Depression is one of the most common mental health disorders and severely impacts one’s physical, psychological, and social functioning. To address access barriers to care, we developed Ascend—a smartphone-delivered, therapist-supported, 8-week intervention based on several evidence-based psychological treatments for depression and anxiety. A previous feasibility study with 102 adults with elevated depression reported that Ascend is associated with a postintervention reduction in depression symptoms.We aimed to examine whether Ascend is associated with a reduction in symptoms of anxiety, and importantly, whether reductions in symptoms of depression and anxiety are maintained up to 12-months postintervention.We assessed whether the previously reported, end-of-treatment improvements seen in the 102 adults with elevated symptoms of depression extended up to 12 months posttreatment for depression symptoms  and up to 6 months posttreatment for anxiety symptoms . We used linear mixed effects models with Tukey contrasts to compare time points and reported intention-to-treat statistics with a sensitivity analysis.P<.001; Hedges g=1.14, 95% CI 0.78-1.49). A total of 60% of the participants with PHQ-9 scores above the cutoff for major depression at baseline (PHQ≥10) reported clinically significant improvement at the 12-month follow-up (at least 50% reduction in PHQ-9 score and postprogram score <10). Participants also reported reductions in symptoms of anxiety that were maintained for at least 6 months after the program .The intervention was associated with reductions in symptoms of depression that were maintained 12 months after the program (6.67-point reduction in PHQ-9 score, 95% CI 5.59-7.75; There is limited evidence on whether outcomes associated with smartphone-based interventions for common mental health problems are maintained posttreatment. Participants who enrolled in Ascend experienced clinically significant reductions in symptoms of depression and anxiety that were maintained for up to 1 year and 6 months after the intervention, respectively. Future randomized trials are warranted to test Ascend as a scalable solution to the treatment of depression and anxiety. Depression is a common mental health disorder and one of the leading causes of disease burden and disability worldwide -3. IndivAlthough there are several effective pharmacological  and psycRecently, digital interventions delivered via smartphone apps have been developed as a means to address this need . SmartphDespite these promising results, several important questions regarding the design, efficacy, and implementation of smartphone-based interventions for common mental health disorders remain unanswered. First, few authors report on the outcomes of commercially developed smartphone-based interventions for depression and anxiety, making it difficult to evaluate their utility ,28. SecoAscend intervention, a novel, 8-week smartphone-based intervention for elevated symptoms of depression and anxiety, assisted by a remote therapist [Ascend is associated with reductions in comorbid symptoms of anxiety, and whether any postintervention reductions in symptoms of anxiety are maintained at a 6-month follow-up. Thus, this study aims to lend important insights into the real-world, long-term impact of smartphone-based interventions for the treatment of common mental health problems. We hypothesized that postintervention reductions in symptoms of depression would be maintained at the 1-year follow-up and that Ascend would be associated with reductions in the symptoms of anxiety, which would persist at the 6-month follow-up. Lastly, as a secondary objective, we aimed to examine whether participant demographics or intervention engagement were predictive of symptom change or attrition at follow-up.We recently evaluated the feasibility of the Meru Health herapist . The intWe used a quasiexperimental research design that included a single-arm, pre- and postintervention assessment of outcomes. Symptoms of depression were measured before the intervention (“baseline”); at the end of the 8-week intervention; and at 1, 3, 6, and 12 months postintervention. Symptoms of anxiety were measured at baseline; week 4 of the intervention; the end of the 8-week intervention; and 1, 3, and 6 months postintervention.Ascend intervention was primarily intended to treat symptoms of depression; however, many individuals with depression also have comorbid symptoms of anxiety [Ascend is also associated with reductions in symptoms of anxiety, Generalized Anxiety Disorder-7 (GAD-7) scale measures were added to the intervention assessment in December 2017. At the time of this study, 102 of the original 197 enrollees with 6-month postintervention PHQ-9 outcomes also had GAD-7 (anxiety) data (recruited between January and June 2018), while 12-month postintervention anxiety data were not yet available for any participants. Note that the 102 participants with 6-month postintervention GAD-7 data are different from the 102 participants with 12-month postintervention PHQ-9 data.This study included adult patients treated at the Meru Health Online Clinic, a national remote health care provider that currently operates in the United States and Finland. The clinic has had a rolling enrolment since March 2017. At the time of this study, 197 enrollees had passed the 6-month postintervention outcome window (recruited between March 2017 and June 2018), and 102 passed the 12-month postintervention outcome window (recruited between March and December 2017). Our primary analysis includes the latter group, although we also report the Patient Health Questionnaire (PHQ-9) results from the former group with 6-month postintervention outcomes only. The Participants were recruited via online Facebook advertisements that sought participants for a smartphone-based intervention for depression that included self-guided smartphone-delivered content, private access to a therapist via messaging, and an anonymous group-chat feature with other participants. Prior to the intervention, participants were given free access to the app and trained on how to use the group-chat feature and communicate with their assigned therapist. Participant demographics  were acquired at the start of the intervention via an intake questionnaire administered online. Since medication status was introduced in July 2017, these data are absent for the first 33 participants. Outcome measures were administered via the app for all within-intervention time points (including immediately postintervention) and via email for all follow-up time points.For inclusion, participants had to provide informed consent via the Meru Health app, own a smartphone, have at least mild symptoms of depression (a score≥5 on the PHQ-9 at baseline), and acknowledge/demonstrate the ability to commit to a minimum of 20 minutes of practice per day for 6 days per week across the 8-week intervention (as judged by both the participant and their assigned therapist). Exclusion criteria included a previous suicide attempt, severe active suicidal ideation with a specific plan, severe self-harm, active substance abuse, and a history of psychosis. Inclusion/exclusion criteria were assessed prior to enrolment via phone interviews between study participants and intervention therapists, as per the standard treatment procedure at the Meru Health Online Clinic. Participants were not compensated for their time but could participate in the intervention for free. All participants provided informed consent for their anonymized data to be used for research purposes prior to engaging with the intervention. All procedures were reviewed by the Pearl Institutional Review Board, which granted exemption for analyses of previously collected and deidentified data. All procedures performed were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.An a priori sample size calculation was performed for comparing patient-reported outcome measures (PHQ-9 and GAD-7 scores) at baseline and follow-up time points. Using an alpha level of .05, a power of 0.8, and a medium effect size of 0.5, 33 subjects were needed. Thus, this study was sufficiently powered to detect a medium effect, even after accounting for substantial dropout over the course of the 1-year follow-up period.Ascend intervention has been described in detail previously [The Meru Health eviously . Brieflyeviously , mindfuleviously , cognitieviously , and beheviously . The conA licensed therapist  provides support to participants via messaging , throughout the intervention. As part of this support, therapists review practice logs using a provider “dashboard” and electronic medical records (that detail participant engagement and patient-reported outcomes to date) to monitor individual participant progress. Therapists aim to spend approximately 20 minutes (on average) supporting each participant per week of the intervention (including initiating contact at least 2-3 times per week), but are at liberty to adjust the levels of support in accordance with each participant’s individual progress. In addition, participants are free (and encouraged) to contact their allocated therapist when they require additional support. Such two-way interaction is designed to create a system of support that is structured while being tailored to each participant’s personal preference and needs. Further, therapists are instructed to conduct a phone-based assessment for any participants that show signs of mental deterioration during or immediately after the intervention. In case of an emergency, such as severe suicidality, the intervention includes a written security plan, which all participants are required to review with their therapist before engaging with the intervention.Participants are enrolled in groups of 10-15 individuals that work through the intervention at the same time and can provide anonymous support to one another via a discussion board within the app. Specifically, participants can post anonymous reflections on practices and lessons to the discussion board, to which their therapist can respond freely, and to which other group members can respond with prewritten empathy statements and emoticons. Free cross-talk between participants is not allowed.In Finland, Meru Health is approved by the Finnish National Supervisory Authority for Welfare and Health  and is compliant with the European Union General Data Protection Regulation. In the United States, Meru Health is compliant with HIPAA  legislation. All protected health information is kept in a HIPAA-compliant electronic medical record, which is housed in cloud-based storage systems hosted by a company named Datica . All datThe PHQ-9 is a 9-item depression scale, derived from the full PHQ and is one of the most widely used instruments to screen for the presence and severity of depression in primary care . ParticiThe GAD-7 is a 7-item scale used extensively in outpatient and primary care settings to screen for the presence and severity of an anxiety disorder . ParticiDescriptive statistics were calculated for participant demographics and week-by-week engagement metrics. For each participant, we calculated a measure of intervention engagement, defined as total days in which >3 minutes of app-based meditation was completed. We used a threshold of 3 minutes, as this corresponds to the shortest meditation session available in the intervention. We used logistic regression to explore whether baseline characteristics were predictive of the completion of outcome measures at 6 and 12 months postintervention. Explanatory variables included age, gender, country, intervention engagement (as defined above), and baseline PHQ-9 or GAD-7 scores.P value.  