Chalcone-Based Synthesis of Tetrahydropyridazines via Cloke-Wilson-Type Rearrangement-Involved Tandem Reaction between Cyclopropyl Ketones and Hydrazines.

A facile and efficient approach for the synthesis of multisubstituted tetrahydropyridazines starting from cyclopropyl ketones and hydrazines has been developed. The transformation is chalcone-based and takes place via a Cloke-Wilson-type rearrangement-involved tandem reaction catalyzed by TfOH in HFIP.

Altered dynamic network interactions in children with ASD during face recognition revealed by time-varying EEG networks.

Although the electrophysiological event-related potential in face processing (e.g. N170) is widely accepted as a face-sensitivity biomarker that is deficient in children with autism spectrum disorders, the time-varying brain networks during face recognition are still awaiting further investigation. To explore the social deficits in autism spectrum disorder, especially the time-varying brain networks during face recognition, the current study analyzed the N170, cortical activity, and time-varying networks under 3 tasks (face-upright, face-inverted, and house-upright) in autism spectrum disorder and typically developing children. The results revealed a smaller N170 amplitude in autism spectrum disorder compared with typically developing, along with decreased cortical activity mainly in occipitotemporal areas. Concerning the time-varying networks, the atypically stronger information flow and brain network connections across frontal, parietal, and temporal regions in autism spectrum disorder were reported, which reveals greater effort was exerted by autism spectrum disorder to obtain comparable performance to the typically developing children, although the amplitude of N170 was still smaller than that of the typically developing children. Different brain activation states and interaction patterns of brain regions during face processing were discovered between autism spectrum disorder and typically developing. These findings shed light on the face-processing mechanisms in children with autism spectrum disorder and provide new insight for understanding the social dysfunction of autism spectrum disorder.

The Association of Supplemental Nutrition Assistance Program Participation and Food Insufficiency among Households with Children in the United States during COVID-19.

BACKGROUND: As the expansion of Supplemental Nutrition Assistance Program (SNAP) benefits and pandemic emergency assistance programs ended in late 2021, little is known about subsequent trends in food insufficiency (FI) among households with children. OBJECTIVES: This research examined the association between SNAP participation and FI among households with children in the United States, particularly non-Hispanic Black (Black) and Hispanic households. METHODS: This cross-sectional analysis used Household Pulse Survey data collected from December 2021 to May 2022. Spatial analysis was conducted to visualize FI and SNAP participation rates across 50 states. With state SNAP policy rules as exogenous instruments and sociodemographic factors as control variables, 2-stage probit models were utilized to assess the SNAP and FI association among all (n = 135,074), Black (n = 13,940), and Hispanic households with children (n = 17,869). RESULTS: Approximately 13.9% [95% confidence interval (CI): 13.85%, 13.99%] of households experienced FI, and 20.4% (CI: 20.35%, 20.51%) received SNAP benefits. Among Black and Hispanic households, higher rates were observed, with 23.3% (CI: 23.12%, 23.4%) and 20.8% (CI: 20.61%, 20.95%) experiencing FI and 36.3% (CI: 36.1%, 36.5%) and 26.9% (CI: 26.61%, 27.13%) receiving SNAP benefits. These rates varied across states, ranging from 8% (Utah) to 21.1% (Mississippi) for FI and from 8.8% (Utah) to 32.7% (New Mexico) for SNAP participation. SNAP participants demonstrated a 12% lower likelihood of FI than nonparticipants (CI: -0.18, -0.05, P < 0.001). Among Black households, SNAP participants had a 29% lower likelihood of FI than nonparticipants (CI: -0.54, -0.03, P < 0.001). However, SNAP participation was not significant among Hispanic households (P = 0.99), nor did it narrow the FI gap between Hispanic and non-Hispanic households (P = 0.22). CONCLUSIONS: SNAP participation was associated with lower levels of FI among households with children, particularly for Black households. However, there was no significant association between SNAP participation and FI among Hispanic households with children.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 µM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

Identifying host-specific amino acid signatures for influenza A viruses using an adjusted entropy measure.

