Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation

Exosomes are 60-100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin alpha 4, CD45, moesin, tubulin (alpha and beta), actin, G(i)alpha(2), and a multitude of other proteins. An alpha 4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergent-resistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.     

Predation by juvenile roach on the calanoid copepod Eudiaptomus gracilis and the cyclopoid copepod Cyclops vicinus: a laboratory investigation with mixed and single prey

We tested the outcome of predation by juvenile roach on the calanoid copepod Eudiaptomus gracilis and the similar sized cyclopoid copepod Cyclops vicinus in laboratory experiments with mixed and single-prey. When ovigerous females and adult non-ovigerous females of the calanoid copepod and the cyclopoid copepod were offered in equal numbers to the fish in the mixed-prey experiments, the cyclopoid copepods were significantly more heavily preyed upon than the calanoids. Between 14 and 16 females of the cyclopoid copepod and only between 2 and 4 calanoids had been consumed after the observation period of two hours. The single-prey experiments revealed that the conspicuousness of the calanoid vs the cyclopoid depended on sex and gravidity. Ovigerous females of the cyclopoid copepod were slightly earlier detected by the fish than ovigerous females of the calanoid, probably a result of the highly visible egg-sacs carried by the cyclopoid females. Females without eggs and males of the cyclopoid copepod were recognized later by the predator than females without eggs or males of the calanoid, probably a result of the different behaviours of the different copepod taxa. Cyclopoids frequently congregated near the aquarium bottom while calanoids were closer to the water surface where they were better visible. The calanoid copepod could better escape the fish's attacks than the cyclopoid copepod. Egg-bearing females of both the calanoid and the cyclopoid copepod could significantly better escape than non-ovigerous females or males. Probably ovigerous females react very early to water disturbances caused by the predator. Activity measurements showed that the cyclopoid copepod displayed 2 to 4 times more hops per time unit than the calanoid. Probably the high number of jerky movements displayed by the cyclopoid attracted attention of the predator and contributed to its greater vulnerability.     

Generation of serum-stabilized retroviruses: reduction of alpha1,3gal-epitope synthesis in a murine NIH3T3-derived packaging cell line by expression of chimeric glycosyltransferases

Retroviral vectors released from mouse-derived packaging cell lines are inactivated in human sera by naturally occurring antibodies due to the recognition of Galalpha1,3Galbeta1,4GlcNAc (alphagal-epitope) decorated surface proteins. In this study, an extensive analysis of the glycosylation potential of NIH3T3-derived PA317 packaging cells using combined MALDI/TOF-MS and HPAE-PAD reveals that 34% of the N-glycan moiety represents alphagal-epitope containing structures. Stable expression of glycosyltransferases and transport signal chimeras has been demonstrated to represent an efficient tool to alter cell- and species-specific glycosylation (Grabenhorst and Conradt, 1999. J. Biol. Chem. 274, 36107-36116). In order to reduce alphagal-epitope synthesis selected chimeric glycosyltransferases were constructed by fusing Golgi-signal sequences for compartment-specific localization with the catalytic domain of alpha2,3-sialyltransferase (ST3). Stable expression of these constructs in these cells resulted in a significant reduced alphagal-epitope synthesis, and moreover, a release of retroviral vectors showing an up to 3.5-fold increase in serum stability. Thus, our results suggest that the stably transfected cells stably transfected with chimeric glycosyltransferases compete efficiently with endogenous alpha1,3-galactosyltransferase. This approach allows favored glycodesign and we anticipate the applicability of such improved retroviral vectors produced by glycosylation engineered host cells for in vivo gene therapy and, furthermore, suggest the therapeutic benefit of this technology for xenotransplantation.     

Damaging UV radiation and invertebrate predation: conflicting selective pressures for zooplankton vertical distribution in the water column of low DOC lakes

In nature most organisms have to manage conflicting demands of food gathering, predator avoidance, and finding a favorable abiotic environment (oxygen, temperature, etc.) in order to maximize their fitness. In the vertical water column of lakes with high solar ultraviolet radiation (UV) and invertebrate predators, zooplankton face two particularly strong and conflicting selective pressures. During daylight hours invertebrate predators often induce an upward vertical migration of zooplankton prey while potentially damaging UV forces a downward migration. We used 2.2 m long columns suspended vertically in a lake to conduct 2×2 factorial experiments to examine patterns of depth selection behavior by zooplankton in the presence and absence of both the invertebrate predator Chaoborus and UV. We hypothesized that Chaoborus and UV both affect the distribution of zooplankton and a combination of both factors would lead to a narrowing of depth distribution. We found that when Chaoborus were present zooplankton tended to be distributed at shallower depths in the columns, while in the presence of UV they exhibited a deeper distribution. Chaoborus themselves were always found near the bottom of the columns regardless of the UV treatment. Simultaneous exposure to predators and UV resulted in a peak of zooplankton (especially Daphnia catawba) distribution at intermediate depths. In a significant number of cases, depth range was narrowed in response to Chaoborus, UV, or both.     