P values<.05 were considered significant. The estimated marginal mean and standard error for each time point was calculated using the “emmeans” package in R.Outcome measures were analyzed using an intention-to-treat analysis in which all participants with outcome measures at baseline were included, regardless of intervention engagement or attrition. We used linear mixed effects models (LMMs) implemented through LME4  and TukeSince LMMs rely on data being “missing at random,” an assumption that is difficult to verify in clinical research, we also implemented an LMM-based pattern-mixture model (PMM) for the analysis of PHQ-9 (depression) scores. PMMs are based on a joint modeling of outcomes and missingness and can account for data that is “missing not at random” . We modig) immediately postintervention and at 6 and 12 months after the intervention as the difference in outcome scores from baseline divided by the pooled weighted SD (based on observed outcome scores only). For the PMM, effect size (Cohen d) was calculated as the estimated reduction in outcome score (from the LMM-based PMM) divided by the SD of observed outcome scores at baseline. We also calculated the percentage of participants who met the definition for clinically significant improvement in PHQ-9 symptoms postintervention and at the 12-month follow-up, defined as either a 5-point reduction in PHQ-9 score from baseline or a 50% reduction in PHQ-9 score combined with a postintervention PHQ-9 score<10 (for participants with baseline scores ≥10).For comparisons with previous literature, we calculated effect size . Approximately one-third of the participants were taking antidepressant medication at the start of the intervention, and 20 participants (19.6%) dropped out of the intervention, where dropout was defined as less than 4 weeks of active participation during the 8-week intervention combined with incomplete PHQ-9 (depression) scores immediately postintervention.Participant demographics for the primary group of participants with 12-month PHQ-9 data are presented in B=–0.11, P=.02) and those who engaged with the intervention on more days  were more likely to complete the PHQ-9 at 12 months postintervention. When including country as a covariate, participants in the United States were less likely to complete PHQ-9 data at 12 months postintervention than participants in Finland , although this may have been driven by differences in baseline PHQ-9 severity.Just over half (51%) of all participants reported a PHQ-9 outcome at 12 months postintervention . LogistiB=–0.07, P<.001), but baseline GAD-7 scores did not (P=.99).We also report participant demographics for a separate group of patients with anxiety outcomes available at the 6-month follow-up in F7,707=42.4, P<.001) and the mean number of days of therapist contact  decreased from week 1 to week 8  and 12 months  postintervention. The improvement at 6 months remained robust when including an additional 95 participants  with outcomes available at 6 months postintervention only .Participants reported clinically significant improvements in depression symptoms (PHQ-9 scores) from baseline to postintervention , 48% reported a clinically significant improvement immediately postintervention, which increased to 60% at the 12-month follow-up (defined as postintervention score<10 combined with ≥50% symptom reduction). When including all participants and using the more liberal definition of a 5-point reduction in PHQ-9 symptoms, 56% reported clinically significant improvements immediately postintervention, which increased to 77% at the 12-month follow-up.P<.001; Cohen d=0.83, 95% CI 0.43-1.24) and 12 months  postintervention.We also estimated PHQ-9 scores at 6 and 12 months postintervention under the conservative assumption that participants with missing data did not experience any long-term benefit from the intervention or benefitted only marginally (see Methods). This approach revealed more modest yet significant reductions in PHQ-9 scores relative to baseline at both 6 months .Participants reported clinically significant improvements in anxiety (GAD-7 scores) from baseline to postintervention  or higher GAD-7 symptoms  at baseline were likely to experience larger reductions in depression (at the 12-month follow-up) and anxiety (at the 6-month follow-up), respectively. However, none of the reported engagement metrics or participant demographics were predictive of score change from baseline to 6- and 12-month follow-ups, for either PHQ-9 or GAD-7 .Individuals with higher PHQ-9 symptoms  than was reported by a recent meta-analysis [g=0.56). However, this study was uncontrolled, and we therefore caution the reader not to overinterpret this difference. Indeed, the large effect reported here is consistent with other within-group (uncontrolled) effect sizes for app- and online-based depression interventions [g=1.29 for the reduction in depression symptoms associated with transdiagnostic, internet-delivered cognitive-behavioral therapy for depression and anxiety at follow-up [Our analysis revealed a larger effect size for the reduction in depression symptoms 12 months postintervention  and 6 months postintervention (g=0.91) than was recently reported in a meta-analysis [g=0.45) [g=1.29).Similarly, our study revealed a slightly higher effect size for the reduction in anxiety symptoms immediately after the intervention  . This di[g=0.45) ,59 and i[g=0.45)  of transAscend was associated with reductions in symptoms of anxiety as well as depression is unsurprising, given that the intervention comprises evidence-based practices that have previously been shown to be effective at treating both conditions [Ascend was slightly larger for depression than for anxiety, likely reflecting the fact that the intervention was designed to address depression as the primary condition. Although previous online psychological interventions have often separated the treatment of depression and anxiety symptoms, there has been a recent trend toward the development of transdiagnostic interventions that target mechanisms common to multiple psychiatric disorders. These results add to a growing body of evidence highlighting the feasibility of such interventions [The finding that nditions . In addinditions . Indeed,ventions  and suggAscend intervention [Ascend did not impact depression or anxiety scores at 6 or 12 months postintervention, although this analysis was likely underpowered due to a large proportion of missing data at these time points. It is also likely that a portion of the variance in scores at these time points was driven by factors not captured by our study. Further research is needed to understand the factors and components of the intervention that are predictive of long-term benefits.Two important but largely unanswered questions are who is likely to benefit the most from app-based psychological interventions and what factors are predictive of the degree to which individuals benefit over time. Our previous feasibility study suggested that a greater volume of app-based practice predicted the occurrence of fewer depressive symptoms 4 weeks after completing the rvention . In thisAs with the former feasibility study , this stAscend to treatment as usual over an extended period are required to fully address these limitations. The Meru Health Online Clinic could also consider collecting information on the antidepressant status at follow-up (and not just at baseline) to better tease apart the long-term effects associated with the current intervention versus antidepressants.Further, approximately one-third of the participants were taking antidepressants during (and presumably after) the intervention, making it difficult to preclude the possibility that the reduction in depression symptoms was caused or maintained by antidepressants  or by thFurther, although engagement with the intervention was high, approximately half of all participants did not complete questionnaires at 6 and 12 months postintervention. Thus, our estimates of the percentage of participants that experienced clinically significant improvements in depression symptoms was based on the participants with complete data only. Further, participants with fewer symptoms of depression postintervention may have been more likely to engage with questionnaires, biasing estimates of the long-term treatment effect. This was addressed by using a robust pattern-mixture model approach and applying the most conservative clinical assumptions about the treatment effect for participants who dropped out or who were lost to follow-up. Nevertheless, such assumptions are inherently unverifiable, and recent guidelines highlight the importance of minimizing the likelihood of incomplete outcome data .Ascend and other app-based interventions for mental health [Although this study suggests that the intervention is associated with reductions in depression and anxiety symptoms, it does not address the underlying mechanisms or mediators of outcome change. Since the intervention encompasses techniques and practices from multiple approaches  as well as remote therapist and peer support, it is difficult to differentiate which components of the intervention are the most effective. Such information could assist with the design and optimization of future iterations of l health .Finally, the study included self-selected participants who may have shown a higher degree of motivation to engage with the intervention. Further, the majority of participants were female, and although the study included individuals from both Finland and the United States, the majority of participants were based in Finland. Moreover, the Meru Health Clinic does not currently track patient race and ethnicity, making it difficult to assess the generalizability of the findings. Together, this suggests that this study sample may not be representative of the wider population of individuals with elevated symptoms of depression or anxiety, and future studies should address this issue by using more robust recruitment strategies and more representative study samples.Ascend are designed to overcome these barriers and may help to significantly reduce the burden of anxiety and depression. Further research is needed to investigate the efficacy of Ascend in comparison to control groups and other established treatments for depression and anxiety.Depression is a serious and growing problem that causes individual suffering and huge economic and societal costs worldwide. Many individuals with depression are unable to access appropriate treatment, with high costs and a lack of trained professionals being major barriers. Scalable, low-cost, app-based interventions such as


Primary gastrointestinal lymphoma is the most common type of extra-nodal lymphoma, representing about 30%-50% of all extra-nodal involvement. The stomach is the most common site, with the colon and rectum accounting for a minority of occurrences. Primary colorectal lymphoma is uncommon, representing only 0.3% of all large intestinal malignancies and approximately 3% of gastrointestinal (GI) lymphomas, with the majority of these being B-cell non-Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL) being the most common subtype. We present a case of an 85-year-old male who presented with symptoms suggestive of bowel obstruction, who, after further evaluation, was diagnosed with primary non-Hodgkin lymphoma of the colon, DLBCL subtype. The gastrointestinal(GI) tract is the most common site of primary extra-nodal lymphoma, with the vast majority being non-Hodgkin lymphomas (NHL). The colon is the primary site in around 17% of cases. Diffuse large B-cell lymphoma (DLBCL) of the colon is the most common subtype of extranodal non-Hodgkin's lymphoma, and while there is conflicting data regarding the most common part of the colon being involved, most studies report the cecum as the most common location. Due to the rarity of reported cases, there is no agreeable protocol on how to manage primary DLBCL of the colon. Currently, surgical resection is the cornerstone of treatment followed by the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen-based chemotherapy and/or radiation therapy. The role of targeted therapy and immunotherapy is still being evaluated but may be a promising alternative to the present standard treatment. We present a case of primary DLBCL of the colon in an 85-year-old male patient followed by a literature review and discussion of the reported cases in the literature.An 85-year-old male with a history of atrial fibrillation presented to the hospital with acute shortness of breath and was subsequently admitted under the diagnosis of acute heart failure exacerbation. At admission, the patient was found to be anemic. His last colonoscopy was five years ago, with findings of lipoma in the ascending colon as well as sigmoid diverticulosis. At the time of admission, he had been more confused per his daughter. He was complaining of progressive fatigue and struggling with constipation, as he had been without bowel movement for several days prior to admission. He also had decreased appetite and lost around six pounds in the past four months. The patient subsequently had a chest X-ray that demonstrated multiple rounded pulmonary nodules throughout the lungs. The patient was clinically diagnosed with bowel obstruction. He subsequently underwent a computed tomography (CT) scan of the abdomen and pelvis, which showed a long apple-core lesion seen in the mid to distal sigmoid colon, which was about six cm in length with well-defined extension into the mesentery and discrete retroperitoneal lymphadenopathy. The patient then underwent a colonoscopy, which showed a near-complete