CasTuner: a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression

Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need for dosage compensation for X-linked genes between the sexes in many species. Understanding dosage-sensitive processes requires the ability to perturb endogenous gene products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that allows analog tuning of endogenous gene expression. In the CasTuner system, activity of Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB repressor domain, which exhibits a digital mode of action. We quantify repression and derepression dynamics for CasTuner and use it to measure dose-response curves between the pluripotency factor NANOG and several of its target genes, providing evidence for target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to perturb gene expression in an inducible, tunable and reversible manner and will be useful to study dose-responsive processes within their physiological contex

Sedative effects of intramuscular alfaxalone in pet guinea pigs (Cavia porcellus)

Objective: To evaluate the efficacy and side effects of alfaxalone administered intramuscularly (IM) as a sedative agent in guinea pigs undergoing survey radiographs

Persistent nonbilious vomiting in a child: Possible duodenal webbing

An association between malrotation and congenital duodenal webbing is rare. We present our experience with four patients at two centers, and a review of published reports. There are currently 94 reported cases of duodenal pathology associated with malrotation. However, only 15 of the 94 cases (15.9%) include patients with malrotation and a duodenal web. We suggest that nonbilious vomiting in a child must prompt the surgeon to consider duodenal pathology even in the presence of malrotation

Histone deacetylase 4 is crucial for proper skeletal muscle development and disease

Epigenetics plays a pivotal role in modulating gene response to physiological or pathological stimuli. Histone Deacetylase inhibitors (HDACi) have been used in the treatment of various cancers1, are ef-fective in several animal models of neurodegenerative diseases, including amyotrophic lateral scle-rosis (ALS), and are currently in clinical trial to promote muscle repair in muscular dystrophies2. However, long-term use of pan-HDAC inhibitors is not tolerated3. The assignment of distinct biologi-cal functions to individual HDACs in skeletal muscle is a prerequisite to improve the efficacy of pharmacological treatments based on HDACi. HDAC4 is a member of class II HDACs that mediates many cellular responses. Clinical reports suggest that inhibition of HDAC4 can be beneficial to cancer cachexia, dystrophic or ALS patients. All the above conditions are characterized by progressive mus-cle wasting and up-regulation of HDAC4 expression in skeletal muscle, suggesting a potential role for this protein in regulating these diseases. To study the role of HDAC4 with a genetic approach, we generated several models of muscle disease in mice lacking HDAC4 in skeletal muscle: cancer ca-chexia, by implanting Lewis lung carcinoma (LLC), muscular dystrophy, by using mdx mice, or ALS, by using SODG93A mice. Lack of HDAC4 worsens skeletal muscle atrophy induced by both LLC and ALS, demonstrated by a reduction in muscle mass and myofibers size. Conversely, dystrophic mice lacking HDAC4 in skeletal muscle show an increased number of necrotic myofibers and run less efficiently than mdx mice. The aggravation of the dystrophic phenotype may be partially due to the impairment in skeletal muscle regeneration observed in mice lacking HDAC4 in skeletal muscle. Our results indi-cate that HDAC4 is necessary for maintaining skeletal muscle homeostasis and function. Current studies aim to investigate the molecular mechanisms underlying the role of HDAC4 in skeletal mus-cle maintenance in response to cancer cachexia, ALS or muscular dystrophy

HDAC4 is necessary for satellite cell differentiation and muscle regeneration

In response to injury, skeletal muscle exhibits high capacity to regenerate and epigenetics controls multiple steps of this process (Giordani et al., 2013). It has been demonstrated in vitro that completion of muscle differentiation requires shuttling of histone deacetylase 4 (HDAC4), a member of class IIa HDACs, from the nucleus to the cytoplasm and consequent activation of MEF2-dependent differentiation genes (McKinsey et al., 2000). In vivo, HDAC4 expression is up-regulated in skeletal muscle upon injury, suggesting a role for this protein in muscle regeneratio

Temporal features of size constancy for perception and action in a real-world setting: A combined EEG-kinematics study

A stable representation of object size, in spite of continuous variations in retinal input due to changes in viewing distance, is critical for perceiving and acting in a real 3D world. In fact, our perceptual and visuo-motor systems exhibit size and grip constancies in order to compensate for the natural shrinkage of the retinal image with increased distance. The neural basis of this size-distance scaling remains largely unknown, although multiple lines of evidence suggest that size-constancy operations might take place remarkably early, already at the level of the primary visual cortex. In this study, we examined for the first time the temporal dynamics of size constancy during perception and action by using a combined measurement of event-related potentials (ERPs) and kinematics. Participants were asked to maintain their gaze steadily on a fixation point and perform either a manual estimation or a grasping task towards disks of different sizes placed at different distances. Importantly, the physical size of the target was scaled with distance to yield a constant retinal angle. Meanwhile, we recorded EEG data from 64 scalp electrodes and hand movements with a motion capture system. We focused on the first positive-going visual evoked component peaking at approximately 90 ms after stimulus onset. We found earlier latencies and greater amplitudes in response to bigger than smaller disks of matched retinal size, regardless of the task. In line with the ERP results, manual estimates and peak grip apertures were larger for the bigger targets. We also found task-related differences at later stages of processing from a cluster of central electrodes, whereby the mean amplitude of the P2 component was greater for manual estimation than grasping. Taken together, these findings provide novel evidence that size constancy for real objects at real distances occurs at the earliest cortical stages and that early visual processing does not change as a function of task demands

Long-term safety and effectiveness of canakinumab therapy in patients with cryopyrin-associated periodic syndrome: results from the β-Confident Registry.

