Molecular basis of gap junctional communication in the CNS of the leech Hirudo medicinalis

Gap junctions are intercellular channels that allow the passage of ions and small molecules between cells. In the nervous system, gap junctions mediate electrical coupling between neurons. Despite sharing a common topology and similar physiology, two unrelated gap junction protein families exist in the animal kingdom. Vertebrate gap junctions are formed by members of the connexin family, whereas invertebrate gap junctions are composed of innexin proteins. Here we report the cloning of two innexins from the leech Hirudo medicinalis. These innexins show a differential expression in the leech CNS: Hm-inx1 is expressed by every neuron in the CNS but not in glia, whereas Hm-inx2 is expressed in glia but not neurons. Heterologous expression in the paired Xenopus oocyte system demonstrated that both innexins are able to form functional homotypic gap junctions. Hm-inx1 forms channels that are not strongly gated. In contrast, Hm-inx2 forms channels that are highly voltage-dependent; these channels demonstrate properties resembling those of a double rectifier. In addition, Hm-inx1 and Hm-inx2 are able to cooperate to form heterotypic gap junctions in Xenopus oocytes. The behavior of these channels is primarily that predicted from the properties of the constituent hemichannels but also demonstrates evidence of an interaction between the two. This work represents the first demonstration of a functional gap junction protein from a Lophotrochozoan animal and supports the hypothesis that connexin-based communication is restricted to the deuterostome clade

Virtual patient design : exploring what works and why : a grounded theory study

Objectives: Virtual patients (VPs) are online representations of clinical cases used in medical education. Widely adopted, they are well placed to teach clinical reasoning skills. International technology standards mean VPs can be created, shared and repurposed between institutions. A systematic review has highlighted the lack of evidence to support which of the numerous VP designs may be effective, and why. We set out to research the influence of VP design on medical undergraduates. Methods: This is a grounded theory study into the influence of VP design on undergraduate medical students. Following a review of the literature and publicly available VP cases, we identified important design properties. We integrated them into two substantial VPs produced for this research. Using purposeful iterative sampling, 46 medical undergraduates were recruited to participate in six focus groups. Participants completed both VPs, an evaluation and a 1-hour focus group discussion. These were digitally recorded, transcribed and analysed using grounded theory, supported by computer-assisted analysis. Following open, axial and selective coding, we produced a theoretical model describing how students learn from VPs. Results: We identified a central core phenomenon designated ‘learning from the VP’. This had four categories: VP Construction; External Preconditions; Student–VP Interaction, and Consequences. From these, we constructed a three-layer model describing the interactions of students with VPs. The inner layer consists of the student's cognitive and behavioural preconditions prior to sitting a case. The middle layer considers the VP as an ‘encoded object’, an e-learning artefact and as a ‘constructed activity’, with associated pedagogic and organisational elements. The outer layer describes cognitive and behavioural change. Conclusions: This is the first grounded theory study to explore VP design. This original research has produced a model which enhances understanding of how and why the delivery and design of VPs influence learning. The model may be of practical use to authors, institutions and researchers

The utility of pulse volume waveforms in the identification of lower limb arterial insufficiency

Background: The ankle brachial index is widely used for non-invasive assessment of lower limb arterial status, but has recognised limitations. The most significant limitation involves arterial calcification, which results in artefactually raised occlusion pressures and uninformative ankle brachial indices. Hypothesis: Analysis of the pulse volume waveform is useful for identification of lower limb arterial insufficiency in the presence of arterial calcification. Method: Individuals (n = 1101) registered at a Welsh general practice were invited to undergo cardiovascular risk assessment. The ankle brachial index was measured using an automated device utilising volume plethysmography and the traditional Doppler ultrasound method. Results: Eight percent of participants (30/368) had an ankle brachial index *1.3, suggesting possible arterial calcification; consideration of the pulse volume waveform in these cases identified possible mild peripheral arterial disease in three cases (10%). Furthermore, in one case, the ankle brachial indices were within the normal range, but the pulse volume waveforms suggested a moderate degree of arterial insufficiency; this participant was subsequently diagnosed with bilateral superficial femoral artery stenoses and treated accordingly. Conclusion: Pulse volume waveforms can be easily utilised as an adjunct to ankle brachial index measurement to identify patients who may benefit from further vascular assessment and interventio

Mutations in shaking-B prevent electrical synapse formation in the Drosophila giant fiber system

The giant fiber system (GFS) is a simple network of neurons that mediates visually elicited escape behavior in Drosophila. The giant fiber (GF), the major component of the system, is a large, descending interneuron that relays visual stimuli to the motoneurons that innervate the tergotrochanteral jump muscle (TTM) and dorsal longitudinal flight muscles (DLMs). Mutations in the neural transcript from the shaking-B locus abolish the behavioral response by disrupting transmission at some electrical synapses in the GFS. This study focuses on the role of the gene in the development of the synaptic connections. Using an enhancer-trap line that expresses lacZ in the GFs, we show that the neurons develop during the first 30 hr of metamorphosis. Within the next 15 hr, they begin to form electrical synapses, as indicated by the transfer of intracellularly injected Lucifer yellow. The GFs dye-couple to the TTM motoneuron between 30 and 45 hr of metamorphosis, to the peripherally synapsing interneuron that drives the DLM motoneurons at approximately 48 hr, and to giant commissural interneurons in the brain at approximately 55 hr. Immunocytochemistry with shaking-B peptide antisera demonstrates that the expression of shaking-B protein in the region of GFS synapses coincides temporally with the onset of synaptogenesis; expression persists thereafter. The mutation shak-B2, which eliminates protein expression, prevents the establishment of dye coupling shaking-B, therefore, is essential for the assembly and/or maintenance of functional gap junctions at electrical synapses in the GFS

The utility of pulse volume waveforms in the identification of lower limb arterial insufficiency

Background: The ankle brachial index is widely used for non-invasive assessment of lower limb arterial status, but has recognised limitations. The most significant limitation involves arterial calcification, which results in artefactually raised occlusion pressures and uninformative ankle brachial indices. Hypothesis: Analysis of the pulse volume waveform is useful for identification of lower limb arterial insufficiency in the presence of arterial calcification. Method: Individuals (n = 1101) registered at a Welsh general practice were invited to undergo cardiovascular risk assessment. The ankle brachial index was measured using an automated device utilising volume plethysmography and the traditional Doppler ultrasound method. Results: Eight percent of participants (30/368) had an ankle brachial index *1.3, suggesting possible arterial calcification; consideration of the pulse volume waveform in these cases identified possible mild peripheral arterial disease in three cases (10%). Furthermore, in one case, the ankle brachial indices were within the normal range, but the pulse volume waveforms suggested a moderate degree of arterial insufficiency; this participant was subsequently diagnosed with bilateral superficial femoral artery stenoses and treated accordingly. Conclusion: Pulse volume waveforms can be easily utilised as an adjunct to ankle brachial index measurement to identify patients who may benefit from further vascular assessment and interventio

Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth

Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types

Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia

Background: The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. Principal Findings: Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D2 (PGD2) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Δ12,14 PGJ2 (15d-PGJ2). BEZ increased PGD2 synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ2 by inhibiting the PGD2 11β -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD2 to 9α11β-PGF2α. B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ2. Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. Significance: Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ2. These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML