Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen.

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism.

Chronic Leptin Deficiency Improves Tolerance of Physiological Damage and Host-Pathogen Cooperation during Yersinia pseudotuberculosis Infection.

To combat infections, hosts employ a combination of antagonistic and cooperative defense strategies. The former refers to pathogen killing mediated by resistance mechanisms, while the latter refers to physiological defense mechanisms that promote host health during infection independent of pathogen killing, leading to an apparent cooperation between the host and the pathogen. Previous work has shown that Leptin, a pleiotropic hormone that plays a central role in regulating appetite and energy metabolism, is indispensable for resistance mechanisms, while a role for Leptin signaling in cooperative host-pathogen interactions remains unknown. Using a mouse model of Yersinia pseudotuberculosis (Yptb) infection, an emerging pathogen that causes fever, diarrhea, and mesenteric lymphadenitis in humans, we found that the physiological effects of chronic Leptin-signaling deficiency conferred protection from Yptb infection due to increased host-pathogen cooperation rather than greater resistance defenses. The protection against Yptb infection was independent of differences in food consumption, lipolysis, or fat mass. Instead, we found that the chronic absence of Leptin signaling protects from a shift to lipid utilization during infection that contributes to Yptb lethality. Furthermore, we found that the survival advantage conferred by Leptin deficiency was associated with increased liver and kidney damage. Our work reveals an additional level of complexity for the role of Leptin in infection defense and demonstrates that in some contexts, in addition to tolerating the pathogen, tolerating organ damage is more beneficial for survival than preventing the damage.

LMBD1 protein participates in cell mitosis by regulating microtubule assembly.

LMBD1 was previously demonstrated to regulate the endocytosis of insulin receptor on the cell surface and to mediate the export of cobalamin from the lysosomes to the cytosol, but little is known about its function in mitosis. In this study, interactome analysis data indicate that LMBD1 is involved in cytoskeleton regulation. Both immunoprecipitation and GST pulldown assays demonstrated the association of LMBD1 with tubulin. Immunofluorescence staining also showed the colocalization of LMBD1 with microtubule in both interphase and mitotic cells. LMBD1 specifically accelerates microtubule assembly dynamics in vitro and antagonizes the microtubule-disruptive effect of vinblastine. In addition, LMBRD1-knockdown impairs mitotic spindle formation, inhibits tubulin polymerization, and diminishes the mitosis-associated tubulin acetylation. The reduced acetylation can be reversed by ectopic expression of LMBD1 protein. These results suggest that LMBD1 protein stabilizes microtubule intermediates. Furthermore, embryonic fibroblasts derived from Lmbrd1 heterozygous knockout mice showed abnormality in microtubule formation, mitosis, and cell growth. Taken together, LMBD1 plays a pivotal role in regulating microtubule assembly that is essential for the process of cell mitosis.

Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues.

Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.

Quantitative display of the redox status of proteins with maleimide-polyethylene glycol tagging.

Cysteine oxidation, either biologically reversible or irreversible, is the main posttranslational modification associated with redox signaling and oxidative stress. Maleimide-polyethylene glycol (m-PEG) has been used to detect reversibly oxidized proteins by reacting to the reduced cysteine residues leading to mobility shift in immunoblots; a method called PEG-switch. With PEG-switch, both reduced and oxidized proteins can be observed on the same immunoblot simultaneously, providing a simple quantitative measurement for protein thiol modifications. In this report, we optimized the assay conditions and exploited the applications of PEG-switch in quantitation of the extent of protein thiol oxidation in cells in response to H2 O2 and insulin. In addition, we have proposed a redox scoring system for measuring the redox status of any given protein from the m-PEG immunoblot. Our results provided quantitative data showing that two cysteine residues of protein tyrosine phosphatase 1B are prone to oxidation following insulin treatment in cultured HeLa cells.

Next generation sequencing identifies abnormal Y chromosome and candidate causal variants in premature ovarian failure patients.

