HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect low-frequency alleles. HIV drug resistance genotyping is a critical tool in the clinical management of HIV infections. Although resistance genotyping has traditionally been conducted using Sanger sequencing, next-generation sequencing (NGS) is emerging as a powerful tool due to its ability to detect low-frequency alleles. However, the clinical value added from NGS approaches to antiviral resistance testing remains to be demonstrated. We compared the variant detection capacity of NGS versus Sanger sequencing methods for resistance genotyping in 144 drug resistance tests (105 protease-reverse transcriptase tests and 39 integrase tests) submitted to our clinical virology laboratory over a four-month period in 2016 for Sanger-based HIV drug resistance testing. NGS detected all true high-frequency drug resistance mutations (>20% frequency) found by Sanger sequencing, with greater accuracy in one instance of a Sanger-detected false positive. Freely available online NGS variant callers HyDRA and PASeq were superior to Sanger methods for interpretations of allele linkage and automated variant calling. NGS additionally detected low-frequency mutations (1 to 20% frequency) associated with higher levels of drug resistance in 30/105 (29%) protease-reverse transcriptase tests and 4/39 (10%) integrase tests. In clinical follow-up of 69 individuals for a median of 674 days, we did not find a difference in rates of virological failure between individuals with and without low-frequency mutations, although rates of virological failure were higher for individuals with drug-relevant low-frequency mutations. However, all 27 individuals who experienced virological failure reported poor adherence to their drug regimen during the preceding follow-up time, and all 19 who subsequently improved their adherence achieved viral suppression at later time points, consistent with a lack of clinical resistance. In conclusion, in a population with low antiviral resistance emergence, NGS methods detected numerous instances of minor alleles that did not result in subsequent bona fide virological failure due to antiviral resistance. Antiretroviral drug therapy (ART) for HIV has been tremendously successful in reducing HIV morbidity, mortality, and transmission . Howeverin vitro diagnostics approval  resistance found a dose-dependent increased risk of virological failure with first-line ART . However26–29–Our reference laboratory provides clinical virology testing for patients in the western Washington region and performed 817 HIV resistance genotyping tests in 2017. Recently, Seattle became one of the first metropolitan areas in the world to attain the 90-90-90 UNAIDS treatment goals, wherein 90% of the patients receiving ART achieve viral suppression . ClearlySerum/plasma samples for HIV antiviral resistance testing were collected as part of routine care and Sanger sequenced for antiviral resistance in our reference clinical virology laboratory using a nested reverse transcription-PCR (RT-PCR) approach, described below . To bestResults of previous genotypic resistance assays performed by our clinical virology laboratory were obtained as the standard-of-care comparison group. Plasma/serum samples were previously processed through the laboratory’s standardized HIV genotypic resistance assay protocol, which involves HIV RNA separated from plasma via the Boom method of silica extraction . Extract2), 8 μg deoxynucleoside triphosphates (dNTPs) (1.25mM each), 1 μl each forward and reverse primer (20 pmol/μg), 0.5 μg Taq polymerase (2.5 U per 10 μl cDNA sample for first-round PCR and 2.5 U per 5 μl PCR product for second-round PCR), and nuclease-free water. A list of primers is provided in Table S1 in the supplemental material. A thermal cycler protocol was programmed for 94°C for 5 min for initial denaturation, followed by 35 cycles of 15 s at 94°C for denaturation, 30 s at 57°C for annealing, 2 min at 72°C for extension, and a final extension at 72°C for 7 min. An annealing temperature of 55°C was used for second-round nested PCR.Nested PCR amplification was performed in 50-μl volume reactions, consisting of 5 μl 10× PCR buffer  after gel electrophoresis  run at 80 mV for 30 min. This PCR product was cleaned by adding 2 μg of exonuclease I and shrimp alkaline phosphatase directly to 10 μg PCR product without changing buffer conditions. The thermal cycler was programmed at 37°C for 15 min, 80°C for 15 min, and 4°C thereafter. Each sample was diluted with nuclease-free water to achieve a final concentration of 10 to 40 ng per reaction mixture. Each sequencing primer (5.0 pmol/μl) was added to 10 μg of the precleaned diluted PCR template in a prelabeled PCR Genemate skirted 96-well PCR plate (T-3107-1).Samples were sent to a centralized sequencing facility. A BigDye terminator kit was used for the sequencing reaction. A Montage SEQ96 Millipore sequence clean-up kit was used for postsequencing. Samples were analyzed using a 3730 XI ABI instrument, and sequence analysis was performed using Sequencher software, v5.2.2.Criteria for acceptable data included raw data peak intensities between 1,000 and 5,000, average noise intensity of <15%, pure base quality value rated high, and no abrupt signal changes, elevated baseline, or spikes. Variant mixes were called when visible in both forward and reverse reads, with minor peaks visible for at least 25% of the surface area of the major peak.pol gene segments  and an interpretation of the resistance profile (what drugs should be avoided) based on the detected mutations. Mutations listed in the genotypic resistance assay report were also interpreted using the Stanford HIV Drug Resistance Database   . For eachttps://www.paseq.org/) and HyDRA (https://hydra.canada.ca/). These two variant callers were selected because they are highly developed, free variant callers with user-friendly web interfaces that require minimal bioinformatics skills and provide robust, reproducible, and easy-to-interpret results that could be implemented in a clinical laboratory  and negative (nuclease-free water) controls to confirm the run. Sequences were preprocessed using cutadapt  and uploboratory . Both caA low-frequency DRM was defined as a resistance-associated mutation between 1% and 20% allele frequency, based on the default 1% minimum allele frequency needed for a mutation to be considered in the drug resistance report produced by both HyDRA and PASeq , 41. A DPatients’ age, sex, and sample date (baseline) were determined based on patient demographics recorded on the genotypic resistance assay report for all patients included in the test cohort. Electronic medical records were reviewed for clinical outcomes. Patients lacking a recorded plasma HIV RNA test result between the sample date and 25 May 2018 were excluded from the patient follow-up cohort due to lack of available follow-up. Laboratory and clinical visit records for patients included in the follow-up cohort were abstracted using a structured data form (information collected is described below), which was designed based on a preliminary in-depth search of five records prior to full record search and abstraction.The following information was recorded for each patient: date of first plasma HIV RNA test in our laboratory information system, plasma HIV RNA load at date of sample (or nearest date), drug regimen prescribed by clinician during clinic visit subsequent to sample date (when both plasma HIV RNA measurement and HIV genotyping results were available to inform clinician actions), and date of most recent office visit where HIV care was received. Adherence (yes/no) was assessed based on clinician visit notes as to whether the patient reported being adherent to the drug regimen prescribed at each clinic visit after baseline. Treatment experience was assessed as naive/experienced based on whether a new diagnosis or initiation of first treatment was noted in the clinical record at the baseline clinic date.The primary outcome of interest, virological failure (yes/no), was determined according to the U.S. Health and Human Services definition, namely, failing to achieve or maintain suppression of viral replication to a plasma HIV RNA level of <200 copies/ml , at a tefmsb package  was analyzed for concordance using percent agreement and Cohen’s Kappa (a standard agreement coefficient for binary ratings). The PASeq and HyDRA allele frequency measures for all called mutations were compared by linear regression analysis. Any variants not called by either caller were assumed to have 0% allele frequency and excluded from the concordance analysis due to absence of quantitation. The linear model coefficient  was also reported. Incidence rate of virological failure was determined based on a count of patients’ first occurrence of HIV RNA level of ≥200 copies/ml divided by time at risk (between sample date and censored date). Patients were censored at date of measured virological failure or last available plasma HIV RNA quantification test prior to 25 May 2018. Relative virological failure rate ratios were calculated using the  package , and allMH789575 to MH789589, MH789591 to MH789610, MH789612 to MH789713, and MH838011 to MH838017 (Table S2).Sequences were submitted to GenBank under accession numbers Of 144 total tests, 29 patients had both Pr-RT and INT tests ordered for them during the sample period, and two patients had a repeat Pr-RT test ordered in the sample period. Therefore, a total of 113 unique patients were ultimately included in the test cohort . PatientUsing the standard-of-care genotyping test by Sanger sequencing, 40 (28%) tests were positive for resistance to at least one drug. By NGS, 63 (44%) tests were positive for resistance by PASeq and 60 (42%) by HyDRA. NNRTI resistance was most common between the methods; PI and INSTI resistance were least common . Of 105 Sanger and NGS showed almost perfect agreement in their detection of high-frequency DRMs (97% of tests). In total, four unexpected differences were observed in a comparison of Sanger- versus NGS-detected variant calls (overview in R2 = 1.00) . Concordance of DRM and accessory allele frequencies  . Of 56 uDRMs on which the two callers disagreed were predominantly very-low-frequency calls ; the medOf 113 patients included in the test cohort, 69 received care at UW Medicine and had obtainable clinical follow-up data. The median follow-up time was 674 days . This cohort was a subset of the test cohort, with a similar age and sex distributions  and prevalence of low-frequency DRMs (32% of patients). Summary statistics for the cohort are provided in Of 69 patients with clinical follow-up, 22 (32%) had low-frequency DRMs detected by NGS that were not found by Sanger. Low-frequency DRMs were most commonly associated with NRTI resistance (9 