Objective. To investigate the relationship between serum 25-hydroxyvitamin D3 [25(OH)D3] levels and carotid intima-media thickness (IMT) as well as carotid atherosclerotic plaque in patients with type 2 diabetes mellitus (T2DM). Methods. 314 patients with T2DM were enrolled in this study. The clinical data and laboratory examinations of subjects were recorded, such as serum 25(OH)D3, hemoglobin A1c (HbA1c), serum lipids, fasting blood glucose (FBG), and other biochemical parameters. Color Doppler ultrasound was used to measure carotid IMT and carotid atherosclerotic plaques. Patients were divided into four quartile groups according to the serum 25(OH)D3 levels from low to high: group Q1~group Q4. Results. From group Q1 to group Q4, carotid IMT and the incidence of plaque were gradually reduced. Serum 25(OH)D3 levels were lower in the plaque group compared with the nonplaque group (P < 0.01). Serum 25(OH)D3 levels were negatively correlated with the carotid IMT . Multiple linear stepwise regression analysis showed that serum 25(OH)D3 was independently associated with carotid IMT . Logistic regression analysis showed that serum 25(OH)D3 levels were independently associated with the presence of carotid plaque in T2DM . Conclusions. Low vitamin D status may contribute to the incidence of carotid atherosclerosis in type 2 diabetic patients. Vitamin D plays an important role in maintaining the integrity of bone. It contributes to the growth and development of bone in children and maintains the bone health in adults. Furthermore, vitamin D helps to prevent osteoporosis and fracture in the elderly . Beyond 314 patients  with T2DM who were hospitalized in the Department of Endocrinology of the First Affiliated Hospital of Zhengzhou University from September 2015 to February 2016 were enrolled. The diagnosis of the T2DM corresponds to the diagnostic criteria made by the Word Health Organization (WHO) in 1999. The exclusion criteria are as follows: patients with acute diabetic complications such as acute infection, ketoacidosis, and hyperosmolar coma; serious liver and kidney dysfunction; thyroid, parathyroid, and other endocrine gland-related diseases; autoimmune diseases; osteoporosis and other bone metabolic diseases; tumor; pregnant women; mental disease; recent surgery; taking any medications known to affect vitamin D metabolism; and patients with a recent history of excess ultraviolet (UV) ray exposure.This study complied with the principles of the Declaration of Helsinki and was approved by the local ethics committee. Written informed consent was obtained from all subjects.General clinical data including sex, age, diabetes duration, smoking history, hypertension history, family history of diabetes, height, weight, systolic blood pressure (SBP), and diastolic blood pressure (DBP) was recorded for all patients. Body mass index (BMI) was calculated as weight in kilograms divided by height per square meter.Blood samples were obtained by venipuncture after an overnight fast. Serum calcium, uric acid, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and other conventional biochemical indicators were measured on an automatic analyzer . FBG was measured by glucose oxidase method. HbA1c was measured by an automated high-performance liquid chromatography analyzer. 25(OH)D3, parathyroid hormone (PTH), and osteocalcin (OSTE) were measured using an electrochemiluminescence immunoassay . The normal reference value of 25(OH)D3 levels in our laboratory was >18.0 ng/mL.α color Doppler diagnostic instrument with a 10–13 MHz probe was used for carotid ultrasonography by the experienced radiologists who were blind to the clinical characteristics of the patients, with subjects in the supine position. The IMT carotid was measured at three different sites on the left and right sides, and then the average of the six measurements were calculated. The carotid atherosclerotic plaque was examined as well.The Aloka-t-test was used to compare two arrays of data. Differences among multiple groups were compared with one-way ANOVA (LSD-t-test). Comparisons of categorical variables between/among the groups were performed with the chi-square test. Correlation analyses between two variables were performed with Pearson's correlation or Spearman's correlation. The risk factors of carotid IMT and carotid plaque were performed with multivariate linear regression analysis and logistic regression analysis. P values less than 0.05 were considered significant.SPSS 21.0 statistic software (SPSS for Windows 21.0) was used to analyse all the data. Continuous variables were presented as mean ± standard deviations The range of 25(OH)D3 levels in all the subjects was (3.0–45.1) ng/mL . There were 233 patients with 25(OH)D3 levels below 18 ng/mL and 81 patients with 25(OH)D3 levels above 18 ng/mL. All the subjects were divided into four quartile groups according to the serum 25(OH)D3 levels: group Q1; group Q2 ; group Q3 ; and group Q4 . As illustrated in P < 0.01). The incidence of carotid plaque in group Q3 , group Q2 , and group Q1  was higher than that in group Q4  (P < 0.05) (Carotid IMT of patients in group Q1 [(1.23 ± 0.40) mm] and group Q2 [(1.19 ± 0.31) mm] was significantly higher than that in group Q3 [(1.06 ± 0.22) mm] and group Q4 [(0.93 ± 0.17) mm], the carotid IMT in group Q3 was significantly higher than that in group Q4 ( < 0.05) .P < 0.05). The number of patients with hypertension history in the plaque group was more than that in the nonplaque group (P < 0.05). However, the levels of serum 25(OH)D3 were significantly lower in the plaque group  compared with the nonplaque group  (P < 0.01). Other parameters were not statistically different between the two groups . Spearman correlation analyses showed that the carotid artery IMT was positively correlated with smoking history and hypertension history (P < 0.05) D3 and calcium ( < 0.05) .Y = 1.12 + 0.01X1 + 0.06X2 − 0.01X3 − 0.39X4. It showed that carotid IMT decreased dose dependently with increasing concentrations of 25(OH)D3. Standardized regression coefficients showed that age was the most important influence factor of carotid IMT, followed by HbA1c, 25(OH)D3, and serum calcium , HbA1c (X2), 25(OH)D3 (X3), and serum calcium (X4) were independently associated with carotid IMT. The regression equation was  calcium .P = 0.004) D3 was independently associated with the incidence of carotid plaques in patients with T2DM  .2D3), the tightly regulated activated molecule of vitamin D3, which can exert biological effect by combining with vitamin D receptor. However, 1,25(OH)2D3 has a very short half-life, so it is not generally used as a clinical biomarker to assess vitamin D status. 25(OH)D3, as the circulating storage form of vitamin D3, has a long-serum half-life, and it is easily to be detected. Accordingly, the quantitation of serum 25(OH)D3 provides a clinically useful assessment of vitamin D status [Vitamin D3, as an essential fat-soluble vitamin, largely obtained from cutaneous exposure to ultraviolet radiation and to a lesser extent from dietary and supplements, is metabolized by the liver and then by the kidney to become 1,25-dihydroxyvitamin D3 91,25(OH)D status . In receDiabetic macrovascular complications are the major causes of mortality and disability among the patients with T2DM, of which the pathological basis is atherosclerosis, and its early change is the increasing artery intima-media thickness, especially the carotid IMT. A large number of studies have demonstrated that carotid IMT can be used as a suitable surrogate marker of macroangiopathy . At presOur study showed that the carotid IMT levels in the lower 25(OH)D3 groups were greater than those in the higher 25(OH)D3 groups. The differences were still statistically significant after adjustment for sex, age, BMI, serum lipids, HbA1c, diabetes duration, blood pressure, smoking history, and other variables. Pearson's correlation analysis showed that the carotid artery IMT was negatively correlated with the levels of serum 25(OH)D3. Multiple linear regression analysis showed that 25(OH)D3 was independently associated with carotid IMT, after adjusting for age, diabetes duration, systolic blood pressure, HbA1c, LDL-C, and serum calcium. Logistic regression analysis showed that low serum 25(OH)D3 was the risk factor of carotid plaque. The results as described above suggested that with the decreasing of serum 25(OH)D3 levels, carotid IMT and carotid plaque showed a trend of increasing.In addition, the correlation analyses showed that carotid IMT correlated positively to age, diabetes duration, SBP, HbA1c, LDL-C, smoking history, and hypertension history. In regression analysis, age, diabetes duration, hypertension history, HbA1c, and LDL-C were also the risk factors of carotid plaque. And the influence of these factors on carotid IMT and atherosclerosis have been confirmed in a number of previous studies. As shown in our study, patients with lower serum 25(OH)D3 levels had a longer diabetes duration, higher SBP, HbA1c, LDL-C, and lower HDL-C than those with higher 25(OH)D3 levels. These results further demonstrated that it is helpful to prevent atherosclerosis by controlling blood glucose, blood pressure, and blood lipids.In our study, serum calcium was independently correlated with carotid IMT. Currently, there is no final conclusion of the relationship between serum calcium status and carotid IMT among type 2 diabetic individuals. A study in north Manhattan showed that subjects with no carotid plaque had lower serum calcium levels within the normal range than those with carotid plaque . Howeverβ cells and/or modulating the balance of calcium homeostasis between intracellular and extracellular. Moreover, vitamin D deficiency is associated with inflammation which has been established as a key pathogenic mechanism in atherosclerosis. (3) Vitamin D can decrease the activity of renin angiotensin aldosterone system (RAAS), which subsequently reduces blood pressure and decreases the risk of atherosclerosis. (4) It is well known that atherosclerosis is associated with dyslipidemia. LDL-C is a recognized risk factor for atherosclerosis, but HDL-C has an antagonistic effect on atherosclerosis. Several studies have suggested that dyslipidemia is associated with vitamin D deficiency. Patients with vitamin D deficiency had higher LDL-C and lower HDL-C levels [The following four aspects might be important for the effects of vitamin D on atherosclerosis: (1) Vitamin D can increase the expression of Ca-ATRase in vascular endothelial cells and smooth muscle cells, elevate cytosolic free calcium concentrations, induce contractile proteins expression, and then promote production of prostacyclin (PGI2), which affects the vascular tone; Vitamin D can improve endothelial function by inhibiting the inflammatory reaction and oxidative injury , 18, andC levels .In conclusion, serum 25(OH)D3 was negatively correlated with carotid IMT and the incidence of carotid plaques in patients with T2DM. When serum 25(OH)D3 concentration is below a certain level, the incidence of atherosclerosis in type 2 diabetic patients may increase. Vitamin D deficiency may predict subclinical atherosclerosis in diabetic patients, and vitamin D supplementation might be a clinical intervention to prevent atherosclerosis in patients with T2DM. Remarkably, our study is a cross-sectional one. It is necessary to carry out further follow-up and researches.


