Utility of Clinical High-Depth Next Generation Sequencing for Somatic Variant Detection in the PIK3CA Related Overgrowth Spectrum

Abstract

Next-generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor associated variants. It is increasingly recognized that post-zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging due to low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly-sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA-Related Overgrowth Spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400x unique read depth for VAF of 10%, and approaching 100% at 1000x. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.

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Effectiveness of structured educational program on knowledge of middle-aged women regarding prevention of osteoporosis

2016-06-15T22-07-27Z
Source: International Journal of Medical Science and Public Health
Shipra Sachan, Kamli Prakash, Upma George.
Background: Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and consequent increase in fracture risk. Objective: To determine the effectiveness of structured educational program on the knowledge of middle-aged women and to determine the association between demographic variables and knowledge score. Materials and Methods: A preexperimental design with one group pretest and posttest approach was selected to carry out the study. A total of 108 middle-aged women were selected by using nonprobability, purposive sampling technique. Structured questionnaire was used for data collection. Data were collected by interview method. The posttest was conducted after 7 days of intervention. Result: The pretest mean knowledge score was 10.44 ± 2.26, which was increased to 19.66 ± 2.28 in posttest. The knowledge of middle-aged women was statistically significantly and it was not associated with their age, marital status, number of children, education, occupation, socioeconomic status, previous knowledge about osteoporosis, and source of information. Conclusion: The structured educational program was effective in increasing the knowledge of middle-aged women regarding prevention of osteoporosis.


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Morphine-induced synaptic plasticity in the VTA is reversed by HDAC inhibition

Dopamine (DA) dysfunction originating from the ventral tegmental area (VTA) occurs as a result of synaptic abnormalities following consumption of drugs of abuse and underlies behavioral plasticity associated with drug abuse. Drugs of abuse can cause changes in gene expression through epigenetic mechanisms in the brain that underlie some of the lasting neuroplasticity and behavior associated with addiction. Here we investigated the function of histone acetylation and histone deacetylase (HDAC2) in the VTA in recovery of morphine-induced synaptic modifications following a single in vivo exposure to morphine. Using a combination of immunohistochemistry, Western blot and whole-cell patch clamp recording in rat midbrain slices, we show that morphine increased HDAC2 activity in VTA DA neurons and reduced histone H3 acetylation at lysine 9 (Ac-H3K9) in the VTA 24 hours following the injection. Morphine-induced synaptic changes at glutamatergic synapses involved endocannabinoid (eCB) signaling to reduce GABAergic synaptic strength onto VTA DA neurons. Both plasticities were recovered by in vitro incubation of midbrain slices with a class I specific HDAC inhibitor (HDACi), CI-994, through an increase in acetylation of histone H3K9. Interestingly, HDACi incubation also increased levels of Ac-H3K9, and triggered GABAergic and glutamatergic plasticities in DA neurons of saline-treated rats. Our results suggest that acute morphine-induced changes in VTA DA activity and synaptic transmission engage HDAC2 activity locally in the VTA to maintain synaptic modifications through histone hypoacetylation.

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Looking for symmetry: fixational eye movements are biased by image mirror symmetry

Humans are highly sensitive to symmetry. During scene exploration, the area of the retina with dense light receptor coverage acquires most information from relevant locations determined by gaze fixation. We characterised patterns of fixational eye movements made by observers staring at synthetic scenes either freely (i.e. free exploration) or during a symmetry orientation discrimination task (i.e. active exploration). Stimuli could be mirror-symmetric or not. Both free and active exploration generated more saccades parallel to the axis of symmetry than along other orientations. Most saccades were small (<2deg) leaving the fovea within a 4-degree radius of fixation. The analysis of saccade dynamics showed that the observed parallel orientation selectivity emerged within 500ms of stimulus onset and persisted throughout the trials under both viewing conditions. Symmetry strongly distorted existing anisotropies in gaze direction in a seemingly automatic process. We argue that this bias serves a functional role in which adjusted scene sampling enhances and maintains sustained sensitivity to local spatial correlations arising from symmetry.

