D-binding Protein Research Articles

Draft Genome of White-blotched River Stingray Provides Novel Clues for Niche Adaptation and Skeleton Formation

The white-blotched river stingray (Potamotrygon leopoldi) is a cartilaginous fish native to the Xingu River, a tributary of the Amazon River system. As a rare freshwater-dwelling cartilaginous fish in the Potamotrygonidae family in which no member has the genome sequencing information available, P. leopoldi provides the evolutionary details in fish phylogeny, niche adaptation, and skeleton formation. In this study, we present its draft genome of 4.11 Gb comprising 16,227 contigs and 13,238 scaffolds, with contig N50 of 3937 kb and scaffold N50 of 5675 kb in size. Our analysis shows that P. leopoldi is a slow-evolving fish that diverged from elephant sharks about 96 million years ago. Moreover, two gene families related to the immune system (immunoglobulin heavy constant delta genes and T-cell receptor alpha/delta variable genes) exhibit expansion in P. leopoldi only. We also identified the Hox gene clusters in P. leopoldi and discovered that seven Hox genes shared by five representative fish species are missing in P. leopoldi. The RNA sequencing data from P. leopoldi and other three fish species demonstrate that fishes have a more diversified tissue expression spectrum when compared to mammals. Our functional studies suggest that lack of the gc gene encoding vitamin D-binding protein in cartilaginous fishes (both P. leopoldi and Callorhinchus milii) could partly explain the absence of hard bone in their endoskeleton. Overall, this genome resource provides new insights into the niche adaptation, body plan, and skeleton formation of P. leopoldi, as well as the genome evolution in cartilaginous fishes.

OR19-3 Differential Interactions of Vitamin D Binding Protein and Vitamin D Receptor in Different Ethnic Groups with Aggressive Thyroid Cancer

Abstract Thyroid cancer affects ethnic groups at different rates and severity. Despite this, thyroid cancer health disparities are still an understudied area. Our laboratory has shown a differential expression of vitamin D binding protein (DBP) in Filipino Americans (FA) versus European Americans (EA) [1]. Higher DBP levels correlate to a better prognosis. A study has revealed a DBP-dependent vitamin D receptor (VDR) promoter activation. In this study, we investigated whether VDR and DBP are expressed differentially in different ethnic groups, such as African Americans (AA), Hispanic Americans (HA), EA, and FA. We were also able to determine the differential DBP polymorphisms, VDR variant expressions, and DBP-VDR interactions within different ethnicities. By PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), we determined a higher frequency of DBP gene polymorphism in FA versus EA. Analyzing the Cancer Genome Atlas (TCGA) thyroid cancer datasets with the UALCAN assay, we found the differential expressions of DBP and VDR genes based on cancer stages, sample types, race, and histological subtypes. By immunohistochemistry, we detected strong nuclear VDR (nVDR) and very low membranous VDR (mVDR) expression that correlated with low DBP in FA thyroid cancer tissues. In contrast, there was a higher expression of both mVDR and DBP in HA versus the other ethnicities. Co-immunoprecipitation analysis revealed a stronger DBP interaction with mVDR in FA compared to other ethnicities. Our data suggest that low DBP correlates with low mVDR in FA, whereas high DBP correlates with high mVDR in the other ethnicities. In conclusion, the strong interaction of DBP with mVDR in FA may implicate the potential role of DBP-mVDR crosstalk in aggressive thyroid cancer. Future research goals will be aimed at determining pathways involved in BDP-mVDR crosstalk in thyroid cancer health disparities. B. Mull, R. Davis, I. Munir, M.C. Perez, A.A. Simental, S. Khan, Differential expression of Vitamin D binding protein in thyroid cancer health disparities, Oncotarget 12(7) (2021) 596-607. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 p.m.

The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS.

Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Serum Vitamin D Biomarkers in Pregnant Women with Preeclampsia: A Prospective Pilot Study

