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At least 19 records

Diagnostic Evaluation of the IS1081-Targeted Real-Time PCR for Detection of Mycobacterium bovis DNA in Bovine Milk Samples

The ability of Mycobacterium bovis (M. bovis) to survive in bovine milk has emerged as a serious public health concern. The first objective of this study was to evaluate the diagnostic utility of IS1081-targeted real-time PCR for the detection of M. bovis DNA in different fractions of bovine milk. In a model study, bovine milk samples were spiked with serially diluted M. bovis BCG to investigate the detection limit of M. bovis DNA in whole milk and milk fractions (cream, pellet, and pellet + cream combined) using IS1081 real-time PCR. The assay was then used to detect M. bovis DNA in whole milk and milk fractions from naturally infected animals. The results showed that the IS1081 real-time PCR was more sensitive when detecting M. bovis DNA in the cream layer alone and cream + pellet combined compared to whole milk or the pellet alone. While PCR-based diagnostic assays for the detection of M. bovis in milk samples provide a quicker diagnostic tool for bovine tuberculosis, safe processing, and handling of M. bovis-infected milk samples remain a challenge and pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to rapidly inactivate infected specimens while preserving nucleic acid for subsequent Molecular analysis. Therefore, the secondary objective of this study was to evaluate the ability of MTM to inactivate M. bovis BCG in spiked milk samples as well as its ability to preserve BCG DNA for the PCR assay. The results showed that MTM can successfully inactivate BCG alone or in spiked milk samples while preserving DNA for the PCR assay. The CT values of M. bovis BCG alone and spiked milk samples aliquoted in MTM and without MTM were similar at various dilutions. Taken together, our results indicate that using DNA extracted from the milk cream fraction alone or combined milk cream and pellet improved the recovery rate of M. bovis DNA in bovine milk samples. MTM has the potential to provide a safe and rapid sample processing tool for M. bovis inactivation in milk samples and preserve DNA for molecular diagnostics.

59 BASIC BIOLOGICAL SCIENCES↗

Associations between SARS-CoV-2 Infection or COVID-19 Vaccination and Human Milk Composition: A Multi-Omics Approach

Background: The risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via human milk-feeding is virtually nonexistent. Adverse effects of coronavirus disease 2019 (COVID-19) vaccination for lactating individuals are not different from the general population, and no evidence has been found that their infants exhibit adverse effects. Yet, there remains substantial hesitation among this population globally regarding the safety of these vaccines. Objectives: Herein, we aimed to determine if compositional changes in milk occur following SARS-CoV-2 infection or COVID-19 vaccination, including any evidence of vaccine components. Methods: An extensive multiomics approach was taken using a subset of milk samples obtained as part of our broad studies examining the effects on milk of SARS-CoV-2 infection and COVID-19 vaccination. Results: We found that compared with unvaccinated individuals, SARS-CoV-2 infection was associated with significant compositional differences in 67 proteins, 385 lipids, and 13 metabolites. In contrast, COVID-19 vaccination was not associated with any changes in lipids or metabolites, although it was associated with changes in 13 or fewer proteins. Compositional changes in milk differed by vaccine. Changes following vaccination were greatest after 1–6 h for the mRNA-based Moderna vaccine (8 changed proteins), 3 d for the mRNA-based Pfizer (4 changed proteins), and adenovirus-based Johnson and Johnson (13 changed proteins) vaccines. Proteins that changed after both natural infection and Johnson and Johnson vaccine were associated mainly with systemic inflammatory responses. In addition, no vaccine components were detected in any milk sample. Conclusions: Together, our data provide evidence of only minimal changes in milk composition because of COVID-19 vaccination, with much greater changes after natural SARS-CoV-2 infection.

60 APPLIED LIFE SCIENCES↗

Campylobacter jejuni resistance to human milk involves the acyl carrier protein AcpP

Campylobacter jejuni is a common foodborne pathogen worldwide that is associated with high rates of morbidity and mortality among infants in low- to middle-income countries (LMICs). Human milk provides infants with an important source of nutrients and contains antimicrobial components for protection against infection. However, recent studies, including our own, have found significantly higher levels of Campylobacter in diarrheal stool samples collected from breastfed infants compared to non-breastfed infants in LMICs. We hypothesized that C. jejuni has unique strategies to resist the antimicrobial properties of human milk. Transcriptional profiling found human milk exposure induces genes associated with ribosomal function, iron acquisition, and amino acid utilization in C. jejuni strains 81–176 and 11168. However, unidentified proteinaceous components of human milk prevent bacterial growth. Evolving both C. jejuni isolates to survive in human milk resulted in mutations in genes encoding the acyl carrier protein (AcpP) and the major outer membrane porin (PorA). Introduction of the PorA/AcpP amino acid changes into the parental backgrounds followed by electron microscopy showed distinct membrane architectures, and the AcpP changes not only significantly improved growth in human milk, but also yielded cells surrounded with outer membrane vesicles. Analyses of the phospholipid and lipooligosaccharide (LOS) compositions suggest an imbalance in acyl chain distributions. For strain 11168, these changes protect both evolved and 11168ΔacpP G33R strains from bacteriophage infection and polymyxin killing. Taken together, this study provides insights into how C. jejuni may evolve to resist the bactericidal activity of human milk and flourish in the hostile environment of the gastrointestinal tract.