obstructing sigmoid mass with ulceration. Multiple biopsies were taken from this sigmoid mass. The pathological evaluation was positive for atypical lymphoid cells Figure  that hadThe gastrointestinal (GI) tract is the most common site of primary extra-nodal lymphoma, with the vast majority being non-Hodgkin lymphomas. In recent years, there has been an increase in the incidence of primary GI non-Hodgkin lymphoma. A retrograde study  done in A retrograde study  that wasThe colon is a rare location for GI lymphoma, representing only 3% of all cases  and the DLBCL staging involves a CT or magnetic resonance imaging (MRI) scan, positron emission tomography (CT-PET) scan, and/or bone marrow biopsy in the case of advanced stages to determine bone marrow involvement. If neurological symptoms are present and central nervous system involvement is suspected, brain imaging and lumbar puncture might also be considered. Recent advances in gene expression profiling and immunohistochemical (IHC) analysis on tissue biopsy have allowed us to differentiate the subtypes of DLBCL and tailor treatment to each type. For example, patients with the activated B-cell (ABC) disease subtype are less likely to respond well to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based regimens than those with germinal center B-cell (GCB) disease . Corners In our review of the literature, we found multiple reported cases and case series, however, we only cited those that were crucial for the purposes of this report. We will give a brief discussion regarding the presenting epidemiology, presentation, and treatment options of these cases. The male to female ratio of the reported cases was 2.2:1, with 13 male patients and six female patients. The mean age was 68 years old. From the 19 cases, 18 mentioned the location of the lymphoma as following: sigmoid 44%, cecum 39%, and the ascending colon 17%. Presenting symptoms include abdominal pain, weight loss, diarrhea, constipation, lower GI bleeding, vesicocolic fistula, and altered mental status. The most common presenting symptoms were bowel obstruction and abdominal pain, present in 40% of 15 cases. Three patients had colonic perforation during admission, representing about 16% of the patient population. Sixty-eight percent of the patients had colectomy or hemicolectomy as part of their treatment with CHOP as the main chemotherapeutic regimen based upon the International Prognostic Index (IPI). The IPI is a common prognostic scoring system used for DLCBL, and it is also useful for primary DLCBL of the colon .Primary DLCBL of the colon is a rare entity representing only 1% of all colon malignancy, with the cecum being the most common location. Diagnosis is often established after a pathological evaluation of colonic biopsy or after colonic resection. Advances in gene expression profiling and immunohistochemical analysis (IHC) have allowed for tailored treatment for each type. Patients with the ABC disease subtype are less likely to respond well to CHOP-based regimens than those with GCB disease. Primary DLBCL of the colon remains an aggressive disease with a poor prognosis. Treatment options include surgical resection, chemotherapy, and radiation therapy. A bone marrow transplant is considered in select cases. Given the current era of rapid advancements in technology, targeted therapy and immunotherapy should also be considered as potential therapeutic options.


Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies. Cell-penetrating peptides (CPPs) are short peptides (<30 amino acids long) that are able to penetrate biological membranes and drive the internalization of a bioactive cargo in cells. An alternative terminology, the protein transduction domain (PTD), was used initially to describe such peptides as the first examples of CPPs were based on natural peptides derived from protein fragments . DiscoveL-amino acids (84.3%)  c. Moreov (84.3%) c 11,12],12L-aminExtensive literature is available on the structure–activity relationship of primary CPPs and the folding properties of amphipathic peptides ,7,12,13.The folding ability of CPPs to form α-helices and β-sheets increases their efficiency to penetrate cells, as described in more detail in 8 and R9 . . 113]. Toperties . In proteins, β-sheet-like secondary structures are dominated by inter-chain hydrogen bonding between closely aligned carbonyl and secondary amino groups on the peptide backbone. They can be formed by proteins, peptides of de novo design, β-hairpins, and aromatic and aliphatic peptide amphiphiles. The formation of β-sheets can occur in two ways. Parallel β-sheets form when the direction of each N (N-terminus) to C (C-terminus) peptide backbone is in the same direction f. Anti-pIn short peptide sequences, β-sheet formation can be obtained by alternating hydrophobic with hydrophilic residues, by introducing aromatic moieties, or by basing the design on known β-sheet-forming moieties. β-sheet-forming peptides based on EAK and RADA peptide sequences, where hydrophobic amino acids residues are alternated with hydrophilic charged ones, have been studied primarily for regenerative medicine applications ,116,117.Folding and secondary structure of CPPs to control translocation. Two main parameters to be considered when studying the effect of secondary structures on CPP translocation efficiency are (a) affinity of CPPs for membranes and (b) their folding capacity in the presence of membranes. These two parameters are interconnected as they are based on the same principle of weak interactions. Their contribution is essential to understand the driving forces of CPP internalization and to improve the design of future CPPs. As already mentioned, the CPP-phospholipid interaction in membranes is based on the interplay of weak interactions, namely electrostatic interactions, hydrophobic effect and hydrogen bonding. As a result of these interactions, CPPs often fold and assume secondary structures. Their ability to fold is also closely related to their amphipathic nature. The folding properties  of CPPs have been studied in detail in membrane models to better understand the role of the interfacial properties and conformational state of these peptides on internalization efficiency. Adsorption experiments at the air–water interface imply that α-helical- and β-sheet-like peptides differ in amphipathic character . . Disulfireported a 133]. . Disulfi library . An impono acids .Recently, the same group has reported on a reversible bicyclization strategy achieved through the formation of a pair of disulfide bonds b 138]. . 138]. CMultivalency. Another parameter to consider is the multivalency of the system. One step in this direction is the development of dimeric branched peptides, as shown by Saludes and co-workers . . 132]. Sx(QL)yKxz or Kx(QW)yKxz. In this case, supramolecular structures form due to the interplay of electrostatic and hydrophobic interactions alongside hydrogen bonding among QL/QW repeating units. The supramolecular assembly of these short cationic peptides into nanostructures with diverse architectures has the advantage of providing an enhanced local charge density. Supramolecular assembly resulted in dense packing of lysine residues, giving rise to high local concentrations of positive charge, i.e., multivalent species able to interact with the cell membrane. This enhanced interaction is beneficial for tuning the membrane activity of FCPPs through programmable physical properties, stability and surface functionality. The design was based on combining tryptophan and lysine at the primary structure level to obtain self-assembly into a variety of nanostructures. Tryptophan was chosen as the hydrophobic aromatic residue through which to increase contacts with membranes but also to drive the assembly. On the other hand, lysine was chosen because of positive charge, thus conferring cell penetration and serving as a DNA complexation moiety. Two molecules with structures K5(QW)6K5 and K10(QW)6 were compared in that study. A total of ten lysine residues were used but they were distributed in a different manner along the sequence. The molecule with a single charged domain (ten lysine residues) at the N-terminus assembled into nanofibrils based on β-sheets, while the other peptide showed random coils and irregular aggregates. The fibrillar K10(QW)6 showed greater capacity to enter Hela cells. DNA delivery with this peptide was 200- and 400-fold higher in the HEK293 and HeLa cell lines, respectively. In this example, the fibrilllar shape of the supramolecular nanostructure with the precise balance of charge distribution and conformational flexibility, was responsible for higher uptake [Dong and coworkers introduced the concept of filamentous CPPs (FCPPs) based on cationic multi-domain peptides, providing examples of supramolecular structure-dependent membrane activity ,159. Ther uptake . This syTryptophan-rich CPPs that assemble into spherical aggregates were reported . Their d6 or RRGRRG) with collagen-like folding domains [n repeating units, which are found in collagen. The exterior of the helices was decorated with the CPPs, a process that led to the accumulation of positive charges, which favored internalization while the helicity improved resistance to proteases. Internalization efficiency (at a concentration below the 10 μM) was shown in Jurkat human leukemia cells using flow cytometry, with the R6-containing helix showing the highest uptake. No uptake was observed when a scrambled version of the helical motif, which was unable to assume the triple helical conformation, was used [Another example worthy of mention comprises triple helical CPPs obtained by combining short oligoarginine CPPs (R domains . In thiswas used .16) tail that drives the self-assembly into nanofibers and serves as hydrophobic pocket for hydrophobic drug encapsulation was added at the N-terminus. Next, a CPP moiety , an enzyme-sensitive moiety and an RGD-based targeting moiety were included. The system was found to self-assemble into fibers that are enzyme-responsive and that protect the CPP sequence. It was observed that RGD drives the accumulation of these fibers in the proximity of the tumor, where they can undergo enzyme-triggered fiber to micelle transition and consequently enter cells on demand as a result of exposure of their CPP moiety  system based on the self-assembly of multidomain peptide sequences. The design consisted of four main structural and functional parameters. A hydrophobic palmitic acid (CP moiety c. The in8 CPP was conjugated at the N-terminus to a negatively charged pyranine-oligoglutamic anion (guest) that was encapsulated by the positively charged polyamine cage (host). The host–guest supramolecular complexes were internalized into Vero cells and showed endosomal localization. This is an example of cell-penetrating systems in which structure and function complexity are achieved through simple design choices [An interesting example of quaternary structure and an alternative approach to control the uptake of oligoarginine-based CPPs was through the formation of host–guest supramolecular complexes based on anion recognition . In this choices . In addiFinally, the recent report from Garcia-Lopez et al. on the design of photo-activatable molecular machines able to drill through cell membranes illustrates the important role that molecular motors will have in the future in the field of intracellular delivery . Despite the variety of CPP design approaches currently available, the translation of CPPs to a clinical setting remains a challenge. From the examples discussed in the text, it can be appreciated that the CPP field is wide and in continuous development . GreaterMany researchers have proposed design strategies that allow control over folding, cyclization, dimerization, stapling, self-assembly and even CPP mimetics with different backbone structures and properties, thus leading to improvements in internalization efficiency and stability. However, due to the concomitant involvement of various internalization mechanisms, the heterogeneity of diseases, and the variety of cell lines available for in vitro studies, there are still no standards through which to rate the internalization efficiency improvements of one design over another. In addition, the heterogeneity of cargoes implies a broadening of the parameters to consider when reporting on CPP efficiency and activity. In this regard, standardized protocols are needed as well as a greater number of comparative studies between different classes of newly designed CPPs. Although huge progress has been made in this field, there is still room for the development of new strategies and approaches.


