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.

Charges for Alcoholic Cirrhosis Exceed All Other Etiologies of Cirrhosis Combined: A National and State Inpatient Survey Analysis.

BACKGROUND: Inpatient charges for patients with cirrhosis are substantial. We aimed to examine trends in inpatient charges among patients with cirrhosis to determine the drivers of healthcare expenditures. We hypothesized that alcoholic cirrhosis (AC) was a significant contributor to overall expense. METHODS: We performed a retrospective analysis of the Health Care Utilization Project Nationwide Inpatient Sample Database 2002-2014 (annual cross-sectional data) and New York and Florida State Inpatient Databases 2010-2012 (longitudinal data). Adult patients with cirrhosis of the liver were categorized as AC versus all other etiologies of cirrhosis combined. Patient characteristics were analyzed using ordinary least squares regression modeling. A random effects model was used to evaluate 30-day readmissions. RESULTS: In total, 1,240,152 patients with cirrhosis were admitted between 2002 and 2014. Of these, 567,510 (45.8%) had a diagnosis of AC. Total charges for AC increased by 95.7% over the time period, accounting for 59.9% of all inpatient cirrhosis-related charges in 2014. Total aggregate charges for AC admissions were $28 billion and increased from $1.4B in 2002 to $2.8B by 2014. In the NIS and SID, patients with AC were younger, white and male. Readmission rates at 30, 60, and 90 days were all higher among AC patients. CONCLUSIONS: Inpatient charges for cirrhosis care are high and increasing. Alcohol-related liver disease accounts for more than half of these charges and is driven by sheer volume of admissions and readmissions of the same patients. Effective alcohol addictions therapy may be the most cost-effective way to substantially reduce inpatient cirrhosis care expenditures.

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.

Increasing Prevalence of Hepatitis C among Hospitalized Children Is Associated with an Increase in Substance Abuse.

OBJECTIVE: To evaluate the impact of substance abuse on pediatric hepatitis C virus (HCV) prevalence, we examined geographic and demographic data on inpatient hospitalizations in children with HCV. STUDY DESIGN: We examined hospitalizations in children using the Kids' Inpatient Database, a part of the Healthcare Cost and Utilization Project. We identified cases using the International Classification of Diseases, 9th edition, codes for HCV infection during 2006, 2009, and 2012. Nonparametric tests for trend were used to calculate trend statistics. RESULTS: From 2006 to 2012 nationally, the number of hospitalizations of children with HCV increased 37% (2.69 to 3.69 per 10 000 admissions; P < .001). The mean age of children hospitalized was 17.6 years (95% CI, 17.4-17.8). HCV cases among those 19-20 years of age represented 68% of the total HCV diagnoses, with a 54% increase over the years sampled (P < .001 for trend). The burden of HCV in children was highest in whites, those in the lowest income quartile, and in the Northeast and Southern regions of the US (all P < .0001). The prevalence of substance use among children with HCV increased from 25% in 2006 to 41% in 2012 (P < .001). CONCLUSION: The increases of HCV in hospitalized children are largely in teenagers, highly associated with substance abuse, and concentrated in Northeast and Southern states. These results strongly suggest that public health efforts to prevent and treat HCV will also need to include adolescents.

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.

Effectiveness and Safety of Sofosbuvir-Based Regimens for Chronic HCV Genotype 3 Infection: Results of the HCV-TARGET Study.

BACKGROUND: Sofosbuvir (SOF) is active against all hepatitis C virus (HCV) genotypes, and SOF-based therapies lead to high rates of sustained virologic response (SVR). However, genotype 3 (GT3) HCV remains a challenge with lower SVR rates reported, particularly in patients with cirrhosis. This study reports the effectiveness and safety of SOF-based therapy in patients with GT3 HCV treated in clinical practice. METHODS: Hepatitis C Virus Therapeutic Registry and Research Network is an international, prospective observational study evaluating patients treated in usual clinical practice. Patients with GT3 HCV were analyzed to assess predictors of treatment response and adverse events using descriptive statistics and multivariable logistic regression. RESULTS: Treatment outcomes were available for 197 patients treated with SOF and ribavirin (RBV), with or without peginterferon, including 54% with cirrhosis and 49% who failed prior therapy. Of 178 patients treated with SOF/RBV, 60% achieved SVR at 12 weeks (SVR12), compared with 84% of 19 patients treated with SOF/peginterferon/RBV. For patients treated with SOF/RBV, the SVR12 rate was 58% in treatment-naive patients with cirrhosis, and 42% in those with cirrhosis who failed prior therapy. In noncirrhotic patients, SVR12 rates were 89% in treatment-naive and 88% in treatment-experienced patients. After controlling for age and sex, absence of cirrhosis (odds ratio [OR], 6.4; 95% confidence interval [CI], 2.78-14.74), albumin levels ≥3.2 g/dL (OR, 12.48; 95% CI, 3.86-40.33), and platelet count >10(5) cells/µL (OR, 7.44; 95% CI, 3.51-15.78) were associated with greater odds of SVR12 CONCLUSIONS: SVR rates were acceptable in patients with GT3 HCV without cirrhosis; however, in those with cirrhosis, treatment with SOF/RBV was suboptimal, highlighting the need for new therapies for this population.

