Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice.

One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genome's impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false-positive results. Here, we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial-related disease and the influence of nutrients on DNA mutation rates.

The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Automated solid phase DNA extraction on a lab-on-a-disc with two-degrees of freedom instrumentation.

BACKGROUND: Lab-on-a-disc (LoaD) technology has emerged as a transformative approach for point-of-care diagnostics and high-throughput testing. The promise of integrating multiple laboratory functions onto a single integrated platform has significant implications for healthcare, especially in resource-limited settings. However, one of the primary challenges faced in the design and manufacture of LoaD devices is the integration of effective valving mechanisms. These valves are essential for fluid control and routing, but their intricacy often leads to complexities in design and increased vulnerability to failure. This emphasizes the need for improved designs and manufacturing processes without complex, integrated valving mechanisms. (96) RESULTS: We describe a fully automated biological workflow and reagent actuation on a LoaD device without an integrated valving system. The Two Degrees-of-Freedom (2DoF) custom centrifuge alters the centre of rotation, facilitating fluid flow direction changes on the microfluidic platform through a custom programmed interface. A novel 360-degree fluid manipulation approach via secondary planetary gear motion enabled sequential assay reagent actuation without embedded valve triggering, with the addition of infinite incubation times and efficient use of platform realty. The simplified LoaD platform uses clever design, with intermediate flow chambers to avoid cross contamination between reagent steps. Notably, the optimized LoaD platform demonstrated a two-fold DNA yield at higher HEK-293 cell concentrations compared to commercially available spin-column kits. This significantly simplified LoaD platform successfully automated a common, complex workflow without inhibiting DNA purification. (129) SIGNIFICANCE: This system exhibits the clever coupling of both 2DoF and centrifugal microfluidics to create an autonomous testing package capable of eradicating the need for complex valving systems to automate biological workflows on LoaDs. This automated system has outperformed commercially available DNA extraction kits for higher cell counts. The platform's elimination of valve requirements ensures unlimited sample incubation times and enhances reliability, making it a straightforward option for automated biological workflows, particularly in diagnostics. (73).

Home values and experiences navigation track (HomeVENT): Supporting decisions about pediatric home ventilation.

Objective: To pilot feasibility and acceptability of HomeVENT, a systematic approach to family-clinician decision-making about pediatric home ventilation. Methods: Parents and clinicians of children facing home ventilation decisions were enrolled at 3 centers using a pre/post cohort design. Family interventions included: 1) a website describing the experiences of families who previously chose for and against home ventilation 2) a Question Prompt List (QPL); 3) in-depth interviews exploring home life and values. Clinician HomeVENT intervention included a structured team meeting reviewing treatment options in light of the family's home life and values. All participants were interviewed one month after the decision. Results: We enrolled 30 families and 34 clinicians. Most Usual Care (14/15) but fewer Intervention (10/15) families elected for home ventilation. Families reported the website helped them consider different treatment options, the QPL promoted discussion within the family and with the team, and the interview helped them realize how home ventilation might change their daily life. Clinicians reported the team meeting helped clarify prognosis and prioritize treatment options. Conclusions: The HomeVENT pilot was feasible and acceptable. Innovation: This systematic approach to pediatric home ventilation decisions prioritizes family values and is a novel method to increase the rigor of shared decision-making in a rushed clinical environment.

Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing.

The analysis of somatic variation in the mitochondrial genome requires deep sequencing of mitochondrial DNA. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. We isolated intact mitochondria using differential centrifugation and alkaline lysis and subjected purified mitochondrial DNA to a sequence-independent and PCR-free method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mitochondrial DNA enrichment. Previously published methods employing mitochondrial DNA purification did not measure mitochondrial DNA enrichment or utilise high coverage short-read sequencing. Here, we describe a protocol that yields mitochondrial DNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are incorrectly identified as heteroplasmy when long-range PCR is used.

A one-step reverse transcription recombinase polymerase amplification assay for lateral flow-based visual detection of PVY.

