Kinetic Resolution of ß-Branched Aldehydes through Peptide-Catalyzed Conjugate Addition Reactions.

The catalytic kinetic resolution of racemic ß-branched aldehydes offers a straightforward stereoselective entry to aldehydes and addition products. Yet, control over stereoselectivity is difficult due to the conformational flexibility of ß-branched aldehydes. Here, we show that the peptide catalyst H-dPro-αMePro-Glu-NH2 resolves ß-branched aldehydes through reaction with nitroolefins and provides γ-nitroaldehydes with three consecutive stereogenic centers in high yields and stereoselectivities. Kinetic, NMR spectroscopic, and computational studies provided insights into the selectivity-determining step and origins of the kinetic resolution.

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms.

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis.

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 16.0 µM and 9.3 µM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks.

BACKGROUND: Ticks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick's central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied. RESULTS: We carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls). CONCLUSIONS: We established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel.

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

Molecular and Functional Characterization of GABA Receptor Subunits GRD and LCCH3 from Human Louse Pediculus Humanus Humanus.

Human louse Pediculus humanus is a cosmopolitan obligatory blood-feeding ectoparasite causing pediculosis and transmitting many bacterial pathogens. Control of infestation is difficult due to the developed resistance to insecticides that mainly target GABA (γ-aminobutyric acid) receptors. Previous work showed that Pediculus humanus humanus (Phh) GABA receptor subunit resistance to dieldrin (RDL) is the target of lotilaner, a synthetic molecule of the isoxazoline chemical class. To enhance our understanding of how insecticides act on GABA receptors, two other GABA receptor subunits were cloned and characterized: three variants of Phh-grd (glycine-like receptor of Drosophila) and one variant of Phh-lcch3 (ligand-gated chloride channel homolog 3). Relative mRNA expression levels of Phh-rdl, Phh-grd, and Phh-lcch3 revealed that they were expressed throughout the developmental stages (eggs, larvae, adults) and in the different parts of adult lice (head, thorax, and abdomen). When expressed individually in the Xenopus oocyte heterologous expression system, Phh-GRD1, Phh-GRD2, Phh-GRD3, and Phh-LCCH3 were unable to reconstitute functional channels, whereas the subunit combinations Phh-GRD1/Phh-LCCH3, Phh-GRD1/Phh-RDL, and Phh-LCCH3/Phh-RDL responded to GABA in a concentration-dependent manner. The three heteromeric receptors were similarly sensitive to the antagonistic effect of picrotoxin and fipronil, whereas Phh-GRD1/Phh-RDL and Phh-LCCH3/Phh-RDL were respectively about 2.5-fold and 5-fold more sensitive to ivermectin than Phh-GRD1/Phh-LCCH3. Moreover, the heteropentameric receptor constituted by Phh-GRD1/Phh-LCCH3 was found to be permeable and highly sensitive to the extracellular sodium concentration. These findings provided valuable additions to our knowledge of the complex nature of GABA receptors in human louse that could help in understanding the resistance pattern to commonly used pediculicides. SIGNIFICANCE STATEMENT: Human louse is an ectoparasite that causes pediculosis and transmits several bacterial pathogens. Emerging strains developed resistance to the commonly used insecticides, especially those targeting GABA receptors. To understand the molecular mechanisms underlying this resistance, two subunits of GABA receptors were cloned and described: Phh-grd and Phh-lcch3. The heteromeric receptor reconstituted with the two subunits was functional in Xenopus oocytes and sensitive to commercially available insecticides. Moreover, both subunits were transcribed throughout the parasite lifecycle.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

A mathematical finance approach to the stochastic and intermittent viscosity fluctuations in living cells.

Here we report on the viscosity of eukaryotic living cells, as a function of time, and on the application of stochastic models to analyze its temporal fluctuations. The viscoelastic properties of NIH/3T3 fibroblast cells are investigated using an active microrheological technique, where the magnetic wires, embedded into cells, are being actuated remotely. The data reveal anomalous transient responses characterized by intermittent phases of slow and fast rotation, revealing significant fluctuations. The time dependent viscosity is analyzed from a time series perspective by computing the autocorrelation functions and the variograms, two functions used to describe stochastic processes in mathematical finance. The resulting analysis gives evidence of a sub-diffusive mean-reverting process characterized by an autoregressive coefficient lower than 1. It also shows the existence of specific cellular times in the ranges 1-10 s and 100-200 s, not previously disclosed. The shorter time is found to be related to the internal relaxation time of the cytoplasm. To our knowledge, this is the first time that similarities are established between the properties of time series describing the intracellular metabolism and the statistical results from a mathematical finance approach. The current approach could be exploited to reveal hidden features from biological complex systems or to determine new biomarkers of cellular metabolism.

