SF162 into the EnvHXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the EnvHXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from Env Surface glycoprotein Env is the main target for neutralizing antibodies elicited by HIV-1 vaccines. Env spontaneously fluctuates among different structures, limiting exposure of many attractive antibody-binding epitopes and, thereby, confounding vaccine development. To characterize these fluctuations, we examined how exposure of the MPER epitope found at the base of Env is regulated by interactions of the V3 loop located in the apex. Starting with an extremely flexible Env with a readily-exposed MPER, we identified two alterations that substantially restricted antibody access to the epitope. The first, a wholesale swap of V3 loops between HIV-1 strains, energetically stabilized Env in a closed structure that restricted access to antibodies throughout the protein. The second, a point mutation that altered V3-loop charge, specifically destabilized the MPER-exposed conformation but had minimal impact on antibody access to Env regions in between the apex and base. The results indicate that MPER exposure is not explicitly tied to the dynamics of Env regions between the apex and base and suggest a new structural fluctuation during Env activation. The Envelope glycoprotein (Env) from human immunodeficiency virus type 1 (HIV-1) is responsible for mediating viral entry through membrane fusion . UnfortuEnv is a homotrimeric, single-pass transmembrane protein with each protomer consisting of two subunits: surface subunit gp120 responsible for binding cellular CD4 and chemokine receptors , and transmembrane subunit gp41 responsible for catalyzing membrane fusion . In the In the presence of target cell membranes, CD4 binding to the gp120 canopy disrupts V1/V2- and V3-loop interactions, leading them to disengage from the conserved Env core as the top of each gp120 subunit rotates and relaxes radially outward –14. ThisRefolding into the TOH supplies the energy required for membrane fusion . AccordiHXB2 and constrained its conformational dynamics through two different modifications to its V3 loop: swapping the entire V3 loop with that from a different Env strain or introducing a charge-altering mutation at V3-loop residue 306. Both modifications substantially reduced anti-MPER antibody potency by shifting MPER exposure from the prefusogenic conformation to a CD4-induced intermediate state. The V3-loop swap produced a chimeric Env variant with the broad neutralization resistance, stability profile and CD4 utilization characteristic of primary isolate Envs. By contrast, the point mutation conferred resistance to sCD4 inactivation and increased the requirement of cellular CD4 for viral entry, but it minimally perturbed exposure of many gp120 epitopes and had no impact on temperature-induced inactivation. The results imply that exposure of the Env gp41 base can be regulated independently from much of the dynamics of the gp120 canopy, motivating us to postulate an additional state along the Env activation pathway leading to membrane fusion.Here, we used NAb sensitivity profiles and stability readouts to interpret how alterations at the Env apex impacted the prefusogenic conformational equilibrium, focusing especially on exposure of the gp41 membrane-proximal external region (MPER) at the trimer base. Previous work has shown that mutations in the V1/V2 and V3 loops of primary isolate Envs can destabilize the closed conformation, increasing temporospatial exposure of neutralizing epitopes throughout the trimer, including the MPER ,53,57–62SF162 (hereafter denoted S5 Env) into the CXCR4-tropic EnvHXB2 (denoted H4 Env) .HXB2 led to a 29-fold reduction in the potency of NAb 2F5, whose extended epitope at the junction of the gp41 C-HR and MPER regions partially overlaps the binding site for 5-Helix  C37-KYI and 5-Helix, implying that the spatiotemporal exposure of the gp41 N-HR and C-HR regions during the PHI were not substantially altered  65,66].,66.65,66 5-Helix  67]. Co. CoHXB2  5-Helix . In fact 5-Helix . Rather,To assess the global impact of the V3-loop swap on Env structure, we compared sensitivities of H4 and H5 Envs to a panel of neutralizing antibodies with epitopes throughout the glycoprotein . H5 Env JRFL, a representative tier 2 clade B primary isolate strain), the enhancement of 10E8 potency matched that for FIs  than for H4 Env (nH = 0.73)  . Since H = 0.73) . In fact = 0.73)  and S4. JRFL, swapping in the EnvHXB2 V3 loop conferred the same increases in potency to FI C37-KYI and 5-Helix  and V3-loop crown 447-52D) . The V3-D . The VNL4-3, which is closely related to H4 Env. Inserting the SF162 V3 loop into the NL4-3 background generated the CCR5-tropic chimeric N5 Env .We also performed a V3-loop swap in CXCR4-tropic EnvEnv Figs  and S1. Env Figs . HoweverEnv Figs . QualitaHXB2 and EnvNL4-3. This residue is an Arg in EnvHXB2, while it is a Ser in EnvNL4-3 and EnvSF162 . However, the properties of H5 Env imply that laboratory adaptation has not fundamentally altered the physiochemical properties of EnvHXB2: in addition to its tier 2-like sensitivity to antibody and sCD4 neutralization, H5 Env displays characteristics of high stability Envs including resistance to both sCD4-induced gp120 shedding and cold inactivation and a requirement for high levels of CD4 on target cells for entry. Interestingly, the single point mutation R306S in the H4 Env V3 loop confers partial stability to the glycoprotein trimer. Like H5 and J5 Envs, H4 R306S Env is resistant to sCD4-induced gp120 shedding and requires high levels of CD4 on target cells for entry. However, H4 R306S Env has the same sensitivity to cold inactivation as wild type H4 Env. These results indicate that the two measures of Env stability reflect different elements of the prefusogenic energy landscape.Laboratory adaptation of HIV-1 results in Envs of lower stability with less-pronounced energetic differences between apo- and receptor-induced conformations . The resHXB2 apex, we posit that fluctuation into the MPER accessible state is tied to the loss of an interaction between V1/V2 and V3 loops regulated by the charge at position 306 and supported by an ionic bond between V1/V2 residue 172 and V3 residue 305. These considerations lead us to propose a new state along the activation pathway in which the V1/V2 and V3 loops disengage from the gp120 core, remaining associated with each other but abrogating intersubunit interactions with loops on adjacent protomers  is likely very destabilizing, especially since these cations are concentrated in the gp120-gp120 interface at the apex of Env. However, introduction of the EnvHXB2 V3 loop into EnvJRFL had only a modest effect on antibody neutralization and no effect on the CoRA-dependence to MPER exposure. It is clear that EnvJRFL regions outside of the V3 loop function to blunt the impact of apical destabilization. Curiously, swapping the EnvHXB2 V3 loop into EnvBG505 did produce a substantial increase in global epitope exposure that rivaled and even exceeded changes observed for the V3-loop swap in EnvHXB2 . OngoinThe following cell lines and reagents were obtained from NIH HIV Reagent Program, Division of AIDS, NIAID, NIH:Cell lines: U87 CD4+CCR5+ cells (ARP-4035) and U87 CD4+CXCR4+ cells (ARP-4036) contributed by Drs. Hong Kui Deng and Dan Littman vitro (ARP-12033) contributed by Dr. John Mascola  supplemented with 100μg/mL DNaseI, 2mM PMSF and 2 μg/mL each of Leupeptin and Pepstatin . Bacteria were lysed using a French press (500psi) and the lysate was diluted 1:2 with PBS500 containing 8 M urea. The solution was centrifuged  at 17,000 x g for 20 minutes at 4°C. Supernatant was applied to Ni-NTA agarose (Qiagen), and protein was renatured by reverse urea gradient over 20 column volumes and ultimately eluted in PBS500 containing 250 mM imidazole. Eluate was concentrated and subjected to size exclusion chromatography (Superdex 75) in 1xPBS to separate monomers from aggregates. D23.2 was concentrated using a 9 kDa MWCO Amicon concentrator to greater than 100 μM, flash frozen in liquid nitrogen and stored at -80°C until use. The identity of the protein was confirmed by SDS-PAGE (molecular weight 14.7 kDa) and HIV-1 inhibition (activity) assays. The concentrations of D23.2, 5-Helix and C37-KYI were determined at 280nm using the method of Edelhoch [The expression plasmid for CD4-antagonist D23.2  was transformed into XL1 Blue cells. A 200mL starter culture of XL1 cells in LB supplemented with 1% (w/v) D-Glucose and 100 μg/ml carbenicillin (Fisher) was grown overnight and used to inoculate 1–2 L of LB plus 1% (w/v) D-Glucose and 100 μg/ml carbenicillin. When the culture achieved an ODEdelhoch .6) with Env-deficient, luciferase-expressing viral vector pNL4-3 E-R-Luc+ (2.5 μg) and Env-expression plasmid pEBB_EnvX (2.5 μg) using linear polyethylenimine . Virus-containing culture supernatant was collected 48hrs post transfection, separated from cellular debris via centrifugation  and stored at -80°C until use. For viral infection, target U87 cells (U87.CD4.CXCR4 or U87.CD4.CCR5) were seeded at 20,000 cells/well  18–24 hours prior to viral inoculation. At the time of infection, culture supernatants were replaced with 50 μl of media with and without antibodies/inhibitors and 50 μl of virus sample. Cultures were incubated at 37°C for 48 hours before supernatant was removed and cells lysed in 1% Triton X-100. Viral infectivity was quantified by luminescence to assess expression of the luciferase reporter .HIV-1 infectivity assays were performed as previously described ,64. HIV-Neutralization potencies were determined by serial dilution of antibodies and inhibitors. Each condition was tested in duplicate or triplicate in each experiment. Averaged data were normalized to the infection level measured in the absence of inhibitors and fit to the Langmuir equation (For the HIV-1 preincubation/dilution experiments in 6) were transfected with 2.5 μg each of pEBB_EnvX and pCMV_Rev using the PEI method described above and incubated for 48 hours. Cells were lifted with EDTA (0.5 mM in 1xPBS) and extensively washed with 1xPBS before being resuspended in 350 μL of 1xPBS containing 2 mg/ml BSA (Sigma-Aldrich), 1 μM PMSF (Sigma-Aldrich), and Complete protease inhibitor (Roche). Aliquots (50 μL) of the cell suspensions were supplemented with sCD4 (50 μL) at various concentrations and incubated for 1 hour at 37°C. Cells were pelleted by gentle centrifugation (10 minutes at 300 x g), and the clarified supernatant was subjected to SDS-PAGE . Separated proteins were transferred to nitrocellulose paper  and probed with sheep anti-gp120 antiserum followed by HRP-conjugated donkey anti-sheep antibody (Jackson Immuno). Protein bands were visualized using ECL detection reagent (Thermo Fisher), imaged using a ChemiDoc system (BioRad), and quantified by densitometry using Adobe Photoshop. Band intensities of shed gp120 were normalized to the intensity of gp120 in a lysate (100 μL of assay buffer supplemented with 2% Triton X-100) of an equal aliquot of untreated cells.HEK293T cells  were immediately flash frozen in liquid nitrogen before being stored at -80°C. At designated timepoints between 96 hours and 1 hour prior to infection, viral aliquots were removed from the -80°C storage, quickly thawed at 37°C for 1 min, and transferred to ice until being applied to target cells. At time of infection, aliquots on ice were collectively warmed to 37°C for 1 min before addition to U87 cells. Viral infection was measured 48 hours later as described above. For each experiment, data were normalized to the infection level of the 1-hour timepoint.S1 TableNumbers reflect the geometric means obtained from viruses with both detected and undetected IC50 values. Orange designates geometric means calculated from groups in which less than 85% of the IC50 values were measurable. Data were procured from the Los Alamos National Laboratory HIV Immunology database (January 2022). For comparison, the mean IC50 values for H4, H5 and H4 R306S Envs found in this study are shown. Red numbers indicate that 50% neutralization was not achieved at the highest concentrations tested.