Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases  have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems. Based on significant efforts, tremendous advances have been achieved in animal genetics and reproductive physiology. These have enabled what is now known as the genome-editing revolution that can be applied to generate gene-edited animals including sheep and goats for various purposes (Generating new and variable phenotypes Dolly from undifferentiated embryonic cells (via pronuclear injection (PNI) was published, announcing the first procedure for the production of transgenic farm animals   have opened the way for the large-scale production of genetically identical individuals.Embryo splitting was also applied to other livestock species including goats for both experimental and commercial purposes . DiffereDespite the family relationship between sheep and goats, hybridization between both genera is an extremely rare event. Natural hybridization between sheep and goats is in most cases accompanied by high mortality rates of hybrid fetuses during the second month of pregnancy, likely as a result of fetal/maternal immunological incompatibility . By usinSheep–goat chimeras differ from sheep–goat hybrids, which can be obtained when a goat naturally mates with a sheep. The phenotypic characteristics of sheep–goat chimeras include regions of both sheep-like wool and goat-like hair. Due to the mosaic nature of goat and sheep tissues in the produced chimeras, chimeric characteristics cannot be transferred to the next generation. Fertile sheep–goat chimeras can either pass on sheep or goat characteristics to their progeny depending on whether the reproductive organs of the chimera formed from caprine or ovine origins . Interspmouse metallothionein-I/human growth hormone (mMT/hGH) transgene, it did not express the integrated gene. The authors speculated that the reasons behind the low efficiencies might be due to the concentration of injected DNA, the composition of the used buffer, the stage of the collected embryos, and other structural aspects of the chromosomes. Further attempts have been made to overcome these obstacles and to facilitate the generation of transgenic animals using this technique. A few years later, in 1991, mouse whey acidic protein/human longer-acting tissue plasminogen activator (mWAP/hLAtPA) transgene using the same technique.PNI was among the first techniques that have been applied to generate transgenic animals. It was the dominant methodology for the generation of transgenic animals during the first decade of animal transgenesis studies. By introducing DNA constructs into the pronuclei of fertilized eggs and transferring the injected eggs to foster mothers, researchers were able to generate transgenic animals , in 1985In both sheep and goats, the reported efficiencies of conventional PNI of DNA constructs were ∼1% of the injected zygotes . Severalvia asexual reproduction and can also be artificially introduced in mammals by using primary techniques such as embryo splitting  gene from transfected fetal fibroblasts (human antithrombin III (hAT) gene  gene . The aimDolly, the sheep from a mammary gland cell of a 6-year-old ewe, taken by Wilmut and his colleagues in 1997  is one of the emerging strategies to conserve genetic diversity and prevent the rapid loss of animal genetic resources. Genetic rescue programs based on iSCNT use nuclei from endangered species in the wild, whose oocytes are difficult to obtain, with oocytes from closely related domesticated species to reconstruct embryos that can then be transferred to foster mothers. The resultant offspring of this process resembles nucleic donors.Ovis and Capra. post mortem somatic cells, and domestic sheep oocytes. This provided an encouraging example of the application of iSCNT for the generation of live founders. Further examples of the implementation of iSCNT using sheep and goats to reconstruct embryos between species within the same genera are listed in Sheep and goats were among the closely related domesticated species that were utilized in the conservation cloning programs of threatened species that belong to the genera Handmade cloning (HMC) is a simplified version of the SCNT technique. This modified technique has emerged to overcome the technical difficulties that obstruct the improvement and widespread application of SCNT. In traditional SCNT, oocyte enucleation is one of the technical steps which encounter major obstacles due to the presence of the zona pellucida  . Thus, imodified nematode mfat-1 gene to enrich muscles and other organs and tissues with omega-3 fatty acids , the applications of HMC in sheep and goats to produce viable founders have not been studied in detail. In general, despite the simplification provided by HMC and other emerging strategies, SCNT remains technically challenging, and few research groups around the world are able to perform it efficiently  gene  from desirable male donors and injects and transplants these into the seminiferous tubules of an infertile recipient males, which results in donor-derived spermatogenesis. In this case, transgenesis can be mediated by manipulating SSCs prior to transplantation into the recipient males or by transferring transgenic donor germ cells from transgenic individuals . This teS2) gene . Homozygin vitro fertilization (IVF), or laparoscopic insemination (LI)  , with vaion (LI)  and tranion (LI)  using maion (LI) . The optlipoprotein lipase (LPL) (solute carrier family 7 member 11 (SLC7A11) (peroxisome proliferator–activated receptor gamma (PPARγ) (myogenin (MyoG) (enhanced green fluorescent protein (eGFP)  , solute SLC7A11) , peroxis (PPARγ) , myogenin (MyoG) , and enhn (eGFP) . In geneViral vectors have been used to mediate transgenesis by delivering and integrating transgenes into the host genome. Viral vectors can be divided into non-integrating viral vectors , and integrating viral vectors that are mostly derived from a retrovirus, lentivirus, and adeno-associated virus (AAV) . Viral vvia the teat canal for the transient production of valuable proteins in milk. Goats are an ideal model for the production of pharmaceutical molecules in milk and have been subjected to this protocol. Human growth hormone (hGH) was among the first published genes to be infused into the goat mammary gland using retroviral vectors  in mammary epithelial cells successfully inhibited PTHrP gene expression (human huntingtin (HTT) in the striatum of transgenic Huntington’s disease (HD) sheep so that these express the full-length HTT gene reduced the mRNA and protein of human HTT by 50–80% in the striatum at 1 and 6 months postinjection  and that target goat pression . Lentivipression Table 6.njection .eGFP marker gene for both zona-free and split embryos and reported potential possibilities for the application of this approach in small ruminants  injection . Both stuminants . Perivitic lambs . Despiteic lambs . Furtherhibitors .via perivitelline space injection of lentiviral vectors encoding shRNAs that have been specifically designed to inhibit the replication of foot-and-mouth disease virus (FMDV), or to silence the expression of myostatin (MSTN) to promote muscle growth  was used to produce transgenic sheep with enhanced fiber growth  transgenic goats that carried both the human lysozyme (LYZ) vector and selectable gene-expressing vector, which contained a left loxP site, neomycin resistance gene Neo, a thymidine kinase gene TK, and a right loxP site. After TAT-Cre protein transduction, one of the TAT-Cre treated cell colonies was used as a nuclear donor to perform nuclear transfer. Two selectable gene-free cloned goats were produced with removed Neo/TK cassette  recognizes and cleaves two FRT recognition sites (FRT site–specific recombination system has been applied in goat somatic cells to mediate the site-specific integration and to eliminate the problematic random integration of transgenes (FRT-docking site into the α1 (I) procollagen (ColA1) locus (HR efficiency of 5.9% “11/185”). Cell clones with successful targeting have been subjected to embryo cloning to achieve rejuvenation or regeneration. Cells with the FRT-homing site were isolated from cloned fetuses and co-transfected with two vectors, an eGFP replacement vector, and an Flp recombinase-expressing vector. After the second round of recombination, transgenic cells exhibited functional expression of eGFP with a gene replacement efficiency of 38.4% (15/39) (The yeast on sites . The Flpansgenes . Briefly (15/39) .Streptomyces bacteriophage–derived ΦC31 (PhiC31) integrase has been applied to mediate HR between attB and corresponding pseudo attP sites  driven by the BLG promoter (Sleeping Beauty transposase resulted in two lambs that carried the transgene 2/7 (29%)  . More anPiggyBac, has been applied by our team in cashmere goats. The PiggyBac transposon system was tested to mediate eGFP gene expression and to generate stably transfected cell lines with the use of goat fetal fibroblasts  were used as donor cells to produce transgenic goats via SCNT  gene to improve the production of fine hair in cashmere goats (keratin-associated protein 6.1 (KAP6.1) promoter was used to construct the Tβ4-overexpressing cassette. Using the same strategy, transfected goat fetal fibroblasts with the integration of Tβ4 gene–expressing cassette have been used as donor cells to generate transgenic goats via SCNT , SCNT, PNI, intratesticular injection, and direct injection into the targeted organ  gene expression by silencing the mRNA of the targeted gene. RNAi has mainly been used to understand the biological and functional roles of specific genes, investigate the inhibitory effects of gene expression, suppress the expression of pathogenic genes, and inhibit the expression of genes that negatively regulate desirable phenotypes. shRNA and small interfering RNA (siRNA) are the main forms of RNAi molecules that have been used to mediate gene knockdown in sheep and goat genomes. In sheep and goats, a set of strategies has been used to deliver RNAi-expressing constructs, to mediate the generation of knockdown cell lines, and to obtain transgenic individuals. These methods include the transfection of RNAi-expressing vectors, viral