Decreasing mortality among patients hospitalized with cirrhosis in the United States from 2002 through 2010.

BACKGROUND & AIMS: It is not clear whether evidence-based recommendations for inpatient care of patients with cirrhosis are implemented widely or are effective in the community. We investigated changes in inpatient outcomes and associated features over time. METHODS: By using the Healthcare Cost and Utilization Project, National Inpatient Sample, we analyzed 781,515 hospitalizations of patients with cirrhosis from 2002 through 2010. We compared data with those from equal numbers of hospitalizations of patients without cirrhosis and patients with congestive heart failure (CHF), matched for age, sex, and year of discharge. The primary outcome was a change in discharge status over time. Factors associated with outcomes were analyzed by Poisson modeling. RESULTS: The mortality of patients with and without cirrhosis, and patients with CHF, decreased over time. The absolute decrease was significantly greater for patients with cirrhosis (from 9.1% to 5.4%) than for patients without cirrhosis (from 2.6% to 2.1%) or patients with CHF (from 2.5% to 1.4%) (P < .01). However, relative decreases were similar for patients with cirrhosis (41%) and patients with CHF (44%). For patients with cirrhosis, the independent mortality risk ratio decreased steadily to 0.50 by 2010 (95% confidence interval, 0.48-0.52), despite patients' increasing age and comorbidities. Hepatorenal syndrome, hepatocellular carcinoma, variceal bleeding, and spontaneous bacterial peritonitis were associated with a higher mortality rate, but the independent mortality risks for each decreased steadily. Sepsis was associated strongly with increased mortality, and the risk increased over time. CONCLUSIONS: Among patients with cirrhosis in the United States, inpatient mortality decreased steadily from 2002 through 2010, despite increases in patient age and medical complexity. Improvements in cirrhosis care may have contributed to increases in patient survival beyond those attributable to general improvements in inpatient care. Further improvements might require an increased use of proven therapies and the development of new treatments-particularly for sepsis.

Burden of Gastrointestinal, Liver, and Pancreatic Diseases in the United States.

BACKGROUND & AIMS: Gastrointestinal (GI), liver, and pancreatic diseases are a source of substantial morbidity, mortality, and cost in the United States. Quantification and statistical analyses of the burden of these diseases are important for researchers, clinicians, policy makers, and public health professionals. We gathered data from national databases to estimate the burden and cost of GI and liver disease in the United States. METHODS: We collected statistics on health care utilization in the ambulatory and inpatient setting along with data on cancers and mortality from 2007 through 2012. We included trends in utilization and charges. The most recent data were obtained from the Centers for Disease Control and Prevention, the Agency for Healthcare Research and Quality, and the National Cancer Institute. RESULTS: There were 7 million diagnoses of gastroesophageal reflux and almost 4 million diagnoses of hemorrhoids in the ambulatory setting in a year. Functional and motility disorders resulted in nearly 1 million emergency department visits in 2012; most of these visits were for constipation. GI hemorrhage was the most common diagnosis leading to hospitalization, with >500,000 discharges in 2012, at a cost of nearly $5 billion dollars. Hospitalizations and associated charges for inflammatory bowel disease, Clostridium difficile infection, and chronic liver disease have increased during the last 20 years. In 2011, there were >1 million people in the United States living with colorectal cancer. The leading GI cause of death was colorectal cancer, followed by pancreatic and hepatobiliary neoplasms. CONCLUSIONS: GI, liver and pancreatic diseases are a source of substantial burden and cost in the United States.

Safety and efficacy of sofosbuvir-containing regimens in hepatitis C-infected patients with impaired renal function.

BACKGROUND & AIMS: Renal clearance is the major elimination pathway for sofosbuvir (SOF). We assessed the safety and efficacy of SOF-containing regimens in patients with varying baseline estimated glomerular filtration rate (eGFR). METHODS: HCV-TARGET database is a multicentre, longitudinal 'real-world' treatment cohort. RESULTS: A total of 1789 patients [genotypes 1 (72%), 2 (17%) 3 (9%), 4-6 (2%)] had baseline eGFR determination: 73 with eGFR≤45 (18 with eGFR≤30, 5 on dialysis) were compared to 1716 with eGFR>45 ml/min/1.73 m(2) . Patients with baseline eGFR≤45 vs. >45 differed in being female (55% vs. 36%), age ≥65 years (24% vs. 16%), Black race (22% vs. 12%), having cirrhosis with decompensation (73% vs. 24%) and being post-transplant (49% vs. 10%), all P < 0.05. All patients with eGFR≤45 were treated with SOF 400 mg/day (including those on haemodialysis) and had median starting ribavirin (RBV) dose of 800 mg (IQR: 400-1200). Sustained virologic response (SVR) frequencies were similar across eGFR groups, ranging from 82-83%. Patients with eGFR ≤45 more frequently experienced anaemia, worsening renal function and serious AEs (all P < 0.05), and these associations persisted when limiting analysis to RBV-free regimens. Patients with baseline eGFR≤30 and eGFR 31-45 had similar frequencies of efficacy and safety outcomes. CONCLUSIONS: Sustained viral clearance was achieved in 83% of patients with renal impairment (eGFR ≤45 ml/min/1.73 m(2) ) treated with SOF-containing regimens. However, these patients had higher rates of anaemia, worsening renal dysfunction and serious adverse events regardless of use of RBV. Patient with renal impairment require close monitoring and should be treated by providers extensively experienced with SOF-containing regimens.

