Backbone and side-chain resonance assignments of (Ca2+)4-calmodulin bound to beta calcineurin A CaMBD peptide.

Calcineurin (CaN) is a heterodimeric and highly conserved serine/threonine phosphatase (PP2B) that plays a critical role in coupling calcium signals to physiological processes including embryonic cardiac development, NF-AT-regulated gene expression in immune responses, and apoptosis. The catalytic subunit (CaNA) has three isoforms (α, ß, and γ,) in humans and seven isoforms in Paramecium. In all eukaryotes, the EF-hand protein calmodulin (CaM) regulates CaN activity in a calcium-dependent manner. The N- and C-domains of CaM (CaMN and CaMC) recognize a CaM-binding domain (CaMBD) within an intrinsically disordered region of CaNA that precedes the auto-inhibitory domain (AID) of CaNA. Here we present nearly complete 1H, 13C, and 15N resonance assignments of (Ca2+)4-CaM bound to a peptide containing the CaMBD sequence in the beta isoform of CaNA (ßCaNA-CaMBDp). Its secondary structure elements predicted from the assigned chemical shifts were in good agreement with those observed in the high-resolution structures of (Ca2+)4-CaM bound to CaMBDs of multiple enzymes. Based on the reported literature, the CaMBD of the α isoform of CaNA can bind to CaM in two opposing orientations which may influence the regulatory function of CaM. Because a high resolution structure of (Ca2+)4-CaM bound to ßCaNA-CaMBDp has not been reported, our studies serve as a starting point for determining the solution structure of this complex. This will demonstrate the preferred orientation of (Ca2+)4-CaM on the CaMBD as well as the orientations of CaMN and CaMC relative to each other and to the AID of ßCaNA.

Toward a Sociology of the Reconciliation of Conflicting Desires.

Desire-based research provides people and communities the opportunity to share their dreams and hopes for a better future. However, conflicting desires are difficult to reconcile. We suggest that sociological research to understand conflicting desires is required to support reconciliation work by Indigenous and non-Indigenous people in Canada. Our contribution begins by identifying much of current and past sociological research about Indigenous people and communities as damaged-centered, that is, identifying problems and obstacles in the hope that the knowledge will lead to change. This model of social change is flawed. We believe that most Canadians desire justice for Indigenous peoples while at the same time desiring land and access to resources, desires that deny that justice. How we as a society reconcile these desires will determine the extent to which true justice for Indigenous peoples will be achieved. We propose a sociology of the reconciliation of conflicting desires and suggest some practical ways that this type of research could move forward.

Density, Salinity, and Entry Depths of Municipal Wastewater in an Urban Lake.

The depths of entry of municipal wastewater into receiving lakes importantly affects associated impacts on water quality. The plunging behavior of two negatively buoyant inflows that carry municipal waste, an urban tributary and an effluent discharge, in Onondaga Lake, NY, is characterized and quantified based on an integrated program of monitoring, density calculations, and modeling. In-lake signatures of plunging from the two inflows are differentiated according to constituents in which each is enriched. Under common contemporary conditions, the summer averages of the fraction of the urban stream and effluent discharge inflows plunging to stratified depths is predicted, with a calibrated hydrodynamic model, to be approximately 0.7 and 0.35, respectively. Recent short-term increases in salinity levels from construction site dewatering caused greater plunging of the effluent discharge and interfered with normal complete fall turnover in the lake.

Changes in the long-term supply of mercury species to the upper mixed waters of a recovering lake.

We quantified internal processes that supply methylmercury from hypolimnetic reducing zones to the upper waters of a Hg-contaminated lake, Onondaga Lake, NY, USA. Diffusive transport continuously supplied methylmercury to the epilimnion under summer stratification, while fall mixing resulted in a pulsed release of methylmercury to the upper mixed waters. These processes were the main internal sources of methylmercury to the epilimnion, and together almost equaled the total external supply. The wind-driven entrainment represented an additional stochastic internal supply of methylmercury of approximately 9% in 2006. Considering more than 15 years of data, we estimate 1.8 wind-driven events occur per year. The mass of methylmercury inputs to the epilimnion exceeded the measured increase, suggesting that loss processes are important in regulating methylmercury accumulation. The relative contribution of internal sources of methylmercury to the epilimnion has decreased in recent years, shifting the importance to the external inputs.

Retrospective analyses of inputs of municipal wastewater effluent and coupled impacts on an urban lake.