Potato virus Y (PVY) is an abundant and damaging virus which reduces crop yield and marketability. Accurate detection of this economically important virus both in-field and in seed potatoes is considered essential in the control of PVY spread. Current detection methods are focused on immunodetection and PCR-based methods, however, identification of PVY through isothermal amplification is a promising avenue for developing accessible, on-site diagnostics with quick turnaround times. In this work, a rapid recombinase polymerase amplification assay was developed which could readily amplify PVY nucleic acids with good sensitivity and specificity. Additionally, this assay was shown to be capable of amplification directly from RNA in a one-step amplification process, without the need for prior reverse transcription. The assay was coupled with lateral flow technology to provide a rapid visual confirmation of amplification. This nucleic-acid lateral flow immunoassay could feasibly be employed in-field, or at any location where testing is required, to aid in the detection and control of PVY.

Monitoring of emerging contaminants of concern in the aquatic environment: a review of studies showing the application of effect-based measures.

Water scarcity is increasingly a global cause of concern mainly due to widespread changes in climate conditions and increased consumptive water use driven by the exponential increase in population growth. In addition, increased pollution of fresh water sources due to rising production and consumption of pharmaceuticals and organic chemicals will further exacerbate this concern. Although surface water contamination by individual chemicals is often at very low concentration, pharmaceuticals for instance are designed to be efficacious at low concentrations, creating genuine concern for their presence in freshwater sources. Furthermore, the additive impact of multiple compounds may result in toxic or other biological effects that otherwise will not be induced by individual chemicals. Globally, different legislative frameworks have led to pre-emptive efforts which aim to ensure good water ecological status. Reports detailing the use and types of effect-based measures covering specific bioassay batteries that can identify specific mode of actions of chemical pollutants in the aquatic ecosystem to evaluate the real threat of pollutants to aquatic lives and ultimately human lives have recently emerged from monitoring networks such as the NORMAN network. In this review, we critically evaluate some studies within the last decade that have implemented effect-based monitoring of pharmaceuticals and organic chemicals in aquatic fauna, evaluating the occurrence of different chemical pollutants and the impact of these pollutants on aquatic fauna with special focus on pollutants that are contaminants of emerging concern (CEC) in urban wastewater. A critical discussion on studies that have used effect-based measures to assess biological impact of pharmaceutical/organic compound in the aquatic ecosystem and the endpoints measurements employed is presented. The application of effect-based monitoring of chemicals other than assessment of water quality status is also discussed.

Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA Profiling.

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.

A review of pharmaceutical occurrence and pathways in the aquatic environment in the context of a changing climate and the COVID-19 pandemic.

Active pharmaceutical ingredients (APIs) are increasingly being identified as contaminants of emerging concern (CECs). They have potentially detrimental ecological and human health impacts but most are not currently subject to environmental regulation. Addressing the life cycle of these pharmaceuticals plays a significant role in identifying the potential sources and understanding the environmental impact that pharmaceuticals may have in surface waters. The stability and biological activity of these "micro-pollutants" can lead to a pseudo persistence, with ensuing unknown chronic behavioural and health-related effects. Research that investigates pharmaceuticals predominantly focuses on their occurrence and effect within surface water environments. However, this review will help to collate this information with factors that affect their environmental concentration. This review focuses on six pharmaceuticals (clarithromycin, ciprofloxacin, sulfamethoxazole, venlafaxine, gemfibrozil and diclofenac), chosen because they are heavily consumed globally, have poor removal rates in conventional activated sludge wastewater treatment plants (CAS WWTPs), and are persistent in the aquatic environment. Furthermore, these pharmaceuticals are included in numerous published prioritisation studies and/or are on the Water Framework Directive (WFD) "Watch List" or are candidates for the updated Watch List (WL). This review investigates the concentrations seen in European Union (EU) surface waters and examines factors that influence final concentrations prior to release, thus giving a holistic overview on the source of pharmaceutical surface water pollution. A period of 10 years is covered by this review, which includes research from 2009-2020 examining over 100 published studies, and highlighting that pharmaceuticals can pose a severe risk to surface water environments, with each stage of the lifecycle of the pharmaceutical determining its concentration. This review additionally highlights the necessity to improve education surrounding appropriate use, disposal and waste management of pharmaceuticals, while implementing a source directed and end of pipe approach to reduce pharmaceutical occurrence in surface waters.

