Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Characterizing the Smell of Marijuana by Odor Impact of Volatile Compounds: An Application of Simultaneous Chemical and Sensory Analysis

Recent US legislation permitting recreational use of marijuana in certain states brings the use of marijuana odor as probable cause for search and seizure to the forefront of forensic science, once again. This study showed the use of solid-phase microextraction with multidimensional gas chromatography—mass spectrometry and simultaneous human olfaction to characterize the total aroma of marijuana. The application of odor activity analysis offers an explanation as to why high volatile chemical concentration does not equate to most potent odor impact of a certain compound. This suggests that more attention should be focused on highly odorous compounds typically present in low concentrations, such as nonanal, decanol, o-cymene, benzaldehyde, which have more potent odor impact than previously reported marijuana headspace volatiles.     

Syringe-push membrane absorption as a simple rapid method of urine preparation for clinical proteomics

Background

The analysis of urinary proteome might reveal biomarkers of clinical value. However, current methods of urine preparation for down-stream proteomic analysis are complicated, time-consuming, and/or expensive. This study aims to develop a robust, simple, inexpensive and readily accessible urine preparation method to facilitate clinical proteomic workflow.

Result

Syringe-push membrane absorption (SPMA) was successfully developed by a combination of 5-ml medical syringe and protein-absorbable membrane. Comparing three membranes i.e., nitrocellulose, polyvinylidene difluoride (PVDF) and Whatman no.1, nitrocellulose combined with SPMA (nitrocellulose-SPMA) provided the greatest quality of proteome profile as demonstrated by 2-DE. The quality of the proteome profile and the performance of nitrocellulose-SPMA were systematically compared with three current methods of urine preparation (i.e., ultrafiltration, dialysis/lyophilization and precipitation). While different methods of urine preparation provided comparable proteome quality, nitrocellulose-SPMA had better working performance due to acceptable recovery yield, less workload, short working time, high accessibility and low unit cost. In addition, protein absorbed on nitrocellulose harvested from the SPMA procedure could be stored as a dried membrane at room temperature for at least 1-month without protein degradation or modification.

Conclusions

SPMA is a simple rapid method of preparing urine for downstream proteomic analysis. Because of it is highly accessible and has long storage duration, this technique holds potential benefit for large-scale multi-center research and future development of clinical investigation based upon urinary proteomic analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9087-4) contains supplementary material, which is available to authorized users.     

Enrichment of the basic/cationic urinary proteome using ion exchange chromatography and batch adsorption

Anionic or acidic proteins are the main compositions of normal urinary proteome. Efforts to characterize human urinary proteome, thus, have focused mainly on the anionic compartment. The information of cationic or basic proteins present in the normal urine is virtually unknown. In the present study, we applied different methods to enrich cationic urinary proteome. Efficacies of these methods were compared using equal volume (1 L) of urine samples from the same pool obtained from 8 normal healthy individuals. Cation exchange chromatography using RESOURCE-S column provided the least amount of the recovered proteins, whereas batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 4.8) provided the greatest yield of protein recovery. The recovered proteins were then resolved with 2-DE (pI 7-11) and identified by peptide mass fingerprinting using MALDI-TOF MS. There were several isoforms of immunoglobulin heavy and light chains enriched by these methods. In addition, three isoforms of interferon alpha-3 (IFNalpha3) and six isoforms of eosinophil-derived neurotoxin (EDN), were also enriched. The enrichment of IFNalpha3 and EDN was particularly effective by batch adsorption using SP Sepharose 4 Fast Flow beads equilibrated with acetic acid (pH 6.0). Initial depletion of anionic components using DEAE batch adsorption reduced the recovery yield of these two proteins and did not improve recovery of any other cationic urinary proteins. We conclude that batch adsorption using SP Sepharose Fast Flow beads equilibrated with acetic acid (pH 6.0) is the method of choice to examine the basic/cationic urinary proteome, as this protocol provided the satisfactory yield of protein recovery and provided the greatest amount as well as maximal number of IFNalpha3 and EDN isoforms. Our data will be useful for further highly focused study targeting on cationic/basic urinary proteins. Moreover, the techniques described herein may be applicable for enrichment of cationic proteomes in other body fluids, cells, and tissues.     

Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood‐feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one‐year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west‐central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00‐23:00 and a smaller peak at 01:00‐03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy.     

