Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1α (~140–150 kDa) and PHLPP1β (~180–190 kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1α/β by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1β in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1α (~145–150 kDa). The other four antibodies detect a non-specific signal at ~150 kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1α by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ~150 kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis. Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a serine/threonine phosphatase with two splice variants. PHLPP1β was first discovered in rat brain2Immuno-based techniques such as Western blot are routinely used to study PHLPP1 in cell death and disease. The first studies characterizing endogenous PHLPP1α by Western blot reported a ~140 kDa migrating protein on SDS-PAGE, using a custom made antibodyHere we used PHLPP1 gene knockout (KO) mice to unambiguously authenticate target specificity of several common, as well as seldom used, PHLPP1 antibodies. Gene deletion in KO mice was confirmed by Western analysis of PHLPP1 in tissue extracts. All antibodies detected the larger ~190 kDa PHLPP1β variant, as confirmed by its absence in KO mice. A presumed ~150 kDa PHLPP1α signal was detected by four of the antibodies but equally present in PHLPP1 KO mice – indicating it is likely a non-specific signal. A fifth relatively new polyclonal commercial antibody uniquely recognized a ~145–150 kDa PHLPP1α signal absent in KO mice. Our findings suggest that antibody selection is a key experimental factor in the study of PHLPP1α (particularly in brain).Brain tissue was harvested from PHLPP1 KO and WT mice. Total protein extracts were probed with anti-PHLPP1 antibodies . AntibodThe PHLPP1β variant was clearly visible after 30 min film exposure using most antibodies (still faint using the Cayman antibody), and absent in KO extracts  or CaymaWe next evaluated the impact of antibody selection by testing if scientific conclusions change by reagent. Protein expression levels of PHLPP1α/β in neurons versus astroctyes were investigated. Performing the experiment with Bethyl laboratories antibody (A300-660A) revealed that “PHLPP1α” (i.e. ~150 kDa band) is highly expressed in neurons and absent in astrocytes . RepeatiImmunohistochemistry (IHC) is commonly used to define distribution/localization patterns of antigens in cells and tissues. The Cosmo antibody appears to be a superior reagent to detect PHLPP1α/β by Western analysis. We tested Cosmos's suitability for IHC applications in brain. A strong fluorescent signal (RED) was observed in both cultured primary WT and KO neurons . ConsistWe next tested PHLPP1 antibodies in human derived cells/tissue. SHSY5Ys are neuronal-like cells which fully differentiate after 7d treatment with 10 μM retinoic acid (RA). We compared protein levels of PHLPP1 variants in undifferentiated (un-Diff) versus 7d RA differentiated (RA-Diff) neuronal cells. All antibodies detected ~190 kDa PHLPP1β in SHSY5Y homogenates, which appeared unaffected by differentiation –4D. In cConfounding detection of non-specific ~150 kDa signal(s) may also be problematic in non-CNS tissues. Bethyl Laboratories antibody (A300-660A) was first used to screen peripheral organs including pancreas, heart, kidney, liver, and lung for the ~150 kDa non-specific band in WT/KO mice . InsulinPHLPP1 is abundant in brain1Here we report that PHLPP1 antibodies accurately detect PHLPP1β (~190 kDa) by Western analysis. In contrast, many commonly used antibodies do not accurately measure PHLPP1α levels (~145–150 kDa). Non-specific band(s) with similar molecular mass to PHLPP1α appear to hide the correct signal. A relatively new commercial PHLPP1 antibody, developed by the Kazusa DNA Research Institute (sold by Cosmo Bio Co Ltd.), accurately reports PHLPP1α levels by avoiding detection of overlapping erroneous ~150 kDa non-specific signals. To the best of our knowledge it has not been used in prior studies.Review of PHLPP1 literature reveal continuing trends to exclude molecular weight standards in data presentation of Western blot figures - which may relate to article space/page restrictions. It is common to find a PHLPP1 figure showing a single cropped band labeled “PHLPP1” but lacking indication of estimated molecular mass  or named variant (α variant versus β variant). Those parameters should be acknowledged. Omitting that vital information make it difficult to retrospectively interpret results in light of our new findings. This is problematic because 1) PHLPP1α and PHLPP1β variants likely have different functions and roles in disease pathology, and 2) work here suggests that a dominant ~150 kDa non-specific signal(s) is preferentially recognized by many PHLPP1 antibodies – thus inaccurately describing PHLPP1α signaling changes.Our results do not contest the validity of past studies using different PHLPP1 antibodies . Rather, we simply caution interpretation of the ~150 kDa band. If using other PHLPP1 antibodies not characterized here, we also recommend including knockdown control experiments to verify target specificity. Notably, some organs such as liver and heart do not appear to express high levels of non-specific ~150 kDa signal(s) – simplifying data interpretation in those tissues . In contrast, the ~150 kDa non-specific signal is especially problematic in brain.Antibody cross-reactivity remains a universal problem with immunoassays131415Tangentially, we do not know why PHLPP1α (as detected by Cosmo) has low expression in cortical neurons. Hippocampus appears to have relatively high levels of PHLPP1α. Future studies need to re-evaluate PHLPP1α signaling in different neuronal populations and glia. Levels may vary by cell type and location.Our second experiment examined the effect of neuronal differentiation on PHLPP1 variants. Results of those studies also show that antibody selection dramatically changes data interpretation. The exact same cell homogenate samples were probed with four PHLPP1 antibodies. Bethyl Laboratories antibody revealed a ~150 kDa band increased after neuronal differentiation. Millipore antibody revealed a ~150 kDa band decreased after neuronal differentiation. Neither the Cosmo or Cayman antibodies detected a ~150 kDa band in SHSY5Y homogenates. The correct finding is most likely that PHLPP1α has low expression in SHSY5Y cells and is not altered by differentiation (i.e. Cosmo results). The ~150 kDa signals detected by Bethyl Laboratories/Millipore antibodies are likely non-specific proteins.The identity of potential cross-reacting ~150 kDa proteins detected by some PHLPP1 antibodies remain unclear. It may be a catenin related signal. A recent report combining immunoprecipitation and mass spectrometry identified a ~90 kDa β-catenin cross-reacting signal detected by some PHLPP1 antibodiesin vitro fails to decrease the ~150 kDa PHLPP1 signal (unpublished observations). Thus while it is reasonable to assume that the ~150 kDa PHLPP1 signal might be PHLPP2, we do not think it the explanation.Cross reactivity with PHLPP2, the second isoform of PHLPP, may be another explanation. PHLPP2 migrates at ~150 kDa by Western analysisPHLPP1α lacks the large n-terminal exon 1 found in PHLPP1β . Lack of exon 1 is the only reported difference between PHLPP1 variants. Exon 4 was chosen for targeted disruption in PHLPP1 gene KO micePHLPP1 Antibodies: Five anti-PHLPP1 antibodies were obtained from commercial sources. Publicly available manufacture information for each antibody is as follows: (1) Bethyl Laboratories Antibody  antigen corresponding to the c-terminal side of human PHLPP1α/β; LPDYYDTPL, 1 mismatch to corresponding mouse sequence). (2) Bethyl Laboratories Antibody  Cayman Chemical Antibody . (4) Millipore . Alpha-Tubulin loading control was purchased from Cell Signaling technology. Goat anti-rabbit – HRP secondary was purchased from Life Technologies. (5) Cosmo Bio Co Ltd.  were collected from three Alzheimer's and three neurologically diseased patients without Alzheimer, who died and samples subsequently stored at −80°C. Patient groups were comparable by age and gender. Tissue samples were homogenized according to procedures detailed below. Brain extracts were stored at −80°C until biochemical analysis.The authors confirm that all experiments were performed in accordance with relevant guidelines and regulations. Animal work methods were carried out in accordance with procedures that were approved by the IACUC of the University of Pittsburgh. Methods of collection for human brain tissues were performed in accordance with procedures that were approved by an Institutional Review Board committee of the University of Pittsburgh Medical Center.Primary Neurons. PHLPP1 WT and KO embryos were collected from timed pregnant females (E14–E16). Embryonic brains were isolated and meninges carefully removed under dissecting microscope. Cortical halves were separated and placed in ice cold hanks balanced salt solution (HBSS). Tissues were minced 1–2 min with sterile scissors in a 1.5 mL tube containing HBSS. Tissue was trypzinized for 8 min at 37°C, protease activity quenched with Neurobasal/B27 supplement + 10% fetal bovine serum (FBS), triturated ~10 times through a fire-polished glass Pasteur pipette, and dissociated cells counted on a hemacytometer. Neurons were seeded onto poly-D-lysine coated 6-well plates in Neurobasal/B27 culture media, and maintained by ½ media exchange every 3 day. ARA-C was added on day in vitro 3 (DIV) to prevent glia proliferation. Neurons were harvested for biochemistry on DIV6. Primary Astrocytes. Postnatal day (PND) 1-2 PHLPP1 WT and KO pups were collected and brains harvested. Meninges were carefully removed under dissecting microscope. Hemispheres dissociated by 10 min incubation in trypsin solution, passed through a 10 mL pipette 10-20X, and protease activity quenched in DMEM/F12/10%FBS (i.e. maintenance media). Total brain mix was seeded onto T75 flasks. Only astrocytes exponentially proliferate over culture days and serial passages. After 2-3 passages on T75 flasks, WT/KO astrocytes were prepared for downstream experiments and seeded onto 6-well plates. Human neuronal SHSY5Ys. Cells were purchased from ATCC. Undifferentiated SHSY5Ys were propagated in Optim-MEM culture media with 10%FBS on T75 culture flasks. Cells were trypsinized and seeded onto 6 well plates. Undifferentiated cells were collected for biochemistry 3d after plating. SHSY5Ys were differentiated over 7d by media replacement with Opti-MEM containing 10 μM RA + 1%FBS. Differentiated neurons were harvest for biochemistry on day 7.Tissues and cells were homogenized in RIPA buffer containing protease inhibitors, phosphatase inhibitors and EDTA. Samples were pulse-sonicated for 20–30s. Protein concentrations were analyzed using the BCA assay. Samples were prepared in Laemmli loading buffer. Protein samples were loaded onto gradient TGX precast SDS-PAGE gels (BioRad): 20 μg/well for human brain tissue, 30 μg/well for mouse WT/KO tissues, 20 μg/well for human neuronal SHSY5Y electrophoresis experiments, and 10–20 μg/well for primary neuron and astrocyte studies. In the latter primary neuron versus astrocyte study, total protein stain was visualized using reversible Swift Membrane Stain (Fisher Scientific). Proteins were electrophoresed at ~150 V for ~1 h. Proteins were transferred to PVDF membranes (100 V/1.15 h/4°C). Blots were blocked 1 h in tris-buffered saline with tween-20 (TBST) + 7.5% milk. Primary antibodies were prepared in TBST/milk and incubated overnight on a rocker at 4°C. Blots were washed 3X in TBS, incubated 2 h in secondary antibody , washed 3X in TBS, incubated 2 min with HRP detection reagent (PIERCE), and films exposed/developed in a dark room. Antibodies were individually optimized for detection of PHLPP1 except for In Vitro Neuron Culture: Sterile 8-well chamber glass culture slides were treated with poly-D-lysine overnight at 37°C. WT and KO neurons were seeded onto glass slides. DIV8 neurons were washed with PBS, fixed ~25 min with 4% paraformaldehyde, washed with PBS, cell membrane permeabilized with 0.1% Trition X-100/PBS, washed with PBS, blocked ~45 min with 20% goat serum + 1%BSA in PBS, and incubated overnight at 4°C with primary antibody dissolved in 3% goat serum/PBS. Neuronal β-tubulin III (neuron marker) was purchased from abcam. Wells were washed with PBS, incubated with Alexa Fluor Goat-anti Rabbit 594 and Alexa Fluor Goat-anti mouse 488 (Life Technologies) for ~1.5 h. Wells were washed with PBS, plastic chamber removed, and glass slide mounted with ProLong Gold Antifade with DAPI (Life Technologies). Images were collected (20× magnification) on a fluorescent microscope  and complied in Photoshop. Ex Vivo Brain Sections: WT and KO mice were anesthetized, transcardially perfused with PBS followed by 10% formalin. Brains were collected and post-fixed in 10% formalin followed by 30% sucrose solution. Brains were sectioned, mounted on glass slides, and incubated with primary rabbit anti-PHLPP1 antibody (Cosmo) as well as either mouse anti-neuronal tubulin III (Neuron Marker) or GFAP (astrocyte marker). Sections were washed and incubated with Alexa Fluor Goat-anti Rabbit 594 (RED) and Alexa Fluor Goat-anti mouse 488 (GREEN). 20× images were collected on a fluorescent microscope.


