DEGAP: Dynamic elongation of a genome assembly path.

Genome assembly remains to be a major task in genomic research. Despite the development over the past decades of different assembly software programs and algorithms, it is still a great challenge to assemble a complete genome without any gaps. With the latest DNA circular consensus sequencing (CCS) technology, several assembly programs can now build a genome from raw sequencing data to contigs; however, some complex sequence regions remain as unresolved gaps. Here, we present a novel gap-filling software, DEGAP (Dynamic Elongation of a Genome Assembly Path), that resolves gap regions by utilizing the dual advantages of accuracy and length of high-fidelity (HiFi) reads. DEGAP identifies differences between reads and provides 'GapFiller' or 'CtgLinker' modes to eliminate or shorten gaps in genomes. DEGAP adopts an iterative elongation strategy that automatically and dynamically adjusts parameters according to three complexity factors affecting the genome to determine the optimal extension path. DEGAP has already been successfully applied to decipher complex genomic regions in several projects and may be widely employed to generate more gap-free genomes.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Enzymatic degradation is an effective means to reduce aflatoxin contamination in maize.

BACKGROUND: Aflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year. RESULTS: In this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be expressing the aflatoxin-degrading enzyme both at the RNA and protein level, were challenged with the aflatoxin-producing strain Aspergillus flavus AF13 and shown to accumulate non-detectable levels of aflatoxin at 14-days post-infection and significantly reduced levels of aflatoxin at 30-days post-infection compared to nontransgenic control Aspergillus-challenged samples. CONCLUSIONS: The expression of an aflatoxin-degrading enzyme in developing maize kernels was shown to be an effective means to control aflatoxin in maize in pre-harvest conditions. This aflatoxin-degradation strategy could play a significant role in the enhancement of both US and global food security and sustainability.

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and ß-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

Characterization and functional biology of the soybean aleurone layer.

BACKGROUND: Soybean is a globally important oil seed crop. Both the high protein and oil content of soybean seeds make this crop a lucrative commodity. As in higher eukaryotic species with available genomes, the functional annotation of most of soybean's genes still remains to be investigated. A major hurdle in the functional genomics of soybean is a rapid method to test gene constructs before embarking on stable transformation experiments. RESULTS: In this paper we describe the morphology and composition of the persistent single-cell aleurone layer that derives from the endosperm of developing soybean seeds. Its composition compared to cotyledonary tissue indicates the aleurone layer plays a role in both abiotic and biotic stress. The potential utility as the aleurone layer as a transient expression system in soybean was shown. As a near transparent single-cell layer it can be used as a transient expression system to study transgene expression and inter- and intra-cellular targeting as it is amenable to microscopic techniques. CONCLUSION: The transparent single cell aleurone layer was shown to be compositionally comparable to cotyledonary tissue in soybean with an enrichment in oxidative response proteins and shown to be a potential transient expression platform.

Aflatoxin-free transgenic maize using host-induced gene silencing.

Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.

Proteome rebalancing in transgenic Camelina occurs within the enlarged proteome induced by ß-carotene accumulation and storage protein suppression.

Oilseed crops are global commodities for their oil and protein seed content. We have engineered the oilseed Camelina sativa to exhibit increased protein content with a slight decrease in oil content. The introduction of a phytoene synthase gene with an RNAi cassette directed to suppress the storage protein 2S albumin resulted in seeds with an 11-24 % elevation in overall protein. The phytoene synthase cassette alone produced enhanced ß-carotene content of an average 275 ± 6.10 µg/g dry seed and an overall altered seed composition of 11 % less protein and comparable nontransgenic amounts of both oil and carbohydrates. Stacking an RNAi to suppress the major 2S storage protein resulted in seeds that contain elevated protein and slight decrease in oil and carbohydrate amounts showing that Camelina rebalances its proteome within an enlarged protein content genotype. In both ß-carotene enhanced seeds with/without RNAi2S suppression, the seed size was noticeably enlarged compared to nontransgenic counterpart seeds. Metabolic analysis of maturing seeds indicate that the enhanced ß-carotene trait had the larger effect than the RNAi2S suppression on the seed metabolome. The use of a GRAS (generally regarded as safe) ß-carotene as a visual marker in a floral dip transformation system, such as Camelina, might eliminate the need for costly regulatory and controversial antibiotic resistance markers. ß-carotene enhanced RNAi2S suppressed Camelina seeds could be further developed as a rapid heterologous protein production platform in a nonfood crop leveraging its enlarged protein content and visual marker.

Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF).

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 µg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

The Potential for Engineering Enhanced Functional-Feed Soybeans for Sustainable Aquaculture Feed.

Aquaculture is the most rapidly growing segment of global animal production that now surpasses wild-capture fisheries production and is continuing to grow 10% annually. Sustainable aquaculture needs to diminish, and progressively eliminate, its dependence on fishmeal-sourced feed from over-harvested fisheries. Sustainable aquafeed sources will need to be primarily of plant-origin. Soybean is currently the primary global vegetable-origin protein source for aquaculture. Direct exchange of soybean meal for fishmeal in aquafeed has resulted in reduced growth rates due in part to soybean's anti-nutritional proteins. To produce soybeans for use in aquaculture feeds a new conventional line has been bred termed Triple Null by stacking null alleles for the feed-relevant proteins Kunitz Trypsin Inhibitor, lectin, and P34 allergen. Triple Null is now being further enhanced as a platform to build additional transgene traits for vaccines, altered protein composition, and to produce high levels of ß-carotene an intrinsic orange-colored aquafeed marker to distinguish the seeds from commodity beans and as the metabolic feedstock precursor of highly valued astaxanthin.

Transgenic soya bean seeds accumulating ß-carotene exhibit the collateral enhancements of oleate and protein content traits.

Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 µg ß carotene g(-1) dry seed weight with a desirable 12:1 ratio of ß to α. The ß carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated ß-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation ß-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated ß-carotene, oleic acid and protein traits in the ß-carotene soya beans confer a substantial additive nutritional quality to soya beans.

Silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome.

The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP-) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP- seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP- soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed's transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP- line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content.

Incidental "rotator cuff tear of the hip" at primary total hip arthroplasty.

Gluteus medius tendon tears are occasionally noted during primary total hip arthroplasty. In this study, we reviewed the cases of 513 total hip arthroplasty patients to determine the incidence of these tears and to report clinical outcomes. We found 8 patients (8 hips) with incidental gluteus medius tendon tears for an incidence of 1.6%. After surgical repair, no patient had a perceptible limp or a Trendelenburg sign postoperatively.

Industrial protein production crops: new needs and new opportunities.

There are many diverse uses for industrial proteins with new opportunities for novel uses frequently emerging. Prominent among these uses are enzymes catalyzing the processing of food/feed and for the production of cellulosic biofuels. Other significant industrial protein uses include antibodies and other binding proteins for purification and/or clean-up of industrial product streams. Enabling technology is needed to produce these now expensive industrial proteins could be produced cost-effectively. Plant-based production of industrial enzymes offers the prospect of massive, scalable production, coupled with low production cost especially if a co-product, such as seed oil or starch, subsidizes the primary crop production costs. High-protein seeds whose composition is remodeled to produce industrial proteins can be a cost-effective means to produce industrial proteins. There are both technical and regulatory issues to resolve in order to deploy plants and seeds as industrial protein production platforms and many of these issues may be more easily resolved by developing nonfood crops specifically for use as industrial production platforms. An emerging industrial plant, Camelina, has potential as a protein-production platform subsidized by the seed oil co-product.

Proteome rebalancing in soybean seeds can be exploited to enhance foreign protein accumulation.