vectors , transposons . The examined brain tissues of a transgenic cloned fetus from lentiviral-shRNA transfected fibroblasts showed a significant decrease in PrP expression  gene. This strategy was applied as a genetic method of sex control and to bias the sex ratio toward females in sheep  that result in gene disruption (knockout). In the presence of donor DNA, homology-directed repair (HDR) functions to mediate more precise modifications such as site-specific integration (knockin) and single-base alteration (point mutation)  meganucleases or homing endonucleases, ii) zinc finger nucleases (ZFNs), iii) transcription activator-like effector nucleases (TALENs), and (iv) CRISPR systems . Of thesutation) . Other futation) .I-SceI of yeast S. cerevisiae . Typically, the ZF domain is composed of three to four individual fingers, each being capable to recognize and bind to an approximately 3-bp-long sequence (triplet bp within the DNA substrate)  . This meMSTN gene in fetal fibroblasts (MSTN exon 1 (MSTN-ZFN mRNA into ovine embryos at the one-cell stage achieved 35% (13/37) efficiency  to investigate the function of this gene in female sex determination  of the sequenced colonies contained the desired insertion  and muscle hypertrophy  into the goat genome using TALEN mRNA and oligonucleotide transfection (BLG gene (BLG knockout and enriched expression of human lactoferrin (hLF)  , and after the second targeting and cloning, five cloned kids were generated (three BLG-/- and two BLG-/hLF) , and the other five goats were BLGhLA/+. BLG expression in the milk of transgenic goats was reduced, while hLA was highly expressed compared to normal goats , and one was alive and healthy (MSTN+/-)   or humanin (hLA)  have beeLG-/hLF) . Furtheral goats . CaprineMSTN+/-) . TALENs Rapid and promising advances have been reported in the field of genetic modification during the past decade. The revolution of genetic engineering has culminated in the emergence of CRISPR systems. These are simple but sophisticated mechanisms derived from nature that act in prokaryotes as an adaptive immune system against phage and foreign DNA infection by the cooperation of CRISPR sequences with Cas proteins . Based oStreptococcus pyogenes CRISPR-Cas9 is the most widely used CRISPR system. The CRISPR/Cas9 system uses two main components: an RNA-directed Cas9 protein and ∼20-nucleotide sgRNA, which leads the Cas9 protein to a user-defined DNA target site as long as it is next to a protospacer adjacent motif (PAM) sequence   showed the mutation with heavier body weight compared to their wild-type counterparts  . Later, terparts . These iMSTN with two other economically important genes, including the agouti-signaling protein (ASIP) and β-carotene oxygenase 2 (BCO2) in sheep  for ASIP, and 27.7% (10/36) for BCO2; 5.6% (2/36) showed the simultaneous targeting of all three genes. No off-target has been detected and founders with MSTN mutations showed enlarged myofibers and enhanced body weight compared to wild-type individuals  has been shown to promote sSMSC differentiation in both number and length. This study has also reported the generation of MSTN-disputed sheep using SCNT from CRISPR/Cas9 transfected ear fibroblasts  gene in sheep  in goat fibroblasts and generated three MSTN knockout goats using SCNT (MSTN or fibroblast growth factor 5 (FGF5) or both genes have been reported by our team  carried a disruption in FGF5, and 10/98 (10.2%) showed simultaneous disruption of both genes. These results confirm the efficient induction of multiplex targeting via CRISPR/Cas9, which is of great importance in farm animals. Especially, since most of the economically important traits are controlled by multiple loci. Further studies have been conducted that used the MSTN mutated founders generated from this experiment to confirm the occurrence of gene disruption and the transmission of the knockout alleles  mutated founders that carried a disruption in FGF5 and showed increased wool length  mutated founders carried both monoallelic and biallelic mutations in FGF5 and showed increased wool length and quantity . This highlights the critical role of the ASIP gene in coat color determination in sheep.Sheep and goats form a valuable source for the production of fibers. Genes associated with fiber quality and quantity are a source of attraction, and many researchers hope to alter these genes in attempts to obtain desired and new fiber properties. The l length . Anotherquantity . Recentlwth rate . The resSPR/Cas9 . The resMSTN/FGF5 knockout goat model  gene knockout goats by SCNT to investigate the disruption effects on the phenotype, hair follicle growth and development   , and latenerated . Four fo in milk . The genturbo GFP (tGFP) in the Rosa26 locus of sheep genome  and acetylserotonin methyltransferase (ASMT) for the expression of melatonin were cytoplasmically co-injected into pronuclear embryos. Of 34 transgenic founders, seven carried AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. Founders carrying these genes produced melatonin-enriched milk. These models might be a good source of melatonin, which has various nutritional and medicinal values and uses.Induction of genetic integration into the genomes is of great interest, especially for the introduction of new expressional characteristics in milk. CRISPR/Cas9 has shown the ability to introduce knockin of the marker gene p genome . This st in milk . In thisstearoyl-CoA desaturase 1 (SCD1) in goat mammary epithelial cells (acetyl CoA acyltransferase 2 (ACAA2) in sheep precursor adipocyte cells  gene has shown to be responsible on an increased ovulation rate and consequently larger litter size  gene, which exerts a large effect on both ovulation rate and litter size  in sheep conceptus elongation using recovered elongating conceptuses  for the simultaneous targeting of PrP and MSTN. Moreover, of the nine dual gene mutant colonies, five had mutations in all four alleles of both genes. These results suggest the use of the CRISPR/Cas9 system to target genes to confer potential disease resistance in farm animals.Gene editing has been suggested as a robust tool that can be used to generate disease-resistant animals. Consequently, genetically modified resistant farm animals have been produced. These offer good models to investigate and understand disease pathogenesis and offer a potential source for the spread of disease resistance traits in commercial flocks. The CRISPR/Cas9 system has been applied in sheep and goats to achieve aims related to improving animal health and welfare. Several disease-related genes have been disrupted using CRISPR/Cas9. The nd goats . PrP-resn of PrP . CRISPR/nt goats . The tarhyaluronidase 2 (HYAL2), which can be used by Jaagsiekte sheep retrovirus as a cell entry receptor, resulting in ovine pulmonary adenocarcinoma syndrome  infection can lead to fetal infection, causing microcephaly and other severe congenital neurological symptoms. Type I interferons (IFNs) are central for host resistance against ZIKV, and nfection . Fan et al model .CF transmembrane conductance regulator (CFTR) gene and CFTR–/– as well as CFTR+/– lambs have been produced with a severe phenotype of CF pathology similar to that of humans.Fan et al. have also provided an interesting sheep model for the investigation of human cystic fibrosis (CF) by employing both CRISPR/Cas9 and SCNT . CF is a has been introduced. The thus generated gene-edited lambs accurately phenocopied human HPP, providing a useful large animal model for the study of rare human bone diseases. The results of these reports corroborate the great potential of the CRISPR/Cas9 system to generate gene-edited sheep and goats that recapitulate human diseases.Additionally, Williams et al. have also reported an interesting sheep model, recapitulating human hypophosphatasia  by applying CRISPR/Cas9 . In thisOne of the most promising strategies to solve the shortage of organ donors worldwide is to generate human organs inside large animals by applying a technique called interspecies blastocyst complementation . In thispancreatic and duodenal homeobox protein 1 (PDX1), which is a necessary gene for pancreatic development. CRISPR/Cas9 combined with dual sgRNAs and direct oocyte microinjection approach was used to generate PDX1 disruption, and a resulting fetus with PDX1-,- mutation lacked a pancreas. The results of these promising attempts highlight the potential of gene-edited sheep to be applied in interspecies organ generation as candidate hosts for the growth of human organs. Integrative programs that employ CRISPR/Cas9 and PSC complementation offer a potential solution for the shortage of organ donors.In this context, an interesting study reported the creation of pancreatogenesis-disabled sheep as a step toward achieving interspecies blastocyst complementation–based xenotransplantation between human and large animals . In thisIn the current era, genome engineering systems have become indispensable tools of basic research, biomedicine, and agriculture. Genome engineering tools, especially the recently emerged CRISPR systems, offer the potential to revolutionize all biological research fields including research on agriculture and farm animals. Applying genome engineering tools to farm animals including sheep and goats is of significant importance. Currently, gene-edited sheep and goats are generated with relative ease using CRISPR systems, providing valuable models for agricultural, veterinary, and biomedical research. The list of sheep and goat genes targeted by CRISPR systems is continuously growing. In the future, a large number of genes will be targeted, and more valuable models will be generated, thus widening our understanding about these genes and their corresponding phenotypes and functions. This basic research is only the beginning; the potent potential of genome engineering will become apparent in the future.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This study was supported by the National Natural Science Foundation of China , and Local Grants . Xiaolong Wang is a Tang Scholar at Northwest A&F University, China.Author TS was employed by Recombinetics Inc. The remaining 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.


