Subtle Defects Research Articles

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation.

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.

Imaging microstructural dynamics and strain fields in electro-active materials in situ with dark field x-ray microscopy.

The electric-field-induced and temperature induced dynamics of domains, defects, and phases play an important role in determining the macroscopic functional response of ferroelectric and piezoelectric materials. However, distinguishing and quantifying these phenomena remains a persistent challenge that inhibits our understanding of the fundamental structure-property relationships. In situ dark field x-ray microscopy is a new experimental technique for the real space mapping of lattice strain and orientation in bulk materials. In this paper, we describe an apparatus and methodology for conducting in situ studies of thermally and electrically induced structural dynamics and demonstrate their use on ferroelectric BaTiO3 single crystals. The stable temperature and electric field apparatus enables simultaneous control of electric fields up to ≈2 kV/mm at temperatures up to 200 °C with a stability of ΔT = ±0.01 K and a ramp rate of up to 0.5 K/min. This capability facilitates studies of critical phenomena, such as phase transitions, which we observe via the microstructural change occurring during the electric-field-induced cubic to tetragonal phase transition in BaTiO3 at its Curie temperature. With such systematic control, we show how the growth of the polar phase front and its associated ferroelastic domains fall along unexpected directions and, after several cycles of electric field application, result in a non-reversible lattice strain at the electrode-crystal interface. These capabilities pave the way for new insights into the temperature and electric field dependent electromechanical transitions and the critical influence of subtle defects and interfaces.

Detection of surface defects on solar cells by fusing Multi-channel convolution neural networks

Manufacturing process defects or artificial operation mistakes may lead to solar cells having surface cracks, over welding, black edges, unsoldered areas, and other minor defects on their surfaces. These defects will reduce the efficiency of solar cells or even make them completely useless. In this paper, a detection algorithm of surface defects on solar cells is proposed by fusing multi-channel convolution neural networks. The detection results from two different convolution neural networks, i.e., Faster R-CNN and R-FCN, are combined to improve detection precision and position accuracy. In addition, according to the inherent characteristics of the surface defects in solar cells, two other strategies are used to further improve the detection performance. First, the anchor points of the region proposal network (RPN) are set by adding multi-scale and multi-aspect regions to overcome the problem of high false negative rate caused by the limitation of anchor points. Second, in view of the subtle and concealed defects of solar cells, the hard negative sample mining strategy is used to solve the problem of low detection precision caused by the negative sample space being too large. The experimental results showed that the proposed method effectively reduced the false negative rate and the false positive rate of a single network, and it greatly improved the accuracy of the locations of defects while improving the object recall rate.

Enhancing die level static fault isolation on power gated devices

Modern SoC design incorporates power management circuits to minimize standby leakages of digital logic circuitry. This isolates the logic blocks from being directly powered by the IC's pad level power supply and ground. As a result, die level static fault isolation became ineffective even for gross defects that are shorting the power lines in the logic blocks due to gated supply. From the foundry standpoint, fault isolation on such devices are even more challenging without the product knowhow, device specific hardware and test program to properly condition the power management circuits. An alternative method is to bypass the power control circuits through microprobing on the top metal traces to directly supply power to the logic blocks. However, the key challenge to this method is the means to differentiate between power gated and non-gated devices and then subsequently verify the relevant power traces to microprobe especially in the absence of layout. This paper illustrates 4 different approaches to overcome this challenge and achieve static fault isolation success on power gated devices. Successful applications of this approach to localize gross defects resulting in short/leakage failures and subtle defects resulting in memory/logic functional failures will be described.