patients), followed by NNRTI (7 patients), PI (6 patients), and INSTI (4 patients). Five (7%) patients received drugs which would have been contraindicated based on the resistance profiles found by NGS.We were not able to detect a difference in the risk of virologic failure between patients with any low-frequency DRMs at baseline compared to those without . Three patients had only level 1 low-frequency DRMs; when we restrict our exposure definition to patients with level 2 or higher resistance , the virological failure rate in patients with any low-frequency DRMs is not significantly higher than in those without . Among five patients with low-frequency DRMs specifically associated with one of their prescribed drugs, four experienced virological failure. Compared to patients without affected regimens, these five patients had a higher rate of virological failure .However, all participants who experienced virological failure and subsequently reported improved adherence to their drug regimen were able to achieve viral suppression, regardless of whether or not they had low-frequency DRMs, even though most (16 of 19 patients) did not change drug regimens in that time period .Our study suggests a limited marginal utility of next-generation sequencing compared to Sanger sequencing for determining HIV antiviral drug resistance in a population with a relatively low prevalence of antiviral resistance. We also demonstrate that two free, online variant callers, PASeq and HyDRA, were marginally superior to Sanger sequencing for detection of high-frequency DRMs. Both NGS callers were highly concordant with each other with respect to their measured allele frequencies for DRMs. Besides HyDRA’s inclusion of accessory variants and polymorphisms in its default report, which PASeq does not include, the two callers provided nearly interchangeable results.PASeq and HyDRA improve upon Sanger sequencing’s manual variant-calling procedures by being free and automated and, thus, faster, easier, and less expensive (once sequencing data are generated). Our study also illustrated two limitations of manual examination procedures for interpreting Sanger chromatograms: missing linkage and reliance on trained laboratory professionals. First, Sanger was unable to impute linkage between two alleles, which manifested as a DRM mixture (L210CW) instead of a polymorphism (L210C). In this example case, the erred call was a false resistance determination that did not negatively impact patient care because the potentially affected drug types are uncommonly prescribed in this care setting, and the erred variant call is associated with only potential and low levels of associated resistance  according to HIVdb. Second, Sanger called three resistance mutations that were below Sanger’s expected 20% threshold of sensitivity. This overcalling is illustrative of how the procedures rely on a highly trained laboratory technician to review DRM regions in a Sanger-sequenced resistance test. Had the technician not been experienced enough to expect a DRM in that location, it may have been missed. Both manifestations of Sanger error resulted from the human element of Sanger variant calling. Given the ease of use and technical capacity of HyDRA and PASeq, these two callers are both pragmatic options for the detection of variants and interpretation of NGS results in clinical laboratory HIV resistance testing.In our comparison of overall DRM detection rates, NGS detected more mutations associated with higher levels of drug resistance in 30/105 (29%) of Pr-RT sequences and 4/39 (10%) of INT sequences due to its capacity to detect low-frequency DRMs. These findings are consistent with numerous studies in which NGS detected more DRMs than Sanger 1114, 16–2016–de novo as part of natural viral diversification in the body . This finding is consistent with previous research , 44, 45 the body . Poor adthe body . The assMore than 80% of patients in our cohort were prescribed a regimen that included an integrase inhibitor at baseline. However, despite this high prevalence of INSTI prescriptions and low prevalence of INSTI resistance mutations, 39% of patients in the cohort experienced virological failure (70% of whom were on an INSTI-based regimen), and just four (15%) failures occurred in patients on drug regimens contraindicated by their NGS resistance profile. Furthermore, the majority of patients never changed drug regimens following their virological failure, and patients who continued to report poor adherence experienced uncontrolled viral loads well over 200 copies/ml for the duration of follow-up, while those with improved adherence achieved suppression . This suggests that poor adherence is a primary factor affecting rates of viral suppression in this cohort irrespective of the prevalence of low-frequency DRMs.By evaluating the utility of NGS in the context of a pragmatic patient population, we provide a real-world example of its potential to inform clinical care and elucidate factors that could affect its implementation. Our focus on a single high-resource setting is a potential limitation of the study. Seattle is unique in already having achieved UNAIDS goals of 90% of patients on antiretroviral drugs with suppressed virological loads, indicating a smaller potential return on investment for NGS antiviral testing and a low overall prevalence of antiviral resistance. In a high-resource clinical setting, any indication of resistance on a Sanger resistance assay report, even at low levels, is sufficient to rule out that drug for an antiretroviral drug regimen. DRMs causing any degree of change to the level of resistance interpreted by HIVdb were therefore considered in this analysis. In less resource-rich settings, however, potential (level 1) and low-level (level 2) resistance may need to be excluded from the analysis because their clinical significance is not well understood and may be overly conservative for prescribing practices in resource-limited settings.Another limitation of our study is its low power. Despite significant efforts exerted to obtain a sufficiently large cohort size for the analysis, more than a third of patients in the test cohort were excluded from the patient follow-up cohort due to lack of follow-up . The exclusion of these individuals is not a major concern as a source of selection bias, because loss to follow-up is a regular occurrence in the course of clinical management and remains representative of a pragmatic population receiving testing in a clinical laboratory. Given the clinical and potential economic importance of this study’s objectives, a long-term cohort study involving many more patients receiving HIV care and repeat sequencing is needed. However, we also caution that such a study may have limited generalizability in the current age of fast-changing HIV clinical pharmacology. As has been the case for efforts to interpret prior similar studies conducted in populations and time periods without integrase inhibitors , 24, evoRegardless, our findings demonstrate that two online, free, and easy-to-use NGS variant callers have high concordance with Sanger in a pragmatic clinical setting and thus may be good candidates for implementation as part of a clinical laboratory analysis pipeline for HIV drug resistance. However, given the growing prevalence of integrase inhibitor regimens with high barriers to resistance and high prevalence of low-frequency DRMs (predominantly NNRTI and NRTI) in patients with poor adherence, we suggest clinical laboratories use caution in overvaluing the low-frequency variant detection afforded by NGS for clinical antiretroviral resistance genotyping, as it may not provide much improvement over standardized Sanger methods for detection of clinically relevant resistance in a real-world context.


The potential of RNA viruses to adapt to new environments relies on their ability to introduce changes in their genomes, which has resulted in the recent expansion of re-emergent viruses. Chikungunya virus is an important human pathogen transmitted by mosquitoes that, after 60 years of exclusive circulation in Asia and Africa, has rapidly spread in Europe and the Americas. Here, we examined the evolution of CHIKV in different hosts and uncovered host-specific requirements of the CHIKV 3’UTR. Sequence repeats are conserved at the CHIKV 3’UTR but vary in copy number among viral lineages. We found that these blocks of repeated sequences favor RNA recombination processes through copy-choice mechanism that acts concertedly with viral selection, determining the emergence of new viral variants. Functional analyses using a panel of mutant viruses indicated that opposite selective pressures in mosquito and mammalian cells impose a fitness cost during transmission that is alleviated by recombination guided by sequence repeats. Indeed, drastic changes in the frequency of viral variants with different numbers of repeats were detected during host switch. We propose that RNA recombination accelerates CHIKV adaptability, allowing the virus to overcome genetic bottlenecks within the mosquito host. These studies highlight the role of 3’UTR plasticity on CHIKV evolution, providing a new paradigm to explain the significance of sequence repetitions. An emergent virus is a virus that has adapted to new hosts or environments through changes in its viral genome. Using as model chikungunya virus, which had explosively spread in the Americas and Europe during the last decade, we studied viral adaptability to mosquito and mammalian hosts. Natural isolates from recent outbreaks display highly different 3’UTRs, which result in different fitness in mosquito cells. Importantly, drastic changes in viral populations occur when the virus jumps between hosts, with different viral variants being positively and negatively selected according to fitness parameters. Frequent events of RNA recombination, in which the viral polymerase switches templates during RNA synthesis, were observed at the viral 3’UTR. RNA recombination explains the high plasticity of this part of the genome and the subsequent emergence of new viral variants that allows the virus to replicate in disparate systems. Our findings support a new model where viral diversity generated by RNA recombination is a potent viral strategy to overcome the fitness barriers imposed during host switching. RNA viruses occur in genetically diverse populations that allow viruses to replicate under unfavorable conditions, evade immune responses, and alternate between different hosts –4. RapidAedes albopictus . Besides, both linages largely differ at their 3’UTRs. To identify sequence repetitions at the 3’UTR from these two distinct re-emergent viruses, RNA matrix comparisons were performed. Local alignment plots confirm the presence of multiple DRs at both 3’UTRs, but with distinct repetition