CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV Delta UL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV DUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing

Rare hemoglobin variant Hb Yaizu observed in Turkey

Objective: To determine the characteristic features of the rare hemoglobin (Hb) variant Hb Yaizu to enable laboratory diagnosis of the hemoglobin variants during screening programs. Materials and Methods: Genomic DNA was obtained from the 4 members of a family living in Denizli province, an Aegean region of Turkey. Blood cell counts, hemoglobin composition, hemoglobin electrophoresis (both alkaline and acid), HPLC analysis, DNA sequencing and beta globin gene cluster haplotypes were done. Results: Hb Yaizu carriers were apparently healthy individuals. Hb Yaizu was slightly faster than Hb S at alkaline pH, but slower than Hb S at acidic pH in hemoglobin electrophoresis. An abnormal hemoglobin peak was observed with a retention time of 4.77 min in HPLC analysis attributed to Hb Yaizu. Two members of the family were heterozygous Hb Yaizu [beta 79(EF3) Asp>Asn] confirmed by DNA sequencing. The mutation was found to be linked with the Mediterranean haplotype I [+ - - ++]. Conclusion: We have presented the details of Hb Yaizu, a rare hemoglobin variant that may be important to hemoglobinopathy screening programs, although its clinical significance is unclear. Copyright © 2008 S. Karger AG

2-[(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methyl­idene]indan-1,3-dione

In the title compound, C21H16N2O2, the five-membered heterocyclic ring makes a dihedral angle of 47.06 (6)° with the attached benzene ring, whereas the indan-1,3-dione ring system and the benzene ring are oriented at a dihedral angle of 21.92 (7)°. In the crystal, inversion dimers linked by pairs of C—H⋯O hydrogen bonds generate R 2 2(22) loops. Aromatic π–π stacking inter­actions [centroid–centroid distances = 3.8325 (12)–3.8600 (12) Å] also occur

Characteristics of patients operated for primary hyperparathyroidism at university hospitals in Türkiye: Differences among Türkiye's geographical regions

Purpose: The aim of this study was to define the clinical and laboratory characteristics of patients operated on for primary hyperpatathyroidism (PHPT) at university hospitals in Türkiye, and to investigate the differences in the clinical presentations of the disease between different geographical regions. Methods: Patients operated on for PHPT in the university hospitals of Türkiye were included in the study. The demographic, clinical, and laboratory findings and the operational data of the patients were investigated according to the whole country and to different geographical regions. Comparisons were performed according to whole country and regions. Results: A total of 1,162 cases were included in the study from different regions and 20 university hospitals. The mean age of patients was 52.4 ± 0.38 (mean ± standard error) in the general population of Türkiye. The rates of hypertension, urolithiasis, bone disease and 25-hydroxyvitamin D insufficiency were 35%, 18.6%, 67.6%, and 63%, respectively. The median parathormone (PTH), serum total calcium (Ca+2) and phosphorus value were 220 pg/mL (range, 70-2,500 pg/mL), 11.2 mg/dL (range, 9.5-11.2 mg/dL), and 2.4 mg/dL (range, 1-4.7 mg/dL), respectively. The median size of the adenomas resected was 16 mm (range, 4-70 mm). Significant differences were observed in the clinical and laboratory findings of the patients operated on due to PHPT between different geographical regions of Türkiye (P < 0.05). Conclusion: The clinical and laboratory characteristics of the patients with PHPT in different geographical regions of Türkiye differ. Furthermore, the general findings of the cases in Türkiye give us a hint that the severity of the disease here is somewhere between Eastern and Western countries. Copyright © 2016, the Korean Surgical Society

Identification of Radiopure Titanium for the LZ Dark Matter Experiment and Future Rare Event Searches

The LUX-ZEPLIN (LZ) experiment will search for dark matter particle interactions with a detector containing a total of 10 tonnes of liquid xenon within a double-vessel cryostat. The large mass and proximity of the cryostat to the active detector volume demand the use of material with extremely low intrinsic radioactivity. We report on the radioassay campaign conducted to identify suitable metals, the determination of factors limiting radiopure production, and the selection of titanium for construction of the LZ cryostat and other detector components. This titanium has been measured with activities of $^{238}$U$_{e}$~$<$1.6~mBq/kg, $^{238}$U$_{l}$~$<$0.09~mBq/kg, $^{232}$Th$_{e}$~$=0.28\pm 0.03$~mBq/kg, $^{232}$Th$_{l}$~$=0.25\pm 0.02$~mBq/kg, $^{40}$K~$<$0.54~mBq/kg, and $^{60}$Co~$<$0.02~mBq/kg (68\% CL). Such low intrinsic activities, which are some of the lowest ever reported for titanium, enable its use for future dark matter and other rare event searches. Monte Carlo simulations have been performed to assess the expected background contribution from the LZ cryostat with this radioactivity. In 1,000 days of WIMP search exposure of a 5.6-tonne fiducial mass, the cryostat will contribute only a mean background of $0.160\pm0.001$(stat)$\pm0.030$(sys) counts.Comment: 13 pages, 3 figures, accepted for publication in Astroparticle Physic

Anatomical classification of the shape and topography of the stomach

The aim of the study was to present the classification of anatomical variations of the stomach, based on the radiological and historical data. In years 2006–2010, 2,034 examinations of the upper digestive tract were performed. Normal stomach anatomy or different variations of the organ shape and/or topography without any organic radiologically detectable gastric lesions were revealed in 568 and 821 cases, respectively. Five primary groups were established: abnormal position along longitudinal (I) and horizontal axis (II), as well as abnormal shape (III) and stomach connections (IV) or mixed forms (V). The first group contains abnormalities most commonly observed among examined patients such as stomach rotation and translocation to the chest cavity, including sliding, paraesophageal, mixed-form and upside-down hiatal diaphragmatic hernias, as well as short esophagus, and the other diaphragmatic hernias, that were not found in the evaluated population. The second group includes the stomach cascade. The third and fourth groups comprise developmental variations and organ malformations that were not observed in evaluated patients. The last group (V) encloses mixed forms that connect two or more previous variations

Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV