Mutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance. Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.

Wild larkspur, Delphinium glaucum S. Watson, grows throughout most of Alaska along roadsides and in forests and is planted as an ornamental. Leaves containing distinct vein-clearing and chlorotic mosaic symptoms were first noticed on several D. glaucum plants during 2000 at the Georgeson Botanical Garden in Fairbanks, AK. Although affected plants continued to produce normal flowers, by 2008, the plants developed overall stunting. Initially, virus presence was determined by a general differential centrifugation extraction and concentration protocol followed by examination of the partially purified virus and leaf sap by electron microscopy. Filamentous particles approximately 725 nm long were observed. Virion protein extractions analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a putative coat protein (CP) of ~35 kDa. Potyvirus identity (family Potyviridae) was confirmed with universal potyvirus antiserum in western blots and ELISA assays (Agdia, Inc., Elkhart, IN). Exotic larkspur plants, D. elatum L., growing next to diseased D. glaucum plants, did not exhibit symptoms nor were they positive for potyvirus when tested serologically as described previously. Total RNA was extracted from potyvirus-infected leaves and used in reverse transcriptase-PCR assays that specifically targeted potyviruses (2,4) to generate genomic segments for identification and sequence analysis. Fragments representing portions of the helper component protease gene, HC-Pro (~700 bp), the cylindrical inclusion gene, CI (~700 bp), and the 3′-end (~1.7 kbp) were purified, cloned, sequenced, and deposited in GenBank (Accession Nos. FJ349329, FJ349328, and FJ349327, respectively). The sequenced 3′-end (1,674 nt) revealed a partial nuclear inclusion protein gene, NIb (1 to 630 nt), a CP gene (631 to 1,443 nt), and a 3′-untranslated region (1,447 to 1,674 nt) attached to a poly (A) tail. Blast searches in GenBank for percent identities of the nucleotide and amino acid comparisons resulted in highest similarities in conserved regions among members in the genus Potyvirus. For example, the highest CI, CP, and HP amino acid identities (0 gaps) were 67% with Potato virus A (Accession No. AF543709), 74% with Araujia mosaic virus (Accession No. EF710625), and 65% with Potato virus A (Accession No. AJ131403), respectively. However, none of the identities were sufficient for inclusion with an existing potyvirus species, whereby the CP amino acid sequence identity must be at least 80% (1). Mechanical transmission of purified virus to Chenopodium amaranticolor, C. quinoa, D. elatum, D. glaucum, and Nicotiana benthamiana seedlings was unsuccessful. We conclude that the isolated virus is a new species in the genus Potyvirus and propose the name Delphinium vein-clearing virus (DeVCV). To our knowledge, this is the first report of a virus isolated from D. glaucum and is representative of the growing number of viruses found in native plants (3). The distribution of DeVCV-infected larkspur is not known in managed or natural ecosystems. Identification of new viruses from native plants is important, in that, the host plant may act as a virus reservoir for transmission to other ornamental and crop plants. References: (1) P. H. Berger et al. Family Potyviridae. Page 819 in: Virus Taxonomy-8th Report of the ICTV. C. M. Fauquet et al., eds. Elsevier Academic Press, San Diego, CA, 2005. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) I. Cooper and A. C. Jones. Adv. Virus Res. 67:1, 2006. (4) C. Ha et al. Arch. Virol. 153:25, 2008.

Peonies (Paeonia sp.) are highly valued for their large showy flowers in home gardens and commercially in the cut flower industry. In 2007, scattered peony (Paeonia lactiflora 'Sarah Bernhardt') plants cultivated on small plots at the University of Alaska Experimental Station in Fairbanks displayed distinct leaf ringspot patterns. Symptoms were more severe during the cooler months of the growing season (June and September), with symptom remission in the intervening warmer months. Leaf samples from six symptomatic plants were collected in July and from 20 symptomatic plants in September and assayed for viruses. Leaf samples (1 g) were assayed with a general protocol for plant virus extraction and partial purification with differential centrifugation followed by protein detection on stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1). No distinct proteins indicative of viral coat protein(s) were detected. Tomato spotted wilt virus (TSWV) and Tobacco rattle virus (TRV), known pathogens of peony, were then specifically targeted. Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and used as the template for reverse transcription (RT)-PCR with random primers. TSWV was not detected by RT-PCR with tospovirus group-specific primers (Agdia, Inc., Elkhart, IN). A nested set of primers designed from the TRV 16-kDa protein gene on RNA1 (4) amplified an ~600-bp fragment from one of the symptomatic plants. This DNA was directly sequenced (GenBank Accession No. FJ357572) and BLAST searches in GenBank revealed as much as 95% nucleotide (nt) identity with TRV accessions J04347 and X03685. Additional primer pairs specific for TRV (2) amplified overlapping fragments with expected sizes of ~818, ~515, and ~290 bp from the 29- and 16-kDa protein genes on the 3'-end of RNA1 that were directly sequenced. Assembly of these sequences in Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI) resulted in a 1,422-nt sequence (Accession No. FJ357571) and Clustal X analysis (3) showed 93 to 94% nt identity to TRV isolates, -ORY (AF034622), -PpK20 (AF314165), -Pp085 (AJ586803), and -SYM (D00155). Mechanical inoculation of partially purified virions from the confirmed TRV-infected peony plant to Nicotiana benthamiana gave no symptoms to occasional ringspots, faintly curled leaves, and chlorotic blotches on N. tabacum 'Samsun', and local lesions on Chenopodium amaranticolor. TRV infection of these hosts was confirmed by RT-PCR. With electron microscopy, rod-shaped particles similar to TRV with a distinct central canal characteristic of TRV were seen occasionally only from inoculated N. benthamiana. On the basis of the biological and molecular data, we have determined the virus in the peony to be an isolate of TRV, tentatively named TRV-Peony. TRV was confirmed in only one other peony based on a sequenced 290-nt PCR fragment with 95% identity with the sequence from the other TRV-infected peony. Lack of TRV detection in the other symptomatic peonies was possibly due to low viral concentrations and interfering plant substances. Documentation of TRV in peonies is especially important to help avoid distribution of virus-infected vegetative propagation material. To our knowledge, this is the first report of TRV in this host in Alaska, but also of this virus in Alaska. References: (1) L. C. Lane. Methods Enzymol. 118:687, 1986. (2) D. J. Robinson. J. Phytopathol. 152:286, 2004. (3) J. D. Thompson et al. Nucleic Acids Res. 24:4882, 1997. (4) F. Van Der Wilk et al. Eur. J. Plant Pathol.100:109, 1994.

Meltwater ponds in the Victoria Valley and in the Labyrinth at the head of the Wright Valley of Victoria Land were sampled in January (summer) and October (late winter) of 2004 to establish their geochemistry and stratification, and to compare this with that of coastal meltwater ponds at a similar latitude near Bratina Island. In summer, vertical profiles were measured in 14 ponds; 10 were thermally stratified (maximum ΔT = 11.5°C) and 12 demonstrated a conductivity increase (∼25x) in the lowest 10–20 cm of the water column. When 11 of these ponds were resampled in October, the ice columns were stratified with respect to conductivity and five ponds had highly saline (up to 148 mS cm−1), oxygenated basal brines present under the ice. Basal brines and summer melt waters were Na-Cl dominated, and Victoria Valley pond meltwaters were enriched in Ca relative to the Labyrinth ponds. Early gypsum precipitation directs the chemical evolution of residual brine during freezing. These ponds were enriched in NO3 relative to the coastal ponds at Bratina Island, due to dissolution of nitrate-bearing soil salts, and the reduced influence of marine aerosols and biological productivity on pond chemistry.

The geochemistry and vertical stratification of shallow meltwater ponds at 78 degrees S near Bratina Island (McMurdo Ice Shelf) have been determined for late winter (October) and summer (January) conditions as part of the Latitudinal Gradient Project. Of the five frozen ponds investigated in October, all were stratified with respect to conductivity, and three had highly saline basal brines beneath the ice at temperatures of -16 to -20 degrees C. In the ice column, inclusions of saline fluid were observed in channels between ice crystals; the abundance increasing with depth and decreasing ice crystal size. In January, seven of the ten ponds investigated (including ponds sampled in October) retained conductivity stratification, whereas significant thermal stratification was observed in only three ponds (maximum Delta T = 5.5 degrees C). Basal brines, ice and meltwaters were Na-Cl or Na-SO4 dominated. FREZCHEM52 modelling, supported by changes in ion ratios, indicated that the precipitation of mirabilite (Na2SO4 center dot 10H(2)O) and gypsum (CaSO4 center dot 2H(2)O) during progressive freezing is an important determinant in chemical evolution of the basal brine. High pH (8.8-11.2) and over-saturation with respect to dissolved oxygen (> 20 mg L-1) in summer, and the presence of sulphide ions in basal brines in winter, occurred in those ponds which experienced high biological productivity during the summer months.

A novel multiple access coding approach for mobile networked systems is presented based on complex quadraphase coding. matched error control coding and channel adaptation. This approach provides an adaptive multiple-access communications capability where residual spreading gain. coding gain. data rates and channel loading are variables. Based on a blind estimate of the channel state. these parameters can be optimised to satisfy user Quality of Service Requirements. With these techniques, the foundation is laid for a new class of Collaboratively Coded Multiple Access Systems that can operate stand-alone or as hybrid CDMA/CCMA communications systems. A principal advantage of this approach is that high user and service density can be accommodated while the overall system complexity can be managed to tractable levels.

