Little is known about how to scale up care for depression in settings where non-physician lay workers constitute the bulk of frontline providers. We aimed to compare a stepped-care intervention package for depression with usual care enhanced by use of the WHO Mental Health Gap Action Programme intervention guide (mhGAP-IG).We did a cluster-randomised trial in primary care clinics in Ibadan, Nigeria. Eligible clinics were those with adequate staffing to provide various 24-h clinical services and with regular physician supervision. Clinics (clusters), anonymised and stratified by local government area, were randomly allocated (1:1) with a computer-generated random number sequence to one of two groups: an intervention group in which patients received a stepped-care intervention  plus enhanced usual care, and a control group in which patients received enhanced usual care only. Patients from enrolled clinics could participate if they were aged 18 years or older, not pregnant, and had moderate to severe depression . The primary outcome was the proportion of patients with remission of depression at 12 months  in the intention-to-treat population. This trial is registered with the International Standard Randomised Controlled Trials Number registry (ISRCTN46754188) and is completed.vs 366 [77%] in the control group; adjusted odds ratio 1·0 [95% CI 0·70–1·40]). At 12 months, 17 (3%) deaths, one (<1%) psychotic illness, and one (<1%) case of bipolar disorder in the intervention group, and 16 deaths (3%) and one (<1%) case of bipolar disorder in the control group were recorded. No adverse events were judged to be related to the study procedures.35 of 97 clinics approached were eligible and agreed to participate, of which 18 were allocated to the intervention group and 17 to the control group. 1178 patients (631 [54%] in the intervention group and 547 [46%] in the control group) were recruited between Dec 2, 2013, and June 29, 2015, among whom 976 (83%) were female and baseline mean PHQ-9 score was 13·7 (SD 2·6). Of the 562 (89%) patients in the intervention group and 473 (86%) in the control group who completed 12-month follow-up, similar proportions in each group had remission of depression (425 [76%] in the intervention group For patients with moderate to severe depression receiving care from non-physician primary health-care workers in Nigeria, a stepped-care, problem-solving intervention combined with enhanced usual care is similarly effective to enhanced usual care alone. Enhancing usual care with mhGAP-IG might provide simple and affordable approach to scaling up depression care in sub-Saharan Africa.UK Medical Research Council. Depression is a substantial cause of disease burden globally, including in sub-Saharan Africa.Several studies have shown the effectiveness of a stepped-care approach for treating common mental health disorders in primary care in low-income and middle-income countries (LMICs).Evidence before this studyWe searched PubMed and PsychINFO from June 1, 2010, to June 30, 2012, for evidence relating to the treatment of depression in primary care in low-income and middle-income countries (LMICs), with no language restrictions. Our search terms included “depression” “treatment/interventions”, “primary care” “non-physician primary care providers”, “low and middle-income countries”, and “lay health workers”. We identified 25 relevant papers, and a further eight by hand-searching the references of those papers. Evidence suggested that primary care providers can, with specialist support, deliver effective interventions for depression that include psychological and medication therapies. However, although the studies showing effectiveness were done in countries classified as LMICs, those countries had much better health-care resources than are obtainable in sub-Saharan countries. For example, studies done in India and Chile were implemented in clinics where routine care was provided by physicians. On the contrary, front-line providers in much of sub-Saharan Africa, including Nigeria, are non-physician lay health workers with much simpler training as health-care providers and with very little experience in the provision of mental health care.Added value of this studyTo our knowledge, this is the largest randomised controlled trial of the effectiveness and cost-effectiveness of a stepped-care intervention for moderate to severe major depression consisting of structured problem-solving therapy delivered within enhanced routine primary care services in sub-Saharan Africa. Usual care was enhanced with WHO's Mental Health Gap Action Programme intervention guide (mhGAP-IG) as a clinical support tool. The primary outcome was assessed at 12 months after trial entry, thereby decreasing the likelihood that the outcomes observed were transient. Remission was observed in similar proportions of patients in the group that received enhanced usual care alone and in the intervention group.Implications of all the available evidenceIn developing models for integration of mental health care into the busy schedule typical of a primary health service, there is a need to explore the most feasible and affordable pathway to scaling up care for depression in low-resource settings. The mhGAP-IG might offer such a prospect by providing evidence-based assessment and treatment algorithms, including the use of medications, appropriate to non-physician primary care workers who are authorised to prescribe. Future research should address practical issues related to implementing this approach within routine primary care, including how to facilitate detection of depression at this level of care.The aim of the STEPCARE study was to evaluate the effectiveness and cost effectiveness, over a 12-month period, of a structured problem-solving therapy delivered within a stepped-care approach by non-physician, lay health workers for moderate to severe depression.As described in the protocol,We recruited consecutive attendees at the enrolled clinics who had a score of 11 or higher on the nine-item patient health questionnaire (PHQ-9),The study was approved by the University of Ibadan and University College Hospital ethics committee and was monitored by an independent trial steering committee. All participants provided written  informed consent.Following recruitment of the initial group of participating clinics, anonymised codes for each clinic were