Anaplastic thyroid carcinoma (ATC) represents one of the most aggressive carcinomas with no consistent survival benefit when treated with conventional radiochemotherapy. Approaches targeting “oncogene addiction” of ATC are increasingly explored and first promising results have been reported in single case studies.To determine the prevalence of mutations in known thyroid oncogenes and signalling pathways amendable to targeted therapy in a large cohort of ATC.BRAF 11.0%, HRAS 4.2%, KRAS 7.6%, NRAS 7.6%, PI3KCA 11.8%) were altered in 33%. Targetable receptor tyrosine kinases were mutated in 11%. The most frequently altered genes were TERT in 86/118 (73%) and p53 in 65/118 (55%) cases. No mutations were found analysing ALK, KIT, MET and mTOR.In 118 ATC  a total of 165 mutations were found. Genes involved in the MAPK/ERK and PI3K pathway  was performed in FFPE samples from 118 ATC using MiSeq (Illumina) and CLC Cancer Research Workbench  for mutation analysis in: To our knowledge this is the largest study analysing mutations for targeted therapy of ATC. We found that 33% of ATC harbour mutations in pathways amendable to targeted therapy. Molecular screening in ATC is suggested for targeted therapies since current conventional treatment for ATC proved mainly futile. Anaplastic thyroid carcinoma (ATC) represents one of the most aggressive human malignancies . TreatmePI3KCA in ATC [BRAF mutations, have been identified in around 60% of papillary thyroid carcinoma (PTC) [Recently targeted therapies have been increasingly explored in ATC, based on the concept that molecular drivers of ATC may represent promising targets to be efficiently tackled. Genetic alterations in ATC enfold genes involved in the PI3K and MAPK/ERK pathway, receptor tyrosine kinases and tumour suppressor genes –5. ActivA in ATC . Gene alma (PTC)  and 15–4ma (PTC) , 9.p53 [p53 cause protein inactivation leading to inactivation of apoptosis and cell cycle progression [TERT) mutations, the C228T and the C250T alterations, conferring increased telomerase activity [et al. showed that coexisting BRAF V600E and TERT C228T alterations in papillary thyroid carcinoma (PTC) are associated with a significantly higher risk of recurrence and poorer outcome as well as older age and distant metastasis in ATC [The most frequently mutated gene in ATC is p53 . Mutatiogression . Alteratgression . Knowledactivity . These mactivity . Furthers in ATC , 13.BRAF [mTOR [ALK [Since the first identification of driver mutations in ATC, several case reports have shown response to targeted therapy in ATC patients. These included the application of inhibitors to BRAF –16, mTORAF [mTOR  and ALK TOR [ALK . In thesTOR [ALK , 20. In TOR [ALK . In a phTOR [ALK .Over the past 2 years, several groups have applied high throughput sequencing either by next generation sequencing (NGS) or whole genome sequencing to unravel the molecular signature of ATC. So far however, these studies were performed on ATC series comprising no more than 33 samples . Hence, In this study the paraffin embedded tumour tissue of 118 ATC patients  with a median age at diagnosis of 65 years (ranging from 26 to 90 years) were analysed for mutations in hotspots of classical oncogenes. Patients characteristics are listed in Table TERT promoter hotspots C250T and C228T were the most frequent alterations found in 86/118 (73%) tumour specimens, followed by p53 alterations in 65/118 (55%), RAS  in 23/118 (19.5%), CDKN2A mutations in 20/118 (16.9%), PI3KCA in 14/118 (11.8%), BRAF in 13/118 (11%) and RET in 9/118 (7.6%) of ATC. EGFR, ERBB2, PDGFRA, RB1 and TSC2 were mutated in less than 2% of all ATC. In ALK, KIT and MET no mutations were found in the investigated exons. Identified mutations are listed in Mutation in the BRAF, PI3KCA and RAS were detected in 40/118 (33%) ATCs. Genetic variations in the BRAF gene were detected in 13 of 118 (11%) ATCs. In 9 ATCs the classical V600E mutation was present, while the G469A variation was detected in 2 tumours, and the G469V and the D594A mutation were found in 1 ATC each.Mutations in classical thyroid oncogenes e.g. PI3KCA were detected in 14/118 (11.8%) ATCs and comprised the E545K variation in 10 tumours, the H1047R variation in 3 tumours and the N1044L variation in 1 tumour.Genetic alterations in HRAS, KRAS and NRAS genes were found in 23/118 (19.5%) of all ATCs. In HRAS the Q61R mutation was detected in 4/118 (3.4%) and the G60S mutation in 1/118 (0.9%) tumours. In KRAS 3 distinct mutations were identified: G12R in 7 (5.9%), H27L and G12V each in 1 (0.9%) ATC sample. In NRAS, the variation Q61R was found in 7 and the G61K mutation was detected in 2 ATCs.Genetic alterations in the BRAF , 3 PI3KCA (two E545K and H1047R) and 1 NRAS (Q61K) variation. In 11 ATCs concurrent mutations in BRAF, PI3KCA and RAS were found: 7 ATCs showed PI3KCA and RAS mutations, 2 BRAF and PI3KCA variations, 1 BRAF and RAS and 1 ATC had mutation in BRAF, PI3KCA and RAS. In the other 21 ATCs concurrent p53 and/or CDKN2A mutations or RET or ERBB2 mutations were detected , PDGFRA  and ERBB2 . Variations in RET were detected in 9/118 (7.6%) ATC: 4 ATC with Y791F, 2 ATC with P596L and 1 ATC each with E818K or E595K.Mutations in the receptor tyrosine kinases TERT promotor (C250T and C228T) were detected in 73% of ATC. The C228T mutation was far more frequent and occurred in 82/118 compared to the C250T mutation in only 3/118 tumours. In 1 ATC sample both mutations were found by Sanger sequencing (Mutations in the quencing .The tumour suppressor gene p53 was mutated in 65/118 (55%) tumour samples and in total 77 mutations occurred: R248Q in 7 ATCs; T125A, R209fs, P278S and R280T in 5 ATCs; S183* and R273C in 3 ATCs each; and C135F, Y220C, G266E, V272L, R282W and E285K in 2 ATCs each .CDKN2A the most frequent mutation was the E88G missense mutation, detected in 8/118 (7%) of ATC samples. Further CDKN2A mutations were L65P, D84G and R98Q (in 2 ATCs each); and G83R, P72L and H66R in 1 ATC 1 each  in four ATC, PI3KCA and RET in three ATC each, NRAS in one ATC and p53 in 27 ATC.In 10/118 (8.5%) ATC no mutations were found in the analyzed genes. In 44/118 (37.3%) only one gene was altered: ALK, KIT, MET or mTOR mutations were detected in the sequenced exons of the ATC series.Furthermore no BRAF and PI3KCA mutations were more prevalent than in de novo ATC samples (22.9% versus 6.3% and 22.9% versus 6.3%). As for TERT and p53 variations described as hallmarks in thyroid cancer dedifferentiation, only p53 mutation prevalence was higher in de novo ATC in comparison to ATC derived DTC (64.1% versus 34.3%).In ATC harbouring residues of DTC, Conventional radiochemotherapy in ATC does mostly not prolong survival, while single case reports have shown impressive tumour shrinkage after targeted drugs inhibiting single mutations in ATC , 17, 18.BRAF, RAS and PIK3CA) involved in thyroid tumourigenesis in 33% (40/118) of all ATC. Moreover, in 11% (13/118) of all ATC, mutations in targetable receptor tyrosine kinases (e.g. RET) were detected. In 40% (47/118) of all ATC mutations involved in the MAPK/ERK or PI3K pathways or receptor tyrosine kinase encoding genes were altered.We found genetic alterations in the classical oncogenes  was consistent with good response to PI3K-inhibitors [et al. [et al. [ in 4.5%  of ATC uutations  and NSCLutations . In theshibitors , 29. Howhibitors . The eff [et al.  showing RAS mutations were detected in 23 ATCs: Among them are mutations that have frequently been found in thyroid tumours [HRAS G60S and KRAS H27L found in 1 ATC each, have so far not been linked to thyroid cancer. RAS mutations are known events in benign and malignant thyroid tumours [RAS mutations may occur concurrently with other mutations including BRAF V600E [RAS-mutated PTC the RAF-MEK-ERK cascade has been shown to confer cell proliferation, however with smaller transcriptional output than BRAF mutations [RAF dimerization [RAS mutated ATC this feedback is mostly not observed [RAS mutations susceptible to downstream MEK-Inhibitors. tumours . The HRA tumours , 33. In  tumours , but in utations . This isrization . In RAS et al. [ALK exons 20 and 23 in 18 ATC. In our series no ALK mutations were detected in exons 20–25. In a recent report of our group [ALK mutations were exclusively detected in exons 1, 3, 16, 28 and 29, which were not addressed in the present NGS approach, suggesting that probably the entire ALK gene has to be sequenced to assess for genetic alterations in ATC.Murugan and Xing et al.  were theur group , ALK mutEGFR, RET, PDGFRA and ERBB2 since targeted therapies for these tyrosine kinases are available for human malignancies. The EGFR T790M variation is well studied in lung cancer and is known to confer EGFR-inhibitor resistance [EGFR mutations were shown to be rare events and in clinical practice mutational screening would be helpful only when treatment with an EGFR-inhibitor is planned.We also analysed genetic variations in receptor tyrosine kinases sistance . In our RET mutations [RET mutations are present [RET mutations are generally rare events [RET gene variations were found in 1/22 (4.5%) ATC investigated [RET mutations in 9/118 (7.6%) ATC: in 4 ATC the Y791F variation was detected, which is described in Multiple endocrine neoplasia type 2A (MEN2A) cases, but is interestingly not associated with MTC development [RET variation (1 ATC) have so far not been described in association with thyroid cancer or other human malignancies and their biological relevance is not known.Hereditary medullary thyroid carcinoma (MTC) is characterized by germline utations  and in 4 present , 40, whie events . In a prn 1/22 4.% ATC invelopment . The P59ERBB2 and PDGFRA were rare events (2/118 and 1/118). This is in accordance with previous reports [Genetic variations in  reports , 7 and hp53 mutations are a molecular hallmark of ATC. In agreement within this and recent large-scale analysis of ATC  p53 was frequenty mutated in our cohort with genetic variations in 65 of 118 tumours. Somatic p53 mutations are very rare in DTC and it has been shown in mouse models that p53 inactivation contributes to the progression from PTC to ATC [et al. reported single mutations in the CDKN1B and CDKN2C encoding genes [CDKN2A mutations in 23 ATC (19.5%). Thereby, the most frequent mutation was the E88G variation, detected in 14 ATC, which has not previously been described in ATC. Reports have shown that co-targeting CDK 4/6 and BRAF is efficient in BRAF mutated tumours to overcome BRAF-inhibitor resistance [Genetic variations in tumour suppressor genes are frequent events in cancer. Inactivating C to ATC . Less wesistance .Weaknesses of our study are the lack of clinical data, rendering correlation of the genetic findings with disease course impossible. Furthermore, only hotspot regions of genes were sequenced, hence we cannot exclude that mutational events were present in other exons. The strength of our study is the application of a NGS which is already routinely applied in patient care at the Essen University Hospital's Comprehensive Cancer Centre underlining its applicability in clinical practice. Moreover, our series presents the largest ATC cohort comprehensively analysed for mutations in targetable oncogenes so far and all tissues were classified as ATC by board certified pathologists of the German Thyroid Pathology Reference Centre at the University Hospital of Essen.In summary we show, that mutations in hotspots of targetable oncogenes are present in approximately 33% of ATC. We therefore propose a panel of oncogenes  that should be screened in clinical practice in patients with newly discovered ATC in order to offer patients targeted therapy treatment as part of a highly individualized treatment approach where feasible and if desired by the patient. The clinical care of ATC patients urgently needs to be improved including central pathology review, national and international networks and accessibility to basket studies testing novel targeted and other therapies.We retrospectively analyzed a study cohort of 118 patients with ATC. FFPE tissue from primary tumour was used to perform NGS. All samples were reviewed by two board-certified pathologists and diagnosis was made according to the WHO classification . When DT® 2.0 Fluorometer dsDNA HS assay kit . All samples showed a concentration between 1–100 ng/μl.After deparaffinization of FFPE tissue with deparaffinization solution  and incubation with 20 μl proteinase K solution (20 mg/ml) over night at 56°C, DNA isolation from the primary tumour tissue was performed using QIAamp DNA FFPE tissue kit  according to the manufacturer's instructions. DNA concentrations were determined by Qubit® AMPure® XP Beads , followed by measurement of total DNA amount by Qubit® 2.0 Fluorometer dsDNA HS assay kit. The library preparation was performed with NEBNext Ultra DNA Library Prep Set for Illumina , according to the manufacturer's recommendations by using 24 different indices per run. The pooled library was sequenced on MiSeq  and analyzed by Cancer Research Workbench .A total amount of 45 ng DNA was used to perform multiplex-pcr (four primer pools with 10 ng/primer pool + 10% excess). Multiplex PCR and purification were performed with the GeneRead DNAseq Custom Panel V2, GeneRead DNAseq Panel PCR Kit V2  and AgencourtFor targeted sequencing a customized ATC-panel was designed containing regions of interest. The ATC-panel contains hot-spot regions out of 17 genes. The analyzed exons are listed in Within CLC Cancer Research Workbench demultiplexed paired-end sequencing data was mapped to human genome (version hg19). A local realignment was performed to reach better quality . All reads, which were mapped outside of targeted-regions, got deleted after mapping process. Within target regions information from different databases  was annotated.Performing a second filtering-step all reference-variants and variants found in dbSNP common, 1000 genome project and HapMap were deleted. All variants found in Cosmic, Clinvar or those which weren't annotated in any database were listed. Therefore, an allele-frequency of minimum 5% and coverage of at least 100 mapped-reads were the selection-parameters.For detection of the two known TERT promotor mutations on chromosome 5 position g.1295250C > T and g.1295228C > T (reference genome hg19) Sanger sequencing was performed. The promoter region of TERT was amplified by PCR, followed by direct sequencing. PCR reaction was carried out with oligonucleotides TERT fwd (CCTGCCCCTTCACCTTCCAG) and TERT rev (AGGACGCAGCGCTGCCTGAA) in a total volume of 50 μl with HotStar Taq Mastermix (Qiagen) according to the recommendations of the supplier. Amplification was performed in a Biometra T3000 Thermocycler  with 10 min of initial enzyme activation at 95°C followed by 45 cycles of denaturation at 94°C for 30 sec, oligonucleotide annealing at 62°C for 60 sec and extension at 72°C for 30 sec. Sequences of both, forward and reverse strand were analyzed on an ABI 3500 Genetic Analyser (LifeTechnologies) using BigDye Terminator chemistry v3.1 (LifeTechnologies).