Background: It has been suggested that hypovitaminosis D is associated with the development of preeclampsia. This study aimed to study the relationship between preeclampsia and various vitamin D serum biomarkers including 25-hydroxyl vitamin D [25(OH)D], vitamin D-binding protein (VDBP), and bioavailable and free 25(OH)D. Methods: This prospective study was conducted with 17 patients with preeclampsia and 38 normal pregnant women as the control group. Total serum 25(OH)D and VDBP concentrations were measured. The levels of bioavailable 25(OH)D and free 25(OH)D were also calculated. Two single nucleotide polymorphisms (rs4588 and rs7041) of the GC gene encoding VDBP were analyzed. Results: VDBP was significantly increased in the preeclampsia group compared to the normal pregnancy group (454.2 vs. 403.4 ng/mL; p = 0.036). When the preeclampsia patients were analyzed by dividing them into early-onset and late-onset, there was no significant difference in the serum vitamin D biomarkers levels. Also, when preeclampsia patients were classified into three subgroups of <2 days, 2–7 days, and >7 days from diagnosis to delivery, free 25(OH)D concentrations were significantly increased in the 2–7 days subgroup compared to the other subgroups (3.5 vs. 6.6 vs. 3.1 pg/mL; p = 0.032). The GC genotype and allele frequency showed no statistically significant different distribution between the preeclampsia and normal pregnancy groups. Conclusions: In the present study, the serum VDBP levels were significantly higher in the patients with preeclampsia than in the normal pregnancy group. Thus, among various serum vitamin D biomarkers, increased VDBP could be associated with the onset and pathogenesis of preeclampsia.

Potential Urine Proteomic Biomarkers for Focal Segmental Glomerulosclerosis and Minimal Change Disease.

Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score—depending on the proteinuria level, the third score—resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more—in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.

Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice.

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-β), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-β and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

Vitamin D-Binding Protein, Bioavailable, and Free 25(OH)D, and Mortality: A Systematic Review and Meta-Analysis.

Introduction: Observational studies reported inverse associations between serum total 25-hydroxyvitamin D (25(OH)D) concentrations and mortality. Evolving evidence indicated, however, that bioavailable or free 25(OH)D may be even better predictors of mortality. We conducted a systematic review and meta-analysis to summarize the epidemiological evidence on associations of vitamin D-binding protein (VDBP), albumin-bound, bioavailable, and free 25(OH)D, with mortality. Methods: We systematically searched PubMed and Web of Science, up to 27 May 2022. Predictors of interest included serum or plasma concentrations of VDBP, albumin-bound, bioavailable, and free 25(OH)D. Assessed health outcomes were all-cause and cause-specific mortality. We included studies reporting associations between these biomarkers and mortality outcomes. We applied random-effects models for meta-analyses to summarize results from studies assessing the same vitamin D biomarkers and mortality outcomes. Results: We identified twelve eligible studies, including ten on VDBP, eight on bioavailable 25(OH)D, and eight on free 25(OH)D. No study reported on albumin-bound 25(OH)D and mortality. In meta-analyses, the highest levels of bioavailable and free 25(OH)D were associated with 37% (hazard ratio (HR): 0.63, 95% confidence interval (CI): 0.46, 0.87), and 29% (HR: 0.71, 95% CI: 0.53, 0.97) decrease in all-cause mortality, respectively, compared with the lowest levels. These estimates were similar to those for total 25(OH)D (HR: 0.67, 95% CI: 0.56, 0.80) observed in the same studies. Higher VDBP levels were associated with lower all-cause mortality in cancer patient cohorts. However, no such association was observed in general population cohorts. Conclusions: Similar inverse associations of total, bioavailable, and free 25(OH)D with mortality suggest that bioavailable and free 25(OH)D do not provide incremental value in predicting mortality.

Mirror image serum lipid carrier protein profiles in pup and lactating mother Atlantic grey seals reflect contrasting resource mobilisation challenges

True, phocid seals have the shortest known lactations relative to body mass, during which mass transfer of adipose stores from mother to offspring occurs at an unrivalled rate and extent. The mothers of most species of seal fast until weaning whilst their pups gorge on the most fat-rich milks known. This results in a dramatic reduction in maternal blubber mass while pups may triple their body weights before weaning. Mothers mobilise their blubber fat, transport it via blood to their mammary glands and into milk, whilst pups transfer fat in the opposite direction, from their intestines, via blood, to their blubber. Using proteomic analysis of mother and pup sera from Atlantic grey seals, we find that this mirror image flux of lipids between mothers and pups is reflected in an almost inverse relationship in the proteins in their blood specialised to transport fats, lipids, and fat-soluble vitamins. For instance, apolipoproteins ApoB-48/100, ApoA-II and ApoA-IV, which are structural components of the main lipid carrier complexes such as chylomicrons and HDL particles, occur at much higher levels in pups than mothers. Meanwhile, carriers of fat-soluble vitamins such as retinol- and vitamin D-binding proteins are lower in pups and gradually build towards weaning. In contrast, sex hormone-binding globulin occurs at remarkably high relative concentrations in pups. There are therefore dramatic differences between, and an unrealised complexity in, the balance of proteins involved in the rapid transfer of fats and other lipids from mother to pups in preparing their offspring for their post-weaning fasts on land and eventual survival at sea before they can feed again.

