A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex.

Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Face, content, and construct validity of a novel VR/AR surgical simulator of a minimally invasive spine operation.

Mixed-reality surgical simulators are seen more objective than conventional training. The simulators' utility in training must be established through validation studies. Establish face-, content-, and construct-validity of a novel mixed-reality surgical simulator developed by McGill University, CAE-Healthcare, and DePuy Synthes. This study, approved by a Research Ethics Board, examined a simulated L4-L5 oblique lateral lumbar interbody fusion (OLLIF) scenario. A 5-point Likert scale questionnaire was used. Chi-square test verified validity consensus. Construct validity investigated 276 surgical performance metrics across three groups, using ANOVA, Welch-ANOVA, or Kruskal-Wallis tests. A post-hoc Dunn's test with a Bonferroni correction was used for further analysis on significant metrics. Musculoskeletal Biomechanics Research Lab, McGill University, Montreal, Canada. DePuy Synthes, Johnson & Johnson Family of Companies, research lab. Thirty-four participants were recruited: spine surgeons, fellows, neurosurgical, and orthopedic residents. Only seven surgeons out of the 34 were recruited in a side-by-side cadaver trial, where participants completed an OLLIF surgery first on a cadaver and then immediately on the simulator. Participants were separated a priori into three groups: post-, senior-, and junior-residents. Post-residents rated validity, median > 3, for 13/20 face-validity and 9/25 content-validity statements. Seven face-validity and 12 content-validity statements were rated neutral. Chi-square test indicated agreeability between group responses. Construct validity found eight metrics with significant differences (p < 0.05) between the three groups. Validity was established. Most face-validity statements were positively rated, with few neutrally rated pertaining to the simulation's graphics. Although fewer content-validity statements were validated, most were rated neutral (only four were negatively rated). The findings underscored the importance of using realistic physics-based forces in surgical simulations. Construct validity demonstrated the simulator's capacity to differentiate surgical expertise.

Examining impact forces during posterior spinal fusion to implement in a novel physics-driven virtual reality surgical simulator.

This study aims to understand the impact forces that surgeons apply to the human spine during a posterior spinal fusion procedure towards the development of a novel spine surgical simulator for training medical residents. The foci of this study are impact forces during graft placement and spinal interbody cage insertion. This study examined the lumbar intervertebral discs of two male cadaveric specimens. Impact forces were collected during graft and spinal cage insertion over multiple levels. An impulse hammer and a camera were used to collect impact forces and displacements, respectively. The results demonstrated a logarithmic relationship between impact forces and cumulative displacement during graft placement. This was also observed between cumulative displacement and number of impacts during spinal cage insertion. A linear relationship was observed for the impact forces and number of impacts during graft placement. Results suggest that surgeons rely on the feedback experienced from impact forces during graft insertion to gauge the amount of graft that was placed in a specific area of the disc. Impact forces during cage insertion provide information about any encountered obstacles. When developing surgical simulators, designing the force feedback system should require modelling these behaviors to effectively impart corresponding skills on a trainee.

On the Mechanism of the Ionizing Radiation-Induced Degradation and Recycling of Cellulose.

The use of ionizing radiation offers a boundless range of applications for polymer scientists, from inducing crosslinking and/or degradation to grafting a wide variety of monomers onto polymeric chains. This review in particular aims to introduce the field of ionizing radiation as it relates to the degradation and recycling of cellulose and its derivatives. The review discusses the main mechanisms of the radiolytic sessions of the cellulose molecules in the presence and absence of water. During the radiolysis of cellulose, in the absence of water, the primary and secondary electrons from the electron beam, and the photoelectric, Compton effect electrons from gamma radiolysis attack the glycosidic bonds (C-O-C) on the backbone of the cellulose chains. This radiation-induced session results in the formation of alkoxyl radicals and C-centered radicals. In the presence of water, the radiolytically produced hydroxyl radicals (●OH) will abstract hydrogen atoms, leading to the formation of C-centered radicals, which undergo various reactions leading to the backbone session of the cellulose. Based on the structures of the radiolytically produced free radicals in presence and absence of water, covalent grafting of vinyl monomers on the cellulose backbone is inconceivable.