Most prior studies characterizing post-transplantation diabetes mellitus (PTDM) have been limited to single-cohort, single-organ studies. This retrospective study determined PTDM across organs by comparing incidence and risk factors among 346 liver and 407 kidney transplant recipients from a single center.Univariate and multivariate regression-based analyses were conducted to determine association of various risk factors and PTDM in the two cohorts, as well as differences in glucometrics and insulin use across time points.There was a higher incidence of PTDM among liver versus kidney transplant recipients (30% vs. 19%) at 1-year post-transplant. Liver transplant recipients demonstrated a 337% higher odds association to PTDM , p<0.01). 1-month FBG was higher in kidney patients , while 1-month insulin use was higher in liver patients . Age, BMI, insulin use, and inpatient FBG were also significantly associated with differential PTDM risk.Kidney and liver transplant patients have different PTDM risk profiles, both in terms of absolute PTDM risk as well as time course of risk. Management of this population should better reflect risk heterogeneity to short-term need for insulin therapy and potentially long-term outcomes. Health care providers face an important tradeoff in managing patients who have undergone solid organ transplantation. Corticosteroids and immunosuppressive agents such as tacrolimus and sirolimus prevent graft rejection. However, these medications bring significantly increased risk for development of impaired glucose tolerance and post-transplantation diabetes mellitus (PTDM), also known as new onset diabetes after transplant (NODAT). PTDM is a potentially serious complication of solid organ transplantation affecting patients without a prior history of a diabetes diagnosis, leading to decreased graft function and survival, increased risk of cardiovascular disease, and increased healthcare costs , 2.Most clinical recommendations on PTDM emphasize the need for prevention of organ rejection while maintaining standards of care for those who develop concurrent PTDM , 4. HoweIn recent years, availability of transplant patient data has allowed for analyses to better understand the pre-transplant risk factors for PTDM –21. HoweWe introduce a dataset of liver transplantation patients to complement a dataset of kidney transplantation patients previously used in published analyses by Boloori et al. . TogetheThis was a retrospective study of a dataset compiled through a de-identified chart review with IRB (Mayo Clinic IRB) approval (Continuing Review #: PR16-008236-03). One dataset consisted of 346 patients who underwent first time liver-only transplant between 2007 and 2012. Information collected on each patient included demographic data and medical history, as well HbA1c, fasting blood glucose (FBG), and diabetes mellitus (DM) status pre-transplant and at 1-, 4-, and 12-months post-transplant. In addition, we collected type of hypoglycemic medication administered to each patient, type of immunosuppressant used, corticosteroid use, and trough level of immunosuppressive agent at each time point post-transplant. We also utilized a previously published data set of 407 kidney transplant patients who underwent transplant between 1999 and 2006 at the same institution . This daIn renal transplants, the standard immunosuppression protocol is induction therapy with either rabbit anti-thymocyte, immumoglobulin, or basiliximab. All patients typically receive a 5-day tapering course of glucocorticoids. Subsequently, most receive a steroid-free maintenance immunosuppression using mycophenolate mofetil, and tacrolimus is instituted. For patients undergoing a liver transplant, an immunosuppression protocol of intravenous corticosteroids, tacrolimus, and mycophenolate mofetil is used postoperatively. Corticosteroid administration follows the regimen of 500 mg intravenous methylprednisolone preoperatively, 50 mg twice daily on postoperative day 0; and 25 mg twice daily on postoperative day 1, then progressively tapered to a treatment stop by 4 months, but may continue in certain patients with autoimmune hepatitis. Mycophenolate mofetil stops at four months unless the patient has renal impairment.Data were reviewed to remove significant outliers that indicated human error in data collection , with only a minimal number (less than 1%) of observations removed from the dataset. In concordance with previous literature , 22, we We report means and standard deviations for available variables of interest determined as those found in previous literature to be predictive of PTDM risk . DiffereA substantial percentage of both renal and liver transplant patients without preexisting DM develop hyperglycemia while still hospitalized. Consequently, insulin is often required at discharge. For example, 87% of renal transplant patients included in this analysis without pre-existing DM exhibited hyperglycemia while hospitalized, and 66% were discharged on insulin . Additioall 3 follow-ups, compared to 8.1% of renal patients. Transient hyperglycemia, that is hyperglycemia which only occurs in the immediate post-transplant period (1-month) followed by remission, accounted for a large percentage of the difference, with 17.5% of liver patients meeting the criteria only in month 1 compared to 6.2% in the kidney group. However, if transient hyperglycemia is not included in the analysis as suggested by the 2014 International Consensus Meeting [Fasting glucose and HbA1c values for patients who did not develop PTDM and those who did are shown in Fasting blood glucose values of patients who did develop PTDM increased over the follow-up period in the liver group, but did not change significantly in the kidney transplant cohort . The 1 mAmong patients who did not develop PTDM , more liLiver transplantation protocols called for tapering off of steroids by 4 months. While 93.6% of liver patients were on steroids at 1 month, this percentage dropped to just 14% at 4 months and 3.6% at 1 year. This contrasts steroid use in the kidney group in which steroid use was relatively stable at all 3 follow-ups . Among patients on steroids, the percentage with hyperglycemia decreased in the kidney cohort , while hyperglycemia in liver transplant steroid users increased over from 1 month to 1 year .Finally, an adjusted analysis was conducted  to deterAll patients undergoing solid organ transplant without a prior history of DM are at risk of developing PTDM. This risk results from a combination of traditional factors  and immunosuppressant medications known to affect glucose metabolism , 28. OncMost studies of PTDM in kidney and liver transplant patients have typically involved analyses of the single organ. To the authors’ knowledge, a comparative study of liver and kidney transplants with regards to PTDM has not been undertaken. The time course for development of PTDM may not be identical between the two, and insulin needs may differ over the short term (1-year) follow-up. Understanding any potential differences can shed insight into how to modify treatment and follow-up algorithms according to the transplant type. Prior analysis by Boloori at al.  demonstrThe analyses demonstrated significant differences in PTDM risk between the two transplant populations. Liver transplant recipients had a 337% greater odds of developing PTDM compared to kidney recipients. There are multiple possible reasons for the differential risk between kidney and liver transplantations that warrant further investigation. First, steroid protocols differ for liver transplant patients compared to renal patients which may impact hyperglycemic effects. Secondly, there may be inherent differences in glucose metabolism between kidney and liver transplant recipients which are not accounted for in this analysis. Finally, lifestyle differences may exist between these populations as a result of different recovery times from transplant allowing for modifications in diet/exercise.Different patterns of hyperglycemia were also noted. Glucose levels were highest at 1 month in the kidney transplant group, and then declined thereafter. In the liver cohort, glucose values did not change over the follow-up time period. These differences in glucose between the two transplant cohorts may well have been due to their respective immunosuppression protocols. We also demonstrated large differences in the time course and remitting/relapsing nature of hyperglycemia between kidney and liver transplant patients. This analysis illustrates further the complex nature of post-transplant hyperglycemic and the need to better understand how the diabetogenic effects of immunosuppressive drugs impact short terms changes in glycemic control.In the adjusted analysis, insulin use at 1 month was predictive of PTDM risk. The proportion of patients on insulin differed between the kidney and liver transplant patients. Among patients who did not develop PTDM, both renal and liver cohorts were able to discontinue insulin after 1 month post-transplant. A similar percentage of renal transplant patients who did have PTDM remained on insulin therapy across the three follow-up periods, while the proportioned declined in liver patients. The reason underlying these different proportions of insulin use cannot be ascertained from this analysis. In addition to HbA1c and blood glucose values, insulin therapy in the outpatient setting is driven by results and patterns of SMBG, which are not available here. It should be noted, however, that both groups had around 30% of recipients on insulin therapy at 1-year, meaning a majority of recipients had their PTDM adequately managed without antidiabetic agents, potentially through diet and exercise.In 2014, the International Consensus Meeting on Post-transplantation Diabetes Mellitus published recommendations and future direction on dealing with PTDM in the medical community . Among tThere are limitations of this study that should be noted. First, the sample size of patients who developed PTDM may be underpowered to detect some of the significant differences in outcomes in the time points where no significant difference was detected. Previous analysis by Boloori et al. , which aThese results provide the first evidence to support differential guideline-making and management between kidney and liver transplant patients to reflect differences in both the risk factors and timeline at which patients may be at risk to develop PTDM. Furthermore, these findings highlight the need for further investigation of PTDM features and outcomes across other transplanted organs, as well as novel strategies to manage PTDM risk. In addition, potential effectiveness of screening strategies to better diagnose and monitor glucose intolerance in this population should be explored. It is likely that hospitals across the United States would benefit from standardization of protocols that optimize PTDM risk management in this patient population to minimize likelihood of poor diabetes outcomes while maximizing the health of the transplanted organ.