Maternal and Neonatal Vitamin D Binding Protein Polymorphisms and 25-Hydroxyvitamin D Cutoffs as Determinants of Neonatal Birth Anthropometry.

Background: Vitamin D-binding protein (VDBP) is a vital regulator of optimal vitamin D homeostasis and bioavailability. Apart from its well-documented role as a key component in vitamin D dynamic transfer and circulation, it has a myriad of immunoregulatory functions related to innate immunity, which becomes particularly critical in states of increased immunological tolerance including pregnancy. In this regard, VDBP dyshomeostasis is considered to contribute to the development of several fetal, maternal, and neonatal adverse outcomes. However, precise physiological pathways, including the contribution of specific VDBP polymorphisms behind such phenomena, are yet to be fully deciphered. Our aim was to assess the combined effect of maternal and neonatal VDBP polymorphism heterogeneity in conjunction with different maternal and neonatal 25(OH)D cutoffs on the neonatal anthropometric profile at birth. Methods: The study included data and samples from a cohort of 66 mother–child pairs at birth. The inclusion criterion was full-term pregnancy (gestational weeks 37–42). Neonatal and maternal 25(OH)D cutoffs were included according to vitamin D status at birth and delivery. Concentrations of 25(OH)D2 and 25(OH)D3 were measured using liquid chromatography–tandem mass spectrometry. Results: The upper arm length of neonates with 25(OH)D ≤ 25 nmol/L was higher in neonate CC carriers for rs2298850. The upper thigh neonatal circumference was also higher in the ones with either 25(OH)D ≤ 50 or ≤75 nmol/L in rs2298850 CG + GG or rs4588 GT + TT carriers. We did not observe any significant effect for maternal VDBP polymorphisms nor for birth maternal 25(OH)D concentrations, on birth neonatal anthropometry. Conclusions: Our findings emphasize a potential role for neonatal VDBP genotypes rs2298850 and rs4588, in conjunction with specific neonatal 25(OH)D cutoffs, in the range of sufficiency on neonatal growth and development.

Megalin and Vitamin D Metabolism-Implications in Non-Renal Tissues and Kidney Disease.

Megalin is an endocytic receptor abundantly expressed in proximal tubular epithelial cells and other calciotropic extrarenal cells expressing vitamin D metabolizing enzymes, such as bone and parathyroid cells. The receptor functions in the uptake of the vitamin D-binding protein (DBP) complexed to 25 hydroxyvitamin D3 (25(OH)D3), facilitating the intracellular conversion of precursor 25(OH)D3 to the active 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). The significance of renal megalin-mediated reabsorption of 25(OH)D3 and 1,25(OH)2D3 has been well established experimentally, and other studies have demonstrated relevant roles of extrarenal megalin in regulating vitamin D homeostasis in mammary cells, fat, muscle, bone, and mesenchymal stem cells. Parathyroid gland megalin may regulate calcium signaling, suggesting intriguing possibilities for megalin-mediated cross-talk between calcium and vitamin D regulation in the parathyroid; however, parathyroid megalin functionality has not been assessed in the context of vitamin D. Within various models of chronic kidney disease (CKD), megalin expression appears to be downregulated; however, contradictory results have been observed between human and rodent models. This review aims to provide an overview of the current knowledge of megalin function in the context of vitamin D metabolism, with an emphasis on extrarenal megalin, an area that clearly requires further investigation.

Differing effects of oral conjugated equine estrogen and transdermal estradiol on vitamin D metabolism in postmenopausal women: a 4-year longitudinal study.