SUN-060 LATE PRESENTATION OF NEPHROTOXICITY DUE TO OXALIPLATIN

Oxaliplatin is a third-generation platinum derivative and considered less nephrotoxic than Cisplatin. It is frequently used as part of chemotherapy to treat various gastrointestinal tumors. Although hypokalemia, ATN and acute nephrotoxicity in the form of renal tubular acidosis due to Oxaliplatin has been reported; long term nephrotoxic effects of Oxaliplatin are not known. We report a novel case of 57- year old male who presented with muscle aches and electrolyte disturbances, several months after receiving oxaliplatin based chemotherapy. Patient had a premorbid history of diabetes mellitus, hypertension and he was diagnosed to have intrahepatic cholangiocarcinoma with neuroendocrine differentiation in September 2018. His creatinine was normal 74 umol/L at baseline. He received five cycles of chemotherapy with FOLFIRINOX (Folinic acid, Flourouracil, Irinotecan and Oxaliplatin). After five cycles he developed Oxaliplatin related ileus and nephropathy causing ATN and required few sessions of hemodialysis. Subsequently his kidney function improved partially with a new elevated baseline creatinine which was stable at 133 umol/L in February 2019. He presented on 28th September 2019 with severe muscle aches to emergency department (ED). His investigations done in ED revealed severe hypokalemia K 1.9 mmol/L, worsening of renal function, baseline creatinine had risen from 133 umol/L to 205 umol/L, he had severe metabolic acidosis with tCO 2 10, with normal anion gap 15 (range 7-16) and hyperchloremia 111 mmol/L, pH was 7.15. His serum phosphorus was low (0.63 mmol/L). Magnesium was normal. Urinary TTKG was calculated and was suggestive of urinary potassium loss. His Urinary Anion Gap was negative. He had glycosuria 4 + even though his blood sugars were well controlled. His urine pH was 5.5. Patient was treated with very high doses of potassium tricitrate and phosphorus supplements. He was discharged home after normalization of electrolytes and acid base status. Proximal renal tubular acidosis (type 2, pRTA) / Fanconi’s syndrome is characterized by bicarbonate wasting and low serum bicarbonate, proximal tubular dysfunction also leads to poor absorption of other substances, like glucose and phosphate. Further investigations were carried out to rule out other causes of Fanconi’s syndrome / Type 2 renal tubular acidosis in this patient. ANA, dsDNA, SPEP, IFE and serum Free light chain assay were all unremarkable. His Lactate was normal. A diagnosis of Fanconi’s syndrome and renal tubular acidosis type 2 (Proximal) due to remote exposure with Oxaliplatin was made. Fanconi’s syndrome due to platinum based drugs like Cisplatin and Oxaliplatin has been reported in Literature. Persistence of subtle partial renal tubular defects is well noted even after many months to years of exposure of Cisplatin but not with Oxaliplatin. We believe this is the first report of Type 2 pRTA and Fanconi’s syndrome presenting several months after exposure to Oxaliplatin. Physicians should be aware of unexpected late nephrotoxicity with Oxaliplatin use.

Single Amino Acid Changes in the Ryanodine Receptor in the Human Population Have Effects In Vivo on Caenorhabditis elegans Neuro-Muscular Function.

The ryanodine receptor mediates intracellular calcium ion release with excitation of nerve and muscle cells. Ryanodine receptor missense variants cause a number of myopathologies, such as malignant hyperthermia, and have been linked with various neuropathologies, including Alzheimer’s disease. We characterized the consequences of ryanodine receptor variants in vivo. Eight Caenorhabditis elegans strains, with ryanodine receptor modifications equivalent to human myopathic RYR1 variants, were generated by genome editing. In humans, these variants are rare and confer sensitivity to the inhalational anaesthetic halothane when heterozygous. Increased sensitivity to halothane was found in both homozygous and heterozygous C. elegans. Close analysis revealed distinct subtle locomotion defects, due to the different single amino acid residue changes, even in the absence of the external triggering agent. Distinct pre- and postsynaptic consequences of the variants were characterized through the responses to cholinergic pharmacological agents. The range of phenotypes reflects the complexity of the regulatory inputs to the ryanodine receptor and the criticality of the calcium ion channel opening properties, in different cell types and with age. Ryanodine receptors with these single amino acid residue changes still function as calcium ion channels, but with altered properties which are likely to have subtle consequences for human carriers of such variants. The long-term consequences of subtly altered calcium ion signalling could be cumulative and may be focussed in the smaller nerve cells rather than the more robust muscle cells. It was important to assess phenotypes in vivo to properly appreciate consequences for a whole organism.

