Analgesia is an important ancillary property of all

Analgesia is an important ancillary property of all anti-inflammatory agents. Most of the anti-inflammatory drugs increased pain threshold in various animal models [44]. This is natural because many endogenous chemical mediators of inflammation play a part in generating pain impulses (for example, histamine, serotonin and prostaglandins) and some other mediators such as bradykinin and cytokines, are involved in prolongation of the sensation of the pain [45].
RA is an inflammatory condition of the joint that is associated with hyperalgesia and functional impairment [46]. Joint inflammation induced by AIA is associated with hyperalgesia and slight compression of the joint which caused leg withdrawal and squeaking. The hyperalgesia associated with arthritis has been reported to involve prostaglandin synthesis [47]. Increased levels of substance P, GDC-0994 gene-related peptide and up-regulation of neurokinin-1 receptor in the dorsal horn of the rat spinal cord have been reported to play an important role in modulating both inflammation and hyperalgesia in the FCA model [48]. However, in the present study, TAPP did not prevent reduction pain latency in Randall–Selitto assay. Therefore, nociception-related mediators (substance-P, calcitonin gene-related peptide and neurokinin-1) are not involved in mechanism of action of TAPP. This also pointed toward the possibility of disease modification as possible mechanism of action of TAPP against RA.
AIA has been generally believed to be the result of a delayed-type hypersensitivity (DTH) response to a disseminated antigen probably derived from the injected bacterial cell wall [49]. AIA in rats is a useful model of inflammatory cachexia that mimics the human pathophysiology in many important ways [50]. AIA induces inflammation as primary lesion which reached to peak after 3–5d with secondary lesions occur after a delay of about 11–12d. Secondary reactions in AIA are characterized by immune responses, inflammation and decrease of weight (cachexia). In our study, the test compound, TAPP significantly protected rats from cachexia because of arthritis induction.
In the past, many plant-derived procyanidines reported to have cytokine inhibitory effect in vitro[51,52]. Similar effects were also attributed to Cinnamon bark extract and polyphenols [16,53]. In vitro pre-challenge with cinnamon extract is reported to suppress lipopolysaccharide (LPS)-induced cytokines expression [54]. Recently, immune regulatory action of TAPP in mouse 3T3-L1 adipocytes is attributed to TTP (tristetraprolin) [55]. Besides, TAPP is capable of affecting immune responses by regulating anti-inflammatory mediators as well as the TTP gene expression in macrophages [16]. Taken together, cytokine inhibition emerges as a probable mechanism of TAPP responsible for cachexia reducing effects in AIA-induced established polyarthritis in rats. This probability is also supported by our result that TAPP reduces serum C-reactive protein (CRP) levels of rats on day-21 compared to day-12 levels (Table 2) whereas AIA control rats showed sustained increase in CRP till day-21 (Table 2).
CRP is an acute-phase protein and has been identified as an important biomarker for various inflammatory, degenerative, and neoplastic diseases [56,57]. Elevated levels of CRP have been found in the blood during almost all diseases associated with active inflammation or tissue destruction, particularly in RA patients [57,58]. Sustained increase in serum CRP levels suggests a lasting production and stimulation of acute-phase proteins during disease progression.
In our study, serum turbidity (showed by absorbance of serum samples) of AIA control rats was found to be significantly reduced compared with normal rats (Fig. 1). Treatment from day-12 to day-21 with TAPP but not diclofenac sodium (an NSAID) prevented AIA-induced fall in turbidity (Fig. 1). The decrease in serum turbidity in AIA control rats, is known to be caused by denatured products and was shown to be correlated with severity [59] and immune response seen in AIA [60]. Further, increased levels of serum lysozyme levels were reported to be strongly correlated with decreases in serum turbidity in AIA [59]. Therapeutically useful anti-inflammatory drugs were shown to reverse this decrease in serum turbidity [32]. Taken together, our results strongly pointed to disease-modifying properties of TAPP and its potential to slow down the RA progression.