Vacuum curette lumbar discectomy mechanics for use in spine surgical training simulators.

Simulation in surgical training is a growing field and this study aims to understand the force and torque experienced during lumbar spine surgery to design simulator haptic feedback. It was hypothesized that force and torque would differ among lumbar spine levels and the amount of tissue removed by ≥ 7%, which would be detectable to a user. Force and torque profiles were measured during vacuum curette insertion and torsion, respectively, in multiple spinal levels on two cadavers. Multiple tests per level were performed. Linear and torsional resistances of 2.1 ± 1.6 N/mm and 5.6 ± 4.3 N mm/°, respectively, were quantified. Statistically significant differences were found in linear and torsional resistances between all passes through disc tissue (both p = 0.001). Tool depth (p < 0.001) and lumbar level (p < 0.001) impacted torsional resistance while tool speed affected linear resistance (p = 0.022). Average differences in these statistically significant comparisons were ≥ 7% and therefore detectable to a surgeon. The aforementioned factors should be considered when developing haptic force and torque feedback, as they will add to the simulated lumbar discectomy realism. These data can additionally be used inform next generation tool design. Advances in training and tools may help improve future surgeon training.

Pneumatosis Intestinalis of the Colon and Greater Omentum following Small Bowel Resection.

Introduction: Pneumatosis intestinalis (PI) is a condition of gas collection within the bowel wall that can represent either a benign clinical finding or a forerunner to potential gastrointestinal catastrophe. As a potentially sinister discovery typically first detected on radiographic imaging, clinicians need to astutely assess the need for additional urgent medical or surgical management in these patients. Apart from portal venous gas, PI outside of the bowel wall is an extremely rare entity that is poorly described. Hence, it is not necessarily clear if PI outside the bowel wall warrants more aggressive management. Case Presentation. We describe a patient with intermittent abdominal pain who presented with PI of the greater omentum in addition to the right and transverse colon nearly two weeks after small bowel resection. Due to his clinical stability, we elected to closely observe him. His condition completely resolved with conservative management. Discussion. PI in the omentum has not been described in a patient who has survived their underlying pathology. Our patient demonstrated PI radiographically in his right and transverse colon and omentum with complete resolution. We did not have to alter our clinical management because of this unique clinical presentation. Conclusion: This case highlights that pneumatosis intestinalis can extend extraluminally and still be managed conservatively with judicious monitoring in the otherwise stable patient.

Analysis of Surgical Forces Required to Gain Access Using a Probe for Minimally Invasive Spine Surgery via Cadaveric-Based Experiments Towards Use in Training Simulators.

INTRODUCTION: Virtual Reality haptic-based surgical simulators for training purposes have recently been receiving increased traction within the medical field. However, its future adoption is contingent on the accuracy and reliability of the haptic feedback. GOAL: This study describes and analyzes the implementation of a set of haptic-tailored experiments to extract the force feedback of a medical probe used in minimally invasive spinal lumbar interbody fusion surgeries. METHODS: Experiments to extract linear, lateral and rotational insertion, relaxation and extraction of the tool within the spinal muscles, intervertebral discs and lumbar nerve on two cadaveric torsos were conducted. RESULTS: Notably, mean force-displacement and torque-angular displacement curves describing the different tool-tissue responses were reported with a maximum force of 6.87 (±1.79) N at 40 mm in the muscle and an initial rupture force through the Annulus Fibrosis of 20.550 (±7.841) N at 6.441 mm in the L4/L5 disc. CONCLUSION: The analysis showed that increasing the velocity of the probe slightly reduced and delayed depth of the muscle punctures but significantly lowered the force reduction due to relaxation. Decreasing probe depth resulted with a reduction to the force relaxation drop. However, varying the puncturing angle of attack resulted with a significant effect on increasing force intensities. Finally, not resecting the thoracolumbar fascia prior to puncturing the muscle resulted with a significant increase in the force intensities. SIGNIFICANCE: These results present a complete characterization of the input required for probe access for spinal surgeries to provide an accurate haptic response in training simulators.

Development and Validation of a Mixed Reality Configuration of a Simulator for a Minimally Invasive Spine Surgery Using the Workspace of a Haptic Device and Simulator Users.

Most surgical simulators leverage virtual or bench models to simulate reality. This study proposes and validates a method for workspace configuration of a surgical simulator which utilizes a haptic device for interaction with a virtual model and a bench model to provide additional tactile feedback based on planned surgical manoeuvers. Numerical analyses were completed to determine the workspace and position of a haptic device, relative to the bench model, used in the surgical simulator, and the determined configuration was validated using device limitations and user data from surgical and nonsurgical users. For the validation, surgeons performed an identical surgery on a cadaver prior to using the simulator, and their trajectories were then compared to the determined workspace for the haptic device. The configuration of the simulator was determined appropriate through workspace analysis and the collected user trajectories. Statistical analyses suggest differences in trajectories between the participating surgeons which were not affected by the imposed haptic workspace. This study, therefore, demonstrates a method to optimally position a haptic device with respect to a bench model while meeting the manoeuverability needs of a surgical procedure. The validation method identified workspace position and user trajectory towards ideal configuration of a mixed reality simulator.

Polymerization Reactions and Modifications of Polymers by Ionizing Radiation.

Ionizing radiation has become the most effective way to modify natural and synthetic polymers through crosslinking, degradation, and graft polymerization. This review will include an in-depth analysis of radiation chemistry mechanisms and the kinetics of the radiation-induced C-centered free radical, anion, and cation polymerization, and grafting. It also presents sections on radiation modifications of synthetic and natural polymers. For decades, low linear energy transfer (LLET) ionizing radiation, such as gamma rays, X-rays, and up to 10 MeV electron beams, has been the primary tool to produce many products through polymerization reactions. Photons and electrons interaction with polymers display various mechanisms. While the interactions of gamma ray and X-ray photons are mainly through the photoelectric effect, Compton scattering, and pair-production, the interactions of the high-energy electrons take place through coulombic interactions. Despite the type of radiation used on materials, photons or high energy electrons, in both cases ions and electrons are produced. The interactions between electrons and monomers takes place within less than a nanosecond. Depending on the dose rate (dose is defined as the absorbed radiation energy per unit mass), the kinetic chain length of the propagation can be controlled, hence allowing for some control over the degree of polymerization. When polymers are submitted to high-energy radiation in the bulk, contrasting behaviors are observed with a dominant effect of cross-linking or chain scission, depending on the chemical nature and physical characteristics of the material. Polymers in solution are subject to indirect effects resulting from the radiolysis of the medium. Likewise, for radiation-induced polymerization, depending on the dose rate, the free radicals generated on polymer chains can undergo various reactions, such as inter/intramolecular combination or inter/intramolecular disproportionation, b-scission. These reactions lead to structural or functional polymer modifications. In the presence of oxygen, playing on irradiation dose-rates, one can favor crosslinking reactions or promotes degradations through oxidations. The competition between the crosslinking reactions of C-centered free radicals and their reactions with oxygen is described through fundamental mechanism formalisms. The fundamentals of polymerization reactions are herein presented to meet industrial needs for various polymer materials produced or degraded by irradiation. Notably, the medical and industrial applications of polymers are endless and thus it is vital to investigate the effects of sterilization dose and dose rate on various polymers and copolymers with different molecular structures and morphologies. The presence or absence of various functional groups, degree of crystallinity, irradiation temperature, etc. all greatly affect the radiation chemistry of the irradiated polymers. Over the past decade, grafting new chemical functionalities on solid polymers by radiation-induced polymerization (also called RIG for Radiation-Induced Grafting) has been widely exploited to develop innovative materials in coherence with actual societal expectations. These novel materials respond not only to health emergencies but also to carbon-free energy needs (e.g., hydrogen fuel cells, piezoelectricity, etc.) and environmental concerns with the development of numerous specific adsorbents of chemical hazards and pollutants. The modification of polymers through RIG is durable as it covalently bonds the functional monomers. As radiation penetration depths can be varied, this technique can be used to modify polymer surface or bulk. The many parameters influencing RIG that control the yield of the grafting process are discussed in this review. These include monomer reactivity, irradiation dose, solvent, presence of inhibitor of homopolymerization, grafting temperature, etc. Today, the general knowledge of RIG can be applied to any solid polymer and may predict, to some extent, the grafting location. A special focus is on how ionizing radiation sources (ion and electron beams, UVs) may be chosen or mixed to combine both solid polymer nanostructuration and RIG. LLET ionizing radiation has also been extensively used to synthesize hydrogel and nanogel for drug delivery systems and other advanced applications. In particular, nanogels can either be produced by radiation-induced polymerization and simultaneous crosslinking of hydrophilic monomers in "nanocompartments", i.e., within the aqueous phase of inverse micelles, or by intramolecular crosslinking of suitable water-soluble polymers. The radiolytically produced oxidizing species from water, •OH radicals, can easily abstract H-atoms from the backbone of the dissolved polymers (or can add to the unsaturated bonds) leading to the formation of C-centered radicals. These C-centered free radicals can undergo two main competitive reactions; intramolecular and intermolecular crosslinking. When produced by electron beam irradiation, higher temperatures, dose rates within the pulse, and pulse repetition rates favour intramolecular crosslinking over intermolecular crosslinking, thus enabling a better control of particle size and size distribution. For other water-soluble biopolymers such as polysaccharides, proteins, DNA and RNA, the abstraction of H atoms or the addition to the unsaturation by •OH can lead to the direct scission of the backbone, double, or single strand breaks of these polymers.