TRPV1 acts as proton channel to induce acidification in nociceptive neurons

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca(2+)-dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na(+), Mg(2+), and Ca(2+). The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.     

Analysing Betula litwinowii encroachment and reforestation in the Kazbegi region,Greater Caucasus,Georgia

Aims

The encroachment of tree and shrub species in high mountains is an increasing worldwide phenomenon, which is expected to dramatically alter high‐mountain ecosystems and their functioning. Moreover it indicates in some cases a reforestation process, which will result in important ecological and social benefits, including carbon sequestration and protection against landslides. We therefore examined the spatial extent of forest growth and shrub encroachment mainly of birch (Betula litwinowii) in the sub‐alpine belt of the Central Greater Caucasus between 1987 and 2010 and its relation to topographic site conditions.

Location

Kazbegi district, Central Greater Caucasus, Georgia.

Methods

We analysed 155 vegetation relevés sampled in 2009, 2011 and 2015, mainly derived from the Caucasus Vegetation Database, to obtain information about topographic site conditions and structure of B. litwinowii stands. B. litwinowii forest growth was assessed by digitizing the forest outlines from aerial and space‐borne imagery (1987, 2005 and 2010). To identify areas of B. litwinowii encroachment as an indicator for different encroachment stages, we modelled the tree and shrub cover using the Random Forest algorithm.

Results

We found four types of B. litwinowii stands, characterized by different tree and shrub coverage (initial Bromus variegatus–Betula litwinowii encroachment indicating the first stage of succession, Aconitum nasutum–Betula litwinowii forest, Rubus idaeus–Betula litwinowii forest and Rhododendron caucasicum–Betula litwinowii tree line scrubs). B. litwinowii forest increased 25% compared to 1987 mainly in an uphill direction. Furthermore the modelled tree and shrub cover (R² = .69) could be related to the four vegetation types.

Conclusions

Our results indicate a recent trend towards shrub encroachment and consequently reforestation in the Kazbegi region.     

Multiple predator defence strategies in Daphnia pulex and their relation to native habitat

Daphnia may respond with an array of anti-predator defences(behavioural, morphological and life history) to a chemicalcue (kairomone) exuded by its predators: fish and Chaoborus.Given the wide array of potential responses, it is an interestingquestion whether anti-predator defences are coupled or independentof each other. Since anti-predator responses are costly andeven possessing the genetic information to respond to a certainpredator might involve a cost, clones may only react to predatorsthey co-occur with in nature. In this study, we provide evidencefor an uncoupling of responses by Daphnia pulex in several anti-predatordefences against Chaoborus. We were unable to detect a correlationbetween behavioural (migration), morphological (neck-spine induction)and life history [growth rate, neonate size and size at firstreproduction (SFR)] responses. Furthermore, anti-predator responsesdid not always comply with what is commonly believed. We foundthat Daphnia clones can migrate up or down when exposed to fishor Chaoborus kairomone and that population growth rate, neonatesize and SFR can increase or decrease in response to Chaoboruskairomone. We also show patterns in anti-predator defences thatseem to relate to the habitat from which clones were derived.Daphnia clones that were collected in habitats with Chaoborusas the dominant predator tended to react strongly to Chaoboruskairomone by migrating upward and producing neck-spines. Themigration behaviour against fish kairomone in these clones wasoften an unexpected upward migration. The Daphnia clone thatco-existed with fish predators showed a downward migration inthe presence of fish as well as Chaoborus kairomone. Clonesthat had occurred with either both or no predators had mixedresponses. We sometimes found an upward migration in combinationwith smaller body size as a response to Chaoborus kairomone.This may be interpreted as a behavioural defence against Chaoborusand a life-history defence against fish. Daphnia seem not toexhibit defence behaviour against predators they do not co-occurwith. It might be costly for Daphnia to maintain genetic informationto respond to these predators and protect that information fromgenetic drift.     

Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP

Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of Pmp22+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in Pmp22+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.     

Development of an image pre‐processor for operational hyperspectral laryngeal cancer detection

Hyperspectral imaging (HSI) is a technology with high potential in the field of non‐invasive detection of cancer. However, in complex imaging situations like HSI of the larynx with a rigid endoscope, various image interferences can disable a proper classification of cancerous tissue. We identified three main problems: i) misregistration of single images in a HS cube due to patient heartbeat ii) image noise and iii) specular reflections (SR). Consequently, an image pre‐processor is developed in the current paper to overcome these image interferences. It encompasses i) image registration ii) noise removal by minimum noise fraction (MNF) transformation and iii) a novel SR detection method. The results reveal that the pre‐processor improves classification performance, while the newly developed SR detection method outperforms global thresholding technique hitherto used by 46%. The novel pre‐processor will be used for future studies towards the development of an operational scheme for HS‐based larynx cancer detection.

RGB image of the larynx derived from the hyperspectral cube and corresponding specular reflections ( a ) manually segmented and ( b ) detected by a novel specular reflection detection method.