Comparing microbial populations from diverse hydrothermal features in Yellowstone National Park: hot springs and mud volcanoes.

Geothermal features, such as hot springs and mud volcanoes, host diverse microbial life, including many extremophile organisms. The physicochemical parameters of the geothermal feature, such as temperature, pH, and heavy metal concentration, can influence the alpha and beta diversity of microbial life in these environments, as can spatiotemporal differences between sites and sampling. In this study, water and sediment samples were collected and analyzed from eight geothermal sites at Yellowstone National Park, including six hot springs, a mud volcano, and an acidic lake within the same week in July 2019, and these geothermal sites varied greatly in their temperature, pH, and chemical composition. All samples were processed and analyzed with the same methodology and taxonomic profiles and alpha and beta diversity metrics determined with 16S rRNA sequencing. These microbial diversity results were then analyzed with respect to pH, temperature, and chemical composition of the geothermal features. Results indicated that predominant microbial species varied greatly depending on the physicochemical composition of the geothermal site, with decreases in pH and increases in dissolved heavy metals in the water corresponding to decreases in alpha diversity, especially in the sediment samples. Similarly, sites with acidic pH values had more similar microbial populations (beta diversity) to one another than to relatively neutral or alkaline pH geothermal sites. This study suggests that pH and/or heavy metal concentration is a more important driver for microbial diversity and population profile than the temperature for these sites and is also the first reported microbial diversity study for multiple geothermal sites in Yellowstone National Park, including the relatively new mud volcano Black Dragon's Caldron, which erupted in 1948.

Comparing Learning Outcomes and Student and Instructor Perceptions of a Simultaneous Online versus In-Person Biochemistry Laboratory Course.

This article compares the learning outcomes and student perceptions of a one semester undergraduate biochemistry laboratory course that was taught using either a fully online or a fully in-person teaching modality. The semester long biochemistry laboratory mimicked the work sequence a researcher would encounter when transforming a plasmid containing a gene for a recombinant protein (superfolder green fluorescent protein, sf-GFP) and then purifying, identifying, and characterizing that protein. The two modalities of the course were completed in the same semester, by the same instructor, in which students self-selected into which modality they preferred at the beginning of the semester. Students in the in-person section reported enjoying the laboratory course more than the online cohort of students and found it to be less time-consuming. Additionally, a survey of biochemistry laboratory instructors from across the United States, who had experience teaching both online and in-person biochemistry laboratories, indicated that the majority of instructors that responded to the survey preferred the in-person modality: believing them to be more effective and engaging for the students, more enjoyable, and less time-consuming for the instructor. Statistical analysis of formative and summative assessments indicated no significant difference in non-hands-on student learning objective and learning goal scores between the two groups, but the small number of students and instructors in this study limits the generalizability of these results.

Validation of a new extraction-free real-time PCR test to detect MPOX virus.

The monkeypox (Mpox) virus has raised significant concerns given its recent spread with an increasing number of confirmed cases worldwide. In this study, we evaluated the performance of a laboratory developed test (LDT) using BioGX Xfree hMPXV/OPXV reagents for the qualitative detection of non-variola Orthopoxviruses and Mpox virus DNA, in swabs from human pustular or vesicular rash specimens. Analytical and clinical testing analysis were carried out on two different platforms: the BD MAX™ System (BD Diagnostics) and the new pixl.16 Real-Time PCR Platform (BioGX), using a synthetic Mpox virus DNA (ATCC VR-3270SD) and residual clinical samples previously identified with an EUA approved Mpox real-time PCR assay. In the end, the Xfree hMPXV/OPXV LDT proved to be a sensitive, specific, and reproducible test for the detection of Mpox on both platforms evaluated with the pixl.16 having an advantage of a small footprint and providing faster TAT facilitated by an extraction-free workflow.

A blueprint for fortification planning and programming: Lessons learned from an analytical review of existing fortification frameworks.

With multiple food fortification frameworks, countries can find it challenging to determine optimal methods for planning and implementing food fortification programmes to combat vitamin and mineral deficiencies, especially without additional technical support. To address this challenge, this study aimed to review existing frameworks to determine consistencies, differences, strengths, and weaknesses across the frameworks, and based on the review findings, formulate an enhanced and streamlined fortification framework. Nineteen frameworks were ultimately examined following a comprehensive literature review and key informant interviews. Generally, the reviewed frameworks amply describe motives and methods for the determination of fortification need and feasibility, industry engagement/quality assurance and quality control, and impact evaluations/surveillance. However, there was limited inclusion or discussion throughout the reviewed frameworks around harmonization of fortification with existing micronutrient interventions; fortification policy and/or strategy; enforcement, incentives, and penalties to ensure producer compliance with industry standards; and periodic fortification programme review and reassessment. The findings were used to develop a comprehensive Fortification Blueprint that aims to provide structured guidance and a library of tools and resources to fortification programme managers and key stakeholders to ensure optimal and sustainable programme design.

Performance evaluation of the Aptima CMV quant assay using plasma and non-plasma samples.

BACKGROUND: Cytomegalovirus (CMV) infection has a major negative impact on transplantation and is associated with increased morbidity and mortality in this patient population. Quantitation of CMV infections using a molecular test is the preferred method for monitoring patients post-transplant. For this analysis, we compared the Aptima CMV Quant Assay (Aptima CMV) on the Panther system to the ELITech MGB Alert® CMV 3.0 ASR (MGB CMV) run on the ELITe InGenius®. METHODS: The analytical performance of the assay was assessed using commercially available CMV reference panels that meet the 1st WHO International Standard for Human Cytomegalovirus for nucleic acid amplification techniques. The clinical performance of the assay was determined using 249 plasma and non-plasma samples. RESULTS: The 95% LOD of the Aptima assay was determined to be 50 IU/mL and 200 IU/mL for the MGB CMV assay. A strong linear correlation with the reference panel (R2 = 0.9945), excellent reproducibility, and accuracy (R2 = 0.986) over the detection range of the assay was observed. Of the 249 clinical samples tested, only 17 (6.8%) yielded discordant results which were at or near the lower limit of quantification of the assays. Although the Aptima CMV assay demonstrated excellent concordance of qualitative results to the MGB CMV assay for all samples, the MGB CMV quantified CMV DNA at an average of 0.5 Log IU/mL higher than Aptima CMV. CONCLUSION: The Aptima CMV assay is both sensitive and accurate in quantifying CMV in both plasma and non-plasma specimens on the fully automated Panther system.

Preventing birth defects, saving lives, and promoting health equity: an urgent call to action for universal mandatory food fortification with folic acid.

July 20, 2021 marked the 30th anniversary of the publication of the landmark trial by the British Medical Research Council showing unequivocally that maternal intake of folic acid (vitamin B9) starting before pregnancy prevents most cases of infant spina bifida and anencephaly-two major neural tube defects that are severe, disabling, and often fatal. Mandatory food fortification with folic acid is a safe, cost-effective, and sustainable intervention to prevent spina bifida and anencephaly. Yet few countries implement fortification with folic acid; only a quarter of all preventable spina bifida and anencephaly cases worldwide are currently avoided by food fortification. We summarise scientific evidence supporting immediate, mandatory fortification with folic acid to prevent the development of spina bifida and anencephaly. We make an urgent call to action for the World Health Assembly to pass a resolution for universal mandatory folic acid fortification. Such a resolution could accelerate the slow pace of spina bifida and anencephaly prevention globally, and will assist countries to reach their 2030 Sustainable Development Goals on child mortality and health equity. The cost of inaction is profound, and disproportionately impacts susceptible populations in low-income and middle-income countries.

Enablers and Barriers of Zinc Fortification; Experience from 10 Low- and Middle-Income Countries with Mandatory Large-Scale Food Fortification.

Adequate zinc nutrition is important for child growth, neurodevelopment, immune function, and normal pregnancy outcomes. Seventeen percent of the global population is estimated to be at risk for inadequate zinc intake. However, zinc is not included in the fortification standards of several low- and middle-income countries with mandatory fortification programs, despite data suggesting a zinc deficiency public health problem. To guide policy decisions, we investigated the factors enabling and impeding the inclusion of zinc as a fortificant by conducting in-depth interviews with 17 key informants from 10 countries. Findings revealed the decision to include zinc was influenced by guidance from international development partners and enabled by the assessment of zinc deficiency, mandatory regional food fortification standards which included zinc, the World Health Organization (WHO) guidelines for zinc fortification, and the low cost of zinc compound commonly used. Barriers included the absence of zinc from regional fortification standards, limited available data on the efficacy and effectiveness of zinc fortification, and the absence of national objectives related to the prevention of zinc deficiency. To promote zinc fortification there is a need to put the prevention of zinc deficiency higher on the international nutrition agenda and to promote large-scale food fortification as a key deficiency mitigation strategy.

