Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation.

Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity.

The conserved multiple PDZ-domain containing protein PATJ stabilizes the Crumbs-Pals1 complex to regulate apical-basal polarity and tight junction formation in epithelial cells. However, the molecular mechanism of PATJ's function in these processes is still unclear. In this study, we demonstrate that knockout of PATJ in epithelial cells results in tight junction defects as well as in a disturbed apical-basal polarity and impaired lumen formation in three-dimensional cyst assays. Mechanistically, we found PATJ to associate with and inhibit histone deacetylase 7 (HDAC7). Inhibition or downregulation of HDAC7 restores polarity and lumen formation. Gene expression analysis of PATJ-deficient cells revealed an impaired expression of genes involved in cell junction assembly and membrane organization, which is rescued by the downregulation of HDAC7. Notably, the function of PATJ regulating HDAC7-dependent cilia formation does not depend on its canonical interaction partner, Pals1, indicating a new role of PATJ, which is distinct from its function in the Crumbs complex. By contrast, polarity and lumen phenotypes observed in Pals1- and PATJ-deficient epithelial cells can be rescued by inhibition of HDAC7, suggesting that the main function of this polarity complex in this process is to modulate the transcriptional profile of epithelial cells by inhibiting HDAC7.

Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis.

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RB responsive microbe Bifidobacterium longum), and non-fermented RB were fed as 10% w/w (diet) to gut microbiota-intactspf or germ-free micegf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed micegf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

Assessment of potentially toxic trace element contamination in urban allotment soils and their uptake by onions: A preliminary case study from Sheffield, England.

Toxic trace element (TTE) contamination in urban soils may pose potential health risks, especially in cities with previous industrial activities. This study aimed to investigate soil contamination in urban allotments in Sheffield, the uptake of TTEs in autumn and spring sown onions (Allium cepa), and their potential risks on human health via consumption of the crops. Paired soil and plant samples were taken in triplicates from four private allotments to assess potentially elevated levels of lead (Pb), zinc (Zn), copper (Cu), arsenic (As), and chromium (Cr). These elements in soils exceeded the ambient background levels for England. Both Pb and As exceeded some UK and EU soil tolerable limits. Concentration factors (CF) were calculated as the ratio of trace element in the plant as compared to that in the soil, and uptake rates were in the order Zn>Cu>Cr>Pb>As. Concentrations were higher for most TTEs in spring sown onions (SSO), and had significantly higher CF (p < 0.05) for Pb and Cr than autumn sown onions (ASO), whereas the opposite was true for As. Toxic elements in plants did not exceed FAO/WHO intake limits when considering TTE content per plant and consumption rates. Human health risk assessment calculations using target hazard quotients (THQ) and hazard indexes (HI) indicated that consuming onions alone did not pose an immediate health risk.

Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds.

This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH = 8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time = 5.0 s, effective capillary length = 55 cm, and run temperature = 20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure.

On the role of monetary incentives in risk preference elicitation experiments.

Incentivized experiments in which individuals receive monetary rewards according to the outcomes of their decisions are regarded as the gold standard for preference elicitation in experimental economics. These task-related real payments are considered necessary to reveal subjects' "true preferences." Using a systematic, large-sample approach with three subject pools of private investors, professional investors, and students, we test the effect of task-related monetary incentives on risk preferences in four standard experimental tasks. We find no significant differences in behavior between and within subjects in the incentivized and non-incentivized regimes. We discuss implications for academic research and forions in the field. Supplementary Information: The online version contains supplementary material available at 10.1007/s11166-022-09377-w.

Integrated Microbiota and Metabolite Changes following Rice Bran Intake during Murine Inflammatory Colitis-Associated Colon Cancer and in Colorectal Cancer Survivors.