Peptide Linkers within the Essential FtsZ Membrane Tethers ZipA and FtsA Are Nonessential for Cell Division.

Bacteria such as Escherichia coli divide by organizing filaments of FtsZ, a tubulin homolog that assembles into dynamic treadmilling membrane-associated protein filaments at the cell midpoint. FtsA and ZipA proteins are required to tether these filaments to the inner face of the cytoplasmic membrane, and loss of either tether is lethal. ZipA from E. coli and other closely related species harbors a long linker region that connects the essential N-terminal transmembrane domain to the C-terminal globular FtsZ-binding domain, and part of this linker includes a P/Q-rich peptide that is predicted to be intrinsically disordered. We found unexpectedly that several large deletions of the ZipA linker region, including the entire P/Q rich peptide, had no effect on cell division under normal conditions. However, we found that the loss of the P/Q region made cells more resistant to excess levels of FtsA and more sensitive to conditions that displaced FtsA from FtsZ. FtsA also harbors a short ∼20-residue peptide linker that connects the main globular domain with the C-terminal amphipathic helix that is important for membrane binding. In analogy with ZipA, deletion of 11 of the central residues in the FtsA linker had little effect on FtsA function in cell division.IMPORTANCEEscherichia coli cells divide using a cytokinetic ring composed of polymers of the tubulin-like FtsZ. To function properly, these polymers must attach to the inner surface of the cytoplasmic membrane via two essential membrane-associated tethers, FtsA and ZipA. Both FtsA and ZipA contain peptide linkers that connect their membrane-binding domains with their FtsZ-binding domains. Although they are presumed to be crucial for cell division activity, the importance of these linkers has not yet been rigorously tested. Here, we show that large segments of these linkers can be removed with few consequences for cell division, although several subtle defects were uncovered. Our results suggest that ZipA, in particular, can function in cell division without an extended linker.

Thyroid Hormone Hyposensitivity: From Genotype to Phenotype and Back.

Thyroid hormone action defects (THADs) have been classically considered conditions of impaired sensitivity to thyroid hormone (TH). They were originally referring to alterations in TH receptor genes (THRA and THRB), but the discovery of genetic mutations and polymorphisms causing alterations in cell membrane transport (e.g., MCT8) and metabolism (e.g., SECISBP2, DIO2) led recently to a new and broader definition of TH hyposensitivity (THH), including not only THADs but all defects that could interfere with the activity of TH. Due to the different functions and tissue-specific expression of these genes, affected patients exhibit highly variable phenotypes. Some of them are characterized by a tissue hypothyroidism or well-recognizable alterations in the thyroid function tests (TFTs), whereas others display a combination of hypo- and hyperthyroid manifestations with normal or only subtle biochemical defects. The huge effort of basic research has greatly aided the comprehension of the molecular mechanisms underlying THADs, dissecting the morphological and functional alterations on target tissues, and defining the related-changes in the biochemical profile. In this review, we describe different pictures in which a specific alteration in the TFTs (TSH, T4, and T3 levels) is caused by defects in a specific gene. Altogether these findings can help clinicians to early recognize and diagnose THH and to perform a more precise genetic screening and therapeutic intervention. On the other hand, the identification of new genetic variants will allow the generation of cell-based and animal models to give novel insight into thyroid physiology and establish new therapeutic interventions.

Vasopressor Therapy and the Brain: Dark Side of the Moon.

