The effect of ADAMTS13 on graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) can potentially cure malignant blood disorders and benign conditions such as haemoglobinopathies and immunologic diseases. However, allo-HSCT is associated with significant complications. The most common and debilitating among them is graft-versus-host disease (GVHD). In GVHD, donor-derived T cells mount an alloimmune response against the recipient. The alloimmune response involves several steps, including recognition of recipient antigens, activation and proliferation of T cells in secondary lymphoid organs, and homing into GVHD-targeted organs. Adhesion molecules on T cells and endothelial cells mediate homing of T cells into lymphoid and non-lymphoid tissues. In this study, we showed that Von Willebrand factor (VWF), an adhesion molecule secreted by activated endothelial cells, plays an important role in mouse models of GVHD. We investigated the effect of the VWF-cleaving protease ADAMTS13 on GVHD. We found that ADAMTS13 reduced the severity of GVHD after bone marrow transplantation from C57BL6 donor to BALB/C recipient mice. A recombinant VWF-A2 domain peptide also reduced GVHD in mice. We showed that ADAMTS13 and recombinant VWF-A2 reduced the binding of T cells to endothelial cells and VWF in vitro, and reduced the number of T cells in lymph nodes, Peyer's patches and GVHD-targeted organs in vivo. We identified LFA-1 (αLß2) as the binding site of VWF on T cells. Our results showed that blocking T-cell homing by ADAMTS13 or VWF-A2 peptide reduced the severity of the GVHD after allo-HSCT, a potentially novel method for treating and preventing GVHD.

Ovarian hyperstimulation syndrome following GnRH agonist trigger for final follicular maturation in a patient undergoing random start ovarian stimulation for egg-donation cycle with an inadvertent concomitant early pregnancy.

We report the first case of OHSS following GnRH agonist trigger for final follicular maturation in random start ovarian stimulation for egg-donation cycles during inadvertent concomitant early pregnancy. As an additional note, the sustained activity exerted by the increasing endogenous hCG production seemed to be responsible for the suboptimal performance in terms of oocyte yield in the current case. OHSS can occur in random-start stimulations protocols even after the use of a GnRH agonist for triggering in case of concomitant unnoticed early pregnancy especially if stimulation is commenced in the periovulatory/luteal phase. The present case report introduces a note of extreme caution when proceeding with this protocol in an otherwise fertile population (egg-donors, elective or oncologic oocyte cryopreservation).

Drosophila Atlastin in motor neurons is required for locomotion and presynaptic function.

Hereditary spastic paraplegias (HSPs) are characterized by spasticity and weakness of the lower limbs, resulting from length-dependent axonopathy of the corticospinal tracts. In humans, the HSP-related atlastin genes ATL1-ATL3 catalyze homotypic membrane fusion of endoplasmic reticulum (ER) tubules. How defects in neuronal Atlastin contribute to axonal degeneration has not been explained satisfactorily. Using Drosophila, we demonstrate that downregulation or overexpression of Atlastin in motor neurons results in decreased crawling speed and contraction frequency in larvae, while adult flies show progressive decline in climbing ability. Broad expression in the nervous system is required to rescue the atlastin-null Drosophila mutant (atl2 ) phenotype. Importantly, both spontaneous release and the reserve pool of synaptic vesicles are affected. Additionally, axonal secretory organelles are abnormally distributed, whereas presynaptic proteins diminish at terminals and accumulate in distal axons, possibly in lysosomes. Our findings suggest that trafficking defects produced by Atlastin dysfunction in motor neurons result in redistribution of presynaptic components and aberrant mobilization of synaptic vesicles, stressing the importance of ER-shaping proteins and the susceptibility of motor neurons to their mutations or depletion.

Surgical Management of Lower Extremity Lymphedema: A Comprehensive Review.

Lymphedema refers to the accumulation of protein-rich fluid in the interstitial spaces. This can occur secondary to congenital malformation of the lymphatic channels or nodes or as a result of an insult that damages appropriately formed channels and nodes. Stagnant, protein-rich lymph initiates an inflammatory response that leads to adipocyte proliferation, fibrous tissue deposition, and increased susceptibility to infections. The end result is permanent disfigurement and dermal changes. Early and accurate diagnosis is essential, since lymphedema is a chronic and progressive problem. When lymphedema affects the lower extremity, it is important to manage it in a way that preserves function and mobility. Early diagnosis also allows for a proactive rather than reactive approach to treatment and utilization of novel physiologic procedures, such as lymphovenous anastomosis and vascularized lymph node transfer. Such interventions slow down disease progression and reduce morbidity by allowing the surgeon to salvage the remaining functional lymphatic channels. When physiologic procedures fail or when faced with a delayed presentation, the addition of excisional procedures can provide a more comprehensive treatment of this debilitating disease. The aim of this article is to review the most current concepts in the surgical management of lower extremity lymphedema.

Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose.

The mechanisms that power the physiological events occurring in cilia, flagella, and microvilli are of fundamental importance for the functions of these important and ubicuous organelles. The olfactory epithelium is mostly populated by ciliated olfactory sensory neurons (OSNs) and surrounding sustentacular cells (SCs) with apical microvilli. The only OSN dendrite extends to the surface forming a knob projecting several chemosensory cilia of ∼50 × 0.2 µm, devoid of inner membranes embedded in a mucus layer. Upon odorant binding, odor receptors couple to G-protein activating adenylyl cyclase, producing cAMP. cAMP opens cyclic nucleotide-gated channels allowing a Ca2+ influx that opens Ca2+-activated Cl- channels, generating the receptor potential. Many enzymes are activated in chemotransduction to hydrolyze ATP. The knob contains approximately two mitochondria; assuming that the cilia ATP is 1 mm and diffuses along it at ∼10 µm in 500 ms, ATP from the knob mitochondria may not fulfill the demands of transduction over the full length of the cilium, which suggests an additional ATP source. We measured millimolar glucose in rat mucus; we detected glucose transporter GLUT3 in rat and toad (Caudiverbera caudiverbera) OSN cilia, SC microvilli, and glycolytic enzymes in rat cilia. We also found that the cilia and knob can incorporate and accumulate 2-deoxyglucose (glucose analog), but not when blocking GLUT. Glucose removal and the inhibition of glycolysis or oxidative phospholylation impaired the odor response. This evidence strongly suggests that glycolysis in the cilia and knob oxidative phosphorylation together fuel chemotransduction.SIGNIFICANCE STATEMENT How processes occurring in cilia and flagella are powered is a matter of general interest. Substantial progress has been made in unraveling the sensory transduction mechanisms, commonly occurring in such structures; however, the energy sources powering them have been scarcely explored. Accessibility to the specialized sensory organelles and their small dimensions have been limiting factors. Olfactory sensory neurons chemosensory cilia are elongated, mucus embedded, fully exposed structures particularly amenable for a multidisciplinary study of this problem, as done here. We demonstrate the occurrence and functionality of glucose uptake and glycolysis in the cilia. We support that odor transduction relies on ATP generated by oxidative phosphorylation in the dendrite and glycolytically in the cilia using glucose internalized from the mucus.

A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl- current when transfected into HEK293.

BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.

Long-term monitoring for conservation management: Lessons from a case study integrating remote sensing and field approaches in floodplain forests.

Implementing long-term monitoring programs that effectively inform conservation plans is a top priority in environmental management. In floodplain forests, historical pressures interplay with the complex multiscale dynamics of fluvial systems and require integrative approaches to pinpoint drivers for their deterioration and ecosystem services loss. Combining a conceptual framework such as the Driver-Pressure-State-Impact-Response (DPSIR) with the development of valid biological indicators can contribute to the analysis of the driving forces and their effects on the ecosystem in order to formulate coordinated conservation measures. In the present study, we evaluate the initial results of a decade (2004-2014) of floodplain forest monitoring. We adopted the DPSIR framework to summarize the main drivers in land use and environmental change, analyzed the effects on biological indicators of foundation trees and compared the consistency of the main drivers and their effects at two spatial scales. The monitoring program was conducted in one of the largest and best preserved floodplain forests in SW Europe located within Doñana National Park (Spain) which is dominated by Salix atrocinerea and Fraxinus angustifolia. The program combined field (in situ) surveys on a network of permanent plots with several remote sensing sources. The accuracy obtained in spectral classifications allowed shifts in species cover across the whole forest to be detected and assessed. However, remote sensing did not reflect the ecological status of forest populations. The field survey revealed a general decline in Salix populations, especially in the first five years of sampling -a factor probably associated with a lag effect from past human impact on the hydrology of the catchment and recent extreme climatic episodes (drought). In spite of much reduced seed regeneration, a resprouting strategy allows long-lived Salix individuals to persist in complex spatial dynamics. This suggests the beginning of a recovery resulting from recent coordinated societal responses to control excessive water extraction in the catchment, highlighting the need for continuing long-term monitoring. The DPSIR framework proved useful as a conceptual tool in analyzing the entire environmental system, while both field and remote sensing approaches complemented each other in quantifying indicator trends, improving the monitoring design and informing conservation plans.