(DOCX)Click here for additional data file.S2 TableNumbers reflect the mean and SEM from three or more independent experiments. The units are ng/ml for all NAbs and nM for sCD4. Red numbers indicate that 50% neutralization was not achieved at the highest concentrations tested. Blank spaces indicate that the antibody or sCD4 was not tested against that Env. Note that B4 R306S Env was nonfunctional.(DOCX)Click here for additional data file.S1 Fig(A) For each virus, infectivity was measured on both U87.CD4.CXCR4 cells (grey bars) and U87.CD4.CCR5 (magenta bars) and normalized to viral content estimated by luciferase expression from proviral DNA in virus-producing cells (see B). Normalized infectivity is reported relative to the values for H4 Env on CXCR4+ cells and H5 Env on CCR5+ cells. Data represent the mean±range-of-mean from two independent experiments. (B) Relationship between luciferase expression from virus producing cells and HIV-1 p24 levels found in culture supernatants. For each experiment, cells were transfected  with differing amounts of pseudotyping vector pNL4-3 E-R-Luc+, Env-expressing vector pEBB_HXB2Env and GFP-expressing vector pBABE_GFP as indicated in the legend. After a 36 hour incubation, supernatants were collected and assayed for viral p24 content by ELISA (TIF)Click here for additional data file.S2 FigA) Experimental timeline of the antibody dilution experiment. HIV-1 was suspended in media with and without NAb 10E8v4 at 2-4x IC50 concentration to form the full-strength samples. A portion of these samples (± antibody) was diluted 10-fold either immediately or after a 1-hour incubation at 37°C. The sample diluted at 1 hour was further incubated at 37°C for 3 hours to allow for antibody unbinding and recovery from neutralization. Culture media over U87 target cells was completely replaced with full strength or diluted samples at the 0-, 1- and 4- hour time points (red circles) to asses viral infectivity in the presence or absence of antibody. (B) Representative data from a single experiment using HIV-1 pseudotyped with H4 Env. Infectivity was measured by luciferase reporter expression for full strength (FS) or diluted (Dil) samples at the indicated time points. Bars and error bars represent the mean ± SEM of three experimental replicates for each sample. (C-F) NAb 10E8v4 neutralization of HIV-1 pseudotyped with H4 (C), H4 R306S (D), H5 (E) and J5 (F) Envs. Fraction infectivity was calculated as the ratio of the luciferase signals recorded in the presence and absence of NAb 10E8v4  under each condition (FS or Dil) at the indicated time point. Antibody concentrations (ng/mL) in the FS and Dil conditions, as well as the IC50 value, are listed in the adjacent box. Data represent the mean ± SEM from four to five independent experiments. The lower fraction infectivity at the 1-hour timepoint compared to the 0-hour timepoint (more easily seen in the Dil samples) indicated that antibody binding to the prefusogenic state occurred during the 0-to-1 hour incubation period under FS conditions. Neutralization was enhanced by approximately the same amount for all Envs tested despite the fact that NAb 10E8 concentrations in H4 R306S, H5 and J5 Env viral samples were approximately 10- to 20-fold higher than that in wild type H4 Env samples. This finding suggested that the antibody association rate constant was much greater for H4 Env than for the other Env species. The lower fraction infectivity in the Dil samples at the 4-hour timepoint compared to the 1-hour timepoint implied that antibody dissociation was slow compared to 10E8-induced Env inactivation Click here for additional data file.S3 FigViral aliquots were incubated at various temperatures for 45 minutes prior to addition to U87 cell cultures at 37°C. Infectivity levels were normalized to the level measured following a 37°C incubation. Data represent the mean±range-of-mean from two independent experiments.(TIF)Click here for additional data file.S4 Fig(A-E) Infectivity levels of pseudotyped HIV-1 were measured in the presence of CD4 antagonist (D23.2) or CoRA (AMD3100 or TAK-779) alone or in combination. U87.CD4.CXCR4 target cells were used for H4 Env (A) and Env89.6 (D), while U87.CD4.CCR5 target cells were used for H5 Env (B), J5 Env (C) and Env89.6 (E). Data were normalized to the level obtained in the absence of both inhibitors. (F-J) The same data in A-E except plotted as D23.2 titrations for each CoRA concentration. Data represent the mean±SEM from seven independent experiments and have been fit to the Hill Equation:0 represents the normalized infection level in the presence of CoRA but the absence of D23.2 and nH is the Hill coefficient. Data were reliably fit for relative infection levels greater than 0.005. (K-O) Relative infection level (Io), IC50 and Hill coefficient (nH) values extracted from the data fits in F-J.(TIF)Click here for additional data file.S5 FigA-C) Inhibitory titrations of C37-KYI (A), 5-Helix (B) and 10E8 (C) against HIV-1 pseudotyped with J4 (open) and J5 (closed) Envs. (D) Table of IC50 values. All data reflect the mean±SEM of four or more independent experiments. For all three inhibitors, J4 Env had significantly lower IC50 values than J5 Env .((TIF)Click here for additional data file.S6 FigA) Ribbon diagram of the V1/V2 and V3 loops in EnvJRFL SOSIP.664  Table of sequence conservation at V1/V2-loop residues 172 and 173 and V3-loop residues 305, 306 and 318 grouped by HIV-1 clade. Numbers represent summed frequencies of the indicated amino acids. (C) NAb 10E8v4 sensitivity of H4, H4 R306S and H5 Envs with the indicated substitutions at residue 318. Data from the Env variants with mutations at residue 318 represent the mean±range-of-mean from two independent experiments. Data from other variants represent mean±SEM of more than three independent experiments. (D) Frequency of a potential ionic interaction between V1/V2-loop residue 172 and V3-loop residue 305 grouped by clade. (E) Ribbon diagram of the V1/V2 and V3 loops in EnvBG505 SOSIP.664 structure  Frequency of a potential cation-pi interaction between V1/V2-loop residue 173 and V3-loop residue 305 grouped by clade. Sequence data obtained from the Los Alamos National Laboratory HIV Sequence database (October 2021).(eference  of main eference  of main (TIF)Click here for additional data file.


Genetically predicted risk of atopic dermatitis increased the levels of 25(OH)D  and 25(OH)D3 . A sensitivity analysis suggested the robustness of these causal associations. Conclusions: This study reported causal relationships between circulating vitamin D levels and the risk of psoriasis, atopic dermatitis, and vitiligo. These findings provide potential disease intervention and monitoring targets.Background: Vitamin D level has been reported to be associated with psoriasis, atopic dermatitis, and vitiligo. However, its causal relationship with the risk of these three diseases remains unclear. Methods: We obtained genome-wide association statistics for three measures of circulating vitamin D levels (25(OH)D in 120,618 individuals, and 25(OH)D3 and epimeric form C3-epi-25(OH)D3 in 40,562 individuals) and for the diseases psoriasis , atopic dermatitis , and vitiligo . We performed Mendelian randomization using inverse-variance weighted, weighted median, MR-Egger, and MR-pleiotropy residual sum and outlier methods. We carried out sensitivity analyses to evaluate the robustness of the results. Results: We showed that elevated vitamin D levels protected individuals from developing psoriasis (OR = 0.995,  Psoriasis, atopic dermatitis, and vitiligo are three chronic inflammatory skin diseases that are commonly characterized by dysregulation of immune function and infiltration of inflammatory cells in skin lesions ,2,3. Vit3) and ergocalciferol (D2) are two most important vitamin D compounds in humans. They are synthesized in the epidermis of the skin or ingested from the diet and supplements. The hydroxylation of D3 and D2 in the liver leads to 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2), respectively. Circulating 25-hydroxyvitamin D (25(OH)D) measures the total amount of 25(OH)D3 and 25(OH)D2 and is regularly used to reflect an individual’s vitamin D status [3 (C3-epi-25(OH)D3), a vitamin D metabolite from 25(OH)D3 through 3β to 3α isomerization transformation, can also affect the amount of bioactive vitamin D conversion [3 levels with psoriasis, atopic dermatitis, and vitiligo [3 levels between atopic dermatitis patients and controls [3 and diseases, but the role of C3-epi-25(OH)D3 in psoriasis, atopic dermatitis, and vitiligo has not been studied yet. Cholecalciferol D3 and the risk of psoriasis, atopic dermatitis, and vitiligo remains unclear. In addition, MR analysis is lacking to investigate whether the incidence of psoriasis and vitiligo changes blood vitamin D levels. Traditional cross-sectional observations are susceptible to potential confounding factors and reverse causality bias, making causal inference challenging . Mendeli3, and C3-epi-25(OH)D3 and the risk of psoriasis, atopic dermatitis, and vitiligo.Here, we performed a bidirectional two-sample MR to comprehensively evaluate the causal relationships between blood levels of three vitamin D measures including 25(OH)D, 25(OH)DTo infer the causal relationship between circulating vitamin D levels and the risk of psoriasis, atopic dermatitis, and vitiligo we performed a bidirectional MR analysis. We summarized our study design in 3 and C3-epi-25(OH)D3 came from a GWAS meta-analysis of 40,562 Europeans [We used genome-wide association statistics for circulating vitamin D levels and the risk of psoriasis, atopic dermatitis, and vitiligo from GWAS with the largest sample sizes. All the summary-level statistics are publicly available. Specifically, genome-wide association summary statistics for blood 25(OH)D were derived from 120,618 healthy adults of European ancestry while the association statistics for 25(OH)Duropeans . We useduropeans , which curopeans . We obtauropeans . We acceuropeans . The deturopeans ,33,34,352r < 0.001 within 1 Mb window) in the GWAS summary statistics for each exposure, using the 1000 Genomes Project Phase 3 (EUR) as the reference panel [p < 5 × 10−8). We calculated an F-statistic for each IV to assess its association strength with exposure [2r > 0.8 with IV) when the IV was missing in the GWAS summary statistics for outcome.To identify IVs for MR analysis, we performed linkage disequilibrium (LD) clumping to identify independent single nucleotide polymorphisms  , weighteTo evaluate the robustness of the causal estimates in MR analyses, we used Cochran’s Q tests  to detechttps://github.com/MRCIEU/TwoSampleMR, accessed on 18 July 2022) and MR-PRESSO  packages. We considered significant causal estimations at p < 0.05 in any of the four MR approaches. We presented the IVW results in this manuscript unless stated otherwise.All analyses were performed in R software version 4.1.1 using TwoSampleMR D3 levels, respectively. The characteristics of these IVs are shown in To evaluate the causal effects of circulating vitamin D levels on the risk of psoriasis, atopic dermatitis, and vitiligo, we performed MR analyses. We identified 14, 9, and 3 IVs for 25(OH)D, 25(OH)Dp = 8.84 × 10−4; 3 and C3-epi-25(OH)D3 levels on increasing the risk of psoriasis D3; OR: 0.998, 95%; CI: 0.996–1.000; p = 0.044 for C3-epi-25(OH)D3; QP > 0.05 in Cochran’s Q test and Pintercept > 0.05 in MR-Egger) nor horizontal pleiotropy effects  D level on developing psoriasis. Each one standard deviation (SD) decrease in circulating 25(OH)D level was associated with approximately 5% increased risk of psoriasis (odds ratio (OR): 0.995, 95% confidence interval (CI): 0.991–0.998, al test) . Leave-oal test) . p > 0.05)  . To check if there is a bidirectional relationship between diseases and vitamin D levels, we further performed reverse two-sample MR analysis. We identified 29, 13, and 48 IVs for the risk of psoriasis, atopic dermatitis, and vitiligo, respectively. The characteristics of these IVs are shown in p = 7.14 × 10−4; 3  but not in the analyses for 25(OH)D3 D D3 . Leave-o25(OH)D3 . Our analyses found the genetically predicted