Cutaneous involvement characterized by urticarial lesions with or without angioedema and itch is commonly observed in routine medical practice. The clinical approach may still remain complex in real life, because several diseases may display similar cutaneous manifestations. Urticaria is a common disease, characterized by the sudden appearance of wheals, with/without angioedema. The term Chronic Urticaria (CU) encompasses a group of conditions with different underlying causes and different mechanisms, but sharing the clinical picture of recurring wheals and/or angioedema for at least 6 weeks. Hereditary Angioedema (HAE) is a rare disorder characterized by recurrent episodes of non-pruritic, non-pitting, subcutaneous or submucosal edema affecting the extremities, face, throat, trunk, genitalia, or bowel, that are referred as “attacks”. HAE is an autosomal dominant disease caused by a deficiency of functional C1 inhibitor, due to a mutation in C1-INH gene (serping 1 gene) characterized by the clonal proliferation of mast cells, leading to their accumulation, and possibly mediator release, in one or more organs. In childhood there are two main forms of mastocytosis, the Systemic and the Cutaneous. The clinical features of skin lesions in urticaria, angioedema and mastocytosis may differ depending on the aetiologic factors, and the underlying pathophysiological mechanisms. The diagnostic process, as stepwise approach in routine clinical practice, is here reviewed for CU, HAE and mastocytosis, resulting in an integrated method for improved management of these cutaneous diseases. Taking into account that usually these conditions have also a relevant impact on the quality of life of children, affecting social activities and behavior, the availability of care pathways could be helpful in disentangle the diagnostic issue achieving the most cost-effective ratio. Cutaneous involvement characterized by urticarial lesions with or without angioedema and itch is commonly observed in routine medical practice, and often this observation poses important diagnostic challenges. In fact, although the diagnostic processes are quite standardized, based on scientific evidences , the cliThis review describes the clinical features of Urticaria, Angioedema, and Mastocytosis in childhood, including epidemiology. The diagnostic process, as stepwise approach, in routine clinical practice is reviewed for chronic urticaria (CU), hereditary angioedema (HAE) and mastocytosis, resulting in an integrated method for improved management of these cutaneous disease.Urticaria is a common disease, affecting 15-25% of the population with at least one episode during lifetime. In children, urticaria appears to be less common. In the United Kingdom the occurence of all forms of childhood urticaria is around 3,4% .A clinical classification of urticaria has been recently published in a European Academy of Allergy and Clinical Immunology (EEACI) guideline  Table 11.Table 1Urticaria is characterized by the sudden appearance of pruritic wheals, with/without angioedema. A wheal consists of three typical features: 1. a central swelling of variable size, almost invariably surrounded by a reflex erythema; 2. itching or, sometimes, burning sensation; 3. a fleeting nature, with the skin returning to its normal appearance, usually within 1–24 h. Urticaria is defined as acute if wheals last less than 6 weeks, and chronic if lasting 6 weeks or more . The terCU in children is caused by physical factors in at least 6% of cases. Less often, infections 4%), foods (4%), additives , aeroallergens  and drugs , are trigger factors %, foods , whereasOther urticarial disorders include cholinergic, acquagenic, contact- and exercise-induced urticaria ,9. The fThere are few epidemiological data about Angioedema in children. In the only published study on children with urticaria, wheals alone were present in 78,4%, angioedema alone in 6,65% and both in 15% [http://www.orpha.net) characterized by recurrent episodes of non-pruritic, non-pitting, subcutaneous or submucosal edema affecting the extremities, face, throat , trunk, genitalia, or bowel, that are referred as “attacks”. Classically edema develops slowly, over a period of up to 36 hours and completely resolve spontaneously within 4 days. Swelling is usually self-limited and painless when affecting the skin, whereas abdominal attacks with diarrhea, vomiting, and pseudo-obstructive syndrome are painful, resulting sometimes in unnecessary surgery. All attacks localized over the shoulders must be considered severe and potentially life-threatening [de novo mutations and exon deletions in the C1inhibitor gene of patients with angioedema without family history, suggesting important and incoming acquisitions in the genetic epidemiology of the disease [Hereditary Angioedema (HAE) is a rare disorder , due to a mutation in C1-INH gene (serping 1 gene) mapped to chromosome 1111q12-q 13.1). Bradykinin is the main mediator implicated in edema . Althoug-q 13.1. Mastocytosis is a heterogeneous disorder characterized by the clonal proliferation of mast cells (MCs), leading to their accumulation, and possibly mediator release, in one or more organs . In chilIn Spain a prevalence of 5.4 cases per 1000 pediatric dermatology patients was found, whereas in Mexico the condition was seen in 1:500 first-time pediatric dermatology patients. Mastocytosis occurs in all ethnic groups and may appear at any age. CM is more common in children; a second smaller peak of incidence is seen in adults in the third to fourth decade .MPCM (urticaria pigmentosa) is the most common clinical variant in which fixed, reddish brown lesions occurring as maculo-papules, plaques, nodules or blisters are found. These lesions urticate in response to physical irritation (Darier’s sign). Urticaria Pigmentosa (UP) lesions tend to be larger, better delineated, and more hyperpigmented in children, as compared to adults, who tend to have numerous small lesions that coalesce to form mottled areas. The trunk and thigh are more commonly involved with sparing of face, palms and soles .DCM is a rare variant of childhood mastocytosis that appears as diffuse infiltrative yellow-orange xanthogranuloma-like subcutaneous nodules, or as a widespread urticarial eruption with bullae and redness. The clinical course of DCM is more severe than that of mastocytoma and MPCM and can even be life-threatening, due to hypovolemic shock, mast cell leukemia, gastrointestinal hemorrhage, and cachexia .A step by step diagnostic approach or chronic urticaria is recommended, starting from the patient history and physical examination, in order to better address the subsequent tests, some of which are performed to exclude severe systemic diseases Table .Table 3Clinical history, including all possible eliciting factors and significant aspects of the origin of urticaria, is the most important issue for a proper diagnostic process. Some of the most frequently asked questions are reported in Table Additional investigations are suggested in the diagnostic approach to CU and CSU. Although allergy is a rare cause of CSU an allergy screening test should be considered in CSU patients with a personal history suggestive of allergic diseases. Chronic persistent bacterial, viral parasitic and fungal infections that may trigger urticarial symptoms in patients with CSU should be considered, only according to the patient’s history . Total iSince autoimmune diseases, like celiac or thyroid disease are commonly reported in children with CU, laboratory tests for these diseases should be done . Recent Some other conditions should be considered in the diagnostic process mainly when an immunocomplex-induced disease is suspected. These include serum sickness or autoimmune diseases Figure . When isfirst line and second line tools  and serum C1-inhibitor protein and its functional levels . TherefoMastocytosis is essentially suspected on clinical ground, (including identification of UP lesions and recognition of compatible MC mediator-related symptoms). Because mastocytosis is due to a clonal proliferation of MCs, the diagnosis is established by detection of MCs with abnormal morphologic features and immunophenotypes in the bone marrow or other extracutaneous organs and investigating specific mutations in its growth factor (c-KIT) receptor .first line and second line of diagnostic tools and the diagnostic pathway process are respectively showed (Table The SCORMA (SCORing Mastocytosis) Index provides a standardized information on the severity of CM. It is a clinical scoring system based on a semi-quantitative analysis of extent, intensity and subjective complains, with no burden for patients, which is particularly important in children. The simultaneous assessment of SCORMA Index and tryptase level is essential in all cases of pediatric mastocytosis . In Tabled Table . In someThe identification and elimination of eliciting factors is the mainstay approach for urticaria treatment, also considered the special issue of the avoidance of physical stimuli for the treatment of physical urticaria (PU). Since the CSU is often reported to be associated with a variety of inflammatory or infectious diseases, the proper treatment for these underlying conditions should be advised. Although, IgE-mediated food allergy is rarely the underlying cause of chronic spontaneous urticaria, after a confirmed diagnostic tests, the specific food allergens need to be omitted as far as possible .In agreement with the most recent international guidelines, non-sedating second generation antihistamines are recommended as first line symptomatic treatment. These drugs have shown to be effective in numerous randomized placebo-controlled clinical trials. First generation antihistamines are equally effective, but they have pronounced anticholinergic and sedative effects which last longer than 12 h, whereas the antipruritic effects last only 4–6 h. In addition, first generation antihistamines can interfere with rapid eye movement sleep, and impact on learning and performance. Thus, the use of first generation antihistamines is discouraged .The second level of therapy includes increasing the dosage of non-sedating second-generation antihistamines. Their mode of action offers the best risk to benefit outcome, in comparison to alternative treatments. At the III level, guidelines recommend to change the non-sedating antihistamine used in level II or to add antileukotriens, as well as a short course of corticosteroids (3 days). At level IV, different treatment options are suggested. H2 antihistamines in combination with H1 antihistamines have a low evidence-based support, whereas more data are available for dapsone and cyclosporine A. However the most effective drug at level IV treatment is Omalizumab .Omalizumab diminished clinical symptoms and signs of chronic idiopathic urticaria in patients who had remained symptomatic despite the use of approved doses of H1-antihistamines. Histamine release from cutaneous mast cells has long been associated with the pathogenesis of urticaria, whereas