60 APPLIED LIFE SCIENCES↗

MILK : a Python scripting interface to MAUD for automation of Rietveld analysis

Modern diffraction experiments ( e.g. in situ parametric studies) present scientists with many diffraction patterns to analyze. Interactive analyses via graphical user interfaces tend to slow down obtaining quantitative results such as lattice parameters and phase fractions. Furthermore, Rietveld refinement strategies ( i.e. the parameter turn-on-off sequences) tend to be instrument specific or even specific to a given dataset, such that selection of strategies can become a bottleneck for efficient data analysis. Managing multi-histogram datasets such as from multi-bank neutron diffractometers or caked 2D synchrotron data presents additional challenges due to the large number of histogram-specific parameters. To overcome these challenges in the Rietveld software Material Analysis Using Diffraction ( MAUD ), the MAUD Interface Language Kit ( MILK ) is developed along with an updated text batch interface for MAUD . The open-source software MILK is computer-platform independent and is packaged as a Python library that interfaces with MAUD . Using MILK , model selection ( e.g. various texture or peak-broadening models), Rietveld parameter manipulation and distributed parallel batch computing can be performed through a high-level Python interface. A high-level interface enables analysis workflows to be easily programmed, shared and applied to large datasets, and external tools to be integrated with MAUD . Through modification to the MAUD batch interface, plot and data exports have been improved. The resulting hierarchical folders from Rietveld refinements with MILK are compatible with Cinema: Debye–Scherrer , a tool for visualizing and inspecting the results of multi-parameter analyses of large quantities of diffraction data. In this manuscript, the combined Python scripting and visualization capability of MILK is demonstrated with a quantitative texture and phase analysis of data collected at the HIPPO neutron diffractometer.

97 MATHEMATICS AND COMPUTING↗

Pharmacokinetics and relative bioavailability study of two cefquinome sulfate intramammary infusions in cow milk

In this study, two intramammary infusions of cefquinome sulfate were investigated for pharmacokinetics and bioavailability. Twelve lactating cows for each group were administered an effective dose of 75 mg/gland for cefquinome, with milk samples collected at various time intervals. The concentrations of cefquinome in milk at different times were determined by the UPLC-MS/MS method. Analyses of noncompartmental pharmacokinetics were conducted on the concentration of cefquinome in milk. Mean pharmacokinetic parameters of group A and group B following intramammary administration were as follows: AUC last 300558.57 ± 25052.78 ng/mL and 266551.3 ± 50654.85 ng/mL, C max 51786.35 ± 11948.4 ng/mL and 59763.7 ± 8403.2 ng/mL, T 1/2 5.69 ± 0.62 h and 5.25 ± 1.62 h, MRT 7.43 ± 0.79 h and 4.8 ± 0.78 h, respectively. Pharmacokinetic experiments showed that the relative bioavailability of group B was 88.69% that of group A. From our findings, group B (3 g: 75 mg) shows a quicker drug elimination process than group A (8 g: 75 mg), which suggests that the withdrawal period for the new formulation may be shorter.

Li, Shuang↗

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

59 BASIC BIOLOGICAL SCIENCES↗

A metagenome-level analysis of a microbial community fermenting ultra-filtered milk permeate

Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.

59 BASIC BIOLOGICAL SCIENCES↗

Bioengineered algal lipids enriched in structured medium- and long-chain triacylglycerols, linoleate, and sn -2 palmitate for human milk fat substitutes

Human milk fat (HMF) contains triacylglycerol (TAG) as its primary component, providing over 50% of the calories for infant nutrition, along with structural and bioactive lipids that are important for immune and nervous system development. Palmitic acid, comprising 20-25% of the fatty acid complement of HMF, is predominantly esterified to the sn -2 position on the glycerol backbone. This regiospecific positioning facilitates absorption as 2-palmitoyl-monoacylglycerol after hydrolysis of the fatty acids at sn -1 and sn -2 by gut lipases. Other features of HMF include enrichment in structured medium- and long-chain triglycerides (MLCTs), and variation in the ratio of oleic acid to linoleic acid with maternal diet and geography. We have engineered Auxenochlorella, an oleaginous green alga, for biosynthesis of an MLCT- and sn -2 palmitate-enriched HMF substitute for infant formula, matching the regioisomeric composition and proportions of the most abundant fatty acids in HMF.