The temporal modulation structure of speech plays a key role in neural encoding of the speech signal. Amplitude modulations  in speech are encoded by neuronal oscillations  that oscillate at matching temporal rates. To date, however, all neural studies have investigated adult-directed speech (ADS) as produced and perceived by highly literate adults. Whether temporal features of ADS vary with the skills of the speaker, for example literacy skills, is currently unknown. Here we analyse the temporal structure of ADS spoken by illiterate, low literate (≤ 4 years of literacy) and highly literate (≥ 12 years of literacy) adults. We find that illiterates produce speech differently. Spontaneous conversational speech produced by illiterate adults showed significantly less synchronised coupling between AM bands (less phase synchronisation) than conversational speech produced by low literate and highly literate adults, and contained significantly fewer syllables per second. There was also a significant relationship between years of literacy and the amount of theta-band energy in conversational speech. When asked to produce rhythmic proverbs learned in childhood, all groups could produce speech with similar AM phase synchronisation, suggesting that the differences in spontaneous conversational speech were not caused by physiological constraints. The data suggest that the temporal modulation structure of spoken language changes with the acquisition of cultural skills like literacy that are usually a product of schooling. There is a cultural effect on the temporal modulation structure of spoken language. Tukey tests showed that high literates produced more syllables per second than illiterates (p = .001) and low literates (p < .001). This would be expected, as a group of highly literate individuals are likely to produce more complex syntax and polysyllabic words , 41. No Using the modulation phase synchronisation analysis approach in , we foun2 = .219]. Post-hoc Tukey tests showed lower delta-theta AM phase synchronisation for illiterates when compared to both low literates (p < .001) and high literates (p = .001), and also lower theta-beta/low-gamma phase AM synchronisation between illiterates and high literates (p = .01), but not between illiterates and low literates (p > .2). This suggests that literacy affects the regularity of the temporal spacing of different phonological units in conversational speech. No group differences were found for the MANOVA for rhythmic speech , showing that all participating adults could produce temporally synchronised speech if required. Accordingly, there were no physiological constraints underpinning the differences in temporal modulation structure found for conversational speech.To explore the effects of literacy on phase synchronisation, and given the speech rate differences for conversational speech but not rhythmic speech, separate MANOVAs were computed for conversational speech and rhythmic speech respectively. In each case, delta-theta PSIs and theta-beta/low gamma PSIs were the dependent variables, with literacy group as the independent variable. The MANOVAs revealed an effect of literacy in conversational speech for both the magnitude of delta-theta and of theta-beta/low gamma phase synchronisation 22], dyslphoneme awareness may appear surprising. In multi-time resolution models of speech processing, faster oscillatory information is thought to support phoneme awareness . Howeveion, see . Changesion, see . AccordiOne limitation of the current study is the relatively old age of the participants, who were screened for general cognitive impairments (via the MMSE) but not specifically matched for hearing. It could be important to access other illiterate populations, ideally younger than the population studied here. This would also enable investigation of whether the growing literature on the core role of theta entrainment for neural speech encoding ,5 is an In conclusion, our data suggest that future neural imaging studies of features of spoken language performance in both perception and production must take into account the literacy skills of participants. Literacy has long been known to change the brain ,13,16. HS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Mycoplasma gallisepticum (MG) has been increasing in virulence. Repeated exposure to low-doses of MG, a proxy for what birds likely experience while foraging, provides significant but incomplete protection against reinfection. Here we sought to determine if the within-host, pathogen load advantage of high virulence is mediated by the degree of prior pathogen exposure, and thus the extent of immune memory. We created variation in host immunity by experimentally inoculating wild-caught, MG-naïve house finches with varying doses and number of exposures of a single pathogen strain of intermediate virulence. Following recovery from priming exposures, individuals were challenged with one of three MG strains of distinct virulence. We found that the quantitative pathogen load advantage of high virulence was strongly mediated by the degree of prior exposure. The greatest within-host load advantage of virulence was seen in hosts given low-dose priming exposures, akin to what many house finches likely experience while foraging. Our results show that incomplete host immunity produced by low-level prior exposure can create a within-host environment that favors more virulent pathogens.The host immune response can exert strong selective pressure on pathogen virulence, particularly when host protection against reinfection is incomplete. Since emerging in house finch populations, the bacterial pathogen  Thus, any factors that alter the ability of a pathogen to replicate within a host can cause strong selection on pathogen virulence4.Pathogens demonstrate extensive variation in the amount of harm, or virulence, they cause their hosts. For directly-transmitted pathogens, high virulence presents a paradox because the increased morbidity and mortality that virulent pathogens cause their hosts should reduce the likelihood of their successful transmission by reducing opportunities for contacts with susceptible hosts. The “trade-off” hypothesis helps to resolve this paradox by considering virulence as an unavoidable consequence of the within-host pathogen replication necessary for ongoing transmission8]. The immune response of a host has been shown to select for more virulent pathogens in cases where the host forms specific immunity against a pathogen, whether via vaccination, previous exposure, or innate immune priming12. If a host has incomplete immune protection, meaning they are able to be reinfected by the same type of pathogen, this provides a particularly strong selective environment for virulence14. As predicted by theory, virulent strains often have higher within-host replication rates16, and thus may be more likely to replicate at levels needed to overcome incomplete host immunity and successfully colonize incompletely-immune hosts. Additionally, when incomplete immunity protects hosts from infection-induced morbidity and mortality, there are fewer evolutionary costs for a pathogen to maintain high levels of virulence7. Thus, virulent pathogens should have an increased likelihood of competing and persisting in a population with a high prevalence of incompletely-immune hosts11.There is growing interest in understanding how the within-host environment modulates selection for virulence . Low-level exposure has been found to produce immunity to a homologous pathogen strain akin to that produced by imperfect vaccination [e.g.20], suggesting that this type of exposure may play a significant role in driving virulence evolution in natural populations.The degree of immunity that a host harbors is strongly mediated by the extent of prior exposure to a pathogen  that an individual receives22 to determine the role of prior exposure level in producing incomplete immunity and altering the within-host environment to favor strains of higher virulence. Free living house finch populations in North America experience seasonal epidemics of conjunctivitis caused by the bacterial pathogen Mycoplasma gallisepticum (MG)23. Since its emergence in 1994, MG has been found to be increasing in virulence in two geographically isolated populations22. This system meets the assumptions of the classic trade-off model, with more virulent strains leading to higher within-host exploitation22 and among-host transmission24. It thus provides a rare opportunity to study the potential ecological factors underlying virulence evolution in an unmanaged, free-living species. Recent work from Fleming-Davies et al.11 provided both empirical and theoretical evidence that incomplete immunity formed from prior exposure to MG can favor the evolution of increased virulence in this system. However, the immunity assessed in Fleming-Davies et al. (2018) was produced from single high dose exposures to MG, which are likely not representative of the range of exposure types experienced in free-living populations. MG is primarily transmitted at feeders while animals forage26, and the pathogen degrades quickly in the environment25. Thus, it is likely that wild birds are exposed to heterogeneous amounts of the pathogen. In addition, wild house finches have been shown to recover from natural infections at rates of over 50%, producing a pool of recovered individuals potentially susceptible to reinfection27. In our previous work, we found that repeated low-level exposure to MG in house finches, a proxy for what birds may experience at feeders in the wild, produces largely incomplete immune protection against secondary high-dose challenge17. Further, host protection against secondary infection becomes progressively more complete with higher degrees of prior host exposure17. Thus, the degree to which host immunity favors virulent pathogen strains in this system is likely to vary with the extent of prior host exposure to the pathogen, and the extent of incomplete immunity.Here we use a natural host-pathogen system where virulence has been shown to be increasing22 in hosts with varying degrees of immunity generated from prior exposure. We predicted that, as previously shown11, the most virulent strain would maintain the highest within-host pathogen load, regardless of host prior exposure. However, we predicted that the level of prior exposure, and thus the degree of pre-existing immunity in hosts, would mediate the extent of the quantitative pathogen load advantage for virulent strains. Specifically, we predicted that virulent strains would have the greatest advantage in individuals that previously received low-level exposure, which generates largely incomplete immunity, as compared to the more complete immunity produced from previous high dose exposure. Finally, we predicted that incomplete immunity would lower the costs of virulence to the pathogen by reducing the degree of infection-induced morbidity caused by virulent strains.In order to test if the level (dose and number of exposures) of prior exposure mediates the quantitative pathogen load advantage of virulence in this system, we created variation in prior exposure by experimentally inoculating wild-caught, MG-naïve house finches with varying doses and number of exposures