risk of neither psoriasis nor vitiligo influenced the circulating vitamin D levels . In this study we carried out a bidirectional MR analysis and tested causal relationships between circulating vitamin D levels and the risk of three common skin inflammatory diseases: psoriasis, atopic dermatitis, and vitiligo. We identified a significant causal effect of elevated blood vitamin D levels on decreasing the risk of psoriasis and suggested that the incidence of atopic dermatitis raise blood vitamin D levels. These findings highlight a role of blood vitamin D levels as potential disease intervention and monitoring targets.3 and C3-epi-25(OH)D3 with psoriasis is yet to be studied. Our study validated the causal effect of 25(OH)D on the risk of psoriasis using a set of IVs which we identified in a different GWAS data of 120,618 Europeans, and performed MR analysis with different psoriasis GWAS summary statistics from the MR base [3 and C3-epi-25(OH)D3 increased the risk of psoriasis. To our best knowledge, we provided the causal evidence of blood 25(OH)D3 and C3-epi-25(OH)D3 with psoriasis for the first time. Their causal effects are in line with the reported one of 25(OH)D levels. Vitamin D receptors are widely present in keratinocytes, dendritic cells, macrophages, and T cells [Early epidemiological studies have shown lower serum vitamin D levels in patients with psoriasis than healthy controls . Adminis MR base ,32. In a T cells . The act T cells . In psor T cells . These r3 and C3-epi-25(OH)D3 and the risk of atopic dermatitis. We found no significant causal effect of blood 25(OH)D3 or C3-epi-25(OH)D3 on the risk of atopic dermatitis but suggest that developing atopic dermatitis raised blood 25(OH)D3 levels. Previous MR identified no causal effects of circulating 25(OH)D or 25(OH)D3 on the risk of vitiligo [3, or C3-epi-25(OH)D3 on vitiligo. In addition, the reverse associations were not statistically significant either. To our best knowledge, this is the first MR analysis to evaluate whether the genetically predicted risk of vitiligo consequently affects blood vitamin D levels. Despite of the common characteristics of immune dysregulation and inflammation among psoriasis, atopic dermatitis, and vitiligo our study identified their heterogeneous causal relationships with blood vitamin D levels. Previous MR analyses using GWAS data of vitamin D in 443,734 individuals suggested no causal effect of blood 25(OH)D on developing atopic dermatitis but a significant causal effect of atopic dermatitis on increasing blood 25(OH)D levels ,45. Our vitiligo . We achiOur study did not support a causal role of vitamin D levels on atopic dermatitis and vitiligo. This finding was in line with previous cross-sectional and perspective studies, which detected no supporting evidence for vitamin D being a risk factor ,15,47. HWe acknowledge that these causal inferences might reflect the mixed effects of insufficient statistical power and unmeasured confounding factors. The interpretation of current MR results needs careful consideration. A recent paper highlighted the potential issue of non-linear genetic effects of vitamin D SNPs on vitamin D . The imp3 and C3-epi-25(OH)D3 levels with the increased risk of psoriasis and consequences of increased 25(OH)D3 levels on atopic dermatitis for the first time. These findings provide intervention targets for psoriasis and disease monitoring biomarkers for atopic dermatitis. In summary, we confirmed the previous reported causal relationship between 25(OH)D level and psoriasis and identified causal associations of lower 25(OH)D


Select HLA and non-HLA SNPs were considered. Non-genetic and genetic factors were assessed by multivariable logistic regression models for autoantibody positivity at initial sampling and autoantibody loss at final sampling. For GADA, IA-2A, and ZnT8A, 70.8%, 76.8%, and 40.1%, respectively, remained positive at the final sampling. Non-genetic predictors of autoantibody loss were low baseline autoantibody titres (P < 0.0001), longer diabetes duration (P < 0.0001), and age-at-onset under 8 years (P < 0.01–-0.05). Adjusting for non-genetic covariates, GADA loss was associated with low-risk HLA class II genotypes (P = 0.005), and SNPs associated with autoimmunity RELA/11q13 (P = 0.017), LPP/3q28 (P = 0.004), and negatively with IFIH1/2q24 (P = 0.018). IA-2A loss was not associated with genetic factors independent of other covariates, while ZnT8A loss was associated with the presence of HLA A*24 (P = 0.019) and weakly negatively with RELA/11q13 (P = 0.049). The largest longitudinal study of islet autoantibody responses from diagnosis of T1D shows that autoantibody loss is heterogeneous and influenced by low titres at onset, longer duration, earlier age-at-onset, and genetic variants. These data may inform clinical trials where post-diagnosis participants are recruited.The clinical usefulness of post-diagnosis islet autoantibody levels is unclear and factors that drive autoantibody persistence are poorly defined in type 1 diabetes (T1D). Our aim was to characterise the longitudinal loss of islet autoantibody responses after diagnosis in a large, prospectively sampled UK cohort. Participants with T1D [ Loss of islet autoantibodies after diabetes onset is heterogeneous but influenced by non-genetic and genetic factors.  Four major type 1 diabetes (T1D)-associated autoantibodies to insulin (IAA), glutamate decarboxylase 65 (GADA), islet antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A) remain primary biomarkers for predicting future diabetes and are distinctive clinical features of islet autoimmunity. At the onset of clinically diagnosed T1D, >90% of people are positive for at least one of these autoantibodies  and to aStudies of autoantibody prevalence after T1D diagnosis spanning 12–56 years (diagnosed between 0 and 56 years of age) generally report GADA as the most frequently detected autoantibody, followed by IA-2A and ZnT8A (where tested) . These sUnderstanding why autoantibody positivity is lost or maintained post-diagnosis may provide insights into the ongoing loss of beta-cells and insulin secretion. Autoantibody positivity is inversely correlated with diabetes duration but does not appear to strongly correlate with C-peptide, a by-product of insulin synthesis and a biomarker for exogenous insulin requirement , 17–20. n = 419 <0.5 years] and at least one longitudinal serum sample at follow-up [median 7.3 years (range 2.0–32.0)] were selected and tested for GADA, IA-2A, and ZnT8A; multiple longitudinal samples were tested until autoantibody negativity was determined.The population-based Bart’s-Oxford (BOX) family study, established in 1985, has recruited and prospectively followed individuals with newly diagnosed T1D under 21 years (with confirmed initiation of exogenous insulin treatment) and their first-degree relatives within the UK’s former Oxford Regional Health Authority. The BOX study is currently approved by the South Central—Oxford C. National Research Ethics Committee. Participants provided informed, written consent and the study was performed according to the principles of the Declaration of Helsinki. All BOX participants that had a serum sample taken close to T1D onset  autoantibody positive individuals at onset were identified. Data for IAA were available from 238 individuals (41.2%) and considered in select analyses where the onset sample was taken within 2 weeks of diagnosis before exogenous insulin treatment. Overall, 290 (50.3%), 183 (31.7%), 87 (15.1%), and 17 (2.9%) individuals provided 1, 2, 3, and 4 follow-up samples, respectively; combinations of samples available for autoantibody detection across categories of follow-up are detailed in Autoantibodies to GAD65 aa1-585), IA-2ic (aa606-aa979), and ZnT8  were detected by radioimmunoassays described previously -585, IA-. LogaritDR3-DQ2/DR4-DQ8; DRB1*03-DQA1*05:01-DQB1*02:01/DRB1*04-DQA1*03:01-DQB1*03:02 (reference variable)], moderate , and low risk (X/X and DQ6/X), where X refers to any other haplotype. Individuals with the protective DQ6/X genotype were categorised as low risk, and only present in four individuals (0.8% of 501). HLA class I genotypes for HLA-A*24 (n = 454), HLA-B*18 (n = 417), and HLA-B*39 (n = 417) determined by PCR-SSP as previously described , and genetic covariates  on autoantibody positivity at initial sampling and loss at final sampling. The Bonferroni correction was applied where applicable for multiple analyses  and an overall loss of 80.5% at a diabetes duration ≥15 years (Pcorr < 0.05 compared with 10–15 years). In contrast, in subjects positive for GADA at onset , 70.8% (340/480 with complete data) remained positive at final sampling [median 7.9 years (range 2–32)], with only 17.3% loss within 5 years of onset (Pcorr< 0.0001) and an overall loss of 47.3% at a diabetes duration ≥15 years (Pcorr<0.001 compared with 10–15 years). Comparable to GADA, in subjects positive for IA-2A at onset , 76.8% (347/452) remained positive at final sampling [median 8.0 years (range 2–32)], with only 11.6% loss occurring within 5 years of onset (Pcorr < 0.0001) and an overall loss of 38.9% at a diabetes duration ≥15 years (Pcorr < 0.01 compared with 10–15 years).Longitudinal autoantibody positivity showed distinct patterns according to antigen specificity. ZnT8A positivity was lost more rapidly than GADA and IA-2A whereas, IA-2A positivity was lost at a rate comparable with GADA over follow-up . In subjP < 0.0001) but high GADA and IA-2A titres were still observed at ≥15 years diabetes duration, which was rare for ZnT8A (n = 68 (14.0%); IA-2A: n = 25 (5.5%)] and/or had evidence of waxing–waning patterns of differing magnitudes from onset over at least two follow-up samples . However, only 6 (1.5%) ZnT8A positive subjects had a higher ZnT8A titre in at least 1 follow-up sample compared to onset, and there was no evidence of waxing–waning patterns (data not shown). Given the testing strategy, the re-emergence or seroconversion of additional autoantibodies after onset cannot be evaluated.In accordance with decreasing autoantibody prevalence, the median autoantibody titre for GADA, IA-2A, and ZnT8A also decreased as a function of increasing diabetes duration (or ZnT8A . Across P < 0.0001–0.001), whereas ZnT8A positivity was most common at diagnosis between 8 and 11 years old and IAA positivity was negatively associated with an older age-at-onset >11 years (P = 0.001–0.023). Females were more likely to have GADA (P = 0.026). Positivity for IA-2A at onset was associated with ZnT8A (P = 4 × 10−6) and IAA (P = 0.003) and ZnT8A positivity at onset was associated with IA-2A (P = 5 × 10−6) and weakly with IAA (P = 0.045) whereas, GADA positivity at onset was not associated with other autoantibodies. An increase in initial sampling (months) from onset was only negatively associated with ZnT8A positivity (P = 0.009), suggesting that loss of ZnT8A can occur within months from onset.Non-genetic factors previously associated with positivity for GADA, IA-2A, and ZnT8A at onset were confirmed in this cohort in adjusted multivariable models . GADA anP < 0.0001–0.05) and was included in all multivariable analyses. Gender was not associated with autoantibody responses at final follow-up independent of other covariates (P > 0.05). Additionally, there was also no evidence of an interaction between baseline age and autoantibody titre on autoantibody loss at final sampling (P > 0.05) and was not included in multivariable analysis.Multivariable analysis on the loss of GADA, IA-2A, and ZnT8A responses at final follow-up adjusted for all non-genetic covariates are detailed in P = 3.8 × 10−14), IA-2A , and ZnT8A  at final follow-up, independent of other covariates. For GADA (P = 3.8 × 10−2 to 1.5 × 10−16) and ZnT8A (P = 6.0 × 10−5 to 6.1 × 10−18), quartiles of baseline autoantibody titre below the highest quartile were strongly associated with increased risk of autoantibody loss; however, for IA-2A, discrimination of risk was only observed between the upper two and lower two quartiles (P = 2.8 × 10−4 to 1.1 × 10−14). Despite the heterogeneity between individuals, autoantibody titres decreased over follow-up across all quartiles