Effects of Foods Fortified with Zinc, Alone or Cofortified with Multiple Micronutrients, on Health and Functional Outcomes: A Systematic Review and Meta-Analysis.

Seventeen per cent of the world's population is estimated to be at risk of inadequate zinc intake, which could in part be addressed by zinc fortification of widely consumed foods. We conducted a review of efficacy and effectiveness studies to ascertain the effect of zinc fortification [postharvest fortification of an industrially produced food or beverage; alone or with multiple micronutrients (MMN)] on a range of health outcomes. Previous reviews have required that the effect of zinc be isolated; because zinc is always cofortified with MMN in existing fortification programs, we did not impose this condition. Outcomes assessed were zinc-related biomarkers (plasma or serum, hair or urine zinc concentrations, comet assay, plasma fatty acid concentrations, and the proportion of and total zinc absorbed in the intestine from the diet), child anthropometry, morbidity, mortality, cognition, plasma or serum iron and copper concentrations, and for observational studies, a change in consumption of the food vehicle. Fifty-nine studies were included in the review; 54 in meta-analyses, totaling 73 comparisons. Zinc fortification with and without MMN increased plasma zinc concentrations (efficacy, n = 27: 4.68 µg/dL; 95% CI: 2.62-6.75; effectiveness, n = 13: 6.28 µg/dL; 95% CI: 5.03-7.77 µg/dL) and reduced the prevalence of zinc deficiency (efficacy, n = 11: OR: 0.76, 95% CI: 0.60-0.96; effectiveness, n = 10: OR: 0.45, 95% CI: 0.31-0.64). There were statistically significant increases in child weight (efficacy, n = 11: 0.43 kg, 95% CI: 0.11-0.75 kg), improvements in short-term auditory memory (efficacy, n = 3: 0.32 point, 95% CI: 0.13-0.50 point), and decreased incidence of diarrhea (efficacy, n = 3: RR: 0.79, 95% CI: 0.68-0.92) and fever (efficacy, n = 2: RR: 0.85, 95% CI: 0.74-0.97). However, these effects cannot be solely attributed to zinc. Our review found that zinc fortification with or without MMN reduced the prevalence of zinc deficiency and may provide health and functional benefits, including a reduced incidence of diarrhea.

Systematic Process Framework for Conducting Implementation Science Research in Food Fortification Programs.

Food fortification has proven to be an effective approach for preventing micronutrient deficiencies in many settings. Factors that lead to successful fortification programs are well established. However, due to the multisectoral nature of fortification and the added complexities present in many settings, the barriers to success are not always evident and the strategies to address them are not always obvious. We developed a systematic process for identifying and addressing gaps in the implementation of a food fortification program. The framework is composed of 4 phases: (1) connect program theory of change to program implementation; (2) develop an implementation research agenda; (3) conduct implementation research; and (4) analyze findings and develop/disseminate recommendations for next steps. We detail steps in each phase to help guide teams through the process. To our knowledge, this is the first attempt to outline a systematic process for applying implementation science research to food fortification. The development of this framework is intended to promote implementation research in the field of food fortification, thus improving access to and effectiveness of this key public health intervention.

Evaluation of Global Experiences in Large-Scale Double-Fortified Salt Programs.

BACKGROUND: Double-fortified salt (DFS) is a vehicle for dual fortification with iron and iodine, to reduce their respective deficiencies. This background article is the third in a series reviewing available research, analyses, and experiences on DFS as an effective delivery vehicle for iron and iodine. OBJECTIVES: The objective of this article is to systematically evaluate current programs distributing DFS around the world and catalogue opportunities, risks, and challenges related to programs that incorporate DFS. We carried out a narrative review of DFS programs from around the world with our data sources deriving from a mix of a nonsystematic literature search and interviews with key informants. METHODS: We assessed programmatic experience with DFS from social safety net programs in India (from the states of Bihar, Madhya Pradesh, and Uttar Pradesh) and from non-social safety net country programs or projects in Argentina, Cote d'Ivoire, Kenya, Morocco, Nigeria, Philippines, and Sri Lanka. RESULTS: Findings revealed color change of the final DFS product was an issue in 9 of the 14 programs or studies reviewed and was the most significant challenge that had a direct impact on consumer acceptance and uptake regardless of type of program (open market or social safety net). Other challenges identified were related to the quality of the salt and lack of DFS formulation standards and regulatory monitoring protocols. CONCLUSIONS: DFS programs need to focus on 1) improved technology with better consumer acceptance and better performance when used with lower-quality salt; 2) elucidation and enforcement of DFS formulation quality standards, along with producer incentives; and 3) strong government backing at the policy level. DFS offers a unique opportunity to leverage an almost universally consumed product with the addition of 2 important nutrients missing in many populations. However, program "maturity" will take time with urgent attention needed for quality production.