Premature ovarian failure (POF) is characterized by heterogeneous genetic causes such as chromosomal abnormalities and variants in causal genes. Recently, development of techniques made next generation sequencing (NGS) possible to detect genome wide variants including chromosomal abnormalities. Among 37 Korean POF patients, XY karyotype with distal part deletions of Y chromosome, Yp11.32-31 and Yp12 end part, was observed in two patients through NGS. Six deleterious variants in POF genes were also detected which might explain the pathogenesis of POF with abnormalities in the sex chromosomes. Additionally, the two POF patients had no mutation in SRY but three non-synonymous variants were detected in genes regarding sex reversal. These findings suggest candidate causes of POF and sex reversal and show the propriety of NGS to approach the heterogeneous pathogenesis of POF.

Mass spectrometric imaging of metabolites in kidney tissues from rats treated with furosemide.

In the kidney, metabolic processes are different among the cortex (COR), outer medulla (OM), and inner medulla (IM). Using matrix-assisted laser desorption/ionization (MALDI) and imaging mass spectrometry (IMS), we examined the change of metabolites in the COR, OM, and IM of the rat kidney after furosemide treatment compared with vehicle-treated controls. Osmotic minipumps were implanted in male Sprague-Dawley rats to deliver 12 mg·day(-1)·rat(-1) of furosemide. Vehicle-treated (n = 14) and furosemide-treated (furosemide rats, n = 15) rats in metabolic cages received a fixed amount of rat chow (15 g·220 g body wt(-1)·day(-1) for each rat) with free access to water intake for 6 days. At day 6, higher urine output (32 ± 4 vs. 9 ± 1 ml/day) and lower urine osmolality (546 ± 44 vs. 1,677 ± 104 mosmol/kgH2O) were observed in furosemide rats. Extracts of COR, OM, and IM were analyzed by ultraperformance liquid chromatography coupled with quadrupole time-of-flight (TOF) mass spectrometry, where multivariate analysis revealed significant differences between the two groups. Several metabolites, including acetylcarnitine, betaine, carnitine, choline, and glycerophosphorylcholine (GPC), were significantly changed. The changes of metabolites were further identified by MALDI-TOF/TOF and IMS. Their spatial distribution and relative quantitation in the kidneys were analyzed by IMS. Carnitine compounds were increased in COR and IM, whereas carnitine and acetylcarnitine were decreased in OM. Choline compounds were increased in COR and OM but decreased in IM from furosemide rats. Betaine and GPC were decreased in OM and IM. Taken together, MALDI-TOF/TOF and IMS successfully provide the spatial distribution and relative quantitation of metabolites in the kidney.

Vasopressin-regulated miRNAs and AQP2-targeting miRNAs in kidney collecting duct cells.

Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10(-9) M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P < 0.05). Nine mature miRNAs exhibiting >1.3-fold changes in expression on the microarray (miR-127, miR-1, miR-873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR-463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR-216b) target the 3'-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3'-untranslated region of mouse AQP2 mRNA. Quantitative real-time PCR and immunoblot analysis demonstrated that dDAVP-induced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis.

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis.

The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

Weight Management Health Note, a Mobile Health Platform for Obesity Management Developed by the Korean Society for the Study of Obesity.

The Weight Management Health Note application, developed by the Korean Society for the Study of Obesity (KSSO), was designed to assist individuals in weight management and enhance overall well-being. The Committee of IT-Convergence Treatment of Metabolic Syndrome of the KSSO designed this application. Committee members reviewed and supervised the application's underlying driving algorithms and scientific rationale. A healthcare-specific application developer subsequently finalized the application. This application encompasses a myriad of features, including a comprehensive food diary, an exercise tracker, and tailor-made lifestyle recommendations aligned with individual needs and aspirations. Moreover, it facilitates connections within a community of like-minded individuals endeavoring to manage their weight, fostering mutual support and motivation. Importantly, the application is rich in evidence-based health content curated by the KSSO, ensuring users access accurate information for effective obesity management. Looking ahead, the KSSO is committed to orchestrating diverse academic research endeavors linked to this application and refining its functionalities through continuous feedback from users. The KSSO aspires for this application to serve as a valuable resource for individuals striving to manage their health and enhance their quality of life.