Lin, Jon Y-T [University of California, Berkeley;]↗

Optimizing dietary rumen-degradable starch to rumen-degradable protein ratio improves lactation performance and nitrogen utilization efficiency in mid-lactating Holstein dairy cows

The dietary rumen-degradable starch (RDS) to rumen-degradable protein (RDP) ratio, denoted as the RDS-to-RDP ratio (SPR), has been proven to enhance in vitro rumen fermentation. However, the effects of dietary SPR in vivo remain largely unexplored. This study was conducted to investigate the effect of dietary SPR on lactation performance, nutrient digestibility, rumen fermentation patterns, blood indicators, and nitrogen (N) partitioning in mid-lactating Holstein cows. Seventy-two Holstein dairy cows were randomly assigned to three groups (24 head/group), balanced for (mean ± standard deviation) days in milk (116 ± 21.5), parity (2.1 ± 0.8), milk production (42 ± 2.1 kg/d), and body weight (705 ± 52.5 kg). The cows were fed diets with low (2.1, control), medium (2.3), or high (2.5) SPR, formulated to be isoenergetic, isonitrogenous, and iso-starch. The study consisted of a one-week adaptation phase followed by an eight-week experimental period. The results indicated that the high SPR group had a lower dry matter intake compared to the other groups ( p < 0.05). A quadratic increase in milk yield and feed efficiency was observed with increasing dietary SPR ( p < 0.05), peaking in the medium SPR group. The medium SPR group exhibited a lower milk somatic cell count and a higher blood total antioxidant capacity compared to other groups ( p < 0.05). With increasing dietary SPR, there was a quadratic improvement ( p < 0.05) in the total tract apparent digestibility of crude protein, ether extract, starch, neutral detergent fiber, and acid detergent fiber. Although no treatment effect was observed in rumen pH, the rumen total volatile fatty acids concentration and microbial crude protein synthesis increased quadratically ( p < 0.05) as dietary SPR increased. The molar proportion of propionate linearly increased ( p = 0.01), while branched-chain volatile fatty acids linearly decreased ( p = 0.01) with increasing dietary SPR. The low SPR group (control) exhibited higher concentration of milk urea N, rumen ammonia N, and blood urea N than other groups ( p < 0.05). Despite a linear decrease ( p < 0.05) in the proportion of urinary N to N intake, increasing dietary SPR led to a quadratic increase ( p = 0.01) in N utilization efficiency and a quadratic decrease ( p < 0.05) in the proportion of fecal N to N intake. In conclusion, optimizing dietary SPR has the potential to enhance lactation performance and N utilization efficiency. Based on our findings, a medium dietary SPR (with SPR = 2.3) is recommended for mid-lactating Holstein dairy cows. Nevertheless, further research on rumen microbial composition and metabolites is warranted to elucidate the underlying mechanisms of the observed effects.

Chen, Panliang↗

Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

Background Incorporating the production of related ligninolytic enzymes into industrial filamentous fungus Aspergillus niger will enhance the bioconversion of lignocelluloses to various chemical products. Results In this study, transgenic expression of Phanerochaete chrysosporium manganese peroxidases (mnps) and Trametes sp. C30 laccase hybrid Lac131 (lac131) were examined and optimized in A. niger 11414 prtT∆ strain. Five mnps (mnp1, mnp2, mnp3, mnp4, and mnp5) and lac131 genes were expressed separately or in combination. The transgenic strain containing the entire mnp2 genomic coding sequence (gmnp2) exhibited the highest mnP activity among the five mnp over-expression strains in the modified minimal medium (mMM) with addition of 5 g/L bovine hemoglobin (bHg). We examined the effects of hemin and bHg on mnP production in the gmnp2 strain cultures and found that at least 1 g/L bHg was required, while hemin was not. Culture conditions for mnP production were further optimized for the gmnp2 strain and the highest mnP activities were detected in the cultures grown at 25 °C and 200 rpm with an initial pH of 4.5. Effects of soy protein, skim milk, and bovine serum albumin on mnP production were investigated; 5 g/L of soy proteins or skim milk had comparable effects to 2.5 g/L bHg, while cultures with bovine serum albumin had diminished mnP activity. Disruption of both prtT and vsm1 substantially augmented the mnP production and its activity reached 575 U/L. Trametes sp. C30 laccase hybrid lac131 was strongly expressed in either A. niger gmnp2 (1975 U/L) or 11414prtT∆ (3895 U/L) strain. Both mnP and laccase in the culture supernatants effectively decolorized selected phenolic compounds (dyes) and cleaved tagged model lignin dimers. Conclusion The mnP was successfully produced in A. niger by optimizing the culture conditions and host strain. Co-expression of all four mnp genes in the same expression host by multiplex CRISPR will lead to the mnP production reaching levels comparable to P. chrysosporium, while only requiring 36 h at 25 °C. The Lac131 activity in transgenic A. niger strain is 4- to 7-times higher than that in previous studies. Co-production of mnP and laccase in A. niger will enhance the lignin bioconversion efficiency.