in patients with chronic idiopathic urticaria, basophils and IgE may also play an important role. Omalizumab, reduces the levels of free IgE and the high-affinity receptor for the Fc region of IgE (FcεRI), both of which are essential in mast-cell and basophil activation. Studies have shown that omalizumab may suppress allergen-mediated skin reactions through its reduction of FcεRI function in basophils and mast cells  Table 77. A studOverall, the disease activity should be evaluated at regular intervals, in order to reduce the therapy. After three months of complete response the intensity treatment might be reduced .The main treatment of HAE is represented by the control of acute attacks, as well as short-term and long-term prophylaxys . Short-tThe first-line therapy for HAE attacks in most countries includes replacement therapy with C1-INH concentrate . BerinerAmong the available new treatment modalities for long-term prophylaxis, plasma-derived C1-esterase inhibitor (pdC1-INH) concentrates represent an alternative treatment for prevention of HAE attacks. Experimental results showed that outcomes with pd C1 INH treatment of HAE in pediatric patients are comparable to adults .The Food and Drug Administration (FDA) has approved the chronic prophylactic use of nano-filtered C1-inhibitor (nfC1-INH) based on limited data, and it will be important that physicians monitor adverse events carefully. Thus far, it appears that the chronic use of C1-INH is safe and effective . Recent The recent literature showed that patients with frequent attacks may benefit from long term prophylaxis. A study evaluated the safety and prophylactic effect of weekly administrations of recombinant C1 INH (rC1 INH). Weekly administrations of 50 U/Kg rhC1INH appeared to reduce the frequency of HAE attacks and were safe and well tolerated .Fresh Frozen Plasma (FFP) is often used for treatment of HAE attacks and for short term prophylaxis. However, the use of FFP should be reserved when C1-INH, icatibant or ecallantide are not available. The appropriate use of disease-specific treatments for HAE improve patients' quality of life and reduce HAE-associated morbidity and mortality. However, many of the newer treatments represent financial challenges due to high costs, which are not always balanced by the reduction of other medical expenses .Usually, isolated CM needs no treatment or action to be taken. CM with cosmetic complaints should be treated by a topical therapy only in children older than 2 years. When CM is confined to about 10% of body surface, occlusive dressing is used. When the extension is larger, (more than 10%) the occlusive dressing is optional, and usually the corticosteroid cream is diluted .In CM with complaints of itch, redness and swelling, avoiding foods with a possible triggering role has to be suggested. Finally, also a systemic therapy with H1-and H2-blocker  may be useful .2/day have been used in children, and reducing the dosage as soon as sustained healing of the skin lesions is noted is sufficient to obtain remission. Monitoring of peripheral blood, liver function, cardiotoxicity, and growth velocity is necessary [DCM is self-limiting, and the need for a treatment is questionable. If DCM causes significant distress, leads to frequent hospitalizations, or is life-threatening, treatment with imatinib is a good option. Imatinib mesylate, a type II kinase inhibitor, inhibits cell proliferation and induces apoptosis by binding the adenosine triphosphate pocket and the adjacent site of the kinase. The nonselectivity of imatinib also explains side effects such as cardiomyopathy and reduced growth velocity, the latter being a relevant side effect in children. Other side effects include nausea, vomiting, diarrhea, increased liver enzymes, hypophosphatemia, edema, skin rash, granulocytopenia, anemia, and thrombocytopenia, but these are rare in children. A dose of 200 mg/mecessary .The clinical features of skin lesions in urticaria, angioedema and mastocytosis may differ depending on the aetiologic factors, and the underlying pathophysiological mechanisms. For this reason, in routine clinical practice itching and cutaneous lesions generally represent a heavy challenge for the pediatrician in terms of scientific knowledge and use of proper diagnostic procedures, taking into account that usually chronic urticaria have also a relevant impact on the quality of life of children, affecting social activities and behavior. Hence, the availability of care pathways for urticaria, angioedema and mastocytosis could be helpful in disentangle the diagnostic issue achieving the most cost-effective ratio.


Stratum Pyramidale (SP) and Stratum Oriens (SO), with very few cells in Stratum Radiatum (SR) and none in Stratum Lacunosum Moleculare (SLM). There was, however, a greater proportion of GAD-positive cells were immunopositive for PV in SP in CA2 than in CA1 or CA3. CA2 SP also contained a larger density of somatostatin-, calbindin-, and VIP-immunopositive somata than CA1 and/or CA3. Like those in CA1 and CA3, CCK-immunopositive somata in CA2 were mostly located in SR. Reelin- and NPY- immunolabeled cell bodies were located in all layers of the three CA regions. However, a higher density of Reelin-positive somata was found in SP and SR of CA2 than in CA1 or CA3.The CA2 region of the mammalian hippocampus is a unique region with its own distinctive properties, inputs and pathologies. Disruption of inhibitory circuits in this region appears to be linked with the pathology of specific psychiatric disorders, promoting interest in its local circuitry, its role in hippocampal function and its dysfunction in disease. In previous studies, CA2 interneurons, including a novel subclass of CA2 dendrite-preferring interneurons that has not been identified in other CA regions, have been shown to display physiological, synaptic and morphological properties unique to this sub-field and may therefore play a crucial role in the hippocampal circuitry. The distributions of immuno-labeled interneurons in dorsal CA2 were studied and compared with those of interneurons in CA1 and CA3. Like those in CA1 and CA3, the somata of CA2 parvalbumin-immunoperoxidase-labeled interneurons were located primarily in  These neurons displayed spike frequency adaptation and a “sag,” in response to hyperpolarizing current injection. These characteristics are strikingly different from those of CA1 PV- and CCK-basket cells, but resemble those of the OLM cells of the CA1 region and horizontally oriented CCK-immunopositive interneurons near the stratum radiatum (SR)/stratum lacunosum moleculare (SLM) border, Schaffer associated cells  of all three CA regions, while the axons of CA2 bistratified cells were very strongly polarized, ramifying in SO and SR of the CA2 and CA1 regions but stopping abruptly at the CA2/CA3 border. The strength and time course of synaptic depression and facilitation were found to correlate with the class of interneuron. Basket and bistratified cells with narrow arbors, like those in CA1, received EPSPs that displayed pronounced depression, while EPSPs elicited in CA2 basket cells with broad dendritic arbor exhibited facilitation and limited augmentation. Another study also revealed a subclass of dendrite-preferring interneurons in CA2 that had not been previously described in any CA regions, CA2 SP-SR interneurons  and fixed overnight . Slices were washed, gelatin-embedded and 50 μm coronal sections cut. Sections were cryoprotected with sucrose and freeze-thawed. Sections were then incubated in 1% Sodium Borohydride (NaBH4) for 30 min and in 10% normal goat serum (NGS) for another 30 min. Sections were incubated overnight at 4°C in a primary antibody solution . Sections were then incubated in a secondary antibody solution, anti-mouse fluorescein isothiocyanate (FITC), made up in PBS. Sections were mounted on slides in Vectashield (Vector Laboratories) and studied by fluorescence microscopy as shown in Figure All procedures used throughout this study were carried out according to the British Home Office regulations with regard to the Animal Scientific Procedures Act 1986. Young adult male Wistar rats weighing 130–160 g were anaesthetized with inhaled Fluothane and then by intra-peritoneal injection of Euthatal  (60 mg/kg). The rats were perfused transcardially with ice cold oxygenated modified artificial cerebrospinal fluid (mACSF) containing in mM: 248 Sucrose, 25.5 NaHCOSections were then washed and incubated overnight at 4°C in a second primary antibody solution made up in PB. The list of primary antibodies used is presented in Table Frog muscle parvalbumin was used to produce the mouse monoclonal parvalbumin antibody (Clone PARV-9). This antibody reacts with human, feline, bovine, rat, frog, goat, fish, pig, canine, rabbit. Studies performed by the manufacturer indicate that this antibody recognizes parvalbumin in a calcium ion-dependent manner and does not react with other members of the EF-hand family, such as calmodulin, intestinal calcium-binding protein, S100A2 (S100L), S100A6 , the α and β chains of S-100. The specificity of this antibody was also tested on tissue from parvalbumin knockout mice and control mice and no staining was observed in mice lacking parvalbumin  was characterized using ELISA and radioimmunoassay . In Western blots, this antibody labeled a single band at 29 kDa corresponding to the molecular mass of calretinin  was prepared against the recombinant reelin residues 164–496 derived from mouse E15–E17 embryonic brain  was raised against somatostatin-14 conjugated to keyhole limpet hemocyanin and was characterized using ELISA and immunocytochemical methods  abolished all tissue staining. Pre-adsorption with the following peptides: Secretin, gastric inhibitory polypeptide, somatostatin, glucagon, insulin, ACTH, gastrin 34, FMRF-amide, rat GHRF, human GHRF, peptide histidine isoleucine 27, rat pancreatic polypeptide, motilin, peptide YY, substance P, neuropeptide Y, and CGRP did not affect the immunstaining. This antibody reacts with buffalo, chicken, guinea pig, hamster, human, monkey, mouse, rat, pig, sparrow, stingray, zebrafish.The polyclonal antibody to VIP was raised in rabbit against porcine VIP conjugated to bovine thyroglobin with carbodiimide. In studies conducted by the manufacturer, preadsorption of the antibody with VIP  staining Figure . The PCPFor quantification in the hippocampus, the number of all GAD-67-, PV-, CCK-, Reelin-, VIP, SOM-, CB-, NPY-immuno-positive somata in CA1, CA2, and CA3 were determined using a DMR microscope (Leica Microsystems) in 5 adjacent coronal 50 μm-thick sections from at least 6 different rats per antibody. Slices used in this study were taken from the dorsal hippocampus in which the width of the CA2 region was on average 405 ± 35 μm to allow comparison of antibodies. The contours of all stained cell bodies in each section were drawn using a drawing tube and then digitized and counted using ImageJ software. The number of immunopositive interneurons was expressed as a percentage of GAD-positive