XIAP deletion sensitizes mice to TNF-induced and RIP1-mediated death.

XIAP is a caspase-inhibitory protein that blocks several cell death pathways, and mediates proper activation of inflammatory NOD2-RIP2 signaling. XIAP deficiency in patients with inflammatory diseases such as Crohn's disease, or those needing allogeneic hematopoietic cell transplantation, is associated with a worse prognosis. In this study, we show that XIAP absence sensitizes cells and mice to LPS- and TNF-mediated cell death without affecting LPS- or TNF-induced NF-κB and MAPK signaling. In XIAP deficient mice, RIP1 inhibition effectively blocks TNF-stimulated cell death, hypothermia, lethality, cytokine/chemokine release, intestinal tissue damage and granulocyte migration. By contrast, inhibition of the related kinase RIP2 does not affect TNF-stimulated events, suggesting a lack of involvement for the RIP2-NOD2 signaling pathway. Overall, our data indicate that in XIAP's absence RIP1 is a critical component of TNF-mediated inflammation, suggesting that RIP1 inhibition could be an attractive option for patients with XIAP deficiency.

Distinctive origin and evolution of endemic thistle of Korean volcanic island: Structural organization and phylogenetic relationships with complete chloroplast genome.

Unlike other Cirsium in Korea, Cirsium nipponicum (Island thistle) is distributed only on Ulleung Island, a volcanic island off the east coast of the Korean Peninsula, and a unique thistle with none or very small thorns. Although many researchers have questioned the origin and evolution of C. nipponicum, there is not much genomic information to estimate it. We thus assembled the complete chloroplast of C. nipponicum and reconstructed the phylogenetic relationships within the genus Cirsium. The chloroplast genome was 152,586 bp, encoding 133 genes consisting of 8 rRNA genes, 37 tRNA genes, and 88 protein-coding genes. We found 833 polymorphic sites and eight highly variable regions in chloroplast genomes of six Cirsium species by calculating nucleotide diversity, as well as 18 specific variable regions distinguished C. nipponicum from other Cirsium. As a result of phylogenetic analysis, C. nipponicum was closer to C. arvense and C. vulgare than native Cirsium in Korea: C. rhinoceros and C. japonicum. These results indicate that C. nipponicum is likely introduced through the north Eurasian root, not the mainland, and evolved independently in Ulleung Island. This study contributes to further understanding the evolutionary process and the biodiversity conservation of C. nipponicum on Ulleung Island.

Patterns of gene and metabolite define the effects of extracellular osmolality on kidney collecting duct.

To investigate the effects of changes in extracellular osmolality on the function of kidney collecting duct cells, particularly on water and sodium reabsorption in the conditions of diuresis and antidiuresis, we generated transcriptome and metabolome profiles of primary cultured inner medullary collecting duct (IMCD) cells. They were grown in hyperosmolar culture medium (640 mOsm) for 4 days and then exposed to either reduced (300 mOsm) or same osmolality for 1 or 2 days more. Integrated analysis of the transcriptome and metabolome revealed that decreased extracellular osmolality was associated with decreased levels of organic osmolytes, glucose, intermediates of citric acid cycle, and branched-chain amino acids (BCAA) in IMCD cells, along with significantly decreased gene expression and protein abundance of P-type transporters (ATP1B1), ABC transporters (ABCC5 and ABCG1), and insulin signaling pathways (IRS2). Quantitative real-time RT-PCR and semiquantitative immunoblotting confirmed the changes of transcript levels of differentially expressed genes and protein levels. Taken together, integrated analysis of omics data demonstrated that water and sodium reabsorption could be reduced by decreased extracellular osmolality per se, through decreased levels of ABC transporters and IRS2, which play a potential role in the transport of organic osmolytes, BCAA, glucose, and trafficking of epithelial sodium channel.

Chromosome-level genome assembly of Plazaster borealis sheds light on the morphogenesis of multiarmed starfish and its regenerative capacity.