Sepsis, a leading cause of morbidity and mortality, is caused by a deregulated host response to pathogens, and subsequent life-threatening organ dysfunctions. All major systems, including the cardiovascular, respiratory, renal, hepatic, hematological, and the neurological system may be affected by sepsis. Sepsis associated neurological dysfunction is triggered by multiple factors including neuro-inflammation, excitotoxicity, and ischemia. Ischemia results from reduced cerebral blood flow, caused by extreme variations of blood pressure, occlusion of cerebral vessels, or more subtle defects of the microcirculation. International guidelines comprehensively describe the initial hemodynamic management of sepsis, revolving around the normalization of systemic hemodynamics and of arterial lactate. By contrast, the management of sepsis patients suffering from brain dysfunction is poorly detailed, the only salient point being mentioned is that sedation and analgesia should be optimized. However, sepsis and the hemodynamic consequences thereof as well as vasopressors may have severe untoward neurological consequences. The current review describes the general neurological complications, as well as the consequences of vasopressor therapy on the brain and its circulation and addresses methods for cerebral monitoring during sepsis.

The Fgf8 subfamily (Fgf8, Fgf17 and Fgf18) is required for closure of the embryonic ventral body wall.

ABSTRACTThe closure of the embryonic ventral body wall in amniotes is an important morphogenetic event and is essential for life. Defects in human ventral wall closure are a major class of birth defect and a significant health burden. Despite this, very little is understood about how the ventral body wall is formed. Here, we show that fibroblast growth factor (FGF) ligands FGF8, FGF17 and FGF18 are essential for this process. Conditional mouse mutants for these genes display subtle migratory defects in the abdominal muscles of the ventral body wall and an enlarged umbilical ring, through which the internal organs are extruded. By refining where and when these genes are required using different Cre lines, we show that Fgf8 and Fgf17 are required in the presomitic mesoderm, whereas Fgf18 is required in the somites. This study identifies complex and multifactorial origins of ventral wall defects and has important implications for understanding their origins during embryonic development.

Cardiac coup and contrecoup following a suicide attempt

A 48-year-old man was admitted as a Level 1 trauma after attempting suicide by jumping 28 m into a river. He was haemodynamically stable with unremarkable physical examination except for...

A locomotor assay reveals deficits in heterozygous Parkinson’s disease model and proprioceptive mutants in adult Drosophila

Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. We introduce a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. We use this inexpensive, fully automated assay to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, we characterize wild-type flies and uncover a hitherto unknown sexual dimorphism in climbing behavior. Second, we study climbing behavior of heterozygous mutants of genes implicated in the fly PD model and reveal previously unreported prominent locomotor defects in some of these heterozygous fly lines. Finally, we study locomotor defects in a homozygous proprioceptory mutation (Trp-γ1 ) known to affect fine motor control in Drosophila Moreover, we identify aberrant geotactic behavior in Trp-γ1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. Our assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects.

Paraoxonase 2 Enables Initiation of B-ALL By Subverting Metabolic Gatekeeper Functions