of baseline titre. Interestingly, some individuals with lower quartiles of baseline titre still maintained autoantibody positivity ≥15 years diabetes duration and some individuals with higher quartiles of baseline titre rapidly lost autoantibody positivity within 5 years diabetes duration . IA-2A loss was only less likely in individuals diagnosed between 11 and 14 years . In contrast, ZnT8A loss was less likely in individuals diagnosed ≥11 years . These effects were independent of baseline titre but the influence of age at onset on both titres and longevity of humoral responses after onset is evident .The presence of co-existing or the number of islet autoantibodies at onset was not associated with the loss of GADA, IA-2A, or ZnT8A at final sampling adjusted for non-genetic covariates . CompareMultivariable models with complete genetic data for autoantibody positivity at initial sampling (<2 years from onset) and autoantibody loss at final sampling adjusted for non-genetic covariates , genetic variables were considered independently as categorical or binary variables as previously stated .DR3  and negatively with DR4  when considered independently. The presence of HLA class I HLA-B*39 was negatively associated with GADA positivity . Investigated non-HLA SNPs were not associated with GADA positivity at initial sampling. However, at final sampling, GADA loss was associated with low-risk HLA class II genotypes , possibly due to the reduced presence of at least one copy of DRB1*03. This was weakly confirmed by the negative association with DR3  when considered independently. Loss of GADA at final sampling was not associated with any HLA class I genotypes but was associated with 3 non-HLA SNPs; positively with the RELA/11q13 T allele  and LPP/3q28 A allele  but was negatively associated with the IFIH1/2q24 A allele .At initial sampling, GADA positivity was not associated with HLA class II risk but was strongly associated with the presence of DR4  and negatively with DR3  when considered independently. The presence of HLA class I HLA-A*24 was negatively associated with IA-2A positivity . IA-2A positivity at initial sampling was also associated with two non-HLA SNPs; positively with the LPP/3q28 A allele  and negatively with the FCRL3/1q23 A allele . IA-2A loss at final sampling was not associated with any of the genotypes considered, independent of non-genetic covariates. However, the presence of at least one copy of DR4 was negatively associated with IA-2A loss  when IA-2A titre at onset was excluded from the model due to an interaction between DR4 and IA-2A titre (data not shown).At initial sampling, IA-2A positivity was not associated with HLA class II genotypes but was strongly associated with the presence of HLA-A*24  and the non-HLA FCRL3/1q23 A allele . The SLC30A8 genotype did not influence overall ZnT8A positivity but strongly influenced the specificity of the ZnT8A response (data not shown). The T allele was strongly associated with the development of ZnT8WA  and strongly negatively associated with the development of ZnT8RA . ZnT8A loss at final sampling was associated with the presence of HLA-A*24 . Only one non-HLA SNP showed a weak negative association with ZnT8A loss at final follow-up, the RELA/11q13 T allele .At initial sampling, ZnT8A positivity was not associated with any HLA class II genotypes but was negatively associated with the presence of HLA class I RELA/11q13, LPP/3q28, and negatively with IFIH1/2q24. IA-2A loss was not linked with any genetic factors considered. ZnT8A loss was associated with the presence of HLA-A*24 and weakly with RELA/11q13. Despite an overall reduction in autoantibody prevalence, number, and titre as a function of increasing diabetes duration, the degree of autoantibody loss was not influenced by the number or combination of autoantibodies present at onset.In this study, we were able to analyse autoantibody loss  because presence at diagnosis was established. The strongest predictor of islet autoantibody loss was lower baseline titres close to onset, while an age-at-onset under 8 years and a diabetes duration over 5 years were also associated. Genetic factors associated with autoantibody loss were antigen-specific and distinct from well-known associations with autoantibody positivity at onset. GADA loss was associated with low-risk HLA class II genotypes and non-HLA loci for P < 0.0001), but the within-subject variance of mean autoantibody titres was unequal across categories of longitudinal follow-up . Similarly, the between-subject variance of mean autoantibody titres was high in all autoantibody responses . Collectively, this indicated that autoantibody responses after onset are highly heterogeneous between individuals, and therefore, predictive modelling using linear trend or imputation would be inappropriate.The strength of our study was the ability to account for baseline autoantibody positivity and titres, which we have shown, have the largest effect size with the persistence of humoral responses in the largest cohort of longitudinally sampled subjects with T1D. This allowed for the analysis of factors that may only be associated with autoantibody responses when baseline positivity and titre are accounted for, enabling the differentiation between prevalence and persistence of humoral responses after onset. A limitation of our current analysis was that the method (multivariable binary logistic regression) only utilised data from the last sample, instead of all available samples. We considered mixed-model effect generalised linear models (GLM) using a Restricted Maximum Likelihood fit and the Geisser–Greenhouse correction to estimate a linear trend accounting for missing values and sphericity in mean autoantibody levels over follow-up, respectively. When applied, this method suggested that there was evidence of a linear trend in decreasing autoantibody titres (After onset (average median 7.3 years diabetes duration), only 40.1% remained positive for ZnT8A compared with, 70.8% and 76.8% for GADA and IA-2A, respectively. The rapid decline in ZnT8A, independent of R325W specificity during the first 10 years of diabetes, compared with GADA and IA-2A, has been reported previously , 13. HowMeasuring islet autoantibodies after diagnosis may also provide important insights into the mechanisms driving ongoing islet autoimmunity and loss of beta-cell function after onset. The results of the present study into autoantibody patterns in addition to, non-genetic and genetic factors may help elucidate these mechanisms. For instance, islet autoantibody loss was strongly influenced by titre around onset but analysis of longitudinal humoral titres after onset showed that periodic enhancement of titres is rare; titres of GADA, IA-2A, and ZnT8A progressively decreased over follow-up in 74.9%, 90.0%, and 98.5%, respectively. This suggests that islet autoantibody responses are predominantly governed by immune memory. However, for GADA our study supports previous reports that GADA titres can peak or stabilize after diagnosis in select individuals, but this may be a distinct feature of GADA responses and the mechanism is not clear , 32, 33.Islet autoantibody positivity at diabetes onset has been linked to age at diagnosis which we have confirmed in this cohort , 34, 35 DRB1*03 and IA-2A with DRB1*04 [DRB1*03 and GADA loss, a positive association between low HLA class II risk and GADA loss, and DRB1*04 was only negatively associated with IA-2A loss when baseline titre was not considered. Type 1 diabetes-associated HLA class I genotypes were not associated with loss of GADA or IA-2A, but the presence of HLA-A*24 was associated with loss of ZnT8A in adjusted models. This is in line with the negative association between HLA-A*24 and ZnT8A positivity which we have confirmed and have previously reported at onset [HLA-A*24 carriers continues after diabetes onset. A previous study has shown a negative association of HLA-A*24 with IA-2A positivity after diagnosis but was unable to account for IA-2A status at onset [HLA-B*39 and progression of autoimmunity has been previously reported [HLA-B*39 and GADA positivity at onset was not observed after onset, suggesting distinct effects of these HLA class I genotypes on GADA and IA-2A responses around onset and after diagnosis.Next, we asked ‘do the genetic drivers of autoantibody production after onset differ from the well-known antigen-specific variants that influence T1D progression and risk?’ For this, we specifically chose to analyse genotypes and genetic loci that have been associated with islet autoantibody positivity before, around, and after diagnosis of T1D. The highly predisposing HLA class II genotypes for T1D risk that strongly influence titres of specific autoantibodies at onset  do not have additional effects on humoral responses after clinical onset. Previous studies investigating prevalence after diagnosis have shown an association of GADA with  DRB1*04 , 37, 38;at onset  and in fat onset , which mat onset . We onlyat onset . An assoreported , but theRELA/11q13 and FCRL3/1q23 SNPs were associated with IA-2A positivity, LPP/3q28 SNPs were associated with GADA positivity, and IFIH1/2q24 was associated with positivity for autoantibodies related to autoimmune gastritis and thyroid disease [FCRL3/1q23 was negatively associated with IA-2A and ZnT8A positivity sampled <2 years of onset [RELA/11q13 and LPP/3q28 were associated with GADA loss, but no SNPs studied were associated with IA-2A loss, the minor allele of IFIH1/2q24  was negatively associated with GADA loss, and RELA/11q13 was weakly negatively associated with ZnT8A loss. Despite the size difference in both cohorts (577 vs ~7000), the different effects of RELA/11q13, LPP/3q28, and IFIH1/2q24 may be related to the present study having a longer follow-up period and correction for baseline autoantibody titre rather than statistical power. Of all the SNPs we studied, only IFIH1/2q24 (rs2111485 in LD with rs1990760) has been associated with both T1D risk [The SNPs investigated in this study and/or genetic loci have also been linked with positivity for specific humoral responses 3–14 years after T1D onset in ~7000 individuals from the T1D genetic consortium  , 27. In of onset , 43, whiT1D risk  and progT1D risk , whereasT1D risk . A limitT1D risk . OverallStudies investigating long-duration >20–50 years) diabetes suggest some individuals have residual beta-cells with the functional capacity to secrete insulin (C-peptide) , 50. Rec years diIf residual antigen remains accessible to immune surveillance either by continued beta-cell death or functional residual beta-cells, the differential antigen expression may provide some rationale as to why ZnT8A is lost more rapidly than GADA or IA-2A. All islet autoantigens are highly expressed in pancreatic islets but ZnT8 expression is almost exclusive to beta-cells and is exposed upon glucose-stimulated insulin secretion whereas, GAD and IA-2 are found in specific cells of the nervous system . HoweverThe clinical usefulness of islet autoantibodies after diagnosis is often questioned but the data from this study present the longest duration of longitudinal autoantibody analysis thus far. Where islet autoantibodies are used to help define diabetes aetiology, testing close to diagnosis is important particularly for ZnT8A, as responses are rapidly lost. This study also suggests that the drivers of islet autoantibody prevalence before diagnosis and persistence after diagnosis are distinct. Whilst islet autoantibody loss is heterogeneous, factors associated with loss are titre, diabetes duration, and age at diagnosis, select HLA Class I/II alleles and non-HLA SNPs. These factors may also be important to consider when interpreting the impact of immunotherapy on islet autoantibodies after the onset of T1D, and what this indicates about the mechanistic actions of the therapy. Therefore, the meaning of islet autoantibodies after T1D onset, merits further investigation, because it has implications for clinical trials and understanding of the ongoing islet autoimmunity after diagnosis in some individuals.uxac087_suppl_Supplementary_MaterialClick here for additional data file.


Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions. Flaviviruses belong to a large family of enveloped viruses with positive-sense single-stranded RNA genomes. The most prominent human viruses in this family are arthropod-borne and transmitted mainly through the bite of an infected mosquito . The previa a cytoplasmic cofactor domain in order to be catalytically active (The endoplasmic reticulum (ER) is the largest organelle of the cell and serves an array of important cellular functions, including production, trafficking, and degradation of proteins, metabolism of carbohydrates, and synthesis of lipids, making it an optimal site for flavivirus replication . After vy active . Thus, ty active . Transmiy active . Severaly active . Thus, vWhile the process of ER membrane remodeling has been studied utilizing the traditional method of imaging fixed cells, visualization of the events of virus-induced host cell manipulation throughout the course of infection has not been well-characterized. Imaging of fixed samples only allows for still snapshots of different cells at specific time points. However, the process of host cell manipulation during infection is very dynamic; therefore, still images cannot provide the full picture of the events that take place throughout the course of infection. To overcome these limitations, long-term time-lapse imaging of living cells infected with recombinant viruses expressing reporter fluorescent proteins has been used in previous reports . However2. C6/36 cells were maintained in a humidified environment at 28°C.U2OS osteosarcoma cells  and HEK-293T cells  were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 IU penicillin/100 µg/mL streptomycin. Vero E6 cells  were cultured in modified Eagle’s medium supplemented with 10% fetal bovine serum and 100 IU penicillin/100 µg/mL streptomycin. C6/36 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 100 IU penicillin/100 µg/mL streptomycin. Human hepatoma cells (Huh7) were maintained in DMEM supplemented with 10% FBS, 9 g/L glucose, and 100U/mL P/S. All mammalian cells were maintained at 37°C in a humidified environment with 5% COvia focus forming assay. Human astrovirus-1 strain Oxford (BEI Resources) was propagated and titrated by focus forming assay as previously described  was propagated in C6/36 cells  incubated at 33°C for 6 days. Supernatants were combined with FBS to a final concentration of 20%, and cell debris was removed through centrifugation at 2300 xg for 15 minutes at 4°C. Clarified supernatants were aliquoted and stored at -80°C for future use. Yellow fever virus strain 17D was obtained from BEI Resources (NR-116) and propagated in VeroE6 cells for 4 days. Supernatants were clarified and aliquoted prior to titration escribed .Flavivirus stocks were titered by focus forming assay as previously described with minor modifications . BrieflyE. coli (Zymo Research).FlavER was cloned through the assembly of three DNA fragments into the NheI and XbaI restriction sites of pcDNA3.1(+) (Invitrogen). The first fragment was generated by amplifying the GFP from pcDNA3.1-GFP  using Q5 high-fidelity DNA polymerase (New England Biolabs) with NheI-GFP_F (5’-GAAGCTAGCCACCATGGTGAGCAAGGGCGAGGAG-3’) and Xhol-GFP_R primers (5’-GAGACTCGAGCTTGTACAGCTCGTCCATGC-3’), followed by digestion with NheI-HF and XhoI (New England Biolabs). The second fragment was synthesized as a gBlock (Integrative DNA Technologies) consisting of 5’-XhoI – simian virus 40 nuclear localization sequence (-TCATCCGATGACGAGGCCACAGCTGATTCCCAGCACTCAACTCCGCCTAAAAAAAAAAGAAAAGTT) – KpnI – a flavivirus protease consensus cleavage sequence (GCAGAGGCTGCAAAAAGGAGGAGTGCTGGACTGAACGAGATG) – BamHI – transferrin receptor transmembrane domain (TATGGGACTATTGCTGTGATCGTCTTTTTCTTGATTGGATTTATGATTGGCTACTTGGGCTAT) – flexible linker (GGTGGATCTGGCGGAGGTTCCGGC) – EcoRI-3’, which was digested with XhoI and EcoRI-HF (New England Biolabs). The third fragment was generated by amplifying the mCherry-KDEL from pAc-mCherry-KDEL  using Q5 high-fidelity DNA polymerase with EcoRI-mCh_F (5’-GTAGGGAATTCATGGTGAGCAAGGGCGAGGAGGATAAC-3’) and XbaI-mCh-KDEL_R (5’-GTAACGTTAGGGGGGGGGGATCTAGATCATAGCTCGTCTTTCTTGTACAGC-3’) to generate an EcoRI and XbaI flanked mCherry-KDEL. The three fragments were digested with the respective terminal restriction sites and ligated using T4 DNA ligase (New England Biolabs) for 3 hours at RT. The ligation was transformed into DH5α E. coli.pLJM1entr was generated to facilitate cloning of FlavER into pLJM1, a lentivirus transfer plasmid. The EGFP transgene in pLJM1-EGFP  were reverse transfected with FlavER plasmid using Fugene HD (Promega) in an 8-well chamber slide (Celltreat). Twenty-four hours post transfection, cells were fixed in 4% PFA in PBS, permeabilized with 0.1% Triton X-100 (Fisher) diluted in PBS, washed with PBS, and incubated with mouse α -V5 epitope tag monoclonal antibody  or anti-dsRNA monoclonal antibody (Kerafast) for 1 hour at RT. The monolayers were washed 3X with PBS for 5 minutes each and probed with goat α -mouse conjugated to Alexa Fluor-647  for 30 minutes at RT. The cells were washed 3X with PBS for 5 minutes each and incubated in PBS containing 300 nM DAPI for 5 minutes at RT. The slide was mounted using Vectashield Antifade Mounting Media (Vector Laboratories) and a 24x50mm Premium Superslip (FisherScientific). Three fields of view per well were captured using an Olympus IX83 inverted fluorescent microscope.U2OS cells were transfected with the indicated plasmids using XtremeGene360 (Roche). When specified, cells were infected with DENV for the indicated timepoints. Cells were lysed in 1x RIPA + protease inhibitor + 0.1% SDS cocktail (Sigma). Lysates were sonicated prior to being separated by SDS PAGE using a 4-20% Tris-glycine polyacrylamide pre-cast gel (BioRad) and transferred to nitrocellulose membranes. Following 30 min of blocking in PBS + 10% non-fat milk, membranes were probed with the indicated primary antibodies: mouse anti-V5 (Invitrogen), rabbit anti-GFP (Proteintech), mouse anti-GFP (Proteintech), mouse anti-actin , and rabbit anti-DENV NS3 (GeneTex) followed by near-infrared dye-conjugated secondary antibodies (LiCor) diluted in PBST + 5% non-fat milk and imaged on an Odyssey CLx imaging system (LiCor).6 cells/well) were reverse transfected with 1 µg pLJM1-FlavER, 0.75 µg psPAX2 (a gift from Didier Trono (Addgene plasmid #12260), and 0.25 µg pCAGGS-G-Kan  using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Lentivirus was harvested by passing the media through 0.2-micron filters  48 hours post transfection. U2OS cells (5 x 104 cells) were reverse transduced with 50 µL FlavER lentiviral vector stock in growth media containing 10 µg/mL polybrene (Sigma Aldrich). Transduced cells were passaged for 48 hours and selected with 5 µg/mL puromycin for 5 days.To produce lentiviral vectors expressing the FlavER transgene, HEK-293T cells (2 x 104 U2OS cells stably expressing FlavER were transferred to an 8-well live-imaging slide (Ibidi). Cells were either left uninfected (mock) or infected with DENV at an MOI of 10. Infections were synchronized at 4°C for 1h, followed by shifting cells to 37°C for 3h prior to onset of imaging. One image per well was taken every 20 minutes over a 20-hour time period. Image series were cropped using ImageJ software , and multi-panel images were constructed using Photoshop CC 2021 .3 x 10Models were made using Adobe Illustrator/Photoshop and Biorender. Percent nuclear translocation of the reporter signal in cells transfected with FlavER and a V5-tagged flavivirus protease was quantified using the counter plugin in ImageJ (NIH). The ImageJ counter plugin was also used to identify the number of the protease expressing cells with the reporter signal in the nucleus. Data was plotted using Prism 9 software .ImageJ was used to quantify relative infection signal from the live-cell time-lapse images. To account for non-specific fluorescence signal in the nucleus from the widefield microscope, the fluorescence signal intensity of the infection reporter was determined relative to the ER signal intensity within the same circular region of interest within the nucleus of individual cells from every image series. Data was plotted using Prism 9 software (GraphPad).One-way ANOVA and nonlinear regression curve analyses were performed using Prism Software 9 software (GraphPad).via the transmembrane domain of the transferrin receptor (type II transmembrane domain) fused to an ER lumen localized fluorescent protein, which is fused to an ER retention signal (KDEL). Thus, we anticipated that the expression of the NS2B3 protease upon flavivirus infection results in cleavage of the consensus cleavage site, allowing for nuclear translocation of the reporter protein from the ER. Post cleavage, the fluorescent ER protein marker will remain in the ER lumen, allowing for visualization of changes to this organelle throughout the course of infection  or catalytically inactive DENV NS2B3 (S135A) and subjected to immunofluorescence (IF) staining or immunoblotting. IF staining of cells expressing FlavER and the active DENV NS2B3 showed localization of the protease-dependent reporter signal in the nucleus, while cells transfected with the catalytically inactive DENV protease (S135A) did not demonstrate this translocation  indicated no difference in viral production over 48 hours of infection , an unrelated positive sense ssRNA virus that also replicates at the ER Figure 4To determine if the consensus sequence in FlavER could be cleaved by multiple flavivirus proteases, we analyzed the lysates of FlavER-expressing cells transfected with the ZIKV, WNV, YFV, and mutant and active DENV proteases , which are not naturally observed in the flavivirus polyprotein sequence . We usedvia protease activity throughout the course of infection.To validate the efficacy of FlavER during infection, long-term time-lapse imaging was used to determine the efficiency of virus-induced nuclear translocation of the reporter signal. U2OS cells stably expressing FlavER were infected with DENV four hours prior to being placed in a humidified chamber set to 37°C for a 20-hour timespan with images taken in 20-minute intervals. DENV infection of cells expressing the reporter resulted in evident translocation of the fluorescent protein-NLS reporter to the nucleus, which was not observed in mock cells, while the ER-KDEL signal was shown to remain localized in the lumen throughout the course of a 24-hour infection  and NIH grant K22AI143963-01 (NL).We thank Carolyne Coyne (Duke University), Kevin Harrod (University of Alabama at Birmingham), and Todd Green (University of Alabama at Birmingham) for reagents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Desemzia sp. strain C1, which was isolated from oil-contaminated soil in South Korea and produces hydrogen peroxide (H2O2). The genome of Desemzia sp. strain C1 contains genes encoding various oxidases involved in H2O2 production and resistance to oxidative stress.Here, we report the draft genome sequence of  Desemzia includes Gram-positive, non-spore-forming, microaerophilic bacteria , was isolated based on the Prussian blue zone-forming reaction of ferric cyanide and H2O2 produced by bacteria (2O2 were grown in brain heart infusion (BHI) broth  and Trypticase soy broth containing 3 g yeast extract (TSBY) (BD Difco) at 30°C under static conditions, and the H2O2 produced was quantified using the Amplex Red hydrogen peroxide/peroxidase assay kit . Desemzia sp. strain C1 produced a maximum of 0.23 mM H2O2 in BHI broth, which was 4 times higher than that of Streptococcus oralis KACC 13048T, a well-known H2O2 producer, in TSBY  from an auto repair shop in Gwangju, South Korea .  