Noninvasive tests predict liver-related events and mortality in patients with nonalcoholic fatty liver disease: sub-analysis of the CLIONE-Asia study.

BACKGROUND AND AIMS: Noninvasive tests (NITs) have prognostic potential, but whether NITs are comparable with liver biopsy is unclear. This study aimed to examine the prognostic accuracy of NITs for liver-related mortality (LRM) and events (LREs) in patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD). METHODS: We investigated 1313 patients with NAFLD. Patients were assigned to low-risk, indeterminate-risk, and high-risk groups using conventional cutoff values of each FIB-4 and NAFLD fibrosis score (NFS) and to stage 0-2 and stage 3-4 groups using the fibrosis stage. Survival and Cox regression analyses of the prognostic potential of NITs for LRM/LREs were conducted. RESULTS: During a median follow-up of 4.5 years, regarding to FIB-4, the incidence rate (/1000 person-years) in the low risk was zero for LRM and 0.5 for LREs. In contrast, the rate in stage 0-2 was 1.3 for LRM and 2.8 for LRE. The adjusted hazard ratios (aHRs) for LREs in the high risk compared with the low risk were 32.85 (P < 0.01). The aHRs in stage 3-4 compared with stage 0-2 were 2.68 (P = 0.02) for LREs and 2.26 (P = 0.582) for LRM. In the same fibrosis stage, the incidence of LRM/LREs was more frequent with a higher risk stratification. The same trend was observed for NFS. CONCLUSIONS: NITs accurately predict LRM and LREs as well as a liver biopsy in Japanese patients with NAFLD. Patients in the low risk may not require close follow-up for at least 5 years. The simple NITs could be an acceptable alternative method to performing a liver biopsy for the prognosis of NAFLD.

Age-dependent effects of diabetes and obesity on liver-related events in non-alcoholic fatty liver disease: Subanalysis of CLIONE in Asia.

BACKGROUND AND AIM: Older age, type 2 diabetes mellitus (T2DM), and obesity are known risk factors for liver-related events (LREs). We investigated the impacts of T2DM and obesity on LRE according to age in Japanese patients with non-alcoholic fatty liver disease (NAFLD). METHODS: We performed a subanalysis of a retrospective cohort study (CLIONE in Asia), including 1395 patients with biopsy-proven NAFLD. The median follow-up was 4.6 years. RESULTS: The median age was 57 years, and 36.2% had T2DM. The median body mass index (BMI) was 27.4, and 28.5% were severely obese (BMI ≥ 30). During follow-up, 37 patients developed hepatocellular carcinoma (HCC), and 58 patients developed LRE. In patients younger than 65 years, advanced fibrosis (hazard ratio [HR] 7.69, P < 0.001) and T2DM (HR 3.37, P = 0.017) were HCC risk factors, and advanced fibrosis (HR 9.40, P < 0.001) and T2DM (HR 2.51, P = 0.016) were LRE risk factors. In patients 65 years and older, advanced fibrosis (HR 4.24, P = 0.010) and obesity (HR 4.60, P = 0.006) were HCC risk factors, and advanced fibrosis (HR 4.22, P = 0.002) and obesity (HR 4.22, P = 0.002) were LRE risk factors. CONCLUSION: Type 2 diabetes mellitus and obesity contributed to LRE in younger and older patients, respectively, along with advanced fibrosis. Therefore, controlling T2DM in patients younger than 65 years and controlling weight in patients 65 years and older could prevent LRE. The development of age-dependent screening and management strategies is necessary for patients with NAFLD.

Transformation of context-dependent sensory dynamics into motor behavior.

The intrinsic dynamics of sensory networks play an important role in the sensory-motor transformation. In this paper we use conductance based models and electrophysiological recordings to address the study of the dual role of a sensory network to organize two behavioral context-dependent motor programs in the mollusk Clione limacina. We show that: (i) a winner take-all dynamics in the gravimetric sensory network model drives the typical repetitive rhythm in the wing central pattern generator (CPG) during routine swimming; (ii) the winnerless competition dynamics of the same sensory network organizes the irregular pattern observed in the wing CPG during hunting behavior. Our model also shows that although the timing of the activity is irregular, the sequence of the switching among the sensory cells is preserved whenever the same set of neurons are activated in a given time window. These activation phase locks in the sensory signals are transformed into specific events in the motor activity. The activation phase locks can play an important role in motor coordination driven by the intrinsic dynamics of a multifunctional sensory organ.