br Methods Retinal nerve fiber layer and macular thickness measurements

Retinal nerve fiber layer and macular thickness measurements were made with the Spectralis SD-OCT (Heidelberg Engineering, Carlsbad, California) using our modified retinal thickness measurement protocol which permits high-density measurement in the macular region. Briefly, macular thickness is measured using 61 lines in the central 20 degree area (centered on the fovea). The retinal thickness is displayed as a color-coded thickness map, modified to reveal retinal thickness differences of 10–15 microns. This modification allows for improved visual detection of small differences in tissue thickness. The thickness maps are displayed with a grid demonstrating retinal thickness values in small 3×3 degree areas with the entire grid tilted to match the fovea-disc axis. Asymmetry analyses between the upper and lower halves of the macula are displayed as a gray scale plot. A black square represents a 30 micron difference at that location and a white square represents no difference between the two macular halves at that location.


Glaucoma is clearly the most studied optic neuropathy using OCT; approximately three quarters of the publications found on a query of “OCT” and “optic neuropathy” focus on glaucoma (personal observation, July 2012). However, recent studies have demonstrated the use of OCT in the management and prognosis of patients with optic neuritis and multiple sclerosis, and other aggressive inflammatory diseases of the central nervous system. In addition, recent publications show that OCT has been used for papilledema and disc edema, optic disc drusen, ischemic optic neuropathies, hereditary optic neuropathies, toxic optic neuropathies, and compressive optic neuropathies.
In a recent and thorough review of “glaucoma look-alikes” by Pasol, he GDC-0994 noted the importance of using the temporal quadrant of RNFL analysis, in differentiating non-glaucomatous optic neuropathies from glaucoma, as seen in our infectious and degenerative cases. As highlighted in our series, ischemic optic neuropathy and optic nerve head drusen have patterns similar to glaucoma, frequently affecting the superior-inferior quadrants as in glaucoma. In a recent prospective cross-sectional study, Suh and colleagues used a combination of optic disc rim area (measured by Heidelberg retinal tomography (HRT)) and RNFL thickness (measured by Cirrus time-domain OCT) to differentiate between glaucoma and non-arteritic ischemic optic neuropathy. In 2007, Chan and Miller examined the OCTs of patients with long-standing non-glaucomatous optic neuropathies and found a residual thickness of about 45 microns, confirming a thickness that could be attributed to the signal generated by the interface between the vitreous and inner retinal surface seen in earlier cadaveric studies.


Traditional descriptions of lymphatic eyelid drainage suggest that the lateral eyelids drain to the preauricular nodes, while the medial portions of the eyelids drain to the submandibular nodes. This understanding of the lymphatic drainage patterns of the eyelids derives from dye studies conducted several decades ago. Other knowledge to date has been extrapolated from investigations conducted postmortem or in animal models.
A sentinel lymph node is often the first site of malignant tumor spread before dissemination to more distant sites, and a SLNB is performed for histopathologic examination to determine the presence of microscopic metastasis. Unlike a lymph node dissection that traditionally involves removing a bulk of tissue with 10–15 lymph nodes, SLNB is less invasive and usually involves the biopsy of one or two lymph nodes.
Pre-operative imaging is an important feature of modern SLNB techniques. In advance of surgical excision, technetium-99m sulfur colloid is injected subcutaneously around the malignancy to identify the sentinel node to generate a lymphoscintography image (Fig. 1). (Intraoperative maneuvers including subcutaneous injection of isosulfan blue and technetium-99m with transcutaneous gamma probe further add to identification of the sentinel node.) With the increasing use of SLNB, the database for in vivo and in situ GDC-0994 drainage pathways from the periocular region is increasing.

GDC-0994 A second area of institutional change and institution building is

A second area of institutional change and institution building is the development of partnerships and governing arrangements in support of climate change GDC-0994 planning. This can involve coordination horizontally among city departments or vertically between the city, civil society, the private sector and other levels of government (Adger et al., 2005; Burch et al., 2013). The inclusion (or lack thereof) of citizen participation and engagement is also part of the city’s institution and capacity building.

A meta-analysis of published research is used to advance our understanding of the institutional changes and strategies cities are initiating in support of climate change adaptation planning. The targeted sources quantitatively and qualitatively document and/or evaluate urban climate change adaptation planning in the U.S. as well as those support programs designed to promote climate change adaptation planning by U.S. cities. Meta-analyses such as this are valuable because they allow a broader and more systematic understanding of important social and political dimensions of global environmental change (Rudel, 2008; Romero-Lankao et al., 2012). To perform the meta-analysis the study identifies relevant high-quality data sources, develops a codebook and codes the data sources, and integrates findings in order to draw conclusions.