Dietary rice bran-mediated inhibition of colon carcinogenesis was demonstrated previously for carcinogen-induced rodent models via multiple anti-cancer mechanisms. This study investigated the role of dietary rice bran-mediated changes to fecal microbiota and metabolites over the time course of colon carcinogenesis and compared murine fecal metabolites to human stool metabolic profiles following rice bran consumption by colorectal cancer survivors (NCT01929122). Forty adult male BALB/c mice were subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated colon carcinogenesis and randomized to control AIN93M (n = 20) or diets containing 10% w/w heat-stabilized rice bran (n = 20). Feces were serially collected for 16S rRNA amplicon sequencing and non-targeted metabolomics. Fecal microbiota richness and diversity was increased in mice and humans with dietary rice bran treatment. Key drivers of differential bacterial abundances from rice bran intake in mice included Akkermansia, Lactococcus, Lachnospiraceae, and Eubacterium xylanophilum. Murine fecal metabolomics revealed 592 biochemical identities with notable changes to fatty acids, phenolics, and vitamins. Monoacylglycerols, dihydroferulate, 2-hydroxyhippurate (salicylurate), ferulic acid 4-sulfate, and vitamin B6 and E isomers significantly differed between rice bran- and control-fed mice. The kinetics of murine metabolic changes by the host and gut microbiome following rice bran consumption complemented changes observed in humans for apigenin, N-acetylhistamine, and ethylmalonate in feces. Increased enterolactone abundance is a novel diet-driven microbial metabolite fecal biomarker following rice bran consumption in mice and humans from this study. Dietary rice bran bioactivity via gut microbiome metabolism in mice and humans contributes to protection against colorectal cancer. The findings from this study provide compelling support for rice bran in clinical and public health guidelines for colorectal cancer prevention and control.

Multi-omics resources for targeted agronomic improvement of pigmented rice.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR-Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

Biocatalytic Foams from Microdroplet-Formulated Self-Assembling Enzymes.

Industrial biocatalysis plays an important role in the development of a sustainable economy, as enzymes can be used to synthesize an enormous range of complex molecules under environmentally friendly conditions. To further develop the field, intensive research is being conducted on process technologies for continuous flow biocatalysis in order to immobilize large quantities of enzyme biocatalysts in microstructured flow reactors under conditions that are as gentle as possible in order to realize efficient material conversions. Here, monodisperse foams consisting almost entirely of enzymes covalently linked via SpyCatcher/SpyTag conjugation are reported. The biocatalytic foams are readily available from recombinant enzymes via microfluidic air-in-water droplet formation, can be directly integrated into microreactors, and can be used for biocatalytic conversions after drying. Reactors prepared by this method show surprisingly high stability and biocatalytic activity. The physicochemical characterization of the new materials is described and exemplary applications in biocatalysis are shown using two-enzyme cascades for the stereoselective synthesis of chiral alcohols and the rare sugar tagatose.

Solutions to Enhance Health with Alternative Treatments (SEHAT) protocol: a double-blinded randomised controlled trial for gut microbiota-targeted treatment of severe acute malnutrition using rice bran in ready-to-use therapeutic foods in Indonesia.

INTRODUCTION: Current formulations of ready-to-use therapeutic foods (RUTFs) to treat severe acute malnutrition (SAM) in children focus on nutrient density and quantity. Less attention is given to foods targeting gut microbiota metabolism and mucosal barrier functions. Heat-stabilised rice bran contains essential nutrients, prebiotics, vitamins and unique phytochemicals that have demonstrated favourable bioactivity to modulate gut microbiota composition and mucosal immunity. This study seeks to examine the impact of RUTF with rice bran on the microbiota during SAM treatment, recovery and post-treatment growth outcomes in Jember, Indonesia. Findings are expected to provide insights into rice bran as a novel food ingredient to improve SAM treatment outcomes. METHODS AND ANALYSIS: A total of 200 children aged 6-59 months with uncomplicated SAM (weight-for-height z-scores (WHZ) <-3, or mid-upper arm circumference (MUAC) <115 mm or having bilateral pitting oedema +/++) or approaching SAM (WHZ<-2.5) will be enrolled in a double-blinded, randomised controlled trial. Children in the active control arm will receive a locally produced RUTF; those in the intervention arm will receive the local RUTF with 5% rice bran. Children will receive daily RUTF treatment for 8 weeks and be monitored for 8 weeks of follow-up. Primary outcomes include the effectiveness of RUTF as measured by changes in weight, WHO growth z-scores, MUAC and morbidity. Secondary outcomes include modulation of the gut microbiome and dried blood spot metabolome, the percentage of children recovered at weeks 8 and 12, and malnutrition relapse at week 16. An intention-to-treat analysis will be conducted for each outcome. ETHICS AND DISSEMINATION: The findings of this trial will be submitted to peer-reviewed journals and will be presented at relevant conferences. Ethics approval obtained from the Medical and Health Research Ethical Committee at the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Madain Yogyakarta Ref. No.: KE/FK/0546/EC/2022 and KE/FK/0703/EC/2023 and from Colorado State University IRB#1823, OHRP FWA00000647. TRIAL REGISTRATION NUMBER: NCT05319717.