of a single pathogen strain of intermediate virulence (VA1994). Following recovery, we assessed quantitative pathogen loads of three MG strains previously shown to have distinct virulence levels1, 102, or 106 color changing units per ml [CCU/mL] of MG or a sham treatment of media alone). All priming inoculations, with the exception of sham controls, were done with a medium virulence strain of MG17. Repeated priming exposures were given every other day, to allow the same recovery window across treatments 17. However, because repeated exposure to 102 CCU/mL produced infection and disease in 100% of individuals and thus may not represent a lower threshold of infective dose in the wild, here we additionally incorporated a dose one order of magnitude lower (6 exposures to 101). For clarity, priming treatments will be referred to using the following format: [number of exposures (priming dose)]. As an example, [6(101)] represents six exposures to a dose of 101 CCU/mL.To determine if low-dose and repeat exposures produce a within-host environment which favors more virulent strains, house finches were given variable priming exposures to generate variation in the degree of pre-existing immunity, and then challenged with one of three MG strains known to vary in virulence Fig. . For pri17 and designed to fall within the window of seasonal epidemics28, birds were challenged with a high dose of one of three strains ranging in virulence. Birds were either given a less virulent, equally virulent (homologous strain), or more virulent strain to test the quantitative pathogen load of strains of varying virulence in previously exposed hosts  prior to and during the experiment. Two weeks before the start of the experiment, birds were moved to individual housing, and remained single-housed throughout the study. All protocols for animal capture, housing and procedures were approved by Virginia Tech’s Institutional Animal Care and Use Committee and performed in accordance with Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals.Hatch-year house finches (n = 104) were captured in Montgomery County and Radford City, VA in June-August 2015 using a combination of mesh wire traps and mist-nets under permits from VDGIF (050352) and USFWS (MB158404-1). After capture, animals underwent a two-week quarantine protocol where they were pair-housed, monitored for visible eye lesions and bled on day 14 post-capture to test for MG-specific antibodies ) were likely of higher actual dose and resulted in stronger protection in this study than in our prior work.We used three strains shown in prior work to vary in both the degree of within-host exploitation22; “CA2006” (2006.052-5 (4P) 1/24/10)) was used as a less virulent strain, VA1994 was used as an equally virulent homologous strain, and “NC2006” (2006.080-5 (4P) 7/26/12) was used as a more virulent strain. Previous work supports a small increase in protection due to homology of strains used across inoculations11. However, in that same study, the effect of virulence of the priming strain was much stronger than the increased protection from homology11, and thus we did not expect strain homology to alter the effects of interest. Additionally, while these strains vary in virulence, they have very low levels of genomic32 and antigenic variability33. All inocula were provided by D.H. Ley, North Carolina State University, College of Veterinary Medicine, Raleigh, NC, USA. For more information regarding inocula and inoculations, see Supplementary Materials.Secondary inoculations were given using three strains known to vary in virulence34. Disease severity following secondary challenge was used a proxy for the costs of virulence experienced by pathogen strains in hosts of varying immune backgrounds. Disease severity directly predicts capture time in mock-predation events  in captivity30 and the presence of conjunctivitis predicts finch mortality in the wild27, and is thus a reasonable proxy for host mortality and the costs associated with pathogen virulence. Disease severity was quantified by scoring clinical signs of conjunctivitis on a scale of 0-3 per eye. Scores vary based on the degree of visible conjunctival inflammation, eversion and exudate35. Clinical scores (“eye scores”) were recorded for each sampling point and summed between eyes for a given sampling date. All scoring was done blind to treatment.Quantitative pathogen load was measured in conjunctival tissue using quantitative PCR (qPCR), with DNA extracted from swabs of both conjunctivae 17 and our main objective was to assess traits key to pathogen fitness during secondary exposure, we have not included primary infection sampling methods here .Animals were sampled for pathogen load and the severity of clinical signs on post-secondary inoculation days 4, 7, 14, 21 and 28. However, only a subset of pathogen load samples that captured the relevant temporal dynamics of infection were analyzed . Additionally, animals were sampled for quantitative pathogen load and clinical signs of infection on pre-inoculation day -15 to ensure that all individuals included in the experiment were not harboring pathogen prior to the study, as well as on post-secondary inoculation day 0 to ensure all animals had cleared infection prior to secondary inoculation. Because responses to priming exposure levels were previously examined36. Quantitative pathogen load and disease severity, measured as scorable eye lesions, were analyzed separately. Although these metrics positively covary22, they represent distinct biological responses that have been found to vary separately in response to exposure level17. For information on primary infection analyses and results, see Supplemental Materials.All analyses were done using the statistical software R37]. Fixed effects included priming treatment, secondary strain, and the interaction between priming treatment and secondary strain, which was our main interaction of interest in this study. Individual ID was included as a random effect. All explanatory variables were treated as categorical. Additional models were run, one excluding control (sham) animals, to assess to what extent effects were driven by priming dose per se, and another including homology (yes/no) to quantify the effect of strain homology on secondary quantitative pathogen loads. Sidak post-hoc pair-wise comparisons were done among priming treatment groups within each secondary strain [R package lsmeans38].Pathogen loads from secondary infection timepoints 7, 14 and 21 from each individual were analyzed using a linear mixed model to account for repeated measures [R package lme439]. Additionally, based on the patterns of variation seen across priming exposure levels and secondary strains during secondary infection, we calculated the coefficient of variation (CV) for the mean of each group. Since these data were lognormally distributed, CV was calculated using the following formula for a lognormal distribution:s is the sample standard deviation of the sample mean for our log transformed pathogen load data40. The 95% confidence intervals for CV were calculated using a nonparametric bootstrap to resample the data 10,000 times in R.Variable level effects of each categorical variable were determined using Type III Wald tests where significant interactions were detected, otherwise Type II tests were used  were able to replicate to levels equivalent to those in immunologically naïve hosts , suggests an effect of waning immunity in this system42. Waning immunity is an important consideration when assessing population-level selection pressure on a pathogen43, as a completely protected individual may become incompletely protected from one epidemic season to another.Our recent work in this system demonstrated that incomplete host immunity produced by prior exposure strongly favors the evolution of more virulent pathogens44. Future work should examine the immunological mechanisms associated with protection from secondary infection, as these mechanisms may shed light on important sources of heterogeneity in susceptibility in this system.Intriguingly, we found that strain virulence and prior exposure level not only altered mean pathogen loads, but also altered the degree of variation in pathogen loads Fig. . The mos30, suggesting that the reduction in disease severity observed in incompletely immune hosts might allow increased pathogen transmission and persistence of virulent strains in wild populations that contain hosts with pre-existing immunity from prior exposure. Future work should integrate the effects of pre-existing immunity on both disease severity and quantitative pathogen load to determine how both together determine optimal pathogen virulence in hosts with varying degrees of prior immunity. One limitation of our study is that we only quantified proxy for within-host fitness of pathogen strains, while the ultimate metric of pathogen fitness is between-host transmission. Although we did not explicitly test how transmission potential changes with a host’s pre-existing immunity, quantitative pathogen loads appear to have important implications for transmission in this system. Previous work showed that as conjunctival pathogen loads increase in house finches, so does pathogen deposition onto feeders41, an important transmission source in the wild26. Thus, differences in quantitative pathogen loads likely have relevant consequences for transmission in this system. However, given that prior work examined how pathogen loads predict transmission potential in immunologically-naïve hosts alone, future work should quantify between-host fitness when determining the role of prior exposure in shaping the disease dynamics of this songbird-bacterium system.In addition to amplifying the quantitative pathogen load advantage of virulence, incomplete immunity formed from prior low-level exposure also reduced the costs of virulence to the pathogen, which we measured as disease severity following secondary challenge Fig. . Under t45], where humans and free-living wildlife are expected to vary in both their distribution in and use of a landscape, altering the potential for pathogen exposure [e.g.47]. Similarly, there is evidence that reinfections, either due to waning immunity [e.g.48] or imperfect immunity [e.g.13], are common across host-pathogen systems. Variation in exposure history is thus an important consideration for anticipating and understanding the evolution of more harmful pathogens in natural populations where reinfections occur.Together, our results indicate that prior exposure level may be an important mediator of within-host pathogen dynamics and selection for higher virulence, particularly where low-level exposures produce incomplete immunity. Incompletely immune house finches appear to provide a within-host environment for MG that both favors virulence and reduces the costs of maintaining virulence, enhancing the fitness potential of virulent pathogens in populations where hosts have repeated infections. This is particularly relevant in systems, like house finches, where there is significant variation in exposure level and where reinfections are possible. There is increasing evidence that exposure level may be an important mediator of host heterogeneity in natural populations [e.g.Supplementary Material.