BACKGROUND: Influenza A viruses (IAV) exhibit vast genetic mutability and have great zoonotic potential to infect avian and mammalian hosts and are known to be responsible for a number of pandemics. A key computational issue in influenza prevention and control is the identification of molecular signatures with cross-species transmission potential. We propose an adjusted entropy-based host-specific signature identification method that uses a similarity coefficient to incorporate the amino acid substitution information and improve the identification performance. Mutations in the polymerase genes (e.g., PB2) are known to play a major role in avian influenza virus adaptation to mammalian hosts. We thus focus on the analysis of PB2 protein sequences and identify host specific PB2 amino acid signatures. RESULTS: Validation with a set of H5N1 PB2 sequences from 1996 to 2006 results in adjusted entropy having a 40% false negative discovery rate compared to a 60% false negative rate using unadjusted entropy. Simulations across different levels of sequence divergence show a false negative rate of no higher than 10% while unadjusted entropy ranged from 9 to 100%. In addition, under all levels of divergence adjusted entropy never had a false positive rate higher than 9%. Adjusted entropy also identifies important mutations in H1N1pdm PB2 previously identified in the literature that explain changes in divergence between 2008 and 2009 which unadjusted entropy could not identify. CONCLUSIONS: Based on these results, adjusted entropy provides a reliable and widely applicable host signature identification approach useful for IAV monitoring and vaccine development.

GAS5/METTL14/ESR1 genetic variants are related to the susceptibility of coronary heart disease.

The prevention and treatment of coronary heart disease (CHD) is a difficult problem to be solved urgently. Genetic factors play a crucial role in CHD development. This study aimed to investigate the association of GAS5/METTL14/ESR1 polymorphisms with CHD susceptibility. We carried out a case-control study that included 506 patients and 506 healthy subjects to detect the correlation between GAS5/METTL14/ESR1 polymorphisms and CHD risk in a Chinese population. Odds ratios (OR) and 95% confidence intervals (CI) were computed to assess the associations. Our study showed that GAS5 rs17359906 (OR 2.32, p = 0.020) and rs75315904 (OR 0.41, p = 0.039) were related to the risk of CHD in females. ESR1 rs6927072 (OR 1.76, p = 0.007) and rs4870061 (OR 0.74, p = 0.036) correlated with CHD risk in age ≤ 60 years. GAS5 rs17359906 (OR 0.10, p = 0.032) and ESR1 rs3020308 (OR 2.73, p = 0.041) were associated with an increased susceptibility to CHD in smokers. We also found that METTL14 rs4834698 (OR 1.57, p = 0.044) and ESR1 rs4870061 (OR 0.62, p = 0.040) were associated with CHD susceptibility in non-drinkers. Besides, METTL14 rs17050450 (OR 0.48, p = 0.029) and ESR1 rs3853248 (OR 1.61, p = 0.018) had the susceptibility of CHD patients with diabetes. Our study indicated that GAS5/METTL14/ESR1 polymorphisms were associated with CHD risk, which might provide a new understanding of CHD in a Chinese population.

Synthesis of Phenolic Coumestans via a Sequential Dehydrogenation/Oxa-Michael Addition Reaction of 2',4'-Dihydroxyl-3-arylcoumarins.

A facile and practical approach for the synthesis of natural coumestans and derivatives starting from 2',4'-dihydroxyl-3-arylcoumarins has been developed. The process involved a seqential intramolecular dehydrogenation/oxa-Micheal reaction efficiently promoted by 1,8-diazabicyclo[5.4.0]undec-7-ene at 40 °C under metal- and ligand-free conditions with good functional group compatibility.

Preliminary Study of Genome-Wide Association Identified Novel Susceptibility Genes for Hemorheological Indexes in a Chinese Population.

