Estimating Skeletal Maturity by Segmented Linear Modeling of Key AP Knee Radiographic Parameters.

BACKGROUND: The recently described Modified Fels knee system allows for accurate skeletal maturity estimation using a single anteroposterior knee radiograph but requires evaluation of 7 parameters. A faster method may have clinical utility in the outpatient setting. METHODS: Seven anteroposterior knee radiographic parameters associated with 90% of the final height (an enhanced skeletal maturity standard compared with peak height velocity) were analyzed in 78 children. Segmented linear regression and generalized estimating equation analyses were used to identify the subsets of parameters most important for accurate skeletal maturity estimation for different patient demographics and parameter scores. This process produced abbreviated skeletal maturity systems, which include fewer parameters and are quicker to use. The accuracy of the resulting abbreviated skeletal maturity systems was evaluated and compared with the full 7-parameter Modified Fels knee system and with the Greulich and Pyle (GP) left-hand bone age. RESULTS: A total of 326 left knee radiographs from 41 girls (range, 7 to 15 y) and 37 boys (range, 9 to 17 y) were included. Models generated by segmented regression and generalized estimating equation analysis required fewer parameters (range, 1 to 5 parameters) than the full Modified Fels knee system (7 parameters). Skeletal age estimates produced by segmented regression models were more accurate than GP (P<0.05) and not significantly different from the full Modified Fels system (P>0.05). The percentage of outlier estimations (estimations >1 y off from actual skeletal age) made by segmented regression models was not significantly different from GP (P>0.05) or the Modified Fels knee system (P>0.05). CONCLUSION: An abbreviated version of the Modified Fels knee system estimates skeletal maturity more accurately than the GP system with just 2 to 3 radiographic knee parameters. CLINICAL RELEVANCE: The abbreviated Modified Fels knee system may allow for rapid skeletal age estimation (~30 s) appropriate for routine outpatient practice.

Chlamydomonas sp. UWO 241 Exhibits High Cyclic Electron Flow and Rewired Metabolism under High Salinity.

The Antarctic green alga Chlamydomonas sp. UWO 241 (UWO 241) is adapted to permanent low temperatures, hypersalinity, and extreme shade. One of the most striking phenotypes of UWO 241 is an altered PSI organization and constitutive PSI cyclic electron flow (CEF). To date, little attention has been paid to CEF during long-term stress acclimation, and the consequences of sustained CEF in UWO 241 are not known. In this study, we combined photobiology, proteomics, and metabolomics to understand the underlying role of sustained CEF in high-salinity stress acclimation. High salt-grown UWO 241 exhibited increased thylakoid proton motive flux and an increased capacity for nonphotochemical quenching. Under high salt, a significant proportion of the up-regulated enzymes were associated with the Calvin-Benson-Bassham cycle, carbon storage metabolism, and protein translation. Two key enzymes of the shikimate pathway, 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase and chorismate synthase, were also up-regulated, as well as indole-3-glycerol phosphate synthase, an enzyme involved in the biosynthesis of l-Trp and indole acetic acid. In addition, several compatible solutes (glycerol, Pro, and Suc) accumulated to high levels in high salt-grown UWO 241 cultures. We suggest that UWO 241 maintains constitutively high CEF through the associated PSI-cytochrome b 6 f supercomplex to support robust growth and strong photosynthetic capacity under a constant growth regime of low temperatures and high salinity.

In vivo diagnostics of early abiotic plant stress response via Raman spectroscopy.

Development of a phenotyping platform capable of noninvasive biochemical sensing could offer researchers, breeders, and producers a tool for precise response detection. In particular, the ability to measure plant stress in vivo responses is becoming increasingly important. In this work, a Raman spectroscopic technique is developed for high-throughput stress phenotyping of plants. We show the early (within 48 h) in vivo detection of plant stress responses. Coleus (Plectranthus scutellarioides) plants were subjected to four common abiotic stress conditions individually: high soil salinity, drought, chilling exposure, and light saturation. Plants were examined poststress induction in vivo, and changes in the concentration levels of the reactive oxygen-scavenging pigments were observed by Raman microscopic and remote spectroscopic systems. The molecular concentration changes were further validated by commonly accepted chemical extraction (destructive) methods. Raman spectroscopy also allows simultaneous interrogation of various pigments in plants. For example, we found a unique negative correlation in concentration levels of anthocyanins and carotenoids, which clearly indicates that plant stress response is fine-tuned to protect against stress-induced damages. This precision spectroscopic technique holds promise for the future development of high-throughput screening for plant phenotyping and the quantification of biologically or commercially relevant molecules, such as antioxidants and pigments.

Advanced genetic tools for plant biotechnology.

Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.

Synergistic reaction of silver nitrate, silver nanoparticles, and methylene blue against bacteria.

In this paper we describe the antibacterial effect of methylene blue, MB, and silver nitrate reacting alone and in combination against five bacterial strains including Serratia marcescens and Escherichia coli bacteria. The data presented suggest that when the two components are combined and react together against bacteria, the effects can be up to three orders of magnitude greater than that of the sum of the two components reacting alone against bacteria. Analysis of the experimental data provides proof that a synergistic mechanism is operative within a dose range when the two components react together, and additive when reacting alone against bacteria.

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations.

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.

Genome-wide association for testis weight in the diversity outbred mouse population.

Testis weight is a genetically mediated trait associated with reproductive efficiency across numerous species. We sought to evaluate the genetically diverse, highly recombinant Diversity Outbred (DO) mouse population as a tool to identify and map quantitative trait loci (QTLs) associated with testis weight. Testis weights were recorded for 502 male DO mice and the mice were genotyped on the GIGAMuga array at ~ 143,000 SNPs. We performed a genome-wide association analysis and identified one significant and two suggestive QTLs associated with testis weight. Using bioinformatic approaches, we developed a list of candidate genes and identified those with known roles in testicular size and development. Candidates of particular interest include the RNA demethylase gene Alkbh5, the cyclin-dependent kinase inhibitor gene Cdkn2c, the dynein axonemal heavy chain gene Dnah11, the phospholipase D gene Pld6, the trans-acting transcription factor gene Sp4, and the spermatogenesis-associated gene Spata6, each of which has a human ortholog. Our results demonstrate the utility of DO mice in high-resolution genetic mapping of complex traits, enabling us to identify developmentally important genes in adult mice. Understanding how genetic variation in these genes influence testis weight could aid in the understanding of mechanisms of mammalian reproductive function.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

Phosphorylation of trihelix transcriptional repressor ASR3 by MAP KINASE4 negatively regulates Arabidopsis immunity.

Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression.

Production of single cell protein from agro-waste using Rhodococcus opacus.

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.