cells in each layer of each of the three CA regions, SO, SP, SR, SL, and SLM. Neuronal density units were also analyzed for each marker in all hippocampal regions and layers using ImageJ software. Statistical analyses were performed using Mann Whitney U test (MW U) for a distinct comparison of the three CA regions. All data are represented as mean ± standard deviation (SD). All antibodies used in this study resulted in a clear staining of immunopositive cell bodies. Some antibodies  only stained somata and/or very proximal dendrites, others  also revealed dendritic and axonal staining. Partial reconstructions were only carried out using a drawing tube (x100 objective) when good antibody staining labeled interneuron dendrites.P < 0.05, UCA1 = 0, UCA3 = 2)  Figure , with a P Figure . PV-posiP < 0.05; UCA1 = 1, UCA2 = 1) . CA1 is not considered to contain an equivalent layer. Partial reconstructions of the dendritic trees of CCK-immunopositive interneurons in CA2 revealed that those with somata located in SP had vertical dendrites that extended from SO to SLM, while those with somata in SO had predominantly horizontal dendrites  Figure  and a his Figure . In thisP < 0.05, UCA1 = 0, UCA2 = 0), with a density of 32 103 ± 2.6 103 reelin-positive cells/mm3 and 19 103 ± 1.2 103 cells/mm3 in CA2 SP and SR respectively  but similar to that in CA3 in all layers . Dendritic trees of SOM-positive interneurons were not labeled with the antibody used in this study.SOM-immunopositive interneurons represented 32.6 ± 7.2% of the total GAD-positive population in CA2, 22.6 ± 4.3% in CA1 and 44.9 ± 14.1% in CA3 Figure . The proP < 0.05, UCA1, CA3 = 0), with densities of 1.2 103 ± 0.7 103 CB-positive cells/mm3, 9.7 103 ± 1.2 103 cells/mm3 and 1.1 103 ± 0.7 103 cells/mm3 in SP of CA1, CA2, and CA3 respectively . CB-immunopositive interneurons with somata located in SO of CA2 had horizontal dendrites that, in some cases, extended into CA3 and/or CA1  but larger than that in SP of the CA3 region . VIP staining labeled only the soma of these interneurons preventing the reconstructions of the cells.VIP-immunopositive interneurons represented 8.7 ± 4.7% of the total interneuronal population in CA2, 8.5 ± 3.8% in CA1 and 9 ± 3% in CA3 Figure . The numP < 0.05, U = 0). SLM of the CA1 and CA3 regions had a greater percentage of GAD- and NPY-positive cell bodies than CA2 .NPY-immunopositive interneurons represented 51.4 ± 3.3% of the total interneuronal population in CA2, 57.2 ± 3.4% in CA1 and 58.1 ± 3.5% in CA3 Figure . There wP > 0.05). There was, however, a larger proportion of GAD- and CR-positive somata in SLM of CA1 than in SLM of the CA2 and CA3 regions . Most CR-positive interneurons with cell bodies located in SO had horizontal dendrites  allowing the comparison of the staining for each antibody used in this study. We can, however, predict that the number of interneurons may vary depending on the species, gender and age of the animals. Immunoperoxidase staining was used to determine the density of different classes of interneurons. The protocol used  was similar for each antibody. Although the dilutions were chosen for an optimal labeling of the cells, we may underestimate the numbers of stained neurons by using the same method for each marker.In this study, interneuronal dendritic trees were only reconstructed when dendritic staining was adequate. Typically, as in CA1 in this and previous studies, the staining of calcium binding proteins labeled somata, dendrites and axons of interneurons whereas staining for other peptides or proteins resulted in poor labeling of dendrites with the exception only of CCK staining. These partial reconstructions did not therefore allow morphological comparisons of immunolabeled cells. To date, only PV-positive basket and bistratified cells and SP-SR cells have been fully characterized in CA2 (Mercer et al., Like other CA regions and indeed, like many other brain regions (Martinez-Cerdeno et al., Between 11 and 21% of interneurons of the CA2 region express parvalbumin and 20–25% calretinin. As in CA1, CA2 PV-interneurons are most likely to be classified as basket, bistratified or axo-axonic cells in SP and OLM cells in SO (Freund and Buzsaki, CCK-immunopositive interneurons of the CA2 region were mainly located in SP and SR with CCK-positive somata representing the largest population of neurons in SR. Dendritic patterns of CA2 CCK-positive cells were similar to those previously described in CA1, where CCK-positive interneurons include basket cells, Schaffer collateral-associated interneurons, apical dendrite innervating cells and perforant path-associated cells (Cope et al., VIP was the least abundant interneuronal marker in the CA2 region. VIP-immunopositive somata were mainly found in SO, SP, and SR of CA2. In CA1, most interneuron-specific interneurons are immunopositive for VIP (Acsady et al., Although no thorny excrescences are present on CA2 pyramidal dendrites (Mercer et al., A receptor subunits appear to be responsible for the re-organization of connections in this region (Loup et al., Growing evidence shows that the CA2 region, and more specifically inhibition in CA2, may play an important role in the hippocampal circuitry. CA2 interneurons target pyramidal cells locally, as well as in CA1 and CA3 and receive inputs from CA2, CA3, the supramamillary body, the amygdala, the entorhinal cortex and the dentate gyrus (Berretta et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text. 2+) entry. Here, we sought understanding of a Synta66-resistant Ca2+ entry pathway activated by VEGF.Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca2+ in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca2+ entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca2+ entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca2+ release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4–synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress.Measurement of intracellular Ca2+ entry and downstream endothelial cell remodeling.VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca Here, we investigated the role of Orai3 and the activation mechanism for these S66-resistant channels.Vascular endothelial growth factor (VEGF) is a primary inducer of endothelial cell function, for example in the regulation of vascular permeability and angiogenesis.Materials and Methods are available in the online-only Data Supplement.2+, VEGF (30 ng/mL) caused a Ca2+ release event, which reached a maximum in 3 minutes and then decayed . We, therefore, investigated tube formation in coculture with fibroblasts and on Matrigel in vitro and in vivo in mice. Orai3 siRNA reduced tube length and number of tube branches Figure H. The da29 which has been previously linked to AA–regulated Ca2+ channels.30 The PLC inhibitor U73122 abolished VEGF-evoked Ca2+ elevation.22 Edelfosine, an alternative PLC inhibitor, had the same effect . Inhibitors of several other signaling elements downstream of VEGFR2 were tested and had no effect . Because AA production is downstream of PLCγ1, we investigated if it was possible to circumvent VEGFR2 by directly applying exogenous AA. The response to exogenous AA included Ca2+ release  and so we investigated if there was an effect on Ca2+ entry independent of release and CRAC channel activation by first depleting Ca2+ stores with thapsigargin and including S66. Ca2+ was present in the extracellular medium. There was robust Ca2+ entry in response to 40 μmol/L AA. Lower AA concentrations generated only small inconsistent effects . To investigate if 40 μmol/L AA caused nonspecific membrane disruption, we applied 40 μmol/L eicosatetraynoic acid, a nonmetabolizeable AA analog, which did not cause Ca2+ entry . Moreover, Orai3 siRNA strongly suppressed the AA-evoked Ca2+ entry, suggesting that exogenous AA activates the Orai3 mechanism without causing membrane disruption  leading to the production of AA by cytosolic group IV phospholipase A2α ,32 We confirmed the expression of MGST2 and knocked down its expression by 2 siRNAs . The data suggest a role for AA metabolism by MGST2 in VEGF-evoked Orai3-dependent Ca2+ entry.We investigated whether AA metabolites are involved in the action of VEGF. It was previously suggested that HUVECs generate AA metabolites, such as LTC4 by enzymatic activity of microsomal glutathione S-transferase 2 (MGST2).2+ entry channel, it has to localize to the plasma membrane. However, we could not detect it at the plasma membrane under basal conditions. We, therefore, tested if Orai3 was at the plasma membrane after stimulation with VEGF. Cells in the control group were treated with sorafenib (1 μmol/L), a multikinase inhibitor, to suppress constitutive ligand-independent VEGF receptor signaling. Co-staining for CD31 protein defined the location of plasma membrane Orai3. Importantly, we could only detect Orai3 at the plasma membrane after VEGF stimulation .This study shows relevance of Orai3 to VEGF signaling and downstream endothelial cell remodeling. It also shows a previously unrecognized mechanism for acute control over Ca22 Second, overexpression of Orai3 in the HEK 293 cell line, a commonly used mammalian cell expression system, leads to constitutive Orai3 at the plasma membrane as shown by previous studies36 and confirmed by us . By contrast, we found no evidence for similar localization of endogenous Orai3 in endothelial cells. There is clearly a technical challenge in studying endogenous low abundance membrane proteins such as Orai3 and so, while we confirmed the specificity of our anti-Orai3 antibody for studies of endogenous Orai3 in endothelial cells , it was important to test our hypothesis without using this antibody. For this work, we expressed exogenous HA-tagged Orai3 in endothelial cells but we were careful to use the minimum expression abundance required for detection, making observations only 6 hours after transfection to reduce the likelihood of overexpression and thus artificial bias of Orai3 to the plasma membrane.It is surprising that Orai3 lacks localization to the plasma membrane in endothelial cells under basal conditions. First, it contrasts with the situation for Orai1, as shown in this study and observed previously.2+ into the cells. Whatever the reason, it presents a mechanistic challenge because, unlike Orai3, STIM1, and Orai1 are constitutively at the plasma membrane, yet, like Orai3, they both contribute to S66-resistant Ca2+ entry.22 This raises a question about how Orai3 integrates with or otherwise influences Orai1 and STIM1. We hope to reveal understanding of the mechanisms through future studies.The reason why there is basal exclusion of Orai3 from the surface membrane is unknown but we speculate that it is important to avoid incorporation of constitutively active Orai3-containing channels that could cause long term, potentially damaging, leak of Ca37 It is notable that all studies have used a relatively high concentration of exogenous AA in efforts to mimic the effect of an endogenous receptor agonist. To generate a robust response we needed