A practical approach is described for adaptively multiplexing in real-time T of N users in a multi-channel communications system using a majoritive logic multiplexer and quasi-othogonal binary coding. The adaptive multiplexer employs the users' class of service and channel noise constraints to optimize T and fidelity criteria.

Silica scaling on the surface of a flat plate and a vertical cylinder has been investigated in a water tunnel, where fluid flow and silica colloid size conditions can be controlled. Development of the test rig is described and some preliminary results are presented. Ultimately, the aim of this work is to characterise the lmk between fluid flow characteristics, silica colloid particle size and the scaling process, and to thereby understand the fundamental processes involved in silica scaling in geothermal systems.

Understanding the biogeochemical behaviour of arsenic ill the weathering and shallow Subsurface environment depends critically upon determining the nature and distribution of the chemical species present in natural waters. To this end, coupled ion chromotography-inductivcly coupled plasma mass spectrometry (IC-ICP-MS) is widely used, though species fractionation during ultrasonic nebulization, and matrix-dependent ionization in the plasma are analytical issues that need to be addressed. Hexapole collision cell technology is shown to be effective ill Suppressing chloride-based polyatomic interferences. Irrespective of the analytical technique used, As(III)/As(V) ratios of natural waters may change Substantially during storage due to (1) differential adsorption of arsenic species oil hydrated ferric oxides (HFOs); and (2) microbial activity. A wide range of apparently contradictory speciation changes observed by various workers call be rationalized in terms of the differences of microbial consortia Present in different water samples. Arsenic speciation in certain water types call be stabilized for days or even weeks by combined filtration, acidification and refrigeration whilst the addition of EDTA and the use of 0.1 mum filters is indicated for iron-rich waters and waters with high activities of redox-active bacteria, respectively. Although the use of hydrochloric acid has been reported elsewhere as resulting ill the apparent oxidation of As(III), we show that for certain water types it acts as all extremely effective preservative of arsenic speciation.

We have developed a new signal-dependent representation which captures, with a few KL vectors and transform coefficients, the perceptually and phonetically important structure of the spectral envelope. Together with a mixed excitation strategy with some novel features, this representation has been applied to the analysis, synthesis, and coding of speech with good results in the 3.5kbps range.

For Part I see ibid., vol.1, no.2, p.180-95 (1993). In Part I of this paper, the authors introduced an approach to the representation of the speech spectral envelope which makes use of the Karhunen-Loeve (KL) transformation of acoustic subword segments. This signal-dependent representation captures, with a few KL vectors and transform coefficients, the perceptually and phonetically important structure of the spectral envelope. Here the authors apply this representation to the analysis, synthesis, and coding of speech. They propose simple quantization and coding strategies for the KL representation vectors as well as for the resulting transform coefficients. The resulting technique is a variable rate encoding scheme which achieves good speech quality at an average rate of 3.5 kb/s. >

An approach to modeling and capturing the time-varying structure of the spectral envelope of speech is reported. Acoustic subword decomposition and the Karhunen-Loeve transform (KLT) are used to extract and efficiently represent the highly correlated structure of the spectral envelope. Integration of the KLT with acoustic subword modeling provides concise representation of both steady-state and dynamic features of the spectra in a unified framework that very effectively captures acoustic-phonetic patterns. The physiological and perceptual basis for the approach, the frame-based and acoustic-subword-based spectral representation, and applications to speaker-dependent recognition are presented. The performance of the recognition algorithm based on this approach compares favorably with that of other techniques.< >

A mathematical model has been developed for tracking spectral transitions within the spectral envelope of a speech signal. This technique incorporates linguistic knowledge into a mathematical framework to determine time-varying acoustic-phonetic features and describe formant transitions. The proposed model is quite robust and is capable of extracting not only rapid spectral movement, but also smoother spectral transitions that occur in vowel and sonorant sequences. This basic approach has been previously used to extract steady-state acoustic-phonetic features across spectrally homogeneous regions and to perform speaker dependent recognition in which quite successful results were attained in clean as well as noisy speech. It has now been augmented to capture the dynamics of spectral acoustic-phonetic features.<>

The authors propose a novel approach to the modeling and estimation of the speech spectral envelope over acoustic subwords that exhibits robust performance in noise. The technique exploits the underlying signal structure of speech to improve parameter estimates, and it uses the perceptual properties of hearing to decrease the computational requirements in a perceptually meaningful way. The approach provides a considerable speech quality improvement over other methods.< >