The aim of this study was to examine the effect of either conjugated equine estrogen or transdermal estradiol on vitamin D metabolism in postmenopausal women. Twenty-five women from the Kronos Early Estrogen Prevention Study who were randomized to conjugated equine estrogen 0.45 mg/d and 20 women who were treated with transdermal estradiol 50 mg/d (patch replaced weekly) were analyzed in the present study. All participants received micronized progesterone for 12 days per month. There was no significant treatment effect on serum total 25-hydroxyvitamin D over 48 months in either study group, and there were no significant differences between treatment arms. In contrast, at 12 months, directly measured free 25-hydroxyvitamin D was significantly higher in the transdermal estradiol group than in the conjugated equine estrogen group. Directly measured free 25-hydroxyvitamin D subsequently increased significantly from 12 to 48 months in both treatment arms. Calculated free 25-hydroxyvitamin D was also significantly higher in the transdermal estradiol group at 36 months. Vitamin D-binding protein decreased significantly in both treatment groups from 12 to 48 months, but at 48 months, least square mean values were no different based on treatment assignment. Directly measured free 25-hydroxyvitamin D levels, but not serum total 25-hydroxyvitamin D levels, are different within the first 12 months of estrogen replacement depending on the preparation. However, this difference is transient, in that there were no differences at 36 or 48 months. These findings suggest that there may be a short-term benefit to prescribing transdermal estradiol for women who are either vitamin D deficient or vitamin D insufficient.

Serum Vitamin D Levels Explored in the Latvian Cohort of Patients with Basal Cell Carcinoma Linked to the Sonic Hedgehog and Vitamin D Binding Protein Cutaneous Tissue Indices.

Ultraviolet radiation is known as one of the major contributors to skin malignancies, including basal cell carcinoma (BCC), which is the most common type of skin cancer. It is a heterogeneous tumor, which presents with various types that are stratified into low- and high-risk tumors. Sunlight is important for overall health and vitamin D synthesis in the skin, whereas deviations from the optimal level of vitamin D are shown to be associated with the risk of the development of BCC. The accumulating evidence suggests the ability of vitamin D to antagonize the Sonic Hedgehog (SHH) signaling, the key tumor pathway, and play a protective role in the development of BCC. Additionally, a vitamin D binding protein (DBP) is shown to be implicated in the complex regulation of vitamin D. Here, we aimed to explore serum vitamin D in patients with different primary and recurrent BCC of the head and neck and investigate cutaneous DBP and SHH indices, confirmed immunohistochemically in these subjects. According to the results, 94.9% of the Latvian cohort of BCC patients were found to be deficient in vitamin D. No significant differences in serum vitamin D levels were found between genders, primary and recurrent tumors, and different types of BCC. Serum vitamin D was inversely associated with tumor size. Susceptible male individuals with low blood vitamin D levels were recognized at risk of developing aggressive and recurrent BCC confirmed by the use of hierarchical clustering analysis. In smaller tumors with a favorable course, such as superficial and nodular BCC, the association between high DBP and low SHH tissue expression was found, providing supportive evidence of the existence of a link between vitamin D, proteins involved in its metabolism, as exemplified by the DBP and SHH signaling pathway. The assumption of a deficiency in the protective effect of vitamin D in patients with high-risk BCCs was proposed in low DBP and high SHH tissue indices. New extensions to existing knowledge and characterization of the BCC signaling pathways and their cross-talk with vitamin D are warranted when searching for a preferential effect of vitamin D on skin cancer.

Consistent Inverse Associations of Total, "Bioavailable", Free, and "Non-Bioavailable" Vitamin D with Incidence of Diabetes among Older Adults with Lower Baseline HbA1c (≤6%) Levels.

Background: Serum 25-hydroxyvitamin (25(OH)D) levels are inversely associated with risk of diabetes. The “free hormone hypothesis” suggests potential effects to be mainly related to concentrations of “bioavailable” and free rather than total 25(OH)D. We assessed associations of serum concentrations of vitamin D-binding protein (VDBP), as well as total “bioavailable”, complementary “non-bioavailable”, and free 25(OH)D, with the risk of developing diabetes among non-diabetic older adults in a large population-based cohort study in Germany. Methods: We included 4841 non-diabetic older adults aged 50–75 years at the baseline exam from the ESTHER cohort conducted in Saarland, Germany, in 2001–2002. Concentrations of “bioavailable” and free 25(OH)D were derived from serum concentrations of VDBP, total 25(OH)D, and albumin. Incidence of diabetes was ascertained during up to 14 years of follow-up. Associations were quantified by multivariable Cox proportional hazards regression models with comprehensive confounder adjustment. Results: During a median follow-up of 10.6 years, 837 non-diabetic participants developed diabetes. We observed similar inverse associations with developing diabetes for VDBP (hazard ratio (HR) for lowest versus highest quintile: 1.37, 95% confidence interval (CI): 1.09, 1.72), total 25(OH)D (HR: 1.31, 95% CI: 1.03, 1.66), and “non-bioavailable” 25(OH)D (HR: 1.30, 95% CI: 1.02, 1.65). Associations were smaller and statistically insignificant for “bioavailable” and free 25(OH)D. However, associations of total “non-bioavailable”, “bioavailable”, and free 25(OH)D with incidence of diabetes were much stronger among, and essentially restricted to, participants with lower baseline HbA1c (≤6%) levels. Conclusions: This large prospective cohort study of older Caucasian adults, in agreement with results from randomized trials and Mendelian randomization studies, supports a protective effect of vitamin D against development of diabetes. The “free hormone theory” may not be relevant in this context. However, our results underline the importance of adequate vitamin D status among those who have not yet shown any sign of impaired glucose tolerance.