to use 40 μmol/L AA. Other studies have used ≤10× less, but such concentrations are still relatively high. This requirement could suggest that exogenous AA is inefficient at mimicking AA generated enzymatically inside cells or that a cofactor is required to activate the mechanism efficiently; activation of VEGFR2 by VEGF generates many signaling factors, some of which might synergize with AA.Studies of other cell types have shown dependence of Orai3 mechanisms not only on Orai1 and STIM1 but also AA and AA metabolism.2+ release-dependent mechanism for activation of this Orai3 system: Ca2+ release activating cPLA2α to catalyze the production of AA. This should not be taken to mean that Orai3 is activated by store depletion in these cells, because it is not . Physiological Ca2+ release does not necessarily cause Ca2+ store depletion. Our previous work on growth factor–activated Ca2+ signaling in vascular smooth muscle cells35 and measurements of stored Ca2+ in endothelial cells during agonist exposure38 have suggested that stores are efficiently maintained replete in the face of Ca2+ release because of reuptake of Ca2+ into stores via smooth endoplasmic reticulum Ca2+ ATPase. Store depletion is a stress to endoplasmic reticulum and so cells will have evolved mechanisms to minimize such stress during physiological signaling.We suggest a Ca2 activity and AA.42The reciprocal relationship between expression of this system and shear stress suggests that it may have greatest functional importance at sites with low or disturbed shear stress. Such sites occur in embryonic development but also at vascular loci in the adult that are vulnerable to disease or directly involved in on-going disease. Our detection of this Orai3 mechanism in endothelial cells from patients with coronary artery disease and cancer suggests relevance in pathophysiological settings; in support of this, atherosclerosis, tissue injury, cancer, and other related conditions have all been associated with increased PLAThis study was supported by research grants from the Medical Research Council, the Wellcome Trust, and British Heart Foundation. B. Hou was supported by a scholarship from the University of Leeds and the China Scholarship Council. P.J. Webster and J.E. Ritchie had fellowships from the LTHT  Charitable Foundation and Cancer Research UK, respectively.None.


Desulfobulbus cultures grown fermentatively with environmentally representative concentrations of dissolved NOM isolates, Hg[II], and HS− were assayed for absolute methylmercury (MeHg) concentration and conversion of Hg(II) to MeHg relative to total unfiltered Hg(II). Results showed the 200Hg tracer was methylated more efficiently in the presence of hydrophobic NOM isolates than in the presence of transphilic NOM, or in the absence of NOM. Different NOM isolates were associated with variable methylation efficiencies for either the 202Hg tracer or ambient 200Hg. One hydrophobic NOM, F1 HpoA derived from dissolved organic matter from the Florida Everglades, was equilibrated for different times with Hg tracer, which resulted in different methylation rates. A 5 day equilibration with F1 HpoA resulted in more MeHg production than either the 4 h or 30 day equilibration periods, suggesting a time dependence for NOM-enhanced Hg bioavailability for methylation.Methylation of tracer and ambient mercury ( Firmicutes phylum may also be important environmental methylators  for Hg methylation in microorganisms  inhibits human fetal and adult neurological development and cellular functionality .Interactions with NOM have been observed to produce variable effects on Hg methylation, including inhibition  grown fermentatively. D. propionicus 1pr3 can methylate Hg at a high rate relative to other SRB and non-SRB methylators  to environmentally realistic concentrations (~10 μM), which could otherwise accumulate under sulfate respiration to influence Hg speciation separately , dissolved NOM, or D. propionicus cells, or interfere with spectrophotometric assays for HS− concentration and cell density. Cell densities for D. propionicus 1pr3 were measured throughout the experiment using optical (cell) density at 600 nm wavelength (“OD600”) absorption measurements on a Bio-Rad SmartSpec spectrophotometer calibrated to D. propionicus 1pr3 cell morphology by direct cell counting on a Neubauer counting chamber slide. All cultures and controls were incubated in 100 mL of medium sparged with N2 and crimp-sealed over butyl rubber septa prior to autoclaving. All culturing experiments were performed in triplicate, while cell-free and killed cell negative control treatments were prepared in experimental triplicate and duplicate, respectively. Cell-free controls consisted of uninoculated medium containing an NOM isolate, while the killed-cell control consisted of cells autoclaved prior to their transfer into experimental medium with NOM isolate “F1 HpoA 30dy.” Cell-free controls displayed neither growth nor Hg methylation during the experiment. The OD600 measurements for this negative control type have been subtracted from those of the respective live cultures  to remove the effect of NOM absorption, and to facilitate inspection of cell growth curves  milli-Q water. Each NOM isolate was injected into autoclaved and cooled experimental media via syringe filtration (0.22 μm pore size cellulose acetate filters) to a final concentration of ~40 mg L−1. Following NOM isolate addition, cultures were amended with 5–10 μM of filter-sterilized aqueous HS− (quantified using the methylene blue method of Cline (− concentrations.NOM isolates were weighed out in a freeze-dried form and dissolved into sterile anoxic (sparged with Nof Cline  on a Hac200Hg(II) was added as a tracer (as 200HgCl2) to each culture or control bottle to a final concentration of 100 ± 10 ng L−1. In addition, the F1 HpoA isolate was used to assay the effect of different 200Hg(II) pre-equilibration times  on MeHg production; these experimental conditions are referenced as “F1 HpoA 4hr,” “F1 HpoA 5dy,” and “F1 HpoA 30dy,” respectively. All other NOM isolates were equilibrated with 200Hg(II) in the dark for 30 days to simulate long exposure periods and/or slow flow rates through anoxic lacustrine or estuarine sediments. After equilibration, pH values were checked in bottles designated as negative (cell-free) controls. With the exception of the “no NOM” cell-free and killed-cell controls (pH 6.9–7.0), all NOM isolate-amended controls showed pH values of 7.1–7.2  to maintain the cultures at an ambient HS− concentration of 5–10 μM. Over the course of the experiment, observed concentrations of aqueous HS− increased by at most a factor of two in most cultures, and a factor of three in a few  transferred at late exponential growth phase. Sampling for concentration of bacterial cells (OD600), total unfiltered Hg(II), and unfiltered MeHg analyses was performed at 4, 24, 48, 72 and 120 h after inoculation for live growth culture bottles, and at 4, 72, and 120 h post-inoculation for negative controls and killed-cell incubations, with 5 and 10 mL of each culture or control taken for unfiltered MeHg and total Hg analyses, respectively. Mercury methylation potential was assessed in three ways. We measured and plotted the unfiltered (“U”) concentrations (in ng L−1) of Me200HgU  and Me202HgU  over time to assess the absolute Hg methylation potential . Total Hg analysis was performed using U.S. EPA Method 1631. Aqueous samples were pre-treated with 1–2 % (v/v) 0.2N bromine monochloride (BrCl) to solubilize and oxidize all forms of Hg to reactive mercuric mercury (Hg[II]). Samples were placed in an oven at 50°C for a minimum of 12 h to accelerate the oxidation reaction. Oxidation was considered complete if excess BrCl was present (faint yellow color) after 12 h. If necessary, additional BrCl was added and allowed to react for another 12 h. Just prior to analysis, a small amount of hydroxylamine hydrochloride (NH2OH–HCl) was added to each sample until any residual color from the BrCl disappeared. Approximately 10 min after BrCl reduction, about 125 mL of sample was poured into a bubbling flask and 0.5 mL of stannous chloride (SnCl2) was added to reduce the Hg(II) to gaseous elemental Hg (Hg[0]). The Hg(0) was then purged from the sample with Hg-free N2 gas and concentrated onto a gold-coated, glass-bead trap. Finally, the gold trap was heated and the Hg(0) thermally desorbed into an argon gas stream and quantified by cold vapor atomic fluorescence spectrometry (CVAFS).All Hg and MeHg analyses were performed at the USGS Mercury Research Laboratory in Middleton, Wisconsin (199Hg) to allow for assessment of recoveries on an individual sample basis. All MeHg in each sample was isolated by water vapor distillation under atmospheric pressure. MeHg in distillates was then derivatized using sodium tetraethylborate and pre-concentrated onto Carbotraps™. Detection and quantification was achieved after thermal desorption, isothermic GC separation, and detection by ICP-MS , and the presence of 5–10 μM aqueous sulfide in all bottles, we acknowledge that a significant portion of the total dissolved D. propionicus was provided with propionate as the carbon substrate and sulfate as the terminal electron acceptor (data not shown).All cultures exhibited a lag phase during the first 24 h of growth Figure . Around 200Hg(II) equilibrated with the F1 HpoA 5dy NOM isolate exhibited the most Hg(II) tracer methylation . Other cultures that exhibited notable 200Hg(II) methylation were the NOM-free, F1 HpoA 4hr, and F1 HpoA 30dy experimental conditions . Less 200Hg was methylated in cultures containing F1 TpiA (~0.3 ng L−1 Me200Hg) and SRHA (~0.4 ng L−1 Me200Hg) NOM isolates. Cell-free controls and killed-cell controls contained less than ~0.25 ng L−1 Me200Hg  was observed in cultures containing WL HpoA and F1 HpoA 5dy NOM isolates .The highest cumulative proportion of methylation of 200Hg methylated cell−1 day−1) at the start of the experiment for the “no NOM” culture, but was higher overall during the experiment for cultures incubated with either F1 HpoA 4hr or F1 HpoA 5dy (~3.5–62 ag 200Hg methylated cell−1 day−1). The slowest overall cell-specific methylation rate (~1.3 ag 200Hg methylated cell−1 day−1) occurred in cultures incubated with F1 TpiA  than tracer Hg (200Hg) was methylated  in the F1 HpoA 5dy NOM isolate-incubated culture and SRHA isolate-containing incubations  methylation rates. Less than ~1 ng L−1 of total Me202Hg was measured in killed-cell incubations  methylation was observed in cultures incubated with F1 TpiA NOM across the whole of the experiment .The greatest cumulative proportion of unfiltered ambient Hg (202Hg) methylation was highest (~58 ag 202Hg methylated cell−1 day−1) near the beginning of the experiment (after ~24 h incubation period) for the culture grown in F1 HpoA 4hr. However, after ~24 h incubation, the cell-normalized methylation rate of this culture was exceeded by the SRHA and F1 HpoA 5dy cultures at rates of ~33–53 ag 202Hg methylated cell−1 day−1. By the final sampling