Seeds possess a high intrinsic capacity for protein production that makes them a desirable bioreactor platform for the manufacture of transgenic products. One strategy to enhance foreign protein production involves exchanging the capacity to produce intrinsic proteins for the capacity to produce a high level of foreign proteins. Suppression of the alpha/alpha' subunit of beta-conglycinin storage protein synthesis in soybean has been shown previously to result in an increase in the accumulation of the glycinin storage protein, some of which is sequestered as proglycinin into de novo endoplasmic reticulum (ER)-derived protein bodies. The exchange of glycinin for conglycinin is quantitative, with the remodelled soybeans possessing a normal protein content with an altered proteome. The green fluorescent protein (GFP)-kdel reporter was transferred in a construct using the glycinin promoter and terminator to mimic glycinin gene expression. When expressed in soybean seeds, GFP-kdel accreted to form ER-derived protein bodies. The introgression of GFP-kdel into the alpha/alpha' subunit of the beta-conglycinin suppression background resulted in a fourfold enhancement of GFP-kdel accumulation to > 7% (w/w) of the total protein in soybean seeds. The resulting seeds accumulated a single population of ER membrane-bound protein bodies that contained both GFP-kdel and glycinin. Thus, the collateral proteome rebalancing that occurs with the suppression of intrinsic proteins in soybean can be exploited to produce an enhanced level of foreign proteins.

In vivo comparison of knee kinematics for subjects having either a posterior stabilized or cruciate retaining high-flexion total knee arthroplasty.

The objective of this study was to determine the in vivo kinematics for subjects having either a fixed posterior stabilized (PS) or cruciate retaining (CR) high-flexion total knee arthroplasty (TKA). Three-dimensional kinematics from full extension to maximum flexion were determined for 30 subjects (15 PS, 15 CR) using fluoroscopy. On average, the PS subjects demonstrated 112 degrees of weight-bearing (WB) flexion, -6.4 mm of posterior femoral rollback, and 2.9 degrees of axial rotation. The CR subjects averaged 117 degrees of WB flexion, -4.9 mm of posterior femoral rollback, and 4.8 degrees of axial rotation. Posterior femoral rollback of the lateral condyle occurred for all PS TKAs and in 93% of the CR TKAs. Only 2 subjects in each group experienced greater than 1.0 mm of condylar lift-off. Subjects in both TKA groups demonstrated excellent WB ranges of motion and kinematic patterns similar to the normal knee, but less in magnitude.

Suppression of soybean oleosin produces micro-oil bodies that aggregate into oil body/ER complexes.

Using RNAi, the seed oil body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-oil bodies about 50 nm in diameter that coalesce with adjacent oil bodies forming a hierarchy of oil body sizes. The oil bodies in the oleosin knockdown form large oil body-ER complexes with the interior dominated by micro-oil bodies and intermediate-sized oil bodies, while the peripheral areas of the complex are dominated by large oil bodies. The complex merges to form giant oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.

Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed.

A mutant Bowman-Birk gene was created that encoded an inactive high-sulfur product. It was used to transform soybean line Asgrow 3237. Transformants bearing the mutant gene were identified by GUS expression, PCR analysis, and Southern analysis. The amount of steady state mRNA from the mutant gene in the transformed plants showed that the gene was highly expressed, but the amount of message from the unmodified Bowman-Birk gene did not change detectably. Proteins synthesized at the direction of the mutant Bowman-Birk gene accumulated in seeds of the transformed plants, and there was a marked decrease in the ability of extracts prepared from these seeds to inhibit trypsin and chymotrypsin despite the presence of Kunitz trypsin inhibitor. The more prevalent mRNA from the mutant gene was considered to out-compete message from the native genes to decrease the amount of active Bowman-Birk inhibitor.

Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine.

The B subunit of the heat labile toxin of enterotoxigenic Escherichia coli (LTB) was used as a model immunogen for production in soybean seed. LTB expression was directed to the endoplasmic reticulum (ER) of seed storage parenchyma cells for sequestration in de novo synthesized inert protein accretions derived from the ER. Pentameric LTB accumulated to 2.4% of the total seed protein at maturity and was stable in desiccated seed. LTB-soybean extracts administered orally to mice induced both systemic IgG and IgA, and mucosal IgA antibody responses, and was particularly efficacious when used in a parenteral prime-oral gavage boost immunization strategy. Sera from immunized mice blocked ligand binding in vitro and immunized mice exhibited partial protection against LT challenge. Moreover, soybean-expressed LTB stimulated the antibody response against a co-administered antigen by 500-fold. These results demonstrate the utility of soybean as an efficient production platform for vaccines that can be used for oral delivery.