Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer''s diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.     

Toxicity of baited spinosad formulations to Ceratitis capitata: from the laboratory to the application

GF‐120, a fruit fly bait designed to attract and kill adult fruit flies, was tested in the laboratory and outdoors to determine effects of pre‐treatment diet and bait aging on mortality of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Two spinosad‐based compounds, GF‐120 and Tracer^® Ultra, had generated two distinctive dose–mortality responds, with LC⁸⁰, LC⁹⁰, and LC⁹⁹ values of 2.4, 2.8, and 4.1 p.p.m., and 255, 479, and 1 143 p.p.m., respectively. The residues of GF‐120 drops, after feeding to the flies, generated 14.3% mortality. The droplet size of the baited spray plays an important role. The toxicity of large drops lasted more than that of small droplets. In the field, exposure to the sun further deteriorates the compound, which lost 50% of its toxicity within 6 days. Disappearance of the compound in the field, due to consumption by various insects, also played a role as 50% of the GF‐120 drops disappeared within 7 days. As mortality was directly related to the amount of insecticide eaten, the effect of GF‐120 depended on the feeding status of the flies: well‐fed flies were almost unaffected compared with starved ones.     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.     

Quality of life following parotidectomy for malignant and benign disease

Parotidectomy is performed for benign or malignant tumors and for selected benign inflammatory and autoimmune conditions. Possible associated complications include facial nerve paralysis, pain, loss of sensation, gustatory sweating, and facial scarring. Global quality of life in patients undergoing parotidectomy has not been reported. The implications of facial surgery with the catastrophic potential of facial nerve paralysis may severely affect quality of life. A quality-of-life study was conducted in patients undergoing parotidectomy for benign and malignant diseases to define the significance of associated morbidity and its impact on quality of life. A quality-of-life instrument was specifically created, based on the principles of the University of Washington Quality of Life questionnaire, and mailed to the patients. Questions addressed recognized complications of parotidectomy. Patient group results were compared for age above and below 45 years, sex, benign versus malignant disease, presence or absence of Frey syndrome, and presence or absence of benign pleomorphic adenoma. Forty-six percent of 125 patients meeting the study criteria fully replied to the questionnaire. The global health score was 3.5, corresponding with "good" to "very good." Except for local sensation, which had a score of 50, all other domains scored above 76. Change in appearance, gustatory sweating, and pain were reported by 70 percent, 57 percent, and 30 percent, respectively. Importance attributed to all domains except facial function was low. Pain was encountered significantly less in patients younger than 45 years of age, and scores for appearance were also highly significant in this age group. Postoperative sequelae were noted in the majority of patients. The dominant sequelae were altered sensation, change in appearance, Frey syndrome, and pain. A degree of permanent postoperative facial nerve impairment was reported by 10 patients. Nevertheless, overall, parotidectomy does not seem to severely affect quality of life.     

Quantitative estimate of mitochondrial [Ca2+] in stimulated motor nerve terminals

Peak values reported for mitochondrial matrix [Ca(2+)] following stimulation have ranged from micromolar to near-millimolar in various cells. Measurements using fluorescent indicators have traditionally used high-affinity dyes such as rhod-2, whose fluorescence would be expected to saturate if matrix [Ca(2+)] approaches millimolar levels. To avoid this potential problem, we loaded lizard motor terminal mitochondria with the low-affinity indicator rhod-5N (K(d) approximately 320 microM). During trains of action potentials at 50Hz, matrix fluorescence transients (measured as F/F(rest)) increased to a plateau level that was maintained throughout the stimulus train. This plateau of matrix [Ca(2+)] occurred in spite of evidence that Ca(2+) continued to enter the terminal and continued to be sequestered by mitochondria. When the stimulation frequency was increased, or when Ca(2+) entry per action potential was increased with the K(+) channel blocker 3,4-diaminopyridine (3,4-DAP), or reduced by lowering bath [Ca(2+)], the rate of rise of matrix [Ca(2+)] changed, but the plateau amplitude remained constant. Calculations demonstrated that the F/F(rest) measured at this plateau corresponded to a matrix [Ca(2+)] of approximately 1 microM. The high K(d) of rhod-5N ensures that this value is not a result of dye saturation, but rather reflects a powerful Ca(2+) buffering mechanism within the matrix of these mitochondria.     

Human Saliva-Derived Exosomes: Comparing Methods of Isolation

ExoQuick-TC^TM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.     

Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (～ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (～ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.     

Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): Temporal patterns and modulation by male condition

We determined the temporal pattern of female remating in the Mediterranean fruit fly, Ceratitis capitata, and how mating with sterile males affects remating. In addition, we examined the hypotheses that sterile male nutrition and age affect the subsequent receptivity of their mates. Temporally, female receptivity varied significantly throughout the experimental period. Relatively high levels of remating (14%) on the days following the first copulation were followed by a decline, with a significantly low point (4.1%) 2 weeks after mating. Subsequently, receptivity is gradually restored (18%) 3 and 4 weeks after the initial copulation. When females were first mated to sterile males, significantly higher remating percentages were recorded. The ability of sterile males to inhibit receptivity of both wild and laboratory reared females on the day of first mating was significantly improved when they were fed a nutrient rich diet. Male age at first mating also affected female receptivity: sterile males of intermediate age (11 days old) inhibited female remating significantly more than younger or older flies. Although further studies are needed to determine the relative roles of natural and sexual selection in modulating patterns of female sexual receptivity, the Sterile Insect Technique may be improved by releasing well nourished, older sterile males.     

Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ～10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ～ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.