Defective DNA Polymerase α-Primase Leads to X-Linked Intellectual Disability Associated with Severe Growth Retardation, Microcephaly, and Hypogonadism.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.

Primary brain calcification: an international study reporting novel variants and associated phenotypes.

Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with a wide spectrum of motor, cognitive, and neuropsychiatric symptoms. It is typically inherited as an autosomal-dominant trait with four causative genes identified so far: SLC20A2, PDGFRB, PDGFB, and XPR1. Our study aimed at screening the coding regions of these genes in a series of 177 unrelated probands that fulfilled the diagnostic criteria for primary brain calcification regardless of their family history. Sequence variants were classified as pathogenic, likely pathogenic, or of uncertain significance (VUS), based on the ACMG-AMP recommendations. We identified 45 probands (25.4%) carrying either pathogenic or likely pathogenic variants (n = 34, 19.2%) or VUS (n = 11, 6.2%). SLC20A2 provided the highest contribution (16.9%), followed by XPR1 and PDGFB (3.4% each), and PDGFRB (1.7%). A total of 81.5% of carriers were symptomatic and the most recurrent symptoms were parkinsonism, cognitive impairment, and psychiatric disturbances (52.3%, 40.9%, and 38.6% of symptomatic individuals, respectively), with a wide range of age at onset (from childhood to 81 years). While the pathogenic and likely pathogenic variants identified in this study can be used for genetic counseling, the VUS will require additional evidence, such as recurrence in unrelated patients, in order to be classified as pathogenic.

Ionizing radiation processing and its potential in advancing biorefining and nanocellulose composite materials manufacturing.

Nanocellulose is a high value material that has gained increasing attention because of its high strength, stiffness, unique photonic and piezoelectric properties, high stability and uniform structure. Through utilization of a biorefinery concept, nanocellulose can be produced in large volumes from wood at relatively low cost via ionizing radiation processing. Ionizing radiation causes significant break down of the polysaccharide and leads to the production of potentially useful gaseous products such as H2 and CO. The application of radiation processing to the production of nanocellulose from woody and non-wood sources, such as field grasses, bio-refining byproducts, industrial pulp waste, and agricultural surplus materials remains an open field, ripe for innovation and application. Elucidating the mechanisms of the radiolytic decomposition of cellulose and the mass generation of nanocellulose by radiation processing is key to tapping into this source of nanocelluose for the growth of nanocellulostic-product development. More importantly, understanding the structural break-up of the cell walls as a function of radiation exposure is a key goal and only through careful, detailed characterization and dimensional metrology can this be achieved at the level of detail that is needed to further the growth of large scale radiation processing of plant materials. This work is resulting from strong collaborations between NIST and its academic partners who are pursuing the unique demonstration of applied ionizing radiation processing to plant materials as well as the development of manufacturing metrology for novel nanomaterials.