Apnea induction for invasive lung function testing in infant olive baboons: Comparison of intravenous propofol versus hyperventilation.

BACKGROUND: In various types of pulmonary research, pulmonary function testing (PFT) is performed to quantify the severity of lung disease. Induction of apnea and positive pressure ventilation are required for accurate PFT measurements in non-cooperative subjects. We compared two methods of apnea induction in infant olive baboons (Papio anubis). METHODS: Pulmonary function testing results were compared during apnea induced by hyperventilation (CO2 washout) vs. intravenous propofol (1 dose 10 mg/kg). PFT was evaluated using a hot-wire pneumotachometer incorporated within an Avea ventilator in nine 1-month-old baboons. RESULTS: Propofol induced apnea faster and more reliably. In both groups, PFT values passed the statistical equivalence test and were not significantly different (Student's t-test). There was a trend toward less data variability after propofol administration. CONCLUSIONS: Intravenous propofol was non-inferior to CO2 washout for apnea induction in infant olive baboons. Propofol induced apnea faster and more reliably and yielded less variable PFT results.

High-Prevalence and Low-Intensity Ichthyophonus Infections in Pacific Halibut.

Ichthyophonus occurred at high prevalence but low intensity in Pacific Halibut Hippoglossus stenolepis throughout the West Coast of North America, ranging from coastal Oregon to the Bering Sea. Infection prevalence in adults was variable on spatial and temporal scales, with the lowest prevalence typically occurring on the edges of the geographic range and highest prevalence consistently occurring inside Prince William Sound, Alaska (58-77%). Additionally, intra-annual differences occurred at Albatross-Portlock, Alaska (71% versus 32% within 2012), and interannual differences occurred along coastal Oregon (50% in 2012 versus 12% in 2015). The infection prevalence was influenced by host age, increasing from 3% or less among the youngest cohorts (age ≤ 6) to 39-54% among age-9-17 cohorts, then decreasing to 27% among the oldest (age-18+) cohorts. There was little indication of significant disease impacts to Pacific Halibut, as the intensity of infection was uniformly low and length at age was similar between infected and uninfected cohorts. These results suggest that Ichthyophonus in Pacific Halibut currently represents a stable parasite-host paradigm in the North Pacific.

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

Discovery of imidazo[1,2-a]pyridine-based anthelmintic targeting cholinergic receptors of Haemonchus contortus.

We report the synthesis of a series of imidazo[1,2-a]pyridine-based molecules as anthelmintic against the livestock parasite Haemonchus contortus. The molecules were tested by using Larval Paralysis Test (LPT), in order to target ionic channels, as most of the prominent marketed anthelminthics present such mechanism of action. The most active compound (5e) displayed paralysis on H. contortus stage 3 larvae until 31.25 µM. Effect of 5e on H. contortus cholinergic receptors (L-AChR1 and 2) was characterized via electrophysiological measurement and a rare antagonist mode of action was unveiled.

Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

Building a Culture of Safety in Ophthalmology.

Patient safety focused on a reduction in both procedural and diagnostic error is the number one concern of the United States healthcare system in the 21st century. The American Board of Ophthalmology has a longstanding interest in patient safety, and in 2015, teamed with the American Academy of Ophthalmology to convene all ophthalmology subspecialties and other prominent national organizations to address patient safety in ophthalmology. This article reviews the topic and highlights concerns for ophthalmologists.

Pre-radiation chemotherapy improves survival in pediatric diffuse intrinsic pontine gliomas.