The study population consistedof 120 students enrolled in the Nursing Care in Healthcare Processes course.The educational intervention was based on theoretical and practical classesabout the evidence-based practice process and the use of the criticalincident technique during the clinical clerkship. Effectiveness was measuredwith the Evidence-Based Practice Competence Questionnaire in three pairedmeasures using repeated-measures analysis of variance. the mean scores of the Evidence-Based Practice Competence Questionnaire were79.83 (CI 95% 78.63-81.03) for the basal measurement, 84.53 (CI 95%83.23-85.83) for the intermediate measurement, and 84.91 (CI 95%83.26-86.55) for the final measurement, with a statistically significantdifference among the three paired measurements (p<0.001). There werestatistically significant differences in Attitudes (p = 0.034) and Knowledge(p <0.001) but not in Skills (p = 0.137). this educational intervention based on theoretical and practical classesabout the evidence-based practice process and the use of the criticalincident technique during the clinical clerkship enhances evidence-basedpractice competence among second-year nursing degree students. Evidence-based medicine (EBM) first appeared as a clinical learning strategy atMcMaster Medical School and, because healthcare professionals from many fieldscurrently adopt this approach in their clinical practice, EBM has evolved intoevidence-based practice (EBP), a wider and more comprehensive concept-EBP involves the integration and implementation of the best available evidence,including clinical expertise and patients’ values and circumstances, in clinicaldecision-making-) as barriers to the incorporation of EBP, probably because they were noteducated on the EBP paradigm,Despite the evidence that EBP improves patient outcomes and performance,incorporating EBP into clinical nursing remains a challenge--,-,,-There are several books on teaching EBP that can help teachers and educatorsimplement EBP education into their curriculaThe nursing degree was introduced at the Universitat Jaume I (Spain) in 2011 afterthe establishment of the European Higher Education AreaTo assess EBP education, some authorsTherefore, the main objective of this study was to evaluate the effectiveness of theeducational intervention implemented in the second year of the nursing degree atUniversitat Jaume I (Spain) on students’ knowledge, skills and attitudes towardsEBP.A quasi-experimental before-and-after study was conducted among a group ofsecond-year nursing students enrolled in the nursing degree programme at UniversitatJaume I (Spain).The study population consisted of 120 students enrolled in the Nursing Care inHealthcare Processes course during the second year of the nursing degree programmeat Universitat Jaume I during the 2013-2014 and 2014-2015 academic years. Thiscourse is taken during the second semester of the second year and is composed of aperiod of theory and clinical simulation and a period of clinical clerkship in adultinpatient units.The sample size was calculated with the GRANMO software package. A minimum samplesize of 65 students was required to detect a difference of 0.2 units in the EBP-COQscore, with a standard deviation of 0.4A student’s failure to give consent to participate was considered an exclusioncriterion. Furthermore, students who did not complete the clinical clerkship, whoabandoned their studies during the clerkship, or who took the course during specialperiods  were excluded. Students who did not complete thethree measures were also excluded from the study.The educational intervention was carried out with second-year nursing degree studentsand was framed within the cross-curricular EBP programme described in This educational intervention consists of two hours of EBP theory and two hours ofcomputer lab sessions. In the first session, terms related to EBP are defined, andstudents are encouraged to reflect on the material and to employ critical thinkingin the use of research tools. The second session is a practical exercise oninformation literacy. During the clinical clerkships (12 weeks), the critical incident technique is used tostimulate the implementation of the acquired knowledge and skills. Students identifya minimum of eight critical incidents related to the content of the course and theirdaily practice and document the critical incident information . Furthermore,students develop a clinical question (following thePopulation-Intervention-Comparison-Outcomes format) for each critical incident andtry to resolve it through a literature search. One lecturer is responsible forproviding support and feedback to a group of 8 to 10 students. Students receive alecture explaining the critical incident technique and its methods before startingthe clinical clerkship. The study was conducted between September 2013 and June2015.The effect of the educational intervention on the students’ knowledge, skills andattitudes towards EBP was measured with the EBP-COQA descriptive analysis of the categorical variables  and continuous variables  was performed.To compare the categorical variables, a Z-proportions comparison test was appliedusing the Bonferroni correction. Repeated-measures ANOVA with the appropriate posthoc contrast was used to compare the three measures of the EBP-COQ, and if normalityof the variables was not met, the Friedman test was used. A p-value <0.05 forbilateral significance was assumed, and the statistical analysis was performed usingSPSS software for Windows v20.This study was approved as an educational innovation project (Grant no.: 045/14) bythe Nursing Department Council of the Universitat Jaume I, and permission to use theEBP-COQ was obtained from its authors. Students were invited to take part in thestudy by means of a cover letter, which included the introduction, objectives, andmethodology of the study and a request for their consent. The questionnaires werecompleted on a voluntary basis and the anonymity of the students was maintainedthroughout the entire process using a previously described procedure in compliancewith Spanish legislation on personal data protection. The ethical principles of theHelsinki Declaration were followed.The response rate was 69.17% (n=83). Two students abandoned their studies during theclinical clerkship, 17 students conducted their clinical clerkship in the specialperiods, 8 students did not complete their clinical clerkship and 10 students didnot respond to the three measurements. Of the participants, 84.3% (n=70) were women,with no significant differences between courses (p= 0.203). The average age was 21.6years (SD ± 5.62), with a minimum of 19 and a maximum of 48, with no significantdifferences between courses .The mean score of the EBP-COQ for the overall sample was 79.83 points  for the baseline measurement, 84.53 points  for the intermediate measurement and 84.91 points  for the final measurement. There were significant differences among thethree measurements (p < 0.001), between the baseline and the intermediatemeasurements (p < 0.001) and between the baseline and the final measurements(p<0.001) but not between the intermediate and the final measurements (p=0.092).All items in the knowledge category showed significant differences among the threemeasurements . Item 1 -The main goal of this study was to evaluate the effectiveness of an educationalintervention carried out in the second-year nursing degree programme at UniversitatJaume I (Spain) on students’ knowledge, skills and attitudes towards evidence-basedpractice. This educational intervention was embedded in the cross-curricularevidence-based practice programme developed in the nursing degree programme at theUniversitat Jaume I. It was based on recommendations from the literature regardingthe use of theoretical and practical classes on the evidence-based practiceprocessOur results show that this educational intervention can improve students’ overallevidence-based practice competence, primarily in the Knowledge and Attitudesdimensions. However, there were no statistically significant differences in theSkills dimension, although the analysis of this dimension by items showedsignificant differences in all cases, which reinforces the educational intervention.The factors that may be involved include the influence of the clinical tutors, theirrelationship with the students and a possible lack of evidence-based practiceknowledge and skills. Students perceive clinical tutors as role models, so involvingtutors in the learning of evidence-based practice is crucial to improving students’evidence-based practice competence,The first part of this educational intervention is conducted at the university, whilethe second occurs during the clinical clerkship. The comparison of means showedsignificant differences between the baseline and intermediate measurements but notbetween the intermediate and final measurements, thus suggesting that the first partof the educational intervention had a greater positive effect compared to the partcarried out during the clinical clerkship. Other studies have evaluated theeffectiveness of specific courses on research or evidence-based practice onstudents’ knowledge, skills and attitudes-It is necessary to consider that this educational intervention was carried out amongsecond-year nursing degree students and was framed within the cross-curricularevidence-based practice programme described earlier. Thus, it is important toconsider that students in our programme had learned statistics and epidemiology intheir first year, as some authorsSome limitations of this study must be considered. No control group was established,and the sample could not be randomised in the assessment of the educationalintervention. This is because it was part of the cross-curricular EBP programme,open to all students enrolled in the Nursing Care in Healthcare Processes course,and because the nursing degree at Universitat Jaume I (Spain) has only recently beenimplemented. In subsequent academic courses, there were new interventions related toevidence-based practice that had to be measured. Moreover, the study sample waslimited to two academic years and was carried out in a single institution.The EBP-COQ is a robust and validated instrument for measuring the effectiveness ofeducational interventions on evidence-based practice, although it is based onstudents’ perceptions rather than on objective data. The use of subjective measuresis suitable, however, because of the correlation between self-reported and objectiveassessments of evidence-based practice competenceThe findings of this study show that an educational intervention based on theoreticaland practical classes on the EBP process and the use of the critical incidenttechnique during clinical clerkship enhances EBP competence among second-yearnursing degree students. However, the effect of the educational intervention islower during clinical clerkships; factors related to clinical tutors, the use oftechnologies or the use of research may affect these results. Nevertheless, thefindings of this study may be of interest to other universities since the existingliterature does not provide sufficient evidence regarding the ideal model or themost appropriate training in EBP.