Non-Targeted Dried Blood Spot-Based Metabolomics Analysis Showed Rice Bran Supplementation Effects Multiple Metabolic Pathways during Infant Weaning and Growth in Mali.

Rice bran contains essential nutrients, antioxidants, and bioactives with anti-inflammatory and diarrheal protective properties important for infants. This 6-month randomized controlled trial investigated the effects of heat-stabilized rice bran supplementation during Malian infant weaning. Fifty healthy 6-month-old infants were randomized to a rice bran intervention (N = 25) or non-intervention control group (N = 25). Intervention infants received dose-escalating rice bran supplementation for 6 months (1-5 g/day). Monthly infant dried blood spot and anthropometric measurements were collected. Dried blood spot metabolite abundances were compared monthly according to diet for six months. Supplementation resulted in favorable weight-for-age and weight-for-length z-score changes. Non-targeted dried blood spot-based metabolomics identified 796 metabolites, of which 33% had significant fold differences between groups (7-12 months). Lipids and amino acids represented 70.6% of the metabolites identified. Rice bran supplementation during infant weaning significantly modulated the metabolites involved in antioxidant defenses and with neuroactive properties including reduced glutathione, glycine, glutamate, cysteinylglycine, tryptophan betaine, and choline. These findings support rice bran as a weaning ingredient to meet infant nutritional requirements and with the potential to reduce oxidative stress and improve cognitive outcomes. This study provides evidence for dried blood spots as a cost-effective tool to detect infant biomarkers of nutritional and metabolic status.

Fine tuning of a biological machine: DnaK gains improved chaperone activity by altered allosteric communication and substrate binding.

DnaK is a member of the Hsp70 family of molecular chaperones. This molecular machine couples the binding and hydrolysis of ATP to binding and release of substrate proteins. The switches that are involved in allosteric communication within this multidomain protein are mostly unknown. Previous insights were largely obtained by mutants, which displayed either wild-type activity or reduced folding assistance of substrate proteins. With a directed evolution approach for improved folding assistance we selected a DnaK variant characterized by a glycine to alanine substitution at position 384 (G384A); this resulted in a 2.5-fold higher chaperone activity in an in vitro DnaK-assisted firefly luciferase refolding assay. Quantitative biochemical characterization revealed several changes of key kinetic parameters compared to the wild type. Most pronounced is a 13-fold reduced rate constant for substrate release in the ATP-bound state, which we assume, in conjunction with the resulting increase in substrate affinity, to be related to improved chaperone activity. As the underlying mechanistic reason for this change we propose an altered interface of allosteric communication of mutant G384A, which is notably located at a hinge position between nucleotide and substrate binding domain.

Mouth-nose masks impair the visual field of healthy eyes.

BACKGROUND: Mouth-nose masks have been requested to prevent the transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The aim of the present study was to investigate, if wearing a mouth-nose mask impairs the visual field function in normals. METHODS: Thirty eyes of 30 subjects were recruited for the present study. White-on-white perimetry (OCTOPUS 900; 90°) was done and sensitivity was analysed in 14 defined test points (P1-P14, inferior visual field) under 3 different test conditions while the subjects were wearing a mouth-nose mask: (I) 1.5 cm under the lower eyelid, nose clip not used (position1.5cm_no_clip); (II) 1.5 cm under the lower eyelid, nose clip correctly positioned (position1.5cm_with_clip); (III) 0.5 cm under the lower eyelid, nose clip correctly positioned (position0.5cm_with_clip). All data were compared to sensitivity without wearing a mouth-nose mask (reference). Mean Δ was calculated, being the difference between the results of each test condition and reference, respectively. RESULTS: Sensitivity was significantly different between position1.5cm_no_clip and reference at 10 test points (p<0.05). Sensitivity at test point P7 was significantly different between position1.5cm_with_clip and position0.5cm_with_clip compared to reference (p<0.001), respectively. Mean Δ increased while wearing a mask at P7: position1.5cm_with_clip (-8.3 dB ± 7.3 dB) < position0.5cm_with_clip (-11.3 dB ± 9.5 dB) < position1.5cm_no_clip (-20.1 dB ± 7.6 dB). CONCLUSION: Visual field function was observed to be significantly impaired in the inferior-nasal sector while persons were wearing a mouth-nose mask, especially when the nose clip was not correctly used.