time, all cultures but the F1 TpiA and F1 HpoA 4hr NOM isolate incubations exhibited cell-specific methylation rates of ~12–20 ag 202Hg methylated cell−1 day−1  was sufficient to describe most or all growth to a late exponential or early stationary phase. Only D. propionicus 1pr3 grown in the presence of the SRHA NOM isolate grew more slowly, e.g., by a factor of 2–4 times between 48 and 72 h. This observation suggests a potentially inhibitive effect on cell growth from the SRHA NOM isolate, possibly also due to its relatively high metals content . We cannot rule out, alternatively, that some NOM isolates actually facilitated cell growth to some degree, possibly by acting as electron acceptors for pyruvate oxidation  when associated with F1 HpoA and to some extent WL HpoA, in contrast to 200Hg(II) equilibrated with, e.g., SRHA or F1 TpiA NOM isolates  and ambient (202Hg) mercury pools, in absolute concentration, was methylated in the presence of F1 HpoA 5dy NOM isolate than with any other NOM isolate  species Figure  demonstr Figures , 3A. In  Figures . These o Figures , which a200Hg was also formed when “no NOM” was used. This result was perhaps a bit surprising as previous workers  methylation relative to 200Hg(II) methylation in the presence of all other NOM isolates .In contrast to the pronounced effect of F1 HpoA, however, it is worth noting that substantial Mes Figure . Possibl200Hg(II) was methylated in the presence of the SRHA NOM isolate  pools were methylated to relatively higher levels in the presence of F1 HpoA Figure , substane Figure . Overalle Figure , but we e Figure . Figure e Figure  and (b) s Figure . Apparen202Hg(II) was methylated than tracer 200Hg(II) by the same NOM isolate cultures /200Hg(II) was observed for the killed-cell control  associated with the NOM isolates was bioavailable for methylation in nearly every culturing condition, possibly after early methylation of the 200Hg tracer  of the ambient200Hg(II) pool associated with each NOM isolate  methylated in association with the F1 HpoA 4hr NOM isolate  for methylation may depend not only on the size and composition of NOM in the environment, but also on the concentration and bioavailability of “aged” Hg  already associated with that NOM. A natural environment likely harbors more than one “pool” of Hg bioavailable for methylation , or Hg(II) that is labile or tightly bound to different NOM functional groups), for which the bioavailability of each Hg pool depends partially on that of one or more others.Generally, we observed that more ambient  Figures , and an l Figure . This obr Figure  and/or re Figure . The exce Figure . One impD. propionicus cells for different time periods  methylation also revealed that the tracer 200Hg was methylated most efficiently in cultures pre-equilibrated for 5 days prior to inoculation. This observation hints at a possible time-dependency for the bioavailability of newly added Hg(II), possibly related to changes in the form of Hg associated with the NOM isolate and/or the conformation of the isolate over time. Although the cumulative proportion of 200Hg(II) converted to Me200Hg in association with the F1 HpoA 30dy condition appears relatively higher than with other NOM isolates or with F1 HpoA 4hr or 5dy . A related problem involves reconciliation of the observation that F1 HpoA 5dy cultures exhibited the greatest amount of ambient Hg methylation  was also achieved, or alternatively that the conformation of the F1 HpoA NOM (5 day) isolate was altered during equilibration and/or incubation in such a way as to facilitate ambient Hg(II) methylation. In any case, we cannot fully explain why naturally aged Hg(II) was associated with a muted response in MeHg production relative to methylation of the 200Hg isotope tracer, but we speculate that the aging process itself may have somehow impacted the bioavailability of Hg(II). Our hypothesis is perhaps consistent with recent findings that aging of nanoparticulate HgS leads to reduced methylation potential  was present in some NOM isolates used in this experiment, then the aggregation state of this phase  should also, in theory, exert influence over its bioavailability for methylation.The observation that different absolute concentrations of methylated ambient Hg(II) were formed when the same NOM isolate was incubated with tracer Hg(II) and live s Figure  is both y Figure , inspects Figure  and celln Figure  reveal tn Figure , althoug201Hg tracer, however, we did not observe any demethylation by D. propionicus 1pr3 (data not shown), although other strains of D. propionicus are known to demethylate MeHg  to Me202Hg was greater than that of any other NOM isolate, despite constituting only a moderate amount of absolute Me202Hg production  may have occurred over the course of the experiment to affect the bioavailability of the 200Hg tracer for methylation.The cumulative proportion of Hg(II) that was methylated was plotted for both tracer and ambient Hg(II) in order to assess the overall efficiency of conversion of Hg(II) to MeHg during our experiment Figure . The sha Figures , 3B, ind−1, is about two orders of magnitude lower than that used by Gerbig et al. , supporting the idea that MeHg production is variably but significantly correlated with the degree of aromatic functionality in each NOM isolate. The aromatic component of NOM has been shown to enhance the dissolution of cinnabar  was fit with a quadratic model  of tracer Hg (200Hg) was added to each bottle for each experimental condition, our analyses in some cases showed considerably less total dissolved Hg(II) was recovered, after filtration through 0.22 μm nylon syringe filters. The observed percentages of MeHg/filtered HgT) could have precipitated as colloidal or nanoparticulate HgS that remained suspended, possibly in association with cells and/or NOM isolates  and other metals . Certainly, several recent studies have shown that Hg(II) readily adsorbs to both live and dead cells, as well as EPS  , relative to older “ambient” Hg(II) in soils and lake waters  for uptake and methylation by a model sulfate-reducing bacterium.In summary, our work has shown that the degree of NOM hydrophobicity  strongly influenced the uptake and/or methylation of newly added (“tracer”) and aged/background (“ambient”) mercury. The observed variability in methylation efficiency probably reflects second-order and presumably kinetic effects on cellular Hg(II) uptake, possibly resulting from variability in the conformation state of the NOM macromolecules to which Hg was partitioned, or from the form of aged Hg-S complexes within the NOM. Other factors such as the relative sizes of new and ambient bioavailable Hg pools may also have influenced methylation efficiency. We note that our results can be considered usefully in the context of JM designed and conducted the experiment with supervision from ER and DK. GA contributed and advised on the NOM isolates. JM and JO conducted Hg and MeHg analyses with supervision from JD and DK. JM wrote the paper with contributions from CG, DK, GA, and ER.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the heart, RyRs play an indisputable role in the process of excitation–contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation–transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP In the heart, these intracellular Ca2+ release channels are essential for normal cell development and indispensable for life, as demonstrated by the fact that genetic ablation of RYR2, the gene encoding for the cardiac isoform of the RyR channel (RyR2), causes death at early embryonic stage in mice (2+) may induce finely graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity or pathological cellular remodeling. This review will concentrate on regulation of RyR2 by phosphorylation, a common post-translational process that confers dynamic functional modulation to a myriad of ion channels and transporters. In the majority of cases, phosphorylation of ion channels or transporters provides an additional layer of regulation without actually being the effector or trigger of activity, i.e., in the case of RyR channels, phosphorylation modulates the effect of Ca2+ on cardiac RyR (RyR2) or depolarization on skeletal RyR (RyR1) without having the inherent ability to open or close the channel per se. In a useful analogy, phosphorylation and Ca2+/depolarization would be similar to the “volume” and “on/off” switches, respectively, of the radio in which RyR songs are played. However, as we will see on several sections of this review, this mode of regulation is so controversial that a definitive role for RyR2 phosphorylation has not been reached yet, despite the paramount importance of this process in physiological and pathophysiological conditions. RyR2 may be phosphorylated in vitro and in vivo by several kinases, but here we will concentrate on CaMKIIδ (the most abundant CaMKII expressed in the heart and referred to here simply as “CaMKII”), and incorporate protein kinase A (PKA) whenever relevant, as these two kinases share some common transduction pathways, bear the most relevance in cardiac diseases and have been studied the most. Similarly, at least two splice variants of CaMKIIδ are expressed in cardiomyocytes, CaMKIIδC (cytosolic) and CaMKIIδB (nuclear), and we will identify their differential effect whenever appropriate.Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of the calcium ions  protrudes into the cytosol to bridge a ~15–20 nm gap between the SR and t-tubule membranes  and inorganic phosphate, metabolites such as pyruvate, fatty acids and polyamines, and ions such as Mg2+, H+, and Cl- , etc. and transduces these cytosolic signals to the channel pore to release appropriate amounts of Ca2+. Furthermore, Ca2+ release is critically influenced by luminal (intra-SR) factors such as Ca2+ content and protein interactions, thus conferring RyR2 channels an additional role as integrative switch-valves that restrict luminal Ca2+ overload, e.g., during sympathetic stimulation. Most of the signal-decoding structures are integral domains of the RyR2 protein, but as if this huge structural assembly were not sufficiently complex, RyR2 channels are also capable of protein–protein interactions that allow them to bind, in some cases steadily and in other cases in a time- and Ca2+-dependent manner, to smaller and independently regulated accessory proteins that add another layer of versatility (and complexity) to regulation of Ca2+ release in vivo. The best known RyR2-interacting proteins are calmodulin (CaM), which tonically inhibits Ca2+ release , forming an intricate multi-protein array . The RyRembranes . The cyt and Cl- . The cyt release  FKBP12.6 release , sorcin,t manner , and the ligands . More re ligands , underscThe multi-protein complex described above that includes kinases and phosphatases associated to the RyR2 strongly suggest that this channel is an avid target of downstream signaling effectors of the β-adrenergic system. PKA is a classical effector of the β-adrenergic pathway and, although the extent of activation of CaMKII by β-adrenergic stimulation and the precise signaling pathways involved are still incompletely understood, it is also accepted that CaMKII is involved in the inotropic and lusitropic effects of sympathetic stimulation . TherefoDespite the presence of multiple phosphorylation consensuses in each RyR2 subunit  and the 2+], only, not at the expected low (diastolic) [Ca2+], where it would cause significant Ca2+ leak  was first discovered by a2+ leak . The laca2+ leak , b, PKA the only CaMKII site in RyR2. As mentioned above, S2808 is also a target for CaMKII, and since there are at least ~4 CaMKII sites for each PKA site, this suggests that there are still other CaMKII sites yet to be discovered. Although S2814 is only six amino acid residues downstream of S2808 and forms part of the same RyR2 “phosphorylation hot spot” (2805RRISQTSQVSV2815 (S2808 and S2814 are underlined). Interestingly, another serine residue within that hot spot, S2811, is also part of a non-canonical CaMKII consensus and yet, it appears to be phosphorylated in vitro by PKA and CaMKII . Whether S2814 phosphorylation contributes to the inotropic effects induced by β-adrenergic stimulation has not been firmly established. However, mice with germline ablation of the S2814 phospho-epitope (RyR2-S2814A) appear more resilient than WT against a variety of cardiac insults. 