This study was performed to test the feasibility of using a biodegradable delivery system for the local controlled release of sodium fluoride as a mechanism for the stimulation of local bone growth. Sodium fluoride grains were mixed with poly-DL-lactic acid (PLA), and the mixture was then dissolved in acetone. After driving off the solvent, sheets of this material were rolled into rods 3.2 mm in diameter and 28.6 mm long. These were inserted into the intramedullary canals of the right femora of adult New Zealand White rabbits. A sham implant, made the same way but containing no fluoride, was inserted into the contralateral left leg. The effects of three different concentrations of sodium fluoride, 0.125 g NaF/g PLA, 0.250 g NaF/g PLA, and 0.500 g NaF/g PLA, were determined after a 4-week implantation period. In a second set of experiments, the concentration was fixed at 0.500 g NaF/g PLA with implantation durations of 4, 8, and 16 weeks. Expressed in terms of property ratios (experimental/control), bending strengths of the femora were significantly raised in most groups with the notable exception of Group 3 (0.500 g NaF/g PLA, 4 weeks). Cross-sectional area ratios in the distal femora were increased in all groups compared to right/left values for a normal unoperated group (p less than 0.05), with the highest ratio being 1.50 (SD = 0.44) for Group 4 (0.500 g NaF/g PLA, 8 weeks). Only Group 4 animals showed significant (p less than 0.05) increases in midshaft area ratio and none had responses proximally. Growth rates, measured by sequential fluorochrome labeling of bone followed the area ratio data and were only significant for the distal femora.

The authors have developed a very successful new approach to automatic speech recognition which incorporates speech knowledge into a mathematical framework and does not require a computationally intensive time alignment/dynamic programming scheme. They transform the speech signal into the spectral domain, segment it into sub-word units and, in turn, perform an additional transformation in the spectral domain to capture the spectral structure within each sub-word unit. The system was shown to perform robustly in hand segmented whole word digit recognition in clean as well as noisy speech. They have now augmented the system with an automatic acoustic sub-word segmentation routine and tested the performance of this integrated system with the TI isolated word database and the confusable E set

CHAG, that is, porous hydroxyapatite hydrothermally converted from the calcium carbonate exoskeleton of a coral (genus Goniopora), has been shown to be effective as a scaffold for bone ingrowth. The large pores in the material, however, resulted in low compressive strengths. Compressive testing was performed to assess the changes in mechanical properties by coating the internal surfaces of CHAG with DL-PLA. Plugs of CHAG with thick (3:1 chloroform to DL-PLA by weight), medium (10:1), and thin (30:1) coatings as well as uncoated CHAG were then implanted transcortically in the proximal third of the diaphysis of rabbit tibiae to assess the in vivo response. The mechanical tests demonstrated significantly improved compressive strength, stiffness, and energy absorption for coated specimens compared with uncoated specimens. Coated specimens were not significantly different from canine tibial cancellous bone in strength and stiffness although they achieved only 36% of the energy absorption capacity. Specimens from rabbit tibiae were harvested at 3, 12, and 24 weeks for interface shear strength determination and contralaterally for histological and histomorphometric assessment. At 12 weeks, uncoated CHAG plugs developed an average ultimate interface shear stress of 26.7 MPa compared with 17 MPa for specimens with 30:1 coatings and 8 MPa for specimens with 10:1 and 3:1 coatings. At 24 weeks, there were no significant differences in shear stress between any of the specimens. Histomorphometric assessments showed that the ratio of area fraction of new bone to area fraction of new bone and void space increased from 68-70% for specimens with 3:1 and 10:1 coatings at 3 weeks to 85.5-89.5% at 24 weeks. In comparison, uncoated and 30:1 specimens had area fraction ratios of about 82% at 3 weeks and 93% at 24 weeks. Histologic sections demonstrated direct apposition of new bone to both the coating and the hydroxyapatite as well as degradation of the coating.

A porous hydroxyapatite material hydrothermally converted from the calcium carbonate exoskeleton of the coral, genus Goniopora (CHAG) was either microcoated using polymethylmethacrylate (PMMA) or polylactic acid (PLA) to cover all internal surfaces, or externally coated to produce a shell, with the objective of reducing the brittleness of the material. Compressive testing showed that while CHAG, externally coated with PMMA, showed the largest increases in strength, stiffness, and energy absorption, the uncoated hydroxyapatite core cracked at low loads although the shell remained intact. CHAG internally microcoated with PMMA showed a 3.84 increase in compressive strength while specimens with internal PLA coatings had 1.81 times the compressive strength of uncoated CHAG specimens. Compared to the mechanical properties of cancellous graft material, specimens internally microcoated with either polymer could be produced having properties equivalent to or greater than those of cancellous graft.