Vitamin D, Vitamin D-Binding Proteins, and VDR Polymorphisms in Individuals with Hyperglycaemia.

Vitamin D reportedly plays an important role in the pathogenesis of diabetes mellitus; however, this role is unclear and debated. This study investigated the association between 25(OH) vitamin D, vitamin D-binding proteins, and vitamin D receptor (VDR) polymorphisms in healthy individuals and those with prediabetes and type 2 diabetes mellitus (T2D) from South Africa. A cross-sectional study was conducted involving subjects of mixed ancestry aged ≥20 years. Males presented with higher mean 25(OH) vitamin D levels than females, while females exhibited significantly higher serum vitamin D-binding protein levels. Significant differences in mean 25(OH) vitamin D levels were observed in normo-glycaemic, prediabetes, screen-detected DM, and known DM individuals. Vitamin D receptor SNPs Fok1 and Taq1 were not associated with glycaemic status. Fok1 was not associated with 25(OH) vitamin D deficiency, while Taq1 was associated with vitamin D insufficiency. This study showed a high prevalence of vitamin D deficiency/insufficiency in this South African population, with decreased vitamin D levels observed in hyperglycaemic individuals, which was not linked to either vitamin D-binding protein or polymorphisms in Fok1 of the VDR gene. These results may be used as a platform for further research into diagnosis and treatment of hyperglycaemia.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Impaired Vitamin D Metabolism in Hospitalized COVID-19 Patients.

There is increasing data regarding the association between vitamin D and COVID-19. This study aimed to reveal the alterations of vitamin D metabolism in the setting of COVID-19. We examined 119 adult COVID-19 inpatients and 44 apparently healthy adult individuals with similar serum 25OH-D3 levels as a reference group. The assessment included serum biochemical parameters (total calcium, albumin, phosphorus, creatinine), parathyroid hormone (PTH), vitamin D-binding protein (DBP), vitamin D metabolites (25OH-D3, 25OH-D2, 1,25(OH)2D3, 3-epi-25OH-D3, 24,25(OH)2D3 and D3) and free 25OH-D. COVID-19 patients had in general very low vitamin D levels (median 25OH-D3 equals 10.8 ng/mL), accompanied by an increased production of the active vitamin D metabolite (1,25(OH)2D3), estimated as higher 1,25(OH)2D3 serum levels (61 [44; 81] vs. 40 [35; 50] pg/mL, p < 0.001) and lower 25OH-D3/1,25(OH)2D3 ratio (175 [112; 260] vs. 272 [200; 433], p < 0.001) which is presumably aimed at preventing hypocalcemia. Patients with COVID-19 also had elevated DBP (450 [386; 515] vs. 392 [311; 433] mg/L, p < 0.001) and low free 25OH-D levels (<LoB vs. 3.9 [3.2; 4.4] pg/mL, p < 0.001). Follow-up assessment of the COVID-19 inpatients showed recovery of the observed changes. Overall, hospitalized patients with an acute course of COVID-19 have not only very low levels of 25OH-D but also profound abnormalities in the metabolism of vitamin D regardless of the clinical course of the disease. These alterations might exacerbate existing vitamin D deficiency and its negative impact.

VDR gene polymorphisms are associated with the increased susceptibility to COVID-19 among iranian population: A case-control study.