2+ waves that were prevalent in the mdx mice (Duchenne muscular dystrophy model), were suppressed by crossbreeding with the RyR2-S2814A mice. In a protocol of ischemia-reperfusion injury, the RyR2-S2814A mice exhibited significantly fewer premature beats (that could be ascribed to delayed afterdepolarizations) than WT, a protection that was not seen in mice with ablation of two phospholamban phospho-sites  was discovered by ot spot”  the roled CaMKII  and in vagonists . Whetheragonists , making n CaMKII . Thus, a PLB-DM; . Respres PLB-DM;  which is2027R-L-L-S2030), oddly this site is preferentially phosphorylated by PKA, at least in vitro. This site, therefore, like the other sites discussed above, does not follow in silico predictions of kinase specificity and conforms instead to the cryptic rules of steric hindrance, topological association to specific kinases, substrate availability, etc. that operate in vivo and separate predicted from actual phosphorylation sites. Experiments in vitro revealed a major role for S2030 in the control of RyR2 activity, and in heart failure patients, it appeared as a reliable marker of RyR2 dysfunction  have just been generated  was found by function , presumafunction . The RyRfunction , in lineenerated  and shouin vitro and in vivo, but a fundamental question still pervades the field: what is the functional effect of RyR2 phosphorylation? This appears as a simple question, set for a straightforward answer, but examining the diverse and apparently opposite effects that have been published in the topic in the last ~2 decades, the only safe response seems to be that phosphorylation does something to RyR2 activity. All potential functional outcomes for RyR2 phosphorylation  have been reported, with tantalizing hints toward, but no clear factors responsible for, the radical differences. Also, the question needs to be framed in a specific integrative context for a defined response to hold some truth. For example, whereas there is compelling evidence that PKA can alter RyR2 activity at the single channel level  and into little effects on steady RyR2 activity. In fact, experiments in which SR Ca2+ load and ICa were kept constant showed that β-adrenergic stimulation of ventricular myocytes accelerated the rate of Ca2+ release  of RyR2s in intact cells and can mitigate effects of phosphorylation in a few beats , form part of a larger “phosphorylation hotspot” that encompasses S2811 and possibly T2810  response of RyR2 to kinases, which could explain the widely variable response detected by investigators to a specific phosphorylation maneuver. Of course, this alternative model based on the phosphorylation hotspot alone does not explain the distinct effect of CaMKII and PKA on RyR2 activity (see below), and to fill this void it is necessary to invoke the participation of S2030 and likely other as-yet-unrecognized phospho-sites. Thus, in an obligatorily more complex model  that we call “multi-phosphosite model”  and exogenous (purified from brain homogenates) CaMKII, and correlated their results with biochemical assays showing additive levels of phosphorylation by endogenous and exogenous CaMKII. This was also the report that unveiled S2808 (S2809 in dogs) as a major CaMKII site, although the proportion of the total RyR2 phosphorylation for which S2808 was solely responsible could not be determined. Thus, if the endogenous CaMKII of inhibition of single RyR2 channel activity by exogenous CaMKII, similar to 3H]ryanodine binding experiments. Nevertheless, multiple CaMKII sites as justification for apparent discrepancies may not be universally applied, as 2+] to keep the RyR2 channels open, and none of them frontally addressed the question of whether CaMKII activates RyR2 at low (diastolic) [Ca2+], a question of paramount importance given the current thinking that CaMKII increases SR Ca2+ leak (see below). This was technically difficult at the time because CaMKII itself was known to require Ca2+ as a cofactor for activation, and the Ca2+-free, auto-phosphorylated active form of the enzyme was not widely known. Hence, more studies are needed to clarify the Ca2+-dependence of CaMKII effect on isolated RyR2 channels. Lastly, in an oversimplified scheme that largely ignored the overwhelming evidence for the aforementioned multiple CaMKII sites, 2+] has not been firmly established yet and needs more refined studies.Although indirect evidence of RyR2 modulation by CaMKII was initially obtained by  find th and the activation of its receptor, the InsP3R. InsP3 originates from the hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C  that propagate as Ca2+ waves through the entire cell first and may ultimately stimulate neighboring cells to form inter-cellular Ca2+ waves. In cardiac myocytes, localization of InsP3Rs in the nuclear envelop underlies their role in excitation–transcription coupling, but recent evidence  suggest that although their abundance is low in the t-tubules and the sarcolemma, InsP3Rs can also rev up excitation–contraction coupling and eventually trigger cellular arrhythmias , nucleotides (ATP), accessory proteins , and also undergo major post-translational modifications such as phosphorylation (3Rs phosphorylation was obtained in the late 80s by Greengard’s and Sydner’s groups from rat cerebella (3Rs and regulate their activity in an isoform- and tissue-specific manner. This second part of this review will focus on the newly discovered CaMKII site (Ser150), on the physiological consequences of CaMKII phosphorylation of InsP3R, and on the mechanisms by which this event may participate in cardiac Ca2+ signaling. Further details on the general properties of InsP3R and their role in cardiac myocytes may be found in excellent reviews  encode for ~300 kDa subunits that assemble as homo- or hetero-tetramers to form a functional InsP3R channel. Similar to RyR2, the low resolution of InsP3Rs crystals shows a mushroom-like structure with a large cytosolic cap constituted by the N-terminal and central regions of the protein and a short stem, inserted into the membrane by the C-terminal domain (3-binding domain (or “core”) is located within the first amino acids of the channel, while up to eight Ca2+ binding sites are distributed throughout its entire sequence . Increasing InsP3 concentrations facilitate the opening of clusters of InsP3Rs, allowing CICR within a cluster. At high concentration, InsP3 promotes CICR between the clusters and intracellular Ca2+ waves  while in similar conditions RyR2 are closed . This activation takes place at intracellular [Ca2+] ranging from 30 to 100 nM. At higher [Ca2+]i, calcineurin activity prevails and dephosphorylation of the channel occurs. In agreement with these results, 3-sensitive Ca2+ flux in rat cerebellum microsomes. However, these outcomes were challenged by observations made in intact HeLa cells stimulated by histamine  dephosphorylates InsP3Rs to restore its activity. This model is particularly elegant since the authors also demonstrated that activated CaMKII facilitates the ER Ca2+ refilling by increasing SERCA activity (likely through PLN phosphorylation). As a consequence, the alternation between phosphorylated and dephosphorylated states of InsP3Rs seemed to be the basic mechanism for histamine-induced intracellular Ca2+ oscillations . Later, similar inhibitory effects of InsP3Rs phosphorylation by CaMKII were reported in channels reconstituted in lipid bilayers  abrogates the inhibitory effects of CaMKII phosphorylation. Conversely, the phospho-mimetic mutant InsP3R-2S150E reproduced the blunted channel activity of phosphorylated WT receptors. Noticeably, S150 belongs to a suppressor domain located prior to the InsP3-binding core. The ablation of that domain increases InsP3R affinity for InsP3 but additionally blocks Ca2+ release by the channel . CaMKIIδB phosphorylates the histone deacetylases (HDACs) to induce their nuclear export and relieve their inhibition on the transcription factor MEF-2  that take place in the nucleus autonomously from the cytosolic [Ca2+]. Interestingly, frequency-dependent Ca2+ oscillations have been shown to increase gene expression via the activation of the calcineurin/NF-AT pathway  from the excitation–contraction coupling. InsP3-dependent oscillations would be created through InsP3Rs activation–inhibition cycles mediated by calcineurin dephosphorylation and CaMKIIδB phosphorylation of S150, respectively .In both atrial and ventricular myocytes, InsPertrophy . Second,ertrophy . This alntration . Finally pathway . Altoget3R-2s is remarkably low compared to RyR2s (for an InsP3R-2:RyR2 ratio of 1:50 to 1:100 in ventricular myocytes). In addition, most of the immunofluorescence studies that have observed partial localization of InsP3R-2s in the sub-sarcolemmal space of atrial myocytes, failed to detect InsP3R-2s in the surface sarcolemma or t-tubules of ventricular myocytes . In agreement with that result, 3R-2 effects are mediated through the activation of the NCX and the increase of the action potential duration rather than a direct effect on RyR2s. Overall, these observations suggest an absence of a direct cross-talk between RyR2s and InsP3Rs and advocate the hypothesis that InsP3Rs mediate their effects on RyR2s by limited Ca2+ diffusion from the InsP3R release sites to the RyR2 sites.In cardiac myocytes, the expression of InsPmyocytes . Activat+ puffs” . Ca2+ puhythmias . Moreove myocyte . All tog3R-3 (Ser930 and Ser2189) for which the targeting kinase(s) remain(s) undetermined. Noticeably, S2189 of InsP3R-3 contains the CaMKII consensus sequence  of the studies indicate that CaMKII phosphorylation of RyR2s and InsP3Rs leads to antithetical outcomes. On one