Temperature compensation of aerobic capacity and performance in the Antarctic pteropod, Clione antarctica, compared with its northern congener, C. limacina.

In ectotherms living in cold waters, locomotory performance is constrained by a slower generation of the ATP that is needed to fuel muscle contraction. Both polar and temperate pteropods of the genus Clione, however, are able to swim continuously by flapping their parapodia (wings) at comparable frequencies at their respective habitat temperatures. Therefore, we expected polar species to have increased aerobic capacities in their wing muscles when measured at common temperatures. We investigated muscle and mitochondrial ultrastructure of Clione antarctica from the Southern Ocean (-1.8°C) and populations of a sister species, Clione limacina, from the Arctic (-0.5 to 3°C) and from the North Atlantic (10°C). We also measured oxygen consumption and the activity of the mitochondrial enzyme citrate synthase (CS) in isolated wings of the two species. The Antarctic species showed a substantial up-regulation of the density of oxidative muscle fibers, but at the expense of fast-twitch muscle fibers. Mitochondrial capacity was also substantially increased in the Antarctic species, with the cristae surface density (58.2±1.3µm(2)µm(-3)) more than twice that found in temperate species (34.3±0.8µm(2)µm(-3)). Arctic C. limacina was intermediate between these two populations (43.7±0.5µm(2)µm(-3)). The values for cold-adapted populations are on par with those found in high-performance vertebrates. As a result of oxidative muscle proliferation, CS activity was 4-fold greater in C. antarctica wings than in temperate C. limacina when measured at a common temperature (20°C). Oxygen consumption of isolated wing preparations was comparable in the two species when measured at their respective habitat temperatures. These findings indicate complete compensation of ATP generation in wing muscles across a 10°C temperature range, which supports similar wing-beat frequencies during locomotion at each species' respective temperature. The elevated capacity in the wing muscles is reflected in the partial compensation of whole-animal oxygen consumption and feeding rates.

Trade-off between aerobic capacity and locomotor capability in an Antarctic pteropod.

At -1.8 degrees C, the waters of Antarctica pose a formidable physiological barrier for most ectotherms. The few taxa that inhabit this zone have presumably made specific adjustments to their neuromuscular function and have enhanced their metabolic capacity. However, support for this assertion is equivocal and the details of specific compensations are largely unknown. This can generally be attributed to the fact that most Antarctic organisms are either too distantly related to their temperate relatives to permit direct comparisons (e.g., notothenioid fishes) or because they are not amenable to neuromuscular recording. Here, as a comparative model, we take advantage of 2 pelagic molluscs in the genus Clione to conduct a broadly integrative investigation on neuromuscular adaptation to the extreme cold. We find that for the Antarctic congener aerobic capacity is enhanced, but at a cost. To support a striking proliferation of mitochondria, the Antarctic species has shed a 2-gear swim system and the associated specialized neuromuscular components, resulting in greatly reduced scope for locomotor activity. These results suggest that polar animals have undergone substantial tissue-level reorganizations to accommodate their environment, which may reduce their capacity to acclimate to a changing climate.

[Single interneuron coordinates wing and tail movements in pteropod mollusk].

Nervous centers that coordinate rhythmical movements with body stabilization in space are well known in vertebrates. Here we report a single identified interneuron CPB3c (cerebropedal neuron c from group B3) that serves the same function ofpostural control during locomotion in a simple animal model--the marine pteropod mollusk Clione limacina. CPB3c interneuron integrates inputs from statocysts and locomotor generator and translates signals to tail motorneurons. So, this neuron has perfect connections to fulfill the coordinative function.

Serotonergic cerebral cells control activity of cilia in the foregut of the pteropod mollusk Clione limacina.

Bilaterally symmetrical pair of serotonergic cells, named C1 in Clione, has been described in the cerebral ganglia of all gastropod species. Here we describe a new role of C1 cells in gastropod mollusks: control of activity of ciliated epithelium in the foregut. Detailed morphological investigation of C1 neurons in the pteropod mollusk Clione limacina revealed that these cells among other destinations send their neurites into foregut where they produce intense arborization with large varicosities along the processes. Intracellular stimulation of a single C1 induced pronounced activation (often followed by inhibition) of cilia lining the foregut. This activation was substantially reduced by serotonin antagonist mianserin. Bath application of serotonin also induced transient increase in ciliary transport rate, followed by inhibition of ciliary activity up to its full cessation in some areas of isolated foregut. These data suggest that C1 in Clione may use serotonin to influence cilia in the foregut. Taking into account high homology of serotonergic cerebral cells across studied species we can speculate that these cells may be involved in the neural control of cilia in the foregut in other gastropod mollusks.