Single Ca(2+)-activated Cl(-) channel currents recorded from toad olfactory cilia.

BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

Climatic events inducing die-off in Mediterranean shrublands: are species' responses related to their functional traits?

Extreme climatic episodes, likely associated with climate change, often result in profound alterations of ecosystems and, particularly, in drastic events of vegetation die-off. Species attributes are expected to explain different biological responses to these environmental alterations. Here we explored how changes in plant cover and recruitment in response to an extreme climatic episode of drought and low temperatures were related to a set of functional traits (of leaves, roots and seeds) in Mediterranean shrubland species of south-west Spain. Remaining aerial green cover 2 years after the climatic event was positively related to specific leaf area (SLA), and negatively to leaf water potential, stable carbon isotope ratio and leaf proline content. However, plant cover resilience, i.e. the ability to attain pre-event values, was positively related to a syndrome of traits distinguished by a higher efficiency of water use and uptake. Thus, higher SLA and lower water-use efficiency characterized species that were able to maintain green biomass for a longer period of time but were less resilient in the medium term. There was a negative relationship between such syndromes and the number of emerging seedlings. Species with small seeds produced more seedlings per adult. Overall, recruitment was positively correlated with species die-off. This study demonstrates the relationship between plant traits and strong environmental pulses related to climate change, providing a functional interpretation of the recently reported episodes of climate-induced vegetation die-off. Our findings reveal the importance of selecting meaningful traits to interpret post-event resilience processes, particularly when combined with demographic attributes.

Transduction for pheromones in the main olfactory epithelium is mediated by the Ca2+ -activated channel TRPM5.

Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl- channels; in TRPM5-GFP+ OSNs, the Ca2+ -activated Cl- ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.

Drosophila light-dependent channels, TRP and TRPL, reside in the light-sensitive microvilli of the photoreceptor's rhabdomere. Phospholipase C mediates TRP/TRPL opening, but the gating process remains unknown. Controversial evidence has suggested diacylglycerol (DAG), polyunsaturated fatty acids (PUFAs, a DAG metabolite), phosphatidylinositol bisphosphate (PIP2), and H(+) as possible channel activators. We tested each of them directly in inside-out TRP-expressing patches excised from the rhabdomere, making use of mutants and pharmacology. When patches were excised in darkness TRP remained closed, while when excised under illumination it stayed constitutively active. TRP was opened by DAG and silenced by ATP, suggesting DAG-kinase (DGK) involvement. The ATP effect was abolished by inhibiting DGK and in the rdgA mutant, lacking functional DGK, implicating DGK. DAG activated TRP even in the presence of a DAG-lipase inhibitor, inconsistent with a requirement of PUFAs in opening TRP. PIP2 had no effect and acidification, pH 6.4, activated TRP irreversibly, unlike the endogenous activator. Complementary liquid-chromatography/mass-spectrometry determinations of DAG and PUFAs in membranes enriched in rhabdomere obtained from light- and dark-adapted eyes showed light-dependent increment in six DAG species and no changes in PUFAs. The results strongly support DAG as the endogenous TRP agonist, as some of its vertebrate TRPC homologs of the same channel family.

Copper enhances cellular and network excitabilities, and improves temporal processing in the rat hippocampus.