hand, CaMKII phosphorylation is presumed to increase RyR2 activity and promote SR Ca2+ leak that, when excessive, may trigger cardiac arrhythmias. On the other hand, CaMKII phosphorylation of nuclear InsP3Rs is presumed to inhibit InsP3Rs-mediated Ca2+ release and to prevent intra-nuclear Ca2+ oscillations . The magnitude of the effect of phosphorylation on these channels is also purportedly different since CaMKII modulates RyR2 channel activity, only, while it appears to play an on/off function on InsP3Rs. The bases of both of these differences are not immediately apparent and in fact, there is no universal agreement that such differences exist. As can be derived from the preceding paragraphs, a unified scheme on the effect of phosphorylation on both, RyR2 and InsP3Rs is yet to be forged. Nonetheless, independent of its precise mechanism of action on these channels, an emerging notion is that excessive CaMKII activity is detrimental for cardiac performance, and the potential salutary effect of blocking its chronic effects appears worth pursuing since its action on RyR2s and InsP3Rs might prevent both Ca2+-mediated arrhythmias and Ca2+-dependent activation of hypertrophic gene programs.CaMKII is a pleiotropic kinase that targets several ion channels and transporters in the heart, and RyR2 and InsPThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes, and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of reactive oxygen species signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.Transient changes in intracellular Ca As sessile organisms, plants encounter various types of environmental stresses, which are generally classified into biotic stresses such as insect and pathogen attacks, and abiotic stresses such as unfavorable temperature, lack of or excessive amounts of water, salinity, heavy metal toxicity, chemical toxicity, and nutrient deficiency. On the other hand, the process of industrialization inevitably brings many detrimental effects to the environment. Poorly controlled release of wastes from industrial processes and human life not only adds various toxic chemicals to our water and soil but also release harmful gasses into the atmosphere. Obviously, human activities are creating environmental challenges, making sustained crop production difficult. Classic agricultural technologies such as irrigation, applications of fertilizer, insecticides, fungicides, and chemical phytoprotectants have helped to improve crop yield, but the effects are limited, the costs are high and the impacts on the ecosystems and human health are undesirable and dangerous. Understanding how plants perceive and respond to various environmental stresses provides the necessary platform to create crop varieties which could fit better into the challenging environments, and has become one of the most important tasks for plant scientists around the world.2+ concentration outside the plasma membrane is usually at millimolar level. Since Ca2+ can form insoluble compounds with phosphate derivatives and complex with macromolecules, high levels of cytosolic Ca2+ are toxic to cells. Ca2+ concentration in the cytoplasm and nucleus is usually maintained at 50–100 nM under resting conditions  through the involvement of Ca2+ pumps and Ca2+/H+ exchangers distributed on the plasma membrane and inner membrane system /CaM-like (CML) proteins, calcium-dependent protein kinases (CDPKs) and calcineurin B like (CBL) proteins , which differ only in one to five amino acid residues GXXXR where I could be replaced with “FLV” and “X” represents any amino acid residue  from plants were identified by screening cDNA expression libraries with labeled CaM as probes , superoxide anion (O2-), and hydroxyl radical (⋅OH) are usually produced in various physiological processes and serve as a class of second messengers  such as hydrogen peroxide  and ascorbate peroxidase, could be regulated by Caular CaM . Later pt manner . The actwith CaM . A peroxCa2+/CaM . Evidencn cloned . The crihibitors .2+/CaM-mediated signaling is also well known to regulate ROS-related signal transduction at various stages. Maize CAP1 encoding a novel form of CaM-regulated Ca2+-ATPase was shown to be induced only during early anoxia, indicating its possible role in oxygen-deprived maize cells (Zea mays) and rice (Oryza sativa), respectively, play a critical role in ABA-induced antioxidant actions  and co-factor (AtBTs) are responsive to H2O2, suggesting that CaM-mediated signaling could directly regulate gene expression in plant responses to oxidative cues  background increased thermotolerance of the transgenic plants. Furthermore, the DNA-binding activity of HS transcription factors and the expression of tested HS genes at both mRNA and protein levels were shown to be down-regulated in atcam3 null mutant and up-regulated in its overexpressing lines upon HS treatment  level in plants was found to be elevated by high temperatures , impose major stress affecting plant growth and productivity. Almost all organisms including plants synthesize HS proteins (HSPs), a class of chaperons to assure normal function of various client proteins under adversely high temperature conditions. It was observed long ago that HS induced a quick and strong increase in cytosolic Ca tobacco . Expressolerance . Liu et ignaling . The Ca2bidopsis . Using mreatment . A role reatment . HS was eratures , and exoeratures . Howeverolerance .pTCB48 encoding a CBP was isolated by screening a cDNA expression library constructed from tobacco cell cultures subjected to HS, and its expression was strongly induced by HS treatment, suggesting a role in HS response  was also found to bind AtCaM2 in the presence of Ca2+, and the binding of Ca2+/CaM decreased the ATPase and foldase activities of this chaperon protein  was identified as a Ca2+-dependent CBP  seedlings expressing aequorin, and the expression of NpCaM-1 is induced by both cold shock and wind but mediated by distinct Ca2+ signaling pathways operating predominantly in the cytoplasm or in the nucleus (Arabidopsis plants overexpressing CaM3 showed decreased levels of COR (cold regulated) transcripts suggesting CaM may function as a negative regulator of cold-induced gene expression , were also found to be induced by cold treatment and thus, likely participate in transducing cold-induced Ca2+ signals  roots was found to be up-regulated by low temperature or salinity stress  regulates various cellular activities such as maintaining pH, ion homeostasis, and protein trafficking. 2+ and pH, and the binding of AtCaM15 to AtNHX1 alters the Na+/K+ selectivity of the exchanger by decreasing its Na+/H+ exchange speed. The interaction between AtCaM15 and AtNHX1 suggests the presence of Ca2+-pH-dependent signaling components in the vacuole, which are involved in mediating plant responses to salt stress. In addition to the above mentioned CaM/CMLs, CML37, CML38, and CML39 are also responsive to various stimuli, including salt, drought, and ABA  TaCCaMK was down-regulated by ABA, salt and PEG treatments, and overexpression of TaCCaMK reduces ABA sensitivity of Arabidopsis, indicating that TaCCaMK is a negative regulator of ABA-mediated signaling  was reported to be a Ca2+/CBP, and its enzymatic function is significantly stimulated by Ca2+/CaM binding , a CaM-binding member of the GTL transcription factor family, was found to be a negative regulator of drought tolerance  responses to Cd2+ toxicity during the early phases of seed germination  cyclic nucleotide gated ion channel (CNGC) called NtCBP4 was identified to be a CBP through protein–protein interaction-based library screening, and shown to be localized to plasma membrane. Transgenic tobacco plants with elevated expression of NtCBP4 displayed tolerance to Ni2+ and hypersensitivity to Pb2+, and consistently showed decreased Ni2+ and increased Pb2+ accumulation, suggesting that NtCBP4 is involved in heavy metal uptake across the plant plasma membrane  and non-essential  heavy metals in the soil can cause toxicity and inhibit plant growth. It was reported that Camination . Ca2+ admembrane . Howevery metals .2+ is a critical second messenger coupling diverse stimuli to various physiological responses in plants. CaM, as well as CML, is one of the most extensively studied Ca2+ sensors, which mediate interpretation of Ca2+ signals in all aspects of plant life, especially in responses to environmental stresses, through interaction with and regulation of various downstream target proteins. Figure 1 depicts an overview of generation and interpretation of Ca2+ signals which are regulated by CaM/CMLs during plant responses to abiotic stresses. One of the most actively regulated class of target proteins are calcium permeable channels, pumps, and antiporters which are actively involved in the generation of intracellular Ca2+ transients. This indicates that the preciseness and accuracy of Ca2+ signal itself is closely monitored by CaM-mediated regulation. Although more than 50 proteins from different plant species have been identified as CBPs with well-defined CaM-binding properties (Table 1), the CaM-mediated regulations of these target proteins are frequently presumptive including SAURs, PCBP, AtBTs, and WRKYIIds (in planta are supported with empirical evidences, such as GAD, CCaMK, AtCAT3, MLO, DWF1, AtSRs/CaMTAs, CRLK1, and CBP60g (2+ transients. In an effort to explain how the simple Ca2+ ion could act as a messenger to couple various environmental stimuli to appropriate physiological responses with astonishing accuracy, 2+ signature” which hypothesized that stimulus-triggered increases in intracellular Ca2+ concentration vary in terms of duration, frequency, amplitude, and spatial distribution, and these carry specific information when they are interpreted into different physiological responses. An obvious support for this hypothesis is that the different Ca2+ spikes triggered by Nod factor from rhizobia and Myc factor from arbuscular mycorrhizal fungi could be interpreted through the action of the same Ca2+, Ca2+/CaM dependent protein kinase, CCaMK, into different physiological responses to support the establishment of root nodulation symbiosis or arbuscular mycorrhization (2+ signals are interpreted into various physiological responses has been made in the last decade, what we know so far may be very limited in scope when one considers the complicated Ca2+ signaling network. Many issues such as specificity, preference and flexibility of interaction between various CaM/CMLs and target proteins in planta are barely understood. The dynamics of Ca2+/CaM mediated regulation, the mechanistic details by which a particular effector detects a difference in Ca2+ signature and initiates distinct signaling pathways, are basically unknown. Progress in addressing these issues will help in understanding the most amazing properties, the versatility, efficiency and accuracy of Ca2+-mediated signaling in plant responses to environmental stresses.CaWRKYIIds . Only a d CBP60g . Hence, hization . AlthougThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.