The application of CRISPR-Cas for single species identification from environmental DNA.

We report the first application of CRISPR-Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR-based techniques to detect a given species from eDNA samples, posing a logistical challenge for on-site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR-Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof-of-concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.

Assessment of candidate folate sensitive-differentially methylated regions in a randomised controlled trial of continued folic acid supplementation during the second and third trimesters of pregnancy.

SCOPE: The aim of this study was to identify if specific regions of the human genome were sensitive to folate status by displaying changes in their DNA methylation patterns in response to continued folic acid supplementation during pregnancy. METHODS AND RESULTS: Samples (n = 119) from a previous randomised controlled trial in pregnancy were used to compare the DNA methylation profiles of the same woman pre- versus post-folic acid intervention. Candidate genes were identified from the literature and a pilot genome wide screen of six women (three from each of the folic acid and placebo arms of the trial). We did not observe consistent DNA methylation changes in response to folic acid intervention at any of our candidate genes (RASA4, DHFR, DHFR2, RASSF1A, EIF2C3, ATPF1). We did identify a 40% decrease in DNA methylation at the RASA4 promoter correlating with a 3.5-fold increase in its mRNA abundance in an in vitro cell culture model. CONCLUSION: Continued folic acid intervention over a 22-week period did not appear to significantly influence the DNA methylation status of six candidate genes in blood samples of women compared to placebo. However, DNA methylation may play a role in the gene expression control of the RASA4 gene.

Evidence from a Randomized Trial That Exposure to Supplemental Folic Acid at Recommended Levels during Pregnancy Does Not Lead to Increased Unmetabolized Folic Acid Concentrations in Maternal or Cord Blood.

BACKGROUND: Exposure to higher intakes of folic acid (FA) from fortified foods and supplements, although largely considered beneficial, is associated with unmetabolized FA in the circulation, which has raised some health concerns. OBJECTIVE: The effect of supplemental FA at a dose of 400 µg/d during pregnancy on unmetabolized FA concentrations in maternal plasma and newborn cord blood plasma was investigated. METHODS: A new analysis was performed of blood samples from participants in a randomized trial in pregnancy. Women aged 18-35 y, who had taken 400 µg FA/d as recommended in the first trimester, were recruited at the start of trimester 2 and randomly allocated to receive either 400 µg FA/d (n = 59) or a placebo (n = 67) throughout the second and third trimesters until delivery. Unmetabolized FA concentrations in maternal and cord blood samples were measured by LC-tandem MS analysis. RESULTS: In response to the intervention from gestational week 14 through delivery, a higher proportion of women in the FA compared with the placebo group had detectable FA (≥0.27 nmol/L) in plasma, but the difference in concentrations was not statistically significant (mean ± SD: 0.44 ± 0.80 compared with 0.13 ± 0.49 nmol/L, P = 0.38). FA treatment throughout pregnancy resulted in higher cord blood plasma total folate (50.6 ± 20.1 compared with 34.5 ± 14.4 nmol/L; P = 0.004) and 5-methyltetrahydrofolate (50.4 ± 20.3 compared with 34.5 ± 14.4 nmol/L; P = 0.005) concentrations, but FA was detected only in 8 of 53 available cord blood samples, and the proportion of samples with detectable FA concentrations was similar in FA-treated and placebo groups. CONCLUSIONS: Plasma concentrations of unmetabolized FA arising from supplemental FA at a dose of 400 µg/d, in addition to FA from fortified foods, were low or undetectable in mothers and newborns. The benefits for mothers and offspring of continuing FA supplementation beyond the first trimester of pregnancy can be achieved without posing any risk of increasing unmetabolized circulating FA, even in those already exposed to FA from fortified foods.

The Dihydrofolate Reductase 19 bp Polymorphism Is Not Associated with Biomarkers of Folate Status in Healthy Young Adults, Irrespective of Folic Acid Intake.