provided by the research team in Ibadan to the study statistician (AAM), who generated the allocation sequence and carried out the random allocation. Primary health-care centres were stratified by local government area and randomly allocated in a 1:1 ratio to the intervention group or the control group. For each local government area, a single balanced block equal to stratum size was generated with use of computer-generated random numbers to ensure balanced allocation to treatment groups. We aimed to recruit 90 participants from each of the 16 clinics initially randomised; however, when recruitment was slower than anticipated, we randomised an additional 19 clinics. Allocation of these additional clinics followed the same procedure.Outcome assessors were blinded to patients' group allocations, were not involved with screening or recruitment of trial participants in the clinic, and were randomly assigned to participants from any clinic in either group of the study. Data were collected and stored electronically using android tablets and downloaded to a secure server located in the research office. Data were kept anonymously using codes to identify individuals. These datasets did not contain the allocation status of the participants which was kept as a separate file and was available only to the trial statistician.In the Nigerian setting, front-line primary care providers consist of nurses, community health officers, and community health extension workers, each of whom has 2–3 years of post-secondary school professional training . Supervision and support to all the primary health-care centres in each local government area  is provided by a general practitioner, acting as primary health-care coordinator, who runs outpatient clinics, provides clinical supervisions on a scheduled regular basis across the clinics, responds to clinical emergency calls, and has administrative management duties. At each of the participating clinics, two front-line primary care providers, of any cadre , were selected and trained to provide treatment appropriate to the study group.Providers in the intervention group and the control group delivered usual care enhanced with the mhGAP-IG,Consecutive patients at participating primary health-care centres were screened on the PHQ-9 by trained research assistants. Each consenting participant who screened positive (scored ≥11 on the PHQ-9) was provided with their PHQ-9 score and referred to one of the workers providing trial treatment in the clinic.In the intervention group, in the first step, primary health-care workers use participants' PHQ-9 screen scores to determine treatment options: those with scores of 11–14 were offered eight sessions of psychological intervention delivered by primary health-care workers, and those with scores of 15 or more at baseline were assessed for additional antidepressant medication in consultation with the supervising general practitioner. Therapy sessions were done in person and individually. During the first session, participants were offered psychoeducation in which information about the symptoms of depression, possible causes, and treatments were discussed. After providing reassurance about the treatability of their condition, the structure of the subsequent sessions was discussed and negotiated. The following five sessions dealt with identified problems, difficulties, and stressors, with the therapist working with the patient to explore potential feasible solutions, including how to harness the assistance of supportive social networks. In the final two sessions, both therapist and patient worked together to integrate the experiences of the previous sessions, draw concrete lessons, and use these to prepare for the future. After the first eight sessions, each participant was reassessed with the PHQ-9, this time by the primary health-care worker. Those with a PHQ-9 score of 11 or more, or greater than 50% of baseline score, proceeded to step two. Step two consisted of additional therapy sessions or a combination of therapy and medication following a review by the supervising physician. All participants who did not improve after step two had their cases discussed with a psychiatrist in the third  step.In the intervention group, all supervision and consultations with doctors were provided on as-needed basis and through mobile phone contact, except when a face-to-face review was deemed necessary and feasible. The components and tasks for each treatment session and the clinical decisions and steps were detailed in manuals and charts provided to the primary health-care worker and the primary care physicians. When medication was required, the first-line antidepressant was amitriptyline, which non-physician primary care providers in Nigeria are authorised to prescribe. The trial manual stipulated that, when antidepressant medications were prescribed, the frontline clinician consults with the general practitioner either face-to-face or via mobile telephone to receive appropriate advice on dosing and monitoring. Other antidepressants could be prescribed by the general practitioners for patients who did not improve or had other contraindications to the use of tricyclic antidepressants. Any emergent medication side-effects were reviewed in consultation with the general practitioner.Participants in the control group received enhanced usual care alone. The choice of intervention  was at the discretion of the primary health-care worker and no specification as to the number of sessions was made.Outcome assessments were done through face-to-face interviews at the respondents' homes. All outcome assessors were college-educated, experienced in the conduct of surveys, and received 2 weeks of training, including trial life interviews, inter-rater exercises, and regular field debriefing. Interviewers assessed patients' disability level using the WHO disability assessment schedule (WHODAS) 2.0,28To assess cost effectiveness at follow-up, changes in service costs were analysed with respect to changes on the PHQ-9 and WHODAS at the 6-month and 12-month follow-up visits. The service costs incurred in each group were collected with the service utilisation questionnaire. A set of unit costs and prices for all inpatient and outpatient service use, as well as the costs of the interventions, were generated using simplified costing templates and local data inputs. Cost-effectiveness