patterns  and another with a drastic deletion (Clone 15). Then, we followed the production of infectious particles in both mammalian and mosquito cells. Clone 25, which carried the deletion of DR3a, showed delayed growth kinetics in mosquito cells while it did not affect viral replication in mammalian cells. Instead, Clone 15, which carried the complete deletion of both DR3 copies, was not able to replicate neither in mammalian nor mosquito cells . TherefoContrarily from CHIKV-Cbn, CHIKV-LR bears the simplest 3’UTR found in nature. Then, we asked whether the 3’UTR of this lineage would also lose DR copies during passaging in cell culture. To this end, we similarly analyzed the 3’UTR from CHIKV-LR passaged populations. In contrast to our observations with CHIKV-Cbn, all the cloned fragments corresponded in size to the parental 3'UTR, for viruses grown in either mammalian or mosquito cells . This reAltogether, these experiments show that CHIKV lineages have different potential to generate genetic variability at their 3’UTRs. While the CHIKV-Cbn (bearing multiple copies of DR elements at the 3’UTR) is greatly dynamic during single-host passaging, CHIKV-LR (with fewer copies) is seemingly stable in cell culture.SacI and NheI restriction sites at positions 1–6 and 858–863 after the translation stop codon. The recombinant CHIKV-Cbn SacI/NheI was infective in both mammalian and mosquito cells  or one DR3 copy from the CHIKV-Cbn infectious clone . DR copies are represented with colored blocks and DR deletions are indicated with light grey blocks. Bottom, comparative growth kinetics (left) and immunofluorescence at 2 days post-infection (right) of Cbn WT and Cbn Δ(1+2)abb’in human fibroblasts. Error bars represent standard deviations from the mean, n = 3.(EPS)Click here for additional data file.S5 FigAlignment of nucleotide sequences of recombinants generated by template switch from (EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Acute kidney injury (AKI) is associated with prolonged hospitalization and mortality following infant cardiac surgery, but therapeutic options are limited. Alkaline phosphatase (AP) infusion reduced AKI in phase 2 sepsis trials but has not been evaluated for cardiac surgery-induced AKI. We developed a porcine model of infant cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) to investigate post-CPB/DHCA AKI, measure serum/renal tissue AP activity with escalating doses of AP infusion, and provide preliminary assessment of AP infusion for prevention of AKI. Infant pigs underwent CPB with DHCA followed by survival for 4 h. Groups were treated with escalating doses of bovine intestinal AP . Anesthesia controls were mechanically ventilated for 7 h without CPB. CPB/DHCA animals demonstrated histologic and biomarker evidence of AKI as well as decreased serum and renal tissue AP compared to anesthesia controls. Only high dose AP infusion significantly increased serum or renal tissue AP activity. Preliminary efficacy evaluation demonstrated a trend towards decreased AKI in the high dose AP group. The results of this dose-finding study indicate that AP infusion at the dose of 25U/kg/hr corrects serum and tissue AP deficiency and may prevent AKI in this piglet model of infant CPB/DHCA. These physiologic derangements complicate recovery and lead to increased post-operative morbidity and mortality7.Approximately 25% of infants with congenital heart disease (CHD) require invasive treatment in the first year of life. Cardiopulmonary bypass (CPB) is utilized for most neonatal and infant cardiac surgeries to maintain non-thoracic organ perfusion during the repair. Additional techniques such as deep hypothermic circulatory arrest (DHCA) and selective cerebral perfusion (SCP), which result in cessation of all blood flow to the thorax and abdomen, are often required for complex repairs. While use of these techniques is necessary for successful repair, these strategies also contribute directly to systemic derangements in the perioperative period, including splanchnic hypoperfusion, ischemia-reperfusion injury, the systemic inflammatory response syndrome, and post-operative organ dysfunction8. The etiology of AKI in this population is complex, but both direct and indirect physiologic effects of cardiac surgery, CPB, and DHCA/SCP are known contributors10. Evidence of proximal tubular injury appears within 2 hours of cardiac surgery in both animal models and human biomarker studies14, followed by evolution to clinical acute kidney dysfunction over a period of hours to days15. Incidence of AKI following pediatric cardiac surgery ranges from 25–64% depending on the specific population studied and definition used18, with an associated mortality between 5–12%20. Development of AKI is also associated with increased duration of mechanical ventilation and length of hospital stay and may increase risk for development of chronic kidney disease22. While substantial progress has been made in identifying early AKI using novel kidney injury biomarkers, options to prevent or treat AKI remain largely limited to supportive care and avoidance of nephrotoxic medications. Novel therapies to decrease the incidence and severity of AKI following cardiac surgery are greatly needed.Acute kidney injury (AKI) is an increasingly recognized and important complication of pediatric cardiac surgery23. These enzymes are well established as biomarkers of liver and bone disease, but their physiologic roles remain incompletely understood. Recent evidence points towards a potential protective effect of AP in the mitigation of AKI through dephosphorylation of nephrotoxic molecules including extracellular adenine nucleotides26 and endotoxin29. Phase 2 human studies have demonstrated efficacy of AP in the treatment of sepsis-induced AKI32. Less is known, though, about the role of AP as a potential therapeutic agent for cardiac surgery-induced AKI. Serum AP activity decreases in adult and pediatric patients during cardiac surgery36 and lower post-operative serum AP activity is independently associated with higher peak post-operative creatinine37. In a phase 2 study of adults undergoing coronary artery bypass grafting, perioperative infusion of bovine AP was well tolerated and decreased post-operative cytokine expression38. To our knowledge, however, there are no preclinical or human studies evaluating dosing or preliminary efficacy of systemic AP infusion for prevention of CPB/DHCA-induced AKI.Alkaline phosphatases (AP) are endogenous metalloenzymes found in serum and in multiple organs throughout the body including bone, liver, intestine, and kidneyin vivo in a dose-dependent manner and would not significantly alter cardiovascular physiology. Finally, we hypothesized that higher doses of BiAP would result in decreased histologic and biomarker evidence of AKI in this model.In this study we sought to establish a porcine model of early infant CPB/DHCA-induced AKI and determine the serum and renal tissue AP activity achieved with escalating doses of bovine intestinal AP (BiAP) infusion in this model. As a secondary aim, we sought to determine optimal dosing and preliminary efficacy data for BiAP infusion to reduce early AKI in this model. We hypothesized that our model would result in AKI as determined by increased histologic and biomarker evidence of proximal tubular damage. We further hypothesized that continuous infusion of BiAP would raise serum and renal tissue AP activity 40 distribution (median 8.85 (range 7–26) vs 0 (range 0–0); p < 0.0001) , there was no difference in heart rate, systolic or mean arterial blood pressure, or base deficit between CPB animals and anesthesia controls . Conversely, kidneys of CPB/DHCA animals demonstrated a range of AKI findings from minimal injury to severe diffuse AKI with proximal tubular dilatation and epithelial flattening . Similarly, we found no significant differences in the distribution of speed of cooling (p = 0.60) or rewarming (p = 0.83) by Mann-Whitney U test in animals with and without AKI.The distribution of serum creatinine at euthanasia was not different between the CPB/DHCA group (median 1.1 mg/dl (range 0.7–2.4)) and anesthesia controls (median 1.1 mg/dl (range 0.7–1.9)) (p = 0.84). CPB/DHCA animals did demonstrate significantly increased serum neutrophil gelatinase-associated lipocalin (NGAL) levels Fig.  as well Biomarkers of early AKI showed mixed associations with kidney injury as assessed by histology scoring. The distribution of serum creatinine at euthanasia (4 hours post-CPB) was not different in animals with and without histologic evidence of AKI (median 1.1 mg/dl (range 0.7–2.4) vs 1.1 mg/dl (range 0.7–1.9); p = 0.37)  demonstrated significantly lower tissue AP activity compared to animals with no AKI Fig. . AnimalsSerum AP activity was measured at induction, cannulation, rewarming and euthanasia (4 hours post-bypass). There were no significant differences in AP activity between intervention groups at baseline (induction of anesthesia) Fig. . Groups Tissue AP activity also did not increase with either the low dose  or medium dose  AP infusions compared to CPB/DHCA animals with no AP infusion  (p = 0.88). Kidney tissue AP activity was significantly lower in the combined group of CPB/DHCA animals with no AP, low dose AP, or medium dose AP infusions compared to anesthesia controls  for comparison versus the high dose BiAP group (n = 5) and anesthesia only control group (n = 7) in order to evaluate the effect of high dose BiAP on physiology and AKI injury in our model.At 4 hours post bypass, high dose BiAP animals demonstrated higher lactate, VIS, and HR than anesthesia controls  among animals receiving high dose BiAP infusion, the remaining combined CPB/DHCA groups, and the anesthesia controls. Incidence of any histologic AKI (AKI score 1–4) was 20% (1/5) in the high dose BiAP group compared to 60% (9/15) in the remaining CPB/DHCA groups and 0% 0/7) in the anesthesia control group (p = 0.019 for a difference among groups). Incidence of severe AKI (AKI score 3–4) was 0% (0/5) in the high dose group, 27% (4/15) in the remaining CPB/DHCA groups, and 0% (0/7) in the anesthesia control group (p = 0.26 for a difference among groups). Histology scores differed significantly among the high dose AP group, remaining combined CPB/DHCA groups, and anesthesia controls, driven by the higher AKI score in the combined CPB/DHCA group  vs 196 ng/ml (range 86–656); p = 0.87) . Preliminary efficacy data demonstrated a trend towards decreased proximal tubular injury in animals treated with high dose BiAP compared to untreated animals or animals receiving lower doses of BiAP. Kidney injury in the high dose BiAP group was comparable to anesthesia-only controls.46. Central cannulation has the advantage of larger venous cannula size, allowing faster and more complete venous