BACKGROUND: Plazaster borealis has a unique morphology, displaying multiple arms with a clear distinction between disk and arms, rather than displaying pentaradial symmetry, a remarkable characteristic of echinoderms. Herein we report the first chromosome-level reference genome of P. borealis and an essential tool to further investigate the basis of the divergent morphology. FINDINGS: In total, 57.76 Gb of a long read and 70.83 Gb of short-read data were generated to assemble a de novo 561-Mb reference genome of P. borealis, and Hi-C sequencing data (57.47 Gb) were used for scaffolding into 22 chromosomal scaffolds comprising 92.38% of the genome. The genome completeness estimated by BUSCO was 98.0% using the metazoan set, indicating a high-quality assembly. Through the comparative genome analysis, we identified evolutionary accelerated genes known to be involved in morphogenesis and regeneration, suggesting their potential role in shaping body pattern and capacity of regeneration. CONCLUSION: This first chromosome-level genome assembly of P. borealis provides fundamental insights into echinoderm biology, as well as the genomic mechanism underlying its unique morphology and regeneration.

E3 ubiquitin-protein ligases in rat kidney collecting duct: response to vasopressin stimulation and withdrawal.

The E3 ubiquitin (Ub)-protein ligases (E3s) play a role as regulators of protein trafficking and degradation. We aimed to integrate the profile of E3s in rat kidney and examine the changes in protein abundance of the selected E3s in response to 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Sprague-Dawley rats were infused with vehicle (n = 13), dDAVP for 5 days (n = 13), or dDAVP was withdrawn for periods (15 min, 30 min, 1, 3, 6, 12, or 24 h) after 5-day infusion (n = 46). Total RNA was isolated from the inner medulla (IM) for transcriptome analysis. Plasma membrane (PM)- or intracellular vesicle (ICV)-enriched fractions of whole kidney were immunoisolated for liquid chromatography-tandem mass spectrometry analysis. dDAVP infusion for 5 days (D5d) significantly increased urine osmolality, which was maintained during 3-h withdrawal of dDAVP after 5-day infusion (D5d-3h). Consistent with this, aquaporin-2 (AQP2) expression in the PM fractions of D5d and D5d-3h increased, whereas AQP2 expression in the ICV fractions of D5d-3h was further increased, indicating internalization of AQP2. Transcriptome analysis revealed 86 genes of E3s and LC-MS/MS analysis demonstrated 16 proteins of E3s. Among these, seven E3s (BRCA1, UBR4, BRE1B, UHRF1, NEDD4, CUL5, and FBX6) were shared. RT-PCR demonstrated mRNA expressions of the seven identified E3s in the kidney, and immunoblotting demonstrated changes in protein abundance of the selected E3s (BRE1B, NEDD4, and CUL5) in response to dDAVP stimulation/withdrawal or lithium-induced nephrogenic diabetes insipidus. The rate of AQP2 degradation was retarded in mpkCCDc14 cells with small interfering RNA-mediated knockdown of NEDD4 or CUL5. Taken together, identified E3s could be involved in the degradation of proteins associated with vasopressin-induced urine concentration.

Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation.

AS160, a novel Akt substrate of 160 kDa, contains a Rab GTPase-activating protein (GAP) domain. The present study examined the role of Akt and AS160 in aquaporin-2 (AQP2) trafficking. The main strategy was to examine the changes in AQP2 translocation in response to small interfering RNA (siRNA)-mediated AS160 knockdown in mouse cortical collecting duct cells (M-1 cells and mpkCCDc14 cells). Short-term dDAVP treatment in M-1 cells stimulated phosphorylation of Akt (S473) and AS160, which was also seen in mpkCCDc14 cells. Conversely, the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002 diminished phosphorylation of Akt (S473) and AS160. Moreover, siRNA-mediated Akt1 knockdown was associated with unchanged total AS160 but decreased phospho-AS160 expression, indicating that phosphorylation of AS160 is dependent on PI3K/Akt pathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocytochemistry revealed that AS160 knockdown in mpkCCDc14 cells was associated with increased AQP2 density in the plasma membrane [135 ± 3% of control mpkCCDc14 cells (n = 65), P < 0.05, n = 64] despite the absence of dDAVP stimulation. Moreover, cell surface biotinylation assays of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of control mpkCCDc14 cells (n = 3), P < 0.05, n = 3]. Taken together, PI3K/Akt pathways mediate the dDAVP-induced AS160 phosphorylation, and AS160 knockdown is associated with higher AQP2 expression in the plasma membrane. Since AS160 contains a GAP domain leading to a decrease in the active GTP-bound form of AS160 target Rab proteins for vesicle trafficking, decreased expression of AS160 is likely to play a role in the translocation of AQP2 to the plasma membrane.