acceptability curves were constructed for a unit improvement on PHQ-9 and WHODAS.The primary outcome was the proportion of patients who had remission of depression (predefined as PHQ-9 score of <6) at 12 months from entry into the trial. Secondary outcomes included depression symptoms as a continuous PHQ-9 score, assessed at 3 months, 6 months, 9 months, and 12 months, as well as level of disability (assessed with the WHODAS),Informed by the results of our pilot study,vs ≥16) by including appropriate interaction terms in the primary regression model. Because the trial was powered to detect overall differences between groups rather than interactions of this kind, these results are to be interpreted with caution. For the cost and cost-effectiveness analyses, 95% CIs were derived through non-parametric bootstrapping techniques owing to the non-normal distribution of mean service costs per study participant (1000 resamples were run). All analyses were done in STATA (version 13.0) software.The primary approach for comparative analyses was to analyse on an intention-to-treat basis at the individual level without imputation of missing data. We used descriptive statistics to assess balance between the study groups at baseline for both clinic and individual participant characteristics. To take appropriate account of the hierarchical nature of the data, we used multivariable mixed-effects regression models to estimate remission from depression at 12 months for the intervention group versus the control group. We present unadjusted and adjusted estimates. In both analyses, we accounted for clustering with use of a mixed-effects model with clinic and local government area as random effects. In the adjusted estimates, we also adjusted for baseline PHQ-9 score, age, and primary health-care centre, and for local government area as a stratification variable. In a sensitivity analysis, we imputed missing primary outcome data with multiple imputation. Similar analyses were repeated for secondary outcomes. For these secondary continuous outcomes, we estimated the difference in mean scores between the intervention and control groups. We also investigated whether between-group differences varied over time by using data from all follow-up visits in repeated-measures analyses. We investigated whether there was any differential effect of the intervention on the primary outcome according to baseline symptom severity  and ten declined to participate. The remaining 35 were randomly allocated: 18 to the intervention group and 17 to the control group . RecruitParticipant characteristics were well balanced between the two groups at baseline except for mean age, which was higher in the intervention group . 976 . 183 (89%) of 205 sessions were rated as substantially adequate in the fidelity assessments.Of the 631 patients who consented to the trial in the intervention group, 602 (95%) received at least one therapy session, 499 (83%) completed four sessions, and 476 (79%) completed the eight prescribed sessions in step one. Two additional therapy sessions in step two were received by 122 (19%) participants. 76 (12%) participants in the intervention group were prescribed antidepressant medications (amitriptyline): 70 (11%) commenced medication because of severe depression PHQ-9 score >15) at trial entry, and six (1%) started medication following poor response after eight therapy sessions, as specified in the protocol. In the control group, 144 (32%) of 448 participants for whom complete treatment details were available were prescribed amitriptyline. Receipt of counselling from other carers  was reported by 27 (4%) participants in the intervention group and 31 (7%) of 448 in the control group. Only two patients were referred to psychiatrists, both in the control group.vs 77% in the control group; adjusted OR 1·0 [95% CI 0·7–1·4]), and this did not change when multiple imputation was used to account for missing outcome data , tuberculosis (six [18%]), diabetes (two [6%]), asthma (two [6%]), liver failure possibly related to alcoholism (three [9%]), typhoid fever (two [6%]), and cancer (one [3%]). The causes were ascribed to old age or were unknown in nine (27%) patients. Only two (6%) of the deceased patients were on antidepressant medication at the time of death. At the 12-month assessment, one (<1%) patient had developed a psychotic illness and one (<1%) other had developed bipolar disorder in the intervention group, and one (<1%) had developed a bipolar disorder in the control group. Suicidal ideation was reported by 57 (10%) participants in the intervention group and by 66 (14%) of those in the control group. No adverse events were judged by the independent trial steering committee to be related to the study procedures.Estimated service costs per participant per month fell from 11 392 naira at baseline to 3002 naira (at 6 months) and 1729 naira (at 12 months) in the intervention group, and from 7254 naira to 1981 naira (at 6 months) and further to 1664 naira (at 12 months) in the control group . Over thOver the follow-up period, the stepped-care intervention group showed marginally better symptomatic and functional status improvements than those of the control group, but considerably greater reductions in service costs . The redIn this study of patients in primary care with major depressive disorder of at least moderate severity, the proportion of patients with remission of depression at 12 months was similar among patients who received a structured, stepped-care intervention consisting of culturally adapted problem-solving therapy and among patients who received usual care enhanced with the use of mhGAP-IG. Participants in the stepped-care intervention group had lower mean PHQ-9 scores at 6 months than those in the control group, a difference that is unlikely to be clinically important. The stepped-care intervention combined with enhanced usual care was found to lower costs more than enhanced usual care alone, with some evidence for a more favourable cost-effectiveness profile for treating depression in this setting. Both groups were similar with respect to functional status (as measured with the WHODAS 2·0) and health-related quality of life (assessed with the WHOQOL-BREF) at the 6-month and 12-month follow-up visits.The essential component of the treatment in the intervention group was a culturally adapted form of problem-solving