Purinergic signaling, which utilizes nucleotides (particularly ATP) and adenosine as transmitter molecules, plays an essential role in immune system. In the extracellular compartment, ATP predominantly functions as a pro-inflammatory molecule through activation of P2 receptors, whereas adenosine mostly functions as an anti-inflammatory molecule through activation of P1 receptors. Neutrophils are the most abundant immune cells in circulation and have emerged as an important component in orchestrating a complex series of events during inflammation. However, because of the destructive nature of neutrophil-derived inflammatory agents, neutrophil activation is fine-tuned, and purinergic signaling is intimately involved in this process. Indeed, shifting the balance between P2 and P1 signaling is critical for neutrophils to appropriately exert their immunologic activity. Here, we review the role of purinergic signaling in regulating neutrophil function, and discuss the potential of targeting purinergic signaling for the treatment of neutrophil-associated infectious and inflammatory diseases. Purinergic signaling is among the most primitive signal transduction systems in evolutionary history . In humavice versa may have important consequences on the immune response outcome . How. How3). Intracellularly, ADO is generated from hydrolysis of AMP by 5-nucleotidase or by hydrolysis of S-adenosyl-homocysteine (SAH) by SAH hydrolase . The intIn the extracellular milieu, ATP and ADP levels are tightly controlled by plasma membrane ectonucleotidases, such as nucleoside triphosphate diphosphohydrolase 1  and ecto-5′-nucleotidase , which convert ATP/ADP to AMP and subsequently convert AMP to ADO, respectively (1–7R), eight P2YRs (P2Y1/2/4/6/11/12/13/14R), and four P1 receptors (A1/A2A/A2B/A3R) have been cloned and characterized (Separate families of receptors for ATP (P2 receptors) and ADO (P1 receptors) were first identified in 1978. According to their transduction mechanism and molecular cloning, P2 receptors are subsequently divided into ionotropic P2XRs and metabotropic P2YRs. At present, seven P2XRs   . TM1 is and TM2) . Followiytokines .1/2/4/6/11Rs, with a sequence homology of 35–52% in amino-acid composition and the presence of a Y–Q/K–X–X–R defining motif in the transmembrane α-helix 7. The second group contains P2Y12/13/14Rs, with a sequence homology of 47–48% and the presence of the K–E–X–X–L motif in transmembrane α-helix 7. Moreover, the two subgroups also differ in their primary coupling to G-proteins  to decrease intracellular cyclic AMP (cAMP). Additional evidence shows that P2Y11R uses Gs to stimulate AC and increase intracellular cAMP.P2YRs belong to the δ-branch of class A G-protein-coupled receptor (GPCR) family and contain seven hydrophobic transmembrane regions connected by three extracellular loops and three intracellular loops . Based oproteins . P2Y1/2/1R, A2AR, A2BR, and A3R. A2AR shares a sequence identity of 46, 37, or 31% with A2BR, A1R, and A3R, respectively . Du. Du25). ing edge . Extrace sensing . Then, rignaling . The act2R, A3R, and A2AR, other purinergic receptors also mediate neutrophil chemotaxis. Activation of P2X1R by extracellular ATP fails to induce neutrophil directional chemotaxis, but it can enhance neutrophil chemokinesis (random cell migration) through Rho kinase signaling  in humans  significantly inhibits fMLP-induced oxidative burst. Monosodium urate crystals can induce neutrophil oxidative burst, but an antagonist to P2Y6R suppresses this monosodium urate crystal-induced neutrophil oxidative burst , secondary granules (SGs), tertiary granules (TGs), and SVs, are formed sequentially during granulopoiesis . Neutropable ATP . Insteadof P2X1R . The des11R-dependent  is an alternative approach to induce SIRS-associated ALI. Performing CLP in −/−P2ry2 and −/−Adora3 mice, investigators observed decreased recruitment of neutrophils into the lungs and attenuated development of ALI compared with WT mice  in myocardial IR injury (2BR in the myeloid lineage or either endothelial or myocardial cells demonstrated that myeloid lineage cells are necessary to provide cardioprotection during IR injury and inhibit neutrophil accumulation (−/−Adora2b neutrophils into neutrophil-depleted mice further confirmed the role of neutrophil-expressed A2BR in cardioprotection against IR injury (2BR appears to promote pulmonary IR injury and inflammatory responses by stimulating the production of cytokines and neutrophil infiltration (2BR are likely specific for resident pulmonary cells, but not BM-derived neutrophils, based on the data obtained from BM chimeric mice (In contrast to the effects of AR injury . The folmulation . AdoptivR injury . In contltration , 94. How7R ameliorates IR-induced renal neutrophil infiltration and tissue injury, and similar results were achieved by knockout of P2X7R (7R deficiency in BM cells further confirm that activation of P2X7R in BM-derived cells is essential for renal neutrophil infiltration during IR injury. Additional studies are needed to further clarify the effects of other P2 receptors on neutrophils during IR injury.Compared with the considerable evidence showing that ADO-driven P1 signaling mediates neutrophil activation and infiltration during IR injury, few studies have focused on the effects of ATP-induced P2R signaling on neutrophil function during IR injury. A recent report demonstrated that antagonism of P2Xof P2X7R . Chimeriin vivo infectious and inflammatory conditions, how purinergic receptors are integrated with other pattern recognition receptors to control neutrophil function and the dynamic biochemistry of ATP and ADO in the extracellular environment.The past 5 years have brought major advances in the identification of numerous types of neutrophil purinergic receptors and in the understanding of their functions in coordinating the appropriate immune response against invading pathogens or diseased tissue. The fine-tuned balance between P2R and P1R signaling appears to be critical for shaping neutrophil plasticity and heterogeneity to orchestrate a complex series of events during inflammation (Figure All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.


D). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (K Clostridium ,,Clostrid, E2, E3 , E9 12]Clostridi, E2, E3 , and E12, E2, E3 ). Within, E2, E3 ,15. All ain (LC) . Howeverain (LC)  and X-raain (LC)   proteins are known to interact and produce pores through which neurotransmitters are released from the nerve terminals to muscles, provoking muscle twitches. Botulinum neurotoxins act at the presynaptic level by cleaving one or more of the SNARE complex proteins, inhibiting neurotransmitter release [Several soluble  release , resulti release ,22.C. butyricum [While the majority of type E cases are foodborne, associated with raw or preserved fish or marine mammal products , rare cautyricum  have beeutyricum ,25.2 product with short serum half-lives (7.5–34.2 h), which eliminates its use for the prevention of botulism and limits its effectiveness as a treatment. Relapses of botulism after treatment have been noted [The current treatment for adult botulism is heptavalent (serotypes A–G) equine botulism antitoxin (BAT) . BAT is en noted . As an aen noted ,30 bindien noted  are beinen noted .Here, we report the generation of a panel of high affinity human BoNT/E mAbs using yeast display and Fluorescence-Activated Cell Sorting (FACS) technologies. mAbs were characterized with respect to ability to bind four BoNT/E subtypes, BoNT domain bound, and epitope overlap. Epitope mapping was compared to previously published data using cryo-EM  and alanH and Vk genes of human volunteers immunized with pentavalent BoNT toxoid . The construction of these libraries has previously been reported [D values of these ten yeast-displayed scFv were measured versus BoNT/E3 by flow cytometry and ranged between 0.10 and 15.4 nM. These ten scFv were further characterized for their binding to BoNT/E1, /E2, /E3, and /E4 (D < 2 nM). Two mAbs (4E17 and 3E6) were identified that bound all four subtypes, with KD values ranging from 15 nM to greater than 100 nM. Four mAbs were identified that bound three subtypes .Yeast-displayed single chain Fv (scFv) antibody libraries were constructed from the Vreported ,36. Yeas and /E4 . One mAbD values of IgG for BoNT/E3 ranged from 7.24 to 0.002 nM. In most cases, improved binding was seen with KD values that were 1.3- to 75-fold lower than the scFv KD  and the human kappa or lambda constant region as previously described for subsequent studies . In the  scFv KD . Improve regions ,36.N (proteolytic and translocation domains), HN (translocation domain), HC (translocation plus binding domain), HC (binding domain), and LC (light chain) as previously described for BoNT/A mAbs [N domain, while 4E13 binds to the HC domain . Each of the other six LC mAbs showed overlap of epitopes by one or more of the other LC mAbs as indicated by values shown in bold in N and was not inhibited by any of the other mAbs.For mAbs binding the same domain, epitope overlap was determined based on whether mAbs could simultaneously bind BoNT/E. Competition for binding was determined by capturing BoNT/E by one of the yeast-displayed scFv followed by probing with each of the IgG . The IgGN at the interface where the LC packs against the HN  of three mAbs at non-overlapping epitopes that are conserved between BoNT subtypes [−8 M) and even poorer affinity for BoNT/E2 and E4  with the highest affinity to BoNT/E1, E2, E3, and E4 (−10 M) lower KD as a yeast-displayed scFv compared to 3E6 (D = 3.9 × 10−11 M). The binding affinity to BoNT/E4 improved, with a KD of 9.7 × 10−8 M  and prior experience with diversifying this CDR in other affinity-maturation campaigns ,36,40. Tan 3E6.1 .50 of BoNT/E3; however, only one to two mice out of ten survived   . This is of mAb) . 3E6.2 bD less than 1.0 × 10−9 M. In vivo BoNT/E3 neutralization by individual mAbs was relatively modest, as expected, with mAb 3E2 showing the most potent neutralization having an ED50 of approximately 500 mouse LD50/50 μg of mAb. The other five individual mAbs tested showed minimal protection against challenge with 100 mouse LD50 of BoNT/E3, including the mAb (4E13) previously shown to bind to the BoNT/E–SV2 binding site and which blocked binding and uptake of BoNT/E into motor neurons [N and LC, it could act by entering the neuron bound to BoNT and preventing or delaying intracellular translocation [N dissociation from the LC-blocking substrate cleavage.Yeast display and sorting of scFv libraries constructed from the V-genes of humans immunized with BoNT toxoid was used to isolate a panel of ten unique human mAbs to BoNT/E. Three of the ten mAbs bound all four BoNT/E subtypes tested and two of these mAbs required affinity maturation in order to achieve our target level binding to the four BoNT/E subtypes tested  with a K neurons . The meclocation  or by pr50 of BoNT/E1 or BoNT/E3 and 50 µg protecting against 10,000 LD50 BoNT/E4. While we did not formally titer the potency of the three-mAb combination at a higher toxin concentration, these antibody combinations could theoretically yield a neutralization capacity of 8 million mouse LD50/mg of mAb combination, which would be the equivalent of 8000 International Units (IU)/mg. Where we have measured the IU potency of a BoNT/A mAb combination, the value determined correlated closely with the value calculated as just described [MAb combinations neutralized BoNT/E much more potently than individual mAbs, with 5.0 µg total of a triple-antibody combination able to completely neutralize 40,000 LDescribed .The greatly increased potency observed for the triple mAb combinations may be due to several different mechanisms. Binding of multiple mAbs could block several steps in the intoxication pathway, for example, receptor binding and translocation ,43. Bind50 of BoNT/E. Similar to the results reported here, 25 μg of a four or six mouse mAb combination synergized to neutralize 20,000 to 40,000 mouse LD50 of BoNT/E [50 were neutralized by 150 mg/kg [50 were neutralized by 2.5 ng of mAb [50, only 80% of BoNT needs to be neutralized for 50% survival while at a challenge dose of 100 LD50, 99% of BoNT must be neutralized for 50% survival. In addition, the murine or macaque mAbs were only evaluated for neutralization against a single subtype.Murine  camelid f BoNT/E . Neutral50 mg/kg  and fiveg of mAb , respectThe BoNT/E mAb combination described here is highly potent, with a projected dose of 0.64 mg to be equivalent to the 5100 IU of BAT in a single vial   and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. The specific national regulations and guidelines to which this animal care and use protocol adheres are the following: (1) 7 USC, Sections 2131–2159, Chapter 54 “Animal Welfare Act”, and  Health Research Extension Act of 1985, Public Law 99-158 “Animals in Research” and the Public Health Service Policy in Humane Care and Use of Laboratory Animals; (3) Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, NIH, Human and Health Services Publication (CDC) 21-112; (4) Army Regulation 40-33 “The Care and Use of Animals in DOD Research, Development, Test and Evaluation or Training Programs”; and (5) DOD Instruction 3216.01 “Use of Animals in DOD Programs”. DOD uses “The Guide for the Care and Use of Laboratory Animals”, 8th Edition, Institute for Laboratory Animal Research, National Research Council, as a guideline for the evaluation and accreditation of a program and it is based on the actual national regulations and guidelines for animal care and use programs. The animals used in this study were euthanized using carbon dioxide gas following the AVMA Guidelines on Euthanasia prior to spleen removal.The University of California, San Francisco (UCSF) Institutional Review Board approved the human use protocol used for the studies described here. Human donors were laboratory workers being immunized to work with BoNT who were recruited via an informational letter and who signed an informed consent form.3E6.1mutH1rev GAC CCA GTG CAT AGC 521 542 542 511 532 GAA TCC AGA GGC TGCwhere:1 = 70% A + 10% T + 10% G + 10% C2 = 70% T + 10% A + 10% G + 10% C3 = 70% G + 10% T + 10% A + 10% C4 = 70% C + 10% T + 10% G + 10% A5 = 50% G + 50% CPYDFor1: CCCCTCAACAACTAGCAAAGGCAGCCCPYDRev: GGTGATGGTGATGATGACCGGTACGCGTAGLink Rev: CGACCCGCCACCGCCAGAGCCACCTCCGCCThe primers for site-directed mutagenesis were designed and synthesized per the QuikChange Site-Directed Mutagenesis Kit  instructions. The primers for human and mouse library construction were synthesized as described previously ,49. The Saccharomyces cerevisiae strain EBY100, SD-CAA, for selection of pYD2-transformed EBY100 and SG-CAA, and for induction of scFv expression on the surface of EBY100. Escherichia coli strain DH5α was used for subcloning and preparation of plasmid DNA. Pure holotoxin BoNT/E3 was purchased from Metabiologics . Crude extract complex for subserotypes E2 and E4 was obtained from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Pure complex subserotype E1 was purchased . All IgGs were expressed from Chinese hamster ovary (CHO) cells, while the mouse anti-SV5 antibody was purified from hybridoma cells and labeled with an AlexaFluo-488 or AlexaFluo-647 labeling kit . The secondary antibody, PE-conjugated goat anti human-Fc, F(ab) was purchased .YPD medium was used for growth of 4S)3 linker to obtain full-length scFv genes by splicing using overlap extension PCR as previously described [To generate human mAbs that bind BoNT/E, total RNA was isolated from the blood of healthy human donors immunized with investigational pentavalent BoNT toxoid . cDNA synthesis, VH, and VK gene repertoire preparation and library construction were performed as described previously . The cDNescribed ,36. The D values of yeast-displayed scFvs were measured by flow cytometry as previously described with modification [6 yeast-displaying scFv were incubated for 1 h at room temperature in FACS buffer with six different concentrations of BoNT/E holotoxin, or complex, that spanned the range 10-fold above and 10-fold below the expected KD. Ice-cold FACS buffer was used to wash the samples, and 2 μg/mL each of an Alexa-647-labeled IgG that bound a different epitope was added plus SV5-488 at 4 °C and incubated for 60 min. Finally, the yeast clones were washed with ice-cold FACS buffer and the mean fluorescence intensity (MFl) of binding was measured by flow cytometry as described previously [The Kfication . Brieflyeviously ,50.Epitopes were classified based on the ability of mAbs to compete with each other for binding to BoNT/E as previously described ,36. BrieC, HN, HC, LC, and LC-HN. The synthetic genes were cloned into the yeast-display vector pYD2 as previously described [C, HN, HC, LC, and LC-HN domains. Domain display was induced and individual IgG tested for binding by incubating yeast-displayed domains with 2 μg/mL IgG for 60 min at room temperature followed by incubation with 1 μg/mL of Alexa-647-labeled anti-human Fc.To determine the BoNT/E domain bound by mAbs, synthetic genes were constructed for the BoNT/E3 Hescribed  for BoNTN, HN, and HC were prepared by introducing random mutations into the domain gene using error-prone PCR with the primers pYD-For/pYD-Rev and DNA polymerase Paq5000  plus 12.5 μM MnCl2 as previously described [To determine the fine epitopes of BoNT/E mAbs, libraries of BoNT/E3 domains LC-Hescribed . The PCRescribed .E. coli XL1-Blue. The alanine mutants for each gene were then individually transformed into EBY100, grown in SD-CAA medium, and induced for expression on the surface of EBY100. DNA sequencing was used to verify each construct. The ΔΔG of binding for each alanine mutant was determined by comparing the IgG KD for the alanine mutant domain to the wild-type KD as previously described [To confirm the energetically important amino acids on the BoNT/E domain critical for mAb binding, single alanine mutants of BoNT/E domains in the putative fine epitope were prepared using the QuikChange II-E Site-Directed Mutagenesis Kit  following the manufacturer’s instructions. Briefly, primers containing the mutation were used for PCR amplification with the plasmid pYD2 containing the corresponding BoNT/E domain gene as a template for 18 cycles. The PCR product was digested by DpnI to remove the parental methylated and hemi-methylated DNA, which was then purified by StrataClean Resin and transformed into escribed .L chain-shuffled scFv library was created by PCR amplifying the Vk gene repertoires from human immune scFv libraries using Pfu polymerase (Stratagene) and the primers LinkFor and PYDRev. To further increase VL diversity, the VL repertoire from a large non-immune scFv phage antibody library transferred from the phagemid vector pHEN1 and cloned into pYD2 was also used [H DNA was amplified from the scFv gene in pYD2 using a 5′ primer that annealed upstream of the VH gene (PYDFor1) and a 3′ primer that annealed in the linker gene between the VH and VL genes (LinkRev). The gel-purified VH gene was mixed with the gel-purified VL repertoires and combined with NcoI- and NotI-digested pYD2 vector DNA. This mixture was used to transform LiAc-treated EBY100 cells by three-fragment gap repair. Library size was measured as >107 transformants. To select higher affinity scFvs, the VL-shuffled library was sorted as described above.To increase the affinity of mAb 3E6, a Vlso used . The 3E67 transformants, and selections and new scFv validation were done as described above.To create a library of CDRH1 mutants of the 3E6.1 scFv, a spiked oligonucleotide (3E6-H1Rev) was used to introduce mutations into five amino acids located in the H1 loop of the 3E6.1 scFv as previously described . The oliFor selected IgGs, the solution phase affinity at equilibrium and binding kinetics were measured using flow fluorimetry in a KinExA as previously described ,50 excep50 of BoNT/E1, /E3, or /E4 in a total volume of 0.5 mL of gelatin phosphate buffer and incubated at room temperature for 30 min. The mixture was then injected intraperitoneally into cohorts of ten mice. The animals were observed multiple times daily for clinical signs of botulism. Moribund animals were euthanized. Surviving mice at the study endpoint were tabulated.The mouse neutralization assay was performed as described previously . Briefly


H mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2  primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VTrial Registration: ClinicalTrials.gov NCT01435135 Developing a successful HIV-1 vaccine remains a high global health priority. Several HIV-1 vaccine trials have been performed with only the RV144 vaccine trial showing vaccine efficacy, albeit modest. No broadly neutralizing antibody activity was identified in RV144 and inducing sterilizing immunity against a complex pathogen like HIV-1 remains a major challenge. Here we characterize the B cell responses after RV144 vaccine-recipients received two additional boosts severals years after the conclusion of the RV144 vaccine trial. Delayed and repetitive boosting of RV144 vaccine-recipients was capable of increasing somatic hypermutation of the Env-reactive antibodies and expanding subdominant pools of neutralizing B cell clonal lineages. These data are pertinent to HIV-1 vaccine-regimen design. Six HIV-1 vaccine efficacy trials have been performed , of whicWhile no broadly neutralizing antibodies (bnAbs) were induced in RV144 ,10 the iHere we sought to determine if a pool of subdominant B cells, such as those that produce long HCDR3 CD4 bs bnAbs, may be expanded when an Env immunogen that binds bnAb UCAs is included in a boosting regimen. In the RV305 clinical trial, RV144 vaccine-recipients who had previously received the initial ALVAC-HIV + AIDSVAX B/E gp120 immunization regimen  and remained HIV-1- uninfected were boosted with ALVAC-HIV, AIDSVAX B/E gp120, or ALVAC-HIV + AIDSVAX B/E gp120 6–8 years later . We founAfter two boosts  with the same immunogens 6–8 years after the completion of the RV144 primary immunizations , plasma H nucleotide mutation frequency increased over 2-fold in each vaccinee from a mean of 3.1% to 6.9%  –28. A ment usage . Coincidused JH6 . To deteIn vaccinee 3053, seven gp120-reactive B cell clonal lineages were present after the initial RV144 vaccine regimen that persisted and had expanded after boosting 6–8 years later in RV305, one of which, DH678, had a long HCDR3. In vaccinee 3043 nine gp120-reactive B cell clonal lineages were identified after RV144 that were also represented in the samples taken after the RV305 boosts. The antibodies in two of these lineages, DH686 and DH576, had long HCDR3s . These dAll antibodies isolated were assayed by ELISA as transient transfection supernatants and we selected twenty-seven Env-binding antibodies derived from blood memory B cells post-RV305 boosts based on HCDR3-length (≥ 22 aa) as a representative set of antibodies for characterization . Nine ofEnv mutations I371, P363, R476 and D368 generally reduce binding by CD4bs Abs . When asIn the TZM-bl cell assay, all neutralizing CD4bs mAbs neutralized not only AE.92TH023 but also the heterologous tier 2 CRF01 isolate AE.CNE8 isolate. DH583 was the broadest neutralizing antibody, also neutralizing the tier 1 viruses B.SF162, B.MN, and the tier 1B  primary isolate C.6644 . Long HCIn RV144, infection risk correlated inversely with V1V2 antibody responses . Two V1VH nucleotide mutations of 10.33%), but the additional mutations did not broaden or strengthen HIV-1 tier 2 CRF01 AE.CNE8 neutralization (H mutations of 7.33%) (50) against the tier 1 AE.92TH023 by over 3 logs, but increased its potency (IC50) against the tier 2 AE.CNE8 by less than 1 log  . To detean 1 log . These dan 1 log .To determine whether neutralization of HIV by a UCA was a common property of HCDR3-loop CD4 bs binding mAbs, we assayed the UCAs of the other vaccine-induced CD4bs mAbs and found that 3 of 8 nAb UCAs neutralized both AE.92TH023 and AE.CNE8 .These daProgression from sporadic tier 2 neutralization to increased tier 2 virus neutralization breadth depends upon the epitope specificity  and the The CD4bs bnAb B12 interaction with gp120 depends upon an aromatic residue at the apex of the HCDR3 loop, aromatic residues around the base of the HCDR3 region, a tyrosine at the apex of the HCDR2 loop and positively charged amino acids in the LCDR1. An aligNegative stain EM of 92Br SOSIP.664 with DH583, the broadest mAb identified, showed that DH583 also binds an open form of the trimer, even though this trimer is stable in the closed form . These oH chain gene mutation frequency and expanded clonal lineages of CD4bs antibodies with long HCDR3 regions. Increased somatic hypermutation and affinity maturation by repetitive immunization with a gp120-protein has previously been reported in humans and non-human primates  ×100. The results were considered positive if ADCC activity was ≥15% specific killing. ADCC activities are reported either as the endpoint concentration (EC), defined as the mAb concentration that intersects the positive cutoff of 15% specific killing, or as positive area under the curve (pAUC), calculated by the trapezoidal rule using the values ≥15% specific killing.ADCC mediated by the mAbs was assessed according to previously published procedures ,58. Brieg frames . CEM.NKRa Trkola  were infbackbone . Whole P508REKR511) was altered to 506RRRRRR511 to enhance cleavage [env gene was obtained from GenScript  and cloned into pVRC-8400 using Nhe1 and NotI restriction sites and the tissue plasminogen activator signal sequence.To generate the autologous HIV-1 CH505 SOSIP.664 and clade B 92Br SOSIP.664 expression constructs we followed established SOSIP design parameters . Brieflycleavage . The resFabs were expressed by transient transfection of HEK 293F suspension cells, using linear PEI following the manufacturer’s suggested protocol. After 5 d, supernatants were clarified by centrifugation and diluted twofold with 1x PBS buffer, and the protein isolated from the diluted spernatant using CaptureSelect LC-Kappa (Hu) affinity matrix , according to manufacturer’s protocols. Fractions containing the protein of interest were pooled, concentrated, and further purified by gel filtration chromatography using a Superdex 200 analytical column  in a buffer of 2.5mM Tris, pH 7.5, 350mM NaCl, and 0.02% sodium azide.Galanthus nivalis) affinity chromatography column overnight at 4°C. The lectin column was washed with 1x PBS, followed with 1x PBS supplemented with 0.5 M NaCl, and proteins were eluted with 1 M methyl-α-D-mannopyranoside dissolved in 1x PBS. The eluate was concentrated and loaded for further purification onto a Superdex 200 10/300 GL column  prequilibrated in a buffer of 5 mM Hepes, pH 7.5, 150 mM NaCl and 0.02% sodium azide for analysis by EM.Each SOSIP.664 construct was transfected into 293F cells together with a plasmid encoding the cellular protease, furin, at a 4:1 Env:furin ratio. The cells were allowed to express the soluble trimer for 5–7 days. Culture supernatants were collected, cells removed by centrifugation at 3800 x g for 20 min, and the supernatant filtered with a 0.2 μm pore size filter. The soluble SOSIP was purified by flowing the filtered supernatant over a lectin   and an FThe DH576 Fab was crystallized at 10–15 mg/mL. Crystals were grown in 96-well format using hanging drop vapor diffusion and appeared after 24–48 h at 20°C. Fab crystals were obtained in the following conditions: 20% PEG 4000, 100mM Hepes, pH 7.0, 1M NaCl. Crystals were harvested and cryoprotected by the addition of 20–25% glycerol to the reservoir solution and then flash-cooled in liquid nitrogen.Diffraction data were obtained at 100 K from beam line 24-ID-C at the Advanced Photon Source using a single wavelength. Datasets from individual crystals were processed with HKL2000. MoleculRefinement was carried out with PHENIX, and allAll statistical analysis was performed in SAS by the Duke Human Vaccine Institute statistical team. The statistical test and p value are recorded where used.H and VL chain genes described have been submitted to Genbank with accessioning numbers KY499910-KY499949.The EM reconstruction has been deposited in the Electron Microscopy Data Bank as EMD-8573. The crystal structure of DH576 has been deposited in the Protein Data Bank as PDB ID5UIX. The VS1 FigThe RV305 clinical trial (NCT01435135) was a placebo-controlled double-blinded HIV-1 vaccine trial that occurred six-eight years after the conclusion of RV144 (NCT00223080). RV144 vaccinees that received the full RV144 immunization regimen and were HIV-1 seronegative were split into three groups and boosted two times with either ALVAC-HIV + AIDSVAX B/E (Group I), AIDSVAX B/E (Group II), or ALVAC-HIV alone (Group III).(EPS)Click here for additional data file.S2 FigThe plasma from two weeks after the second boost in RV305 (wk. 26) from vaccinees boosted with AIDSVAX B/E (Group I), AIDSVAX B/E + ALVAC-HIV (Group II), or ALVAC-HIV (Group III) were screened in the A3R5 neutralization assay for neutralization of a panel of HIV-1 CRF_01 AE isolates. (A) ID50 in A3R5 cells of each CRF_01 isolate per group (Grp I and III n = 23 and Grp II n = 24). (B) Individual ID50 in A3R5 neutralization assay of vaccines from Group I and Group II when assayed against the 11 CRF_01 AE viruses in (A). Red arrows denote vaccinees selected for antibody isolation.(EPS)Click here for additional data file.S3 FigThe plasma of the four vaccinees selected for antibody isolation were longitudinally assayed for (A) IgG binding to the autologous isolate AE.A244gp120Δ11 and heterologous isolate B.6240gp120Δ11 by Luminex and (B) plasma neutralization of tier 1 and tier 2 viruses in the TZM-bl neutralization assay.(EPS)Click here for additional data file.S4 FigAE.A244gp120-double positive (upper right quadrant)memory B cells from two weeks after the second RV305 boost (wk. 26) were antigen-specific single-cell sorted with fluorophore labeled conjugates for RT-PCR.(EPS)Click here for additional data file.S5 FigH/VL chain genes were PCR-amplified and screened for Env-reactivity by ELISA. The VH chain gene mutation frequency and HCDR3 lengths of 145 Env-reactive antibodies from RV135/144 and 242 Env-reactive antibodies from RV305 were analyzed with Cloanalyst[PBMCs from (A)14 RV135/144 vaccinees and (B) 4 RV305 vaccinees were antigen-specific single-cell sorted with fluorophore labeled conjugates. The Vloanalyst.(EPS)Click here for additional data file.S6 FigH/VL chain genes were PCR-amplified and analyzed with Cloanalyst [PBMCs from two vaccinees that were immunized in both the RV144 and RV305 vaccine trials were antigen-specific single-cell sorted with fluorophore labeled conjugates. The Voanalyst .(EPS)Click here for additional data file.S7 FigAntibodies were diluted 3-fold starting at 100 ug/mL and assayed by ELISA for blocking of the CD4bs bnAbs VRC01 and CH31 binding to AE.A244gp120.(EPS)Click here for additional data file.S8 FigThe RV305 antibodies were assayed for ADCC against WITO, 1086C and CM235 infected cells. Shown is the antibody (A) end point concentration and (B) area under the curve. For comparison other CD4 bs and the C1/C2 antibody A32 are include.(EPS)Click here for additional data file.S9 FigThe Fourier shell correlation curve for the complex is shown along with the resolution determined using FSC = 0.5.(EPS)Click here for additional data file.S10 Fig(A) The HCDRH3 loop is highlighted to indicate the different conformation of this loop for the two Fabs. The panel on the right shows a superposition with the B12-gp120 complex and the arrow points to where the HCDR3 loop of DH576 clashes with gp120 when the two antibodies are superimposed onto gp120 at the same angle. (B) A heavy chain alignment of the DH576 unmutated common ancestor and naturally occurring DH576 clonal lineage members with the CD4 binding site bnAb B12. The * highlights conserved aromatic residues critical for B12 binding .(EPS)Click here for additional data file.S11 Fig2D class averages of the complex are shown (top two rows) and unliganded trimer in the closed form (bottom row).(EPS)Click here for additional data file.S1 TableH/VL chain gene sequences were analyzed with Cloanalyst[PBMCs from four vaccinees post-RV305 were antigen-specific single-cell sorted, PCR-amplified and sequenced. The Vloanalyst.(EPS)Click here for additional data file.S2 TableThe frequency of gp120-reactive (RV144 boosts) or gp140-reactive (HVTN 505 and GSK PRO HIV-002) mAbs with HCDR3s ≥ 22 amino acids were compared using the Fisher’s Exact Test. The RV144 boosted vaccinees had a greater frequency of long HCDR3s compared to either the HVTN505 or GSK PRO HIV -002 clinical trials.(EPS)Click here for additional data file.S3 TableH/VL chain gene sequences were analyzed with Cloanalyst[The Vloanalyst.(EPS)Click here for additional data file.S4 TableThe antibodies were recombinantly expressed and assayed for neutralization of AE.92TH023 in the TZM-bl neutralization assay.(EPS)Click here for additional data file.S5 TableThe antibodies were recombinantly expressed and assayed for neutralization of four tier 2 CRF_01 clade AE isolates in the TZM-bl neutralization assay.(EPS)Click here for additional data file.S6 Tabled).The unmutated common ancestor and intermediate antibody sequences were inferred with Cloanalyst. The ant(EPS)Click here for additional data file.S7 TableThe UCAs of the eight CD4 bs clonal lineages were inferred with Cloanalyst. The ant(EPS)Click here for additional data file.S8 TableStatistics for highest-resolution shell are shown in parentheses.(EPS)Click here for additional data file.