Inflorescence capacity plays a crucial role in reproductive fitness in plants, and in production of hybrid crops. Maize is a monoecious species bearing separate male and female flowers . The switch from open-pollinated populations of maize to hybrid-based breeding schemes in the early 20th century was accompanied by a dramatic reduction in tassel size, and the trend has continued with modern breeding over the recent decades. The goal of this study was to identify selection signatures in genes that may underlie this dramatic transformation. Using a population of 942 diverse inbred maize accessions and a nested association mapping population comprising three 200-line biparental populations, we measured 15 tassel morphological characteristics by manual and image-based methods. Genome-wide association studies identified 242 single nucleotide polymorphisms significantly associated with measured traits. We compared 41 unselected lines from the Iowa Stiff Stalk Synthetic (BSSS) population to 21 highly selected lines developed by modern commercial breeding programs, and found that tassel size and weight were reduced significantly. We assayed genetic differences between the two groups using three selection statistics: cross population extended haplotype homozogysity, cross-population composite likelihood ratio, and fixation index. All three statistics show evidence of selection at genomic regions associated with tassel morphology relative to genome-wide null distributions. These results support the tremendous effect, both phenotypic and genotypic, that selection has had on maize male inflorescence morphology. Zea mays L.) is a branched structure that displays remarkable morphological diversity. Tassels vary in length, number of branches, compactness, and curvature, all of which are capable of affecting the quantity and dispersal of pollen. Inflorescence architecture is crucial to reproduction in wind pollinated plants  and teosinte glume architecture1 (tga1), which were targets of domestication that changed plant and inflorescence architecture between teosinte and modern maize (barren infloresence2 (bif2) locus (Previous studies have identified quantitative trait loci (QTL) and quantitative trait nucleotides associated with different aspects of tassel morphology and development that colocalize with 2) locus . The sam2) locus .Until 1935, most of the maize grown in the North American Corn Belt consisted of open-pollinated populations, in which plants are allowed to reproduce freely with each other. Within 4 years, however, >90% of maize grown in Iowa had transitioned to higher-yielding hybrids created by controlled crosses, and the rest of the Corn Belt soon followed .The shift in breeding methods to single-cross hybrids placed an emphasis on developing inbred lines with characteristics that enhance their potential to serve as parents. These parents are subsequently crossed to each other to produce vigorous hybrid offspring. In open-pollinated populations, a competitive advantage was conferred by greater pollen production and dispersal . Inbred a priori selection candidates, and compared selection statistics at those SNPs to their genome-wide distribution. If selection by breeders between the BSSS and modern ex-PVPs acted to reduce tassel size, we expect that loci associated with tassel morphology phenotypes would show enrichment for selection signatures relative to the rest of the genome. Indeed, we observe an enrichment of selection statistic scores in regions containing SNPs associated with tassel morphological traits, a finding that is consistent across three different selection statistics.Among the germplasm used in this study are inbred lines derived from the original BSSS population, as well as a number of commercially developed inbred lines for which the Plant Variety Protection certificates have recently expired (ex-PVPs). These ex-PVP lines represent the most modern germplasm from private seed companies that is available to the public, and they can be traced back to founder lines from the BSSS population. These two sets of materials afford us an opportunity to evaluate changes in maize tassel architecture caused by the accelerated evolutionary process of modern breeding. We used single nucleotide polymorphisms (SNPs) identified as significant in association mapping as 2 of the original parents.This study used two populations of maize inbred lines: a diversity panel and a nested association mapping (NAM) population. The diversity panel (WiDiv-942) consists of 942 diverse lines that typically reach grain physiological maturity in the Midwest regions of the United States, and represents an expansion of the 503 line Wisconsin Diversity panel , tassel length (TL), and spike length (SL) on three representative plants per plot during or after flowering. BN was quantified as the number of primary tassel branches, TL was measured as the distance in cm from the lowest tassel branch to the tip of the spike, and SL was measured as the distance in centimeter from the uppermost tassel branch to the tip of the spike. All traits were measured in all environments with the exception of SL, which was not measured in the PHW65 NAM at West Madison in 2014. Three derived tassel traits can be calculated from these measurements: branch zone length (BZ) as TL minus SL, branch density (BD) as BN divided by BZ, and spike proportion (SP) as SL divided by TL.In addition to manual measurements, tassels measured in 2015 were also imaged and measured automatically using TIPS  for all manually measured traits, except TW, were calculated as the random genotypic effect predictions from the linear model Heritability was calculated on an entry-mean basis for all manually measured traits except for TW as Genotypic and phenotypic correlations between all traits were calculated for each population. Because of differing numbers of phenotyped lines and differing numbers of environments in which the traits were evaluated, the correlations were only calculated using data from the West Madison 2015 location, and only with lines that were phenotyped for all 15 traits in that environment .The WiDiv-942 lines were genotyped by RNA sequencing as described in Inbred lines in the PHW65 NAM population were genotyped using genotyping-by-sequencing (GBS) as described in The four parents of the PHW65 NAM population were resequenced and genotyped at 23.3 million SNPs  with theWe identified lines that represent early Stiff Stalk germplasm, recent ex-PVP Stiff Stalk germplasm, and publicly developed Stiff Stalk germplasm that represents a temporally intermediate group between the two.The WiDiv-942 contains 47 inbreds derived through self-pollination from the original BSSS population (referred to hereafter as BSSSC0), which represent maize Stiff Stalk germplasm from the early 20th century.R2 value >0.1 within the window were discarded, leaving 109,605 SNPs. The pruned marker set was used in ADMIXTURE without seeding founding individuals. Ten-fold cross-validation of the errors was minimized at K groups equal to eight as an appropriate model for this marker set. These eight groups represent the resulting subpopulations; B14, B37, B73, Oh43, Lancaster, Iodent, Iodent-Lancaster, and Flint. To identify lines that both display evidence of descent from the BSSS as well as exclude lines with evidence of introgression from other germplasm pools, individuals were required to have a sum of >95% identity to the B14, B37, and B73 groups, as well as <2% identity to each of the other groups. Of the lines that met this requirement, 21 had been phenotyped and were kept for subsequent analysis. Those BSSS-descended ex-PVP lines are referred to hereafter simply as ex-PVPs. Hierarchical clustering of the ex-PVPs and BSSSC0 lines revealed six of the BSSSC0 lines did not cluster with the others. Those six were removed from further analysis, leaving 41 BSSSC0 lines.From all ex-PVP lines in the WiDiv-942, inbreds were chosen that showed genotypic evidence of being directly descended from the BSSS population (File S1). Although pedigree information was available for some ex-PVPs, the number of individuals that could be traced by pedigree back to BSSS-derived inbreds was low. Additionally, using genotypic information to identify BSSS-derived ex-PVP lines allows exclusion of individuals showing evidence of contamination from other breeding material. Population substructure within all the ex-PVP lines was estimated using ADMIXTURE  for all lines in the WiDiv-942 were calculated by the same models used to calculate BLUPs, but with genotype fit as a fixed rather than random effect. BLUEs for the BSSSC0, Public, and ex-PVP lines were compared for each of the 15 tassel morphological traits, and significant differences were evaluated by Tukey’s honest significant difference. .farmCPU  for the WiDiv-942 were performed in R  using th−5 to 1.5 × 10−4) and allowed terms to exit the model based on a limit set at twice the entry threshold. Residuals from the final stepwise model were calculated separately for each chromosome, with any SNPs on that chromosome excluded from the model. The residuals for each chromosome were used as the dependent variable in a resampling-based mapping method similar to that described in −4. This process was repeated 100 times for each combination of trait and chromosome, and each SNP fitted by the resampling GWAS method was assigned a resampling model inclusion probability (RMIP) between 0 and 1, reflecting the proportion of models in which that SNP was included. A permutation-based threshold for RMIP was calculated in a manner similar to that described in The PHW65 NAM was mapped using stepwise model selection with the GBS SNPs followed by resampling GWAS with the 10.6 million projected parental SNPs, as described in We used the 41 BSSSC0 and 21 ex-PVP lines to perform genome-wide scans for selection. The BSSSC0 lines represent maize Stiff Stalk germplasm from the early 20th century, while the ex-PVP lines represent the product of ∼60 years of commercial maize breeding. We calculated cross population extended haplotype homozogysity  on the same set of SNPs as XP-EHH, with a grid size of 100 bp, a genetic window size of 0.1 cM, a maximum of 50 SNPs per window, and correlation level of 0.7. The genetic positions of the WiDiv SNPs were estimated by linear interpolation of the NAM genetic map  between the two groups was calculated on the same set of SNPs as XP-EHH and XP-CLR, using http://beissingerlab.github.io/docs/vectorFst.R , we used 10,000 iterations of a circular permutation method , recombination rate (±20%), frequency of alleles in the BSSSC0 lines (±15%), and frequency of alleles in the ex-PVP lines (±15%) were randomly sampled without replacement. For tassel-associated windows with <50 matching windows genome-wide, the allele frequency criteria were omitted.P-value  and genotypic data for the WiDiv-942 can be found at https://doi.org/10.5061/dryad.6tg35t6 . Code is available on GitHub at github.com/joegage/tassel_selection. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.7097813.Tassel images of both populations and genotypic data for the PHW65 NAM are available on the CyVerse Data Store  and an NAM population (PHW65 NAM) with PHW65 as the common parent and PHN11, MoG, and Mo44 as founder parents. These materials were grown in three environments between 2013 and 2015 in south central Wisconsin. The WiDiv-942 association panel used in this study is comprised of 942 diverse inbred lines, while the PHW65 NAM consists of three nested biparental populations of 200 individuals each. The WiDiv-942 was chosen to capture broad phenotypic and genetic diversity, whereas the PHW65 NAM leverages controlled biparental crosses between individuals with contrasting tassel morphologies. All tassels manually measured in 2015 were also photographed and measured using the image-based tassel phenotyping system TIPS  and those that contribute to tassel size by contributing to branchiness . Although TW and TWp are measures of mass rather than branching, both traits are affected more by changes in branchiness than changes in TL and therefore are included in the former group.For most traits, the PHW65 NAM population has a higher or equivalent mean to the WiDiv-942 population, with the exception of TWp (0.79 g lighter), BN (1.83 fewer branches), BNp (1.07 fewer branches), and BD (0.24 fewer branches/cm). As expected, given the allelic representation contained in each of the populations, phenotypic variability within the PHW65 NAM is lower than in the WiDiv-942, with the exception of TW (SD 0.3 g greater) and BNp (SD 0.07 branches greater).Heritabilities for manually measured traits ranged from 0.93  to 0.97 (BN) in the WiDiv-942 population and from 0.72 (BZ and SP) to 0.94 (BN and TW) in the PHW65 NAM population. Because images and TW were taken only at one location, repeatability was calculated for image-based traits and TW in place of heritability. Repeatabilities ranged from 0.72 (PR) to 0.86 (TWp) in the WiDiv-942 population and 0.63 (SLp and TLp) to 0.72 (SK) in the PHW65 NAM population . The higGenotypic and phenotypic correlations were calculated within the single environment in which all traits were measured . With the exception of CP, branchiness traits were generally positively correlated with each other in both populations. Similarly, length-related traits were all positively correlated with each other in both populations. Phenotypic and genotypic correlations were similar to each other in both populations, partly due to relatively high heritability of all traits and partly due to having been calculated within a single location.The WiDiv-942 association panel contains 41 inbred lines developed by selfing without selection from cycle 0 of the BSSS population (BSSSC0), 16 public inbred lines with pedigrees that trace entirely back to lines derived from the BSSS, and 21 ex-PVPs with genotypic evidence for descent from the BSSS (File S1). These three groups of lines allow comparison of tassel morphology over the course of modern maize breeding. The BSSSC0 lines represent the earliest material, the ex-PVPs represent the most recent and heavily selected material, while the public lines form an intermediate group.Comparison of BLUEs for each group revealed that the mean values of public inbred BLUEs for each trait showed between a 2% (FD) and a 16% (TWp and BNp) change relative to the BSSSC0 line, while ex-PVP lines displayed between a 4% (TLp) and a 45% (TWp) change in mean relative to the BSSSC0 lines . For allAll 15 traits were mapped in the WiDiv-942 association panel using the multiple locus linear mixed model implemented in FarmCPU . Generally, there is more concordance between SNPs associated with manual and image-based measurements for traits with higher heritabilities/repeatabilities (BN and TW) than lower heritabilities/repeatabilities (SL and TL). This is to be expected, as genetic signal is stronger when heritability is high, but the pattern seen in this study may be due only to chance as BN and TW are only marginally more heritable than SL and TL.a priori candidates for regions having undergone selection between the BSSSC0 lines and the ex-PVP lines. Scans for selection were conducted using LD-focused methods XP-EHH , as well as genome-wide null distributions generated by randomly sampling windows with similar characteristics , suggesting enrichment for selection signals in regions associated with tassel morphology.To test for genomic signatures of selection on tassel morphology, SNPs associated with tassel characteristics in the WiDiv-942 and PHW65 NAM lines were used as -EHH (ST . Genome-P = 2.9 × 10−3). Branchiness traits have more influence on tassel size and biomass than tassel and spike length. With the exception of BZ, all the length traits increased from the BSSSC0 to the ex-PVPs, whereas all the branchiness traits decreased. As such, most of the SNPs showing evidence of selection in the ex-PVPs are associated with traits that control overall tassel size and biomass and have decreased significantly since the 1930s.XP-EHH scores for tassel-associated windows  were enrSimilar to XP-EHH, XP-CLR is a haplotype-based method for identifying selective sweeps. Rather than identify alleles that have recently arisen to substantial frequency, as XP-EHH does, XP-CLR identifies alleles that have changed in frequency more quickly than expected under neutrality  show enrST, a measure of population differentiation based on allele frequencies, differs from the previous two selection statistics in that it is not haplotype based. FST values for tassel-associated windows . The lack of any difference could be due to the low number of SNPs we were able to directly test, or perhaps due to noise introduced by identifying SNPs that are not causative themselves but in LD with variants that directly affect tassel morphology.None of these three selection statistics show strong, systematic relationships with each other on a genome-wide basis  or among the tassel-associated windows . For the 101 significant associations that were genotyped in the WiDiv-942 , we hypothesized that alleles with higher frequency in the ex-PVPs would generally have allelic effects conferring more ex-PVP-like phenotypes, that is, positive values for TL, SL, TLp, SLp, and SP, and negative values for all other traits. However, we found no difference between the effect sizes of alleles with higher frequency in the ex-PVPs and alleles with higher frequency in the BSSSC0s  by breeders aiming to reduce overall tassel size. The enrichment for positive XP-EHH scores, although not as extreme as for negative scores, indicates a number of tassel-associated loci with slower LD decay in the BSSSC0 lines than in the ex-PVP set. One possible explanation for this observation is that prior to the creation of the BSSS, during a period characterized by open-pollinated populations, heavier tassels with more branches had a reproductive advantage due to higher pollen production. During the last century of maize breeding the selective pressure on high pollen production has been removed by a hybrid breeding scheme in which inbred combining ability and hybrid grain yield are prioritized. It is possible that the BSSSC0 lines show extended haplotypes due to selection favoring alleles that confer large tassel size, whereas modern breeding schemes have removed that selective pressure and allowed or encouraged recombination in previously selected regions.unbranched2 and unbranched3 have been shown to simultaneously decrease branch number and increase kernel row number, a trait that directly affects grain yield (zea floricaula/leafy 2 decrease both branch number and kernel row number (The results presented here provide evidence for selection by 20th century maize breeders on regions associated with maize tassel morphology. Whether these changes are the result of direct or indirect selection on tassel size and shape is unclear. Modern, commercially developed maize inbreds display a suite of characteristics that have systematically changed since the open-pollinated varieties of the 1930s, and it is difficult to disentangle selection for changes in tassel morphology from selection for traits that are regulated by loci pleiotropically involved in tassel morphology. Because the tassel and the ear follow a shared developmental path early in development, genes affecting morphology of one may very well have effects on the other. QTL have been discovered that exhibit pleiotropic behavior, conferring a positive relationship between tassel and ear length (w number . There iw number . AlthougChange in tassel size and shape between the early 20th century and modern day is a well-characterized phenomenon but the causes of that phenomenon and its potential causal effects on other agronomic traits and productivity are not fully known. Our results present evidence for enriched signals of selection in genomic regions associated with tassel morphological traits. Further, this selection took place in ∼60 years between the creation of the BSSS in the 1930s and the release of ex-PVP lines in the 1980s and 1990s, a tremendous example of the efficiency of modern crop breeding in imposing drastic genetic and phenotypic changes.