therapy which, in a pilot study, had been shown to be acceptable to both providers and patients. In the current study, most participants in the intervention group received eight structured sessions of the therapy in the first step and a few went on, depending on symptom relief, to receive further therapy sessions in the second step or were prescribed antidepressants in consultation with a physician. On the other hand, participants in the control group received a few sessions of basic psychosocial treatment, while many more were prescribed antidepressants than were those in the intervention group. The two groups of participants had essentially the same outcomes.Few studies in sub-Saharan Africa are available to which the results of our study could be compared, the most comparable being one conducted in Zimbabwe.To our knowledge, our study is the largest in an LMIC setting to evaluate the effectiveness and cost effectiveness of interventions for depression delivered by non-physician primary care workers in which follow-up was over a 12-month period, thus increasing the likelihood that the observed outcomes were not transient. Trial participants had a rigorously defined diagnosis of major depression of at least moderate severity, with scores on a validated screening tool complemented with a structured diagnostic interview. The follow-up rate was high and, with a narrow 95% CI in the primary analysis (0·7–1·4) that excluded the effect the trial was designed to detect (OR 2·1), there is assurance of adequate precision. We used a form of problem-solving therapy that had been previously culturally adapted and piloted in our setting. Treatments were delivered by currently serving primary care workers and no new cadres of staff were specially recruited. This has the advantage of enhancing appropriateness to routine primary care in LMICs, thus making sustainability and policy adoption of the findings more likely. Outcomes were assessed by assessors masked to group allocation, with validated tools that had been subjected to cultural adaptation. However, given the complex nature of the treatments in both groups, it is difficult to be sure about the specific aspects of the interventions that were responsible for the change in the depression status of the patients. This is a common observation in studies exploring complex interventions in primary care.The results of this trial should be interpreted with due consideration for the limitations. First, about one in five eligible clinics declined participation in the trial. Second, the intervention was compared with enhanced usual care rather than no treatment. This comparison is similar to the approach taken in many other studies.In summary, for patients presenting with moderate to severe depression in primary care settings in sub-Saharan Africa, a structured problem-solving therapy delivered within a stepped-care approach combined with usual care (enhanced with use of the WHO mhGAP-IG) might provide similar benefit in terms of remission at 12 months, and might represent a more cost-effective intervention compared with enhanced usual care alone. Further research is warranted to confirm these findings and to elucidate whether, using the mhGAP-IG, non-physician health workers in routine primary health-care settings in LMICs could detect and assess depression and offer appropriate treatment.ogureje@comui.edu.ng, and after a signed data access agreement with the principal investigator.The deidentified participant data on which this report is based will be made available, following publication, upon request to


Felis catus), dog (Canis lupus familiaris), and grey wolf (Canis lupus lupus), providing the opportunity to examine the red fox (Vulpes vulpes) Y-chromosome in the context of closely related species. Here we present a data-driven approach to identifying Y-chromosome sequence among the scaffolds that comprise the short-read assembled red fox genome. First, scaffolds containing genes found on the Y-chromosomes of cats, dogs, and wolves were identified. Next, analysis of the resequenced genomes of 15 male and 15 female foxes revealed scaffolds containing male-specific k-mers and patterns of inter-sex copy number variation consistent with the heterogametic chromosome. Analyzing variation across these two metrics revealed 171 scaffolds containing 3.37 Mbp of putative Y-chromosome sequence. The gene content of these scaffolds is consistent overall with that of the Y-chromosome in other carnivore species, though the red fox Y-chromosome carries more copies of BCORY2 and UBE1Y than has been reported in related species and fewer copies of SRY than in other canids. The assignment of these scaffolds to the Y-chromosome serves to further characterize the content of the red fox draft genome while providing resources for future analyses of canid Y-chromosome evolution.While the number of mammalian genome assemblies has proliferated, Y-chromosome assemblies have lagged behind. This discrepancy is caused by biological features of the Y-chromosome, such as its high repeat content, that present challenges to assembly with short-read, next-generation sequencing technologies. Partial Y-chromosome assemblies have been developed for the cat ( Over the last decade, the emergence of next-generation sequencing (NGS) technologies has catalyzed a proliferation of reference assemblies, including those of non-traditional model species . HoweveWork in several species has indicated that Y-chromosome information can be extracted from genomes sequenced with short-read technologies. De novo contigs constituting a partial assembly (186 Kbp) of the horse Y-chromosome were assembled using Roche 454 reads to conduct targeted resequencing of horse BAC clones that had been selected based on homology to Y-chromosome genes in other mammals . In the Vulpes vulpes) is a non-traditional mammalian genomic model in which characterization of Y-chromosome diversity is of particular interest. The red fox is the world’s widest-spread terrestrial carnivore [The red fox  markers would provide higher resolution than microsatellite markers and allow for phylogenetic comparisons over longer timescales . IdentifFelis catus; KP081775.1) and dog , are available [The present analysis characterizes MSY sequence using three complementary approaches: analysis of gene content within scaffolds, identification of male-specific sequence