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.

SHORT syndrome due to a novel de novo mutation in PRKCE (Protein Kinase Cɛ) impairing TORC2-dependent AKT activation.

SHORT syndrome is a rare, recognizable syndrome resulting from heterozygous mutations in PIK3R1 encoding a regulatory subunit of phosphoinositide-3-kinase (PI3K). The condition is characterized by short stature, intrauterine growth restriction, lipoatrophy and a facial gestalt involving a triangular face, deep set eyes, low hanging columella and small chin. PIK3R1 mutations in SHORT syndrome result in reduced signaling through the PI3K-AKT-mTOR pathway. We performed whole exome sequencing for an individual with clinical features of SHORT syndrome but negative for PIK3R1 mutation and her parents. A rare de novo variant in PRKCE was identified. The gene encodes PKCε and, as such, the AKT-mTOR pathway function was assessed using phospho-specific antibodies with patient lymphoblasts and following ectopic expression of the mutant in HEK293 cells. Kinase analysis showed that the variant resulted in a partial loss-of-function. Whilst interaction with PDK1 and the mTORC2 complex component SIN1 was preserved in the mutant PKCε, it bound to SIN1 with a higher affinity than wild-type PKCε and the dynamics of mTORC2-dependent priming of mutant PKCε was altered. Further, mutant PKCε caused impaired mTORC2-dependent pAKT-S473 following rapamycin treatment. Reduced pFOXO1-S256 and pS6-S240/244 levels were also observed in the patient LCLs. To date, mutations in PIK3R1 causing impaired PI3K-dependent AKT activation are the only known cause of SHORT syndrome. We identify a SHORT syndrome child with a novel partial loss-of-function defect in PKCε. This variant causes impaired AKT activation via compromised mTORC2 complex function.

Oral-facial-digital syndrome type I cells exhibit impaired DNA repair; unanticipated consequences of defective OFD1 outside of the cilia network.

Defects in OFD1 underlie the clinically complex ciliopathy, Oral-Facial-Digital syndrome Type I (OFD Type I). Our understanding of the molecular, cellular and clinical consequences of impaired OFD1 originates from its characterised roles at the centrosome/basal body/cilia network. Nonetheless, the first described OFD1 interactors were components of the TIP60 histone acetyltransferase complex. We find that OFD1 can also localise to chromatin and its reduced expression is associated with mis-localization of TIP60 in patient-derived cell lines. TIP60 plays important roles in controlling DNA repair. OFD Type I cells exhibit reduced histone acetylation and altered chromatin dynamics in response to DNA double strand breaks (DSBs). Furthermore, reduced OFD1 impaired DSB repair via homologous recombination repair (HRR). OFD1 loss also adversely impacted upon the DSB-induced G2-M checkpoint, inducing a hypersensitive and prolonged arrest. Our findings show that OFD Type I patient cells have pronounced defects in the DSB-induced histone modification, chromatin remodelling and DSB-repair via HRR; effectively phenocopying loss of TIP60. These data extend our knowledge of the molecular and cellular consequences of impaired OFD1, demonstrating that loss of OFD1 can negatively impact upon important nuclear events; chromatin plasticity and DNA repair.

The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Novel Hemi-Staple for the Fusionless Correction of Pediatric Scoliosis: Influence on Intervertebral Disks and Growth Plates in a Porcine Model.