Coronavirus disease 2019 (COVID‐19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), but the pathogenesis is unclear. Host genetic background is one of the main factors influencing the patients' susceptibility to several viral infectious diseases. This study aimed to investigate the association between host genetic polymorphisms of two genes, including vitamin D receptor (VDR) and vitamin D binding protein (DBP), and susceptibility to COVID‐19 in a sample of the Iranian population. This case‐control study enrolled 188 hospitalized COVID‐19 patients as the case group and 218 suspected COVID‐19 patients with mild signs as the control group. The VDR (rs7975232, rs731236 and rs2228570) and DBP (rs7041) gene single nucleotide polymorphisms (SNPs) were genotyped by Polymerase Chain Reaction Restriction – Fragment Length Polymorphism (PCR‐RFLP) method. A significant association between rs2228570 SNP in the VDR gene and the susceptibility of COVID‐19 was found between case and control groups. The CT genotype (Heterozygous) of rs2228570 C > T polymorphism showed significant association with a 3.088 fold increased odds of COVID‐19 (p < .0001; adjusted OR: 3.088; 95% CI: 1.902–5.012). In addition, a significant association between CC genotype of rs2228570 CT polymorphism and increased odds of COVID‐19 in male and female groups (p = .001; adjusted OR: 3.125; 95% CI: 1.630–5.991 and p = .002; adjusted OR: 3.071; 95% CI: 1.485–6.354 respectively) were determined. Our results revealed no significant differences in the frequency of genotype and allele of VDR (rs7975232 and rs731236) and DBP (rs7041) between SARS‐CoV‐2‐infected patients and controls (p > .05). Our results showed that polymorphism of VDR (rs2228570) probably could influence individual susceptibility to COVID‐19. The polymorphisms of VDR (rs7975232 and rs731236) and DBP (rs7041) were not associated with SARS‐CoV‐2 infection susceptibility.

D-VITylation: Harnessing the biology of vitamin D to improve the pharmacokinetic properties of peptides and small proteins

Peptides have great potential to be potent and specific therapeutics, yet their small size leads to rapid glomerular filtration, which severely limits therapeutic applications. Although conjugation of small proteins to large polymers typically results in longer residence times, these conjugates often have a significant loss of biological activity due to steric hindrance. Here, we improve the pharmacokinetics (PK) of peptide therapeutics by harnessing the biology of vitamin D. Attachment of a small vitamin D-based molecule (D-VITylation) protects the conjugated peptide or protein from renal clearance by virtue of reversible binding to the serum-circulating vitamin D binding protein (DBP), without compromising bioactivity. Varying the conjugation site on vitamin D affects the binding to DBP, with higher affinity corresponding to a longer plasma half-life. We also demonstrate the important contribution of the peptide to the overall PK, likely due to alternative clearance mechanisms such as protease degradation and receptor-mediated cellular uptake. With a Fab antibody fragment, for which these alternate clearance mechanisms are not significant, D-VITylation increases the half-life of elimination from 14 to 61 h in rats. The PK profile in minipigs and projected lifetime in humans suggest that D-VITylation is a viable strategy to achieve once-weekly dosing of peptide therapeutics in humans.

Hypovitaminosis D Is Prevalent in Patients With Renal AL Amyloidosis and Associated With Renal Outcome.

IntroductionVitamin D deficiency is common, but no data have been reported on vitamin D levels in light chain (AL) amyloidosis.Patients and MethodsIn this exploratory study, stored serum samples from 173 patients with newly diagnosed AL amyloidosis were analyzed for vitamin studies which included 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D] and vitamin D binding protein (DBP). Measurements were made by liquid chromatography-tandem mass spectrometry. Kidney survival and overall survival (OS) were assessed in association to vitamin D status.ResultsCardiac and kidney involvement occurred in 69% and 63% of patients, respectively. 25(OH)D deficiency (<20 ng/mL) was seen in 56.6% of the patients and was notably found among patients with heavy proteinuria (96%), hypoalbuminemia (84.3%) and morbidly obese patients (68.3%). Heavy proteinuria (>5 gr/24-h) and vitamin D supplementation were independent predictors of 25(OH)D level on nominal multivariate regression analysis. 1,25(0H)2D deficiency was noted in 37.6% of patients and was independently associated with low eGFR and hypoalbuminemia. Progression to ESRD occurred in 23.7% of evaluable patients. Patients who progressed to ESRD had lower serum 25(OH)D and 1,25(OH)2D levels compared to those who did not progress to ESRD. On a multivariate analysis, severe 25(OH)D deficiency was an independent predictor of progression to ESRD as was renal stage, while 1,25(OH)2D deficiency was not.ConclusionsHypovitaminosis D is common in AL amyloidosis, particularly among patients with heavy proteinuria. Severe 25(OH)D deficiency at time of diagnosis predicts progression to ESRD.