Changes in wingstroke kinematics associated with a change in swimming speed in a pteropod mollusk, Clione limacina.

In pteropod mollusks, the gastropod foot has evolved into two broad, wing-like structures that are rhythmically waved through the water for propulsion. The flexibility of the wings lends a tremendous range of motion, an advantage that could be exploited when changing locomotory speed. Here, we investigated the kinematic changes that take place during an increase in swimming speed in the pteropod mollusk Clione limacina. Clione demonstrates two distinct swim speeds: a nearly constant slow swimming behavior and a fast swimming behavior used for escape and hunting. The neural control of Clione's swimming is well documented, as are the neuromuscular changes that bring about Clione's fast swimming. This study examined the kinematics of this swimming behavior at the two speeds. High speed filming was used to obtain 3D data from individuals during both slow and fast swimming. Clione's swimming operates at a low Reynolds number, typically under 200. Within a given swimming speed, we found that wing kinematics are highly consistent from wingbeat to wingbeat, but differ between speeds. The transition to fast swimming sees a significant increase in wing velocity and angle of attack, and range of motion increases as the wings bend more during fast swimming. Clione likely uses a combination of drag-based and unsteady mechanisms for force production at both speeds. The neuromuscular control of Clione's speed change points to a two-gaited swimming behavior, and we consider the kinematic evidence for Clione's swim speeds being discrete gaits.

Shell density of planktonic foraminifera and pteropod species Limacina helicina in the Barents Sea: Relation to ontogeny and water chemistry.

Planktonic calcifiers, the foraminiferal species Neogloboquadrina pachyderma and Turborotalita quinqueloba, and the thecosome pteropod Limacina helicina from plankton tows and surface sediments from the northern Barents Sea were studied to assess how shell density varies with depth habitat and ontogenetic processes. The shells were measured using X-ray microcomputed tomography (XMCT) scanning and compared to the physical and chemical properties of the water column including the carbonate chemistry and calcium carbonate saturation of calcite and aragonite. Both living L. helicina and N. pachyderma increased in shell density from the surface to 300 m water depth. Turborotalita quinqueloba increased in shell density to 150-200 m water depth. Deeper than 150 m, T. quinqueloba experienced a loss of density due to internal dissolution, possibly related to gametogenesis. The shell density of recently settled (dead) specimens of planktonic foraminifera from surface sediment samples was compared to the living fauna and showed a large range of dissolution states. This dissolution was not apparent from shell-surface texture, especially for N. pachyderma, which tended to be both thicker and denser than T. quinqueloba. Dissolution lowered the shell density while the thickness of the shell remained intact. Limacina helicina also increase in shell size with water depth and thicken the shell apex with growth. This study demonstrates that the living fauna in this specific area from the Barents Sea did not suffer from dissolution effects. Dissolution occurred after death and after settling on the sea floor. The study also shows that biomonitoring is important for the understanding of the natural variability in shell density of calcifying zooplankton.

A hyperpolarization-activated inward current alters swim frequency of the pteropod mollusk Clione limacina.

The pteropod mollusk, Clione limacina, exhibits behaviorally relevant swim speed changes that occur within the context of the animal's ecology. Modulation of C. limacina swimming speed involves changes that occur at the network and cellular levels. Intracellular recordings from interneurons of the swim central pattern generator show the presence of a sag potential that is indicative of the hyperpolarization-activated inward current (I(h)). Here we provide evidence that I(h) in primary swim interneurons plays a role in C. limacina swimming speed control and may be a modulatory target. Recordings from central pattern generator swim interneurons show that hyperpolarizing current injection produces a sag potential that lasts for the duration of the hyperpolarization, a characteristic of cells possessing I(h). Following the hyperpolarizing current injection, swim interneurons also exhibit postinhibitory rebound (PIR). Serotonin enhances the sag potential of C. limacina swim interneurons while the I(h) blocker, ZD7288, reduces the sag potential. Furthermore, a negative correlation was found between the amplitude of the sag potential and latency to PIR. Because latency to PIR was previously shown to influence swimming speed, we hypothesize that I(h) has an effect on swimming speed. The I(h) blocker, ZD7288, suppresses swimming in C. limacina and inhibits serotonin-induced acceleration, evidence that supports our hypothesis.

Pedal serotonergic neuron clusters of the pteropod mollusc, Clione limacina, contain two morphological subtypes with different innervation targets.

