Evaluation and Validation of a Real-Time PCR Assay for Detection and Quantitation of Human Adenovirus 14 from Clinical Samples

In 2007, the Centers for Disease Control and Prevention (CDC) reported that Human adenovirus type 14 (HAdV-14) infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10² to 10⁷ copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.     

Life history and host range of Sauris nr. purpurotincta,an unsuitable biological control agent for Chinese tallowtree

Foreign surveys in China discovered a defoliating insect species feeding on the leaves of Chinese tallowtree (Triadica sebifera), an invasive weed of the southeastern U.S.A. The life history of this species, Sauris nr. purpurotincta (Lepidoptera: Geometridae), was examined and larval no-choice and adult multiple-choice host range tests were conducted in quarantine to evaluate their suitability for biological control of Chinese tallowtree. The results indicated that the larvae have five instars and require approximately 22 days to complete development to the adult stage. Host range tests indicated that the larvae could not feed and complete development on most species tested. However, 40% of the larvae survived when fed leaves of Hippomane mancinella, a state-listed endangered species in Florida, and all larvae survived when fed Morella cerifera, a common native species of the southeastern U.S.A. Multiple-choice oviposition tests indicated eggs were laid on leaves of both a south Florida native plant Gymnanthes lucida and Chinese tallowtree. Considering this broad host range, this species will not be considered further for biological control of Chinese tallowtree in the U.S.A.     

我国口岸截获芒果象属检疫性有害生物疫情分析

【目的】芒果象属昆虫是我国禁止进境的植物检疫性有害生物,广泛分布于非洲、北美洲和东南亚,寄主为芒果。其生活隐蔽,羽化孔未出现时看不出危害状,危害率可达30%~80%,严重影响芒果的产量和质量。近年来,我国口岸截获的芒果象甲的数量日益增多,表明该类检疫性有害生物传入我国的风险越来越大。【方法】运用Microsoft Excel工作表对2003—2015年全国口岸截获芒果象属有害生物的疫情进行统计分析。【结果】2003—2015年全国口岸共截获芒果象4156批次,22个直属局有截获记录。其中,截获芒果果核象甲3028批次,数量最多,占截获芒果象总批次的72.86%;芒果果实象甲和芒果果肉象甲相对较少,分别为837和291批次。截获芒果象的来源国主要为非洲和东南亚芒果象疫情分布国家和地区,且主要自进境旅客随身携带物中检出。【结论】我国口岸应加大对芒果象的检疫力度,保护我国农林业生产安全。     

Host range testing and life history of the defoliator Hymenomima nr. memor: an unsuitable biological control agent for Schinus terebinthifolia in the U.S.A.

Host range of larvae of Hymenomima nr. memor (Lepidoptera: Geometridae) was examined in quarantine to evaluate its suitability as a biological control of Brazilian peppertree, Schinus terebinthifolia. Brazilian peppertree, S. terebinthifolia is an environmental and agricultural weed from South America that had invaded many subtropical and tropical areas of the world including Florida and Hawaii, USA. Laboratory life history and quarantine host range studies of H. memor were conducted with no-choice feeding tests. These tests included eight species of the Anacardiaceae and one species of Sapindaceae. Larvae of H. memor had five to six instars with each head capsule width increasing by 1.68-X. Development time from neonate to adult was 46.7?±?2.2 days. In host range tests, neonates completed development to the adult stage on all non-target species, except Toxicodendron radicans. Moreover, developmental times were delayed and pupal weights were reduced for larvae fed Spondias purpurea leaves. Due to the broad host range exhibited by H. memor larvae, this species will not be considered as a biological control agent of S. terebinthifolia in the continental U.S.A.     