The empirical sources identified in the review process included 22 sources that use either survey data or other comprehensive review methods, 25 sources that use case studies, and seven relevant support programs. These sources included global, national, and sub-national surveys and support programs; international comparisons; regional analyses; and case studies of individual cities. Less than half of the sources (40%) are journal articles (Appendix A).
Fig. 1 reveals the frequency with which each source refers to the topics of interest. Research (case studies, reviews, and surveys) and support program sources exhibit very similar patterns. The most frequently discussed topics include the horizontal and vertical coordination taking place to facilitate adaptation planning, public participation and stakeholder engagement strategies, financing sources external to city budgets, the importance of leaders, and building existing city programs as part of an adaptation strategy. Beyond simple count tallies, the qualitative patterns and consistent themes in each of the three institutional categories are discussed in greater detail below.

Second, this GDC-0994 analysis finds that the current focus of city governments on protecting assets and reducing vulnerability typically excludes the consideration of equity concerns, social vulnerability, and the role of non-climatic conditions. Decision makers must find ways to engage with and incorporate new information and expertise. Indeed, incorporating information about future climatic conditions is a necessary component of effective adaptation planning and is what distinguishes adaptation from other policy areas such as disaster risk reduction (Bulkeley and Tuts, 2013). However, an over-reliance on technical information in the planning process can also serve to exclude other considerations (Eden and Tunstall, 2006). Future research should examine not only the credibility and usability of future climate projections but also the incorporation of equity concerns, social vulnerability, and non-climatic conditions in adaptation planning. What role do existing sources of climate change information and expertise play in shaping the priorities and focus of urban adaptation planning? For example, vulnerability assessments may be done through contracts; stakeholder task forces may be dominated by physical scientists or engineers without a background in the social and human dimensions of adaptation.


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The general mechanism on polysulfide pathway

The general mechanism on polysulfide pathway can be explained by the following equations and the leaching mechanism of the thiosulfate pathway and the polysulfide pathway are showed as Fig. 4[13,108].
The schematic diagram of the leaching mechanism of the thiosulfate pathway and…
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Fig. 4. The schematic diagram of the leaching mechanism of the thiosulfate pathway and the polysulfide pathway.
3.3. Attached and planktonic effect
The process of the attached and planktonic effect of the iron(Ⅱ)- and S-oxidizing bacteria and transfer of electrons in At. ferrooxidans is graphed as Figs. 5 and 6.
The contact, un-contact and cooperative leaching mechanisms of the bacteria
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Fig. 5. The contact, un-contact and cooperative leaching mechanisms of the bacteria.
The downhill pathway of transfer of electrons in At
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Fig. 6. The downhill pathway of transfer of electrons in At. ferrooxidans.
3.4. Extracellular polymeric substances, biofilm formation and passivation
The components of EPS of different ferrous- and S-oxidizing bacteria coupling with different leaching conditions have been widely studied. Gehrke et al. verified that GDC-0994 the EPS of At. ferrooxidans consists of the sugars glucose, rhamnose, fucose, xylose, mannose, C12–C20 saturated fatty acids, glucuronic acid, and ferric ions, on the surface of pyrite [123,124]. The compositions and amount of components of EPS would change when the bacteria adapted to the new substrate in the solution. Sharma et al. found the surface charges were different between the bacteria grown in the solution with ferrous ions and those dwell at the surface of the metal sulfide or sulfur due to the difference of protein content [125]. Arredondo et al. demonstrated that the attachment functionality of the bacteria was assisted and enhanced by lipopolysaccharides and some specific cell surface proteins [126]. The ferric ions was combined by uronic acids through complexation in EPS, which facilitated the biooxidation. Cells grown on the surface of elemental sulfur do not effectively attach to the surface of FeS2 due to a potentially changed EPS composition compared with that of the pyrite-grown cells. Pronk et al. showed that, in aerobic conditions, the ferric ions in the EPS could be reduced by the attached bacteria in the process of bioleaching of metal sulfide [127]. Rodriguezleiva and Tributsch detected that the range of the thickness of the EPS was from 10 nm to 100 nm and the EPS thickness of At. ferrooxidans was estimated to be 28.7 nm (±13.5) based on the analysis of AFM [128]. Ohmura et al. found the Acidithiobacillus ferrooxidans was more likely to attach to sulphides that contain iron [129]. Solari et al. proposed that the adhesion rate of inoculum would be elevated if the pH was reduced due to the change of the bacterial GDC-0994 hydrophobicity in specific pH environment. Edwards and Rutenberg summarized that the small alterations of local surface in according to bacterial metabolism could strongly affect the parameters of local adhesion [130]. Flemming and Wingender presented that the formation of bacterial biofilm was accompanied by the obvious augment in production of EPS [131]. Microbial attachment and biofilm formation provide a mechanism through which the microorganism can locate itself near an energy source. It is widely accepted that the passivation of the surface of metal sulfide (e.g., chalcopyrite) is the main reason for the low leaching rate. The elemental S and jarosite are vital components for the formation. S can be formed by oxidizing the surface of sulphide and following intermediate through using Fe3+ and S-oxidizing bacteria. Actually, in low redox conditions, elemental S in chalcopyrite surfaces can also be formed through reduction reactions [132]. The equations of the reduction of chalcopyrite are listed as followed,

br Our study demonstrated that the average excursions of

Our study demonstrated that the average excursions of the bilateral GDC-0994 during tidal breathing (right: 11.0 mm, 95% CI 10.4 to 11.6 mm; left: 14.9 mm, 95% CI 14.2 to 15.5 mm) were numerically less than those during forced breathing in previous studies using other modalities 2; 7 ;  8. Using fluoroscopy, Alexander reported that the average right excursion was 27.5 mm and the average left excursion was 31.5 mm during forced breathing in the standing position in 127 patients (2). Using ultrasound, Harris et al. reported that the average right diaphragm excursion was 48 mm during forced breathing in the supine position in 53 healthy adults (7). Using MR fluoroscopy, Gierada et al. reported that the average right excursion was 44 mm and the average left excursion was 42 mm during forced breathing in the supine position in 10 healthy volunteers (8). The difference in diaphragmatic excursion during tidal breathing versus forced breathing is unsurprising.

Our study showed that the excursion and peak motion speed of the left diaphragm are significantly greater and faster than those of the right. With regard to the excursion, the results of our study are consistent with those of previous reports using fluoroscopy in a standing position 2 ;  3. However, in the previous studies evaluating diaphragmatic motion in the supine position, the asymmetric diaphragmatic motion was not mentioned 7 ;  8. The asymmetric excursion of the bilateral diaphragm may be more apparent in the standing position, but may not be detectable or may disappear in the supine position. Although we cannot explain the reason for the asymmetry in diaphragmatic motion, we speculate that the presence of the liver may limit the excursion of the right diaphragm. Regarding the motion speed, to the best of our knowledge this study is the first to evaluate it. The faster motion speed of the left diaphragm compared to that of the right diaphragm would be related to the greater excursion of the left diaphragm.

We found that higher BMI and higher tidal volume were independently associated with the increased excursions of the bilateral diaphragm by both univariate and multivariate analyses, although the strength of these associations was weak. We cannot explain the exact reason for the correlation between BMI and the excursion of the diaphragm. However, a previous study showed that BMI is associated with peak oxygen consumption (23), and the increased oxygen consumption in an obese participant may affect diaphragmatic movement. Another possible reason is that lower thoracic compliance due to higher BMI may cause increased movement of the diaphragm for compensation. Regarding the correlation between tidal volume and excursion of the diaphragm, given that diaphragmatic muscle serves as the most important respiratory muscle, the result is to be expected. Considering our results, the excursion evaluated by dynamic X-ray phrenicography could potentially predict tidal volume.

Our study has several limitations. First, we included only 172 volunteers, and additional studies on larger participant populations are required to confirm these preliminary findings. Second, we evaluated only the motion of the highest point of the diaphragms for the sake of simplicity, and three-dimensional motion of the diaphragm could not be completely reflected in our results. However, we believe that this simple method would be practical and more easily applicable in a clinical setting.