drainage, as well as the ability to directly arrest the heart with the use of aortic cross-clamping and cardioplegia. The primary disadvantage of central cannulation is the high level of surgical expertise needed by the investigator team, potentially limiting feasibility of these large animal models as well as increasing cost in many cases.To our knowledge, this study demonstrates the first use of a peripheral cannulation technique in an infant piglet model of CPB or DHCA-induced AKI. Multiple groups have published neonatal or infant swine models of CPB +/− DHCA utilizing central cannulation techniqueset al. previously described the use of a minimally invasive cannulation strategy for CPB in adult pigs using the right internal carotid artery and right external jugular vein47. We found this approach to be straightforward in infant piglets and conducive to adequate venous drainage for cooling on CPB. In addition, we found that DHCA could consistently be achieved using this peripheral approach without the use of aortic cross-clamping and cardioplegia. Our animals were able to separate from bypass after rewarming with a level of heart failure/inotropic support similar to that of infants undergoing cardiac surgery for the repair or palliation of CHD. Overall, a peripheral cannulation strategy proved to be highly feasible in infant animals and we believe it represents an attractive alternative to central cannulation, particularly for the study of non-cardiac multi-organ injury post-CPB/DHCA.Patel, 48. Additionally, interpretation of a rise in serum creatinine can be quite complicated in neonates and infants undergoing cardiac surgery due to fluid overload, differences in muscle mass, the presence of maternal creatinine, and physiologic changes in glomerular filtration rate49.Our model produces histologic findings consistent with injury to the proximal tubules including epithelial thinning with bleb formation, tubular dilation, and brush border loss. These histologic findings of acute tubular injury are complemented by increased urinary NGAL/Cr ratio and NGAL mRNA expression in CPB/DHCA animals, but as expected no difference in serum creatinine. While serum creatinine and urine output represent the standards for clinical diagnosis of AKI given the inability to routinely measure injury directly through biopsy, the rise in serum creatinine is a late finding, generally occurring hours to days after the initial injury50. Biomarker evidence of proximal tubular injury appears in children as early as 2 hours after the initiation of cardiac surgery with CPB, even earlier than the time point used in our study20. In an elegant study based on recommendations of the Acute Dialysis Quality Initiative Consensus Conference, Basu et al. examined the  relative importance of early kidney dysfunction (measured by 2-hour plasma cystatin C) versus early tubular injury (measured by 2-hour urine NGAL) in the evolution to subsequent severe clinical AKI in pediatric cardiac surgery patients15. Patients were divided into 4 categories based on these early biomarker assessments: 1) no evidence of injury or dysfunction, 2) dysfunction without injury, 3) injury without dysfunction, or 4) combined dysfunction and injury. Early dysfunction alone (elevated cystatin C) demonstrated poor sensitivity and specificity for identifying patients who would ultimately develop AKI. This early dysfunction was hypothesized to represent a transient or physiologic change in glomerular filtration without damage to the tubules themselves. Presence of biomarker evidence of tubular injury (elevated NGAL), however, was highly specific (98%) for the subsequent development of severe AKI although with low sensitivity . The finding of combined early injury and dysfunction maintained high specificity with modestly increased sensitivity. Collectively these findings support the use of proximal tubular injury as an important early renal outcome measure following pediatric cardiac surgery.Recent research points towards the importance of early proximal tubular injury as a critical step prior to the subsequent development of renal dysfunction51. Wang, et al. found similar qualitative  and quantitative histologic changes  at 2 hours post-CPB in their neonatal piglet model of CPB without DHCA, utilizing the previously described low magnification scoring system42. Tirilomis et al. also described this pattern of proximal tubular injury in neonatal piglets 6 hours after CPB compared to minimal histologic changes in the kidneys of piglets undergoing sham sternotomy and laparotomy44. Previous adult porcine models using either central13 or peripheral51 cannulation techniques also demonstrated consistent but heterogeneous early proximal tubular injury following CPB. Taken together, our study and others demonstrate a highly reproducible pattern of early, heterogeneous renal cortical injury in swine of diverse ages undergoing variations of CPB with or without DHCA. Furthermore, we and others do not find evidence of excessive injury  or substantial inflammatory cell infiltrate with these early injury models51. Although the paucity of human renal histology from the early post-CPB period makes direct comparison to porcine models challenging, a single study of ischemic injury in renal transplant grafts demonstrated similar tubular injury52 and we believe that the level of proximal tubular injury present in these models is consistent with the common finding of biomarker evidence of proximal tubular injury seen in adults and children following cardiac surgery50.Our findings of proximal tubular injury are consistent with previously published porcine models of CPB +/− DHCA induced AKI20. Early in the course of ischemia/reperfusion injury NGAL renal expression increases, particularly in the proximal tubule as demonstrated by a series of experiments in rodents50. This increased expression leads to elevated urine and serum levels within an hour after CPB, although interpretation of measurements is complicated by alternative sources of NGAL (notably neutrophil production) as well as filtration and reabsorption of soluble NGAL53. While data concerning NGAL and AKI in porcine models of kidney ischemia-reperfusion are relatively rare, Goebel, et al. have demonstrated increased serum NGAL in adult pigs as early as 2 hours after CPB. Similarly, Mei, et al. showed a rise in both serum and urine NGAL peaking at 6 hours after ischemia-reperfusion injury in their porcine model of ventricular fibrillation54. In contrast, human data on urine and serum NGAL levels after CPB are robust and demonstrate consistent increases in both serum and urine NGAL following CPB, especially in neonates and infants undergoing repair of CHD55.From a biomarker perspective our model demonstrated a mix of expected and unexpected findings. Our most expected finding was an early increase in serum and urine NGAL as well as tissue NGAL mRNA expression in animals exposed to CPB/DHCA. NGAL is a 25 kDa protein typically expressed at low levels in multiple tissues including kidneyet al. did find decreased urinary NGAL concentrations with increased length of ischemic time in a porcine model of unilateral renal ischemia53. These findings support the idea that NGAL expression may occur primarily in stressed but not highly injured proximal tubules and could help explain some of the inconsistency in NGAL prediction of subsequent AKI seen in recent multicenter clinical studies21.Our model also demonstrated two unexpected, novel findings regarding biomarkers of AKI. First, we found that the majority of tissue NGAL staining occurred in otherwise healthy appearing proximal tubules, with progressive loss of staining in tubules with histologic evidence of injury. Similarly, there was a non-significant trend towards higher NGAL mRNA expression in mild to moderately injured kidneys compared to either healthy kidneys or severely injured kidneys. To our knowledge, this pattern of NGAL expression has not previously been described, although Pedersen, 58 and humans57. While the exact function of KIM-1 is only partially understood, studies have demonstrated increased levels in both regenerating proximal tubular cells and cellular debris late after ischemic injury, suggesting a role in both cellular regeneration and potentially as a marker for phagocytosis of dead cells57. Most studies report minimal KIM-1 expression in healthy kidneys58 and little is known about tissue expression of KIM-1 early after ischemia-reperfusion injury. To our knowledge ours is the first report of depressed KIM-1 mRNA expression in injured kidneys in the early post-ischemic period; additional studies are warranted to understand the physiologic significance of this finding.Our second unexpected finding was the consistent suppression of KIM-1 mRNA expression in injured kidneys early after CPB/DHCA. KIM-1 is a cell membrane glycoprotein that is highly expressed in the kidney 12–24 hours after ischemic renal injury in rodent models36 and children37 undergoing cardiac surgery have consistently demonstrated decreased serum AP activity in the immediate postoperative period. Previous studies in our center point towards a combination of early depletion secondary to low AP content in the blood prime of the CPB circuit (unpublished data) as well as ongoing loss of AP activity in the postoperative period37. The etiology of this ongoing loss in humans is not fully understood. It has been suggested that during the process of dephosphorylation, AP may form complexes with pathogen or damage associated molecular pattern molecules, which are subsequently cleared by endothelial cells and macrophages leading to decreased circulating AP59, however other studies including those from our group have shown dephosphorylation of key pathologic molecules (particularly adenine nucleotides) without complex formation61. Similar to findings in humans, our model produced an early decrease in serum AP activity after initiation of CPB due to depletion of AP with introduction of the low-AP activity blood prime, and persistently low activity through rewarming. Serum AP activity in our animals then gradually increased through 4 hours post-CPB/DHCA, a finding similar to that in adult patients36 but different from the more prolonged deficiency seen in our pediatric patients. As our study was not designed to explore the mechanism of ongoing AP depletion, further studies are required to understand if the prolonged deficiency in pediatric patients is secondary to ongoing consumption of AP in the face of persistent post-operative pathophysiology or decreased capacity to produce new AP compared to adult humans or our infant piglets.Observational cohort studies in adultset al. demonstrated marked decrease in the renal tissue AP activity from biopsies of human kidneys with a variety of pathophysiology including