速生槐叶蘋入侵风险评估及管理措施分析

[目的]速生槐叶蘋是一种原产于巴西东南部的多年生漂浮型水生植物,广泛引种至世界各地后逃逸,侵入湖泊、河流和稻田等各种水生生境。通过开展速生槐叶蘋入侵风险评估,进而提出其管理措施,可为其检疫决策及防治提供科学依据。[方法]通过查阅梳理文献,分别对速生槐叶蘋的国内外分布、传播方式及入侵扩散历史、防治方法、生态适应性和抗逆性、生物学和遗传特性、危害性及利用价值等方面进行定性分析。并基于外来植物风险指标体系和判断标准,对4个指标层的17个指标和32个问题进行量化赋值,完成速生槐叶蘋的定量分析。[结果]速生槐叶蘋通过自然和人为传播方式已经入侵全世界57个国家地区,可通过无性繁殖快速分裂生长建立种群,其环境适应性和抗逆性强、竞争能力强、检疫鉴定和根除难度大,对经济、生态和社会造成巨大负面影响。速生槐叶蘋的入侵性R₁为72,适应性R₂为53,扩散性R₃为82,危害性R₄为81,风险值R为73.4,应归为严格禁止引入的植物。[结论]尽管速生槐叶蘋在中国的分布范围有限,但属于高入侵风险物种,其潜在的逃逸扩散风险和危害性不可忽视,应严格禁止引入。考虑到该种已经传入我国且有作为观赏植物的市场需求,应制定相应的风险管理制度,定期开展检疫、监测和灭除,防止其逃逸至野外定殖于其他还未发生的地区。     

An autoradiographic analysis of hypothalamic magnocellular neurons

Summary A light and electron microscopic autoradiographic analysis revealed that H³-valine infused into the lateral ventricle of normal and acutely dehydrated cats is preferentially taken up by the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus. Grain counts for these magnocellular neurons in normal unstressed cats were highest at one hour post infusion with a significant fall off by three hours. Uptake by the SON and PVN at one hour exceeded neighboring nuclear groups by a factor of 7 and 4 fold respectively. Electron microscopic autoradiographs from acutely dehydrated cats revealed the presence of emission grains in association with rough endoplasmic reticulum and large osmiophilic neurosecretory vesicles. In view of statistically significant uptake values and rapid turnover of H³-valine by SON and PVN in normal animals, coupled with emission tracks in direct association with underlying neurosecretory product in acutely dehydrated ones, it is speculated that valine may be an amino acid component of one or both of the neurophysins to which neurohypophyseal hormones are non-covalently linked.Supported by U.S.P.H.S. Grant NS 08171.U.S.P.H.S. Career Development Awardee K04 GM70001.The authors are deeply indebted to Dr. Finley P. Gibbs and Dr. Sandy Sorrentino, Jr. for their advice and assistance in statistically quantifying autoradiographic data.     

Available sulphydryl groups of mammalian ribosomes in different functional states

The numbers of sulphydryl groups on NH₄Cl-washed rat liver polyribosomes in different functional states were measured under carefully standardized conditions with ¹⁴C-labelled N-ethylmaleimide and ³⁵S-labelled 5,5-dithio-bis(2-nitrobenzoic acid). Ribosomes denatured with urea had 120 titratable sulphydryl groups, 60 on each subunit, whereas native ribosomes invariably showed fewer available sulphydryl groups. Ribosomes stripped of transfer RNA (S-type ribosomes) had 55 available sulphydryl groups. Ribosomes bearing the growing peptidyl-tRNA at the acceptor site had 41 sulphydryl groups available. If these A-type ribosomes were labelled with ¹⁴C-labelled N-ethylmaleimide and dissociated into subunits, 23 of the labelled sulphydryl groups were found on the 60 S subunit and 19 on the 40 S subunit. After translocation of the peptidyl-tRNA to the donor position on ribosomes (D ribosomes), the number of available sulphydryl groups increased to 72, of which 43 were on the 60 S subunit and 29 on the 40 S subunit. This demonstrates that both subunits participate in the change of peptidyl-tRNA from the A to D positions. When the D ribosomes were reacted with EF2 (elongation factor) and GTP, the available sulphydryl groups increased to 82; addition of EF2 alone or with GDP, GDPCP or ATP failed to cause this increase, which has accordingly been attributed to an energy-dependent conformational change in the ribosome.Ribosomes were reconstructed from subunits with poly(U) and Phe-tRNA. In the presence of poly(U) only, a ribosome with 55 available SH groups was formed, thus corresponding to the stripped ribosomes. When both poly(U) and Phe-tRNA were present, a ribosome was formed with 44 available sulphydryl groups, corresponding approximately to an A-type ribosome. Since no EF1 or GTP was used in reconstructing this ribosome, these data indicate that the conformation of A-type ribosomes is not dependent on EF1 or GTP, but is due to the presence of tRNA at the acceptor site.We therefore incline to the view that the observed changes in available SH groups reflect conformational changes, with an opening up of ribosome structure as it progresses from having the peptidyl-tRNA at the A position to the D position and then binds EF2 and GTP, followed by a restoration of the more compact from when the incoming aminoacyl-tRNA is then bound.     