STUDY DESIGN: In vivo porcine model utilized to evaluate the influence of an intravertebral fusionless growth modulating device (hemi-staple) on intervertebral disks and growth plates. OBJECTIVE: To evaluate the radiographic and histologic changes in disks and growth plates with the purpose of measuring influence of the explored hemi-staple. SUMMARY OF BACKGROUND DATA: Fusionless growth modulation for the early treatment of scoliosis should insure the long-term viability of the intervertebral disk and successfully reduce or arrest local growth. A novel hemi-staple that proved effective in the control of coronal spinal alignment warranted further analyses of its influence on the disk health and growth-plate morphology. METHODS: A hemi-staple that inhibited local vertebral growth exclusive of the disk was introduced over T5-T8 in 4 immature pigs (16 vertebrae; experimental), whereas 3 underwent surgery without instrumentation (sham) and 2 had no intervention (control). Three-month follow-up before animal euthanasia provided radiographic (disk height and health) and histologic (growth plate morphology, disk health, and type X collagen distribution) analyses. RESULTS: No postoperative complications were experienced. Radiographic data returned inverse disk wedging (greater disk height adjacent to device, 2.6±0.7 mm compared with the noninstrumented side, 1.8±0.5 mm) in experimental segments and suggested disk viability. Histologic data confirmed device growth modulation through significant local reduction of growth plate hypertrophic zone (125.64±16.61 µm and 61.16±8.25 µm in noninstrumented and instrumented sections, respectively) and cell height (16.14±1.87 µm and 9.22±1.57 µm in noninstrumented and instrumented sections, respectively). A variability of disk health, dependant of device insertion location, was observed. Type X collagen was consistently identified in experimental growth plates and absent from intervertebral disks. CONCLUSIONS: Hemi-staples decreased growth plate hypertrophic zone and cell height, and, depending on device insertion site, showed positive signs of disk health sustainability. Spinal growth modulation achieved exclusive of disk compression, as practiced by this method, offers unique advantages over other fusionless techniques. This technique may provide a suitable and attractive alternative for the early treatment of idiopathic scoliosis.

Identifying candidate genes for 2p15p16.1 microdeletion syndrome using clinical, genomic, and functional analysis.

The 2p15p16.1 microdeletion syndrome has a core phenotype consisting of intellectual disability, microcephaly, hypotonia, delayed growth, common craniofacial features, and digital anomalies. So far, more than 20 cases of 2p15p16.1 microdeletion syndrome have been reported in the literature; however, the size of the deletions and their breakpoints vary, making it difficult to identify the candidate genes. Recent reports pointed to 4 genes (XPO1, USP34, BCL11A, and REL) that were included, alone or in combination, in the smallest deletions causing the syndrome. Here, we describe 8 new patients with the 2p15p16.1 deletion and review all published cases to date. We demonstrate functional deficits for the above 4 candidate genes using patients' lymphoblast cell lines (LCLs) and knockdown of their orthologs in zebrafish. All genes were dosage sensitive on the basis of reduced protein expression in LCLs. In addition, deletion of XPO1, a nuclear exporter, cosegregated with nuclear accumulation of one of its cargo molecules (rpS5) in patients' LCLs. Other pathways associated with these genes (e.g., NF-κB and Wnt signaling as well as the DNA damage response) were not impaired in patients' LCLs. Knockdown of xpo1a, rel, bcl11aa, and bcl11ab resulted in abnormal zebrafish embryonic development including microcephaly, dysmorphic body, hindered growth, and small fins as well as structural brain abnormalities. Our multifaceted analysis strongly implicates XPO1, REL, and BCL11A as candidate genes for 2p15p16.1 microdeletion syndrome.

The Effects of a Home-Based Connective Tissue Targeting Therapy on Hip Development in Children With Cerebral Palsy: Six Case Reports.

Hip subluxation in children with Cerebral Palsy (CP) has an incidence of 10-30 %, and children with severe CP having the highest incidence. The condition deteriorates if left untreated. Surgery is the most common method used in managing hip subluxation because standard conservative therapies do not improve it. Surgery may have to be repeated and comes at a biological cost to the child. A new home-based CAM, Advanced Biomechanical Rehabilitation (ABR), has shown encouraging results leading to improved spinal stability and stability in sitting in children with severe CP. This case report examines hip development over time in six children with severe CP in the ABR Program. Changes in their clinical picture and pelvic X-Rays are reported. ABR appeared to help stabilize and improve hip subluxation, resulting in these children not requiring further surgical intervention. These findings warrant further investigation of ABR as a noninvasive therapy for hip subluxation.