High-resolution impedance manometry (HRiM) is the test of choice to diagnose esophageal motility disorders and is particularly useful for identifying achalasia subtypes, which often guide therapy. HRiM is typically performed without sedation in the office setting. However, a substantial number of patients fail this approach. We report our single-center experience on endoscopy-assisted HRiM under monitored anesthesia care (MAC) in adults to demonstrate the feasibility and effectiveness of this approach. Patients who had failed prior HRiM attempts received propofol under MAC. Patients then underwent an upper endoscopy, followed immediately by passage of a Diversateck HRiM motility catheter through the nares and under direct visualization into the stomach, often using the tip of the endoscope to guide the catheter. We then awakened the patients and asked them to perform 10 saline swallows. We successfully completed HRiM studies in 14 consecutive patients. Six patients had achalasia; two had esophagogastric junction outflow obstruction; two had absent contractility; one had distal esophageal spasm; one had ineffective esophageal motility; and one had a normal study. The majority of these patients were treated successfully with targeted interventions, including per oral endoscopic myotomy, gastrostomy, botox injection, medical therapy, and dietary modifications. Esophageal manometry has become the gold-standard test to diagnose esophageal motility disorders and is also useful in the evaluation of gastroesophageal reflux disease (GERD), noncardiac chest pain, or systemic conditions that may lead to esophageal dysmotility. High-resolution impedance manometry (HRiM) with topography plotting incorporates impedance and manometry sensors, providing information on esophageal peristaltic patterns and pressures. Identification of specific esophageal motility disorders, especially subtypes of achalasia, is important, since this often guides therapeutic options . The proPrevious reports trialed using through-the-scope manometric assessment revealed a good correlation between LES pressures obtained by standard manometry. However, these reports were limited by reduced peristaltic wave amplitudes due to the use of dry swallows . AnotherWe report our single-center experience on endoscopy- and monitored anesthesia care- (MAC-) assisted high-resolution esophageal impedance manometry in adult patients to demonstrate the feasibility and effectiveness of this technique.We evaluated patients who had failed prior attempts at manometry in the office setting for this study. All MAC- and EGD-assisted HRiM studies were performed within one week of prior failed attempts of unsedated HRiM tests. Subjects arrived at the endoscopy suite after fasting for a minimum of 6 hours. Topical anesthesia was provided with Cetacaine  spray to the throat and 5 mL of viscous lidocaine 2% solution to the nares. Patients were then sedated using propofol at the anesthetist's discretion in a monitored anesthesia care (MAC) setting. First, while, in the supine position, patients underwent a standard upper endoscopy using an Olympus GIF H180 or H190 gastroscope; an Olympus GIF XP190 scope was required in some cases. A Diversateck HRiM motility catheter was then passed through the right or left nostril into the esophagus and proximal stomach, under direct visualization and guidance with an endoscope. The tip of the catheter was centered directly above the esophagogastric junction (EGJ) prior to entering the stomach. One patient required insertion of a nasopharyngeal trumpeted airway to allow passage of the manometry catheter through the nares due to a history of prior craniofacial surgery. Once the manometry probe was in the correct position, the endoscope was withdrawn. To avoid damaging the motility catheter, endoscopic tools such as biopsy forceps or snares were not used. In some cases, simple endoscopic maneuvers such as opening the EGJ with the neonatal endoscope or gently nudging the tip of the catheter with an endoscope to redirect the motility probe were required to successfully place the motility catheter into the stomach. There was no visible damage to the motility catheter. After allowing sufficient time to awaken , patients were given 10 consecutive 5-mL boluses of normal saline, followed by 5 mL of a viscous solution  as needed, while being in supine position for a total of 10 to 20 swallows. Esophageal muscle functions were recorded using the Diversatek ZVU software. The motility catheter was removed at the end of the procedure. On average, the EGD-assisted HRiM study took about 30 minutes versus 15 to 20 minutes for conventional HRiM test.As shown in Patient 1 was a 32-year-old woman with a history of achalasia with a Heller myotomy at age of eight years. She developed recurrent dysphagia that had been either refractory or with only temporary response, to multiple pneumatic dilations at other institutions. She was unable to tolerate unsedated manometry. The manometry catheter was successfully placed at the time of sedated endoscopy with findings of patent esophagogastric junction (EGJ)/LES, severe ineffective motility and distal esophageal spasm, and a normal IRP consistent with her history of previously treated type III achalasia.Patient 2 was a 51-year-old woman with prior laparoscopic hiatal hernia repair and Toupet fundoplication with solid and liquid dysphagia and nausea who was being considered for surgical revision. Due to a history of craniofacial fractures and postsurgical anatomy and refusal to undergo unsedated manometry, she required direct visual guidance with an EGD scope for proper passage and positioning of the catheter; her manometry was normal.Patient 3 was a 62-year-old woman with a prior Nissen fundoplication for GERD followed by repair of type III paraesophageal hernia with Belsey-Mark IV fundoplication two years later who developed dysphagia and regurgitation. She was unable to tolerate unsedated manometry due to severe gagging. Subsequently, the motility catheter was successfully advanced into the proximal stomach with MAC-assisted endoscopic guidance and showed findings of severe ineffective esophageal motility.Patient 4 was a 71-year-old woman with reflux symptoms and pulmonary fibrosis undergoing evaluation for lung transplantation. Unsedated manometry was attempted but unsuccessful due to inability to traverse the EGJ with the motility catheter. Under endoscopic guidance, the catheter was visualized abutting the distal esophagus just above the EGJ and was successfully guided into the proximal stomach with the assistance of a neonatal endoscope (Olympus GIF XP190). Her motility findings were consistent with type II achalasia. After discussing risks and benefits with the transplant team, patient deferred Heller myotomy and transplant listing.Patient 5 was a 52-year-old woman with a six-month history of progressively worsening solid and liquid dysphagia associated with a >50 lbs weight loss. Patient discomfort and inability to traverse the EGJ precluded her from completing unsedated manometry. Via endoscopy-assisted manometry, a diagnosis of type II achalasia was made and the patient proceeded to POEM with improvement in Eckardt score from 10 (preop) to 3 (postop).Patient 6 was an 18-year-old man with progressive dysphagia and an associated 65-lb weight loss who was unable to tolerate insertion of the manometry probe due to severe gagging and discomfort. He underwent MAC-assisted probe placement; direct visualization was needed due to a dilated esophagus and tight gastroesophageal junction. He was diagnosed with type II achalasia and proceeded to POEM with improvement in Eckardt score from 9 to 0.Patient 7 was an 85-year-old man with solid and liquid dysphagia and an inability to traverse the gastroesophageal junction during prior attempts at manometry. He had undergone prior endoscopies with balloon dilation of the mid esophagus with minimal relief. Barium esophagram revealed a corkscrew appearance. He underwent endoscopy-assisted manometry that showed distal esophageal spasm; he was referred for Botox injection 5 cm above the EGJ with significant symptomatic improvement.Patient 8 was a 63-year-old man with a history of a bilateral lung transplant due to silicosis with episodes of recurrent aspiration and a barium esophagram concerning for a gastroesophageal junction stricture. Due to this known abnormality, direct visualization following standard upper endoscopy exam was used to help advance the manometry probe past a tight lower esophageal sphincter. A diagnosis of EGJ outflow obstruction was made; due to fragile pulmonary status following frequent aspiration events, a percutaneous endoscopic gastrostomy tube was placed following this diagnosis.Patient 9 was a 65-year-old man with a history of dysphagia who failed awake manometry due to inability to pass the probe through his nares with looping in the posterior oropharynx. He had a history of prior craniofacial surgery. After MAC sedation, attempt to insert the motility catheter via either nostril failed. A trumpeted nasal airway was used to allow passage of the probe through his nares and into the esophagus. Due to difficulty traversing the EGJ, an Olympus GIF XP190 scope was used to help guide the probe into proximal stomach. Manometry revealed EGJ outflow obstruction and he was treated successfully with a calcium channel blocker.Patient 10 was a 66-year-old man with two years of worsening dysphagia and 40-lb weight loss referred from another institution after an EGD showed a dilated esophagus and inability to traverse the EGJ with the endoscope, suggestive of achalasia. He underwent repeat EGD at our institution, noting a tight EGJ/LES that was traversed with moderate pressure. Under direct visualization, the manometry catheter was placed into the distal esophagus but was unable to be advanced further due to patient's oxygen desaturation that resolved after propofol infusion was discontinued. Manometry revealed aperistalsis with panesophageal pressurization that, in conjunction with the EGD results and barium esophagram showing tapering of the distal esophagus, was strongly suggestive of type II achalasia. The patient was referred for and underwent successful POEM.Patient 11 was a 58-year-old man with a history of bilateral lung transplant due to cryptogenic organizing pneumonia referred for recurrent aspiration pneumonia and abnormal esophagram with poor antegrade peristalsis and distal intraesophageal reflux. After failing unsedated probe placement, he underwent EGD with endoscopic placement of the motility catheter into the proximal stomach. Manometry revealed ineffective esophageal motility.Patient 12 was a 24-year-old woman with a history of Type I achalasia who had undergone Heller myotomy seven years earlier and complained of progressively worsening dysphagia. Unsedated manometry was unsuccessful, as the probe was not able to enter the esophagus. During sedated probe placement with direct visualization, the probe was not able to enter esophagus via the right pyriform sinus. The probe was repositioned to the left pyriform sinus and able to be advanced into proximal stomach. EGD showed evidence of prior myotomy and patent EGJ/LES. Postsurgical type I achalasia was diagnosed based on manometry findings and dietary modifications were recommended.Patient 13 was a 61-year-old man with a two-year history of solid and liquid dysphagia. An esophagram revealed smooth narrowing of the gastroesophageal junction with incomplete relaxation, a bird's beak appearance, and esophageal dysmotility suggestive of achalasia. Unsedated manometry was attempted and revealed absent peristalsis. However, the probe had looped in the distal esophagus and did not traverse the EGJ. Due to the uncertainty in diagnosis, the patient underwent endoscopy-guided placement of the motility catheter into the proximal stomach under MAC. EGD showed patent EGJ/LES. Manometry confirmed absent contractility and dietary modifications were recommended.Patient 14 was a 75-year-old woman with a history of atypical chest pain and dysphagia who did not tolerate unsedated manometry due to gagging. She underwent successful placement of the manometry catheter into the proximal stomach with EGD guidance under MAC. EGD showed patent EGJ/LES. Manometry findings were consistent with absent contractility and she was currently treated with Bethanechol.We report our experience with MAC/endoscopy-assisted HRiM and clinical outcomes in 14 patients in whom unsedated manometry had previously failed or was contraindicated. The approach was used in a subset of our patients in whom manometric data was required prior to referral for possible invasive procedures or in whom there was diagnostic uncertainty. Notably, all procedures were successful without any peri- or postprocedural complications.This technique effectively overcame anatomical issues related to esophageal tortuosity, distal esophageal dilation, tight LES, deviated septum, or prior craniofacial surgery, respectively. Additionally, patient-related issues such as excessive coughing or gagging and inability to complete the study due to excessive discomfort were managed. Lastly, direct visualization was able to resolve uncertainty in cases with questionable prior manometric findings related to uncertain probe placement. Examples of nasopharyngeal/laryngeal and esophageal/EGJ issues of manometry probe placement are shown in Figures In our experience, the use of endoscopy-assisted HRiM allowed for the successful diagnosis of dysphagia and subsequent treatment with a procedural intervention in a substantial portion of our cohort , as well as medical therapy in three patients (21.4%), or dietary modifications in four (28.6%). In addition, in four patients (28.6%), the manometry findings supported the decision to defer further interventional procedures, including two patients with previously treated achalasia found to have a normal IRP, and two patients with prior hiatal hernia repair under consideration for surgical revision. Lastly, in one pre-lung transplant patient (Patient #4), a new diagnosis of achalasia assisted the patient in deciding not to pursue lung transplantation after a risk-benefit discussion with the transplant team.Limitations of this study include the small sample size, retrospective review of the cases, and lack of a controlled comparative group. However, our study does show the potential benefit of pursuing objective HRiM data and a definitive diagnosis in patients who fail initial attempts at unsedated manometry. Due to the additional expense and small increase in risk of complication due to the endoscopic procedure and required sedation, we only selected a subset of our patients in whom there was a particularly high suspicion of a major motility disorder in which a procedural intervention might be beneficial. Also, we selected patients in whom the decision to recommend a surgical procedure was, in large part, dependent on the manometry findings. Furthermore, obtaining a definitive manometric diagnosis for spastic esophageal disorders as the cause of dysphagia becomes increasingly important as POEM emerged as an attractive treatment option , 9.Another potential limitation of this technique is that the effect of anesthesia on esophageal motility is uncertain. Topical anesthetics do not influence the pharyngeal phase of swallowing, once the swallowing reflex is triggered . PropofoIn conclusion, endoscopy- and MAC-assisted HRiM can ensure completion of esophageal motility studies in patients otherwise unable to achieve definitive manometric diagnoses. In selected patients with suspected major motility disorders such as achalasia, EGJOO, or spastic esophageal motility disorders, our approach can help guide therapy and result in successful treatment outcomes.


