Gastric GDF15 levels are regulated by age, sex, and nutritional status in rodents and humans.

AIM: Growth differentiation factor 15 (GDF15) is a stress response cytokine that has been proposed as a relevant metabolic hormone. Descriptive studies have shown that plasma GDF15 levels are regulated by short term changes in nutritional status, such as fasting, or in obesity. However, few data exist regarding how GDF15 levels are regulated in peripheral tissues. The aim of the present work was to study the variations on gastric levels of GDF15 and its precursor under different physiological conditions, such as short-term changes in nutritional status or overfeeding achieved by HFD. Moreover, we also address the sex- and age-dependent alterations in GDF15 physiology. METHODS: The levels of gastric and plasma GDF15 and its precursor were measured in lean and obese mice, rats and humans by western blot, RT-PCR, ELISA, immunohistochemistry and by an in vitro organ culture system. RESULTS: Our results show a robust regulation of gastric GDF15 production by fasting in rodents. In obesity an increase in GDF15 secretion from the stomach is reflected with an increase in circulating levels of GDF15 in rats and humans. Moreover, gastric GDF15 levels increase with age in both rats and humans. Finally, gastric GDF15 levels display sexual dimorphism, which could explain the difference in circulating GFD15 levels between males and females, observed in both humans and rodents. CONCLUSIONS: Our results provide clear evidence that gastric GDF15 is a critical contributor of circulating GDF15 levels and can explain some of the metabolic effects induced by GDF15.

Orthostatic blood pressure recovery associates with physical performance, frailty and number of falls in geriatric outpatients.

OBJECTIVE: Blood pressure (BP) recovery after orthostatic hypotension might be important to prevent cerebral hypoperfusion episodes in older adults, and be related to better clinical outcome. The objective was to study the relationship between BP recovery and clinical outcome, that is physical and cognitive performance, frailty and falls, in geriatric outpatients. METHODS: One hundred and sixty-eight geriatric outpatients underwent continuous (beat-to-beat) BP measurements during standing up, and a comprehensive geriatric assessment, including assessment of physical performance (chair stand test), cognitive performance (Mini Mental State Examination), frailty (Fried criteria) and falls in the previous year. BP recovery was evaluated at 15-30, 30-60, 60-120 and 120-180 s after standing up and defined as mean SBP and DBP in the respective time intervals minus baseline BP. Associations with clinical outcome were assessed using linear (physical and cognitive performance and frailty) and logistic (falls) regression, adjusting for age, sex, baseline BP and initial BP drop. RESULTS: SBP recovery was associated with frailty (30-60 s interval; ß = 0.013, P = 0.02) and falls (30-60 s interval; odds ratio = 1.024, P = 0.02). DBP recovery was associated with physical performance (30-60 s interval; ß = 0.215, P = 0.01), frailty (30-60 s interval; ß = 0.028, P = 0.02) and falls (30-60 s interval; odds ratio = 1.039, P = 0.04). Neither SBP nor DBP recovery was associated with cognitive performance. CONCLUSION: DBP recovery was particularly associated with clinical outcome in geriatric outpatients, suggesting BP recovery to be of clinical interest.

Nano gel filtration reveals how fish hemoglobins release oxygen: The Root Effect.

The Root Effect is to many species of fish what the Bohr Effect is to humans regarding the release of O2 from their hemoglobins at low pH. However, Root Effect hemoglobins accomplish this more extensively than human adult hemoglobin in order to satisfy the diverse oxygen requirements in fish. To understand this difference between fish and human hemoglobins, we studied their subunit interface strengths using very low (nanomolar) concentrations, referred to as nano gel filtration. Root Effect hemoglobins in their CO form dissociate in a tetramer-monomer equilibrium. In contrast, tetramers and dimers but no monomers are found for adult human hemoglobin consistent with its well known tetramer-dimer equilibrium. By analogy to the human variant Hb Kansas and a similar recombinant Hb, both of which readily release oxygen due to an unstable oxygenated structure, the mechanism proposed is that oxygenated Root Effect tetramers release their oxygen to form energetically stable deoxygenated tetramers rather than dissociate to energetically unfavorable oxygenated dimers with labile interfaces. In contrast, the strong binding of CO permits observation of dissociation to monomers, thus revealing an intrinsic property of Root Effect fish hemoglobins enabling it to function as an oxygen pump.

Embryonic and Fetal Human Hemoglobins: Structures, Oxygen Binding, and Physiological Roles.