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

4-Thiouridine (s⁴U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s⁴U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s⁴U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s⁴U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s⁴U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s⁴U. In the absence of an RLD, MMP1354 uses Cys²⁶⁵ and Cys²⁶⁸ located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S²⁻ is a physiologically relevant sulfur donor for s⁴U formation catalyzed by MMP1354 (K_m for Na₂S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s⁴U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.     

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m⁵C and m⁶₂A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m²G which is not affected at all and m³U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.     

Concurrent application of ethyl formate and 1‐methylcyclopropene to control Tetranychus urticae on exported sweet persimmons (Diospyros kaki Thunb. ‘Fuyu’)

Extending the shelf life of fruits during post‐harvest storage and eradicating pests associated with quarantine issues could together comprise the key steps toward expanding the exportation of sweet persimmon in South Korea. Here we firstly investigated the concurrent application of ethyl formate (EF), a methyl bromide alternative fumigant, which is currently considered a beneficial and safe fumigant in quarantine use, and 1‐methylcyclopropene (1‐MCP), an anti‐ethylene compound that is broadly used in post‐harvest systems, on sweet persimmon. We also suggest the proper fumigation methods to be follow when using these compounds. Tetranychus urticae, an important quarantine pest, was inoculated under the calyx of sweet persimmon, and the fruits were then fumigated using 35.0 mg L^?1 of EF for 6 h before and after treating with 1.0 μL L^?1 of 1‐methylcyclopropene for 24 h under storage conditions of 5°C. Our results showed that concurrent treatments with 1‐MCP and EF could be suitable for commercial purposes by extending shelf life, delaying color changes and softening, and offering complete control of the target pest, Tetranychus urticae.     

Purification and partial characterization of two genetic variants of placental alkaline phosphatase

The two most common variants of placental alkaline phosphatase, the F and S variants, were purified to homogeneity and characterized. Their molecular weights were determined by equilibrium ultracentrifugation and sodium dodecylsulfate polyacrylamide gel electrophoresis, which gave almost identical values for the two variants, 118,000 (F) and 119,000 (S). The amino acid compositions of the F and S variants presented here are found to be very similar. Differences between the two variants were found in specific activity (160 U/mg for F and 250 U/mg for S), isoelectric point (IP=4.5 for F and 4.7 for S), sedimentation coefficient (6.5×10^?13 sec for F and 6.4×10^?13 sec for S). Thus the structural differences observed for these enzyme variants seem to affect both the active site and the protein conformation.     

Covalent attachment of poly (U) template to 40S - mammalian ribosomal subunits

When 40S subunits are irradiated at 254nm in presence of [³H] poly (U), formation of a 40S subunit-poly (U) complex can be demonstrated either by filtration technique at low Mg⁺⁺ concentration or by polyacrylamide gel electrophoresis. No stable complex was detected using unirradiated samples under the same conditions. Electrophoresis of this complex in the presence of dodecyl sulfate showed that part of the poly (U) directly associates with 18S RNA. This association is not through proteins, since it is not disrupted by pronase treatment.     

Endo-β-1,4-d-mannanase is efficiently produced by Sclerotium (Athelia) rolfsii under derepressed conditions

A number of wild-type isolates of Sclerotium (Athelia) rolfsii and S. coffeicola were studied for their ability to produce endo-β-1,4-mannanase, endo-β-1,4-xylanase, and endo-β-1,4-glucanase activity when grown on cellulose- or glucose-based media. Whereas the presence of the inducer cellulose was strictly necessary for increased xylanase and endoglucanase production by both S. rolfsii (208 and 599 U ml⁻¹, respectively) and S. coffeicola (102 and 330 U ml⁻¹, respectively), elevated activities of mannanase (up to 96.6 U ml⁻¹) were formed even when employing glucose as the only carbohydrate substrate. Significant production of mannanases as well as of auxiliary mannan-degrading enzymes (β-mannosidase, β-glucosidase, α-galactosidase, acetyl esterase) was only observed, however, under derepressed conditions, i.e. after glucose had been consumed from the medium. By applying a fed-batch strategy, in which a glucose solution was continuously fed to a cultivation of S. rolfsii CBS 191.62 so that the glucose concentration in the medium never exceeded a certain low, critical value, production of mannanase could be almost doubled as compared to a batch cultivation on glucose (462 versus 240 U ml⁻¹). Mannanase preparations produced by several S. rolfsii and S. coffeicola strains under inductive and noninductive conditions (i.e. using cellulose or glucose as the substrates, respectively) were further analyzed with respect to the patterns of isoformic mannanases formed under these different growth conditions. Multiple mannanases were secreted by all isolates investigated. Certain mannanase isoenzymes were only formed by S. rolfsii in the presence of the inducer cellulose, indicating a complex and separated regulation of the synthesis of mannanase isoenzymes in this strain.     

Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation

In order to obtain an industrial strain with higher chitosanase yield, the wild strain Bacillus sp. S65 cells were mutated by a novel mutagen, nitrogen ion beam, with energy of 15 keV and dose ranging from 2.6 × 10¹⁴ to 5.2 × 10¹⁵ ions/cm². One mutant, s65F5 with high yield of chitosanase was isolated. Results showed that the production of chitosanase of s65F5 was dramatically increased from 4.1 U/ml in s65 to 25 U/ml by ion beam implantation, while the fermentation time was shortened from 72 to 56 h, both of which greatly increased efficiency and reduced the cost of industrial production. Besides, the mutagenic effects of low-energy ion beam on survival rate showed characteristic down–up–down pattern, which was different from the traditional mutagens such as UV and γ-ray and the possible mutation mechanism was discussed.     

Energy Sprawl or Energy Efficiency: Climate Policy Impacts on Natural Habitat for the United States of America

Concern over climate change has led the U.S. to consider a cap-and-trade system to regulate emissions. Here we illustrate the land-use impact to U.S. habitat types of new energy development resulting from different U.S. energy policies. We estimated the total new land area needed by 2030 to produce energy, under current law and under various cap-and-trade policies, and then partitioned the area impacted among habitat types with geospatial data on the feasibility of production. The land-use intensity of different energy production techniques varies over three orders of magnitude, from 1.9–2.8 km²/TW hr/yr for nuclear power to 788–1000 km²/TW hr/yr for biodiesel from soy. In all scenarios, temperate deciduous forests and temperate grasslands will be most impacted by future energy development, although the magnitude of impact by wind, biomass, and coal to different habitat types is policy-specific. Regardless of the existence or structure of a cap-and-trade bill, at least 206,000 km² will be impacted without substantial increases in energy efficiency, which saves at least 7.6 km² per TW hr of electricity conserved annually and 27.5 km² per TW hr of liquid fuels conserved annually. Climate policy that reduces carbon dioxide emissions may increase the areal impact of energy, although the magnitude of this potential side effect may be substantially mitigated by increases in energy efficiency. The possibility of widespread energy sprawl increases the need for energy conservation, appropriate siting, sustainable production practices, and compensatory mitigation offsets.     

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA^Lys _UUU, tRNA^Glu _UUC and tRNA^Gln _UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm⁵s²U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm⁵ side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm⁵s²U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm⁵U and s²U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm⁵s²U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA^Lys _UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm⁵ and s² wobble uridine modification that could account for growth impairment and mainly originates from tRNA^Lys _UUU hypomodification and malfunction.     

Flow of microbially fixed nitrogen in a model ecosystem

Summary The preceding observations showed that both Azotobacter and a mixed flora with Azotobacter are about equally capable of fixing atmospheric nitrogen. During subsequent transfer of the nitrogen there was a distinct differentiation between movement of the newly fixed nitrogen and the nitrogen that was already present in tissues introduced in the beginning of the experiment. Pathways and transfer coefficients of both N¹⁴ and N¹⁵ were different in the presence of the different microbial populations. Turnover was more complex and in general slower in the presence of a mixed population than with pure Azotobacter. The lower transfer coefficients in the mixed populations may reflect more recycling within these populations than in pure cultures of Azotobacter. Denitrification losses upon acidification were negligible for N¹⁴ but appreciable for N¹⁵. The effect of acidification tended to be greater in systems with pure cultures of Azotobacter than in systems with a mixed culture.Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.