Aspergillus niger↗

Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection

Generating top-down tandem mass spectra (MS/MS) for complex mixtures of proteoforms has become possible through improvements in fractionation, on-line separation, dissociation, and mass analysis. The algorithms to match tandem mass spectra to sequences have undergone a parallel evolution, with both spectral alignment and peak matching being paired with diverse methods for scoring proteoform-spectral matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification through three distinct challenges. The first is identifying a large yield of PrSMs while controlling false discovery rate (FDR) in identifying thousands of proteoforms from complex cell lysates via four software workflows: ProSight Proteome Discoverer, TopPIC, Informed Proteomics, and pTop. The second is the deconvolution of data from both Thermo Orbitrap-class and Bruker maXis Q-TOF instruments to produce consistent precursor charge and mass determinations while generating fragment mass lists to optimize identification. The third attempts to detect diverse post-translational modifications (PTMs) in proteoforms from cow milk and human ovarian tissue. The data demonstrate that existing software suites produce admirable sensitivity, in some cases identifying a third of collected tandem mass spectra with FDR controlled below 2%; the overlap in these PrSMs, however, illustrates real value in searching data with multiple search engines. Differences among identification workflows seem to result from each search algorithm incorporating its own deconvolution algorithm. By transmitting deconvolution data from multiple deconvolution routes (Thermo Xtract, Bruker Auto MSn, Mascot Distiller, TopFD, and FLASHDeconv) to the downstream TopPIC search algorithm, we were able to detect common causes of deconvolution disagreement. The detection of PTMs was very inconsistent among search algorithms, with some workflows suggesting as little as 1% of PrSMs from cow’s milk were singly-phosphorylated while other workflows found that 18% of PrSMs were singly-phosphorylated. Taken together, these results make a strong argument for top-down researchers to adopt a standard practice of analyzing each MS/MS experiment with at least two different search engines.

59 BASIC BIOLOGICAL SCIENCES↗

Limited effects of tannin supplementation on the dairy cattle fecal microbiome with modulation of metabolites

Tannins are plant secondary metabolites that bind organic carbon (C) and nitrogen (N), potentially altering substrate bioavailability for enteric fermentation in ruminants. This interaction may reduce greenhouse gas (GHG) emissions and influence nitrogen partitioning. Given tannins' resistance to ruminal degradation and persistence through the gastrointestinal tract, this study investigated the effects of a tannin-based feed additive on fecal microbial diversity, fecal chemical composition, and GHG emissions. Twenty-four early- to mid-lactation dairy cows were randomized to receive either a tannin-based feed additive (TRT; containing condensed and hydrolyzable tannins from Schinopsis quebracho-colorado [Schltdl.]) or a control diet (CON) for 64 days. Cows were blocked by parity, dry matter intake, milk yield, body weight, and days in milk. Fecal samples were collected on days 0, 16, 32, and 64 and analyzed using 16S rRNA gene amplicon sequencing. Fecal C, N, and indole-3-lactate were measured, and GHG emissions (N2O, CH4, CO2) were assessed via 14-day laboratory incubation. A total of 1,538 amplicon sequence variants were identified, with Firmicutes as the dominant phylum. Fecal phylogenetic diversity showed a significant treatment × day interaction (p < 0.01), with TRT cows exhibiting reduced microbial diversity from day 16 to 64. Fecal C and N concentrations were significantly lower (p < 0.01) in TRT cows on day 16, while indole-3-lactate levels were higher on day 64 (p = 0.02). GHG emissions did not differ significantly between treatments. The tannin-based feed additive influenced fecal microbial community structure and select chemical parameters but did not significantly affect GHG emissions from feces. These findings suggest that dietary tannins may modulate gut microbial ecology with minimal impact on downstream manure-related emissions.

