A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

Revealing parental mosaicism: the hidden answer to the recurrence of apparent de novo variants.

Mosaicism refers to the presence of two or more populations of genetically distinct cells within an individual, all of which originate from a single zygote. Previous literature estimated the percentage of parental mosaicism ranged from 0.33 to 25.9%. In this study, parents whose children had previously been diagnosed with developmental disorders with an apparent de novo variant were recruited. Peripheral blood, buccal and semen samples were collected from these parents if available for the detection of potential parental mosaicism using droplet digital PCR, complemented with the method of blocker displacement amplification. Among the 20 families being analyzed, we report four families with parental mosaicism (4/20, 20%). Two families have maternal gonosomal mosaicism (EYA1 and EBF3) and one family has paternal gonadal mosaicism (CHD7) with a pathogenic/ likely pathogenic variant. One family has a paternal gonosomal mosaicism with a variant of uncertain significance (FLNC) with high clinical relevance. The detectable variant allele frequency in our cohort ranged from 8.7-35.9%, limit of detection 0.08-0.16% based on our in-house EBF3 assay. Detecting parental mosaicism not only informs family with a more accurate recurrence risk, but also facilitates medical teams to create appropriate plans for pregnancy and delivery, offering the most suitable care.

Actionable pharmacogenetic variants in Hong Kong Chinese exome sequencing data and projected prescription impact in the Hong Kong population.

Preemptive pharmacogenetic testing has the potential to improve drug dosing by providing point-of-care patient genotype information. Nonetheless, its implementation in the Chinese population is limited by the lack of population-wide data. In this study, secondary analysis of exome sequencing data was conducted to study pharmacogenomics in 1116 Hong Kong Chinese. We aimed to identify the spectrum of actionable pharmacogenetic variants and rare, predicted deleterious variants that are potentially actionable in Hong Kong Chinese, and to estimate the proportion of dispensed drugs that may potentially benefit from genotype-guided prescription. The projected preemptive pharmacogenetic testing prescription impact was evaluated based on the patient prescription data of the public healthcare system in 2019, serving 7.5 million people. Twenty-nine actionable pharmacogenetic variants/ alleles were identified in our cohort. Nearly all (99.6%) subjects carried at least one actionable pharmacogenetic variant, whereas 93.5% of subjects harbored at least one rare deleterious pharmacogenetic variant. Based on the prescription data in 2019, 13.4% of the Hong Kong population was prescribed with drugs with pharmacogenetic clinical practice guideline recommendations. The total expenditure on actionable drugs was 33,520,000 USD, and it was estimated that 8,219,000 USD (24.5%) worth of drugs were prescribed to patients with an implicated actionable phenotype. Secondary use of exome sequencing data for pharmacogenetic analysis is feasible, and preemptive pharmacogenetic testing has the potential to support prescription decisions in the Hong Kong Chinese population.

Heart transplantation after total artificial heart bridging-Outcomes over 15 years.

BACKGROUND: Data are limited on outcomes after heart transplantation in patients bridged-to-transplantation (BTT) with a total artificial heart (TAH-t). METHODS: The UNOS database was used to identify 392 adult patients undergoing heart transplantation after TAH-t BTT between 2005 and 2020. They were compared with 11 014 durable left ventricular assist device (LVAD) BTT patients and 22 348 de novo heart transplants (without any durable VAD or TAH-t BTT) during the same period. RESULTS: TAH-t BTT patients had increased dialysis dependence compared to LVAD BTT and de novo transplants (24.7% vs. 2.7% vs. 3.8%) and higher levels of baseline creatinine and total bilirubin (all p < .001). After transplantation, TAH-t BTT patients were more likely to die from multiorgan failure in the first year (25.0% vs. 16.1% vs. 16.1%, p = .04). Ten-year survival was inferior in TAH-t BTT patients (TAH-t BTT 53.1%, LVAD BTT 61.8%, De Novo 62.6%, p < .001), while 10-year survival conditional on 1-year survival was similar (TAH-t BTT 66.8%, LVAD BTT 68.7%, De Novo 69.0%, all p > .20). Among TAH-t BTT patients, predictors of 1-year mortality included higher baseline creatinine and total bilirubin, mechanical ventilation, and cumulative center volume <20 cases of heart transplantation involving TAH-t BTT (all p < .05). CONCLUSION: Survival after TAH-t BTT is acceptable, and patients who survive the early postoperative phase experience similar hazards of mortality over time compared to de novo transplant patients and durable LVAD BTT patients.

Social isolation, inflammation, and cancer mortality from the National Health and Nutrition Examination Survey - a study of 3,360 women.

