Protein Phosphatase 1 (PP1) Is a Post-Translational Regulator of the Mammalian Circadian Clock

Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock

Induction of general anaesthesia by blowpipe darting in a fractious companion horse

a fractious nine-year-old, 520-kg, neutered Swiss Warmblood was presented with a history of anorexia, progressive weight loss and mild hindlimb lameness. Because of its temperament, standard physical examination was considered to be only feasible under general anaesthesia. For safety reasons, general anaesthesia was planned to be induced by blowpipe darting. two attempts are described and discussed in the present report. the first attempt, using a combination of medetomidine and tiletamine-zolazepam, was unsuccessful. Conversely, detomidine combined with butorphanol, followed by a second dart of detomidine and tiletamine-zolazepam, proved to be adequate to induce anaesthesia. Factors that could have influenced the outcome, such as different therapeutic approach, drug protocol and dosages, stress level, or genetic mutations, are presented and discussed

VGLUT1 binding to endophilin or intersectin1 and dynamin phosphorylation in a diurnal context

Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins.VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A–E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light–dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1

Preparation and deep characterization of composite/hybrid multi-scale and multi-domain polymeric microparticles

Polymeric microparticles were produced following a three-step procedure involving (i) the production of an aqueous nanoemulsion of tri and monofunctional acrylate-based monomers droplets by an elongational-flow microemulsifier, (ii) the production of a nanosuspension upon the continuous-flow UV-initiated miniemulsion polymerization of the above nanoemulsion and (iii) the production of core-shell polymeric microparticles by means of a microfluidic capillaries-based double droplets generator; the core phase was composed of the above nanosuspension admixed with a water-soluble monomer and gold salt, the shell phase comprised a trifunctional monomer, diethylene glycol and a silver salt; both phases were photopolymerized on-the-fly upon droplet formation. Resulting microparticles were extensively analyzed by energy dispersive X-rays spectrometry and scanning electron microscopy to reveal the core-shell morphology, the presence of silver nanoparticles in the shell, organic nanoparticles in the core but failed to reveal the presence of the gold nanoparticles in the core presumably due to their too small size (c.a. 2.5 nm). Nevertheless, the reddish appearance of the as such prepared polymer microparticles emphasized that this three-step procedure allowed the easy elaboration of composite/hybrid multi-scale and multi-domain polymeric microparticles

The nuclear receptor REV-ERBα regulates Fabp7 and modulates adult hippocampal neurogenesis

The function of the nuclear receptor Rev-erbα (Nr1d1) in the brain is, apart from its role in the circadian clock mechanism, unknown. Therefore, we compared gene expression profiles in the brain between wild-type and Rev-erbα knock-out (KO) animals. We identified fatty acid binding protein 7 (Fabp7, Blbp) as a direct target of repression by REV-ERBα. Loss of Rev-erbα manifested in memory and mood related behavioral phenotypes and led to overexpression of Fabp7 in various brain areas including the subgranular zone (SGZ) of the hippocampus, where neuronal progenitor cells (NPCs) can initiate adult neurogenesis. We found increased proliferation of hippocampal neurons and loss of its diurnal pattern in Rev-erbα KO mice. In vitro, proliferation and migration of glioblastoma cells were affected by manipulating either Fabp7 expression or REV-ERBα activity. These results suggest an important role of Rev-erbα and Fabp7 in adult neurogenesis, which may open new avenues for treatment of gliomas as well as neurological diseases such as depression and Alzheimer

TSEN54 missense variant in Standard Schnauzers with leukodystrophy

We report a hereditary leukodystrophy in Standard Schnauzer puppies. Clinical signs occurred shortly after birth or started at an age of under 4 weeks and included apathy, dysphoric vocalization, hypermetric ataxia, intension tremor, head tilt, circling, proprioceptive deficits, seizures and ventral strabismus consistent with a diffuse intracranial lesion. Magnetic resonance imaging revealed a diffuse white matter disease without mass effect. Macroscopically, the cerebral white matter showed a gelatinous texture in the centrum semiovale. A mild hydrocephalus internus was noted. Histopathologically, a severe multifocal reduction of myelin formation and moderate diffuse edema without inflammation was detected leading to the diagnosis of leukodystrophy. Combined linkage analysis and homozygosity mapping in two related families delineated critical intervals of approximately 29 Mb. The comparison of whole genome sequence data of one affected Standard Schnauzer to 221 control genomes revealed a single private homozygous protein changing variant in the critical intervals, TSEN54:c.371G>A or p.(Gly124Asp). TSEN54 encodes the tRNA splicing endonuclease subunit 54. In humans, several variants in TSEN54 were reported to cause different types of pontocerebellar hypoplasia. The genotypes at the c.371G>A variant were perfectly associated with the leukodystrophy phenotype in 12 affected Standard Schnauzers and almost 1000 control dogs from different breeds. These results suggest that TSEN54:c.371G>A causes the leukodystrophy. The identification of a candidate causative variant enables genetic testing so that the unintentional breeding of affected Standard Schnauzers can be avoided in the future. Our findings extend the known genotype-phenotype correlation for TSEN54 variants.Peer reviewe

HOX11L2/TLX3 is transcriptionally activated through T-cell regulatory elements downstream of BCL11B as a result of the t(5;14)(q35;q32).

International audienceThe t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation

Hematopoietic Stem Cell Transplant for the Treatment of X-MAID

We report outcomes after hematopoietic stem cell transplant for three patients with X-MAID, including 1 patient from the originally described cohort and two brothers with positive TREC newborn screening for SCID who were found to have a T-B-NK+ SCID phenotype attributable to X-linked moesin associated immunodeficiency (X-MAID). A c.511C>T variant in moesin was identified via exome sequencing in the older of these siblings in the setting of low lymphocyte counts and poor proliferative responses consistent with SCID. He received reduced intensity conditioning due to CMV, and was transplanted with a T-depleted haploidentical (maternal) donor. His post-transplant course was complicated by hemolytic anemia, neutropenia, and sepsis. He had poor engraftment, requiring a 2nd transplant. His younger brother presented with the same clinical phenotype and was treated with umbilical cord blood transplant following myeloablative conditioning, has engrafted and is doing well. The third case also presented with severe lymphopenia in infancy, received a matched related bone marrow transplant following myeloablative conditioning, has engrafted and is doing well. These cases represent a novel manifestation of non-radiosensitive X-linked form of T-B-NK+ SCID that is able to be detected by TREC based newborn screening and effectively treated with HCT