Brain metastases in non-small cell lung cancer (NSCLC) patients are more often detected due to imaging modalities improvements but also emerge because of improved treatments of the primary tumor which lead to a longer survival. In this context, development of leptomeningeal metastases (LM) is a devastating complication and its prognosis remains poor despite advances in systemic and local approaches. Histology characterization of NSCLC and molecular expression influence LM management. For those with “oncogene addiction,” new generation epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) were developed to strongly penetrate the blood-brain barrier (BBB) with the aim to prevent central nervous system cancer dissemination, eventually impacting on LM appearance and its subsequent management. Systemic chemotherapy, often combined with intrathecal chemotherapy (when possible), was one of common indications for lung cancer patients affected by LM, without driver mutations and a good performance status but currently, with the advent of innovative systemic approaches treatment solutions in this subgroup of patients are rapidly evolving. Whole brain radiation therapy (WBRT) is the conventional treatment for patients with brain metastases. Furthermore, modern radiation techniques, as stereotactic radiotherapy (SRT), improve outcomes in those cases with a limited number of lesions. However, LM represent a minority of CNS metastases and few literature data are available to drive the radiotherapy approach. Considering all relevant progress made in this setting, after a literature review, the aim of this paper is to discuss about recent developments and therapeutic options in LM management of non-oncogene addicted NSCLC. Non-small cell lung cancer (NSCLC) is characterized by a high incidence of central nervous system (CNS) dissemination, with approximately 40% of patients developing brain metastases (BM) in the course of their disease , 2 and lThe blood–brain barrier (BBB) is constituted by a continuous stratum of endothelial cells connected by tight junctions surrounded by pericytes and perivascular end feet of astrocytes, thus being a highly selective barrier which separates systemic circulation from cerebrospinal fluid (CSF) .BBB maintains CNS homeostasis by enabling the transport of selected substances only, through a combined result of influx and efflux mechanisms. Therapeutic efficacy in this area is determined by whether drug concentrations can be achieved in the CSF and these differ as a result of multiple conditions. The ability of a drug to cross the BBB is substantially improved by particular physic-chemical properties, including low potential for active efflux, few rotatable bonds, small polar surface area, few hydrogen bond donors . For insLM involve penetration of inner layers of meninges and subarachnoid space in which CSF circulates. Its diagnosis is specifically based on three different assessments: clinical signs and symptoms, CSF cytological examinations and neuro-radiological imaging.Early clinical presentation can be subtle and may include headaches and back pain, cranial nerve deficits, cauda equine symptoms or signs, visual disturbances, diplopia, hearing loss and neurocognitive syndromes. In later stages, symptoms related to elevated intracranial pressure could occur , 29. CytMagnetic resonance imaging (MRI), ideally with a 3 Tesla scanner is the most useful imaging modality for the detection of LM. Both T1 with and without contrast enhancement and high resolution T2 fluid attenuated inversion recovery sequences post-contrast are important in establishing a radiological diagnosis of LM.Particularly the disruption of BBB in presence of CNS metastases is often evidenced by peritumoral edema and accumulation of contrast during the imaging scans and, as more recently observed, penetration of CNS metastases is identified by nuclear medicine tracers, such as 18-Sodium Fluoride .The EANO-ESMO clinical practice guidelines propose to classify LM by using two major criteria, being “type I” those LM when the diagnosis has been verified citologically or histologically and “type II” in the absence of verification. While on the basis of the neuroimaging findings: linear leptomeningeal disease (type A), nodular leptomeningeal disease (type B), both (type C) or neither nor, e.g., no neuroimaging evidence of LM except possibly hydrocephalus (type D) .Treatment of LM is preferentially multidisciplinary and mostly indicated when diagnosis is unequivocal or symptoms are strongly suggestive, in case of negative cytology.The goals of treatment in patients with LM are to improve or stabilize the neurologic status of the patient, maintain or regain quality of life and optimally to prolong survival together with marginal toxicity. Limited data are available to establish treatment recommendations in the management of LM: no randomized trials proved a survival benefit of a specific treatment modality and, accordingly, the optimal strategy is still poorly defined, particularly in non-oncogene addicted NSCLC. In this last setting of patients, palliative radiotherapy to symptomatic sites of disease, cytotoxic chemotherapies, intrathecal therapy  are traditionally considered, with the new innovative immunotherapy chance –39.In the era of improved radiation modalities, local treatment of BM is rapidly improving: a growing amount of data support an integrate use of WBRT or stereotactic radiosurgery (SRS) with systemic treatment in a variety of clinical scenarios, together with alternative radiation approaches, such as intensity modulated radiation therapy (IMRT) or even proton beam therapy, when applicable –42.LM represent a minority of CNS metastases (11–20%) , 44 and WBRT is typically used in cases of concurrent brain metastases or major meningeal cerebral involvement . For BM However, craniospinal irradiation (CSI) is generally not recommended, as it is assumed to cause substantial myelotoxicity. In fact, in his review, Chamberlain stated that “whole neuroaxis radiation of the craniospinal axis is rarely indicated in the treatment of LM in solid tumors” .Conformal radiotherapy may help to limit bone marrow and neurotoxicity making focal radiotherapy better tolerated. Proton therapy is only available in a few centers, but this approach promises further reduction of toxicity and effectiveness from CSI , 52.Focal RT administration in fractionated regimens, such as involved-field or stereotactic RT or administered in single fractions (radiosurgery), can be used to treat nodular disease and symptomatic cerebral or spinal sites . By contInvolved field radiotherapy (IFRT) is considered to be the standard of care for palliative treatment of LM. Relevant to this, the US National Comprehensive Cancer Network (NCCN) 2017 guidelines for management of LM recommend Intrathecal Chemotherapy (IT) in combination with IFRT in patients with good prognosis disease . Patients not meeting criteria for good prognosis are recommended to undergo IFRT to symptomatic sites or best supportive care . Up to nTable In the last few years, understanding of immune system's role in the response to ionizing radiation is progressively raising, novel opportunities to study how to combine immunotherapy with radiation-induced cell killing are revolutionizing cancer treatment. Accumulating preclinical and clinical data showed that combination of radiation techniques with immunotherapy stimulates immune response, improves locoregional and distant control finally resulting in better OS. Radiation appears to stimulate the immune system through multiple mechanisms, including the increase of the tumor-associated antigens (TAAs) availability, improving antigen presentation and subsequent stimulation of effector T cells, and enhancing infiltration of dentritic cells and T cells into the tumor microenvironment. The limited evidence for immunotherapy to date in the treatment of BM and LM stems from the deliberate exclusion of patients with active brain metastases from many large randomized trial assessing drug efficacy –63. Howe131I) and yttrium-90 (90Y) with radiolabeled antibodies as HMFG1, 3F8 and 8H9 have been utilized, but further improvement in the pharmacokinetic modeling of CNS RIT modality should refine this emerging therapy to fit the clinical context  consisting of intra CSF administration of radioisotopes like iodine-131  has improved the prognosis of NSCLC patients harboring EGFR mutations on both CNS metastases and extracranial disease, by contrast non-oncogene addicted NSCLC prognosis remains extremely poor.programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) values ≥ 50%  is the mainstay of treatment in advanced non-small cell lung cancer (aNSCLC) with BM and or LM NSCLC at diagnosis without oncogenic driver mutations or es ≥ 50% –73. Tables ≥ 50% –73.Pemetrexed is a compound currently approved both in combination with platinum in first-line setting and as a single agent in maintenance or second line setting for the treatment of non-squamous cell carcinoma –77. AlthFor both squamous and non-squamous cell carcinoma without oncogene-addiction and TPS ≥ 50%, pembrolizumab represents the standard of care in the first-line setting. Data on the efficacy of immunotherapy for BM or LM are currently limited, because of the exclusion of these patients from clinical trials , 87–89. Looking at other type of compounds, a phase II trial with abemaciclib   is ongoiIntrathecal administration is the most common method to deliver chemotherapeutic agents in non-nodular and non-bulky LM in solid tumors, although efficacy compared to systemic administration and choice of regimen are poorly understood due to limited randomized controlled trials . SystemiSeveral retrospective studies demonstrated a survival benefit from IT-therapy. Because of the paucity of available patients, LM studies often accrue multiple primary histologies. Most reports of intrathecal LM treatments include patients who simultaneously receive systemic agents , 95–98. LM is undoubtfully a serious complication in NSCLC patients. Prognosis remains poor, even with the use of personalized treatments, principally due to low penetration into the CSF of currently used agents. To our knowledge no randomized trial has demonstrated a clear survival benefit of any single modality in the treatment of LM. The optimal treatment strategy involves a multi- disciplinary approach. The increasing prevalence of LM warrants further investigation into therapies and prognostic variables to serve as a guide for treatment recommendations and patients counseling. An improved understanding of the biologic mechanisms underlying tumor CNS metastases and novel available diagnostic tools will allow for patient-tailored treatment strategies. Future well-designed randomized controlled studies are needed to evaluate the efficacy of chemotherapeutics, immunotherapies and radiation treatment in this specific subgroup of patients and, more specifically, in those without an “oncogene addiction.”AT and AM wrote the manuscript with support from TV. SN supervised the manuscript.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.