The time-resolved quantitative analysis of the diaphragms with dynamic X-ray phrenicography is feasible. The average excursions of the diaphragms are 11.0 mm (right) and 14.9 mm (left) during tidal breathing in a standing position in our health screening center cohort. The diaphragmatic motion of the left is significantly larger and faster than that of the right. Higher tidal volume and BMI are associated with increased excursions of the bilateral diaphragm.

br The model summaries provided in Table xA

The model summaries provided in Table 2 ;  Table 3 show a clear pattern over time, with the ScHARR model commissioned by NICE [23]; [25] ;  [26] clearly a pivotal point in model evolution. There is GDC-0994 in modeling technique, model structure, input data, and assumptions across the six models developed before the ScHARR model [14]; [15]; [16]; [17]; [18]; [19]; [20]; [21] ;  [22]. In contrast, models developed after the ScHARR model converge with respect to the technique and basic structure. In line with other recent reviews [7] ;  [9], one feature common to all included models was the use of EDSS to model disability progression, in spite of criticism of this instrument for its inability to capture relevant clinical milestones [34]. As one would expect in a review of models taking a UK perspective, the impact of changes in guidance issued by NICE over time is also apparent, both in terms of the recommended discount rates for costs and utilities and also in terms of the balance between the risks of extrapolation of limited clinical effectiveness data versus the desire for a lifetime horizon. Time horizons varied, with 5, 8, 10, 20 years, and lifetime (represented as 50 years in two models) all used; more recent models have used longer time horizons. Short-term studies would have a tendency to underestimate the cost-effectiveness of DMTs because the m### advantage of using DMTs is to postpone the development of severe MS, which occurs after a longer time period.

Of the models published before the ScHARR model in 2003 [23], two appeared to be decision tree models (though were not explicit in stating their model type) [14]; [15]; [16] ;  [18], one described itself as semi-Markov [21], one simulated individual patients [19] ;  [20], whereas two used forms of regression analysis [17] ;  [22]. The decision tree and Markov-type models [14]; [15]; [16]; [18] ;  [21] used various cohort structures based on different subsets of EDSS scores. The regression models attempted to consider the area under the EDSS score–time curve, in spite of the EDSS score being a series of ordered categories rather than a cardinal number amenable to such analysis [17] ;  [22]. Only one of the early models attempted to incorporate progression from RRMS to SPMS into its structure in any form [21]. This model applied inputs from the SPMS population to all patients with an EDSS score of 4.5 or above [21], which is not consistent with disease progression in clinical practice [35]. This same model was also the only early example to incorporate death and treatment withdrawal [21]. None of the early models reported incorporating the disutility or costs of adverse events (AEs), subgroup analyses, or probabilistic sensitivity analysis. All early studies provided results from an NHS and PSS perspective, though it was not the primary perspective for all the studies—MS has considerable effects on productivity for both patients and carers; therefore, some models provided results from a societal perspective [17]; [18] ;  [21]. NICE, however, specifies an NHS and PSS perspective as the basis for decision making, and UK models tend therefore to follow this approach; greater variation in perspective would be expected had other countries been included in this review. Costs were all discounted by the then-standard 6%, but utilities were not consistently discounted by the then-standard 1.5%.