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.

Application of crystallization-induced asymmetric transformation to a general, scalable method for the resolution of 2,8-disubstituted Tröger's base derivatives.

A general method for the gram scale resolution of 2-substituted and 2,8-disubstituted Tröger's base (TB) derivatives in 63-91% yield has been achieved through the application of crystallization-induced asymmetric transformation (CIAT). Enantiomeric ratios of the resolved TB derivatives range from 99.1:0.9 to >99.5:0.5. Among the Tröger's base compounds resolved are four synthetically valuable bromo and iodo derivatives.

Heart failure management at home: a non-randomised prospective case-controlled trial (HeMan at Home).

BACKGROUND/OBJECTIVES: Heart failure (HF) is a growing clinical and economic burden for patients and health systems. The COVID-19 pandemic has led to avoidance and delay in care, resulting in increased morbidity and mortality among many patients with HF. The increasing burden of HF during the COVID-19 pandemic led us to evaluate the quality and safety of the Hospital at Home (HAH) for patients presenting to their community providers or emergency department (ED) with symptoms of acute on chronic HF (CHF) requiring admission. DESIGN/OUTCOMES: A non-randomised prospective case-controlled of patients enrolled in the HAH versus admission to the hospital (usual care, UC). Primary outcomes included length of stay (LOS), adverse events, discharge disposition and patient satisfaction. Secondary outcomes included 30-day readmission rates, 30-day ED usage and ED dwell time. RESULTS: Sixty patients met inclusion/exclusion criteria and were included in the study. Of the 60 patients, 40 were in the HAH and 20 were in the UC group. Primary outcomes demonstrated that HAH patients had slightly longer LOS (6.3 days vs 4.7 days); however, fewer adverse events (12.5% vs 35%) compared with the UC group. Those enrolled in the HAH programme were less likely to be discharged with postacute services (skilled nursing facility or home health). HAH was associated with increased patient satisfaction compared with Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) score in North Carolina. Secondary outcomes of 30-day readmission and ED usage were similar between HAH and UC. CONCLUSIONS: The HAH pilot programme was shown to be a safe and effective alternative to hospitalisation for the appropriately selected patient presenting with acute on CHF.

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.

Clostridium difficile testing algorithms: what is practical and feasible?

There has been renewed interest in the laboratory diagnosis of Clostridium difficile infections due in large measure to the increase in both numbers and severity of cases of this disease. For the past two decades, enzyme-immunoassays (EIAs) for the detection of first C. difficile toxin A and then toxins A and B have been the most widely used diagnostic test for diagnosis of C. difficile infections. Recently this diagnostic approach has been called into question by the recognition that a screening test which detects glutamate dehydrogenase, a cell wall antigen of C. difficile, was significantly more sensitive than toxins A and B EIAs making it an effective screening test for C. difficile infection. Although sensitive, GDH lacks specificity and so if this test was utilized, a confirmatory test to differentiate false positives from true positives was needed. Studies to date have used cytotoxin neutralization or toxigenic culture as confirmatory tests but both of these have their limitations. A testing algorithm using rapid immunochromatographic devices for detection of GDH and toxins A and B as screening tests will give an accurate test result in approximately 90% of specimens within one hour when using cytotoxin neutralization as a reference method. For the other 10% of specimens, a third test would be needed in the algorithm. This test could be cytotoxin neutralization, toxigenic culture, or PCR for toxin or toxin operon genes.

Impact of tetracycline-clay interactions on bacterial growth.

Antibiotics are extremely effective against bacterial infections due to their selective toxicity for bacteria rather than the host. Extensive use and misuse of antibiotics resulted in significant increases in antibiotic levels in aquatic and soil environments. Bacteria exposed to antibiotics with low concentrations may develop antibiotic resistance. In this study a swelling 2:1 clay mineral montmorillonite (MMT) and a non-swelling 1:1 clay mineral kaolinite were premixed with tetracycline (TC) of varying concentrations. The gram-negative bacteria Escherichia coli (E. coli) and Salmonella enterica (S. enterica) of both TC sensitive and TC resistant strains were tested for their growth in the presence TC-loaded clay minerals of different amounts and under different physico-chemical conditions. The antimicrobial activity of TC was significantly decreased in the presence of MMT. In the absence of MMT, no bacteria growth was found at a TC concentration 0.25mg/mL and above. On the contrast, in the presence of MMT, 50% growth was still found for a TC resistant E. coli at a TC concentration of 5mg/g. The influence of kaolinite was to a lesser degree. These results suggest that antimicrobial agents present in clayey soils could be responsible for possible mutation of bacteria of high antibiotic resistance.

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.