Absence of the enteric nervous system in the newborn: presentation of three patients and review of the literature

Three patients suffering from an absence of the enteric nervous system are reported. Two sisters presented with severe vomiting shortly after birth and dilatation of the intestine proximal to a stenosis. There was an absence of the enteric nervous system throughout the entire length of the intestine distal to the duodenum. A boy presenting an ileus was found to suffer from an aganglionosis of the entire colon. There was also an absence of neuronal bodies and nerve fibers in the small intestine. The final diagnosis was made by histochemical and immunocytochemical stains for acetylcholinesterase, lactate hydrogenase, neuron-specific enolase, protein S-100, and substance P. In the literature, 13 other patients have been reported. On the basis of differences of symptoms, incidence, sex ratio, genetics, and, presumably, pathogenesis between absence of the enteric nervous system and aganglionosis, it is assumed that the two diseases are separate entities.     

Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock

Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.Circadian clocks are biological timing systems that allow organisms to anticipate and prepare for daily changes in the environment. A hallmark of a circadian oscillator is its ability to drive self-sustained rhythms in gene expression and behavior with a period close to 24 h, even in the absence of environmental cues (1). A general challenge for the biochemical machinery that generates rhythms is to precisely define the duration of the day in the face of perturbations, including fluctuations in the cellular abundance of the molecular components. The importance of maintaining precise circadian timing is underscored by experiments showing that mismatch between the clock period and the rhythms in the external environment results in health problems and fitness defects (2, 3).Although circadian clocks are found across all kingdoms of life, the Kai oscillator from cyanobacteria presents a uniquely powerful model system to study the design principles inherent in the molecular interactions that generate rhythms. A mixture of the purified proteins KaiA, KaiB, and KaiC results in stable oscillations in the phosphorylation state of KaiC in vitro that persist for many days and share many of the properties of circadian clocks in vivo (4–6). In particular, the oscillator can successfully generate near–24-h rhythms over a range of concentrations of the clock proteins both in vivo and in vitro (7–9), so fine-tuning of gene expression is not needed to support a functional clock. Much has been learned about the behavior of the isolated Kai proteins, including the determination of high-resolution crystal structures of all three components (10–12). A critical challenge that remains is to understand how the properties of the Kai proteins are integrated together in the full system to generate precisely timed rhythms.KaiC appears to be the central hub of timing information in the oscillator. Each KaiC molecule consists of two AAA+ family ATPase domains that consume the free energy of ATP hydrolysis to drive oscillations. Like many other members of this family, KaiC forms hexamers, and the enzymatic active sites are formed at the subunit interfaces where nucleotides are bound. The C-terminal, or CII, domain of KaiC has additional phosphotransferase activities that are unusual for the AAA+ family: it can phosphorylate and dephosphorylate two residues near the subunit interface, Ser431 and Thr432 (13). KaiC autokinase and autophosphatase activities occur at the same active site (14, 15). In isolation, KaiC has high phosphatase activity, but the enzyme is pushed toward kinase activity by the activator protein KaiA, which interacts directly with the KaiC C-terminal tail (16, 17). Roughly speaking, kinase activity predominates during the day, and phosphatase activity predominates during the night (18). Thus, understanding the feedback mechanisms that generate a precise time delay between these modes is crucial to understanding timing in the oscillator (19).Inactivation of KaiA and a transition from kinase to phosphatase mode occur when KaiB•KaiC complexes form, closing a negative feedback loop by sequestering KaiA in a ternary complex and leaving it unable to act on other KaiC molecules (20, 21). By temporarily removing KaiA molecules from their activating role, this molecular titration mechanism may act to synchronize the activity of all KaiC hexamers in the reaction (20, 22, 23). Phosphorylation and dephosphorylation proceed in a strongly ordered fashion so that in response to a change in KaiA activity, Thr432 is (de)phosphorylated first, followed later by Ser431 (18, 20, 21). It is known that phosphorylated Ser431 is important for allowing the formation of KaiB•KaiC complexes. However, recent work has made it clear that the binding of KaiB involves both KaiC domains—in particular, the slow ATPase activity of the N-terminal CI domain, which is not phosphorylated, is required for KaiB interaction (24, 25).Because of the importance of precisely timing negative feedback via KaiB•KaiC complex formation for generating appropriate rhythms (22), we wanted to understand the role of phosphorylation of the KaiC hexamer in controlling this process. The involvement of both KaiC domains suggests that information about phosphorylation in CII is communicated allosterically through changes in hexamer structure to the CI domain, potentially through ring–ring stacking interactions (24, 26). We therefore hypothesized that the KaiC phosphorylation sites on each subunit might act as allosteric regulators in the context of a hexameric ring so that phosphorylation of one subunit would alter the ability of all other subunits in the ring to engage with KaiA and KaiB, providing a cooperative mechanism to control the timing of these interactions.We conducted a series of biochemical experiments and perturbations to study the effect of altering the status of each phosphorylation site on the KaiC hexamer. To interpret these results, we then developed a mathematical model analogous to classical models of allosteric transitions in multimeric proteins. We constrain the kinetic parameters in this model using experimental measurements of rate constants, allowing us to compare the predictions of the model directly with data. We conclude that maintenance of circadian timing over a range of protein concentrations requires an effectively ultrasensitive switch in each KaiC hexamer from an exclusively KaiA-binding state to a state that can bind to KaiB as phosphorylation proceeds. This effect requires that KaiC hexamers consist of mixtures of differentially phosphorylated subunits, as would be produced by stochastic autophosphorylation of a hexamer. Ultrasensitivity results from opposing effects of phosphorylation on Thr432 and Ser431 in controlling a concerted transition within a given KaiC hexamer. Including this mechanism in the model is necessary to explain the experimentally observed tolerance of the system to altered protein concentrations.     