motifs, and comparison of sequencing depth between males and females. The first approach, similarity between the scaffold sequence content and known Y-chromosome genes, has been used to identify MSY sequence in other mammals  was characterized with CNV-seq [Two additional methods are used to complement the syntenic analysis. These methods are not restricted to regions that contain genes, but instead examine sex-specific patterns in whole genome resequencing data (WGS) mapped onto the reference assembly. Specifically, 15 male and 15 female red foxes bred on the same farm as the reference genome donor fox were resequenced at a depth of 2.5× per fox . The WGS CNV-seq  to identTETY2 or FLJ36031Y) were more recently transposed to the Y-chromosome from the X-chromosome or an autosome [DYNG, which is a novel Y-chromosomal gene identified in dog [AMELY, FLJ36031Y, RPS4Y, and TETY1) and for EIF2S3Y, whose canine protein sequence has not been deposited. First, we sought to identify red fox orthologs of genes located on the MSY of dog and cat, which are two carnivores closely related to the red fox ,7,35,38 −5 was specified. Hits to autosomes and the X-chromosome were removed based on the chromosomal positions assigned to the scaffolds [Gene sequences were then mapped against all scaffolds longer than 5 Kbp in the draft red fox genome  using trcaffolds   Y-chromosome [TMSB4Y, AP1S2Y, and WWC3Y) along with a wolf ortholog of the dog gene DYNG —were alk-mers across two groups [k-mers in the YGS.pl output were excluded from downstream analysis. The percent V_SC_UK  on each scaffold was normalized by calculating the standard score .Male-specific sequence motifs were then identified using a pipeline for comparing o groups . The pipo groups  was follo groups , which ho groups  with theo groups . YGS.pl In order to compare sequencing depth across the red fox draft genome between males and females, the male and female reads were aligned using Bowtie2  to the 6a priori chromosomal origins assigned [k-means clustering algorithm [k, or the number of centers, set to 3. k-means clustering is an unsupervised machine learning algorithm that assigns individual data points to one of k clusters and adjusts the positions of the centers of the clusters to minimize the point-to-center distance across all points. The particular implementation used here was R’s native k-means clustering function [k-mers on the scaffold that were valid, single-copy, and unmatched in the female reads (as identified with YGS.pl). The maximum number of sets of random centers to be selected (nstart) was set to 100. The scores corresponding to each window from CNV-Seq (sex-based depth) and from YGS.pl  were plotted, first, for only the windows on scaffolds with assigned ,28,41, algorithm  to the dfunction . Clusterk-means clustering were then evaluated to determine how likely they were to represent the three expected classes of chromosomes. Some scaffolds had been previously assigned to a position on the X-chromosome or autosomes [The clusters identified by utosomes  or identutosomes ,28). TheThe scaffolds assigned to the Y-chromosome using this process were then examined to ensure that all metrics were consistent with what was expected for the Y-chromosome. SAMTools depth was used to estimate the sequencing depth along the putative Y-chromosome scaffolds for the male and female WGS reads mapped to the fox reference assembly. Depth along each scaffold was visualized with ggplot2, as described above, in windows of 5 Kbp or 10% of scaffold length, whichever was smaller.The next step was to reassemble the putative male-specific sequence using a different alignment algorithm than was used in the red fox reference genome assembly project. The alignment of the male resequencing data to the fox genome, as described above, was filtered using SAMTools view with the -L parameter to extract only the reads that mapped to putative Y-scaffolds. The 15 libraries from the genome project , which pk (k-mer size) to optimize for N50 and assembly size. The values of k tested ranged from 50 to 96.ABySS version 2.1.5 ,59 was tThe ABySS assembly and putative Y-chromosome draft genome scaffolds were then compared: first, to each other ; then to the dog Y-chromosome assembly ; and finally to the cat Y-chromosome assembly  using MashMap . The resTETY1 and FLJ36031Y) that have not been previously reported in canids and TSPY, which has been reported to occur in multiple copies on the MSY of cat, dog, and wolf [Based on the BLAST results, 22 out of the 25 genes previously identified on the dog, wolf, and/or cat MSY were assigned to a position on one or more red fox scaffold(s) not previously assigned to an autosome or the X-chromosome . The genand wolf ,14,35. BTSPY was identified in the red fox scaffolds. Two scaffolds contained sequence similar to the dog BCOR-derived MSY genes (BCORY1 and BCORY2). The BLAST results indicated that scaffold310 contained one copy and scaffold360 the other, with most exons mapping to both scaffolds. A blastn search of the NCBI’s online repository indicated that BCORY2 is more similar to the sequence of scaffold310 and BCORY1 to scaffold360  [SHROOM2, consistent with findings in other carnivores [SHROOM2 is located at approximately chrX: 6,642,728–6,499,660 bp in dogs and corresponds to the drop in male sequence mapping depth  18-mers were identified on 10,522 of the 12,625 largest scaffolds, and the number of 18-mers on each scaffold that were unmatched by the female reads (VSC_UK) ranged from 0 to 15,900,208. Across these scaffolds, male-specific 18-mers (VSC_UK) were, on average, 57.1% (sd: 34.8) of all valid, single-copy 18-mers (VSC). As the proportion of ltimodal , with leIn the CNV analysis, each scaffold was examined in 10-Kbp windows to compare coverage in the female (reference) versus male (test) sequencing. The average genome-wide depth of coverage from the male resequencing was 72.133 and from the female was 63.564. After removing windows with a combined coverage of less than 0.01×, an average of 52.9% of reads mapping to the remaining windows came from the male resequencing data. The density of the mapped reads was expected to be modulated by sex and by the ploidy of the chromosome . Because autosomes are equally represented in males and females, approximately half of the reads mapping to autosomal windows should come from males. In