A retrospective review and analysis are presented of the evolution of treatment, point of discharge considerations, and constituent loading from the Metropolitan Syracuse Wastewater Treatment Plant (Metro), and the coupled water quality effects on the receiving urban lake (Onondaga Lake, New York) from the early 1970s to 2010. The analysis is based on long-term monitoring of the discharge, Onondaga Lake, and a nearby river system considered as a potential alternate to receive the effluent. The Metro discharge is extraordinarily large relative to the lake's hydrologic budget, representing approximately 25% of the total inflow, greater than for any other lake in the United States. The large loads of nitrogen and phosphorus received from the facility resulted in severe water quality effects in the lake during the early portion of record, including (1) violations of standards to protect against toxic effects of ammonia and nitrite, (2) violations of the water clarity standard for swimming safety, (3) exceedances of a limit for the summer average concentration of total phosphorus in the upper waters, and (4) lakewide violations of the oxygen standard during fall turnover. The effects of Metro were compounded by effects of discharges from soda ash/chlor-alkali and pharmaceutical manufacturing facilities. The sedimentary record of the lake indicates that even greater levels of cultural eutrophication prevailed before the monitoring commenced. Dramatic improvements in the water quality of the lake were achieved in recent years by implementing advanced treatment technologies. Exceedances of receiving water limits in the lake were eliminated, with the exception of the total phosphorus limit. A zebra mussel invasion compromised the oxygen resources and assimilative capacity of the nearby river for more than 15 years. This eliminated an option, previously supported by managers, of full diversion of the Metro effluent to the river.

Nurses' perceptions and attitudes towards new ADU technology and use.

BACKGROUND: The introduction of a new technology in hospitals - Automated Dispensing Units (ADUs) - aims to contribute to more secure, safe, efficient and cost effective health services. Several studies highlight the beneficial effects of similar technologies as well as their cost-savings potential but there is little literature exploring nurses' perceptions and attitudes towards technology acceptance and the impact on technology use in a healthcare unit. OBJECTIVE: This research aimed to explore nurses' perceptions and attitudes towards current technology use on their units and towards the introduction of ADU technology and use with nursing staff in two different hospitals in South-East New-Brunswick, Canada. METHODS: Semi-structured interviews were realized with the collaboration of nursing staff from two hospitals which were in urban and rural settings, prior to the introduction of ADUs in hospital wards. RESULTS: Findings in this study highlight the fact that missing medications (i.e., doses not available in cart) are inherently related to the completion of nursing staff's medication distribution routine. Missing doses cause delays in medication delivery which may increase the occurrence of medication errors. Participants described current technology use as an intricate part of their routine. The latter is mainly utilized for patient monitoring and information retrieval. Overall, interview data indicated that ADU technology introduction is positively perceived by nursing staff particularly if the technology reduces missing doses events. CONCLUSIONS: Findings in this study underscore important concerns expressed by nursing staff regarding ADU technology integration into the current medication process and its impact on time management. Pre-implementation training and technical support were identified as important factors in facilitating technology acceptance and proper technology use.

Factors diminishing the effectiveness of phosphorus loading from municipal effluent: critical information for TMDL analyses.

Factors that diminish the effectiveness of phosphorus inputs from a municipal wastewater treatment facility (Metro) in contributing to phosphorus levels and its availability to support algae growth in a culturally eutrophic urban lake (Onondaga Lake, NY) were characterized and quantified. These factors included the bioavailability and settling characteristics of particulate phosphorus from this effluent, the dominant form (70%) of phosphorus in this input, and the plunging of the discharge to stratified layers in the lake. Supporting studies included: (1) chemical and morphometric characterization of the phosphorus-enriched particles of this effluent, compared to particle populations of the tributaries and lake, with an individual particle analysis technique; (2) conduct of algal bioavailability assays of the particulate phosphorus of the effluent; (3) conduct of multiple size class settling velocity measurements on effluent particles; and (4) determinations of the propensity of the discharge to plunge, and documentation of plunging through three-dimensional monitoring of a tracer adjoining the outfall. All of these diminishing effects were found to be operative for the Metro effluent in Onondaga Lake and will be integrated into a forthcoming phosphorus "total maximum daily load" analysis for the lake, through appropriate representation in a supporting mechanistic water quality model. The particulate phosphorus in the effluent was associated entirely with Fe-rich particles formed in the phosphorus treatment process. These particles did not contribute to concentrations in pelagic portions of the lake, due to local deposition associated with their large size. Moreover, this particulate phosphorus was found to be nearly entirely unavailable to support algae growth. While substantial differences are to be expected for various inputs, the effective loading concept and the approaches adopted here to assess the diminishing factors are broadly applicable.