Reducing the Burden of Anemia and Neural Tube Defects in Low- and Middle-Income Countries: An Analysis to Identify Countries with an Immediate Potential to Benefit from Large-Scale Mandatory Fortification of Wheat Flour and Rice.

Using a predetermined set of criteria, including burden of anemia and neural tube defects (NTDs) and an enabling environment for large-scale fortification, this paper identifies 18 low- and middle-income countries with the highest and most immediate potential for large-scale wheat flour and/or rice fortification in terms of health impact and economic benefit. Adequately fortified staples, delivered at estimated coverage rates in these countries, have the potential to avert 72.1 million cases of anemia among non-pregnant women of reproductive age; 51,636 live births associated with folic acid-preventable NTDs (i.e., spina bifida, anencephaly); and 46,378 child deaths associated with NTDs annually. This equates to a 34% reduction in the number of cases of anemia and 38% reduction in the number of NTDs in the 18 countries identified. An estimated 5.4 million disability-adjusted life years (DALYs) could be averted annually, and an economic value of 31.8 billion United States dollars (USD) generated from 1 year of fortification at scale in women and children beneficiaries. This paper presents a missed opportunity and warrants an urgent call to action for the countries identified to potentially avert a significant number of preventable birth defects, anemia, and under-five child mortality and move closer to achieving health equity by 2030 for the Sustainable Development Goals.

The Unfinished Agenda for Food Fortification in Low- and Middle-Income Countries: Quantifying Progress, Gaps and Potential Opportunities.

Large-scale food fortification (LSFF) is a cost-effective intervention that is widely implemented, but there is scope to further increase its potential. To identify gaps and opportunities, we first accessed the Global Fortification Data Exchange (GFDx) to identify countries that could benefit from new fortification programs. Second, we aggregated Fortification Assessment Coverage Toolkit (FACT) survey data from 16 countries to ascertain LSFF coverage and gaps therein. Third, we extended our narrative review to assess current innovations. We identified 84 countries as good candidates for new LSFF programs. FACT data revealed that the potential of oil/ghee and salt fortification is not being met due mainly to low coverage of adequately fortified foods (quality). Wheat, rice and maize flour fortification have similar quality issues combined with lower coverage of the fortifiable food at population-level (< 50%). A four-pronged strategy is needed to meet the unfinished agenda: first, establish new LSFF programs where warranted; second, systems innovations informed by implementation research to address coverage and quality gaps; third, advocacy to form new partnerships and resources, particularly with the private sector; and finally, exploration of new fortificants and vehicles (e.g. bouillon cubes; salt fortified with multiple nutrients) and other innovations that can address existing challenges.

Addressing the Fortification Quality Gap: A Proposed Way Forward.

Large-scale food fortification is an effective, sustainable, and scalable intervention to address vitamin and mineral deficiencies, however, pressing gaps exist globally around ensuring the quality of fortified foods. This paper summarizes the global challenges and gaps faced in monitoring the quality of fortified foods, the guidance produced in response to these challenges, where we are today in terms of effective implementation, and what approaches and opportunities may be usefully applied to enhance the quality of fortified foods moving forward.

Analyzing protein glycosylation using UHPLC: a review.

Ultrahigh performance liquid chromatography (UHPLC) uses small stationary-phase particle size (<2 µm) and high pressure in order to achieve rapid and efficient separations. The speed and high resolution of this method has made it a valuable tool for analyzing the complex glycosylation patterns found in post-translationally modified proteins. This article highlights the differences between UHPLC and HPLC and reviews recent UHPLC applications and developments for detecting glycosylated proteins (e.g., glycomics studies) and characterizing glycosylated pharmaceuticals (e.g., monoclonal antibodies).

Q-X1-P-X2 motif search for potential celiac disease risk has poor selectivity.