As discussed in the Introduction, the controversy surrounding the first NICE appraisal of beta interferons and glatiramer acetate led to the commissioning of a new economic model by NICE. The commissioned model was produced by a consortium led by ScHARR and is available on the NICE website [25] along with a short addendum addressing new utility data that became available after the main report had been submitted but before the appraisal process was completed [26]. The effect of this process on those modeling DMTs in MS from a UK perspective is apparent from the results presented in Table 2 ;  Table 3, and after the ScHARR model all models describe themselves explicitly as being based on the ScHARR model. This new model defined a Markov structure based on the full set of EDSS scores (including the half-number scores as well as the whole-number scores) incorporating RRMS EDSS states 0–10, SPMS EDSS states 2–10, and a general death state. Mortality was modeled either as disease progression to EDSS state 10 (MS death), or as a result of other causes at any model stage (general death). Patients could transition from RRMS to SPMS at any point, and adverse effects and withdrawal from treatment were explicitly considered. Extrapolation of the short-term clinical efficacy data available at the time was balanced with the long-term nature of the disease through the choice of a 20-year time horizon. Comprehensive scenario analyses and probabilistic sensitivity analyses were also reported, addressing a criticism of many of the previously submitted models that had presented limited uncertainty analyses. Subsequent models have clearly converged to a standardized structure based on that used in the ScHARR model, but with two modifications. First, the number of states has been reduced from the full EDSS set used in the ScHARR model down to a set based on whole-number states, with each half-number state grouped with the one above. The exception to this is the EDSS 9.5 state, which is grouped in with EDSS state 9 (and 8.5). Second, the separate MS death (EDSS state 10 in each of RRMS and SPMS) and general death states have been combined into a single absorbing state. In all the models developed after the ScHARR model, costs and utilities were both discounted by the current UK standard of 3.5%. In spite of the adoption of this standardized structure, variation has been apparent in the model assumptions, as will be discussed below, and also with respect to some aspects of the data in which there are problems with lack of consensus and openness of data, with consequences for replicability.

GDC-0994 AcknowledgementsPreliminary results were presented as

AcknowledgementsPreliminary results were presented as an abstract at the 15th International Congress of the European Society for Veterinary Clinical Pathology, Berlin, 6-9 November 2013. The study was financially supported by the Faculty Research Committee and the Department of Companion Animal Clinical Studies of the University of Pretoria, the South African Veterinary Foundation and the South African National Research Foundation (grant no. N00175). The authors also wish to thank Becton-Dickinson and The Scientific Group (South Africa) for sponsoring the FACSCalibur for the duration of the study.
Chlamydia abortus; Enzootic abortion; Sheep; Intranasal infection; Intratracheal infection; Pneumonia
Chlamydia abortus is an obligate intracellular bacterial pathogen that infects the placenta of several species of mammals, leading to abortion during the last phases of pregnancy. The infection mainly affects small ruminants and is the cause of ovine enzootic abortion (OEA), which causes GDC-0994 major economic losses in sheep and goat-breeding countries in Europe. C. abortus is also recognised as a zoonotic pathogen involved in an acute influenza-like illness followed by miscarriage or stillbirth in pregnant women, especially those in contact with infected flocks ( Longbottom and Coulter, 2003).
Early studies have shown that ewes or lambs become infected during the lambing season, when their environment is contaminated by chlamydial elementary bodies from fetal membranes and vaginal discharges from previously infected animals (Nietfeld, 2001). The infection of non-pregnant sheep leads to a state of latency influenced by interferon (IFN)-纬 (Brown et al., 2001). No clinical signs are observed until the infected ewes become pregnant, after which time the GDC-0994 colonises the placenta and subsequently multiplies, eventually causing abortion in the last 2-3 weeks of gestation (Nietfeld, 2001). After abortion, the animals acquire protective immunity, which allows successful rebreeding, but they also become carriers and will shed the organism in the following oestrus (Nietfeld, 2001).
In studies of OEA, two animal models have been developed using mice and sheep. Although mice are not a natural host for C. abortus, this model has been widely used to shed light on immune pathological events of the disease and to test vaccine candidates because of the similarities between the experimental infection in mice and the natural disease in small ruminants ( Caro et al., 2009). Moreover, the mouse model provides economical, handling and research advantages (e.g. a shorter gestation period) compared with the sheep model. Despite these benefits, the transfer of research results to the natural host is still necessary to reflect reality and to provide the agricultural industry with solutions to the problem of OEA.
There have been many studies using experimental pregnant sheep to study the immunopathology of OEA (Buxton et al, 1990, Papp, Shewen, 1996, Navarro et al, 2004, Sammin et al, 2006, Guti茅rrez et al, 2011 and Longbottom et al, 2013) and to evaluate the effectiveness of live and attenuated vaccines (Rodolakis, Souriau, 1983, Souriau et al, 1988 and Garc铆a de la Fuente et al, 2004). The use of this experimental model however has many disadvantages such as high economic costs, poor animal welfare, the need for facilities for reproductive handling and lambing and, above all, the long time required for the pregnancy to reach term. The development of a new non-pregnant natural host model that provides greater simplicity and is cheaper than the pregnant model would be valuable.