cm from  in TSBY . TherefoN50, 8,836 bp). The draft genome of Desemzia sp. strain C1 was constructed based on PacBio sequencing data , with an overall G+C content of 38.7%. The genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using the best-placed reference protein set method (GeneMarkS-21) , pyruvate oxidase, and three distinct NADH oxidases (Genomic DNA (gDNA) was extracted using the MagAttract high-molecular-weight (HMW) DNA kit  after culture in BHI broth for 36 h at 30°C under static conditions. Extracted gDNA was sheared using the Megaruptor 3 , and small fragments of less than 3 kb were removed using AMPure XP beads . The DNA library was constructed by using the SMRTbell Express template preparation kit v2.0  . The SMRN50, 2,69,877 bp, 2O2 production and resistance mechanisms in the bacterial system. In addition, the H2O2-producing putative gene encoding LOX could be a good candidate involved in the bacterial enzyme-mediated advanced oxidation processes to apply for degradation and detoxification of various organic pollutants.The current genome information can shed light on the understanding of HDesemzia sp. strain C1 has been deposited in GenBank under the BioProject accession number PRJNA777376, the BioSample accession number SAMN22852026, and the GenBank accession number JAJIZP000000000. The raw reads can be accessed under the SRA accession number SRR17868029. The version described in this paper is the first version, JAJIZP010000000.The draft genome of


The concentration of PB/Ti3C2Tx was optimized and the optimal detection potential of PB/Ti3C2Tx/GOx/Nafion/SPIL-GE is −0.05 V. The performance of PB/Ti3C2Tx/GOx/Nafion modified SPIL-GE was characterized by cyclic voltammetry and chronoamperometry technique. This paper-based platform integrated with nanomaterial composites were realized for glucose in the range of 0.0–15.0 mM with the correlation coefficient R2 = 0.9937. The limit of detection method and limit of quantification were 24.5 μM and 81.7 μM, respectively. In the method comparison, this PB/Ti3C2Tx/GOx/Nafion/SPIL-GE exhibits a good correlation with the reference hexokinase method. This novel glucose sensing platform can potentially be used for the good practice to enhance the sensitivity and open the opportunity to develop paper-based electroanalytical devices.As glucose biosensors play an important role in glycemic control, which can prevent the diabetic complications, the development of a glucose sensing platform is still in needed. Herein, the first proposal on the in-house fabricated paper-based screen-printed ionic liquid/graphene electrode (SPIL-GE) modified with MXene (Ti Diabetes mellitus, a leading non-communicable disease-related health issue, is a risk factor for a variety of vascular illnesses . By 20452O2, this byproduct may be detected by highly sensitive electrochemical techniques and is proportional to the amount of glucose in the blood ), potassium ferrocyanide trihydrate (K4[Fe(CN)6]·3H2O), iron (III) chloride (FeCl3), potassium chloride (KCl), hydrochloric acid (HCl), lithium fluoride (LiF), D-(+)-glucose, Nafion 117 (~5%), sodium di-hydrogen phosphate monohydrate (NaH2PO4·H2O), di-sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), sodium chloride (NaCl), ascorbic acid, uric acid, and hemoglobin were obtained from Sigma-Aldrich . Whatman No. 1 filter paper was purchased from Whatman International Ltd.  and A4 230-g card paper was purchased in Bangkok, Thailand. In this work, total amounts of 0.2 mM stock solution of glucose were prepared by dissolving D-(+)-glucose in 0.1 M PBS (pH 7.4). The solution of 10 mg/mL glucose oxidase enzyme was prepared by dissolving it in 50 mM sodium acetate (pH 5.1). The deionized water used in this study was obtained in the laboratory by using ELGA LabWater purification system .Glucose oxidase from 2 laser cutting machine (60 Watt.)  was purchased from the MIT group, Thailand. Field emission scanning electron microscope (FESEM) analysis were performed at the National Science and Technology Development Agency, Thailand. HITACHI SU8030 FESEM  were used to study the morphology of the modified electrode.All electrochemical characterization was carried out using a portable PalmSens4  . For the3C2Tx/GOx/Nafion on SPIL-GE were characterized by cyclic voltammetry (CV) with 5 mM ferri/ferrocyanide in 0.1 M KCl solution. The chronoamperometry technique was used for characterization to find the optimal concentration of modified nanomaterial and selection of optimal detection potential conditions for quantitative determination of glucose.The electrochemical properties of PB/TiTo Fabricated of SPIL-GEs on paper based, The commercial ionic liquid graphene ink (IL-GP ink) was composed of a conductive carbon paste, exfoliated graphene powder in poly polystyrene sulfonate (PEDOT/PSS) solution and 3-methyl-1-propylpyridinium bis(trifluoromethyl sulfonyl)imide (PMPlm) as a weight ratio of 100:2.5:1 . In Figu3C2Tx flakes, Ti3C2Tx were synthesized by the modified minimally intensive layer delamination (MILD) method using in situ HF formation from HCl and LiF to prepare both etchant and intercalation in exfoliation process which is less hazardous and provides an easier step than using HF where the flakes are isolated by manual shaking without further intercalation  were dissolved in 10 mL of 0.1 M KCl (pH 1.5) for 30 min. A total 10 mL of 2 mg/mL Ti3C2Tx suspension was slowly added into the mixture solution of FeCl3 and K3[Fe(CN)6]. After continuous stirring for 30 min, the precipitated PB/Ti3C2Tx were filtrated by vacuum assisted filtration and washed with deionized water for 5 times. The resulting PB/Ti3C2Tx nanocomposites were obtained after being dried at 60 °C for 12 h.For PB/Ti studies . The PB 3C2Tx nanocomposites on the electrode surface, 3 mg/mL PB/Ti3C2Tx was dispersed in deionized water and the suspended solution was sonicated for 90 min to acquire a homogeneous mixture. In 3C2Tx mixture was then dropped cast on paper-based SPIL-GE and dried at 60 °C for 30 min. After the electrode was cooled for 10 min, 6 μL of 10 mg/mL GOx in sodium acetate (pH 5.1) was dropped and dried at 25 °C for 2 h. Subsequently, 2 μL of 0.5% Nafion was dropped at room temperature for 1 h. Finally, the designed Whatman No.1 was cut by using CO2 laser cutting machine and applied on the working electrode area with transparent adhesive tape to assist the delivery of sample by capillary force, as showed in To fabricate the devices, paper-based SPIL-GEs were electrochemically cleaned to remove contaminants on electrode surfaces . BrieflyThe paper-based SPIL-GE device was used to determine blood glucose level by using human blood plasma (n = 10) and compared with enzymatic glucose assay (Hexokinase) using automated analyzer ARCHITECT ci4100 . The concentration of ascorbic acid (0.1 mM), uric acid (0.1 mM), and hemoglobin (0.06 mM) in PBS were used to study the effects of the main interferences in human blood plasma.3C2Tx/GOx/Nafion/SPIL-GE was used to measure blood glucose concentration. In this work, separated blood plasma was carefully collected from sodium fluoride (NaF) containing tube after drawing the whole blood sample from volunteers and centrifuged at 3000 rpm for 10 min. The human blood plasma was then determined to have glucose concentration in plasma using paper-based PB/Ti3C2Tx/GOx/Nafion/SPIL-GE that was connected to Sensit Smart. In the process of real sample assay, the sample matrix is one of the crucial factors that effects the obtained signal because it contains lots of interference, including ascorbic acid, and uric acid. To minimize the sample matrix effects, we diluted the plasma sample with PBS at a ratio of 1 to 3. Furthermore, the Nafion was utilized to trap the negatively charged interference species that may affect the real sample of plasma glucose measurements.To verify the electrochemical method for real sample analysis, the prepared paper-based PB/Ti6]3−/4− in 0.1 M KCl by cyclic voltammetry (CV) at a potential range between −0.5 to +1.0 V/s with a scan rate of 50 mV/s. As shown in 3C2Tx nanocomposites on SPIL-GE was distinctly enhanced due to the excellent electroactivity of PB nanoparticles and the multilayer of Ti3C2Tx sheets. Furthermore, the redox peak currents of PB/Ti3C2Tx/GOx/Nafion/SPIL-GEs were also studied. Immobilized GOx and Nafion on PB/Ti3C2Tx showed a significant decrease in the redox peak currents, which may be affected by the fact that GOx is a protein and Nafion also has an insulation property that may block the electron transfer between the redox probe and electrode surfaces 3−/4− in 0.1 M KCl solution showed a pair of redox peaks which were the oxidation and reduction peak currents. To investigate the electrochemical behaviors, 2) of 0.9743 and 0.9948, respectively. Indicating the diffusion control behavior.The electrochemical behavior of modified electrodes in this work was investigated in the presence of a 5 mM redox probe [Fe(CN)surfaces ,31. To cIn addition, A plot of logarithm of redox peak currents vs. logarithm of scan rate were investigated. 3C2Tx and PB/Ti3C2Tx. The micrographs of Ti3C2Tx and PB/Ti3C2Tx were recorded under the accelerating voltage of 5.0 kV and magnifications of 50,000× as showed in FESEM was employed to investigate the morphological analysis of Ti3C2Tx and PB/Ti3C2Tx nanocomposites. 3C2Tx after delamination and further confirmed the elemental analysis of Fe and N elements in PB/Ti3C2Tx nanocomposites as showed in The elemental mapping analysis was carried out using EDS analysis to study elemental distribution in Ti3C2Tx on paper-based SPIL-GE, varying concentrations of PB/Ti3C2Tx nanocomposites were prepared as mentioned above. . As shown in 3C2Tx on SPIL-GE the current response was increases until the concentration reaches to 4 mg/mL. However, the background and standard deviation (error bar) at a concentration of 4 mg/mL PB/Ti3C2Tx were also increased. While the concentration of 3 mg/mL PB/Ti3C2Tx gives the highest signal/background ratio as showed in 3C2Tx for modified SPIL-GE due to the lower in standard deviation and the highest current of signal/background ratio.To optimize the modified PB/Tid above.  To selec3C2Tx on SPIL-GE with chronoamperometry, the detection potential for determining glucose concentration was also optimized. To select the optimal detection potential for acquiring the lowest detection limit, the highest current response with the lowest background current was considered. Therefore, the final concentration of 0.5 mM glucose in 0.1 M PBS pH 7.4 was added and measured at fixed different potentials of 0.00, −0.05, −0.10, −0.15, and −0.20 V (vs Ag/AgCl) compared with 0.1 M PBS pH 7.4 as the background current response. In After optimizing the concentration of PB/Ti2 = 0.9937 (LOD = 24.5 μM) and 0.9878 (LOD = 62.1 μM), respectively. These results indicate that, the paper-based PB/Ti3C2Tx/Gox/Nafion/SPIL-GE showed an excellent electrochemical response for glucose determination.To determine the analytical performance of the paper-based SPIL-GE biosensor, the different concentrations of glucose were prepared and measured by using chronoamperometry with an applied detection potential of −0.05 V a and the3C2Tx/Gox/Nafion/SPIL-GE and automated analyzer (Hexokinase method) were compared. The result demonstrated that no bias for measurement of glucose in any samples was obtained, since the difference between the two methods fell within the ±1.96 standard deviation (SD) as shown in In this study, blood samples with spiked glucose at different concentrations in the range of 4.28–12.61 mM (n = 10) were analyzed using diluted procedure as mentioned above. The results were compared with the reference hexokinase/glucose-6-phosphate dehydrogenase (G6PD) method using automated analyzer (Architect ci4100). As shown in 0.05, 9) was calculated to be ±2.262. Paired sample statistical analysis revealed that the experimental value of |t| was 1.022, a significantly lower than the critical t-value so the null hypothesis assumed no differences in means between the results of the two methods. This implies that our proposed electrochemical paper-based device can be used in the application of glucose analysis with the portable electrochemical device.A scatter plot of the results obtained from both methods was constructed in which a correlation coefficient of 0.9846 b was obt3C2Tx nanocomposites for easy electrode surface modification based on the process of reacting reeducation between Ti3C2Tx and Prussian Blue precursors [In addition, this research is the first to use screen-printed ionic liquid electrode on a paper substrate. The development is further studies of previous work on disposable screen-printed ionic liquid/graphene on polyethylene (PET) substrates . A proceecursors , which iecursors ,33,34. TTo investigate the selectivity of the proposed electrode toward glucose detection, the potential interferences in blood plasma including uric acid, ascorbic acid, and hemoglobin at their significant interfering concentration were added into 5 mM of glucose. The amperometric measurements were carried out and the % recoveries were reported as shown in 3C2Tx/GOx and Nafion was successfully fabricated for glucose sensing in real plasma samples. The use of ionic liquid/graphene ink to create an electrode on a paper substrate was described for the first time. With the simple modification process, the PB/Ti3C2Tx/GOx/Nafion SPIL-GE can be applied to manufacture scale in the future. the advantageous properties of MXene such as good in conductivity and catalytic activity synergies with the outstanding properties of PB which can be measured glucose at low potential. This strategy has been applied in glucose determination to avoid the disturbed signal from interference species at high potential and enhance sensitivity. This promising platform can potentially be used for in-field analysis and applied to monitor other reactions that produce H2O2 as a by-product.A novel paper-based screen-printed ionic liquid/graphene modified with PB/Ti


The levels of 25(OH)D3 were significantly low in T2DM patients compared to the control. T2DM patients with hypovitaminosis D displayed significantly increased FBG, insulin, and HOMA-IR compared to normovitaminosis. Their lifetime and 10-year ASCVD risk scores were significantly higher regardless of vitamin D deficiency levels  in comparison to patients with sufficient levels of vitamin D. Among patients, the lifetime and 10 years of ASCVD risk showed a significant negative correlation with serum 25(OH)D3 and HDLc  (P=0.0001), respectively, and significant positive correlation with T2DM duration, serum insulin, and HOMA-IR   , respectively. The 10-year ASCVD risk exhibited a significant positive correlation with FBG (P=0.003) and HbA1c (P=0.009). T2DM duration was a predictor of vitamin D deficiency among T2DM patients . There is a considerable association between lifetime and 10 years of ASCVD risk with hypovitaminosis D in T2DM, regardless of the deficiency levels which could be predicted by the diabetes duration.We evaluated the prevalence and association of Vitamin D deficiency with glycemic control and CVD risk in T2DM patients. Serum 25 (OH)D Furthermore, hydroxylation occurs in the kidney and to less extent in monocytes, placenta, and macrophages, by the enzyme (CYP27B1)25(OH)D31α-hydroxylase yielding 1,25-hydroxyvitamin D  [The deficiency of vitamin D has been identified lately as a major health concern . As a mu2D3 may increase insulin sensitivity, thus resulting in the cellular responsiveness for transportation of glucose [There is growing evidence linking pathognomonic and molecular mechanisms of deficient vitamin D status with the development of T2DM, and it has been postulated that 1,25 dihydroxycholecalciferol or 1,25(OH) glucose . Alpha,  glucose . In addi glucose .Vitamin D deficiency has been linked to several pathological events besides T2DM, such as obesity, dyslipidemia, endothelium dysfunctions, and hypertension . TherefoSaudi Arabia has been ranked as the seventh worldwide and as the second middle east country for the incidence of T2DM . Since r2). The BMI categories for normal, overweight, and obesity were 18–24.9, 25–29.9, and ≥30, respectively [This study enrolled 93 T2DM patients, who were recruited from the Diabetes and Endocrinology clinic, at Al Noor hospital in Makkah according to the following criteria aged > 30 years, not pregnant or lactating, under dialysis or affected by other types of diabetes, chronic disease, or cancer. The control group comprised 69 healthy individuals who were matched for age and gender to the T2DM group. All subjects were exposed to detailed history-taking, especially focused on the duration of diabetes, smoking, medical history, and full clinical examination. The diagnosis of hypertension in the studied group was confirmed if the measurement of at least the systolic blood pressure (SBP) was 140 mmHg or more and/or the diastolic blood pressure (DBP) was 90 mmHg or more on two successive and separate occasions or if the patient was on antihypertensive treatment [ectively .From each subject, two blood samples were collected, a morning fasting sample and another 2-h postprandial sample. A 7 ml of venous blood was withdrawn under complete aseptic condition. Then, the whole blood was placed into two sterile vacutainer tubes, one plain tube, and one EDTA tube. The EDTA tube was used for the assessment of whole blood HbA1c. The plain tube was centrifuged at 1500 rpm for 15 min; the resulting serum was stored at −20°C till further assessment of the studied parameters.The stored sample was utilized for assessing the following parameters using the standardized enzymatic colorimetric kits for fasting blood glucose (FBG) and 2-h postprandial glucose (2H-PPG) by the glucose oxidase method , triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDLc) and high-density lipoprotein cholesterol (HDLc) , urea, and creatinine , and C-reactive protein was assessed using standardized and automated assays.The glycemic control for each subject was evaluated by assessment of HbA1c in the whole blood sample via full automated high-performance liquid chromatography (HPLC) supplied by TecoDiagnostics, Lakeview Ave, Anaheim, CA, USA .Serum insulin levels were determined using the enzyme-linked immunosorbent assay (ELISA) , insulin resistance was assessed by homeostasis model assessment of insulin resistance (HOMA-IR) = fasting serum glucose (mg/dl) × serum insulin (uIU/mL)/405 .The levels of 25(OH)D3 were measured by the electrochemiluminescence assay on a Cobas immunoassay analyzer . Levels of 25-hydroxyvitamin D3 were stratified according to the classification of the Endocrine Society Clinical Practice Guidelines in 2011, the deficiency cut-off level was below 20 ng/mL, the insufficiency range was 20–29 ng/mL, and the adequate cut-off level was 30 ng/mL [For assessment of the cardiovascular disease risk in the studied groups, we used both the lifetime atherosclerotic cardiovascular disease risk (lifetime ASCVD) and the 10-year ASCVD risk assessment tools. For the calculation of the ASCVD score, the following data were included; demographic variables were included such as age and gender, besides the clinical and biochemical laboratory variants such as SBP, TC, HDLc, and history of diabetes, hypertension, or smoking. The calculated values for both risk scores were expressed as percentages. The optimal risk for lifetime ASCVD was estimated at cut off 5% for men and 8% for women, while the optimal risk for 10-year ASCVD was 1.2%, and patients with higher scores than 1.2% were at high risk for CVD development, whereas those with scores less than 1.2% were at low risk .N = 162), comparing the diabetic group to controls. The effect size (ES) for 25(OH)D3 in this study was 0.59, considered to be moderate using Cohen's (1988) criteria, with an alpha = 0.05 and sample size = 93 in the T2DM group and 69 in controls, a posthoc power analysis was conducted with these effect sizes (G Power 3.1), and it was approximately (1−β) = 0.962 [A statistical power analysis was performed after sample size estimation, based on data from the current study  and sex (P=0.42). Concerning anthropometric measures of all participants, the BMI was significantly higher in T2DM patients (32.57 ± 5.16 kg/m2) as compared to controls (29.28 ± 2.09 kg/m2) (P=0.001); 74.2% of T2DM patients were obese, 24.7% were overweight, and 1% was normal weight vs. 47.8%, 43.5%, and 8.7% in the control group (P=0.001), respectively. The median of the duration of T2DM diagnosis was 4 years. As expected, the FBG, 2H-PPG, HbA1C, insulin, HOMA-IR, ESR, CRP levels, and blood pressure were significantly higher among patients with T2DM in comparison to the control subjects (P=0.0001). 