MicrobeTrace: Retooling molecular epidemiology for rapid public health response

Outbreak investigations use data from interviews, healthcare providers, laboratories and surveillance systems. However, integrated use of data from multiple sources requires a patchwork of software that present challenges in usability, interoperability, confidentiality, and cost. Rapid integration, visualization and analysis of data from multiple sources can guide effective public health interventions. We developed MicrobeTrace to facilitate rapid public health responses by overcoming barriers to data integration and exploration in molecular epidemiology. MicrobeTrace is a web-based, client-side, JavaScript application (https://microbetrace.cdc.gov) that runs in Chromium-based browsers and remains fully operational without an internet connection. Using publicly available data, we demonstrate the analysis of viral genetic distance networks and introduce a novel approach to minimum spanning trees that simplifies results. We also illustrate the potential utility of MicrobeTrace in support of contact tracing by analyzing and displaying data from an outbreak of SARS-CoV-2 in South Korea in early 2020. MicrobeTrace is developed and actively maintained by the Centers for Disease Control and Prevention. Users can email vog.cdc@ecarteborcim for support. The source code is available at https://github.com/cdcgov/microbetrace.     

Comparative analysis of inverted repeats of polypod fern (Polypodiales) plastomes reveals two hypervariable regions

Background

Ferns are large and underexplored group of vascular plants (~ 11 thousands species). The genomic data available by now include low coverage nuclear genomes sequences and partial sequences of mitochondrial genomes for six species and several plastid genomes.

Results

We characterized plastid genomes of three species of Dryopteris, which is one of the largest fern genera, using sequencing of chloroplast DNA enriched samples and performed comparative analysis with available plastomes of Polypodiales, the most species-rich group of ferns. We also sequenced the plastome of Adianthum hispidulum (Pteridaceae). Unexpectedly, we found high variability in the IR region, including duplication of rrn16 in D. blanfordii, complete loss of trnI-GAU in D. filix-mas, its pseudogenization due to the loss of an exon in D. blanfordii. Analysis of previously reported plastomes of Polypodiales demonstrated that Woodwardia unigemmata and Lepisorus clathratus have unusual insertions in the IR region. The sequence of these inserted regions has high similarity to several LSC fragments of ferns outside of Polypodiales and to spacer between tRNA-CGA and tRNA-TTT genes of mitochondrial genome of Asplenium nidus. We suggest that this reflects the ancient DNA transfer from mitochondrial to plastid genome occurred in a common ancestor of ferns. We determined the marked conservation of gene content and relative evolution rate of genes and intergenic spacers in the IRs of Polypodiales. Faster evolution of the four intergenic regions had been demonstrated (trnA- orf42, rrn16-rps12, rps7-psbA and ycf2-trnN).

Conclusions

IRs of Polypodiales plastomes are dynamic, driven by such events as gene loss, duplication and putative lateral transfer from mitochondria.

     

Regulation of Epithelial Differentiation in Rat Intestine by Intraluminal Delivery of an Adenoviral Vector or Silencing RNA Coding for Schlafen 3

Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.     

Phorbol ester-induced lymphocyte adherence: selective action on NK cells

Treatment of human peripheral blood lymphocytes (PBL) with phorbol dibutyrate (PDBU) for 20 to 45 min at 37 degrees C induces adherence of 5 to 30% of the cells to plastic. The adherent cells (pAd) were highly enriched in NK cells on the basis of the following findings: 1) they exhibit high NK and ADCC activity but do not lyse the NK-resistant cell line, Daudi; 2) cytotoxic activity is enhanced by pretreatment with interferon-alpha (IFN-alpha); 3) the surface markers of these cells, as determined with monoclonal antibodies, are consistent with NK cells; and 4) they are enriched with cells morphologically similar to large granular lymphocytes. Conversely, the PDBU-nonadherent cells were substantially depleted of NK cells. The fact that the pAd cells do not lyse the Daudi line and that their NK activity can be further augmented by IFN-alpha would suggest that the pAd are enriched for NK cells rather than changed in their characteristics as a result of the separation procedure. Moreover, no consistent and appreciable modulation of NK activity induced by PDBU was observed. This report therefore demonstrates that PDBU selectively induces NK adherence of NK cells, which may have practical as well as biological implications.     

Non-linear regression models for time to flowering in wild chickpea combine genetic and climatic factors

Background

Accurate prediction of crop flowering time is required for reaching maximal farm efficiency. Several models developed to accomplish this goal are based on deep knowledge of plant phenology, requiring large investment for every individual crop or new variety. Mathematical modeling can be used to make better use of more shallow data and to extract information from it with higher efficiency. Cultivars of chickpea, Cicer arietanum, are currently being improved by introgressing wild C. reticulatum biodiversity with very different flowering time requirements. More understanding is required for how flowering time will depend on environmental conditions in these cultivars developed by introgression of wild alleles.

Results

We built a novel model for flowering time of wild chickpeas collected at 21 different sites in Turkey and grown in 4 distinct environmental conditions over several different years and seasons. We propose a general approach, in which the analytic forms of dependence of flowering time on climatic parameters, their regression coefficients, and a set of predictors are inferred automatically by stochastic minimization of the deviation of the model output from data. By using a combination of Grammatical Evolution and Differential Evolution Entirely Parallel method, we have identified a model that reflects the influence of effects of day length, temperature, humidity and precipitation and has a coefficient of determination of R²=0.97.