Mental health professionals' perspectives of telemental health with remote and rural First Nations communities.

We conducted an online survey and interviews amongst mental health workers in Canada who reported experience in working with rural and remote First Nations (although not necessarily telemental health). Sixty-three respondents (of the 164) to the online survey reported experience in working with clients in remote and rural First Nations. Only 16 of the online survey respondents with remote and rural First Nations experience reported having received training in videoconferencing use. When asked how frequently they used videoconferencing with clients, 51% reported never using it, 19% used it once every few months and 10% reported using it a few times a month. Approximately 50% of participants reported finding it useful. Approximately 38% found the technology easy or very easy to use, and 15% found it very difficult. Individual in-depth interviews were also conducted with professionals who had First Nations telemental health experience specifically (n = 5). A quantitative data analysis was used to explore their perceptions of usefulness and ease of use of telemental health, as well as the relationships among these constructs. Advantages, disadvantages and challenges in using the technology were identified from the qualitative data. Promising ways forward include incorporating traditional practices and the Seven Teachings into telemental health services.

Conversations on telemental health: listening to remote and rural First Nations communities.

INTRODUCTION: Telemental health involves technologies such as videoconferencing to deliver mental health services and education, and to connect individuals and communities for healing and health. In remote and rural First Nations communities there are often challenges to obtaining mental healthcare in the community and to working with external mental health workers. Telemental health is a service approach and tool that can address some of these challenges and potentially support First Nations communities in their goal of improving mental health and wellbeing. Community members' perspectives on the usefulness and appropriateness of telemental health can greatly influence the level of engagement with the service. It appears that no research or literature exists on First Nations community members' perspectives on telemental health, or even on community perspectives on the broader area of technologies for mental health services. Therefore, this article explores the perspectives on telemental health of community members living in two rural and remote First Nations communities in Ontario, Canada. METHODS; This study was part of the VideoCom project, a collaborative research project exploring how remote and rural First Nations communities are using ICTs. This current exploration was conducted with the support of Keewaytinook Okimakanak (KO), our partner in Northwestern Ontario. With the full collaboration of the communities' leadership, a team involving KO staff and VideoCom researchers visited the two communities in the spring of 2010. Using a participatory research design, we interviewed 59 community members, asking about their experiences with and thoughts on using technologies and their attitudes toward telemental health, specifically. A thematic analysis of this qualitative data and a descriptive quantitative analysis of the information revealed the diversity of attitudes among community members. Finally, based on a discussion with the community telehealth staff, a 'ways forward' section was proposed as a way to begin addressing certain issues that were raised by community members. RESULTS: This article explores the continuum of community members' perspectives that range from interest and enthusiasm to hesitancy and concern. One participant reported personal experience with using telemental health and found the approach helpful in increasing her comfort in the therapeutic situation. In addition, concerns relating to appropriateness and safety were voiced. A variety of advantages (eg facilitation of disclosure, increased access to services, usefulness) and disadvantages or concerns (eg interference with capacity building, concerns about privacy) are reported and discussed. Following a coding procedure, a descriptive quantitative analysis demonstrated that 47% of the participants were categorized as having a positive response toward telemental health, 32% as having a negative response, and 21% as being neutral or undecided. CONCLUSIONS: Valuing Indigenous knowledge can help us understand community members' experiences of and concerns with telemental health and inform more successful and appropriate initiatives. With the invaluable support of the KO Telemedicine co-authors, we offer ways forward to address concerns identified by the community members. Most importantly, any ways forward for community telemental health initiatives need to be community driven and community led.

Recognition of ß-calcineurin by the domains of calmodulin: thermodynamic and structural evidence for distinct roles.