72% and 42% of the T2DM patients showed HbA1c > 7.5% (poor glycemic control) and were hypertensive (not shown). A significant increase in TC, LDLc, TG, and decreased HDLc levels were noted in T2DM patients compared to the control group (P=0.0001), (P=0.01), (P=0.008), and (P=0.0001), respectively. Consequently, LDLc/HDLc was significantly higher in T2DM patients compared to the control group (P=0.0001).3 level was significantly low in T2DM patients as compared to the control group (18 Vs. 21) (P=0.0001). The overall prevalence of 25(OH)D3 status among patients was 51.6%, 26,9%, and 21.5 for deficiency, insufficiency, and sufficiency versus 40.6%, 29%, and 30.4% for control subjects.The median of 25(OH)DP=0.02) and obese diabetics (79.5%) (P=0.035). The management plan revealed a nonsignificant difference (P=0.4) between T2DM regarding 25(OH)D3 status P=0.0001), serum insulin (P=0.01), and HOMA-IR (P=0.001) compared to those with sufficient D levels but not for the HbA1c level (0.3). Furthermore, TG and LDLc levels were significantly higher in T2DM patients with hypovitaminosis 25(OH)D3 as compared to those with sufficient 25(OH)D3 levels (P=0.045) and (P=0.04), respectively.The demographic, clinical, and biochemical data among T2DM patients with vitamin D hypovitaminosis versus those with sufficient levels were shown in 3 levels. For the CVD risk score, T2DM patients with hypovitaminosis revealed significantly increased scores of lifetime and 10-year ASCVD risk when compared to those with sufficient 25(OH)D3 level (P=0.006) and (P=0.023), respectively. Furthermore, T2DM patients with 25(OH)D3 insufficiency and deficiency revealed significantly increased scores for lifetime ASCVD risk when compared to those with 25(OH)D3 sufficiency , respectively. Moreover, T2DM patients with 25(OH)D3 deficiency exhibited a significant increase of 10 years of ASCVD risk as compared to those with a sufficient level (P=0.012) The frequency of 10 years of ASCVD risk among T2DM patients with hypovitaminosis was 54.8% vs. 5% in those with normal 25(OH)D3 ;  and HDLc ;  respectively and significant positive correlation with body weight ; , T2DM duration ; , serum insulin , , HOMA-IR , , TC , , and LDLc , . In addition, 10-year ASCVD risk exhibited a significant positive correlation with FBG , 2H-PPG , and HbA1c  Among T2DM patients, the lifetime and 10 years of ASCVD risk showed significant negative correlation with serum 25(OH) D3 deficiency among patients (P=0.03) Linear regression analysis showed that T2DM duration was a predictor of 25(OH)D3 was significantly low in T2DM patients in comparison to the control group. Several studies in various ethnic groups were in agreement with our findings [3 levels were significantly decreased in T2DM but not in T1DM patients when compared with healthy controls [In the current study, the median level of 25(OH)Dfindings , 21. Piecontrols . Vitamincontrols .3 status was assessed via measurement of serum 25(OH)D3 levels for all studied groups as its clearance rate is slower than 1,25(OH)2D, so the former is a preferable biomarker for vitamin D status, while the latter is clinically useful for follow-up and monitoring of certain genetic or acquired conditions linked to calcium, phosphate, or vitamin D [3 status among T2DM patients was 51.6%, 26,9%, and 21.5% for deficiency, insufficiency, and sufficiency versus 40.6%, 29%, and 30.4% for control subjects, 25(OH)D3 hypovitaminosis was higher among the T2DM group (78.5%) than control subjects (69.6%); however, it did not reach a significant level. Most T2DM patients with hypovitaminosis (86%) were among the age group of (45–55 years). In agreement with our findings, a previous study in North India reported a prevalence of 81% vitamin D deficiency among T2DM patients [2D3 when the chemiluminescence immunoassay was used [In the current study, 25(OH)Ditamin D . Our respatients . In anotwas used . Evermor3 displayed significantly higher levels of FBG, serum insulin, HOMA-IR, TG, and LDLc compared to those with sufficient 25(OH)D3 levels. HbA1c levels were higher as well; however, they did not reach a significant value. In agreement with these findings, several studies reported that the levels of 25(OH)D3 were inversely correlated with impairment of glucose homeostasis and insulin resistance [3 levels were found to be related to glycemic control in T2DM [3 levels as compared to those with good glycemic control [3 with T2DM or suggest that deficiency of vitamin D might be the result of diabetes, not the cause. Thus, being a cause or consequence, hypovitaminosis vitamin D is strictly associated with poor glycemic control [In this study, T2DM patients with hypovitaminosis 25(OH)Dsistance , HOMA-IRsistance , whereas in T2DM . In a pr control . An inve control . These f control .In fact, vitamin D affects pancreatic beta cell function and insulin secretion and resistance directly and indirectly via genomic and nongenomic cellular events. Directly, vitamin D induces the release of insulin from beta cells of the pancreas via binding to its specific vitamin D receptors (VDR) as its responsive elements have been identified at the promotor region of the insulin receptor gene. Indirectly, vitamin D contributes to insulin release from beta cells through regulation of calcium flux as well as extracellular calcium to ensure an adequate calcium pool , and suc3 deficiency levels (<20 ng/ml) revealed significantly increased both lifetime and 10-year ASCVD risk scores when compared to those with a sufficient 25(OH)D3 level (≥30 ng/ml); moreover, T2DM patients with 25(OH)D3 insufficiency (20–29 ng/ml) showed only a significantly increased lifetime ASCVD risk score in comparison to those with sufficient 25(OH)D3 levels. The studied cohort with hypovitaminosis 25(OH)D3 also displayed significantly increased serum insulin, LDLc, and HOMA-IR with poor glycemic control indices as FBG compared to T2DM patients with adequate 25(OH)D3 levels regardless of the management modalities whether oral therapy, insulin, or combined. Considering these findings altogether, they might point toward the possible atherogenic role of 25(OH)D3 hypovitaminosis in the development of CVD in poorly controlled T2DM patients. In line with our finding, Alaidarous and his colleagues found that hypovitaminosis of vitamin D is significantly associated with CVD risk in T2DM patients especially in uncontrolled cases regardless of the duration of the disease; however, they used different assessment units of vitamin D deficiency as a cut off (<50 nmol/l) which is equivalent to the cut off we used in our study (<20 ng/ml) [In the current study, T2DM patients with 25(OH)D0 ng/ml) .Recently, the rise of serum levels of vitamin D is found to be associated with improvement of glucose tolerance and better control of diabetes; each rise of 1 ng/ml of vitamin D levels is associated with the decline of HbA1c percentage by 0.09%, signifying the key value of the rise of vitamin D in declining the risk of CVD complications in T2DM patients .3 and HDLc, while both risk scores exhibited a direct significant correlation with weight, duration of T2DM, fasting insulin level, HOMA-IR, LDLc, and TC. Linear regression analysis showed that T2DM duration was a significant predictor of vitamin D deficiency in T2DM patients. Along the same line as our findings, Alaidarous and his colleagues reported a converse correlation between vitamin D levels with lifetime ASCVD risk scores in uncontrolled patients with long duration of diabetes but a positive association with newly-diagnosed uncontrolled T2DM patients [Among the T2DM patients, the lifetime and 10 years of ASCVD risk showed a significant inverse correlation with serum 25(OH)Dpatients . In contpatients , 39.Numerous studies and metanalysis have recently pointed to the complex connection of hypovitaminosis D with obesity, diabetes, hypertension, and endothelium dysfunction which explains the possible impact of low levels of vitamin D as a risk factor for CVD in T2DM patients , 40–43; 3 deficiency was high in Saudi patients with T2DM mainly among the age group of (45–55 y), predicted by the duration of T2DM, regardless of the treatment strategy. In addition, there was a direct association of lifetime and 10 years of ASCVD risk with 25(OH)D3 hypovitaminosis in T2DM. Moreover, there is an inverse relationship between FBG, HOMA-IR, and 25(OH)D3 status which account for the 10 years of ASCVD risk solely. Thus, maintaining adequate vitamin D status and avoiding deficiency may help to prevent CVD complications among patients with T2D. Furthermore, it is valuable to study if vitamin D should be given to only vitamin D deficient patients or all T2DM patients.The prevalence of 25(OH)D3 and CVD, the effect of vitamin D supplementation on CVD, and T2DM patients. Secondly, this study is limited to Asian people, so our findings may not be applicable to other ethnic populations with different nutritional and behavioral aspects. Finally, the observational nature of the data and all these factors may affect the generalizability of this study.The present study had a few limitations. Firstly, a causal relationship cannot be established due to the cross-sectional study design. Further studies are required to evaluate the causative relationships between 25(OH)D3 level was significantly low in T2DM patients as compared to the control group, and there is a considerable association between lifetime and 10-year ASCVD risk with 25(OH)D3 hypovitaminosis in T2DM, regardless of the deficiency levels which could be predicted by the diabetes duration, revealing the importance of early detection of vitamin D deficiency and supplementation.25(OH)D