OBJECTIVE: To report the long-term safety and effectiveness of canakinumab, a fully human anti-interleukin 1β monoclonal antibody, in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disease (NOMID), in a real-world setting. METHODS: From December 2009 to December 2015, the β-Confident Registry prospectively enrolled patients with CAPS and non-CAPS conditions who received canakinumab per routine care and were prospectively followed for up to 6 years. The registry protocol did not mandate specific visits or procedures; however, all observed adverse events (AEs) and serious adverse events (SAEs) had to be recorded. Canakinumab effectiveness was evaluated by Physician's Global Assessment (PGA). RESULTS: Of 288 patients enrolled, 3 were excluded due to missing informed consent. Among the remaining 285 patients, 243 (85.3%) were patients with CAPS and 42 (14.7%) had atypical CAPS (6.3%) or other conditions (8.4%). The median age was 26.6 years. Based on PGA, 58 of 123 (47.2%) patients with CAPS had no disease activity at 48 months, and 65 of 123 (52.8%) experienced mild/moderate disease activity at 48 months. Among CAPS phenotypes, AE incidence rates per 100 patient-years were lowest for FCAS (73.1; 95% CI 60.3 to 87.8) compared with those with MWS (105.0; 95% CI 97.2 to 113.2) or NOMID (104.6; 95% CI 86.6 to 125.2). One hundred twenty-eight SAEs were reported in 68 patients with CAPS (incidence rate/100 patient-years, 14.0; 95% CI 11.6 to 16.6). One death (metastatic rectal adenocarcinoma in a patient with MWS) was reported. CONCLUSIONS: The response to canakinumab was sustained for up to 6 years. Canakinumab demonstrated a favourable safety profile over long-term treatment in patients with CAPS. TRIAL REGISTRATION NUMBER: NCT01213641

Planck pre-launch status: HFI beam expectations from the optical optimisation of the focal plane

Planck is a European Space Agency (ESA) satellite, launched in May 2009, which will map the cosmic microwave background anisotropies in intensity and polarisation with unprecedented detail and sensitivity. It will also provide full-sky maps of astrophysical foregrounds. An accurate knowledge of the telescope beam patterns is an essential element for a correct analysis of the acquired astrophysical data. We present a detailed description of the optical design of the High Frequency Instrument (HFI) together with some of the optical performances measured during the calibration campaigns. We report on the evolution of the knowledge of the pre-launch HFI beam patterns when coupled to ideal telescope elements, and on their significance for the HFI data analysis procedure

Three-dimensional evaluation of masseter muscle in different vertical facial patterns: a cross-sectional study in growing children.

The aim of the present study was to analyze the anatomical three-dimensional (3D) characteristics of masseter muscle in growing subjects with different vertical patterns by using an ultrasound (US) method. The sample comprised 60 prepuberal subjects (33 males, 27 females) with a mean age of 11.5 ± 1.6 years with late mixed or permanent dentition and Class I molar and skeletal relationship. For each subject, a lateral cephalogram was required, and according to the mandibular plane angle (Frankfort horizontal plane/mandibular plane angle [FMA]), the subjects were divided into three groups of different underlying vertical facial patterns: brachyfacial: FMA &lt; 22°, mesofacial: 22° ≤ FMA ≤ 28°, and dolichofacial: FMA &gt; 28°. For each subject, an US scan was carried out to analyze the width, the thickness, the cross-sectional area, and the volume of the masseter muscle. Mean differences in measurements between vertical facial subgroups were contrasted by means of analysis of variance (ANOVA) with Tukey’s post hoc tests ( p &lt; 0.05). Measurements of the whole masseter in dolichofacial patients were significantly smaller when compared with brachyfacial and mesofacial individuals during relaxation and contraction. The volume of the masseter decreased significantly by 10% going from the brachyfacial group to the mesofacial group and from the mesofacial group to the dolichofacial group with no difference between the left and the right sides. A significant negative correlation was found between the US measurements and the divergency (FMA°). Ultrasound is a technique indicated in children for evaluating muscles of mastication in vivo. Growing patients with a dolichofacial vertical pattern present with a reduced dimension of the masseter when compared with brachyfacial and mesofacial subjects. </jats:p