Background: Genome-wide association studies for various hemorheological characteristics have not been reported. We aimed to identify genetic loci associated with hemorheological indexes in a cohort of healthy Chinese Han individuals. Methods: Genotyping was performed using Applied Biosystems Axiom™ Precision Medicine Diversity Array in 838 individuals, and 6,423,076 single nucleotide polymorphisms were available for genotyping. The relations were examined in an additive genetic model using mixed linear regression and combined with identical by descent matrix. Results: We identified 38 genetic loci (p < 5 × 10-6) related to hemorheological traits. In which, LOC102724502-OLIG2 rs28371438 was related to the levels of nd30 (p = 8.58 × 10-07), nd300 (p = 1.89 × 10-06), erythrocyte rigidity (p = 1.29 × 10-06), assigned viscosity (p = 6.20 × 10-08) and whole blood high cut relative (p = 7.30 × 10-08). The association of STK32B rs4689231 for nd30 (p = 3.85 × 10-06) and nd300 (p = 2.94 × 10-06) and GTSCR1-LINC01541 rs11661911 for erythrocyte rigidity (p = 9.93 × 10-09) and whole blood high cut relative (p = 2.09 × 10-07) was found. USP25-MIR99AHG rs1297329 was associated with erythrocyte rigidity (p = 1.81 × 10-06) and erythrocyte deformation (p = 1.14 × 10-06). Moreover, the association of TMEM232-SLC25A46 rs3985087 and LINC00470-METTL4 rs9966987 for fibrinogen (p = 1.31 × 10-06 and p = 4.29 × 10-07) and plasma viscosity (p = 1.01 × 10-06 and p = 4.59 × 10-07) was found. Conclusion: These findings may represent biological candidates for hemorheological indexes and contribute to hemorheological study.

Knockdown of Long Noncoding RNA SNHG14 Protects H9c2 Cells Against Hypoxia-induced Injury by Modulating miR-25-3p/KLF4 Axis in Vitro.

ABSTRACT: Cyanotic congenital heart disease (CCHD) is the main cause of death in infants worldwide. Long noncoding RNAs (lncRNAs) have been pointed to exert crucial roles in development of CHD. The current research is designed to illuminate the impact and potential mechanism of lncRNA SNHG14 in CCHD in vitro. The embryonic rat ventricular myocardial cells (H9c2 cells) were exposed to hypoxia to establish the model of CCHD in vitro. Quantitative real-time polymerase chain reaction was conducted to examine relative expressions of SNHG14, miR-25-3p, and KLF4. Cell viability was determined by the MTT assay. Lactate dehydrogenase (LDH) was measured by an LDH assay kit. Apoptosis-related proteins (Bax and Bcl-2) and KLF4 were detected by Western Blot. The targets of SNHG14 and miR-25-3p were verified by the dual-luciferase reporter assay. SNHG14 and KLF4 were upregulated, whereas miR-25-3p was downregulated in hypoxia-induced H9c2 cells and cardiac tissues of patients with CCHD compared with their controls. Knockdown of SNHG14 or overexpression of miR-25-3p facilitated cell viability, while depressing cell apoptosis and release of LDH in hypoxia-induced H9c2 cells. MiR-25-3p was a target of SNHG14 and inversely modulated by SNHG14. MiR-25-3p could directly target KLF4 and negatively regulate expression of KLF4. Repression of miR-25-3p or overexpression of KLF4 reversed the suppression impacts of sh-SNHG14 on cell apoptosis and release of LDH as well as the promotion impact of sh-SNHG14 on cell viability in hypoxia-induced H9c2 cells. Sh-SNHG14 protected H9c2 cells against hypoxia-induced injury by modulating miR-25-3p/KLF4 axis in vitro.

VdOGDH is involved in energy metabolism and required for virulence of Verticillium dahliae.

Verticillium dahliae, a soil-borne fungus, can invade plant vascular tissue and cause Verticillium wilt. The enzyme α-oxoglutarate dehydrogenase (OGDH), catalyzing the oxidation of α-oxoglutarate in the tricarboxylic acid cycle (TCA), is vital for energy metabolism in the fungi. Here, we identified the OGDH gene in V. dahliae (VdOGDH, VDAG_10018) and investigated its function in virulence by generating gene deletion mutants (ΔVdOGDH) and complementary mutants (ΔVdOGDH-C). When the ΔVdOGDH mutants were supplemented with different carbon sources, vegetative growth on Czapek Dox medium was significantly impaired, suggesting that VdOGDH is crucial for vegetative growth and carbon utilization. Conidia of the ΔVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches. Mutants ΔVdOGDH-1 and ΔVdOGDH-2 were highly sensitive to H2O2 in the medium plates and had higher intracellular ROS levels. ΔVdOGDH mutants also had elevated expression of oxidative response-related genes, indicating that VdOGDH is involved in response to oxidative stress. In addition, the disruption of VdOGDH caused a significant increase in the expression of energy metabolism-related genes VdICL, VdICDH, VdMDH, and VdPDH and melanin-related genes Vayg1, VdSCD, VdLAC, VT4HR, and VaflM in the ΔVdOGDH mutants; thus, VdOGDH is also important for energy metabolism and melanin accumulation. Cotton plants inoculated with ΔVdOGDH mutants exhibited mild leaf chlorosis and the disease index was lower compared with wild type and ΔVdOGDH-C strains. These results together show that VdOGDH involved in energy metabolism of V. dahliae, is also essential for full virulence by regulating multiple fungal developmental factors.