SLC13A1 . SLC13A1 encodes the apical sodium-sulfate cotransporter (NaS1) responsible for sulfate (re)absorption in the kidneys and intestine. SLC13A1 R12X and W48X were independently associated with a 27.6% (P = 2.7 × 10−8) and 27.3% (P = 6.9 × 10−14) decrease in serum sulfate, respectively (P = 8.8 × 10-20 for carriers of either SLC13A1 nonsense SNV). We further performed the first exome- and genome-wide association study (ExWAS/GWAS) of serum sulfate and identified a missense variant (L348P) in SLC26A1, which encodes the basolateral sulfate-anion transporter (Sat1), that was associated with decreased serum sulfate (P = 4.4 × 10−12). Consistent with sulfate’s role in xenobiotic detoxification and protection against acetaminophen-induced hepatotoxicity, SLC13A1 nonsense SNV carriers had higher aminotransferase levels compared to noncarriers. Furthermore, SLC26A1 L348P was associated with lower whole-body bone mineral density (BMD) and higher serum calcium, consistent with the osteochondrodysplasia exhibited by dogs and sheep with naturally occurring, homozygous, loss-of-function mutations in Slc13a1. This study demonstrates the power and translational potential of systematic identification and characterization of rare, loss-of-function variants and warrants additional studies to better understand the importance of sulfate in human physiology, disease, and drug toxicity.Using genomic applications to glean insights into human biology, we systematically searched for nonsense single nucleotide variants (SNVs) that are rare in the general population but enriched in the Old Order Amish (Amish) due to founder effect. We identified two nonlinked, nonsense SNVs (R12X and W48X) in  Population isolates have been critical in elucidating the genetic basis of both classical Mendelian and complex diseases. While many loss-of-function variants associated with disease are rare in the general population, genetic drift in founder populations like the Old Order Amish (Amish) increases the probability that many individuals will share the same rare, disease-causing variant. Recent advances in genome-wide genotyping and sequencing technologies have made possible the systematic identification of these variants and their subsequent investigation.SLC13A1, which encodes the apical membrane, sodium-sulfate cotransporter (NaS1) responsible for sulfate (re)absorption in the intestines and kidneys.In this study, we systematically interrogated nonsense single nucleotide variants (SNVs) from the Illumina Human Exome BeadChip for minor allele frequency (MAF) enrichment in the Amish compared to outbred, European-derived populations. Using this approach, we observed an enrichment of two nonsense SNVs  in 42-) is an important micronutrient vital for numerous cellular and metabolic processes in human development and physiology  identify additional novel genetic contributors through completion of the first exome-wide association study (ExWAS) and genome-wide association study (GWAS) of serum sulfate, and (4) investigate associations between sulfate-altering genotypes and relevant clinical phenotypes including aminotransferase levels, bone mineral density (BMD), and serum calcium.Given the potential importance of sulfate in the development of several disorders, we comprehensively characterized serum sulfate in 977 Amish individuals in order to (1) determine the heritability of serum sulfate, (2) examine the relationship between This report is based on the Amish community living in Lancaster County, PA, whom our research group has been studying since 1993. This community was founded by several hundreds of individuals who immigrated to Lancaster County, PA from central Europe during the early 18th century, with the present-day Lancaster County Amish community comprised of their descendants -approved studies conducted at the University of Maryland School of Medicine Amish Research Clinic (ARC). Written informed consent was obtained from each participant.SLC13A1 R12X (rs28364172), W48X (rs138275989), N174S (rs2140516), and R237C (rs139376972), was performed on 1725 Amish subjects using the Illumina Human Exome BeadChip . Genotyping was performed according to the manufacturer’s instructions and genotypes were assigned using Illumina GenomeStudio software (Illumina Inc.).Exome-wide genotyping, including SLC13A1 nonsense SNVs, R12X and W48X, was performed on additional Amish subjects using TaqMan single nucleotide polymorphism (SNP) genotyping assays . In total, SLC13A1 R12X and W48X genotypes were available in 3924 and 3782 unique individuals, respectively. For both SNPs, the TaqMan genotype concordance was > 99.8% in a subset of duplicate samples. For subjects genotyped using the Illumina Human Exome BeadChip and TaqMan SNP genotyping assays, genotype concordance between platforms was 100% for both SLC13A1 R12X and W48X.In subsequent follow-up investigations, genotyping of For 917 of the 977 subjects for which serum sulfate was measured, genome-wide genotyping was performed using the Affymetrix GeneChip Human Mapping 500K or 1M (version 6.0) arrays according to the manufacturer’s instructions . Genotype calls were performed using BRLMM (500K array) or Birdseed version 2 (1 M array). SNVs present on both arrays with an MAF > 1% were included in the analyses.SLC13A1 SNVs, R12X, W48X, and R237C, for whom fasting, serum aliquots were available, were selected for sulfate measurement. Such subjects consisted of participants from the Amish Pharmacogenomics of Antiplatelet Intervention (PAPI) Study (File S1). All other subjects consisted of randomly selected participants of the Amish PAPI (All known Amish carriers of the rare ◦. Sulfate concentration was determined by turbidimetry according to Dodgson and Price (1962) using a Quantichrom Sulfate Assay Kit . In order to improve accuracy, a quadratic least squares fit was used instead of a linear fit to generate the standard curve (NIST/SEMATECH 2013). All standards and samples were measured in duplicate. For each sample, sulfate concentration was calculated as the mean of the duplicate measurements. Samples with an absolute difference > 20% were considered discordant duplicate measurements and were not included in the analysis. Of the 1003 samples in which serum sulfate was measured, 26 subjects’ samples were excluded due to discordant duplicate measurements, resulting in valid sulfate concentration measurements for 977 subjects’ samples.Sulfate measurements were completed on frozen, fasting serum aliquots stored at −80Serum ALT, AST, calcium, and albumin measurements were collected as part of the Amish PAPI (BMD was measured as part of the Amish PAPI Study (n = 669) and 6728 in ESP-EA (n = 3364). For each nonsense SNV, the MAF in the Amish was compared to the MAF in 1000g-EUR and ESP-EA (when available) using a chi-squared test.The MAF for each nonsense SNV was obtained from the Total European Ancestry population from 1000 Genomes (1000g-EUR) and the European American population from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) (ESP-EA). For each SNV, the total allele count was assumed to be 1338 in 1000g-EUR  program . For eacIn accordance with our human subjects’ research protocols, we are unable to release the individual-level data set. We continue to deposit aggregate-level data into the National Institutes of Health (NIH) database of Genotypes and Phenotypes (dbGaP); however, given the Old Order Amish are a founder population with publically available pedigree data, depositing individual-level data on our subjects creates the potential for personal identification of our research subjects. In the past, the NIH has provided exemptions for this reason. Furthermore, all of our analyses were performed using the MMAP program  which mon = 1725), 43,459 of the >240,000 markers on the Illumina Human Exome BeadChip were polymorphic, 266 of which were nonsense SNVs. For 45 of the 266 nonsense SNVs, no MAF was reported in 1000g-EUR nor ESP-EA. For 131 of the 266 nonsense SNVs, the MAF was greater in this Amish cohort compared to the MAF in 1000g-EUR averaged with the MAF in ESP-EA. Of these 131 SNVs, 67 and 103 were significantly enriched (P < 0.05) compared to 1000G-EUR and ESP-EA, respectively (Table S1). With an intent to discover novel biology, we filtered out variants in genes with an associated Online Mendelian Inheritance in Man (SLC13A1 (vs. 0.23%) and 4.4-fold (0.87% vs. 0.20%), respectively, compared to ESP-EA allele frequencies. In addition, two missense SNVs  in SLC13A1 were also present on the Illumina Exome BeadChip and enriched 1.2-fold (39% vs. 32%) and 10.7-fold (1.7% vs. 0.16%), respectively, compared to ESP-EA allele frequencies (Table S2).In this Amish cohort , the allele frequencies were enriched 1.2-fold (0.29% vs. 0.023%) and 3.7-fold (0.74% vs. 0.20%), respectively (Table S2). All four SLC13A1 SNVs conformed to Hardy-Weinberg expectations (Table S2); no R12X homozygotes, W48X homozygotes, or R12X/W48X compound heterozygotes were observed. None of the four SNVs were in significant linkage disequilibrium (LD) with one another with the exception of N174S and R237C  .Upon further genotyping of SLC13A1 in sulfate (re)absorption, we measured sulfate levels in 977 individuals. Amish research subjects selected for sulfate measurement consisted of 481 males (49.2%) and 496 females (50.8%), with a mean age of 46.0 ± 14.4 yr, and a mean BMI of 27.1 ± 4.8 kg/m2 . Serum sulfate was associated with increasing age , but not with gender . The total variance in serum sulfate explained by age, gender, and study was 13% (Table S3a). The residual heritability of serum sulfate after adjustment for age, gender, and study was 0.40 (P = 1.8 × 10−16).Given the role of .8 kg/m2 . Serum sSLC13A1 SNVs and serum sulfate independently (Table S3a–b). Specifically, we observed strong associations between R12X and W48X, and lower serum sulfate. SLC13A1 R12X was associated with a 26.7% lower serum sulfate   and SLC13A1 W48X was associated with a 26.5% lower serum sulfate  . We evaluated the influence of N174S and R237C on serum sulfate levels in 900 of these individuals. In contrast to R12X and W48X, N174S was associated with a 3% higher serum sulfate per allele  (P = 0.15)  . No asso = 0.15) . The estSLC13A1 R12X and W48X were not correlated with each other, we performed conditional association analyses to evaluate whether these variants represented independent association signals (Table S2a). Indeed, both R12X and W48X were independently associated with a 27.6%  and 27.3%  decrease in serum sulfate, respectively  as a covariate (Table S3a). We observed a 27% decrease in serum sulfate among SLC13A1 nonsense SNV carriers compared to subjects who do not carry R12X or W48X   . The estn = 900) , demonstrated genome-wide significance (P < 5.0 × 10−8) for W48X , and suggestive-significance for R12X  in SLC13A1 on chromosome 7q31.32 (Table S3b). The ExWAS also revealed a novel cluster of four SNVs spanning ∼3 megabases on chromosome 4p16.3 demonstrating genome-wide significance , with L348P in SLC26A1  being the most significant, associated with a 12% decrease in serum sulfate per allele . SLC26A1 encodes the basolateral membrane, sulfate-anion transporter 1 (Sat1), consistent with this locus as a true determinant of serum sulfate . No other genomic region revealed association signals that met or exceeded genome-wide significance (Table S3).An ExWAS of serum sulfate, performed using genotype data from the Illumina Human Exome BeadChip  . No genomic region revealed association signals that met or exceeded genome-wide significance .A secondary ExWAS of serum sulfate, adjusting for n = 917) , demonstrated genome-wide significance for a variant on chromosome 7 (rs17684427) and genome-wide suggestive-significance for a variant on chromosome 4 (rs362272) in strong LD with SLC13A1 W48X and SLC26A1 L348P, respectively . A secondary GWAS of serum sulfate, adjusting for rs17684427 and rs362272 genotypes, was also performed (n = 917) . No genomic region revealed association signals that met or exceeded genome-wide significance .A GWAS of serum sulfate, performed using genotype data from the Affymetrix GeneChip  was used to further estimate residual heritability (Table S3b). In this model, SLC13A1 R12X, W48X, and N174S, and SLC26A1 L348P were all independently associated with decreased serum sulfate while SLC13A1 R237C was not. The estimated residual heritability after adjustment for SLC13A1 R12X, W48X, N174S, and R237C, and SLC26A1 L348P genotype was 0.31 with these variants, together, explaining ∼29% of the additive variance and 13% of the total variance in serum sulfate.A model including all five SNV genotypes  and aspartate aminotransferase (AST) in 684 participants from the Amish PAPI Study . Similarly, a trending association was observed between SLC13A1 nonsense carriers and increased AST    . No assoctively) .SLC13A1 nonsense SNV carriers than in noncarriers   (We were able to replicate these associations in an independent Amish cohort consisting of 264 participants from the AFLS . ALT andctively) . Additio × 10−3) .SLC13A1 nonsense SNV carriers and whole-body BMD. No association was observed between SLC13A1 nonsense carriers and whole-body BMD (P = 0.19); however, SLC26A1 L348P was significantly associated with decreased whole-body BMD  . Furthermore, SLC26A1 L348P was significantly associated with increased total serum calcium , and showed a nearly significant association with albumin-adjusted serum calcium    . Upon fu = 0.06) . No assoctively) .2, P = 0.03) and lower intertrochanter BMD  (Table S8).Serum sulfate concentration by itself was not associated with any of the aforementioned clinical phenotypes with the exception of trochanter and intertrochanter BMD. Lower serum sulfate was marginally associated with lower trochanter absorption, and perhaps other traits for which sulfate may play a role. We further conducted the first ExWAS and GWAS of serum sulfate in an attempt to identify other genetic contributors of serum sulfate, a trait not measured clinically despite its important physiological role in human development, disease, and drug toxicity. To the best of our knowledge, our group is the first to establish that serum sulfate is heritable . In addition, these studies have identified novel associations between R12X, W48X, and N174S in SLC13A1, and L348P in SLC26A1, and serum sulfate levels. Further clinical characterization of the three sulfate-lowering variants implicates sulfate-wasting in liver function, and perhaps BMD and calcium homeostasis.Using genomic approaches to unveil new human biology, we systematically identified nonsense SNVs that are rare in the general population but enriched in the Amish due to founder effect. Two nonsense SNVs (R12X and W48X), as well as two missense SNVs (N174S and R237C), in SLC13A1 R12X, our findings are consistent with work performed by Bowling et al., which demonstrated an association between R12X and increased urinary fractional excretion index (FEI) of sulfate in a cohort with ASD . We attribute the increased effect of sulfate-lowering variants in participants of the AFLS to routine medication/supplement use, as well as increased participant age which may render hepatocytes more susceptible to damage from xenobiotics. This phenomenon may partially explain the lack of an association between SLC26A1 L348P and elevated aminotransferase levels in the participants of the younger Amish PAPI Study , there was far less power to detect such associations with SLC13A1 nonsense SNVs in this study due to the lower allele frequency  and the lack of homozygotes. The lack of an association with SLC13A1 nonsense variants may also be explained by differential tissue expression of SLC13A1 and SLC26A1.Sulfate’s physiological role in bone and cartilage is well known. Sulfated proteoglycans are a critical component of extracellular matrices in cells throughout the body, specifically in connective tissues, and are required for maintaining normal bone and cartilage structure  function and is seemingly independent of serum sulfate. These findings are consistent with mouse models in which hyposulfatemic, Slc26a1-knockout mice exhibit nephrocalcinosis and increased calcium oxalate kidney stone formation while hyposulfatemic, Slc13a1-knockout mice do not exhibit these manifestations (SLC26A1 has been suggested to play a role in humans with recurrent calcium oxalate kidney stones (SLC26A1 L348P allele decreases Sat1 function, decreasing sulfate transport into the blood as well as decreasing anion transport into cells. As a result, serum oxalate and thus urine oxalate concentrations may increase resulting in the formation of calcium oxalate crystals which either accumulate to form stones and/or increase serum calcium levels. We do not have an adequate number of subjects with renal stones to address this hypothesis further.The association between increased serum calcium and stations , 2012. ASLC13A1, which encodes the apical membrane, sodium-sulfate cotransporter, NaS1. Through our ExWAS of serum sulfate, we further identified a novel association with L348P in SLC26A1, which encodes the basolateral membrane, sulfate-anion transporter, Sat1. We also investigated associations between sulfate-lowering SNVs, aminotransferase levels, BMD, and serum calcium, results of which implicate sulfate and these SNVs in important human metabolism, physiology, and disease processes.Sulfate is an essential micronutrient, as impaired sulfation capacity has been reported in a number of human conditions including reduced xenobiotic clearance, skeletal dysplasias, premature pubarche, ASD, and neurological disease . DespiteIn conclusion, this study demonstrates the power and translational potential of leveraging genetic data in founder populations like the Old Order Amish to unveil and better understand the phenotypic consequences of rare, deleterious variants enriched by genetic drift. Further, our clinical findings warrant additional studies to better understand the importance of sulfate in human physiology, disease, and drug toxicity.


These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4 H1)-like response [The mammalian immune system is a complex network of cells regulated by signals transmitted by many secreted and receptor proteins. These include the human tumor necrosis factor (TNF) superfamily that comprises at least 19 members and represents a major class of stimulatory proteins. TNF-like 1A (TL1A) is a newly described member of the TNF superfamily that was shown to be involved in a range of autoimmune inflammatory diseases, including inflammatory bowel diseases (IBD), rheumatic arthritis (RA), and asthma . TL1A isresponse ,7. It waresponse . Subsequresponse .+ T cells and natural killer cells, with such expression being increased upon T cell activation [DR3, the receptor for TL1A, is expressed by CD4tivation . Althougtivation ,6,9. Inttivation ,11. Thistivation ,12. ThusTL1A gene contribute to susceptibility to IBD in Japanese and European populations [Using two distinct animal models for Chron's disease (CD), it was shown that the induction of intestinal inflammation is associated with significant up-regulation of TL1A and DR3 in the inflamed mucosa ,13. Subsulations . Finallyulations ,6,16.The importance of the TL1A pathway in promoting inflammation suggests that blocking TL1A-DR3 interaction could lead to the abolishment of downstream signaling effects and thus prevent various inflammatory disorders. At the same time, the use of antibodies or soluble DR3 receptor able to bind TL1A with high affinity could lead to efficient blocking of TL1A binding to the endogenous DR3 receptor. The advantages of utilizing soluble DR3 receptor are the small molecular weight of the DR3 extracellular domain (ECD) and its perfect recognition of the TL1A binding surface. Moreover, the engineering of DR3 ECD for increased affinity and stability would enhance its ability to compete with the endogenous DR3 receptor for TL1A binding and increase its serum half-life following administration. Indeed, we have previously utilized directed evolution to engineer soluble IL-17A receptor for efficient blocking of IL-17A-mediated inflammatory responses. Our engineered IL-17 receptor exhibits high biological activity and effectively inhibits psoriasis plaque formation in a humanized mice model [Over the past two decades, a directed evolution approach has been extensively used for improving the function of various proteins. This approach is based on the generation of mutants libraries followed by effective screening for the isolation of mutants with improved functions ,19. Dire+ cells and apoptosis in TF-1 cells, relative to the native receptor. These results suggest that the engineered soluble DR3 variants can serve as drug candidates for IBD.In the present work, we utilized directed evolution to generate soluble DR3 ECD variants that show improved stability, augmented TL1A binding affinity and potent inhibition of TL1A-induced cytokine secretion from T cells or apoptosis in a TF-1 cell line. As a first step, we generated a DR3 mutant library containing diversity based on DR3 sequence alignment. We used yeast surface display (YSD) followed by screening of single DR3 mutants expressed in mammalian cells to isolate two improved DR3 variants . These vE. coli Clooni strain (Lucigen) was used for cloning and plasmid extraction. For the display of DR3 on the yeast cell surface, the DR3 variants were cloned into the pCTCON plasmid using the NheI and BamHI sites, as previously described [6/ml in RPMI 1640, with 10% FBS, 2 mM glutamine, 1x Pen/strep solution supplemented with 10% heat inactivated FBS . TF-1 cells were grown in RPMI medium supplemented with 10% FBS, 2 mM glutamine, 1 x Pen/step solution  and 2 mM GM-CSF (Peprotech). The culture was maintained between 3*104 and 5*105 viable cells/ml. TF-1 cells were obtained from the ATCC, HEK293F cells were obtained from Invitrogen and HEK293T cells were a kind gift from Dr. Ran Taube .We amplified the human DR3 extracellular domain from an EST cDNA clone of human DR3 (Open Biosystems). The escribed ,22. For 2HPO4 and 8.6 g NaH2PO4) at 30°C to logarithmic phase and induced as previously described [The DR3 variants were displayed on the yeast cell surface of EBY100 strain cells and analyzed the cells by flow cytometry, essentially as described ,22. For escribed . The celescribed .A DR3 back-to-consensus library was generated following amplification of the human DR3 gene and digestion of a 5 μg sample with DNaseI to yield 50–125 bp fragments, as previously described ,23. The 7 of cells displaying the DR3 mutants were labeled and sorted using a FACS (Synergy iCyt). We sorted the top 1–2% of the green and red fluorescence intensity area. These cells (3–5*104) were collected into rich medium and plated for a new round of sorting. The naïve library was sorted using a sorting gate that included the top 5% of fluorescent cells. Three iterative rounds of enrichment were performed.For fluorescence-activated cell sorting (FACS), the naïve DR3 library was induced and labeled with antibodies against c-myc and biotinylated TL1A, as described above. More than 10DR3 was PCR-amplified from DNA extracted from the last round of FACS sorting as previously described [escribed , cloned escribed  with minescribed .3SO4, 50 mM Tris-HCl, pH 7.3 and then loaded on a butyl HIC column (GE). The