Control of ventricular ciliary beating by the melanin concentrating hormone-expressing neurons of the lateral hypothalamus: a functional imaging survey

The cyclic peptide Melanin Concentrating Hormone (MCH) is known to control a large number of brain functions in mammals such as food intake and metabolism, stress response, anxiety, sleep/wake cycle, memory, and reward. Based on neuro-anatomical and electrophysiological studies these functions were attributed to neuronal circuits expressing MCHR1, the single MCH receptor in rodents. In complement to our recently published work (1) we provided here new data regarding the action of MCH on ependymocytes in the mouse brain. First, we establish that MCHR1 mRNA is expressed in the ependymal cells of the third ventricle epithelium. Second, we demonstrated a tonic control of MCH-expressing neurons on ependymal cilia beat frequency using in vitro optogenics. Finally, we performed in vivo measurements of CSF flow using fluorescent micro-beads in wild-type and MCHR1-knockout mice. Collectively, our results demonstrated that MCH-expressing neurons modulate ciliary beating of ependymal cells at the third ventricle and could contribute to maintain cerebro-spinal fluid homeostasis.

Melanin-concentrating hormone regulates beat frequency of ependymal cilia and ventricular volume

Ependymal cell cilia help move cerebrospinal fluid through the cerebral ventricles, but the regulation of their beat frequency remains unclear. Using in vitro, high-speed video microscopy and in vivo magnetic resonance imaging in mice, we found that the metabolic peptide melanin-concentrating hormone (MCH) positively controlled cilia beat frequency, specifically in the ventral third ventricle, whereas a lack of MCH receptor provoked a ventricular size increase.

Prospective ex-vivo study on thermal effects in human skin phototypes II, IV and VI: a comparison between the 808, 1064, 1210 and 1320-nm diode laser

Laser Assisted Skin Healing (LASH) was first introduced in 2001 by Capon and Mordon to prevent keloids and hypertrophic scars. LASH requires homogenous heating throughout the full thickness of the skin around the wound. However, LASH therapy with 808-nm diode laser is deemed to be only applicable for phototype I-IV due to melanin absorption. This prospective ex-vivo study aims to evaluate the thermal effects of different wavelengths (808, 1064, 1210 and 1320 nm) on human skin phototype II, IV and VI.

Black hair follicular dysplasia in Large Münsterländer dogs: clinical, histological and ultrastructural features

Four Large Münsterländer cross-bred dogs affected with black hair follicular dysplasia (BHFD) and one unaffected control littermate were observed, and skin was sampled weekly over the first 19 weeks of life. Affected dogs were born with silvery grey hair, a consequence of melanin clumping in the hair shafts. Hair bulb melanocytes were densely pigmented, and contained abundant stage IV melanosomes but adjacent matrix keratinocytes lacked melanosomes. Melanin clumping was not prominent in epidermal melanocytes in the haired skin but occurred in the foot pads. Follicular changes progressed from bulbar clumping, clumping in the isthmus/infundibulum and finally to dysplastic hair shafts. Alopecia developed progressively in pigmented areas. Silver-grey hair, melanin clumping, accumulation of stage IV melanosomes within melanocytes and insufficient melanin transfer to adjacent keratinocytes are also classic features of human Griscelli syndrome. The underlying cause in Griscelli syndrome is a defect of melanocytic intracellular transport proteins leading to inadequate and disorganized melanosome transfer to keratinocytes with resultant melanin clumping. In view of the correlation in the phenotype, histology and ultrastructure between both disorders, a defect in intracellular melanosome transport is postulated as the pathogenic mechanism in BHFD.

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.

Effect of a chitosan additive to a Sn(2+)-containing toothpaste on its anti-erosive/anti-abrasive efficacy-a controlled randomised in situ trial