Each pedal ganglion of the pteropod mollusc Clione limacina contains a cluster of serotonin-immunoreactive neurons that have been shown to modulate contractions of the slow-twitch musculature of the wing-like parapodia, and contribute to swim accelerations. Each cluster has a variable number of neurons, between 5 and 9, but there is no significant difference between right and left ganglia. In experiments with electrophysiological recordings followed by dye-injection (carboxyfluorescein), the clusters were found to contain two subsets of neurons. The majority innervate the ipsilateral wing via nerve n4. Two of the neurons in each cluster send processes out of the pedal ganglion in nerves n3 and n8. The processes in nerve n3 innervate the body wall of the neck region, while those in nerve n8 innervate the body wall of the tail. The baseline electrophysiological activity of the two subsets of neurons was different as "wing" neurons had constant barrages of small synaptic activity, while the "body wall" neurons had few synaptic inputs. The potential roles of the Pd-SW cluster in swim acceleration (wing neurons) and control of fluid pressure in the body and wing hemocoelic compartments (body wall neurons) are discussed.

Buccal neurons activate ciliary beating in the foregut of the pteropod mollusk Clione limacina.

Beating of cilia lining the foregut of gastropods facilitates the swallowing of food and, therefore, plays a role in feeding behavior. Despite the fact that neural control of feeding is well studied in mollusks, no neurons controlling ciliary beating in the foregut have been identified to date. Here we describe for the first time a pair of buccal neurons innervating the foregut of Clione. Intracellular stimulation of these neurons induced vigorous activation of cilia lining the foregut in a semi-intact preparation. Using immunochemistry labeling, buccal foregut cells were found to contain peptides similar to CNP neuropeptides of the terrestrial snail Helix lucorum. Application of DYPRL-amide, a member of the Helix CNP peptide family, mimicked the effect of buccal foregut cell stimulation on ciliary activity. Induction of fictive feeding in an isolated CNS preparation resulted in the activation of buccal foregut cells suggesting that these cells control ciliary beating in the foregut during feeding. Thus, cilia-activating buccal neurons may represent a new intrinsic element of the neural control of feeding in gastropods.

Organization of Buccal Cone Musculature in the Pteropod Mollusc Clione limacina.

The pteropod mollusc Clione limacina is a feeding specialist, preying on shelled pteropods of the genus Limacina. Specialized prey-capture structures, called buccal cones, are hydraulically everted from within the mouth to capture the prey. Once captured, the prey is manipulated so the shell opening is over the mouth of Clione. Analyses of high-speed cine sequences of prey capture suggest that the mouth is actively opened rather than passively forced open by buccal cone eversion. The inflated buccal cones are initially straight and form a wide angle (maximum, 113°) prior to prey contact. Individual buccal cones bend orally following prey contact, suggesting a sensory trigger. To determine the muscular basis of buccal cone movements, the musculature of the buccal cones is described. Three distinct muscle fiber types include circular smooth muscle, longitudinal smooth muscle, and longitudinal striated muscle. The organization, distribution, and innervation of the muscle types suggest that circular muscle is used during buccal cone eversion, longitudinal smooth muscle is used for buccal cone withdrawal, and longitudinal striated muscle is used for oral bending of the buccal cones after prey contact and for manipulation of the prey.

Neural control of heartbeat during two antagonistic behaviors: whole body withdrawal and escape swimming in the mollusk Clione limacina.

Two cardioexcitatory and one cardioinhibitory neural groups have been previously identified as the central cardioregulatory system in the pteropod mollusk Clione limacina. We describe in this study one additional element of the central cardioregulatory system, which consists of a large intestinal neuron named Z-cell with a novel effect on the heart activity. Intracellular stimulation of the Z-cell induced only auricle contractions with no effect on the ventricle activity. The Z-cell processes were traced down to the heart, and vigorous branching was found in the auricle tissue. Specific patterns of activity of the Z-cell as well as intestinal heart excitatory and inhibitory neurons were studied during initiation of two behaviors--whole body withdrawal and escape swimming. It was found that initiation of both behaviors was accompanied by activation of Z-cell and intestinal heart excitor neurons. The firing rate of neurons induced by sensory stimuli was sufficient to trigger auricle contractions in the semi-intact preparations. Video analysis of heart activity revealed that auricle indeed was activated during both active and passive avoidance reactions, though the intensity and delay of the activation were different. The possible physiological role of the auricle contractions during antagonistic forms of behavior is discussed.