The autonomic nervous system (ANS) has a significant influence on the structural integrity and electrical conductivity of the atria. Aberrant activation of the sympathetic nervous system can induce heterogeneous changes with arrhythmogenic potential which can result in atrial tachycardia, atrial tachyarrhythmias and atrial fibrillation (AF). Methods to modulate autonomic activity primarily through reduction of sympathetic outflow reduce the incidence of spontaneous or induced atrial arrhythmias in animal models and humans, suggestive of the potential application of such strategies in the management of AF. In this review we focus on the relationship between the ANS, sympathetic overdrive and the pathophysiology of AF, and the potential of sympathetic neuromodulation in the management of AF. We conclude that sympathetic activity plays an important role in the initiation and maintenance of AF, and modulating ANS function is an important therapeutic approach to improve the management of AF in selected categories of patients. Potential therapeutic applications include pharmacological inhibition with central and peripheral sympatholytic agents and various device based approaches. While the role of the sympathetic nervous system has long been recognized, new developments in science and technology in this field promise exciting prospects for the future. The heart is extensively innervated and effectively regulated by the autonomic nervous system (ANS) through its sympathetic and parasympathetic branches . The carThe extrinsic nerves run through the hilum of the heart along the great cardiac vessels and divide into seven epicardial subplexi, the intrinsic neural pathways of the ANS . Small nAutonomic nervous system activation has been well-known as a central determining factor of atrial arrhythmogenesis, . Studies2+-inward current and upregulation of inward rectifier K+ currents   . Both co2+) . During 2+) . On the 2+) .Intense atrial fibrosis formation and cardiomyocyte hypertrophy are common features of structural remodeling. Atrial tissue fibrosis damages electrophysiological cell-to-cell coupling and conduction . Long-teSympathetic activation also plays a major role in atrial arrhythmias following cardiac surgeries and post-operative (post-op) AF is associated with reduced long term survival with cardiac surgeries such as the coronary bypass and valvular surgeries especially the aortic valve replacement . Often fGiven the contribution of sympathetic overdrive in the development of AF, sympathetic inhibition represents a logical therapeutic approach in the management of AF and the frequently coexisting hypertension, thereby alleviating the associated cardiovascular mortality and morbidity. Autonomic dysfunction characterized by sympathetic overdrive in scenarios of organic heart disease exhibits both electrical and structural remodeling of atrial myocardium in AF. Pharmacological sympatholysis can be achieved by using drugs such as clonidine and moxonidine. Clonidine is a centrally acting α2-adrenergic agonist that supresses the sympathetic outflow in the lower brainstem , the preMoxonidine seems specifically useful in obesity related hypertension where the sympathetic overdrive sustains both obesity and BP elevation. With the well-established relationship between abdominal obesity, the metabolic syndrome, and the development of AF, obese hypertensives are at a greater risk for AF. Studies have identified that autonomic dysfunction occurs early in the pathophysiology of AF and precedes the development of metabolic abnormalities such as insulin-resistance and obesity  which inSympathetic blockade at the periphery can also be achieved by beta-adrenergic blockers. Though beta-blockers are not generally regarded as membrane stabilizing agents, they delay atrial repolarization and protect against AF. It was speculated that beta-blockers suppress the pulmonary vein ectopy that triggers AF  as well Other interventions include device-based therapeutic approaches to modulate ANS to lower central sympathetic nerve activity such as renal denervation (RDN) and carotid body ablation. These approaches are primarily aimed at reducing sympathetic activity and BP in resistant hypertensive patients but are now investigated as potential treatment strategies in the context of AF.Recently, the AFFORD study, a single-arm pilot study including 20 patients with symptomatic paroxysmal or persistent AF, suggested that RDN alone was safe and able to decrease AF burden in min/day as measured using an implantable cardiac monitor (ICM) at 12-month follow-up accompanied by an improvement in quality of life . A cliniP = 0.0021) and ventricular rate response in pigs with permanent AF. The authors described a reduction of 24% in ventricular rate response in the treated pigs vs. sham, as well shorter AF episodes post-RDN compared with sham  (Mechanistic studies demonstrated that modulation of the SNS via RDN improved AF control . Linz et 0.0091) ,b. Effec 0.0091) . RDN pre 0.0091) . Recentl 0.0091) .P = 0.0251) or non- CKD patients who underwent PVI  over 22.4 ± 12.1 months following intervention. In addition, they found an improvement in echocardiographic parameters such as indexed left atrial volume, left ventricular end-diastolic diameter, and left ventricular mass index in patients who underwent RDN compared to baseline and to other groups. Another important finding was that patients with CKD stage 4 who underwent only PVI (64.41%) demonstrated a higher rate of AF recurrence than those subjected to PVI + RDN  , Kiuchi et al., applying an extensive ablation technique reported the incidence of AF recurrence. It was higher in CKD patients treated with PVI alone (61.5%) than in CKD patients treated with PVI + RDN  .Chronic kidney disease and AF share risk factors and putative mechanisms suggesting that common pathophysiologic processes may drive both pathologies. One possible common link between AF and CKD is activation of the renin–angiotensin–aldosterone system (RAAS) . Angiote18FDG (fluorodeoxyglucose) – uptake in the brainstem, left stellate ganglion nerve activity and atrial tachyarrhythmic events. It was proposed that neural remodeling in the brain stem and stellate ganglion may partially explain the described antiarrhythmic effects of RDN (Trans-synaptic degeneration is a phenomenon in the central and peripheral nervous system that may remain active both at the level of the insult and in remote brain structures for as long as 1 year following trauma (s of RDN . Trans-sg trauma  resulting trauma .trans-synaptic degeneration (Since the sympathetic preganglionic neurons that project to the stellate ganglion are dispersed in spinal cord segments sections T1–T10 , they haneration , as the neration . RDN mayneration .Pulmonary vein isolation with ganglionated plexi ablation has been used in autonomic modulation of interactions between the systemic and intrinsic cardiac ANS. PVI combined with autonomic ganglia modification significantly improved success rates after PVI compared to PVI alone . In animRecent studies have reported that an abundance of epicardial fat is associated with direct adipocyte infiltration into the underlying atrial myocardium . MoreoveStudies demonstrate that stimulation of the stellate ganglion increased sinus rate and predisposes to atrial arrhythmias ,b. IncreIn patients undergoing cardiac surgery and cardiopulmonary bypass, high thoracic epidural anesthesia was found to markedly reduce sympathetic tone  and the Hypersensitive chemoreceptors result in sympathetic excitation. Enhanced chemoreflex sensitivity potentially increasing central sympathetic drive was observed in animal models of pacing-induced congestive heart failure . SurgicaEnhanced sympathetic activation increases circulating catecholamines and causes hypertension and associated complication such as AF and congestive heart failure. Hypertension often coexists with comorbidities like obesity, metabolic syndrome, and obstructive sleep apnea that further exaggerate and sustain the sympathetic overdrive. Substantial evidence indicates that targeting sympathetic overactivity either by pharmacotherapy or device-based interventions seems to be a logical and useful approach for the management of AF. Sympathetic ablation by RDN appears to be a promising strategy to achieve atrial antiarrhythmic effects in animal models and humans. While carotid body ablation is a proven strategy to attenuate sympathetic overdrive, the potential anti-arrhythmic effects are yet to be investigated in future studies. Other techniques employed to reduce sympathetic activity are applied in specific conditions such as stellate blockade in high-risk patients and following myocardial infarction, ganglionic plexus ablation to lower AF recurrence in patients following circumferential pulmonary vein isolation, and high thoracic epidural anesthesia in post-surgical AF management. However further clinical studies are needed to substantiate the role of various sympatholytic approaches in the management of AF.RC and MS have drafted the manuscript. MK, JH, and VM have researched data, contributed to inidvidual sections, and critically reviewed the manuscript.MS is supported by an NHMRC Research Fellowship and has received consulting fees, and/or travel and research support from Medtronic, Abbott, Novartis, Servier, Pfizer, and Boehringer-Ingelheim. The remaining 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.