Klein, Matthew L↗

Overexpression of the Mas1 gene mitigated LPS-induced inflammatory injury in mammary epithelial cells by inhibiting the NF-κB/MAPKs signaling pathways

Breast infection is the primary etiology of mastitis in dairy cows, leading to a reduction in the quality of dairy products and resulting in substantial economic losses for animal husbandry. Although antibiotic treatment can eliminate the pathogenic microorganisms that induce mastitis, it cannot repair the inflammatory damage of mammary epithelial cells and blood milk barrier. Mas1 is a G protein-coupled receptor, and its role in lipopolysaccharide (LPS) -induced inflammatory injury to mammary epithelial cells has not been studied. LPS treatment of EpH4 EV cells led to a significant downregulation of Mas1 transcript levels, which attracted our great interest, suggesting that Mas1 may be an important target for the treatment of mastitis. Therefore, this study intends to verify the role of Mas1 in the inflammatory injury of EpH4 EV cells by gene overexpression technology and gene silencing technology. The findings demonstrated that the overexpression of the Mas1 gene effectively reversed the activation of the nuclear factor-κB/mitogen-activated protein kinase (NF-κB/MAPK) signaling pathways induced by LPS, while also suppressing the upregulation of pro-inflammatory mediators. Furthermore, overexpression of the Mas1 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, and Claudin-3 caused by LPS, suggesting that Mas1 could promote to repair the blood-milk barrier. However, the silencing of the Mas1 gene using siRNA resulted in a contrasting effect. These results indicated that Mas1 alleviated the inflammatory injury of mammary epithelial cells induced by LPS.

Yan, Shuping↗

Tailoring the rheological properties of high protein suspension by thermal-mechanical treatment

The viscoelasticity of concentrated protein suspensions associates closely with the mixing efficiency and cleaning frequency of facility during high-protein food development. This study investigated the effects of thermal-mechanical treatment on the viscoelasticity of milk protein isolate (MPI) suspensions and their underlying mechanisms to develop protein ingredient with low viscoelasticity. MPI suspensions (20%) were treated at 25, 50 and 85°C for 10-60 min under constant shear (100 s -1 ), followed by storage at 4°C. The viscosity (η) of MPI suspension treated at 50°C and 85°C was similar to 1-10% as those treated at 25 ° C. After four days of storage at 4°C, η showed the least value in 50°C-treated samples compared to those at 25 ° C and 85 ° C. The η and storage modulus (G') was decreased with prolonged treatment at 25 and 50°C, whereas opposite trend was found in 85°C treated samples. Differential scanning calorimetry found proteins in 50°C treated samples had smaller enthalpy than those in the control and 25°C treated samples. Protein surface hydrophobicity was increased slightly from 25°C to 50°C, but remarkably in 85°C treated samples. Ultra-small angle x-ray scattering showed the radius of gyration (R-g) of casein micelle was similar to 38 nm at 25°C and 50°C treated samples but increased to similar to 44 nm 85°C treated samples with reduced compactness. A new sphere-like structure with R-g of 18 nm was generated in 85°C treated samples. These findings suggested modulating temperature during thermal-mechanical treatment is essential to alter protein structures and morphology for desirable rheological properties.

59 BASIC BIOLOGICAL SCIENCES↗

Idaho National Laboratory Site Environmental Surveillance Report: First Quarter 2022

This report for the first quarter of 2022 contains results from the INL Site environmental surveillance program’s monitoring of the U.S. Department of Energy’s Idaho National Laboratory (INL) Site’s onsite, boundary and distant location environment, January 1 through March 31, 2022. All sample types (media) and the sampling schedule followed during 2022 are listed in Appendix A. This report contains results for the following sample types: • Air, including particulate air filters, charcoal cartridges, and atmospheric moisture, • Quarterly composites, • Precipitation, • Milk, and • Large game animals

99 GENERAL AND MISCELLANEOUS↗

Idaho National Laboratory Site Environmental Surveillance Program Report: Third Quarter 2022

This report for the third quarter of 2022 contains results from the INL Site environmental surveillance program’s monitoring of the U.S. Department of Energy’s Idaho National Laboratory (INL) Site’s onsite, boundary and distant location environment, July 1 through September 30, 2022. All sample types (media) and the sampling schedule followed during 2022 are listed in Appendix A. This report contains results for the following sample types: • Air, including particulate air filters, charcoal cartridges, and atmospheric moisture, • Precipitation, • Milk, • Lettuce, • Grain, and • Large Game Animal Sampling.

99 GENERAL AND MISCELLANEOUS↗