OBJECTIVES It is well known that Sn(2+) is a notable anti-erosive agent. There are indications that biopolymers such as chitosan can enhance the effect of Sn(2+), at least in vitro. However, little information exists about their anti-erosive/anti-abrasive in situ effects. In the present in situ study, the efficacy of Sn(2+)-containing toothpastes in the presence or absence of chitosan was tested. METHODS Ten subjects participated in the randomised crossover study, wearing mandibular appliances with human enamel specimens. Specimens were extraorally demineralised (7 days, 0.5 % citric acid, pH 2.6; 6 × 2 min/day) and intraorally exposed to toothpaste suspensions (2 × 2 min/day). Within the suspension immersion time, one half of the specimens were additionally brushed intraorally with a powered toothbrush (5 s, 2.5 N). Tested preparations were a placebo toothpaste (negative control), two experimental toothpastes (F/Sn = 1,400 ppm F(-), 3,500 ppm Sn(2+); F/Sn/chitosan = 1,400 ppm F(-), 3,500 ppm Sn(2+), 0.5 % chitosan) and an SnF2-containing gel (positive control, GelKam = 3,000 ppm Sn(2+), 1,000 ppm F(-)). Substance loss was quantified profilometrically (μm). RESULTS In the placebo group, tissue loss was 11.2 ± 4.6 (immersion in suspension) and 17.7 ± 4.7 (immersion in suspension + brushing). Immersion in each Sn(2+)-containing suspension significantly reduced tissue loss (p ≤ 0.01); after immersion in suspension + brushing, only the treatments with GelKam (5.4 ± 5.5) and with F/Sn/chitosan (9.6 ± 5.6) significantly reduced loss [both p ≤ 0.05 compared to placebo; F/Sn 12.8 ± 6.4 (not significant)] CONCLUSION Chitosan enhanced the efficacy of the Sn(2+)-containing toothpaste as an anti-erosive/anti-abrasive agent. CLINICAL RELEVANCE The use of Sn(2+)- and chitosan-containing toothpaste is a good option for symptomatic therapy in patients with regular acid impacts.

The Immune System of Spiders

Spiders, as all other arthropods, have an open circulatory system, and their body fluid, the hemolymph, freely moves between lymphatic vessels and the body cavities (see Wirkner and Huckstorf 2013). The hemolymph can be considered as a multifunctional organ, central for locomotion (Kropf 2013), respiration (Burmester 2013) and nutrition, and it amounts to approximately 20 % of a spider’s body weight. Any injury includes not only immediate hemolymph loss but also pathogen attacks and subsequent infections. Therefore spiders have to react to injuries in a combined manner to stop fluid loss and to defend against microbial invaders. This is achieved by an innate immune system which involves several host defence systems such as hemolymph coagulation and the production of a variety of defensive substances (Fukuzawa et al.2008). In spiders, the immune system is localised in hemocytes which are derived from the myocardium cells of the heart wall where they are produced as prohemocytes and from where they are released as different cell types into the hemolymph (Seitz 1972). They contribute to the defence against pathogens by phagocytosis, nodulation and encapsulation of invaders. The humoral response includes mechanisms which induce melanin production to destroy pathogens, a clotting cascade to stop hemolymph loss and the constitutive production of several types of antimicrobial peptides, which are stored in hemocyte granules and released into the hemolymph (Fukuzawa et al.2008) (Fig.7.1). The immune system of spiders is an innate immune system. It is hemolymph-based and characterised by a broad but not very particular specificity. Its advantage is a fast response within minutes to a few hours. This is in contrast to the adaptive immune system of vertebrates which can react to very specific pathogens, thus resulting in much more specific responses. Moreover, it creates an immunological memory during the lifetime of the species. The disadvantage is that it needs more time to react with antibody production, usually many hours to a few days, and needs to be built up during early ontogenesis.

Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus.

Rapid-eye movement (REM) sleep correlates with neuronal activity in the brainstem, basal forebrain and lateral hypothalamus. Lateral hypothalamus melanin-concentrating hormone (MCH)-expressing neurons are active during sleep, but their effects on REM sleep remain unclear. Using optogenetic tools in newly generated Tg(Pmch-cre) mice, we found that acute activation of MCH neurons (ChETA, SSFO) at the onset of REM sleep extended the duration of REM, but not non-REM, sleep episodes. In contrast, their acute silencing (eNpHR3.0, archaerhodopsin) reduced the frequency and amplitude of hippocampal theta rhythm without affecting REM sleep duration. In vitro activation of MCH neuron terminals induced GABAA-mediated inhibitory postsynaptic currents in wake-promoting histaminergic neurons of the tuberomammillary nucleus (TMN), and in vivo activation of MCH neuron terminals in TMN or medial septum also prolonged REM sleep episodes. Collectively, these results suggest that activation of MCH neurons maintains REM sleep, possibly through inhibition of arousal circuits in the mammalian brain.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.