During the past two decades, significant advances have been made in our understanding of the human fetal and embryonic hemoglobins made possible by the availability of pure, highly characterized materials and novel methods, e.g., nano gel filtration, to study their properties and to correct some misconceptions. For example, whereas the structures of the human adult, fetal, and embryonic hemoglobins are very similar, it has generally been assumed that functional differences between them are due to primary sequence effects. However, more recent studies indicate that the strengths of the interactions between their subunits are very different leading to changes in their oxygen binding properties compared to adult hemoglobin. Fetal hemoglobin in the oxy conformation is a much stronger tetramer than adult hemoglobin and dissociates to dimers 70-times less than adult hemoglobin. This property may form the basis for its protective effect against malaria. A major source of the increased strength of fetal hemoglobin resides within the A-helix of its gamma subunit as demonstrated in studies with the hybrid hemoglobin Felix and related hybrids. Re-activating fetal hemoglobin synthesis in vivo is currently a major focus of clinical efforts designed to treat sickle cell anemia since it inhibits the aggregation of sickle hemoglobin. The mechanisms for both the increased oxygen affinity of fetal hemoglobin and its decreased response to DPG have been clarified. Acetylated fetal hemoglobin, which makes up 10-20% of total fetal hemoglobin, has a significantly weakened tetramer structure suggesting a similar role for other kinds of protein acetylation. Embryonic hemoglobins have the weakest tetramer and dimer structures. In general, the progressively increasing strength of the subunit interfaces of the hemoglobin family during development from the embryonic to the fetal and ultimately to the adult types correlates with their temporal appearance and disappearance in vivo, i.e., ontogeny.

Apoptotic cell-derived extracellular vesicles: structure-function relationships.

Apoptosis is an essential process for normal physiology and plays a key role in the resolution of inflammation. Clearance of apoptotic cells (ACs) involves complex signalling between phagocytic cells, ACs, and the extracellular vesicles (EVs) they produce. Here, we discuss apoptotic cell-derived extracellular vesicles (ACdEVs) and how their structure relates to their function in AC clearance and the control of inflammation, focussing on the ACdEV proteome. We review the current knowledge, ongoing work and future directions for research in this field.

Phosphorylation of Serine Induces Lysine p Ka Increases in Histone N-Termini and Signaling for Acetylation. Transcription Implications.

The mild acetylating agent, methyl acetyl phosphate, is used to estimate the p Ka values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys ε amines have p Ka values in the range of 7.8-8.3, which are much lower than the currently assumed values. When Ser-1 is phosphorylated, the p Ka values of these Lys amines are elevated to the range of 8.8-10.3, thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed p Ka values of Lys ε amines by Ser phosphorylation represents the basis for signaling in histone N-terminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating p Ka values based on a linkage between the Henderson-Hasselbalch and Michaelis-Menten equations indicates that the p Ka values of the Lys ε amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on the location and proximity to the phosphorylation site.

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities.

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate-Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding.

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect.

Effectiveness of a structured curriculum focused on recognition and response to acute patient deterioration in an undergraduate BSN program.

The study purpose was to evaluate the effectiveness of a structured education curriculum with simulation training in educating undergraduate Baccalaureate of Science in Nursing (BSN) students to recognize and respond to patients experiencing acute deterioration as first responders. Researchers have demonstrated a lack of adequate clinical reasoning skills in new graduate nurses is a factor in critical patient incidents. A mixed methods design using a quasi-experimental, repeated measures and a descriptive, qualitative approach was used. A convenience sample of 48 BSN students was recruited. Statistically significant increases were shown in knowledge, self-confidence, and perceptions of teamwork. Six categories emerged from the qualitative data analysis: sources of knowledge, knowledge as a person, knowledge as a group, reasoning under pressure, feelings, real person versus simulation, and values. Nursing educators need to use innovative teaching strategies to ameliorate or even eliminate the theory-practice gap in nursing.

Intrinsic regulation of hemoglobin expression by variable subunit interface strengths.

The expression of the six types of human Hb subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the 'extrinsic' component of regulation). Here, we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the 'intrinsic' component of regulation). The adult Hb dimers have the strongest subunit interfaces and the embryonic Hbs the weakest, with fetal Hbs being of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these Hb types form functional O(2) -binding tetramers consistent with gene switching.

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces.

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low-Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.

Energetic differences at the subunit interfaces of normal human hemoglobins correlate with their developmental profile.