Assembly of a reference transcriptome for the gymnosome pteropod Clione limacina and profiling responses to short-term CO2 exposure.

The gymnosome (unshelled) pteropod Clione limacina is a pelagic predatory mollusc found in polar and sub-polar regions. It has been studied for its distinctive swimming behavior and as an obligate predator on the closely related thecosome (shelled) pteropods. As concern about ocean acidification increases, it becomes useful to compare the physiological responses of closely-related calcifying and non-calcifying species to acidification. The goals of this study were thus to generate a reference transcriptome for Clione limacina, to expose individuals to CO2 for a period of 3days, and to explore differential patterns of gene expression. Our Trinity assembly contained 300,994 transcripts of which ~26% could be annotated. In total, only 41 transcripts were differentially expressed following the CO2 treatment, consistent with a limited physiological response of this species to short-term CO2 exposure. The differentially expressed genes identified in our study were largely distinct from those identified in previous studies of thecosome pteropods, although some similar transcripts were identified, suggesting that comparison of these transcriptomes and responses may provide insight into differences in responses to ocean acidification among phylogenetically and functionally distinct molluscan lineages.

The role of sensory network dynamics in generating a motor program.

Sensory input plays a major role in controlling motor responses during most behavioral tasks. The vestibular organs in the marine mollusk Clione, the statocysts, react to the external environment and continuously adjust the tail and wing motor neurons to keep the animal oriented vertically. However, we suggested previously that during hunting behavior, the intrinsic dynamics of the statocyst network produce a spatiotemporal pattern that may control the motor system independently of environmental cues. Once the response is triggered externally, the collective activation of the statocyst neurons produces a complex sequential signal. In the behavioral context of hunting, such network dynamics may be the main determinant of an intricate spatial behavior. Here, we show that (1) during fictive hunting, the population activity of the statocyst receptors is correlated positively with wing and tail motor output suggesting causality, (2) that fictive hunting can be evoked by electrical stimulation of the statocyst network, and (3) that removal of even a few individual statocyst receptors critically changes the fictive hunting motor pattern. These results indicate that the intrinsic dynamics of a sensory network, even without its normal cues, can organize a motor program vital for the survival of the animal.

The contribution of the pleural type 12 interneuron to swim acceleration in Clione limacina.

The pteropod mollusc, Clione limacina, swims by alternate dorsal-ventral flapping movements of its wing-like parapodia. The basic swim rhythm is produced by a network of pedal swim interneurons that comprise a swim central pattern generator (CPG). Serotonergic modulation of both intrinsic cellular properties of the swim interneurons and network properties contribute to swim acceleration, the latter including recruitment of type 12 interneurons into the CPG. Here we address the role of the type 12 interneurons in swim acceleration. A single type 12 interneuron is found in each of the pleural ganglia, which contributes to fast swimming by exciting the dorsal swim interneurons while simultaneously inhibiting the ventral swim interneurons. Each type 12 interneuron sends a single process through the pleural-pedal connective that branches in both ipsilateral and contralateral pedal ganglia. This anatomical arrangement allowed us to manipulate the influence of the type 12 interneurons on the swim circuitry by cutting the pleural-pedal connective followed by a "culture" period of 48 h. The mean swim frequency of cut preparations was reduced by 19% when compared to the swim frequency of uncut preparations when stimulated with 10(-6) M serotonin; however, this decrease was not statistically significant. Additional evidence suggests that the type 12 interneurons may produce a short-term, immediate effect on swim acceleration while slower, modulatory inputs are taking shape.

Toward an organismal neurobiology: integrative neuroethology.

Overt behavior is generated in response to a palette of external and internal stimuli and internal drives. Rarely are these variables introduced in isolation. This creates challenges for the organism to sort inputs that frequently favor conflicting behaviors. Under these conditions, the nervous system relies on established and flexible hierarchies to produce appropriate behavioral changes. The pteropod mollusc Clione limacina is used as an example to illustrate a variety of behavioral interactions that alter a baseline behavioral activity: slow swimming. The alterations include acceleration within the slow swimming mode, acceleration from the slow to fast swimming modes, whole body withdrawal (and inhibition of swimming), food acquisition behavior (with a feeding motivational state), and a startle locomotory response. These examples highlight different types of interaction between the baseline behavior and the new behaviors that involve external stimuli and two types of internal drives: a modular arousal system and a motivational state. The investigation of hierarchical interactions between behavioral modules is a central theme of integrative neuroethology that focuses on an organismal level of understanding of the neural control of behavior.