butyl column was washed for more than 20 CVs with 1 M NH3SO4, 200 mM NaCl, 25 mM Na-citrate, pH 5.8 and eluted by applying a 21 CV gradient to buffer containing 50 mM citrate, 25 mM NaCl, pH 5.8, and elution fractions were collected and analyzed by SDS PAGE. Fractions containing DR3-Fc at a purity of 90% or higher were pooled, dialyzed against PBS containing 1.5 mM DTT and flash-frozen in liquid nitrogen in small aliquots for future use.Plasmid pFUSE containing the DR3 variants was transfected into 500 ml of 293F cells in Freestyle medium according to the manufacturer’s procedure. Six days post-transfection, the media were collected and concentrated five-fold using a 10K Amicon ultrafiltration device, the concentrated supernatant was subsequently diluted six-fold with 30 mM Tris-HCl, pH 8.3 (buffer A) and the supernatant was loaded onto a MonoQ column (GE). Next, the column was washed with 20 column volumes of buffer A and the protein was eluted by applying a gradient of 20 column volumes (CVs) of buffer A to buffer B , with fractions being collected during the entire elution process. Fraction activity was measured by ELISA, and active fractions were pooled dialyzed against PBS containing 1.5 mM DTT and then diluted ten-fold into 50 mM Na-citrate, 25 mM NaCl, pH 5.8 (buffer C). The diluted active fractions were directly loaded on a SP column (GE), and the column was washed with buffer C until the OD 280 was stable. The SP column was eluted by applying a gradient from buffer C to a buffer containing 50 mM Na-citrate, 450 mM NaCl, pH 5.8 (buffer D) with a gradient length of 23 CVs, with fractions being collected during the whole elution process. Samples were run on an SDS PAGE gel and fractions that contained a major band of approximately 60 kDa corresponding to DR3-Fc fusion protein were pooled. The pooled fractions were diluted two-fold into 2 M NHTo measure the binding affinity of DR3 variants to TL1A, SPR was performed using the ProteOn XPR36 (Bio-Rad) instrument as previously described . All samTo examine the stability of the DR3 variants in serum, 20 μg/ml of DR3-FC, H3 and O6 mutants were incubated in human serum type AB (Sigma) at 37 or 42°C. Samples were taken following 30 min of incubation, centrifuged and added to TF-1 cells together with 8 ng/ml TL1A, 10 μg/ml cycloheximide . Following six hours of incubation, the cells were lysed and cleavage of DEVD-AMC by caspase-3 was monitored in a Tecan Infinite M200 plate reader with the excitation filter set at 350 nm and the emission filter set at 450 nm.To measure thermal denaturation using CD, 120 μg/ml (2.58 μM) of DR3-Fc and mutants in PBS containing 1 mM DTT were denatured by raising the temperature from 30°C to 70°C at a heating rate of 0.37°C/min and a pitch of 0.5°C using a JASCO Peltier PTC 423S apparatus. The CD signal at 215 nm was measured during the denaturation process using a JASCO J815 CD spectrometer. Full spectrum (190–260 nm) measurements were taken at 30°C and 70°C to determine the level of secondary structure in DR3-Fc and mutants. The data were normalized to a fraction of folded protein and the melting temperature (Tm) was derived by fitting the normalized data to a logistic model using SigmaPlot software.+ T cell subset was performed using a CD4+ isolation kit  as described by the manufacturer. PBL or CD4+ cells were incubated with IL-12 (2 ng/ml) and IL-18 (50 ng/ml) with or without TL1A (100 ng/ml) or DR3 variants at different concentrations for 72 h. The cultured media were collected and IFN-γ levels were quantitated using ELISA kits (PeproTech) according to the manufacturer’s procedure.PBMCs  were isolated from the blood of normal healthy volunteers using Lymphoprep  according to the manufacturer’s instructions. The PBL  fraction was isolated by incubation of the PBMCs in complete RPMI medium in a flask at 37°C for three hours, with the non-adherent fraction being designated as the PBL fraction. The isolation of the CD45/ml) in RPMI medium containing 1% FBS  in a black 96-well plate with a clear bottom (Greiner bio-one) at a final volume of 100 μl. Eight ng/ml TL1A, 10 μg/ml cycloheximide, and 0.625–20 μg/ml DR3-Fc (native or mutants) were added, as indicated. After six hours, 100 μl of lysis buffer  were added to each well and the plate was incubated for ten minutes at 37°C. Subsequently, cleavage of DEVD-AMC by caspase-3 was monitored in a Tecan Infinite M200 plate reader with the excitation filter set at 350 nm and the emission filter set at 450 nm.TF-1 cells were seeded at 75,000 cells/well , hinders high expression of this protein in a bacterial system. Previously, our attempts to express a soluble IL-17 receptor led to a high level of aggregated protein that was not active in cell-based assays [DR3 is a receptor containing an N-terminal signal peptide followed by an ECD composed of 171 amino acids. The ECD contains 28 cysteines which constitute ~15% percent of the domain and likely form a complex network of disulfide bonds. The large number of cysteines, as well as the two predicated N-glycosylation and three predicated O-glycosylation sites  to induce caspase-3 release that can lead to apoptosis in TF-1 cells . We explectively . Such deEarlier analysis of engineered proteins containing back-to-consensus mutations indicated a significant increase in the stability of the target protein. We have previously shown that back-to-consensus mutations in selected IL-17R or sulfotransferase 1E1 variants led to significant increases in stability . To evalhttp://zhanglab.ccmb.med.umich.edu/I-TASSER/) [In this work, we employed directed evolution to generate DR3 variants with improved TL1A affinity, stability and biological activity. Sequence analysis of the six mutants that were initially analyzed showed that some of the mutations were highly abundant (e.g. V47P), while others (e.g. E38K) are found in only one or two variants . The H3 TASSER/) . This moTASSER/) . The DR3TASSER/) . Mapping6 mutants, followed by screening of a manageable size of ~250 individual mutants following expression in mammalian cells. Of the ~250 mutants screened, only two mutants (<1%) exhibited improved activity, suggesting that it would have been extremely difficult to isolate these variants solely based on yeast surface display or HEK293T-based screening systems.As demonstrated before, the ECD of many receptors is highly glycosylated and such glycosylation can significantly contribute to receptor conformation and binding to the target ligand . Thus, a+ and TF-1 cells showed the high potency of H3 in inhibiting TL1A-induced IFN-γ secretion and apoptosis, respectively  Alignment of mammalian DR3 proteins identifies residues that deviate from the family consensus. Highlighted are the V44, D48 and W53 positions of human DR3 that deviate from the family consensus. (B) The percentage of identity of each DR3 orthologue to human DR3. (C) The oligonucleotide spiking process for obtaining back-to-consensus mutations in the DR3 gene (see ((TIF)Click here for additional data file.S2 Fig(A) Single clones PCR analysis from DR3 library following 1, 3 and 4 rounds of enrichment. A significant enrichment for shorter genes is observed at the 4th round of enrichment impeding subsequent screening using the yeast surface display system. (B) Flow cytometry histogram analysis of a cell population displaying a naïve DR3 mutant library (R0- black), and libraries following one round of enrichment (R1-light blue), two rounds of enrichment (R2- orange) and three rounds of enrichment (R3-green), left- assessing the level of DR3 display with anti-DR3 antibodies and right—assessing the display of the full length DR3 using anti-myc antibodies to a myc tag located on the C-terminal of DR3. (C) Dot-plot analysis of the TL1A binding analyzed using streptavidin-APC conjugated against biotinylated TL1A and display levels using anti-myc antibodies. The data indicate no significant increase in DR3 display in the third round of enrichment (D) Cloning of the full length genes of the FACS-enriched library in the mammalian expression vector. Sub-cloning was performed to avoid contamination of short DR3 variants as false positives (see main text for details).(TIF)Click here for additional data file.S3 FigA) Schematics of the ELISA for DR3 binding to TL1A. The ELISA plate is coated with anti-TL1A antibodies (green) and subsequently, TL1A (blue). Different DR3 variants (red) are then added to the plate and binding to TL1A is detected using specific biotinylated anti-DR3 antibodies as the primary antibody (yellow) and streptavidin-HRP (red). (B) DR3 calibration curve. Commercially available native DR3 at five different concentrations was used in the TL1A-binding ELISA assay, as described in Material and Methods.((TIF)Click here for additional data file.S4 FigELISA binding signals are presented as fold increase relative to the ELISA signal obtained with native DR3, used as a control during the screening.(TIF)Click here for additional data file.S5 FigA) The molecular weight (MW) of the DR3 variants is ~60 kDa, while the calculated MW is 45 kDa. (B) Deglycosylation of native DR3, and the H3 and O6 variants using Endo-H enzyme. Following incubation with the enzyme, a ~10 kDa reduction in the MW of the proteins was observed, indicating the contribution of N-linked glycosylation to the MW of the proteins. The blue error points to the DR3 band on the gel.((TIF)Click here for additional data file.S6 FigCells were incubated for 72 hours with 100 ng/ml TL1A, 20 ng/ml IL-12 and 50 ng/ml IL-18 and different concentrations of soluble native DR3 and the H3 and O6 variant receptors. The 1:10 diluted cell supernatant was analyzed by ELISA for detection of IFN-γ levels. The IFN-γ levels presented here were calculated according to an IFN-γ calibration curve. Black stars denote measurements that are statistically different from no receptor (DR3 = 0) with a p < 0.03 while red stars are measurements that are statistically different between the O6 and native versions of the protein (p < 0.05).(TIF)Click here for additional data file.S7 FigS4 Fig and S6 Fig). Cells were incubated for 72 hours with 100 ng/ml TL1A, 20 ng/ml IL-12 and 50 ng/ml IL-18 and different concentrations of soluble native DR3 and the G6, N8, I12 variants (A) or native DR3 and H3 and A7 variant (B) receptors. The 1:10 diluted cell supernatant was analyzed by ELISA for detection of IFN-γ levels. The IFN-γ levels presented here were calculated according to an IFN-γ calibration curve. Black stars denote measurements that are statistically different (p < 0.05) from no receptor (DR3 = 0).In contrast, the H3 and G6 variants are improved, relative to the native protein Click here for additional data file.S8 FigCells were incubated for 72 hours with 100 ng/ml TL1A, 20 ng/ml IL-12 and 50 ng/ml IL-18 and different concentrations of soluble DR3 variants. The 1:10 diluted cell supernatant was analyzed by ELISA for detection of IFN-γ levels. The IFN-γ levels presented here were calculated according to an IFN-γcalibration curve. Black stars denote measurements that are statistically different (p < 0.05) from no receptor (DR3 = 0).(TIF)Click here for additional data file.S9 Fighttp://zhanglab.ccmb.med.umich.edu/I-TASSER/). The structure shown was generated using the UCSF Chimera program and is presented from the surface view. The positions mutated in the engineered variants are located on the surface of the DR3 model structure and are located at two faces of the molecules (A- front view and B-back view). The I18, V47, D51, N61, K93, Q101, Q104, L129 (Table 1) positions are highlighted in purple.The structural model of DR3 was generated using the I-TASSER server ((TIF)Click here for additional data file.