Measurement of breath acetone concentrations by selected ion flow tube mass spectrometry in type 2 diabetes

Selected ion flow tube-mass spectrometry (SIFT-MS) can measure volatile compounds in breath on-line in real time and has the potential to provide accurate breath tests for a number of inflammatory, infectious and metabolic diseases, including diabetes. Breath concentrations of acetone in type 2 diabetic subjects undertaking a long-term dietary modification programme were studied. Acetone concentrations in the breath of 38 subjects with type 2 diabetes were determined by SIFT-MS. Anthropomorphic measurements, dietary intake and medication use were recorded. Blood was analysed for beta hydroxybutyrate (a ketone body), HbA1c (glycated haemoglobin) and glucose using point-of-care capillary (fingerprick) testing. All subjects were able to undertake breath manoeuvres suitable for analysis. Breath acetone varied between 160 and 862 ppb (median 337 ppb) and was significantly higher in men (median 480 ppb versus 296 ppb, p = 0.01). In this cross-sectional study, no association was observed between breath acetone and either dietary macronutrients or point-of-care capillary blood tests. Breath analysis by SIFT-MS offers a rapid, reproducible and easily performed measurement of acetone concentration in ambulatory patients with type 2 diabetes. The high inter-individual variability in breath acetone concentration may limit its usefulness in cross-sectional studies. Breath acetone may nevertheless be useful for monitoring metabolic changes in longitudinal metabolic studies, in a variety of clinical and research settings.     

Combined anti-PD-1 and anti-CTLA-4 checkpoint blockade: Treatment of melanoma and immune mechanisms of action

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.     

Living with heart failure: An integrative review

Objectives

To conduct an integrative review that explores the experiences and perceptions of patients who have heart failure to find any commonality that might enhance care giving to this patient group and areas for future research.

Background

Patients are expected to take responsibility for much of the day-to-day management of their heart failure and modify their behaviour to maintain and improve their health. The degree of responsibility which patients can take is to a large extent dependant on how they see themselves in the context of their new situation. Limited research has been undertaken about how patients come to terms with their new condition and subsequent ‘new self’, but this has not been linked to patients’ ability to take more responsibility. This review will integrate this notion of self-care, personal responsibility and the implications of this for nursing care, as this is not identified in current guidelines.

Design and data sources

Using an integrative review method, Ovid MEDLINE (R), CINAHL, EMBASE, AMED, HMIC, BNI and PsycINFO databases were searched from January 1985 to May 2008. 18 papers were selected that most clearly reflected the concept of understanding the patient's experience of living with heart failure.

Results

Patients appear to undergo a process of taking on a new identity, ‘a new self’. From the review five conceptual categories emerge: diagnosis and manifestations of heart failure; perceptions of day-to-day life; coping behaviours; role of others and concept of self which influences all of the other categories. This maps a journey, as the individual experiences heart failure; their success or failure at adapting to this new sense of self, will influence self-care behaviours.

Conclusions

Literature that explores understanding the patient's experiences of living with heart failure, taking on a new identity and the influence of this on their self-care behaviour, is limited. The integration of the new condition with patient's sense of self is likely to a have a major impact on the effectiveness of treatments offered. An essential and ethical care component for patients with heart failure should be an ongoing assessment of their experience of the situation that they live with.