Design, synthesis and biological evaluation of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives as potential antitumor agents for the treatment of multiple myeloma (MM).

A series of novel (E)-N-phenyl-4-(pyridine-acylhydrazone) benzamide derivatives were designed, synthesized, and evaluated for their anti-proliferative activity against two different human cancer cell lines and one human normal cell line. Compound 8b had the best anti-proliferative activity (IC50 = 0.12 ± 0.09 µM, RPMI8226 cells) than the other compounds. And compound 8b had lower toxicity than imatinib. Flow cytometry analysis showed that compound 8b could arrest the cell cycle at the G0/G1 phase, and induce apoptosis of RPMI8226 cells by promoting mitochondrial ROS release, thereby effectively inhibiting cell proliferation. Our findings provided a promising lead compound 8b for further structural optimization and will be instructive for the discovery of more potent antitumor drugs with high selectivity and low toxicity.

Comparative skin transcriptome of two Oujiang color common carp (Cyprinus carpio var. color) varieties.

Body color variation has long been a hot research topic in evolutionary and functional biology. Oujiang color common carp (Cyprinus carpio var. color) is a well-known economical and ornamental fish. Three main types of pigments and four distinct color patterns are typical characters of Oujiang color common carp, which makes it an excellent fish model to study body coloration. In this study, skin transcriptome assembly and comparisons were conducted in two Oujiang color common carp varieties: whole red and whole white. Transcriptome comparison revealed that more differentially expressed energy metabolism genes were upregulated in whole white compared to whole red. The results indicated that energy metabolism genes might be strongly associated with environmental adaption and growth performance and likely affect the red and white color formation in Oujiang color common carp. Our study provided direct guidance for the aquaculture industrials of Oujiang color common carp and presented valuable genetic resources for body color research in fish.

Adaptive evolution to a high purine and fat diet of carnivorans revealed by gut microbiomes and host genomes.

Carnivorous members of the Carnivora reside at the apex of food chains and consume meat-only diets, rich in purine, fats and protein. Here, we aimed to identify potential adaptive evolutionary signatures compatible with high purine and fat metabolism based on analysis of host genomes and symbiotic gut microbial metagenomes. We found that the gut microbiomes of carnivorous Carnivora (e.g., Felidae, Canidae) clustered in the same clade, and other clades comprised omnivorous and herbivorous Carnivora (e.g., badgers, bears and pandas). The relative proportions of genes encoding enzymes involved in uric acid degradation were higher in the gut microbiomes of meat-eating carnivorans than plant-eating species. Adaptive amino acid substitutions in two enzymes, carnitine O-palmitoyltransferase 1 (CPT1A) and lipase F (LIPF), which play a role in fat digestion, were identified in Felidae-Candidae species. Carnivorous carnivorans appear to endure diets high in purines and fats via gut microbiomic and genomic adaptations.

Effects of Vip3AcAa+Cry1Ac Cotton on Midgut Tissue in Helicoverpa armigera (Lepidoptera: Noctuidae).