BACKGROUND: This study evaluates the role of social isolation on inflammation and cancer mortality among women. METHODS: Data were abstracted from the U.S. National Health and Nutrition Examination Survey from 1988 to 1994. The Social Network Index was used to assess participants' degree of social isolation. C-reactive protein and fibrinogen levels were included as markers of inflammation. We used the National Death Index to identify causes and dates of mortality. Chi-square and multivariable Cox regressions were employed for statistical analyses. RESULTS: Of 3360 women (median age: 54 years), the most isolated, very isolated, somewhat isolated, and not isolated comprised 14.5, 30.2, 37.1, and 18.2% of the sample, respectively. The most isolated participants were more likely to have low income (56.8% vs 12.2%, p < 0.001), have fewer years of education (40.8% vs 12.3%; p < 0.001), have low physical activity (27.3% vs 14.7%; p < 0.003), be obese (32.5% vs 24.4%; p = 0.02), and be current smokers (34.2% vs 10.3%; p < 0.001) compared to the not isolated ones. Mean fibrinogen levels increased with degree of social isolation (p = 0.003), but C-reactive protein showed no association (p = 0.52). Kaplan-Meier estimates indicated higher cancer mortality rates among participants with elevated fibrinogen levels, though not with statistical significance (p = 0.08). Furthermore, there was no association between social isolation and cancer mortality (p = 0.54). On multivariate analysis, obesity (HR = 1.56; 95% CI: 1.11-2.18), higher education (HR = 1.36; 95% CI: 1.01-1.83), and smoking (HR = 4.42, 95% CI: 2.84-6.88) were independent predictors for cancer mortality, while high physical activity predicted for lower mortality from cancer (HR = 0.67, 95% CI: 0.51-0.87). However, social isolation was not a predictor. CONCLUSION: Social isolation among women was associated with an increased level of fibrinogen, but not associated with cancer mortality. The relationship between inflammation and cancer mortality warrants further investigation.

Mandibular asymmetry as a possible etiopathologic factor in temporomandibular disorder: a prospective cohort of 134 patients.

OBJECTIVES: The present study aimed to investigate mandibular asymmetry as a possible etiopathologic factor in temporomandibular disorder (TMD). MATERIALS AND METHODS: A prospective cross-sectional study of patients with dentofacial deformities seeking corrective orthognathic surgery was conducted. The pre-operative prevalence of TMD in patients with mandibular asymmetry and other dentofacial deformities was assessed using the Diagnostic Criteria for TMD (DC/TMD) Axis I protocol. RESULTS: A total of 134 patients were recruited - 82 with mandibular asymmetry and 52 without. There was a significantly higher prevalence of TMD in those with mandibular asymmetry (67.1%; 95% CI 59 to 75%) compared to those without (40.4%; 95% CI 32 to 49%, p = 0.002). The overall pre-operative prevalence of TMD in this population of patients was 56.7% (95% CI 48 to 65%). Pain disorder only was present in 9.7%, TMJ disorder only in 29.9%, and both pain and TMJ disorders in 17.2%. The most prevalent type of TMD is disc displacement with reduction (77.6%), followed by myalgia (35.5%) and arthralgia (21.1%). CONCLUSION: The prevalence of TMD in those with mandibular asymmetry was significantly higher than those without, suggesting that mandibular asymmetry could be a possible etiopathologic factor in TMD. CLINICAL RELEVANCE: The significantly higher prevalence of temporomandibular disorder in those with mandibular asymmetry suggests that we need to be especially cognizant of this condition in our pre-operative, surgical, and post-operative management of this group of orthognathic patients.

Income, inflammation and cancer mortality: a study of U.S. National Health and Nutrition Examination Survey mortality follow-up cohorts.

BACKGROUND: To estimate the relationship between inflammatory biomarkers and cancer mortality in a nationally representative sample of the U.S. population while controlling for education, occupation, and income. METHODS: Data were obtained from the U.S. National Health and Nutrition Examination Survey from 1988 to 1994 (N = 7817) and 1999-2002 (N = 2344). We fit Cox proportional hazard models to examine the relationship between C-reactive protein (CRP) and fibrinogen with cancer mortality. RESULTS: In the full Cox multivariate model, clinically raised CRP was associated with cancer mortality in NHANES 1988-1994 (> 0.99 mg/dL: 95%CI: 1.04-2.13). However, across two inflammatory biomarkers (CRP and Fibrinogen), two NHANES time periods (1998-1994 and 1999-2002) and three income levels (12 strata in total), Hazard ratio confidence intervals did not include the null only for one association: CRP and cancer mortality among low income participants from 1988 to 1994 (HR = 1.83, 95% CI: 1.10-3.04). CONCLUSIONS: We find evidence that only in one unique stratum is earlier life CRP, and not fibrinogen, associated with prospective cancer mortality. After more complete control for socioeconomic confounding, CRP and fibrinogen do not predict cancer mortality in most subpopulations.