Background and significance: The B-lymphoid transcription factors IKZF1 and PAX5 are essential for early B-cell development but also function as metabolic gatekeepers by restricting glucose uptake across the cell membrane (Chan et al., Nature 2017; Xiao et al., Cell 2018). Paraoxonase 2 (PON2) is used as a diagnostic marker and included in a 15 gene diagnostic LDA panel (NCT02883049) for the identification of Ph+ and Ph-like ALL, a B-ALL subgroup with poor outcome and frequent deletion of IKZF1. Pon2 is a member of detoxifying enzymes that are located to the mitochondrial membrane, and hydrolyze lactone metabolites. While PON2 is typically not expressed in normal B-cell precursors, we studied the functional role of high expression levels of Pon2 in B-ALL. Results: Indeed, high Pon2 mRNA levels at diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003). While PON2 mRNA and protein levels are barely detectable in normal B-cell precursors we found >10-fold increased mRNA and protein levels in multiple B-ALL. Pon2-deficient mice have a number of subtle defects, including propensity to neurogenerative disease and atherosclerosis. However, deletion of Pon2 had profound effects in two models for B-ALL (BCR-ABL1- and NRASG12D). Compared to Pon2 wildtype, Pon2-/- B-ALL cells failed to form colonies in semisolid agar, were prone to cell cycle arrest in G0/G1 phase. Consistent with these observations, Pon2-/- B-ALL cells had a substantially reduced proliferation rate and expressed Arf and p21 checkpoint proteins at substantially increased levels compared to Pon2+/+ ALL cells. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in mouse transplant experiments for BCR-ABL1 and NRASG12D B-ALL models. Pon2-deficiency substantially prolonged survival of recipient mice of BCR-ABL1 B-ALL cells (P=0.0001). Strikingly, NSG mice transplanted with Pon2-deficient NRASG12D B-ALL cells did not develop any leukemia and survived for indefinite periods of time. Based on engineered expression of Cas9 and PON2-specific cRNAs, genetic deletion of PON2 replicated these findings in patient-derived B-ALL xenografts (PDX). Mechanism: Reflecting PAX5 and IKZF1 function, B-lymphoid cells exhibit transcriptional repression of glucose transport (e.g. repression of INSR, GLUT1). Interestingly, deletion of PON2 decreased glucose uptake even further. Intracellular ATP levels in Ph+ ALL cells were drastically reduced and activated the AMPK energy-stress sensor in both murine BCR-ABL1 and NRASG12D B-ALL models and human Ph+ B-ALL cells. Reconstitution of PON2 restored ATP production as early as 3 hours after PON2 induction. Mechanistically, PON2 was previously identified as an inhibitor of STOM and GLUT1 interactions. By disrupting STOM: GLUT1 interactions, PON2 can restore glucose transport. STOM-knockdown in PON2-/- ALL cells replenished cellular ATP levels as did PON2 reconstitution. We conclude that PON2 serves a critical role in B-ALL by subverting the metabolic gatekeeper function of IKZF1 and PAX5 through interference with STOM:GLUT1 interactions. Therapeutic targeting of PON2: While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts. Hence, we tested the concept of leveraging PON2 lactonase activity in a prodrug-approach as a synthetic lethal in Pon2+ B-ALL cells. 3OC12 had profound cytotoxic effects in wildtype but not Pon2-/- B-ALL cells while reconstitution or overexpression of PON2 restored cytotoxic responses to 3OC12. Conclusion: We describe the previously unrecognized function of the detoxifying PON2 lactonase as a central factor in glucose and energy supply in B-ALL, hence subverting metabolic gatekeeper functions of PAX5 and IKZF1. In addition to identifying PON2 as the biomarker for for Ph+ and Ph-like ALL and outcome predictor for patients with B-ALL, our results indicate PON2 facilitate glucose uptake and ATP production via its interaction with STOM. PON2 protects B-ALL cells against glucose depletion and enables malignant growth. On the other hand, we demonstrate a targeted approach that leverages PON2-lactonase as a synthetic lethal to the lactone-prodrug 3OC12. Disclosures No relevant conflicts of interest to declare.

Characterization of YIG thin films and vacuum annealing effect by polarized neutron reflectometry and magnetotransport measurements

Nanometer-thick Y3Fe5O12 (YIG) films epitaxially grown on (111) Gd3Ga5O12 with a magnetic dead layer as thin as about 1.2 nm are quantified by polarized neutron reflectivity and magnetization measurements. Vacuum annealing on YIG at 300–400 °C leads to substantial reduction in the anomalous Hall effect, spin Hall magnetoresistance, and spin pumping in YIG/Pt bilayers but causes large enhancement in the spin Seebeck effect. The structural, static, and dynamic magnetic measurements show that the annealing has no discernible influence on the global oxidization states and saturation magnetization of YIG films but introduces subtle defects possibly in the form of oxygen vacancies. This study suggests that subtle defects in thin YIG films have multiple effects on the spin transport properties, and caution should be taken in annealing YIG in vacuum.

Recurrent visual loss in a 64-year-old man

A 64-year-old Indian man presented to the emergency department with a 3-day history of worsening blurred vision in his right eye. This was accompanied by dull right periocular pain exacerbated...

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy.

Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a high-throughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.

Shared roles for Scl and Lyl1 in murine platelet production and function

AbstractThe stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEβ), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.