Metabolomics of Rice Bran Differentially Impacted by Fermentation With Six Probiotics Demonstrates Key Nutrient Changes for Enhancing Gut Health.

The consumption of rice bran has been shown to have a positive effect on nutritional status and prevention of chronic diseases related to hundreds of metabolites with bioactivity. Consumption after fermentation can lead to specific beneficial effects, yet is lacking complete characterization when fermented with diverse strains. The objective of this study was to examine the effect of fermentation on the rice bran metabolite profile. Bacterial probiotics (Bifidobacterium longum, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus and, Escherichia coli) were used to ferment rice bran alone or after incubation with yeast probiotic Saccharomyces boulardii. Fermented rice bran was methanol extracted and analyzed by UPLC-MS/MS. The metabolome of the two fermentation types was deeply modified when compared with non-fermented rice bran. The two-step fermentation provided alternative substrate to the bacteria in a few cases. Key metabolites of high nutritional value (essential amino acids, vitamins) and gut health (arabinose, maltotriose) were identified.

Arsenic speciation in rice bran: Agronomic practices, postharvest fermentation, and human health risk assessment across the lifespan.

Arsenic (As) exposure is a global public health concern affecting millions worldwide and stems from drinking water and foods containing As. Here, we assessed how agronomic practices and postharvest fermentation techniques influence As concentrations in rice bran, and calculated health risks from consumption. A global suite of 53 rice brans were tested for total As and speciation. Targeted quantification of inorganic As (iAs) concentrations in rice bran were used to calculate Target Hazard Quotient (THQ) and Lifetime Cancer Risk (LCR) across the lifespan. Mean iAs was highest in Thailand rice bran samples (0.619 mg kg-1) and lowest in Guatemala (0.017 mg kg-1) rice bran samples. When comparing monosodium-methanearsonate (MSMA) treated and the Native-soil counterpart under the irrigation technique Alternate Wetting and Drying (AWD) management, the MSMA treatment had significantly higher total As (p = 0.022), and iAs (p = 0.016). No significant differences in As concentrations were found between conventional and organic production, nor between fermented and non-fermented rice bran. Health risk assessment calculations for the highest iAs-rice bran dosage scenario for adults, children and infants exceeded THQ and LCR thresholds, and LCR was above threshold for median iAs-rice bran. This environmental exposure investigation into rice bran provides novel information with food safety guidance for an emerging global ingredient.

Daily Rice Bran Consumption for 6 Months Influences Serum Glucagon-Like Peptide 2 and Metabolite Profiles without Differences in Trace Elements and Heavy Metals in Weaning Nicaraguan Infants at 12 Months of Age.

BACKGROUND: Environmental enteric dysfunction (EED) is associated with chronic gut inflammation affecting nutrient absorption and development of children, primarily in low- and middle-income countries. Several studies have shown that rice bran (RB) supplementation provides nutrients and modulates gut inflammation, which may reduce risk for undernutrition. OBJECTIVE: The aim was to evaluate the effect of daily RB dietary supplementation for 6 mo on serum biomarkers in weaning infants and associated changes in serum and stool metabolites. METHODS: A 6-mo randomized-controlled dietary intervention was conducted in a cohort of weaning 6-mo-old infants in León, Nicaragua. Anthropometric indices were obtained at 6, 8, and 12 mo. Serum and stool ionomics and metabolomics were completed at the end of the 6-mo intervention using inductively coupled plasma MS and ultra-high performance LC-tandem MS. The ɑ1-acid glycoprotein, C-reactive protein, and glucagon-like peptide 2 (GLP-2) serum EED biomarkers were measured by ELISA. RESULTS: Twenty-four infants in the control group and 23 in the RB group successfully completed the 6-mo dietary intervention with 90% dietary compliance. RB participants had higher concentrations of GLP-2 as compared with control participants at 12 mo [median (IQR): 743.53 (380.54) pg/mL vs. 592.50 (223.59) pg/mL; P = 0.04]. Metabolite profiles showed significant fold differences of 39 serum metabolites and 44 stool metabolites from infants consuming RB compared with control, and with significant metabolic pathway enrichment scores of 4.7 for the tryptophan metabolic pathway, 5.7 for polyamine metabolism, and 5.7 for the fatty acid/acylcholine metabolic pathway in the RB group. No differences were detected in serum and stool trace elements or heavy metals following daily RB intake for 6 mo. CONCLUSIONS: RB consumption influences a suite of metabolites associated with growth promotion and development, while also supporting nutrient absorption as measured by changes in serum GLP-2 in Nicaraguan infants. This clinical trial was registered at https://clinicaltrials.gov as NCT02615886.