Copper, an ion with many important metabolic functions, has also been proposed to have a role as modulator on neuronal function, mostly based on its effects on voltage- and neurotransmitter-gated conductance as well as on neurological symptoms of patients with altered copper homeostasis. Nevertheless, the mechanisms by which copper exerts its neuromodulatory effects have not been clearly established in a functional neuronal network. Using rat hippocampus slices as a neuronal network model, the effects of copper in the range of 10-100 nm were tested on the intrinsic, synaptic and network properties of the CA1 region. Most of the previously described effects of this cation were in the micromolar range of copper concentrations. The current results indicate that copper is a multifaceted neuromodulator, having effects that may be grouped into two categories: (i) activity enhancement, by modulating synaptic communication and action potential (AP) conductances; and (ii) temporal processing and correlation extraction, by improving reliability and depressing inhibition. Specifically it was found that copper hyperpolarizes AP firing threshold, enhances neuronal and network excitability, modifies CA3-CA1 pathway gain, enhances the frequency of spontaneous synaptic events, decreases inhibitory network activity, and improves AP timing reliability. Moreover, copper chelation by bathocuproine decreases spontaneous network spiking activity. These results allow the proposal that copper affects the network activity from cellular to circuit levels on a moment-by-moment basis, and should be considered a crucial functional component of hippocampal neuronal circuitry.

A new semisynthetic derivative of sauroine induces LTP in hippocampal slices and improves learning performance in the Morris Water Maze.

Two semisynthetic acetyl derivatives of the alkaloid sauroine from Huperzia saururus, monoacetyl sauroine, and diacetyl sauroine (DAS) were obtained and their chemical structures were analyzed by NMR. While monoacetyl sauroine is the typical product of acetylation, DAS is an unexpected derivative related to the keto-enol formation of sauroine. Recordings of field excitatory post-synaptic potentials from the CA1 region of rat hippocampal slices showed that only DAS acutely applied induced chemical long-term potentiation (LTP) in a dose-dependent manner with an EC50 of 1.15 ± 0.09 µM. This effect was blocked by 10 µM D(-)-2-amino-5-phosphonopentanoic acid (AP5), suggesting dependence on the NMDA receptor. DAS significantly increased NMDA receptor-dependent excitatory post-synaptic currents without affecting α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-dependent currents. Repetitive administration of DAS improved visuo-spatial learning in the Morris Water Maze. In slices from rats tested in the Morris Water Maze, LTP resulting from electrical synaptic stimulation was 2.5 times larger than in controls. Concentration of DAS measured in the brain after repetitive administration was 29.5 µM. We conclude that slices perfused with DAS display a robust NMDA receptor-dependent chemical LTP. During chronic treatment, DAS enhances learning abilities through a metaplastic mechanism as revealed by the augmentation of LTP in slices. DAS, therefore, may be a promising compound as a nootropic therapeutic drug. A semisynthetic derivative of sauroine, diacetyl sauroine (DAS), induces chemical long-term potentiation in rat hippocampal slices increasing the NMDA receptor-dependent current. 2 mg/kg prior to each session in a Morris Water Maze (MWM) improves behavior performance. In slices prepared from the tested rats the electrical stimulation-dependent long-term potentiation (LTP) was greatly enhanced. Therefore, DAS may have potency as a nootropic drug against the memory decline.

Overexpression of hyperpolarization-activated cyclic nucleotide-gated channels into the ventral tegmental area increases the rewarding effects of ethanol in UChB drinking rats.

BACKGROUND: A number of studies have shown that ethanol (EtOH) activates dopamine neurocircuitries and is self-administered into the ventral tegmental area (VTA) of the rat brain. In vitro and in silico studies have showed that hyperpolarization-activated cyclic nucleotide-gated (HCN) ionic channels on VTA dopamine neurons may constitute a molecular target of EtOH; however, there is no in vivo evidence supporting this assumption. METHODS: Wistar-derived University of Chile Drinking (UChB) rats were microinjected into the VTA with a lentiviral vector coding for rat HCN-2 ionic channel or a control vector. Four days after vector administration, daily voluntary EtOH intake was assessed for 30 days under a free-access paradigm to 5% EtOH and water. After EtOH consumption studies, the effect of HCN-2 overexpression was also assessed on EtOH-induced conditioned place preference (CPP); EtOH-induced locomotion, and EtOH-induced dopamine release in the nucleus accumbens (NAcc). RESULTS: Rats microinjected with the HCN-2 coding vector into the VTA showed (i) a ~2-fold increase in their voluntary EtOH intake compared to control animals, (ii) lentiviral-HCN-2-treated animals also showed an increased CPP to EtOH (~3-fold), (iii) a significant higher locomotor activity (~2-fold), and (iv) increased dopamine release in NAcc upon systemic administration of EtOH (~2-fold). CONCLUSIONS: Overexpression of HCN-2 ionic channel in the VTA of rats results in an increase in voluntary EtOH intake, EtOH-induced CPP, locomotor activity, and dopamine release in NAcc, suggesting that HCN levels in the VTA are relevant for the rewarding properties of EtOH.

Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse.

Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, we investigate whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, we found that both pyruvate and L-lactate, as the only carbon sources in the synapses bathing-solution, cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 pre-synaptic loading ability, however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. SIGNIFICANCE STATEMENT: The question of whether monocarboxylic acids can fuel the Drosophila motoneuron NTR was challenged. Our findings show that exogenously supplied monocarboxylates trigger a large transient synaptic enhancement just under extreme glycolysis reduction but fail to maintain NTR under sustained synaptic demand, still at low frequency stimulation, driven to the synapses to a synaptic depression state. Glycolysis activation, by adding sucrose to the monocarboxylate bath solution, restores the motoneuron NTR ability, giving place to a hexoses role in SV recruitment. Moreover these results suggest exogenously supplied C3 carbon sources could have an additional role beyond providing energetic support for neural activity.

Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors.

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase. This complex enzyme cascade ultimately opens the light-dependent transient receptor potential channels, TRP and TRPL. PIP2, DAG, H+ and 2-LG are possible channel activators, either individually or combined, but their direct participation in channel-gating remains unresolved. Molecular interaction with the channels, modification of the channels' lipid moiety and mechanical force on the channels by changes in the membrane structure derived from light-dependent changes in lipid composition are possible gating agents. In this regard, mechanical activation was suggested, based on a rapid light-dependent contraction of the photoreceptors mediated by the phototransduction cascade. Here, we further examined this possibility by applying force to inside-out patches from the microvilli membrane by changing the pressure in the pipette or pulling the membrane with a magnet through superparamagnetic nanospheres. The channels were opened by mechanical force, while mutant lacking both channels was insensitive to mechanical stimulation. Atomic Force Microscopy showed that the stiffness of an artificial phospholipid bilayer was increased by arachidonic acid and diacylglycerol whereas elaidic acid was ineffective, mirroring their relative effects in channel activity previously observed electrophysiologically. Together, the results are consistent with the notion that light-induced changes in lipid composition alter the membrane structure, generating mechanical force on the channels leading to channel opening.

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Fatty acid composition of Drosophila photoreceptor light-sensitive microvilli.

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

[Colorectal carcinoma under the age of 50 years]. / Vastagbélrák-elofordulás 50 év alattiak körében.

INTRODUCTION: Colorectal cancer is the second most common cause of cancer related death in Hungary. AIM: The authors examined retrospectively the ratio and the clinical course of patients under the age of 50 years operated by themselves between January, 2006 and May, 2012. METHOD: During this period of time 667 patients underwent surgery for colorectal carcinoma and 44 (6.6%) were less than 50 years old. 22.2% of the operations were performed for emergency reasons. The surgical technique, TNM stage, differentiation state and localization of the tumours were analyzed as well as the histological types with unfavourable prognosis. In order to analyse risk factors a questionnaire was sent to patients of young age group. RESULTS: The majority of patients, including those under 50 years of age were diagnosed as having TNM stages III and IV. Only half of the patients returned the questionnaire. CONCLUSIONS: Due to a relatively low response rate, the importance of risk factors for colorectal carcinoma in patients under 50 years of age remains to be further investigated.

Reconstruction of cervico-thoracic defect with bipedicled deep inferior epigastric perforator free flap following resection of a giant recurrent thyroid tumor: a case report and review of literature.

The bipedicled Deep Inferior Epigastric Perforator (DIEP) flap, originally described and primarily utilized in autologous breast reconstruction for specific cases, has expanded its applications to encompass diverse anatomical regions in recent years. This report presents the case of a 69-year-old woman with a recurrent giant thyroid tumor who underwent surgical resection, resulting in a large cervico-thoracic defect effectively reconstructed using a bipedicled DIEP flap. The patient's postoperative recovery was uneventful, and the follow-up assessments revealed a healthy, well-perfused flap that provided sufficient coverage to critical structures, adequate restoration of the region contour, and enough volume to offset potential adverse effects of subsequent radiation therapy. In addition, this report incorporates a concise literature review highlighting the expanding indications of the bipedicled DIEP flap beyond breast reconstruction, showing the versatility and efficacy of the bipedicled DIEP flap in addressing complex soft-tissue defects in various anatomical areas.