The European Food Safety Authority (EFSA) recently published guidelines for assessment of potential celiac disease risk for newly expressed proteins in genetically modified (GM) crops. This novel step-wise approach prescribes, in part, how to conduct sequence identity searches between a newly expressed protein and known celiac disease peptides including a Q/E-X1-P-X2 amino acid motif. To evaluate the specificity of the recommended sequence identity searches in the context of risk assessment, protein sequences from celiac disease causing crops, as well as from crops not associated with celiac disease, were compared with known HLA-DQ restricted epitopes and searched for the presence of motifs followed by peptide analysis. Searches for the presence of the Q/E-X1-P-X2-motif were found to generate a high proportion of false-positive hits irrelevant to celiac disease risk. Identification of a 9mer exact match between a newly expressed protein and the known celiac disease peptides (recommended by the guideline) along with a supplementary sequence comparisons (suggested by FARRP/AllergenOnline) is considered better suited to more specifically capture the potential risk of a newly expressed protein for celiac disease.

Review of Grain Fortification Legislation, Standards, and Monitoring Documents.

OBJECTIVE: Analyze the content of documents used to guide mandatory fortification programs for cereal grains. METHODS: Legislation, standards, and monitoring documents, which are used to mandate, provide specifications for, and confirm fortification, respectively, were collected from countries with mandatory wheat flour (n=80), maize flour (n=11), and/or rice (n=6) fortification as of January 31, 2015, yielding 97 possible country-grain combinations (e.g., Philippines-wheat flour, Philippines-rice) for the analysis. After excluding countries with limited or no documentation, 72 reviews were completed, representing 84 country-grain combinations. Based on best practices, a criteria checklist was created with 44 items that should be included in fortification documents. Two reviewers independently scored each available document set for a given country and food vehicle (a country-grain combination) using the checklist, and then reached consensus on the scoring. We calculated the percentage of country-grain combinations containing each checklist item and examined differences in scores by grain, region, and income level. RESULTS: Of the 72 country-grain combinations, the majority of documentation came from countries in the Americas (46%) and Africa (32%), and most were from upper and lower middle-income countries (73%). The majority of country-grain combinations had documentation stating the food vehicle(s) to be fortified (97%) and the micronutrients (e.g., iron) (100%), fortificants (e.g., ferrous fumarate) (88%), and fortification levels required (96%). Most (78%) stated that labeling is required to indicate a product is fortified. Many country-grain combinations described systems for external (64%) monitoring, and stated that industry is required to follow quality assurance/quality control (64%), though detailed protocols (33%) and roles and responsibilities (45%) were frequently not described. CONCLUSIONS: Most country-grain combinations have systems in place for internal, external, and import monitoring. However, documentation of other important items that would influence product compliance to national standard, such as roles and responsibilities between agencies, the cost of regulating fortification, and enforcement strategies, are often lacking. Countries with existing mandatory fortification can improve upon these items in revisions to their documentation while countries that are beginning fortification can use the checklist to assist in developing new policies and programs.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

Regulatory Monitoring of Fortified Foods: Identifying Barriers and Good Practices.

While fortification of staple foods and condiments has gained enormous global traction, poor performance persists throughout many aspects of implementation, most notably around the critical element of regulatory monitoring, which is essential for ensuring foods meet national fortification standards. Where coverage of fortified foods is high, limited nutritional impact of fortification programs largely exists due to regulatory monitoring that insufficiently identifies and holds producers accountable for underfortified products. Based on quality assurance data from 20 national fortification programs in 12 countries, we estimate that less than half of the samples are adequately fortified against relevant national standards. In this paper, we outline key findings from a literature review, key informant interviews with 11 fortification experts, and semi-quantitative surveys with 39 individuals from regulatory agencies and the food fortification industry in 17 countries on the perceived effectiveness of regulatory monitoring systems and barriers to compliance against national fortification standards. Findings highlight that regulatory agencies and industry disagree on the value that enforcement mechanisms have in ensuring compliance against standards. Perceived political risk of enforcement and poorly resourced inspectorate capacity appear to adversely reinforce each other within an environment of unclear legislation to create a major hurdle for improving overall compliance of fortification programs against national standards. Budget constraints affect the ability of regulatory agencies to create a well-trained inspector cadre and improve the detection and enforcement of non-compliant and underfortified products. Recommendations to improve fortification compliance include improving technical capacity; ensuring sustained leadership, accountability, and funding in both the private and the public sectors; and removing political barriers to ensure consistent detection of underfortified products and enforcement of applicable fortification standards. Only by taking concrete steps to improve the entire regulatory system that is built on a cooperative working relationship between regulatory agencies and food producers will a nutrition strategy that uses fortification see its intended health effects.