Protein Stability Buffers the Cost of Translation Attenuation following eIF2α Phosphorylation.

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital response to many forms of stress, including protein-misfolding stress in the endoplasmic reticulum (ER stress). It is believed to cause a general reduction in protein synthesis while enabling translation of few transcripts. Such a reduction of protein synthesis comes with the threat of depleting essential proteins, a risk thought to be mitigated by its transient nature. Here, we find that translation attenuation is not uniform, with cytosolic and mitochondrial ribosomal subunits being prominently downregulated. Translation attenuation of these targets persists after translation recovery. Surprisingly, this occurs without a measurable decrease in ribosomal proteins. Explaining this conundrum, translation attenuation preferentially targets long-lived proteins, a finding not only demonstrated by ribosomal proteins but also observed at a global level. This shows that protein stability buffers the cost of translational attenuation, establishing an evolutionary principle of cellular robustness.

Blood Lead Levels in Females of Childbearing Age in Flint, Michigan, and the Water Crisis.

OBJECTIVE: To compare blood lead levels in females of childbearing age, 12-50 years, living within and adjacent to Flint, Michigan, before, during, and after the Flint River water exposure and compare the levels to those that have been shown to cause fetal loss and preterm birth. METHODS: The switch in the community water source to the Flint River occurred on April 25, 2014, and was reverted to the original source on October 15, 2015. Using a retrospective cross-sectional study design using geocoded blood lead levels obtained from all females of childbearing age available from a single hospital database, we compared blood lead levels for the following 18-month time periods: April 25, 2012-October 15, 2013 (PRE), April 25, 2014-October 15, 2015 (DURING), and April 25, 2016-October 15, 2017 (POST). RESULTS: Results are reported as geometric mean (95% CI). Within Flint, PRE blood lead levels in females of childbearing age were 0.69 micrograms/dL (95% CI 0.63-0.75), DURING blood lead levels were 0.65 micrograms/dL (95% CI 0.60-0.71), and POST blood lead levels were 0.55 micrograms/dL (95% CI 0.54-0.56). DURING Flint River water exposure blood lead levels were not significantly different than the PRE Flint River water time period. POST Flint River water exposure blood lead levels were significantly lower than both PRE and DURING levels. Overall, lower blood lead levels were found outside the Flint boundary in all cohorts. CONCLUSION: Blood lead levels in Flint females of childbearing age did not increase during the Flint River water exposure and subsequent 18-month time period. Mean blood lead levels during the Flint River water exposure are not consistent with the markedly higher blood lead levels reported in the literature to be associated with fetal loss, low birth weight, or preterm birth.

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast.

We have engineered the chloroplast of eukaryotic algae to produce a number of recombinant proteins, including human monoclonal antibodies, but, to date, have achieved expression to only 0.5% of total protein. Here, we show that, by engineering the mammalian coding region of bovine mammary-associated serum amyloid (M-SAA) as a direct replacement for the chloroplast psbA coding region, we can achieve expression of recombinant protein above 5% of total protein. Chloroplast-expressed M-SAA accumulates predominantly as a soluble protein, contains the correct amino terminal sequence and has little or no post-translational modification. M-SAA is found in mammalian colostrum and stimulates the production of mucin in the gut, acting in the prophylaxis of bacterial and viral infections. Chloroplast-expressed and purified M-SAA is able to stimulate mucin production in human gut epithelial cell lines. As Chlamydomonas reinhardtii is an edible alga, production of therapeutic proteins in this organism offers the potential for oral delivery of gut-active proteins, such as M-SAA.