Conclusions

We used our model to test two important hypotheses. We propose that chickpea phenology may be strongly predicted by accession geographic origin, as well as local environmental conditions at the site of growth. Indeed, the site of origin-by-growth environment interaction accounts for about 14.7% of variation in time period from sowing to flowering. Secondly, as the adaptation to specific environments is blueprinted in genomes, the effects of genes on flowering time may be conditioned on environmental factors. Genotype-by-environment interaction accounts for about 17.2% of overall variation in flowering time. We also identified several genomic markers associated with different reactions to climatic factor changes. Our methodology is general and can be further applied to extend existing crop models, especially when phenological information is limited.

     

Whole Genome Analysis of Epidemiologically Closely Related Staphylococcus aureus Isolates

The change of the bacteria from colonizers to pathogens is accompanied by a drastic change in expression profiles. These changes may be due to environmental signals or to mutational changes. We therefore compared the whole genome sequences of four sets of S. aureus isolates. Three sets were from the same patients. The isolates of each pair (S1800/S1805, S2396/S2395, S2398/S2397, an isolate from colonization and an isolate from infection, respectively) were obtained within <30 days of each other and the isolate from infection caused skin infections. The isolates were then compared for differences in gene content and SNPs. In addition, a set of isolates from a colonized pig and a farmer from the same farm at the same time (S0462 and S0460) were analyzed. The isolates pair S1800/S1805 showed a difference in a prophage, but these are easily lost or acquired. However, S1805 contained an integrative conjugative element not present in S1800. In addition, 92 SNPs were present in a variety of genes and the isolates S1800 and S1805 were not considered a pair. Between S2395/S2396 two SNPs were present: one was in an intergenic region and one was a synonymous mutation in a putative membrane protein. Between S2397/S2398 only one synonymous mutation in a putative lipoprotein was found. The two farm isolates were very similar and showed 12 SNPs in genes that belong to a number of different functional categories. However, we cannot pinpoint any gene that explains the change from carrier status to infection. The data indicate that differences between the isolate from infection and the colonizing isolate for S2395/S2396 and S2397/S2398 exist as well as between isolates from different hosts, but S1800/S1805 are not clonal.     

Influence of the spacer region between the Shine–Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine–Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine–Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.     

Sexually Antagonistic “Zygotic Drive” of the Sex Chromosomes

Genomic conflict is perplexing because it causes the fitness of a species to decline rather than improve. Many diverse forms of genomic conflict have been identified, but this extant tally may be incomplete. Here, we show that the unusual characteristics of the sex chromosomes can, in principle, lead to a previously unappreciated form of sexual genomic conflict. The phenomenon occurs because there is selection in the heterogametic sex for sex-linked mutations that harm the sex of offspring that does not carry them, whenever there is competition among siblings. This harmful phenotype can be expressed as an antagonistic green-beard effect that is mediated by epigenetic parental effects, parental investment, and/or interactions among siblings. We call this form of genomic conflict sexually antagonistic “zygotic drive”, because it is functionally equivalent to meiotic drive, except that it operates during the zygotic and postzygotic stages of the life cycle rather than the meiotic and gametic stages. A combination of mathematical modeling and a survey of empirical studies is used to show that sexually antagonistic zygotic drive is feasible, likely to be widespread in nature, and that it can promote a genetic “arms race” between the homo- and heteromorphic sex chromosomes. This new category of genomic conflict has the potential to strongly influence other fundamental evolutionary processes, such as speciation and the degeneration of the Y and W sex chromosomes. It also fosters a new genetic hypothesis for the evolution of enigmatic fitness-reducing traits like the high frequency of spontaneous abortion, sterility, and homosexuality observed in humans.     

Evolutionary genomics of Culex pipiens: global and local adaptations associated with climate,life-history traits and anthropogenic factors

We present the first genome-wide study of recent evolution in Culex pipiens species complex focusing on the genomic extent, functional targets and likely causes of global and local adaptations. We resequenced pooled samples of six populations of C. pipiens and two populations of the outgroup Culex torrentium. We used principal component analysis to systematically study differential natural selection across populations and developed a phylogenetic scanning method to analyse admixture without haplotype data. We found evidence for the prominent role of geographical distribution in shaping population structure and specifying patterns of genomic selection. Multiple adaptive events, involving genes implicated with autogeny, diapause and insecticide resistance were limited to specific populations. We estimate that about 5–20% of the genes (including several histone genes) and almost half of the annotated pathways were undergoing selective sweeps in each population. The high occurrence of sweeps in non-genic regions and in chromatin remodelling genes indicated the adaptive importance of gene expression changes. We hypothesize that global adaptive processes in the C. pipiens complex are potentially associated with South to North range expansion, requiring adjustments in chromatin conformation. Strong local signature of adaptation and emergence of hybrid bridge vectors necessitate genomic assessment of populations before specifying control agents.     

Genetic variation in six species of the genus Oxytropis DC. (Fabaceae) from Kamchatka Peninsula

Using the isozyme analysis, genetic variations in six species of the genus Oxytropis DC. (Fabaceae) from Kamchatka was assessed. It was demonstrated that diploid species from the section Arctobia were characterized by a low level of variations typical of endemic plant species. At the same time, polyploid species from the Orobia section demonstrated very high values of the heterozygosity parameters (H _o varied from 0.200 to 0.274). It has been suggested that the level of polymorphism of the oxytropes from Kamchatka was shaped as a result of the interaction of a number of factors, among which the most important are the ecological confinedness of the species, the specific features of the reproductive system, and gene drift. In the species of Orobia section, it is also the presence of the polyploid genome.