S641 contributes HERG K+ channel inactivation

The kinetics of voltage-dependent inactivation of the rapidly activating delayed rectifier, I _Kr, are unique among K⁺ channels. The human ether-a-gogo-related gene (HERG) encodes the pore-forming subunit of I _Kr and shares a high degree of homology with ether-a-gogo (EAG) channels that do not inactivate. Within those segments thought to contribute to the channel pore, HERG, possesses several serine residues that are not present in EAG channels. Two of these serines, S620 and S631, are known to be required for inactivation. We now show that a third serine, S641, which resides in the outer portion of the sixth transmembrane segment, is also critical for normal inactivation. As with the other serines, S641 is also involved in maintaining ion selectivity of the HERG channel and alters sensitivity to block by E4031. Larger charged or polar substitutions (S641D and S641T) disrupted C-type inactivation in HERG. Smaller aliphatic and more conservative substitutions (S641A and S641C) facilitated C-type inactivation. Our data show that, like S620 and S631, S641 is another key residue for the rapid inactivation. The altered inactivation of mutations at S620, S631, and S641 were dominant, suggesting that a network of hydroxyl side chains is required for the unique inactivation, permeation, and rectification of HERG channels.     

Abdominal ultrasound in the diagnostic work-up of visceral leishmaniasis and for detection of complications of spleen aspiration

IntroductionAbdominal ultrasound (US) is increasingly used in the diagnostic work-up of infectious diseases, but studies on its diagnostic value in visceral leishmaniasis (VL) are lacking. US could help to identify complications of spleen aspiration (SA). We aimed to assess the diagnostic value of US and the evolution of findings after VL treatment; the incidence and degree of splenic injury; and the pain perceived during SA.Methodology/resultWe conducted a cross-sectional prospective study at the Leishmaniasis Research and Treatment Center, Gondar, Ethiopia between Oct 2017 and Dec 2018. We enrolled VL suspects undergoing tissue aspiration; US were conducted before and after SA, and at the end of VL treatment. Splenic injury was graded using the American association of surgery trauma injury scale (grade 1–4). The pain perceived during SA was graded using a visual analogue scale. Out of 392 VL suspects, 192 (49%) were confirmed VL cases. The median age was 25 years (IQR 21–30). Massive splenomegaly and hepatomegaly were the most common US findings. Splenic nodules were seen in 3.7% of the 190 VL cases and 1.5% of the 197 non-VL cases. Ascites was more common in VL (16.4%) than in non-VL cases (9.1%). The frequency of US abnormalities decreased with treatment. None of the US findings had sufficient sensitivity and specificity to justify its use as a diagnostic test. US detected splenic injury in four of the 318 patients who had post-SA US. All four patients remained clinically stable. Pain was perceived as moderate or severe in 51% of patients.ConclusionThe diagnostic value of abdominal US for VL was low but found useful to detect subclinical splenic injury. SA caries a risk of splenic injury and was perceived painful by most. Further research on less invasive diagnostic tools is needed.     

An antagonist to GnRH in the control of reproduction in teleost fish: dopaminergic inhibition. Ancestral origin and differential conservation within vertebrates?

In mammals, the neurohormonal control of the pituitary gonadotropes is provided by the gonadoliberin GnRH. Several studies on teleost fish indicate that a single positive control by GnRH is not a general rule among vertebrates. Peter and colleagues presented the first evidence of an inhibitory neurohormonal factor, "GRIF" (gonadotropin-release inhibiting factor). They induced a preovulatory LH surge by injuring particular brain areas in the goldfish. Subsequent in vivo and in vitro studies identified dopamine as GRIF, and neuroanatomical investigations have demonstrated that dopaminergic neurones in the anterior preoptic area projecting to the pituitary represent the anatomical substrate for GRIF activity. An inhibitory role of dopamine on the control of LH and ovulation/spermiation has been evidenced in many adult teleosts, including its implications for aquaculture. However, dopamine does not play an inhibitory role in all adult teleosts. As regards the early stages of gametogenesis and especially the control of puberty, a role for dopamine has been suggested or rejected depending on species. The European eel has a unique life cycle with a long prepubertal stage, which has made it a useful model to demonstrate the key-role of dopamine in the control of puberty. Data from tetrapods suggest that the role of dopamine as a GRIF is not restricted to the teleosts, but that it may have an ancient evolutionary origin, and has been differentially conserved throughout vertebrate evolution.     

Use of GnRHa-delivery systems for the control of reproduction in fish

The most commonly observed reproductivedysfunctions in cultured fish are theunpredictability of final oocyte maturation(FOM) in females, and the diminished volume andquality of sperm in males. Gonadotropin-releasing hormone agonists (GnRHa)have been used extensively in order tostimulate the release of pituitary luteinizinghormone (LH) required to induce FOM, ovulationand spermiation. Because multiple hormonaltreatments are often necessary for a successfulresponse, fish must be monitored and handledextensively, which is labor intensive,stressful to the fish and can often result inbroodstock mortalities. To ameliorate thisproblem, sustained-release delivery systems forGnRHa have been developed during the last twodecades and have been increasingly applied incontrolling reproduction of a variety ofcultured fish. Solid implants of cholesterolor poly[ethylene-vinyl acetate], andbiodegradable microspheres ofpoly[lactide-glycolide] or poly[fatty aciddimer-sebasic acid] release GnRHa for a periodof time (from a few days to many weeks.) GnRHa-delivery systems do not causedesensitization of the pituitary gonadotrophsin fish, and by stimulating a sustainedelevation of plasma LH they induce the naturalprogression of plasma steroid increasesassociated with FOM and spermiation. Thismethod has been used with very encouragingresults in females of more than 40 culturedspecies and has been effective in inducing FOM,ovulation or spawning in fish with synchronous,group-synchronous and asynchronous ovariandevelopment. In males, GnRHa-delivery systemshave been tested in more than 20 species,producing significant increases in miltproduction for up to 5 weeks. Future researchshould focus on the optimization of thistechnology in terms of (a) using the mostpotent GnRHa, (b) identifying the mostappropriate GnRHa release kinetics according tothe reproductive biology of different species,and (c) determining minimum effective doses. Developments in these areas will greatlyenhance the effectiveness and efficiency ofGnRHa-delivery systems, while at the same timereducing their cost thus making them moreaffordable to the aquaculture industry.     

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50–60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40–100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging.     

Isolation,Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca²⁺ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.     

Stalk phase formation: effects of dehydration and saddle splay modulus

One of the earliest lipid intermediates forming in the course of membrane fusion is the lipid stalk. Although many aspects of the stalk hypothesis were elaborated theoretically and confirmed by experiments it remained unresolved whether stalk formation is always an energy consuming process or if there are conditions where the stalks are energetically favorable and form spontaneously resulting in an equilibrium stalk phase. Motivated by a recent breakthrough experiments we analyze the physical factors determining the spontaneous stalk formation. We show that this process can be driven by interplay between two factors: the elastic energy of lipid monolayers including a contribution of the saddle splay deformation and the energy of hydration repulsion acting between apposing membranes. We analyze the dependence of stalk formation on the saddle splay (Gaussian) modulus of the lipid monolayers and estimate the values of this modulus based on the experimentally established phase boundary between the lamellar and the stalk phases. We suggest that fusion proteins can induce stalk formation just by bringing the membranes into close contact, and accumulating, at least locally, a sufficiently large energy of the hydration repulsion.