BACKGROUND: Dihydrofolate reductase (DHFR) is essential for the conversion of folic acid to active folate needed for one-carbon metabolism. Common genetic variation within DHFR is restricted to the noncoding regions, and previous studies have focused on a 19 bp deletion/insertion polymorphism (rs70991108) within intron 1. Reports of an association between this polymorphism and blood folate biomarker concentrations are conflicting. OBJECTIVE: In this study, we evaluated whether the DHFR 19 bp deletion/insertion polymorphism affects circulating folate biomarkers in, to our knowledge, the largest cohort to address this question to date. METHODS: Healthy young Irish individuals (n = 2507) between 19 and 36 y of age were recruited between February 2003 and February 2004. Folic acid intake from supplements and fortified foods was assessed with the use of a customized food intake questionnaire. Concentrations of serum folate and vitamin B-12, red blood cell (RBC) folate, and plasma total homocysteine (tHcy) were measured. Data were analyzed with the use of linear regression models. RESULTS: Folic acid intake was positively associated with serum (P < 0.0001) and RBC (P = 0.0005) folate concentration and was inversely associated with plasma tHcy (P = 0.001) as expected. The DHFR 19 bp polymorphism was not significantly associated with either serum (P = 0.82) or RBC (P = 0.21) folate, or plasma tHcy (P = 0.20), even in those within the highest quintile of folic acid intake (>326 µg folic acid/d; P = 0.96). A nonsignificant trend toward lower RBC folate by genotype (P = 0.09) was observed in the lowest folic acid intake quintile (0-51 µg/d). CONCLUSION: In this cohort of healthy young individuals, the DHFR 19 bp deletion allele did not significantly affect circulating folate status, irrespective of folic acid intake. Our data rule out a strong functional effect from this polymorphism on blood folate concentrations.

An active second dihydrofolate reductase enzyme is not a feature of rat and mouse, but they do have activity in their mitochondria.

The identification of a second functional dihydrofolate reductase enzyme in humans, DHFRL1, led us to consider whether this is also a feature of rodents. We demonstrate that dihydrofolate reductase activity is also a feature of the mitochondria in both rat and mouse but this is not due to a second enzyme. While our phylogenetic analysis revealed that RNA-mediated DHFR duplication events did occur across the mammal tree, the duplicates in brown rat and mouse are likely to be processed pseudogenes. Humans have evolved the need for two separate enzymes while laboratory rats and mice have just one.

Replication and exploratory analysis of 24 candidate risk polymorphisms for neural tube defects.

BACKGROUND: Neural tube defects (NTDs), which are among the most common congenital malformations, are influenced by environmental and genetic factors. Low maternal folate is the strongest known contributing factor, making variants in genes in the folate metabolic pathway attractive candidates for NTD risk. Multiple studies have identified nominally significant allelic associations with NTDs. We tested whether associations detected in a large Irish cohort could be replicated in an independent population. METHODS: Replication tests of 24 nominally significant NTD associations were performed in racially/ethnically matched populations. Family-based tests of fifteen nominally significant single nucleotide polymorphisms (SNPs) were repeated in a cohort of NTD trios (530 cases and their parents) from the United Kingdom, and case-control tests of nine nominally significant SNPs were repeated in a cohort (190 cases, 941 controls) from New York State (NYS). Secondary hypotheses involved evaluating the latter set of nine SNPs for NTD association using alternate case-control models and NTD groupings in white, African American and Hispanic cohorts from NYS. RESULTS: Of the 24 SNPs tested for replication, ADA rs452159 and MTR rs10925260 were significantly associated with isolated NTDs. Of the secondary tests performed, ARID1A rs11247593 was associated with NTDs in whites, and ALDH1A2 rs7169289 was associated with isolated NTDs in African Americans. CONCLUSIONS: We report a number of associations between SNP genotypes and neural tube defects. These associations were nominally significant before correction for multiple hypothesis testing. These corrections are highly conservative for association studies of untested hypotheses, and may be too conservative for replication studies. We therefore believe the true effect of these four nominally significant SNPs on NTD risk will be more definitively determined by further study in other populations, and eventual meta-analysis.

Methods to study translated pseudogenes: in vitro translation, fusion with a tag/reporter gene, and complementation assay.