Calcineurin (CaN, PP2B, PPP3), a heterodimeric Ca(2+)-calmodulin-dependent Ser/Thr phosphatase, regulates swimming in Paramecia, stress responses in yeast, and T-cell activation and cardiac hypertrophy in humans. Calcium binding to CaN(B) (the regulatory subunit) triggers conformational change in CaN(A) (the catalytic subunit). Two isoforms of CaN(A) (α, ß) are both abundant in brain and heart and activated by calcium-saturated calmodulin (CaM). The individual contribution of each domain of CaM to regulation of calcineurin is not known. Hydrodynamic analyses of (Ca(2+))4-CaM(1-148) bound to ßCaNp, a peptide representing its CaM-binding domain, indicated a 1:1 stoichiometry. ßCaNp binding to CaM increased the affinity of calcium for the N- and C-domains equally, thus preserving intrinsic domain differences, and the preference of calcium for sites III and IV. The equilibrium constants for individual calcium-saturated CaM domains dissociating from ßCaNp were ∼1 µM. A limiting K(d) ≤ 1 nM was measured directly for full-length CaM, while thermodynamic linkage analysis indicated that it was approximately 1 pM. ßCaNp binding to ¹5N-(Ca(2+))4-CaM(1-148) monitored by ¹5N/¹HN HSQC NMR showed that association perturbed the N-domain of CaM more than its C-domain. NMR resonance assignments of CaM and ßCaNp, and interpretation of intermolecular NOEs observed in the ¹³C-edited and ¹²C-¹4N-filtered 3D NOESY spectrum indicated anti-parallel binding. The sole aromatic residue (Phe) located near the ßCaNp C-terminus was in close contact with several residues of the N-domain of CaM outside the hydrophobic cleft. These structural and thermodynamic properties would permit the domains of CaM to have distinct physiological roles in regulating activation of ßCaN.

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin.

Trifluoperazine (TFP; Stelazine) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca(2+))(4)-CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the "open" conformation seen in CaM-kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca(2+))(4)-CaM and explore differential effects on the N- and C-domains of CaM, stoichiometric TFP titrations of CaM were monitored by (15)N-HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca(2+))(4)-CaM. In both cases, the preferred site was in the C-domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP-binding sites in apo CaM appeared distinct from those in (Ca(2+))(4)-CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ-motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N-domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the "closed," "semi-open," and "open" domains of CaM. In physiological processes, apo CaM, as well as (Ca(2+))(4)-CaM, needs to be considered a potential target of drug action.

Thermodynamics and conformational change governing domain-domain interactions of calmodulin.

Calmodulin (CaM) is a small (148 amino acid), ubiquitously expressed eukaryotic protein essential for Ca(2+) regulation and signaling. This highly acidic polypeptide (pI<4) has two homologous domains (N and C), each consisting of two EF-hand Ca(2+)-binding sites. Despite significant homology, the domains have intrinsic differences in their Ca(2+)-binding properties and separable roles in regulating physiological targets such as kinases and ion channels. In mammalian full-length CaM, sites III and IV in the C-domain bind Ca(2+) cooperatively with ~10-fold higher affinity than sites I and II in the N-domain. However, the difference is only twofold when CaM is severed at residue 75, indicating that anticooperative interactions occur in full-length CaM. The Ca(2+)-binding properties of sites I and II are regulated by several factors including the interplay of interdomain linker residues far from the binding sites. Our prior thermodynamic studies showed that these residues inhibit thermal denaturation and decrease calcium affinity. Based on high-resolution structures and NMR spectra, there appear to be interactions between charged residues in the sequence 75-80 and those near the amino terminus of CaM. To explore electrostatic contributions to interdomain interactions in CaM, KCl was used to perturb the Ca(2+)-binding affinity, thermal stability, and hydrodynamic size of a nested set of recombinant mammalian CaM (rCaM) fragments terminating at residues 75, 80, 85, or 90. Potassium chloride is known to decrease Ca(2+)-binding affinity of full-length CaM. It may act directly by competition with acidic side chains that chelate Ca(2+) in the binding sites, and indirectly elsewhere in the molecule by changing tertiary constraints and conformation. In all proteins studied, KCl decreased Ca(2+)-affinity, decreased Stokes radius, and increased thermal stability, but not monotonically. Crystallographic structures of Ca(2+)-saturated rCaM(1-75) (3B32.pdb) and rCaM(1-90) (3IFK.pdb) were determined, offering cautionary notes about the effect of packing interactions on flexible linkers. This chapter describes an array of methods for characterizing system-specific thermodynamic properties that in concert govern structure and function.

Effects of processing on immunoreactivity of cashew nut (Anacardium occidentale L.) seed flour proteins.

Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.

A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation.

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

Production and characterization of protective human antibodies against Shiga toxin 1.

Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(kappa)() [IgM(kappa)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(kappa) elements specific for subunit B) were isolated. Two IgM(kappa) Hu-MAbs (2D9 and 15G9) and three IgG1(kappa) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.

Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli O157:H7 infection.

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies. Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.