What is the association between low levels of low-density lipoprotein cholesterol and risk of sepsis in patients admitted to the hospital with serious infection?In this cohort study of deidentified electronic medical records of patients admitted to the hospital with infection, measured levels of low-density lipoprotein cholesterol in 3961 patients and a low-density lipoprotein cholesterol genetic risk score in 7804 patients were not associated with increased risk of sepsis after adjusting for comorbidities.Levels of low-density lipoprotein cholesterol are not directly associated with the risk of sepsis or poor outcomes in patients hospitalized with infection. This cohort study of medical records assesses whether low-density lipoprotein cholesterol (LDL-C) levels are associated with an increased risk of sepsis among patients admitted to the hospital with infection. Whether low levels of low-density lipoprotein cholesterol (LDL-C) are associated with increased risk of sepsis and poorer outcomes is unknown.To examine the association between LDL-C levels and risk of sepsis among patients admitted to the hospital with infection.International Classification of Diseases, Ninth Revision, Clinical Modification, and received an antibiotic within 1 day of hospital admission (N = 61 502). Data were collected from January 1, 1993, through December 31, 2017, and analyzed from January 24 through October 31, 2018.Cohort study in which deidentified electronic health records were used to define a cohort of patients admitted to Vanderbilt University Medical Center, Nashville, Tennessee, with infection. Patients were white adults, had a code indicating infection from the Clinically measured LDL-C levels  and a genetic risk score (GRS).The primary outcome was sepsis; secondary outcomes included admission to an intensive care unit (ICU) and in-hospital death.P = .001) and ICU admission , but not in-hospital mortality ; however, none of these associations were statistically significant after adjustment for age, sex, and comorbidity variables . The LDL-C GRS correlated with measured LDL-C levels  but was not significantly associated with any of the outcomes.Among the 3961 patients with clinically measured LDL-C levels  and the 7804 with a GRS for LDL-C , lower measured LDL-C levels were significantly associated with increased risk of sepsis (odds ratio [OR], 0.86; 95% CI, 0.79-0.94; Results of this study suggest that lower measured LDL-C levels were significantly associated with increased risk of sepsis and admission to ICU in patients admitted to the hospital with infection; however, this association was due to comorbidities because both clinical models adjusted for confounders, and the genetic model showed no increased risk. Levels of LDL-C do not appear to directly alter the risk of sepsis or poor outcomes in patients hospitalized with infection. No specific effective treatments are available for sepsis; thus, interest in new approaches to prevent sepsis and treat patients is great.In the United States, sepsis is a common cause for admission to an intensive care unit (ICU) and contributes to 1 in every 2 to 3 in-hospital deaths.6 Studies that manipulated LDL levels in animals6 suggested that LDL protected against LPS-induced mortality.The effect of lipoproteins on sepsis and its outcomes is 1 such area of interest. Lipoproteins, including low-density lipoproteins (LDL), bind toxic bacterial products such as lipopolysaccharide (LPS) that mediate many of the manifestations of sepsis such as vasodilation, increased capillary permeability, and decreased peripheral vascular resistance.9; however, these studies do not answer the question whether LDL-C modifies the risk of sepsis and poor outcomes directly, or if it does so indirectly through the effects of comorbid illness. A study performed in community-dwelling adults10 showed that low LDL-C levels at entry to the cohort were associated with an increased risk of future sepsis, suggesting that LDL-C levels could affect the risk of sepsis directly.Studies in humans also suggest that LDL is protective against sepsis but are less clear. Patients with low levels of LDL cholesterol (LDL-C) have an increased risk of sepsis and worse outcomes12 Also, administering lipoproteins to patients at risk for sepsis is a therapeutic strategy that could potentially prevent or ameliorate the disease. Thus, we tested the hypothesis that low LDL-C levels are directly associated with increased risk of sepsis and poorer outcomes using an epidemiologic approach with careful attention to confounders and a genetic approach that is less prone to confounding. We used a deidentified electronic health record (EHR) repository linked to a DNA biobank to define baseline measured LDL-C levels and an LDL-C genetic risk score (GRS). To define the association between LDL-C and sepsis, we examined the association between measured LDL-C levels as well as the LDL-C GRS and the following 3 outcomes in patients admitted to the hospital with infection: (1) sepsis, (2) admission to an ICU, and (3) in-hospital mortality.Understanding the link between LDL-C levels and sepsis is important because newer medications to lower lipid levels  can reduce LDL-C concentrations to very low levels.15 We used codes from the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) for outcomes and ICD-9-CM codes for cohort construction and covariates. The study was approved by the institutional review board of the VUMC, which waived the need for informed consent for the use of deidentified data. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.Data were obtained from the Synthetic Derivative, a database that contains a deidentified copy of the EHR for every patient in the Vanderbilt University Medical Center (VUMC) system (>2.5 million patients).ICD-9-CM codes for infection based on the criteria of Angus et al16 and Donnelly et al,17 excluding viral, mycobacterial, fungal, and spirochetal infections. The qualifying antibiotics are shown in eTable 2 in the We identified white adult patients (≥18 years) who had been admitted to VUMC with an infection from January 1, 1993, through December 31, 2017 . The dayBaseline LDL-C levels were measured to reduce the confounding effects of illness. We excluded individuals with evidence of severe chronic illness in the year before the index hospital admission .17 and a definition of sepsis as concurrent infection and organ dysfunction identified using a validated algorithm to detect sepsis in the EHR with minor modifications . Surveillance studies have shown that most sepsis cases (86.8%) are present on admission to the hospital.1 In brief, individuals with infection met the definition of sepsis if they had septic shock or severe sepsis or met any organ dysfunction criterion. Septic shock and severe sepsis were defined by ICD-9-CM and ICD-10 codes that are highly specific (99.3%).1 Criteria for organ dysfunction included (1) cardiovascular failure, defined as the use of the vasopressor norepinephrine bitartrate (Levophed) or use of the vasopressors dobutamine hydrochloride or dopamine hydrochloride with a billing code for administration of a vasopressor; (2) respiratory failure, defined as the use of codes for ventilation and admission to an ICU; (3) renal failure, defined as doubling or a greater increase of baseline creatinine level ; (4) hepatic failure, defined as total bilirubin level of 2.0 mg/dL  and doubled from baseline ; (5) hematologic failure, defined as a platelet count of less than 100 × 103/μL  and at least a 50% decline from a baseline that must have been at least 100× 103/μL   were not present in any individual in the subcohort in whom baseline LDL-C levels had been measured because of the exclusions applied to that group. Each comorbidity group was included as an independent covariate in the analysis. We manually reviewed a sample of EHRs to confirm phenotyping , high-density lipoprotein cholesterol (HDL-C), and triglyceride values in the EHR for each individual; age was ascertained at the time of the index hospital admission. We also established the presence or absence of the comorbidities that constitute the Charlson/Deyo comorbidity index, with modifications.P < 5 × 10−8) with LDL-C in the meta-analysis of genome-wide studies performed by the Global Lipids Genetics Consortium  weighted for the effect size (β) of the single-nucleotide polymorphism–LDL-C association. The association between the LDL-C GRS and measured LDL-C levels was validated in an independent cohort of 4313 white patients, none of whom were included in the LDL-C GRS cohort.We generated a GRS for LDL-C using 81 single-nucleotide polymorphisms independently associated  and outcomes. We estimated odds ratios (ORs) per 1-SD increase and 95% CIs for the association between baseline LDL-C levels or LDL-C GRS and sepsis outcomes  adjusting for age, sex, and comorbidities in the measured LDL-C analyses and in the GRS analyses for age and sex. Sensitivity analyses in the subcohort with measured LDL-C level included (1) additionally adjusting for HDL-C and triglyceride levels, EHR length, and BMI; (2) excluding individuals (n = 473) who had a hospital admission for infection before a qualifying LDL-C measurement; and (3) imposing no exclusions on the LDL-C measurement and using the value closest to admission and imposing no exclusions for comorbidities (n = 12 334). In secondary analyses, we estimated ORs and 95% CIs for quartiles of LDL-C levels and LDL-C GRS using the highest quartile as reference and adjusting as in the primary analyses. Because we tested 3 outcomes, we considered 2-sided A total of 61 502 patients met the definition of infection. A total of 3961 patients had clinically measured LDL-C levels , and 7804 had a GRS for LDL-C  . A totalAmong the 3961 patients with qualifying baseline LDL-C measurements , 594 patP = .001) and ICU admission  but not in-hospital mortality . These associations were not significant after adjustment for age, sex, and comorbidity variables  . An analysis that excluded individuals with a previous admission to hospital for infection  was consistent with the main analysis   . Sensitir = 0.24; P < 2.2 × 10−16), accounted for 5.8% of the variability in LDL-C, and was associated with hyperlipidemia  and coronary atherosclerosis . However, the GRS was not significantly associated with sepsis, ICU admission, or in-hospital mortality  and with adjustment for age and sex .The GRS was significantly associated with LDL-C levels  and ICU admission  compared with those in the highest quartile of measured LDL-C levels; however, after adjustment for age, sex, and comorbidity variables, none of measured LDL-C level quartiles differed significantly A and B. We used epidemiologic and genetic approaches to define the association between LDL-C levels and sepsis and found that lower LDL-C levels measured in patients at least 1 year before they were admitted to the hospital with infection were associated with increased risk of sepsis, ICU admission, and in-hospital mortality. However, when comorbidities were considered, no association between LDL-C levels and sepsis or its outcomes occurred. Concordant with this finding, genetic risk factors for LDL-C were not associated with sepsis or its outcomes.Sepsis is a life-threatening and costly condition, which is difficult to anticipate and treat. No specific treatment for sepsis other than early antibiotic therapy and supportive care is available. Strategies to determine which patients with infection are likely to develop sepsis are important because these patients can be targeted for early intensive monitoring. Also, new therapies to prevent or treat sepsis are an important but elusive goal. Thus, LDL-C levels represent an interesting candidate for prevention and treatment of sepsis.6 In murine models, high endogenous LDL levels (induced by deleting the LDL receptor) protected against the lethal effects of LPS and gram-negative infection.5 In contrast, mice that were rendered hypolipidemic had increased LPS-induced mortality that could be reversed by administering exogenous lipoproteins that increased serum lipid levels back to within the physiologic range.6 Despite the animal studies suggesting that LDL could affect the outcomes of sepsis directly, few studies have defined this association in patients.In vitro studies and studies in animals showed that LDL binds toxic bacterial products such as LPS and has other immunologic effects that prevent or ameliorate many of the manifestations of sepsis.9 and with increased risk of sepsis in hospitalized patients7; also, sepsis outcomes were worse than those of patients with high LDL-C levels.10 However, these studies do not necessarily