BackgroundPositive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors.ResultsWe developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity.ConclusionsThe advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.

Heterodera glycines, the soybean cyst nematode (SCN), is a subterranean root pathogen that causes the most damaging disease of soybean in the USA. A novel nematode virus genome, soybean cyst nematode virus 5 (SbCNV-5), was identified in RNA sequencing data from SCN eggs and second-stage juveniles. The SbCNV-5 RNA-dependent RNA polymerase and RNA helicase domains had homology to pestiviruses in the family Flaviviridae, suggesting that SbCNV-5 is a positive-polarity ssRNA virus. SbCNV-5 RNA was present in all nematode developmental stages, indicating a transovarial mode of transmission, but is also potentially sexually transmitted via the male. SbCNV-5 was common in SCN laboratory cultures and in nematode populations isolated from the field. Transmission electron microscopy of sections from a female SCN showed virus particles budding from the endoplasmic reticulum and in endosomes. The size of the viral genome was 19 191 nt, which makes it much larger than other known pestiviruses. Additionally, the presence of a methyltransferase in the SbCNV-5 genome is atypical for a pestivirus. When cDNA sequences were mapped to the genome of SbCNV-5, a disproportionate number aligned to the 39 NTR, suggesting that SbCNV-5 produces a subgenomic RNA, which was confirmed by RNA blot analysis. As subgenomic RNAs and methyltransferases do not occur in pestiviruses, we conclude that SbCNV-5 is a new flavivirus infecting SCNs.

The effects of room temperature and light intensity before breeding and into early gestation were evaluated on the reproductive performance and well-being of gilts housed individually in crates. In eight replicates, estrus was synchronized in mature gilts (n = 198) and after last feeding of Matrix were randomly assigned to a room temperature of 15°C (COLD), 21°C (NEUTRAL), or 30°C (HOT) and a light intensity of 11 (DIM) or 433 (BRIGHT) lx. Estrous detection was performed daily and gilts inseminated twice. Blood samples were collected before and after breeding for determination of immune measures and cortisol concentrations. Gilt ADFI, BW, and body temperature were measured. On d 30 postbreeding, gilts were slaughtered to recover reproductive tracts to evaluate pregnancy and litter characteristics. There were no temperature × light intensity interactions for any response variable. Reproductive measures of follicle development, expression of estrus, ovulation rate, pregnancy rate (83.2%), litter size (14.3 ± 0.5), and fetal measures were not affected by temperature or lighting (P > 0.10). Gilts in COLD (37.6°C) had a lower (P < 0.05) rectal temperature than those in NEUTRAL (38.2°C) and HOT (38.6 ± 0.04°C). Both BW gain and final BW were greater (P < 0.0001) for gilts kept in HOT than those in NEUTRAL or COLD environments. Cortisol was greater (P < 0.01) for gilts kept in COLD compared with those kept in the HOT room. Gilts housed in the HOT environment made more postural changes (P < 0.05) than did those kept in either COLD or NEUTRAL temperatures. Gilts kept in the HOT temperature spent more total time lying and more time lying ventrally compared with those gilts housed in the NEUTRAL or COLD rooms. Total white blood cells and the percentage of neutrophils as well as neutrophil-to-lymphocyte ratio were all influenced (P < 0.05) by temperature but there was no effect (P > 0.10) of light or interaction with temperature on other immune cells or measures. These results indicate that temperatures in the range of 15 to 30°C or light intensity at 11 to 433 lx do not impact reproduction during the follicular phase and into early gestation for mature gilts housed in gestation crates. However, room temperature does impact physiological, behavioral, and immune responses of mature gilts and should be considered as a potential factor that may influence gilt well-being during the first 30 d postbreeding.

A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

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