To determine cellular changes caused by the chimeric protein Vip3AcAa against Helicoverpa armigera, we used transmission electron microscopy to examine ultrastructural changes in midgut cells of third-instar larvae of Cry1Ac-susceptible H. armigera after feeding on an artificial diet containing the Vip3AcAa toxin. Midgut epithelial cells of Cry1Ac-resistant H. armigera larvae that had fed on an artificial diet containing Vip3AcAa or on Bt cotton expressing Vip3AcAa+Cry1Ac were also examined using optical microscopy and hematoxylin-eosin staining. In the midgut cells of H. armigera larvae fed with Vip3AcAa, microvilli were swollen and broken; inner cristae of the mitochondria were indistinct and vacuolated; endoplasmic reticulum was swollen, fractured, and disordered; boundaries of karyotheca in the nucleus were indistinct and chromatin underwent pyknosis and was pressed close to the karyotheca. Histopathological changes and the time of onset in midgut tissues of H. armigera larvae fed on Vip3AcAa or Cry1Ac were similar. Vip3AcAa and transgenic cotton expressing Vip3AcAa+Cry1Ac caused the goblet cell cavity and microvilli pathological changes in the midgut epithelial cells of the Cry1Ac-susceptible and Cry1Ac-resistant H. armigera larvae that eventually killed the larvae.

ABP9, a maize bZIP transcription factor, enhances tolerance to salt and drought in transgenic cotton.

MAIN CONCLUSION: ABP9 , encoding a bZIP transcription factor from maize, enhances tolerance to multiple stresses and may participate in the ABA signaling pathway in transgenic cotton by altering physiological and biochemical processes and stress-related gene expression. Abiotic stresses, such as soil salinity and drought, negatively affect growth, development, and yield in cotton. Gene ABP9, which encodes a bZIP transcription factor, binds to the abscisic acid (ABA)-responsive-element (ABRE2) motif of the maize catalase1 gene. Its expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses, but little is known about its role in cotton. In the present study, the ABP9 gene was introduced into upland cotton (Gossypium hirsutum L.) cultivar R15 by Agrobacterium tumefaciens-mediated transformation, and 12 independent transgenic cotton lines were obtained. Cotton plants over-expressing ABP9 have enhanced tolerance to salt and osmotic stress. Under stress, they developed better root systems in a greenhouse and higher germination, reduced stomatal aperture, and stomatal density in a growth chamber. Under drought conditions, survival rate and relative water content (RWC) of transgenic cotton were higher than those of R15 plants. Under salt and osmotic stresses, chlorophyll, proline, and soluble sugar contents significantly increased in transgenic cotton leaves and the malondialdehyde (MDA) content was lower than in R15. Overexpression of ABP9 also enhanced oxidative stress tolerance, reduced cellular levels of reactive oxygen species (ROS) through increased activities of antioxidative enzymes, and alleviated oxidative damage to cell. Interestingly, ABP9 over-expressing cotton was more sensitive to exogenous ABA than R15 at seed germination, root growth, stomatal aperture, and stomatal density. Moreover, ABP9 overexpression upregulated significantly the transcription levels of stress-related genes such as GhDBP2, GhNCED2, GhZFP1, GhERF1, GhHB1, and GhSAP1 under salt treatment. Conjointly, these results showed that overexpression of ABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton. The stress-tolerant transgenic lines provide valuable resources for cotton breeding.

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm.

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for pest resistance management in China.

Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China.

Model-based clustering with certainty estimation: implication for clade assignment of influenza viruses.

BACKGROUND: Clustering is a common technique used by molecular biologists to group homologous sequences and study evolution. There remain issues such as how to cluster molecular sequences accurately and in particular how to evaluate the certainty of clustering results. RESULTS: We presented a model-based clustering method to analyze molecular sequences, described a subset bootstrap scheme to evaluate a certainty of the clusters, and showed an intuitive way using 3D visualization to examine clusters. We applied the above approach to analyze influenza viral hemagglutinin (HA) sequences. Nine clusters were estimated for high pathogenic H5N1 avian influenza, which agree with previous findings. The certainty for a given sequence that can be correctly assigned to a cluster was all 1.0 whereas the certainty for a given cluster was also very high (0.92-1.0), with an overall clustering certainty of 0.95. For influenza A H7 viruses, ten HA clusters were estimated and the vast majority of sequences could be assigned to a cluster with a certainty of more than 0.99. The certainties for clusters, however, varied from 0.40 to 0.98; such certainty variation is likely attributed to the heterogeneity of sequence data in different clusters. In both cases, the certainty values estimated using the subset bootstrap method are all higher than those calculated based upon the standard bootstrap method, suggesting our bootstrap scheme is applicable for the estimation of clustering certainty. CONCLUSIONS: We formulated a clustering analysis approach with the estimation of certainties and 3D visualization of sequence data. We analysed 2 sets of influenza A HA sequences and the results indicate our approach was applicable for clustering analysis of influenza viral sequences.

Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants.

Background: Pork processing plants in the United States (US) cease operations for 24-48 h every six or twelve months to perform intense sanitization (IS) using fogging, foaming, and further antimicrobial treatments to disrupt natural biofilms that may harbor pathogens and spoilage organisms. The impact such treatments have on short-term changes in environmental microorganisms is not well understood, nor is the rate at which bacterial communities return. Methods: Swab samples were collected from floor drains to provide representative environmental microorganisms at two US pork processing plants before, during, and after an IS procedure. Samples were collected from four coolers where finished carcasses were chilled and from four locations near cutting tables. Each sample was characterized by total mesophile count (TMC), total psychrophile count (TPC), and other indicator bacteria; their biofilm-forming ability, tolerance of the formed biofilm to a quaternary ammonium compound (300 ppm, QAC), and ability to protect co-inoculated Salmonella enterica. In addition, bacterial community composition was determined using shotgun metagenomic sequencing. Results: IS procedures disrupted bacteria present but to different extents depending on the plant and the area of the plant. IS reduced TPC and TMC, by up to 1.5 Log10 CFU only to return to pre-IS levels within 2-3 days. The impact of IS on microorganisms in coolers was varied, with reductions of 2-4 Log10, and required 2 to 4 weeks to return to pre-IS levels. The results near fabrication lines were mixed, with little to no significant changes at one plant, while at the other, two processing lines showed 4 to 6 Log10 reductions. Resistance to QAC and the protection of Salmonella by the biofilms varied between plants and between areas of the plants as well. Community profiling of bacteria at the genus level showed that IS reduced species diversity and the disruption led to new community compositions that in some cases did not return to the pre-IS state even after 15 to 16 weeks. Discussion: The results found here reveal the impact of using IS to disrupt the presence of pathogen or spoilage microorganisms in US pork processing facilities may not have the intended effect.

Transcriptome analysis of two buffalograss cultivars.

BACKGROUND: Buffalograss [Buchloë dactyloides (Nutt.) Engel. syn. Bouteloua dactyloides (Nutt.) Columbus] is a United States native turfgrass species that requires less irrigation, fungicides and pesticides compared to more commonly used turfgrass species. In areas where water is limited, interest in this grass species for lawns is increasing. While several buffalograss cultivars have been developed through buffalograss breeding, the timeframe for new cultivar development is long and is limited by a lack of useful genetic resources. Two high throughput next-generation sequencing techniques were used to increase the genomic resources available for buffalograss. RESULTS: Total RNA was extracted and purified from leaf samples of two buffalograss cultivars. '378' and 'Prestige' cDNA libraries were subjected to high throughput sequencing on the Illumina GA and Roche 454 Titanium FLX sequencing platforms. The 454 platform (3 samples) produced 1,300,885 reads and the Illumina platform (12 samples) generated approximately 332 million reads. The multiple k-mer technique for de novo assembly using Velvet and Oases was applied. A total of 121,288 contigs were assembled that were similar to previously reported Ensembl commelinid sequences. Original Illumina reads were also mapped to the high quality assembly to estimate expression levels of buffalograss transcripts. There were a total of 325 differentially expressed genes between the two buffalograss cultivars. A glycosyl transferase, serine threonine kinase, and nb-arc domain containing transcripts were among those differentially expressed between the two cultivars. These genes have been previously implicated in defense response pathways and may in part explain some of the performance differences between 'Prestige' and '378'. CONCLUSIONS: To date, this is the first high throughput sequencing experiment conducted on buffalograss. In total, 121,288 high quality transcripts were assembled, significantly expanding the limited genetic resources available for buffalograss genetic studies. Additionally, 325 differentially expressed sequences were identified which may contribute to performance or morphological differences between 'Prestige' and '378' buffalograss cultivars.