chronic ischemia62. In the same study, the authors also demonstrated decreased renal tissue AP activity in rats with ischemia reperfusion injury62. Other groups have demonstrated similar findings in rodent models including a dose-response of lower renal tissue AP activity with longer periods of ischemia64. This decrease in AP activity is thought to be secondary to loss of brush border AP during proximal tubular injury. Decreased tissue AP activity has also been reported in an animal model of intestinal ischemia/reperfusion, again due to loss of brush border AP65, suggesting a common response of multiple epithelial-lined abdominal organs to ischemia-reperfusion injury. Interestingly, rodent models of renal endotoxin exposure result in increased renal tissue AP expression66, demonstrating that tissue AP may have different responses depending on the mechanism and severity of renal injury67.To our knowledge the effects of CPB/DHCA on kidney tissue AP activity have not been previously described. We found decreased tissue AP activity after CPB/DHCA, especially in kidneys with histologic evidence of proximal tubular injury. Heinert, et al. to reduce inflammation and improve pulmonary function (without apparent adverse effects) in a sheep model of sepsis68. The high dose BiAP infusion also was the only dose to result in increased renal tissue AP activity in our model. This finding is consistent with prior distribution studies performed using a human recombinant AP molecule, where injected AP was preferentially delivered to the blood and liver with lower doses achieved in the kidney and other abdominal organs, therefore requiring higher serum levels to result in increased renal tissue levels69.As the final goal of our study, we sought to increase AP activity in both serum and renal tissue using escalating doses of BiAP infusion as well as gather preliminary efficacy data on the incidence and severity of post-CPB/DCHA AKI. We found that only our highest dose of BiAP (75U/kg bolus followed by and infusion of 25U/kg/hr) significantly increased serum AP activity by a factor of ~5 fold above baseline. This dose is similar to the regimen used by Su, 70. Since there are no published studies to provide an estimate for the effect size of AP for the prevention post-CPB/DHCA induced AKI, we did not expect this dose-finding study to be adequately powered to establish efficacy. Instead, these results will be used to power future studies aimed at establishing potential efficacy of BiAP for prevention of AKI in this piglet model of infant CPB/DHCA.From an efficacy standpoint, we found a trend towards decreased proximal tubular injury in animals treated with the highest dose BiAP infusion. To our knowledge this study is the first to evaluate AP therapy in an animal model of CPB with or without DHCA, although rodent models of isolated renal ischemia-reperfusion injury have suggested a decrease in tubular injury with AP therapy32. The first two studies utilized BiAP, with a similar bolus dose (67.5U/kg) but a lower infusion rate (5.5U/kg/hr) compared to our compared to our study31. This dosing resulted in an increase in creatinine clearance as well as a decrease in cytokines and urinary AKI biomarkers without any reported adverse effects31. Serum AP activity was not tracked in either study as a means to assess successful drug delivery or dose response. In the largest study to date, Pickkers, et al. demonstrated a non-significant increase in creatinine clearance in the first 7 days of treatment (primary outcome) and significant increases in day 21 and 28 creatinine clearance (secondary outcomes) in subjects treated with recombinant human AP compared to placebo32. AP-treated subjects also demonstrated lower mortality in this study (secondary outcome). While not definitive, these studies likely support further evaluation in the form of adequately powered phase 3 clinical trials for sepsis-induced AKI. To date, no study has utilized any form of AP to attempt to reduce AKI after cardiac surgery. The direct translation of any protective effects of AP infusion in sepsis-induced AKI to cardiac surgery-induced AKI is inappropriate given the significant differences in timing and physiologic mechanisms of injury. However, it is possible that CPB-induced AKI may be more amenable to treatment than sepsis-induced AKI, as the timing of injury is known and AP could be delivered as prophylaxis prior to initiation of injury rather than as treatment following development of AKI. With this goal in mind, future studies should focus on firmly establishing the efficacy of BiAP infusion to prevent AKI in large animal models of CPB with or without DHCA and, if warranted, phase 1 clinical trials in at-risk cardiac surgery populations.Three recent phase 2 studies have demonstrated promising findings regarding the use of either BiAP or recombinant human AP for the treatment of sepsis-induced AKI72. Large animal models produce more heterogeneous injury13 that is similar to human disease52, but may require larger numbers of animals to demonstrate efficacy of novel therapeutic agents. In an attempt to adhere to guidelines advocating for the reduction the number of animals used to achieve a scientific goal, we chose as a first step to identify the optimal dose from a delivery standpoint to correct the underlying deficiency of AP after CPB/DHCA. We therefore designed our project as a dose finding study with the primary goals of identifying the level of injury in the model, the biochemical changes in the molecule of interest (AP), and the ability to correct this biochemical change with increasing doses of BiAP . The study also generated preliminary efficacy data with which to appropriately power a subsequent larger study using the optimal dose identified from this current work.The primary limitation of our study is the relatively small number of animals in each experimental group in the setting of heterogeneous kidney injury. While small animal models of ischemic AKI utilizing renal pedicle clamping produce more consistent levels of AKI, it has generally been difficult to translate these findings to humansAdditional limitations include the use of a single early time point for the determination of tissue-level injury and biochemistry. Although AKI is thought to occur early after infant cardiac surgery, injury likely continues to evolve in the first 12–24 hours post-operatively. Future studies should incorporate longer post-CPB observation times with the use of serial noninvasive biomarkers to help track injury throughout the post-CPB period, ultimately to include survival models. Also, as we and others have identified, the natural history of renal biomarkers as well as their mechanistic link to tissue damage remain incompletely defined in both large animal models and humans. The field would greatly benefit from mechanistic natural history studies of AKI throughout the post-CPB period to further expand our understanding of noninvasive markers of AKI. In this study, we also focused on injury to a single  organ. Post-CPB/DHCA injury is a multi-system problem and future research should continue to explore the integrated systems biology of this multi-organ injury as well as the systemic effects of BiAP therapy (efficacy and safety). The current study also was not designed to further define the mechanisms of action of AP in prevention of AKI beyond those already assessed in the scientific literature. Further studies are needed to evaluate the relative contributions of particular AP targets (most notably adenine nucleotides and endotoxin) to the kidney injury seen in our model. Finally, to minimize variability in this early pre-clinical study, we chose to focus on a single sex with a relatively narrow weight range. Additional studies are needed to establish the effects of sex, age, and weight on both the biology of post-CPB/DHCA injury as well as the pharmacology and efficacy of BiAP infusion for the prevention of AKI in this setting.The animal protocol used in our studies was approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus in accordance with the Guide for the Care and Use of Laboratory Animals  and the ARRIVE guidelines. Under isoflurane anesthesia, infant female pigs (5–10 kg) underwent peripheral CPB through cannulation of the internal carotid artery and external jugular vein using a 10fr DLP pediatric arterial cannula and 14fr Bio-Medicus one-piece venous cannula . Cannulation was performed after standard surgical dissection of the vessels via a right neck approach and ligation of the cranial end of each vessel. After gaining caudal control of each vessel, the vessel was incised and dilated, followed by cannula placement. The external jugular vein was selected for this model due to its large size in our animals  and distance from the internal carotid, which simplified subsequent placement of the arterial cannula.The CPB circuit consisted of a D101 pediatric oxygenator , low volume tubing, and a standard roller pump. The animals were cooled using the CPB circuit to 22 °C rectal temperature to induce circulatory arrest without the need for aortic cross-clamp and cardioplegia. Once the target temperature was reached with accompanying asystole, CPB was stopped and the animals were left in DHCA for 75 min. Following the period of DHCA, CPB was restarted and the animals were rewarmed over ~30 min to 36 °C. Inotropic support was initiated at 34 °C using a standardized starting dose of epinephrine, dopamine, and milrinone at 0.05 µg/kg/min, 2 µg/kg/min, and 0.5 µg/kg/min respectively. The piglets were then separated from CPB and provided ICU care for 4hrs including full mechanical ventilation and inotropic/vasoactive support . An additional group of animals were ventilated under anesthesia for 7hrs without undergoing CPB/DHCA to serve as anesthesia controls.67.Five animals underwent the CPB/DHCA protocol without BiAP infusion to serve as the baseline model. Medical grade BiAP  was then given to three groups of piglets (n = 5 per group) in escalating doses to assess the effect on serum AP activity and identify the best candidate dose for reduction of AKI in this model. BiAP was administered as a bolus prior to initiation of CPB along with simultaneous initiation of a continuous infusion via a femoral central venous catheter. The infusion was continued through euthanasia. Dosing was provided at the following levels: low (3U/kg bolus followed by 1U/kg/hr infusion); medium (15U/kg bolus followed by 5U/kg/hr infusion); high (75U/kg bolus followed by 25U/kg/hr infusion). Doses were chosen base on extrapolation from phase 2 human sepsis studies (15U/kg bolus followed by 5U/kg/hr infusion-moderate dose group) as well as 5-fold lower and higher doses40. Fresh whole arterial blood was collected at cannulation, rewarming, and euthanasia to measure arterial lactate levels using iSTAT point of care testing .All animals underwent continuous cardiovascular monitoring including femoral arterial blood pressure, central venous pressure, and electrocardiographic monitoring. Heart rate and blood pressure were recorded hourly along with doses of inotropic and inodilator medications and the combined VISFive ml of whole blood was collected from the femoral arterial line at induction, cannulation, rewarming and euthanasia and placed in a standard red top serum tube for processing. Once serum was separated it was frozen and stored at −70 °C for batch analysis. Serum AP activity was measured using a commercially available DRI-CHEM analyzer . To determine kidney tissue AP activity, 50 mg of tissue was homogenized in 500 µl of AP assay buffer  then centrifuged at 11,000 × g for 20 min. Supernatant was diluted 1:20 and analyzed using DRI-CHEM analyzer using the same methods as performed for serum analysis.52. At low magnification (2X), sections were evaluated for tubular dilatation and epithelial flattening and scored 0–4 based on the percent of the parenchyma affected, as follows: 0 = none to less than 5%, 1 = 5–25%, 2 = 26–50%, 3 = 51–75%, 4 = greater than 75%.Kidney tissue was collected from each animal at euthanasia and fixed overnight in 10% formalin, paraffin-embedded, and sectioned at 4 µm. Sections were oriented to demonstrate a radial section of cortex and medulla. Serial sections were stained with hematoxylin-eosin (H&E) and evaluated for severity of kidney injury by a blinded board certified pediatric pathologist with a specialization in pediatric kidney disease (Dr. Treece) utilizing previously published methodologySections were also evaluated for NGAL by immunohistochemistry. After deparaffinization and rehydration, sections were blocked with 1.5% goat serum  in phosphate-buffered saline for 30 min, then incubated with rabbit polyclonal NGAL  antibody overnight, washed with phosphate-buffered saline, and incubated with goat anti-rabbit biotinylated secondary antibody  for 30 min. Vectastain Elite ABC reagent (Vector Laboratories) was then applied, followed by Nova red as substrate. Sections were counterstained with hematoxylin, dehydrated and cover-slipped.Serum and urine samples were taken immediately prior to euthanasia for measurement of serum NGAL and urine NGAL/creatinine ratios. Pig NGAL ELISA kit  was used to analyze serum and urine samples by a Biotek plate reader . Creatinine assay kit  was used to determine urine concentrations by Biotek plate reader . Serum creatinine was measured using a commercially available DRI-CHEM analyzer .2) using the RNeasy Mini Kit  as described in the manufacturer’s protocol. RNA concentrations were quantified at 260 nm, and the purity and integrity were determined using a NanoDrop. RT and real-time PCR assays were performed to quantify steady-state mRNA levels of NGAL, kidney KIM-1, and IL-6. cDNA was synthesized from 0.5 µg of total RNA. Predeveloped TaqMan primers and probes  were used for detection. Reporter dye emission was detected by an automated sequence detector combined with ABI Prism 7300 Real Time PCR System . Real-time PCR quantification was performed with TaqMan b-actin controls and relative mRNA expression calculated using the 2−ΔΔCT method.Total RNA was isolated from kidney tissue  to assess for a difference among groups. If indicated, subsequent pairwise testing was performed using either Student’s t-test or Mann-Whitney U test, as appropriate for the data distribution, to determine specific differences between groups. P < 0.05 was considered statistically significant. GraphPad Prism 6  was used for data analyses and graphics production.Supplementary Figures


Het), which present with approximately 30% less synaptic ACh compared to control mice. Because ACh is constitutively decreased in VKDHet, we first analyzed developing NMJs and muscle fibers. We found no obvious morphological or molecular differences between NMJs and muscle fibers of VKDHet and control mice during development. In contrast, we found that moderately reducing ACh has various effects on adult NMJs and muscle fibers. VKDHet mice have significantly larger NMJs and muscle fibers compared to age-matched control mice. They also present with reduced expression of the pro-atrophy gene, Foxo1, and have more satellite cells in skeletal muscles. These molecular and cellular features may partially explain the increased size of NMJs and muscle fibers. Thus, moderately reducing ACh may be a therapeutic strategy to prevent the loss of skeletal muscle mass that occurs with advancing age.In addition to driving contraction of skeletal muscles, acetylcholine (ACh) acts as an anti-synaptogenic agent at neuromuscular junctions (NMJs). Previous studies suggest that aging is accompanied by increases in cholinergic activity at the NMJ, which may play a role in neuromuscular degeneration. In this study, we hypothesized that moderately and chronically reducing ACh could attenuate the deleterious effects of aging on NMJs and skeletal muscles. To test this hypothesis, we analyzed NMJs and muscle fibers from heterozygous transgenic mice with reduced expression of the vesicular ACh transporter (VAChT; VKD In ChAT null mice, the formation and subsequent stability of nAChR clusters at the postsynaptic region of the NMJ are similar to wild type mice . Specifically, in the gastrocnemius and diaphragm muscles, the amplitude of miniature endplate potentials (MEPPs), which represents the spontaneous release of vesicles at active zones, was found to be larger in old mice compared to young mice , which have decreased expression of VAChT that results in approximately 30% less ACh loaded and released from synaptic vesicles, and a concomitant decrease in the MEPP amplitude  and littermate Thy1-YFP16 controls. Genotyping was carried out using RT-PCR following proteinase K DNA extraction with subsequent purification using a QIAquick PCR purification kit (Qiagen 28104). All transgenic animals were allowed free access to food and water and housed with a 12-h light/dark cycle. Mice were anesthetized using isoflurane and either dissected immediately for fresh tissue or perfused transcardially with 1× PBS (pH 7.4) and 4% paraformaldehyde (PFA) for fixed tissue. All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.Heterozygous VAChT-KD mice . After washing once more with 1× PBS, muscles were whole-mounted onto slides using Vectashield (Vector Labs).The extensor digitorum longus (EDL) muscle was dissected from VKDNMJs from P9 EDL muscles were blocked for 1 h with 3% BSA, 5% goat serum, and 0.5% Triton-X. Samples were then incubated with primary anti-VAChT  for 24 h at 4°C. After three washes with 1× PBS, samples were incubated with secondary antibody Alexa 488 goat anti-guinea pig  and Alexa 555 conjugated α-bungarotoxin  for 2 h at room temperature. Then samples were washed three additional times with 1× PBS and mounted using Vectashield (Vector Labs). Intensity analysis was done using ZEN software (Zeiss) with background correction.To analyze structural features at NMJs, maximum intensity projections of confocal stacks were created using ZEN software (Zeiss). We analyzed structural features as previously described  and cut in 16 μm slices using a cryostat.The sections were stained with Alexa-488-conjugated wheat germ agglutinin  and 4′,6-diamidino-2-phenylindole  diluted in 1× PBS for 2 h at room temperature. The sections were then washed three times in 1× PBS and mounted using Vectashield. Images were acquired using a Zeiss LSM 700 confocal microscope and maximum intensity projections from z-stacks were created with Zen Black (Zeiss). The area outlined by WGA was measured and Feret’s diameter was determined using ImageJ. At least 100 muscle fibers per cross-section were randomly selected for analysis. From the selected fibers, the percent of muscle fibers with centrally located myonuclei was recorded. At least three cross-sectional images were analyzed and averaged together to represent an individual mouse. At least three animals per genotype were analyzed to generate the represented data.The sections were stained with Alexa-488-conjugated  and DAPI  diluted in 1× PBS for 2 h at room temperature. The sections were then washed three times in 1× PBS and mounted using Vectashield. Images were acquired using a Zeiss LSM 700 confocal microscope and maximum intensity projections from z-stacks were created with Zen Black (Zeiss). The total cross-sectional area (CSA) of the TA muscles was outlined by WGA and measured using ImageJ. The average CSA was calculated from at least three cross-sectional images per individual animal. At least three animals per experimental group were analyzed to generate the represented data.Het = 1,775 ± 156, 17 month control = 1,596 ± 147, 17 month VKDHet = 1621 ± 356).An antibody against Pax7, obtained as a gift from Dr. Julia Von Maltzahn, was used according to the protocol described in Ahrens et al. . In brieTA and EDL muscles and spinal cord were dissected and flash-frozen in liquid nitrogen. Total RNA was prepared using Aurum Total RNA Mini kit (Bio-Rad) following the manufacturer’s instructions. cDNA was synthesized from 500 ng of total RNA using the iScript cDNA synthesis kit (Bio-Rad) and subsequently diluted to 100 ng. PCR amplification was performed on the Bio-Rad CFX Connect Real-Time System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). All primers used in this study are listed in t-tests were used to compare differences between two groups. For statistical analysis of multiple groups, a one-way ANOVA with post hoc Bonferroni test was used to determine significance. A Kolmogorov–Simirnov test was used to compare differences between sample distributions. Data are expressed as the mean ± SEM (standard error measurement) with individual data points represented along the error bar. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism software.Student’s Het, to determine the impact of moderately and chronically reducing synaptic ACh on NMJs and muscle fibers. First, we validated previous findings showing that VAChT mRNA is significantly lower (−34.83 ± 2.092%) in the spinal cord of VKDHet mice compared to litter mate control mice , which binds selectively to nAChRs located on the postsynaptic region of the NMJ. Quantification of fluorescence intensity revealed a marked reduction in VAChT (−53.5 ± 7.63%) but not fBTX at NMJs of the EDL muscle of VKDHet mice compared to control mice  specifically in neurons, including alpha-motor neurons  isoforms , cyclin dependent kinase 5 (CDK5), docking protein 7 (Dok7), LDL receptor related protein 4 (LRP4), and Rapsyn in the TA and EDL muscles of 4- and 17-month-old VKDrol mice . Unexpecrol mice . We nextrol mice . This isHet mice suggested that reducing ACh may have a similar effect on muscle fibers. To visualize and measure the area of muscle fibers, we stained 16 μm muscle cross-sections from the TA muscle of 4- and 17-month-old mice with WGA  presenting with reduced expression of the VAChT, and thus decreased loading of ACh into synaptic vesicles. VKDHet mice present with a mild reduction in MEPP amplitude  the increased size of the NMJ may be a consequence of reduced internalization of nAChRs, thus leading to larger postsynaptic sites. In this case, muscle fibers may not be under pressure to alter the expression of nAChR subunits since the moderate loss of ACh may simply cause nAChR subunits to redistribute. For example, decreasing ACh may cause nAChR subunit protein to accumulate at the peripheral membrane resulting in decreased levels in vesicles destined for degradation or recycling. (2) The increased expression of AChE may result from skeletal muscles attempting to maintain AChE density to account for the expanded size of NMJs. In support of this hypothesis, AChE has been shown to increase in differentiating and thus growing muscle fibers (Fuentes and Taylor, In stark contrast, decreasing ACh markedly affected adult NMJs and muscle fibers. In young adult and aged VKDHet mice compared to age-matched control mice. In young adult VKDHet mice, we found that reduced expression of Foxo1 contributes to the increased size of muscle. In aged VKDHet mice, we found that increased expression of Pax7 and an associated increase in the number of satellite cells contributes to the increased size of muscle fibers. This finding is significant as it demonstrates that a moderate decrease in ACh slows sarcopenia, or the atrophy of skeletal muscles with advancing age, by increasing the number of satellite cells. However, our study does not address whether the effects of reducing ACh on satellite cells is direct or indirect. It is possible that ACh acts in both manners given that satellite cells have been shown to respond to changes in activity patterns (Tatsumi et al., We also found that muscle fibers are larger in young adult and aged VKDThis study demonstrates that moderately, chronically, and ubiquitously reducing ACh can mitigate the deleterious effects of aging on NMJs and muscle fibers. However, the use of a transgenic mouse line (Prado et al., Additionally, it remains possible that other components of the cholinergic system could become dysregulated at the presynaptic and postsynaptic regions of the NMJ, in addition to Terminal Schwann cells, independently of changes in ACh levels. In addition to cellular constituents of the NMJ, it may be necessary to devise combinatorial strategies to modulate the cholinergic system in other skeletal muscle resident cells to confer additional protection to NMJs and muscle fibers from aging. This could include, but is not limited to, activating the alpha7 nAChRs in macrophages to reduce the adverse effects of inflammation (Lee and Vazquez, All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.SV and NS contributed to data collection, data analysis, and writing the manuscript. GV designed the study, prepared the manuscript, and procured funding. All authors read and approved 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.


At least 90% of human genes are alternatively spliced. Alternative splicing has an important function regulating gene expression and miss-splicing can contribute to risk for human diseases, including Alzheimer’s disease (AD).n = 24) and cognitively normal elderly controls  and identified three exon skipping events in two genes (RELN and NOS1) as significantly associated with AD . Next, we identified single-nucleotide polymorphisms (SNPs) affecting exon skipping events using the splicing decision model and then performed an association analysis of SNPs potentially affecting three exon skipping events with a global cortical measure of amyloid-β deposition measured by [18F] Florbetapir position emission tomography (PET) scan as an AD-related quantitative phenotype. A whole-brain voxel-based analysis was also performed.We developed a splicing decision model as a molecular mechanism to identify functional exon skipping events and genetic variation affecting alternative splicing on a genome-wide scale by integrating genomics, transcriptomics, and neuroimaging data in a systems biology approach. In this study, we analyzed RNA-Seq data of hippocampus brain tissue from Alzheimer’s disease  adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-β levels . This SNP is within the splicing regulatory element, i.e., intronic splicing enhancer. The minor allele of rs362771 conferred decreases in cortical amyloid-β levels in the right temporal and bilateral parietal lobes.Two exons in Our results suggest that exon skipping events and splicing-affecting SNPs in the human hippocampus may contribute to AD pathogenesis. Integration of multiple omics and neuroimaging data provides insights into possible mechanisms underlying AD pathophysiology through exon skipping and may help identify novel therapeutic targets.The online version of this article (10.1186/s12920-018-0453-8) contains supplementary material, which is available to authorized users. CLU and CD33 were reported to be associated with AD pathogenesis  Florbetapir position emission tomography (PET) scans. These results provide a new link between alternative splicing changes and AD.This study explores how transcriptomics, genomics and neuroimaging endo-phenotypes can be leveraged as a means to clarify our understanding of the genetic architecture of AD. Using RNA-Seq data from cognitively normal elderly controls and AD-affected human hippocampal tissues, alternative splicing isoforms were evaluated by measuring exon skipping. A computational pipeline to identify exon skipping events using RNA-Seq data and a splicing decision model to identify actionable loci among common SNPs for gene regulation were applied in this study to gain insights into the functionality of the variations and emphasized their importance for the AD pathology . We idenhttp://human.brain-map.org/). RNA was isolated from the hippocampus tissue of brains of AD patients  and non-AD elderly controls  from the Adult Changes in Thought (ACT) study. The ACT study is a longitudinal population-based prospective cohort study of brain aging and incident dementia in the Seattle metropolitan area, as described in detail in previous studies  Florbetapir PET scans downloaded from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) were pre-processed as described [18F] Florbetapir PET scans were intensity normalized by the whole cerebellum. The normalization yielded standardized uptake value ratio (SUVR) images. The ongoing ADNI study was launched in 2003 to test whether serial magnetic resonance imaging (MRI), PET, other biological markers, and clinical and neuropsychological assessment could be combined to measure the progression of MCI (mild cognitive impairment) and early AD, as published previously [www.adni-info.org. Genotyping for ADNI was performed using three different Illumina genotyping platforms. We imputed un-genotyped SNPs separately in each platform using MACH and the HRC (Haplotype Reference Consortium) data as a reference panel after standard sample and SNP quality control procedures and selection of only non-Hispanic Caucasian participants [[escribed . [18F] Feviously , 39 and icipants .http://www.fil.ion.ucl.ac.uk/spm/software/spm8/) [First, an association analysis was performed for SNPs that potentially affect identified exon skipping events with a global cortical measure of amyloid-β deposition as measured by PET scans in an AD-related quantitative phenotype. The global cortical amyloid-β level was calculated as a mean regional SUVR value extracted for the frontal, parietal, temporal, limbic, and occipital lobes using the MarsBaR toolbox implemented in the Statistical Parametric Mapping 8 (SPM8) software (e/spm8/) . Also, aThis study analyzed RNA-Seq data of hippocampus brain tissues from 74 participants from the ACT study. Participants were diagnosed as cognitively normal elder controls (CN) or AD patients. As shown in Table RELN and NOS1, as significantly associated with the AD . Two exons in RELN, exons 24 and 37, showed significantly lower expression levels in AD patients compared to CN participants, suggesting that the exons tend to be skipped more in the AD , suggesting that the exon tends to be skipped more in higher Braak stages of AD participants. In NOS1, exon 23 had lower expression levels in AD patients compared to CN participants . The adjacent exon 23 and 25 showed similar levels of difference but not significant . The transcript (transcript1) retaining the exon 24, which encodes the hEGF, likely results in a functional version of the RELN gene, while the transcript which lacks exon 24 will lose the hEGF domain and thus produce the truncated protein product due to the out-of-frame of the exon length . The transcript (transcript1) retaining the exon 23, which encodes a part of the NO_synthase, can be translated into the protein with normal functions of NOS1. In contrast, the transcript with the skipping of exon 23 may not only lose the NO_synthase domain but also produce the truncated protein due to the out-of-frame of the exon length. We also showed the partial loss of the protein structure due to the skipped exon using the protein 3D structure analysis  from HRC-based imputed ADNI GWAS data. We identified one SNP (rs362771) in intron adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-β levels . Furthermore, we performed an unbiased whole-brain-based imaging association analysis using age, sex, years of education as covariates to assess the effect of rs362771 on whole-brain amyloid-β deposition and identified significant associations after adjustment for multiple comparisons using cluster-wide FDR procedure. The minor allele of rs362771 conferred decreases in cortical amyloid-β levels in the right temporal and bilateral parietal lobes , suggesting that the SNP may affect the exon skipping and the skipped exon may be associated with AD pathogenesis.Next, we performed an association analysis of SNPs affecting exon skipping events with a global cortical measure of amyloid-β deposition as an AD-related quantitative phenotype. Using the splicing decision model, we first identified 46 and 11 SNPs in RELN and NOS1 in the human hippocampus affected by Alzheimer’s disease (AD) were identified. Interestingly, expression levels of identified alternatively spliced exons are decreased in the AD and the minor allele of one SNP in RELN potentially affecting exon skipping negatively correlate with a global cortical amyloid-β burden. Our results indicate that essential exon regions for the RELN and NOS1 genes are alternatively spliced in the AD hippocampus compared to cognitively normal