comparison, the male resequencing should contribute only 33% of the reads mapping to the X-chromosome because males are hemizygous. Windows containing MSY sequence were expected to have very low (~0) coverage in females, with all of the reads mapping to these regions expected to come from the male sequence. The proportion of reads mapping to each window from the female and male resequencing data supported this pattern .k-mers unmatched in females). These patterns were examined in windows located on scaffolds with a known fox chromosomal position and then in all windows  for each window were plotted against the YGS.pl output , definek (corresponding to k-mer size) revealed that, as expected, k influenced the assembly statistics  and BCORY2 (assumed to be the copy on scaffold310) revealed that the increase in the depth of mapped reads occurred only on scaffold310 (BCORY2 (scaffold310) was associated with a corresponding loss of coverage on scaffold360 , in general, this was not the case. In order to visualize depth in the exonic regions of the genes only, the genomic position of each dog exon in the fox genome was estimated using the BLAST output, and then the average depth over the nearest 15 bp was plotted, corresponding to the estimated position of the codon itself and two codons in either direction. In the case of fragmented genes, the best hit for each amino acid in the protein was used regardless of its scaffold of origin. Comparing ffold310 . While iDDX3Y, USP9Y, UTY, and UBE1Y have previously been used as single-copy controls [CUL4BY and SRY—which are multi-copy in dogs and cats—and HSFY, which is multi-copy in cats but single-copy in dogs and wolves [DDX3Y, UTY, and USP9Y all showed an average per-individual depth of approximately 0 in females and approximately 2.5 in males   suggests that these segmental replications, including additional copies of BCORY2 and ZFY, may have evolved on a short timescale and may distinguish European and North American red foxes. Similarly, rapid changes in segmental replications have been reported in grey wolf populations and haplogroups [The opposite may have occurred on scaffold310: although, unlike in wolf, the genes f BCORY2  is consiRY2 (ZFY , which clogroups .With the red fox adding to the recent increase in the availability of comparative carnivore Y-chromosome resources, studies examining the timing and effect of Y-chromosome evolution, including replication events and gene loss and gain, have become increasingly feasible. The red fox sequence information presented here serves to refine the timing of differences in the Y-chromosome sequences of dogs and cats and also provides an outgroup for studies of Y-chromosome evolution during the evolution of dogs from wolves. These results support the set of Y-chromosome markers available for the red fox ,28 and pBCORY2 and ZFY. The red fox also appears to carry many copies of the gene UBE1Y, and findings suggest that replication of SRY in the dog and wolf occurred subsequent to divergence with the fox 6–9 million years ago. This work demonstrates the potential for information pertinent to Y-chromosome tool development to be extracted through the bioinformatic analysis of even a highly fragmented draft genome, and supports previous findings suggesting recent segmental replications occurred on the Y-chromosome of North American foxes [Sequence from the Y-chromosome was identified among the scaffolds comprising the short-read-assembled red fox draft reference genome. These scaffolds contained all 11 of the core Y-chromosome genes, as well as all but one of the genes previously identified on the dog MSY. The red fox Y-chromosome shows evidence of recent segmental replication that have resulted in an increase in copy number for the gene an foxes . These g


Barcode swapping results in the mislabelling of sequencing reads between multiplexed samples on patterned flow-cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays; however, the severity and consequences of barcode swapping remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in two plate-based single-cell RNA-sequencing datasets. We found that approximately 2.5% of reads were mislabelled between samples on the HiSeq 4000, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Furthermore, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA-sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10x Genomics experiments, allowing the continued use of cutting-edge sequencing machines for these assays. DNA barcode swapping results in mislabelling of sequencing reads between multiplexed samples. Here, the authors investigate the severity and consequences of barcode swapping for single-cell RNA-seq data, and develop a computational method to exclude swapped reads. This results in mislabelling whereby reads assigned to one sample are derived from molecules in another, thus compromising the interpretation of many 'omics assays  and the amount of sequencable cDNA (400-800 bp) in the multiplexed library from each plate. However, we did not observe a strong correlation of swapping fraction estimates with the ratio of free barcode to sequencable cDNA , we considered this as the sample-of-origin, and removed the molecule count from all other samples. Where this was not the case, we removed the molecule from all samples Fig. , as an uWe applied our method to the aforementioned mouse epithelial cell dataset sequenced on the HiSeq 4000. In one experiment, two samples  appeared to contain many cells with expression profiles that were distinct from those of the cells in the other samples Fig. . We obseThese results demonstrate the importance of excluding swapped reads prior to further analysis. Failure to do so would have resulted in misleading biological conclusions if the artefactual cells were used in analyses such as clustering and detection of differentially expressed genes. Indeed, the artefactual cells form their own cluster Fig. , and cou14. Here, our method removed a negligible number of molecules  for large-scale droplet scRNA-seq experiments while avoiding the confounding effects of barcode swapping.Using plate-based scRNA-seq datasets, we have reproducibly estimated the fraction of barcode-swapped reads on the HiSeq 4000 to be approximately 2.5%, which is lower than previously reported6 with minor modifications . For each plate, we fitted a linear model to quantify contributions of different cells in the HiSeq 2500 data to the swapping-affected transcriptomes of the HiSeq 4000 data  contains a detailed report that expands on the analyses described herein, describing models and showing results. The repository also contains a script to download the processed data, and the code used to generate the report.A Github repository (E-MTAB-6843 for plate-prepared mouse T-cells, and E-MTAB-6854 for droplet-prepared HiSeq 2500- and HiSeq 4000-sequenced mouse epithelial cells.Raw data can be acquired from ArrayExpress accession codes Supplementary Information