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described, i.e. self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although many mutant human hemoglobins are known to have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strengths, i.e., the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3 orders of magnitude and display an undulating profile similar to the transitions ("switches") of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile. Embryonic hemoglobins are the weakest; fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O(2) affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its level of expression diminishes and adult hemoglobin A formation begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection among genetics, thermodynamics, and development.

From ethics to palliative care: a community hospital experience.

One of the most vulnerable and voiceless groups of patients within American hospitals and institutions today are those who are dying. Health care institutions struggle with the challenge of providing excellent palliative and end-of-life care to patients while providing curative therapies at the same time. This article describes the efforts and accomplishments of the ethics committee of a community hospital system to provide for the palliative and end-of-life needs of its patients.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell.

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

Collaborative development of an insulin nomogram for intensive insulin therapy.

A significant reduction in morbidity and mortality can be achieved by using intensive insulin therapy to achieve normoglycemia in the critically ill patient. Nurses hourly titrate insulin infusion rates using protocols, most often in chart form, to guide practice. Despite the reported development of several intensive insulin protocols, information about the accuracy, practicality, ease of calculation, and preferences of critical care nurses have not been studied. The purpose of this study was to test a newly developed nomogram to determine if it was clinically practical, easily understood, and more quickly and accurately used than existing protocols in chart form. Three cohorts of nurses (N = 153), with an average of 9 years experience in critical care, performed calculations of insulin doses on the basis of hypothetical patient data, using chart and nomogram protocols. Time, accuracy, preference, and feedback were examined over all 3 phases. No statistical significance was found between the chart and nomogram methods regarding time to complete the calculations nor was there any significant effect of experience on the time it took to complete the calculations or the accuracy of the resultant answers. Overall, the accuracy of the calculations improved with each revision of the nomogram. The nurses did not have a preference of one tool over another but gave extensive feedback that helped refine the nomogram. The resultant nomogram was shown to be easy to use, helpful in improving the accuracy of insulin dosage calculation, and judged to be practical by the nurses who would use the tool at the bedside.

N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity.

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

The benefits of serving on a hospital ethics committee: a faculty perspective.

Community hospitals ethics committees rarely have formal support from persons within the academic community. The author discusses a liaison between a suburban hospital system and a nearby university school of nursing that garners positive educational, service, and programmatic benefits, related to ethics, for both. The lessons learned and insights gained by a faculty member serving on the ethics committee are examined.

A combined cognitive and behavioral intervention for cocaine-using methadone clients.

Treating cocaine use by opiate-dependent clients in methadone programs is a well-documented challenge. Both behavioral (contingency management) and cognitive (relapse prevention) interventions have shown promise in helping engage these clients in treatment. In this study, the effectiveness of combining contingency management with a cocaine-specific relapse prevention counseling module was examined. Sixty-one cocaine-using methadone clients were randomly assigned to one of four treatment conditions to participate in the eight-week intervention and eight-week follow-up period. Using analysis of variance (ANOVA), differences in cocaine use and treatment retention were examined. Contingency management was significantly related to reductions in cocaine use and the counseling module was positively related to six-month retention rates. Both interventions were associated with positive treatment response but the effects were reflected in different behavioral outcomes.

This new house: building knowledge through online learning.

The purpose of this study was to understand from baccalaureate-degree nursing students the experience of virtual learning in nursing and to understand how online education enhanced and detracted from learning. Three in-depth, focus-group interviews were conducted with a total of 27 baccalaureate nursing students. Hermeneutic phenomenology was employed to analyze the narrative data. Seven themes were identified from the data. The first theme was "making the framework": (1) providing flexibility; (2) enabling students through observing, guiding, and modeling; (3) faculty as facilitators; and (4) questioning as method. The second theme was "building incredible dialogue": (1) having great discussions, (2) sharing ideas/knowledge, and (3) not being inhibited. The third theme was "critical thinking--the 3-D effect": (1) sitting with the words, (2) using references/research, (3) seeing a variety of perspectives, (4) critiquing self and others, and (5) using selective language. The fourth theme was "personal and professional growth": (1) being connected, (2) feeling valued and respected, and (3) having intimate relationships. The fifth theme, representing the outcomes for online courses, was "more comprehensive learning": (1) deeper understanding and (2) better retention of knowledge. The sixth theme was "being overwhelmed", and the seventh theme was "being frustrated". As a result of this study, an understanding of the phenomenon of online learning was developed. The findings provide direction for future development of the pedagogy of distance learning.