Bacteria can promote calcium oxalate crystal growth and aggregation

Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 10⁵ CFU/ml E. coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation–sedimentation study) confirmed their promoting effects on both growth (20.17 ± 3.42, 17.55 ± 2.27, 16.37 ± 1.38, and 21.87 ± 0.85 % increase, respectively) and aggregation (57.45 ± 2.08, 51.06 ± 5.51, 55.32 ± 2.08, and 46.81 ± 3.61 % increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E. coli, K. pneumoniae, S. aureus, and S. pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.     

Antimicrobial resistance markers as a monitoring index of gonorrhoea in Thailand

Multiplex PCR was applied to explore the antimicrobial-resistance profiles of 145 gonococci isolated from Bangrak Hospital, Thailand in 2007. All isolates were clearly identified for the plasmid-mediated resistant types of penicillin (Asia, Africa and Toronto) and tetracycline (American and Dutch). This method can also predict the decreased susceptibility to ciprofloxacin by detection of Ser-91 mutation. Prevalence rates of penicillinase-producing Neisseria gonorrhoeae (PPNG) and high-level tetracycline-resistance N. gonorrhoeae (TRNG) were shown to be high as 82.1% and 84.1%, respectively. Most PPNG carried the Africa-type (78.2%) while the American-type (61.8%) was harboured in most TRNG. Mono- and triple-resistance patterns were presented in 2.6% and 79.5% of male, 20.7% and 62.1% of men who have sex with men (MSM), 0% and 75.0% of female, and 10% and 70% of female sex workers (FSW). Additionally, the rate of the Dutch type was high in patients among the age of 35-44 years (57.1%) and female patients (43.8%). The changing types of plasmids have been noticed during the time period of study. The multi-resistance patterns of the gonococcal isolates can be used as an epidemiological index of gonorrhoea and human sexual behaviours. This information will support the management of individual patients as well as the public health surveillance.     

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?1 at 30 °C. Since adenine is deaminated ～103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.     

Human papillomavirus genotypes among infected Thai women with different cytological findings by analysis of E1 genes

Cervical cytological data may not be sufficient for cervical cancer screening and prevention. In this project, we determined HPV genotype among infected Thai women with different cytological findings by characterization of E1 genes. Five hundred and thirty-five specimens were tested by PCR amplification of the E1 genes. HPV genotypes were determined by sequencing, comparison with the GenBank database and were analyzed in relation to different cytological findings. HPV-DNA by PCR were typed and revealed 32 different genotypes. HR-HPV (HPV16, 18 or 52) was detected in all samples with cervical cancer cytology. HPV16 was most prevalent irrespective of cervical cytology. Moreover, HPV31 and 52 were most prevalent in the HSIL and LSIL groups whereas HPV66 was found mostly in the LSIL group. The LSIL group displayed the highest variation of HPV genotypes. Moreover, HPV31 and 52 predominated in the HSIL and LSIL groups especially HPV52 which was found in cancer samples. We hoped that these data of HPV genotypes can be used as preliminary data of HPV in Thailand and can serve as basic data for future research into the HPV genotype in south-east Asia.     

Optical chemosensors for Cu(II) ion based on BODIPY derivatives: an experimental and theoretical study

Two BODIPY derivatives for Cu²⁺ ion chemosensors containing 4-[2-(diethylamino)-2-oxoethoxy]phenyl (BDP1) and 3,4-bis[2-(diethylamino)-2-oxoethoxy]phenyl (BDP2) were synthesized by coupling appropriate N,N-diethyl-2-(4-formylphenoxy)acetamide and 2,4-dimethylpyrrole moieties in the presence of trifluoroacetic acid and anhydrous dichloromethane at room temperature. The binding abilities between these chemosensors and 50 equivalents of Na⁺, K⁺, Ag⁺, Ca²⁺, Fe²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺ and Pb²⁺ ions were studied using UV-vis and fluorescence spectrophotometry. The results show that, compared to other ions, both the UV-vis absorption and fluorescence emission intensity of BDP2 decreased dramatically when Cu²⁺ ion was added. To explain this behavior, ab initio quantum chemical calculations were performed using correlated second-order Møller-Plesset perturbation theory (MP2/LanL2DZ). The calculated orbital energies indicated that the decrease in UV-vis absorption intensity and the quenching of fluorescene emission were due to the single-electron reduction of Cu²⁺ to Cu⁺ ion.