Meanwhile, a backward stepwise elimination method was used to confirm the optimal multi-temporal combination for KNN monitoring model. A classification and regression tree (CART) and back propagation neural networks (BPNN) approaches were used for comparison and validation of initial results. VIs of all periods except 1 and 3 provided the best multi-temporal data set for winter wheat powdery mildew monitoring. Compared with the traditional single-date (period 6) image, the multi-temporal images based KNN approach provided more disease information during the disease development, and had an accuracy of 84.6%. Meanwhile, the accuracy of the proposed approach had 11.5% and 3.8% higher than the multi-temporal images-based CART and BPNN models’, respectively. These results suggest that the use of satellite images for early critical disease infection periods is essential for improving the accuracy of monitoring models. Additionally, satellite imagery also assists in monitoring powdery mildew in late wheat growth periods.Powdery mildew is one of the dominant diseases in winter wheat. The accurate monitoring of powdery mildew is important for crop management and production. Satellite-based remote sensing monitoring has been proven as an efficient tool for regional disease detection and monitoring. However, the information provided by single-date satellite scene is hard to achieve acceptable accuracy for powdery mildew disease, and incorporation of early period contextual information of winter wheat can improve this situation. In this study, a multi-temporal satellite data based powdery mildew detecting approach had been developed for regional disease mapping. Firstly, the Lansat-8 scenes that covered six winter wheat growth periods  were collected to calculate typical vegetation indices (VIs), which include disease water stress index (DSWI), optimized soil adjusted vegetation index (OSAVI), shortwave infrared water stress index (SIWSI), and triangular vegetation index (TVI). A multi-temporal VIs-based  Blumeria graminis) is one of the most destructive foliar diseases infecting winter wheat and occurs in areas with cool or maritime climates [Powdery mildew  algorithm [k training samples in the feature space. This instance-based learning algorithm is very simple, popular, efficient and effective for pattern recognition, and has been widely used for classification [The lgorithm ,29,30 isfication ,32. The fication ,32. HoweIn this study, we used early multi-temporal satellite imagery to develop a model that focuses on monitoring the occurrence of powdery mildew in the late winter wheat growth period (filling period) at regional scales. Multi-temporal Landsat-8 imagery was adopted in this study. The objectives of this study were (1) to identify the optimal multi-temporal data set for the monitoring of powdery mildew occurrence in winter wheat from a number of different multi-temporal combinations; (2) to assess the feasibility of using imagery containing information for early critical disease infection periods to monitor the occurrence of powdery mildew in late winter wheat growth period; (3) to evaluate the performance of the multi-temporal indices-based KNN disease monitoring approach and its capability for mapping powdery mildew occurrence.The study area encompassed two typical regions affected by the disease (region 1 and region 2), located in the western Guanzhong plains, Shaanxi Province, China . The areDI was calculated using:x is the value of incidence level, f is the total number of leaves for each degree of disease severity, and n is the value of highest disease severity gradient.A total of 62 field survey plots were collected to evaluate the damage severity caused by winter wheat powdery mildew as ground truth data in region 1 and region 2 during 10 May 2014 . Five 1-DI = 0), slight infection (0 < DI ≤ 30%), and severe infection (DI > 30%). The criteria of DI = 30% for these classifications was suggested by China’s national plant protection department (NY/T 613-2002) [DI are generally lower than 3. Therefore, data collected in region 2 was just used for validating in this case.For simplicity, in this study, disease occurrence severity was grouped into three classes for subsequent analysis. These classes included: normal (13-2002) . The ovePowdery mildew is wind-dispersed and infects volunteers after harvest and autumn-sown crops. The disease cycles slowly but can continue during the winter if temperatures are mild. In spring, the growth increases rapidly in tandem with rising temperature and high humidity, and infects leaves ,27. GeneThe spectral divergences among different ground objects, such as farmland, forests, water bodies, and impervious areas are always greater than that between healthy and diseased areas in crop fields. Thus, it is necessary to extract information from the winter wheat planting area before conducting disease monitoring, and a decision tree method was successfully applied to the extraction of winter wheat planting area . Based oIt is reported that the plant growth status and habitat characteristics are associated with the susceptibility of plants to diseases, and the physiological and biochemical characteristics will be changed once the plant is infected by the diseases ,8,36,37.k is a user-defined constant, a query or test point (unlabeled vector) is classified by assigning a label, which is the most recurrent among the k training samples nearest to that query point. No actual model or learning is performed during the training phase, although a training data set is required, it is used solely to populate a sample of the search space with instances whose class is known, for this reason, this algorithm is also known as lazy learning algorithm. It means that the training data points are not used to do any