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow expression of the protein of interest either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homologue.

An NTD-associated polymorphism in the 3' UTR of MTHFD1L can affect disease risk by altering miRNA binding.

Maternal folate levels and polymorphisms in folate-related genes are known risk factors for neural tube defects (NTDs). SNPs in the mitochondrial folate gene MTHFD1L are associated with the risk of NTDs. We investigated whether different alleles of SNP rs7646 in the 3' UTR of MTHFD1L can be differentially regulated by microRNAs affecting MTHFD1L expression. We previously reported that miR-9 targets MTHFD1L and now we identify miR-197 as an additional miRNA regulator. Both of these miRNAs have predicted binding sites in the MTHFD1L 3' UTR in the region containing SNP rs7646. We have determined whether the alleles of SNP rs7646 (A/G) and miRNA expression levels affect miRNA binding preferences for the MTHFD1L 3' UTR and consequently MTHFD1L expression. Our results indicate that miR-9 and miR-197 specifically downregulate MTHFD1L levels in HEK293 and MCF-7 cells and that SNPrs7646 significantly affects miR-197 binding affinity to the MTHFD1L 3' UTR, causing more efficient posttranscriptional gene repression in the presence of the allele that is associated with increased risk of NTDs. These results reveal that the association of SNP rs7646 and NTD risk involves differences in microRNA regulation and, highlights the importance of genotype-dependent differential microRNA regulation in relation to human disease risk.

Lung transplantation assessment in a patient with COPD: a case study.

This article focuses on the lung transplant assessment process for a patient with chronic obstructive pulmonary disease (COPD). It explains the investigations undertaken and their relevance in the context of transplantation. For a patient to be accepted onto the lung transplant waiting list, it is important to establish that their lung disease is severe enough to warrant transplantation, while ensuring that the patient is well enough to undergo the procedure with the potential for a good subsequent quality of life. The article also explores the importance of supportive care, symptom management and psychosocial assessment in potential lung transplant recipients.

Anti-reflux surgery for lung transplant recipients in the presence of impedance-detected duodenogastroesophageal reflux and bronchiolitis obliterans syndrome: a study of efficacy and safety.

BACKGROUND: The aim of this study was to determine the safety of anti-reflux surgery for lung transplant recipients and assess its effect on lung function. METHODS: We retrospectively collected and analyzed data from all lung transplant recipients who underwent anti-reflux surgery at St Mary's Hospital London from July 2005 to May 2012. The indications for surgery were histologic evidence of gastroesophageal reflux aspiration on bronchoscopy biopsy specimens or a positive impedance study with symptomatic reflux or a consistent decline/fluctuating forced expiratory volume in 1 second (FEV(1)). We studied the difference in mean FEV(1) and rate of change of FEV(1), before and after fundoplication. The safety of anti-reflux surgery was determined by post-operative morbidity and mortality and compared with predicted figures, using a risk prediction model based on the P-POSSUM (Portsmouth Modification of the Physiological and Operative Severity Score for Enumeration of Mortality and Morbidity) assessment. RESULTS: Forty patients underwent laparoscopic Nissen fundoplication. Overall, mean FEV(1) declined from 2119 ± 890 to 1967 ± 1027 ml (p = 0.027), and mean rate of change in FEV(1) improved from -2.42 ± 4.40 to -0.41 ± 1.77 ml/day (p = 0.007). Patients referred for fundoplication based on histologic evidence of reflux (n = 9) showed an improvement in rate of change of FEV(1) from -3.39 ± 6.00 to -0.17 ± 1.50 ml/day (p = 0.057), and those with positive impedance study and consistent decline in FEV(1) (n = 13) showed a significant improvement from -3.62 ± 3.35 to -0.74 ± 2.33 ml (p = 0.021). Actual and predicted morbidity was 2.5% and 31%, respectively. Actual and predicted 30-day mortality was 0% and 1.9%, respectively. CONCLUSIONS: Anti-reflux surgery is safe for lung transplant recipients and results in an improvement in the rate of change in FEV(1) despite a decline in mean FEV(1) post-operatively.