Microbiota control of maternal behavior regulates early postnatal growth of offspring.

Maternal behavior is necessary for optimal development and growth of offspring. The intestinal microbiota has emerged as a critical regulator of growth and development in the early postnatal period life. Here, we describe the identification of an intestinal Escherichia coli strain that is pathogenic to the maternal-offspring system during the early postnatal stage of life and results in growth stunting of the offspring. However, rather than having a direct pathogenic effect on the infant, we found that this particular E. coli strain was pathogenic to the dams by interfering with the maturation of maternal behavior. This resulted in malnourishment of the pups and impaired insulin-like growth factor 1 (IGF-1) signaling, leading to the consequential stunted growth. Our work provides a new understanding of how the microbiota regulates postnatal growth and an additional variable that must be considered when studying the regulation of maternal behavior.

Chemiluminescent dual-enzyme immunoassays capable of simultaneously quantifying carbohydrate antigen 19-9 and carcinoma embryonic antigen in a sample.

Using the internal chemiluminescence resonance energy transfer (Inter-CRET) of luminescent dye and the high-energy intermediate formed in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction, we developed for the first time dual-enzyme immunoassays operated with alkaline phosphatase (ALP) and horseradish peroxidase (HRP) for the simultaneous quantifications of carbohydrate antigen 19-9 (CA19-9) and carcinoma embryonic antigen (CEA) in a sample. Fluorescein formed from the ALP enzyme immunoassay emits green light in ODI-CL reaction, while resorufin formed from the HRP enzyme immunoassay emits red light. Green and red CL lights emitted in a detection cell were measured individually with two photomultiplier tubes with an optical filter capable of sensing green or red emission without interferences. The limits of detection for CA 19-9 (0.09 U/ml) and CEA (0.11 ng/ml) determined using the accurate and reproducible dual-enzyme immunoassays were as low as those calculated using the conventional single-enzyme immunoassays capable of sensing CA 19-9 or CEA in a sample. In conclusion, we confirmed that the highly selective dual enzyme immunoassays can be applied as a new analytical method capable of early diagnosing and monitoring colon and pancreatic cancers with the simultaneous analyses of CA 19-9 and CEA.

Highly sensitive chemiluminescence enzyme immunoassay for the quantification of carcinoembryonic antigen in the presence of an enhancer and a stabilizer.

Using the advantages of phenol red, a signal enhancer, and bovine serum albumin (BSA), a stabilizer of horseradish peroxidase (HRP), added in HRP enzyme reaction of Amplex Red and H2O2, highly sensitive 1,1'-oxalyldiimidazole chemiluminescence enzyme immunoassay (ODI-CLEIA) was developed to rapidly quantify trace levels of carcinoembryonic antigen (CEA) in human serum. Phenol red acts as an enhancer in ODI-CLEIA while BSA supported rapid and stable activation of HRP. The CL emission of resorufin formed from the HRP enzyme reaction in the presence of BSA and phenol red was about 70-fold brighter than that in the absence of both materials. ODI-CLEIA in the presence of BSA (1.5 mg/ml).and phenol red (1 mM) was able to rapidly analyze CEA in human serum with the wide linear calibration curve (2.5-100 ng/ml). The limit of detection (LOD = 3σ/slope) of ODI-CLEIA was as low as 0.19 ng/ml. Additionally, it was confirmed that the accuracy, precision, and reproducibility of ODI-CLEIA in the presence of BSA and phenol red were good with the statistically acceptable error range.