BACKGROUND: The median survival of patients with diffuse intrinsic pontine glioma (DIPG) remains less than 1 year. The BSG 98 pre-irradiation chemotherapy protocol showed a significant increase in overall survival. In contrast to current treatment strategies, patients did not have to undergo surgical stereotactic biopsy, which can sometimes lead to complications, to be included in this protocol. MATERIALS AND METHODS: We retrospectively reviewed all the cases of DIPG that were treated in our department from September 15, 2004 to September 15, 2014. We compared the group of patients who followed our BSG 98 protocol to those who were treated with new targeted therapy protocols where systematic biopsy was required. RESULTS: Patients in the BSG 98 protocol were treated with BCNU, cisplatin, and methotrexate, followed by radiation at disease progression. Targeted therapy protocols included radiation therapy along with treatment by erlotinib, cilengitide, or an association of nimotuzumab and vinblastine. Sixteen patients were treated with the BSG 98 protocol, and 9 patients were treated with new targeted therapy protocols. Median overall survival was significantly higher in the BSG 98 group compared to the targeted therapy group (16.1 months (95 % CI, 10.4-19.0) vs 8.8 months (95 % CI 1.4-12.3); p = 0.0003). An increase in the median progression-free survival was observed (respectively, 8.6 vs 3.0 months; p = 0.113). CONCLUSION: The present study confirms that the BSG 98 protocol is one of the most effective current treatment strategies for DIPG. It may be used as the control arm in randomized trials investigating the use of innovative treatments and may be proposed to families who are averse to biopsy.

Combined therapy in children and adolescents with classical Hodgkin's lymphoma: A report from the SFCE on MDH-03 national guidelines.

Hodgkin's lymphoma (HL) in children and adolescents is highly curable, but children are at risk of long-term toxicity. The MDH-03 guidelines were established in order to decrease the burden of treatment in good-responder patients, and this report should be considered a step toward further optimization of treatment within large collaborative trials. We report the therapy and long-term outcomes of 417 children and adolescents treated according to the national guidelines, which were applied between 2003 and 2007 in France. The patients were stratified into three groups according to disease extension. Chemotherapy consisted of four cycles of VBVP (vinblastine, bleomycin, VP16, prednisone) in localized stages (G1/95 pts/23%), four cycles of COPP/ABV (cyclophosphamide, vincristine, procarbazine, prednisone, adriamycin, bleomycin, vinblastine) cycles in intermediate stages (G2/184 pts/44%) and three cycles of OPPA (vincristine, procarbazine, prednisone, adriamycin) plus three cycles of COPP in advanced stages (G3/138 pts/33%). Radiation therapy of the involved field was given to 97% of the patients, with the dose limited to 20 Gy in good responders (88%). With a median follow-up of 6.6 years, the 5-year event-free survival (EFS) and overall survival (OS) were 86.7% (83.1-89.7%) and 97% (94.5-98.1%), respectively. EFS and OS for G1, G2, and G3 were 98% and 100%, 81% and 97%, and 87% and 95%, respectively. Low-risk patients treated without alkylating agents and anthracycline had excellent outcomes and a low expected incidence of late effects. Intensification with a third OPPA cycle in high-risk group patients, including stage IV patients, allowed for very good outcomes, without increased toxicity.

Cybervictimization of Young People With an Intellectual or Developmental Disability: Risks Specific to Sexual Solicitation.

BACKGROUND: Studies demonstrate that youth are vulnerable to online sexual solicitation. However, no study has estimated this risk for youth diagnosed with an intellectual or developmental disability (IDD). METHODS: A literature review of the risk factors associated with online sexual solicitation in youths was done using electronic databases, such as PsychInFO, ERIC, MEDLINE and Scopus. RESULTS: Fifty-seven published papers were found relevant. However, only two pertained to the population with IDD. Sexual and physical abuse, social isolation, loneliness, depression, and chatting were found to increase the risk of being prey to sexual solicitation on the Internet. Many of these risk factors are even more prevalent in youth with IDD than in the general population. CONCLUSION: Recommendations are made for future research to help understand and prevent sexual cybersolicitation.

Controlling the spin of co atoms on pt(111) by hydrogen adsorption.

We investigate the effect of H adsorption on the magnetic properties of individual Co atoms on Pt(111) with scanning tunneling microscopy. For pristine Co atoms, we detect no inelastic features in the tunnel spectra. Conversely, CoH and CoH2 show a number of low-energy vibrational features in their differential conductance identified by isotope substitution. Only the fcc-adsorbed species present conductance steps of magnetic origin, with a field splitting identifying their effective spin as Seff=2 for CoH and 3/2 for CoH2. The exposure to H2 and desorption through tunnel electrons allow the reversible control of the spin in half-integer steps. Because of the presence of the surface, the hydrogen-induced spin increase is opposite to the spin sequence of CoHn molecules in the gas phase.

Effect of the alcohol consumption on osteocyte cell processes: a molecular imaging study.

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups: Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.