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs.

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.

Regulatory barriers to xenotransplantation.

PURPOSE OF REVIEW: There is a grave discordance between supply and demand for patients with failing organs largely due to an insufficient donor pool for transplantation. Xenotransplantation has been proposed as a solution to bridge this gap. RECENT FINDINGS: Recent success over the last decade in nonhuman primate models, due to emerging gene-editing technologies combined with novel immunosuppression regimens, has produced promising results in pancreatic islet cell, heart, lung, kidney and liver xenotransplantations. SUMMARY: As the prospect of xenotransplantation is realized, safety and ethical considerations have come to the forefront of discussion. The WHO and World Health Assembly have encouraged member states to form regulatory bodies to govern human xenotransplantation studies with the highest standards. Here, we summarize the current regulatory landscape governing preclinical advances toward the first human clinical trials.

CD4+CD25Hi FoxP3+ regulatory T cells in long-term cardiac xenotransplantation.

BACKGROUND: CD4+CD25Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors. METHODS: Heterotopic cardiac xenotransplantation was performed on baboons using hearts obtained from GTKO.hCD46 (n = 8) and GTKO.hCD46.TBM (n = 5) genetically modified pigs. Modified immunosuppression regimen included antithymocyte globulin (ATG), anti-CD20, mycophenolate mofetil (MMF), cobra venom factor (CVF), and costimulation blockade (anti-CD154/anti-CD40 monoclonal antibody). FACS analysis was performed on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies (mAb) to analyze the percentage of Treg cells in six baboons that survived longer than 2 months (range: 42-945 days) after receiving a pig cardiac xenograft. RESULTS: Total WBC count was low due to immunosuppression in baboons who received cardiac xenograft from GTKO.hCD46 and GTKO.hCD46.hTBM donor pigs. However, absolute numbers of CD4+CD25Hi FoxP3 Treg cells in PBLs of long-term xenograft cardiac xenograft surviving baboon recipients were found to be increased (15.13 ± 1.50 vs 7.38 ± 2.92; P < .018) as compared to naïve or pre-transplant baboons. Xenograft rejection in these animals was correlated with decreased numbers of regulatory T cells. CONCLUSION: Our results suggest that regulatory T (Treg) cells may contribute to preventing or delaying xenograft rejection by controlling the activation and expansion of donor-reactive T cells, thereby masking the antidonor immune response, leading to long-term survival of cardiac xenografts.

Consideration of appropriate clinical applications for cardiac xenotransplantation.

The field of cardiac xenotransplantation has entered an exciting era due to recent advances in the field. Although several hurdles remain, the use of rapidly evolving transgenic technology has the potential to address current allogeneic donor pool constraints and mechanical circulatory system device limitations. The success of xenotransplantation will undoubtedly be dependent on specific patient selection criteria. Defining these particular indications for xenotransplantation is important as we approach the possibility of clinical applications.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model.

BACKGROUND: Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six-gene modifications within a pig-to-baboon cardiac xenotransplantation model. METHODS: Baboons (8-15 kg) underwent heterotopic cardiac transplantation using xenografts obtained from genetically engineered pigs. Along with previously described modifications (GTKO, hCD46), additional expression of human transgenes for thromboregulation (endothelial protein C receptor, tissue factor pathway inhibitor, thrombomodulin), complement inhibition (decay accelerating factor), and cellular immune suppression (hCD39, hCD47) was used. Immunosuppression consisted of targeted T-cell and B-cell depletion and conventional anti-rejection agents. RESULTS: Heterotopic cardiac transplantations were performed without complication. Flow cytometry and immunohistochemistry on donor biopsies confirmed transgenic phenotype. In contrast to the prior three-gene generation, significant coagulopathy or consumptive thrombocytopenia has not been observed in the six-gene cohort. As a result, these recipients have experienced decreased bleeding-related complications. Pro-inflammatory responses also appear to be mitigated based on cytokine analysis. Baboons survived the critical 30-day post-operative period when mortality has historically been highest, with no evidence of graft rejection. CONCLUSIONS: The inclusion of additional human genes in genetically engineered pigs appears to confer superior xenograft outcomes. Introduction of these genes has not been associated with adverse outcomes. This multifactorial approach to genetic engineering furthers the prospect of long-term cardiac xenograft survival and subsequent clinical application.

Heart xenotransplantation.

PURPOSE OF REVIEW: Cardiac xenotransplantation has entered an exciting era, marked by numerous considerable advances in the field, and continues to be of great interest because of its potential ability in ameliorating the current constraints of limited allograft availability. This review aims to examine recent progress in this rapidly changing discipline. RECENT FINDINGS: Although several hurdles remain, the use of rapidly evolving transgenic technology, in combination with novel immunosuppression regimens, has the potential to address current allogenic donor pool constraints and mechanical circulatory system device limitations. Furthermore, innovative uses of biomarker tools, such as miRNA, serve as a method for improved monitoring of xenograft status. These tools may allow for more targeted immunosuppressive strategies as well as earlier interventions to mitigate xenograft rejection. Finally, coinciding with these remarkable advances, preliminary consideration of the clinical application for cardiac xenotransplantation has begun, particularly regarding specific patient criteria for these initial clinical trials. SUMMARY: The studies in this review highlight efforts in multiple disciplines to optimize perioperative and postxenotransplant outcomes. In examining these individual topics, this article reflects the exciting and ongoing progress in the field of cardiac xenotransplantation.

The influence of age and other prognostic factors associated with survival of ovarian immature teratoma - A study of 1307 patients.

OBJECTIVE: To determine impact of age and other prognostic factors on the survival of ovarian immature teratoma (IT) patients. METHODS: Data obtained from the SEER database between 1973 and 2012. Kaplan-Meier methods and multivariate Cox regression models were used for statistical analyses. RESULTS: Of 1307 patients (median: 24years; range: 0-93), 78%, 5%, 13%, 4% were stages I, II, III and IV, respectively. 25%, 35%, and 40% had grades 1, 2, and 3. Whites were less likely to be diagnosed, and Asians had a nearly 3-fold higher proportion of IT compared to the proportion of Asians in the U.S. census. The 5-year disease-specific survival (DSS) was 91.2%. Those with stages I, II, III and IV disease had survivals of 99.7%, 95%, 81%, and 71.8% (p<0.001) and grades 1, 2, and 3 had DSS of 98.7%, 95.8%, and 91% (p<0.001), respectively. Of those who underwent fertility-preserving surgery, the DSS was 98.8%. Over time from 1973 to 1986, to 1987-1999, to 2000-2012, the survivals were 76.4%, 92.8%, and 94.7% (p<0.001). Of stage I patients, no patient <18years (n=214, used as adult cutoff) and 2 of 283 patients >18years died of cancer, with corresponding 5years DSS of 100% vs. 99.6% (p>0.05). Older age (by year, HR: 1.05; 95% CI: 1.04-1.06; p<0.0001) and higher stage (HR: 11.52; 95% CI: 4.08-32.48; p<0.0001) were independent factors indicating poorer survival. CONCLUSION: The outcome of patients with stage I disease was excellent at 99.7%, with children and adults having corresponding survivals of 100% and 99.6%.

Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

Impact of ethnicity on human umbilical cord blood banking: a systematic review.

BACKGROUND: How ethnicity impacts characteristics of umbilical cord blood collected by cord blood banks remains unclear. STUDY DESIGN AND METHODS: After a systematic search, we identified 11 studies for analysis that reported on various variables of collected cord blood units. RESULTS: Non-Caucasian ethnicity of the cord blood donor was associated with a higher risk of failing to meet banking criteria, lower cord blood volume, reduced total nucleated cell count, fewer CD34+ cells, and reduced colony-forming units. CONCLUSIONS: Non-Caucasian ethnicity is associated with reductions in hematopoietic measures of collected cord blood units. Public banking efforts will have to balance issues related to building ethnic diversity within the bank and maintaining the characteristics of banked units that remain desirable by transplant centers.

Simpson-Golabi-Behmel syndrome type 1 with normal birth parameters.

A newborn baby born at 34 weeks and 5 days gestation was admitted for prematurity, dysmorphic features and congenital heart defects. Antenatal scan at 21 weeks showed a large-for-gestational-age foetus with a large abdominal circumference and liver, ventricular septal defect, right prominent renal pelvis and echogenic bowel. Antenatal genetic tests for overgrowth syndromes were negative. The mother had early onset pre-eclampsia. After birth, an overgrowth syndrome was still suspected despite the baby having normal birth parameters. Raw data of the trio whole exome sequencing from the amniocentesis sample were manually inspected. Hemizygous exon 7 deletion in the GPC3 gene was found, and a postnatal diagnosis of Simpson-Golabi-Behmel syndrome, a rare overgrowth syndrome, was made. This case report discusses the significance of antenatal findings, an atypical presentation of a rare syndrome and the obstacles of diagnostic genetic testing.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.