Although CAs catalyse a similar reaction as other

Although β-CAs catalyse a similar reaction as other CA isoforms there are some important structural differences between these classes. In α-, γ- and δ-CAs the active Zn-binding site is coordinated by three histidines (Fig.2uanduFig.3), whereas in β-CAs it is linked with one histidine and two cysteine residues (Cox et al., 2000). In spite of this structural difference, β-CAs basically follow the same molecular mechanism for reversible hydration of CO2 into H+ and HCO3u like α-CAs (Strop et al., 2001). Our limited gene expression data suggest no major function of β-CAs in pH balance in freshwater crayfish. However, to confirm the activities of β-CAs in crayfish and other decapod crustaceans, more detailed physiological and biochemical investigations are needed.
The two partial CA transcripts identified, ChqCA-p1 and ChqCA-p2 showed greatest protein similarity with the GPI-linked CA isoform (see Fig.4 in Ref (Ali et al., accepted for publication)). As these genes show greatest similarity with the GPI-linked CA isoform we hypothesise that these transcripts may represent duplicated copies of ChqCAg. GPI-linked carbonic anhydrase genes show evidence of duplication in a range of species including fish and other vertebrates (Tolvanen et al., 2013). In fact, it has been hypothesised that the extensive duplication of GPI-linked carbonic anhydrase genes in fish has resulted in different isoforms undertaking slightly different roles in breathing and GDC-0994 excretion (Tolvanen, 2013). As crayfish also inhabit an aquatic environment the duplication of GPI-linked carbonic anhydrase genes in C. quadricarinatus may also have different functions in similar physiological processes in these species.
4.2. Other candidate osmoregulatory genes in C. quadricarinatus
We have found a number of candidate genes that have previously been identified as playing a role in osmoregulation and pH balance in crustaceans. Many of these genes have been shown to be important for adaptation in response to salinity gradients and also in stress response to abrupt changes in pH or water chemistry (Henry et al., 2002, Serrano and Henry, 2008, Liu et al., 2010uanduHavird et al., 2013). One group of candidate genes of particular interest are those involved in ion transport and systemic acidabase balance. These genes included sodiumapotassium ATPase, sarco/endoplasmic reticulum Ca2u+-ATPase, arginine kinase, calreticulin, Na+/H+ exchanger, Na+/HCO3 cotransporter, alkaline phosphatase, multiple subunits of vacuolar proton pump and a number of different genes from the solute carrier gene family. In this section, we will discuss two of these genes for which we had gene expression data, V-type H+-ATPase and arginine kinase, and whether they may play an important role in acidabase balance in freshwater crayfish.
The V-type H+-ATPase is a key enzyme that regulates osmoregulation and pH balance in many organisms (Forgac, 1998, Saliba and Kirk, 1999, Beyenbach, 2001, Kirschner, 2004uanduCovi and Hand, 2005). Its involvement in freshwater osmoregulation has also been shown by a number of studies of gene expression and protein activity (Faleiros et al., 2010, Lee et al., 2011uanduTowle et al., 2011). This protein pumps protons to acidify intracellular organelles and in this process it influences HCO3 and Cl exchange and Na+ uptake (Weihrauch et al., 2004; Martin Tresguerres et al., 2006) and also plays a vital role in branchial NH3 excretion (Weihrauch et al., 2002, Weihrauch et al., 2009, Martin et al., 2011uanduWeihrauch et al., 2012). The large number of V-type H+-ATPase subunits expressed in the gills of C. quadricarinatus suggests that it may play an important role in this tissue. The gene expression data for V-type H+-ATPase subunit A was close to significant; so it may play a role in acidabase balance in crustaceans. Further gene expression studies on this gene with greater replication than used in the current study may elucidate whether V-type H+-ATPase subunit A is involved in acidabase balance in C. quadricarinatus. Examining the expression of other V-type H+-ATPase subunits may also shed further light on other members of this gene family in acidabase balance in freshwater crayfish.