elderly controls. It was previously reported that RELN delays amyloid-β fibril formation and rescues cognitive deficits in an AD model [RELN and NOS1 were associated with neurologically related traits, the methodology presented here suggests new possible mechanisms by which they may influence AD pathology [Here we developed a computational pipeline for the identification of exon skipping and a splicing decision model for the identification of SNPs affecting exon skipping. Altered expression patterns (exon skipping events) within two distinct exon regions of AD model . Two majathology –45.RELN is an extracellular matrix glycoprotein that plays a number of important roles in the central nervous system (CNS) and its dysfunction is associated with AD [RELN and neurological traits, often using case-control study design [RELN. The function of the RELN protein is to help mediate cell migration during brain development by activating a signaling pathway through binding of cell surface proteins [RELN expression can significantly accelerate amyloid-β deposition in transgenic AD mice [RELN protein that potentially accelerates amyloid-β deposition and/or mis-regulation of tau phosphorylation, both leading to AD-related phenotypes. Specific exonic expression appears to play an important role. Thus, understanding the role of exon skipping may lead to more consistent conclusions with respect to the role of RELN and other proteins [ with AD –48. Manyy design . Charactproteins , 50. Theproteins .NOS1, the other gene identified in this work, has also been of significant interest in relation to heart and neuronal function since nitric oxide (NO) is an important signaling molecule that is produced by NO-producing enzymes, such as NOS1 [NOS1 could lead to increased ROS, and thus increased protein mis-folding and aggregation associated with neurodegenerative diseases (i.e. AD). Both increased and decreased expression of NOS1 have been associated with a cognitive disruption in relation to the AD [ as NOS1 . NOS cano the AD . TherefoNOS1 and RELN. Since both RELN and NOS1 could exacerbate different mechanisms by which AD arises, it would be interesting for future work to investigate the co-occurrence of variants in these genes and how it impacts neurological function. While many advantages of the methodology employed here have been described in detail, it is also valuable to point out some limitations to avoid over-interpretation and reflect on what improvements need to be addressed in future work.Although both genes had previous connections with neuronal development and splicing , 51, thiRELN and NOS1 genes were identified as having differential exon usages with respect to the AD. This work also suggests applying an imaging genetics approach, along with utilizing SRE variants, will help shed light on previously unidentified gene-trait relationships.For instance, our sample size was moderate for this study with respect to the expression analysis. Thus, we may have missed some genes with alternative splicing in SRE SNPs associated with AD due to limited detection power. Previous work has suggested sex differences in AD . The othAPP [PSEN1 [PSEN2 [APOE [MAPT [p-value < 0.05 and fold changes between − 1.5 and 1.2, which might be due to the following reasons, 1) alternative splicing events occur in the tissue- and cell type- specific manner and are even more specific to brain regions [Our study was not able to recapitulate a statistically significant exon skipping or spliced isoforms in any of the historical genes . How regions , 57, 2) RELN and NOS1, three exons are altered in their expressions in the human hippocampus affected by Alzheimer’s disease. It also suggests that the functional relationship between exon skipping and protein structures for RELN and NOS1 may be altered in AD pathology. Further studies are needed to better understand the functional role that identified exon skipping and SNPs affecting exon skipping play in AD pathophysiology. Integration of multiple omics and neuroimaging data in a systems biology approach will provide valuable insights into a possible mechanism underlying AD pathology through exon skipping, thus potentially helping identify novel therapeutic targets.In conclusion, this study provides evidence that our novel approach can identify significant exon skipping events and genetic variation potentially affecting exon skipping in Alzheimer’s disease. Between Additional file 1:Figure S1. Functional impact of the AD-associated exon skipping event (exon 37) in RELN. (A) Schema of the potential functional implication of exon skipping and splicing-associated SNP. (B) Normalized expression levels for exon 37 between AD and CN participants. (C) Structure alignment of the pair of transcript1 retaining exon 37 (green) and transcript 2 with the exon skipping (red). (PDF 58 kb)


We report a patient with multiple endocrine neoplasia type 1 with pancreatic polypeptide (PP) secreting subcentimeter pancreatic neuroendocrine tumors (pNETs) treated with octreotide and review the current literature that pertains to the management of these patients. Clinical data, laboratory results, and imaging were reviewed. A literature search was performed in PUBMED using combinations of the terms “multiple endocrine neoplasia type 1,” “somatostatin,” octreotide,” “pancreatic polypeptide,” and “pancreatic tumor.” Relevant references were selected and reviewed. A 43-year-old male with a history of MEN1 and multiple subcentimeter neuroendocrine tumors with elevation of PP was treated with octreotide therapy leading to a reduction and normalization of PP levels. The patient tolerated octreotide therapy but self-discontinued octreotide after 24 months with a rise in PP levels off therapy. Tumors remained stable in size through 40 months of imaging follow-up. In patients with MEN1 and subcentimeter pNETs, octreotide therapy is well tolerated and can lead to a significant drop in PP levels with no change in lesion size. There is insufficient data to suggest long-term benefit with octreotide therapy but it may be considered versus standard conservative management. Multiple endocrine neoplasia type 1 (MEN1) is a rare, autosomal dominant syndrome causing a predisposition to tumors of the parathyroid glands, anterior pituitary, and enteropancreatic endocrine cells with an estimated prevalence of 2 to 20 per 100,000 individuals , 4. ThesCurrent recommendations for screening for enteropancreatic neuroendocrine tumors include both biochemical evaluation and radiological screening . Cost efThe management of pNETs is variable due to the heterogeneity of the disease. The role of surgery for nonfunctional pancreatic tumors (NFPETs) in MEN1 is controversial with the endocrine society clinical practice guideline suggesting surgery for tumors greater than 1 cm and/or demonstrating significant growth over 6-12 months . AdditioHere we describe a middle-aged patient with a history of MEN1 who presented with a markedly elevated serum PP level, leading to the discovery of multiple subcentimeter pancreatic lesions found on screening CT, and with prolonged stability on SSA medical therapy.μg 3 times daily, and this was later transitioned to a long-acting octreotide 20 mg monthly. Following the octreotide treatment, the serum pancreatic polypeptide levels normalized to 62 pg/mL in approximately 6 weeks . A CT scan of the abdomen showed multiple subcentimeter lesions. The patient refused any treatment initially; 11 months later the serum pancreatic polypeptide levels rose to 1198 pg/mL (range 912-1588), and 5 weeks later the value was 1215 pg/mL . At this 6 weeks . A follo 6 weeks . The pat 6 weeks  performeUse of somatostatin analogs in the treatment of pNETs is based on investigations showing that the natural 14-amino-acid peptide binds to G-protein-coupled somatostatin receptors (SSTRs) that are expressed on most NETs with SSTR-2 expressed in approximately 80% of pNETs , 12. OctAdditionally, there is insufficient evidence to clarify whether a decline in PP level correlates with a reduction in tumor burden. We would argue that the significant decline in PP level, a nonspecific tumor marker, in addition to the lack of tumor growth and stable follow-up PP level is indicative of a treatment effect as has been seen with other hormonally active NETs treated with SSA therapy. We speculate that if the level of PP continues to rise off therapy, the patient would experience further nodular growth, which could have adverse health consequences. Typical management of subcentimeter NFPETs has typically involved serial imaging and biochemical testing. SSA therapy in addition to serial testing represents a reasonable treatment option that is well tolerated in this patient population although its high-cost may hinder its utilization.Furthermore, basal and meal-stimulated pancreatic polypeptide has been shown to be useful for early detection of pancreatic involvement in 75% of patients with MEN-1. Our patient's basal fasting pancreatic polypeptide levels were not only elevated but actually doubled at one-year follow-up. The stability of the pancreatic tumors along with the normalization of serum PP level confirms the effectiveness of SSA therapy in this patient. It may be stated that the pancreatic lesions would have remained stable without octreotide treatment; however, octreotide was effective in our patient for several reasons: (1) the serum PP level doubled over the 11-month observation period when he did not receive octreotide treatment; (2) there was an increase in the size of pancreatic tumors during this observation period; and (3) a gallium-68 DOTATATE scan showed stability of tumor size over the course of treatment. Therefore, these findings support the effectiveness of octreotide treatment. As the benefits of pancreatic surgery for small lesions (<2cm) are not clear, the morbidity, mortality, and long-term complications of surgery must be considered. Our patient was thus offered medical therapy with long-acting octreotide, which was recently shown to provide 90% objective tumor response and stability in MEN-1 patients with early nonfunctional pancreatic NET . DiscontThe malignant potential of pNETs is the primary life-threatening manifestation of MEN1 but treatment for NFPET <1 cm has not been clearly defined by the guidelines. We describe the case of a middle-aged patient with a history of MEN1 who presented with elevated serum PP levels and multiple subcentimeter pancreatic lesions found on imaging who was treated with SSA therapy for 24 months leading to a marked reduction in PP level during treatment with stability in lesion size after 40 months of follow-up. SSA therapy represents a reasonable and potential alternative to watchful waiting in this patient population but further research is necessary to better define long-term outcomes.