implicate LDL-C directly as a modifier of sepsis because the confounding effects of comorbid illness could account for the findings . An epidemiologic study of approximately 30 000 community-dwelling adults was less prone to confounding and showed that lower LDL-C levels at entry to the cohort were associated with increased risk of future sepsis.10 This finding suggested that LDL levels could affect the risk of sepsis directly; however, individuals in the lowest LDL-C quartile were more likely to have preexisting comorbidities, and the possibility of residual confounding remained a concern.10Low LDL-C levels were associated with increased risk of death in patients with community-acquired pneumonia23 However, no association between LDL-C level and risk of sepsis was found after statistical adjustment that included the comorbidity variables.To define the association between LDL-C levels and sepsis more clearly, we took 2 approaches to minimize the confounding effects of comorbid illness. The first used an epidemiologic approach that accounted for comorbidities, and the second used a GRS that should be independent of the confounding effects of illness. Lower measured LDL-C levels were indeed associated with higher risk of sepsis and its complications, although we took steps to exclude LDL-C levels obtained when patients were acutely ill and excluded patients with several comorbid illnesses associated with low LDL-C levels and increased risk of infection. Results of this unadjusted analysis were concordant with those of previous reports.The study design sought to reduce the confounding effects of preceding and concomitant illness and thus only included LDL-C measurements obtained more than 1 year before the index admission and excluded individuals with many comorbidities that could lower LDL-C levels and increase the risk of infection. However, this design could have introduced bias and reduced the generalizability of the findings. Two findings suggest that the findings were robust. First, the GRS, which does not anchor on LDL-C measurement and thus provides a way of assessing the validity of the measured LDL-C approach, yielded concordant findings. Second, the sensitivity analysis that imposed no time exclusion on the LDL-C and used the value closest to admission and imposed no exclusions for comorbidities yielded results  and the risk of cardiovascular disease22; and between low LDL-C levels and the risk of diabetes.28 Similarly, an LDL-C GRS approach suggested that low LDL-C levels were not associated with Alzheimer disease, although this had been a clinical association of concern.29 In the present study, the Global Lipids Genetics Consortium–based LDL-C GRS was significantly associated with LDL-C levels extracted from the EHR in patients who had had them measured, and with hyperlipidemia and coronary atherosclerosis, but was not significantly associated with the risk of sepsis or its adverse outcomes.The genetic approaches further supported the interpretation that LDL-C levels are not directly associated with sepsis. The genetic architecture of circulating lipid levels is well understood; the Global Lipids Genetics Consortium has published a series of large-scale genome-wide association studies,The present study has several strengths. Large clinical trials in patients with sepsis are difficult to perform; we were able to use existing clinical and genetic information from a large EHR system linked to a DNA bank that provided longitudinal information, including information about drugs, diagnoses, and laboratory findings in a large number of patients to address the questions of interest. The extensive clinical data available allowed us to restrict the measured LDL-C cohort to individuals who had their LDL-C levels measured at least 1 year before the index admission to hospital. This restriction not only minimized the effect of the current illness on LDL-C levels but also provided clinical diagnoses in the preceding year to adjust for comorbidities. In addition, using a clinical and a genetic approach maximized our ability to distinguish whether any association between LDL-C levels and sepsis was likely to be direct or indirect.25; however, the genetic component has been strong enough for the reliable detection of several disease associations.28 Nevertheless, those associations were between LDL GRSs and chronic diseases; the association with acute illness may be more difficult to detect. Fourth, the LDL GRS did not capture LDL changes due to environmental factors, such as diet and lifestyle. Fifth, we cannot exclude the possibility of unrelated competing effects of LDL-C levels and comorbidities on sepsis risk, although little evidence supports it. Sixth, we only studied LDL-C levels and the relatively common variants reported to be associated with them in genome-wide association studies. Concentrations of other lipids such as HDL-C9 and the presence of other variants in genes such as PCSK9 that affect LDL-C but were not identified in genome-wide association studies could be important because they may affect the outcomes of sepsis.23 Seventh, we studied white patients admitted to a tertiary-level US hospital with infection who had LDL-C levels measured at least 1 year before admission or had undergone genotyping, which limits the generalizability of the findings.However, this study also has several limitations. First, the EHR provides information about a range of variables that facilitates statistical adjustment; however, we did not perform a randomized clinical study. Thus, results could be influenced by unmeasured confounders. This possibility seems unlikely to be an important consideration because the confounders included accounted for much of the association between LDL-C levels and sepsis. Second, patients could have received care outside of the VUMC system. Thus, covariates could be incomplete, and although we set out to capture the first hospitalization due to infection, a patient could have been treated elsewhere previously for a serious infection that was not captured. Third, although the GRS was significantly associated with measured LDL-C levels, it explained only a small percentage of overall LDL-C variation in the population. This is the case even for large-scale genetic analyses of circulating LDL-C levelsIn this study, lower measured LDL-C levels were significantly associated with increased risk of sepsis and admission to ICU in patients admitted to hospital with infection, but this finding was owing to comorbidities, because adjusted clinical models and genetic models showed no increased risk. Levels of LDL-C do not appear to alter the risk of sepsis or poor outcomes directly in patients hospitalized with infection.


Trio studies, which involve the testing of samples from a proband and both parents, are often used by clinical laboratories to help with the classification of genetic variants, including copy number variants. In order for the results of the trio study to be valid, the mother and father must be the true biological parents of the proband. As such, non-paternity and sample mix-ups are potential sources of error. To address these potential issues, we developed a computer script to accurately assess maternity and paternity using single nucleotide polymorphism (SNP) data generated by Agilent chromosomal microarrays, a platform-of-choice for clinical copy number testing.We assessed the performance of the script on 10 putative trios tested at our laboratory, and found that the numbers and proportions of discordant SNPs were useful for determining parental relationships. The results of the assessment also confirmed maternity and paternity in the 10 trios tested, and by doing so essentially excluded pre-analytical sample switching in these 30 samples.Computational analysis of SNP data can be implemented as a quality control measure for trio testing performed on Agilent microarrays.The online version of this article (10.1186/s12911-018-0684-9) contains supplementary material, which is available to authorized users. Array comparative genomic hybridization (aCGH) is a first-line diagnostic tool for the post-natal evaluation of patients with intellectual disability, autism spectrum disorders, and complex syndromes with multiple congenital anomalies . A signiThe scripts were written for R (version 3.2.2), a free statistical computing software. In order to develop and test our algorithm, we identified 10 putative trios  who were tested in our laboratory over a period of several months on the Agilent Human Genome CGH + SNP 4x180K Microarrays . Briefly, the testing platform determines (in addition to copy number data) the genotypes  at approximately 30 thousand polymorphic SNP loci across the genome, and records the data as a text table file.The scripts assess the relationships between a proband and the putative parents in the following manner: 1) data files from a proband and one of his/her putative parents (either the mother or father) are merged; 2) SNP loci are then filtered to only include those that are homozygous in both the proband and the parent ; 3) for each remaining (2-copy) homozygous locus, the scripts determines if the genotypes of the proband and parent are identical. If these are identical , the SNP locus is “concordant.” If these are not identical, the SNP locus is “discordant” . The premise is that correct proband-parent pairings  should have very few discordant SNP loci , while incorrect proband-parent pairings  should have significantly greater numbers of discordant loci, since SNPs included are polymorphic. Since the numbers of SNP loci remaining after filtering (homozygous in both the proband and the putative parent) may vary depending on a number of factors (e.g. may be higher when there is a higher degree of relatedness), we considered the proportions of discordant SNPs (rather the absolute numbers of discordant SNPs) to be the most reliable values for assessing parental relationships.Two versions of the script were developed, a test script for assessing performance on putative trios (as described above) and a clinical script for potential clinical use. In the clinical script, a true-pair is called only if the proportion of discordant SNP loci is < 0.015  using real patient data from the 10 putative trios, and determined the numbers of 1) discordant SNP loci, 2) concordant SNP loci, and 3) proportion of discordant SNP loci across 20 possible putative true-pairs  and 180 possible putative false-pairs . This analysis  and SNPTrio [Our clinical script performs quality control functions that overlap with the quality control modules of the Affymetrix GeneChip Genotyping (GTYPE) Analysis Software  the degree-of-relatedness between the putative parent and the proband, 2) the frequencies of the alleles for each SNPs on the array in different populations, and 3) the quality metrics of the specimens, which may vary from laboratory to laboratory. In view of these considerations, we cannot guarantee that the script will reliably distinguish parents from close relatives in all conceivable laboratory settings. Also, the script may be unreliable in other situations. For example, the algorithm will fail to detect a sample switch between the proband and his/her brother or sister, since sibling-parent pairs are true-pairs. Overall, we would recommend that clinical laboratories consider independent internal validation studies (possibly involving samples from close relatives) prior to using script; further, any unusual results reported by the script  should be treated with caution.A second potential issue is that some aspects of our relatively simple script may not be optimized. For example, the cut-off for calling true-pairs (proportion of discordant SNPs < 0.015) in this study is not the result of the statistical analyses of well-defined distributions. The value was selected based on the proportions of discordant SNPs observed for the 20 true proband-mother and proband-father pairs  and protocol/instructions are available as Additional files Computational analysis of SNP data, originally intended for the assessment of absence of heterozygosity, can be implemented as a quality control measure for trio studies performed using Agilent microarrays.Project name: Trio-R: A Script for Assessing Maternity and Paternity in Trio Studies Performed on Agilent Chromosomal Microarrays.Project home page:https://sites.google.com/site/trioscript/Operating system: Platform independent.Programming language: R.Other requirements: R (developed using version 3.2.2).Any restrictions to use by non-academics: none.Additional file 1:Protocol for implementing the R-script. (DOCX 167 kb)Additional file 2:Clinical version of the R-script. (R 7 kb)