Cochlear basal turn patency in unrecognized perilymph gushers

ObjectivesPerilymph gusher (PLG), an uncommon complication of otologic surgery, has been attributed to communication between the cochlea and the internal auditory canal (IAC). Subtle osseous defects may be missed on routine review of computed tomography (CT). This study aimed to quantify cochlear basal turn patency not seen on axial CT in patients with PLG and compare those against patients without intraoperative PLG. MethodsEars that underwent cochlear implantation or stapedotomy with preoperative helical CT that was interpreted as “normal” at a tertiary referral center. An otologist and a radiologist independently and in a blinded fashion measured the dimensions of cochlear basal turn patency on CT images in oblique plane and parasagittal planes along the interface of the cochlea and IAC fundus. ResultsSixty-one ears were reviewed, including 3 with surgically confirmed PLGs and 12 with apparent dehiscence without a PLG. Mean defect width with PLG was 0.83 mm (range 0.75–0.9 mm) and without PLG was 0.43 mm (range 0.3–0.65 mm, p = 0.011). A greater proportion of PLGs occurred in ears with defects (3 of 15) than in ears without (0 of 46, p = 0.013). Using a cutoff of 0.75 mm, a greater proportion of PLGs occurred with defect width >0.75 mm (3 of 3) than in defects <0.75 mm (0 of 12, p = 0.022). ConclusionsCT dehiscence between the IAC and cochlear basal turn, particularly with a width > 0.75 mm, should be considered a risk for PLG with stapedotomy or cochlear implantation.

1918-P: Carbohydrate and Fat Oxidation Fasting and in Response to a Hyperinsulinemic Clamp is Similar in Obese Youth Regardless of PCOS Status

Whole-body metabolic flexibility (MF), i.e., switching substrate oxidation between fasting and fed states often becomes abnormal with insulin resistance (IR). Data regarding MF in women with PCOS, whom often have IR, are mixed and confounded by varied diets and habitual physical activity. No study has assessed MF in response to a multi-phase hyperinsulinemic-euglycemic clamp in PCOS females relative to non-PCOS with similar habitual physical activity and controlled diet. Adolescent girls with obesity and PCOS (age 14.9 ± 1.5 years; BMI 35 ± 4 kg/m2), 10 obese controls (age 13.5 ± 1.3; BMI 32 ± 2) and 13 normal BMI PCOS (age 16.2 ± 1.5; BMI 22.8 ± 2) were included. All girls were sedentary per questionnaire and 7 day accelerometry. A 4-phase hyperinulinemic-euglycemic clamp (basal, 10, 16 and 80 mU/m2/min of insulin) including glucose and glycerol tracers was performed. Glucose rate of disappearance (Rd), glycerol rate of appearance (Ra) and respiratory quotient (RQ) were assessed during each clamp phase. MF was defined as a change in RQ from baseline to each phase (ΔRQ). Basal RQs were not different between groups. Lean PCOS had higher ΔRQ at phase 10 compared to obese PCOS (p=0.005) and obese controls (p=0.003). All groups had similar ΔRQs at 16 and 80 phases. In multivariable regression models, Δ 10 glycerol Ra (Δ from baseline to 10 phase) was associated with phase 10 ΔRQ, independent of age and BMI (p=0.04); Δ 80 glycerol Ra and Δ 80 glucose Rd were associated with phase 80 ΔRQ, independent of age and BMI (p=0.048). Sedentary obese adolescents regardless of PCOS status have lower MF compared to lean PCOS during the low insulin dose which was associated with whole body lipolysis. However, this difference is not found during the high insulin dose, suggesting obese girls have only subtle defects in MF. Further, PCOS is not associated with lower MF once diet and habitual activity are matched. Disclosure P. Wiromrat: None. C.A. Rynders: None. K.J. Nadeau: None. M. Cree-Green: None. Funding American Diabetes Association (7-11-CD-08 to K.J.N.); American Heart Association (13CRP14120015); Thrasher Research Fund; National Center for Research Resources Colorado (TL1TR002533); Pediatric Endocrinology Society; National Institute of Diabetes and Digestive and Kidney Diseases (T32DK063687, K23DK107871)