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Self-reinnervated muscles lose autogenic length feedback but intermuscular feedback can recover functional connectivity

In this study, we sought to identify sensory circuitry responsible for motor deficits or compensatory adaptations after peripheral nerve cut and repair. Self-reinnervation of the ankle extensor muscles abolishes the stretch reflex and increases ankle yielding during downslope walking, but it remains unknown whether this finding generalizes to other muscle groups and whether muscles become completely deafferented. In decerebrate cats at least 19 weeks after nerve cut and repair, we examined the influence of quadriceps muscles' self-reinnervation on autogenic length feedback, as well as intermuscular length and force feedback among the primary extensor muscles in the cat hindlimb. Effects of gastrocnemius and soleus self-reinnervation on intermuscular circuitry was also evaluated. We found that autogenic length feedback was lost after quadriceps self-reinnervation indicating loss of the stretch reflex appears to be a generalizable consequence of muscle self-reinnervation. However, intermuscular force and length feedback evoked from self-reinnervated muscles was preserved in most of the interactions evaluated with similar relative inhibitory or excitatory magnitudes. These data indicate intermuscular spinal reflex circuitry has the ability to regain functional connectivity, but the restoration is not absolute. Explanations for the recovery of intermuscular feedback are discussed based on identified mechanisms responsible for lost autogenic length feedback. Functional implications due to permanent loss of autogenic length feedback and potential for compensatory adaptations from preserved intermuscular feedback are discussed.

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{alpha}1- and {alpha}2-adrenergic receptors in the retrotrapezoid nucleus differentially regulate breathing in anesthetized adult rats

Norepinephrine (NE) is a potent modulator of breathing that can increase/decrease respiratory activity by α₁-/ α₂-adrenergic receptors (AR) activation, respectively. The retrotrapezoid nucleus (RTN) is known to contribute to central chemoreception, inspiration and active expiration. Here we investigate the sources of catecholaminergic inputs to the RTN and identify respiratory effects produced by activation of ARs in this region. By injecting the retrograde tracer FluorGold into the RTN we identified back-labeled catecholaminergic neurons in the A7 region. In urethane-anesthetized, vagotomized and artificial ventilated male Wistar rats unilateral injection of NE or moxonidine (α₂-ARagonist) blunted Dia_EMG frequency and amplitude, without changing AbdEMG. Those inhibitory effects were reduced by pre-application of yohimbine (α2-AR antagonist) into the RTN. Conversely, unilateral RTN injection of phenylephrine (α₁-AR agonist) increased Dia_EMG amplitude, frequency and facilitated active expiration. This response was blocked by prior RTN injection of prazosin (α₁-AR antagonist). Interestingly, RTN injection of propranolol (β-AR antagonist) had no effect on respiratory inhibition elicited by applications of NE into the RTN, however, the combined blockade of α₂- and β-ARs (co-application of propranolol and yohimbine) revealed an α₁-AR-dependent excitatory response to NE that resulted in increase in Dia_EMG frequency and facilitation of active expiration. However, blockade of α₁-, α₂-, or β-ARs in the RTN had minimal effect on baseline respiratory activity, on central or peripheral chemoreflexes. These results suggest that NE signaling can modulate RTN chemoreceptor function; however, endogenous NE signaling does not contribute to baseline breathing or the ventilatory response to central or peripheral chemoreceptor activity in urethane-anesthetized rats.

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Validating silicon polytrodes with paired juxtacellular recordings: method and dataset

Cross-validating new methods for recording neural activity is necessary to accurately interpret and compare the signals they measure. Here we describe a procedure for precisely aligning two probes for in vivo "paired-recordings" such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micro-pipette. Our new method allows for efficient, reliable, and automated guidance of both probes to the same neural structure with micron resolution. We also describe a new dataset of paired-recordings, which is available online. We propose that our novel targeting system, and ever expanding cross-validation dataset, will be vital to the development of new algorithms for automatically detecting/sorting single-units, characterizing new electrode materials/designs, and resolving nagging questions regarding the origin and nature of extracellular neural signals.

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