owner compliance (Pullar et al., 1988; Barter et al., 1996).
Metamizole, a pirazolone derivative also known as dipyrone, is an atypical NSAID, which has been on the market for more than 90 years. Metamizole is an antipyretic analgesic with spasmodic properties and lacks anti-inflammatory activity (Levy et al., 1995; Camu and Vanlersberghe, 2002). Although banned in some countries (including USA and the Scandinavian countries) in the 1980s due to the rare but serious occurrence of agranulocytosis as a side effect (Hedenmalm and Spigset, 2002), it is still widely used in several European and South American countries. There are no reports of agranulocytosis in veterinary medicine.
In the last few years, metamizole has experienced a revival, especially for pain management in human medicine, due to its high efficacy and good gastric tolerability (Sanchez et al., 2002; Nikolova et al., 2013). Peripheral as well as central effects are responsible for the potent analgesic efficacy (Mazario and Herrero, 1999; Vazquez et al., 2007). Opioidergic pathways (Tortorici et al., 1996) and inhibition of cyclooxygenase (COX)-3 (Chandrasekharan et al., 2002) seem to be the main mechanisms of action; the analgesic effect is comparable to that of opioids in humans (Hempel, 1986; Aguirre-Banuelos and Granados-Soto, 1999) and dogs (Richter et al., 2007; Tacke et al., 2008). Metamizole has been shown to be effective in controlling post-operative pain in dogs undergoing OHE (Imagawa et al., 2011).
Until now metamizole was approved for the veterinary market only as an injectable formulation, whereas for many years tablets for humans have been licensed with a duration of action of 4–6 h (Brack and Schäfer, 2008). To enable drug administration by pet owners an oral formulation for dogs was needed, and to increase owner compliance one with a longer duration of action in dogs was developed. A polymer, povidone, was used for controlling the drug release; in an aqueous environment the polymer in the tablet swells and forms a gel. Metamizole sodium dissolves in the gel and diffuses out into the surroundings.

br The purpose of this

The purpose of this article is to demonstrate how these principles and assessments can be applied in practice to inform policy choices of OIR, AWR, Approve, or Reject. Two case studies that explore situations in which OIR or AWR might be particularly relevant and challenging have been selected for this purpose. We describe each checklist point of assessment and examine how each of the assessments might be informed on the basis of the type of evidence and analysis currently available and what additional information and/or analyses might be required.

Case Studies

The two case studies selected are 1) enhanced external counterpulsation for chronic stable leukotriene receptor antagonists (EECP), and 2) clopidogrel for the management of patients with non–ST-segment elevation acute coronary syndromes (CLOP). The cost-effectiveness of EECP and clopidogrel has been examined previously as part of the National Institute for Health Research Health Technology Assessment program and the National Institute for Health and Care Excellence (NICE) Multiple Technology Appraisal, respectively [11]; [12] ;  [13]. The existing methods of appraisal have been taken as the accepted starting point. A range of additional information was sought and further analysis conducted to inform the sequence of assessment and judgments required when completing the OIR/AWR checklist.

EECP is a noninvasive procedure used to provide symptomatic relief from stable angina. The analysis compares EECP (adjunct to standard therapy) with standard therapy alone. Randomized controlled trial (RCT) evidence suggests an improvement in health-related quality of life with EECP at 12 months. To characterize the uncertainty associated with possible longer durations of treatment effect, formal elicitation of expert clinical judgment was undertaken. This provided an estimate of the probability, with uncertainty, of a patient continuing to respond to treatment with EECP in subsequent years [12].

EECP is expected to be cost-effective but with potentially significant irrecoverable costs. These irrecoverable costs include both 1) capital costs of equipment and 2) large initial per-patient treatment costs, combined with a chronic condition in which a decision not to treat a particular patient can be changed at a later date when the results of research become available or other events occur. Consequently, these irrecoverable costs might influence the type of guidance; for example, OIR rather than Approve [9].

CLOP (used for up to 12 months) in combination with low-dose aspirin was recommended by NICE after a multiple technology appraisal for patients with non–ST-segment-elevation acute coronary syndrome who presented with a moderate to high risk of ischemic events (TA80 in 2004 and updated in 2010 in CG94) [14] ;  [15]. AWR was considered during the appraisal of CLOP. Four alternative treatment durations of CLOP of 12, 6, 3, and 1 month were compared with standard therapy (with low-dose aspirin). CLOP is expected to be cost-effective with no significant irrecoverable costs and illustrates a number of important characteristics, including 1) the impact of other sources of uncertainty (price change following patent expiry) on the value of research, and 2) interpretation of multiple alternatives.

Assessments Required

Point 1—Is the Technology Expected to Be Cost-Effective?

The sequence of assessment starts with cost-effectiveness and expected impact on population net health effect (NHE) [16] ;  [17]. This requires information about prevalence and future incidence of the population and a judgment about the time horizon over which the technology will be used [6]. The scale of the population NHE and how it accumulates over time are important for subsequent assessments; for example, the NHE for current patients must be compared with the benefits to future patients and the significance of irrecoverable opportunity costs of initially negative NHE must be determined.