generalization and all the training data is needed during the testing phase. When an instance whose class is unknown is presented for evaluation, the algorithm computes its k closest neighbors, and the class is assigned by voting among those neighbors [The KNN algorithm was implemented to monitor powdery mildew based on multi-temporal Landsat-8 imagery and its performance was evaluated. This algorithm consists of training phase and classification phase. In training phase, the training examples are vectors (each with a class label) in a multidimensional feature space. The feature vectors and class labels of training samples are stored in this phase. In the classification phase, eighbors . The maieighbors . One of eighbors . Compareeighbors . Thus, w2) and the root mean square error (RMSE) were used as accuracy measurements of multi-temporal combination group estimation.Although VIs of the six different periods were chosen as the primary input variables for the construction of multi-temporal VIs-based KNN monitoring model, whether the performance of this multi-temporal combination was optimal or not was uncertain. Hence, a back stepwise elimination  method wSeveral statistical accuracy indicators  were used to evaluate the performance of the KNN disease monitoring models. Additionally, an overall accuracy (OA), user’s accuracy (UA), producer’s accuracy (PA), and kappa coefficient were calculated for the monitoring results based on field truths. These accuracy assessments were used to evaluate the model’s performance in monitoring disease occurrence severity. For comparison and validation, a classification and regression tree (CART)  and backThe spectral changes induced by powdery mildew infection forms the basis for remote sensing monitoring. Generally, the three visible channels  display a higher reflectance in diseased samples than in healthy samples, but the NIR channel exhibits the opposite pattern . This spThe DSWI and SIWSI provided the lowest values for severely diseased samples in six periods. The OSAVI and TVI provided the lowest values for normal samples in the first three periods, and which provide the lowest values for severely diseased samples during the remaining three periods. Additionally, the indices among the three samples exhibited different changing patterns over time.2 and highest RMSE. Meanwhile, the other period groups’ assessment results showed that removing VIs of period 1 and period 3 significantly improved the performance of the model. Additionally, the remaining four periods  were all important; the absence of any one period VIs would lead to a decline in the accuracy of the model. Therefore, the VIs of periods 2, 4, 5, and 6 composed the optimal multi-temporal input variables for disease monitoring model.For backward stepwise elimination analysis, the results evaluation of different multi-temporal combination groups are listed in Optimal multi-temporal  VIs-based models were constructed using the CART, BPNN, and KNN methods to detect the importance of early period information for disease monitoring. In the three multi-temporal models, powdery mildew occurred extensively in region 1, and slightly occurred in region 2 . The monThe CART, BPNN, and KNN monitoring models were also constructed through traditional single-date VIs (period 6), and used to map powdery mildew for region 1 and region 2 . These mFor example, normal and severely infected wheat were distributed throughout the study area in the CART single-date VIs-based model. In the BPNN and KNN models, slightly infected and normal wheat covered most of the entire crop area. Several statistical parameters were used to evaluate the performance of the single-date VIs-based models . The CARCompared to the traditional single-date VIs, all three models produced higher statistical parameters for multi-temporal VIs. The multi-temporal VIs models produced the higher values for Somers’ D, Kendall’s Tau-c, Goodman-Kruskal Gamma, and Spearman correlation. However, the BPNN method had lower values for Kendall’s Tau-c and Goodman-Kruskal Gamma. The results can be seen in We found that classification using multi-temporal VIs produced higher accuracies than the traditional single-date VIs when monitoring for powdery mildew in winter wheat. Not all selected periods were useful for monitoring of disease; in six different periods, only the VIs of period group which composed with periods 2, 4, 5, and 6 were the optimal multi-temporal data set for monitoring of powdery mildew in winter wheat. Furthermore, only the KNN model obtained acceptable results among the three methods using traditional single-date VIs. The KNN method was also observed to produce the most accurate classification using the multi-temporal VIs approach. The overall monitoring accuracy of multi-temporal VIs was obviously higher than that of the traditional single-date VIs. This suggests that the methodology employed by the multi-temporal VIs improved overall monitoring accuracy.Winter wheat undergoes a series of physiological and biochemical changes when infected with powdery mildew. These changes can alter the spectral response characteristics of winter wheat in the visible and NIR spectral ranges . For insSix periods for the extraction of VIs in this study represent the development of powdery mildew infection in winter wheat ,34. The In this study, KNN method has been successfully used for the monitoring of powdery mildew occurrence in winter wheat. No matter based on single-date or multi-temporal VIs, the KNN method outperformed the CART and BPNN methods, which demonstrated that the KNN  model peOverall, the multi-temporal VIs-based KNN model performed better than existing traditional models in monitoring occurrence of powdery mildew in winter wheat based on Landsat-8 imagery data, with a good accuracy of 84.6%. We expect the selection periods to include the whole key periods of powdery mildew development and to observe the disease’s entire evolution in the entire growth period of winter wheat. However, due to data limitations, the characteristics of remote sensing in critical periods of disease infestation were only considered from a phenological perspective in this study. The occurrence, development, and dispersal of plant diseases are closely associated with local weather conditions ,61,62. TThis study developed a multi-temporal VIs-based KNN approach based on Landsat-8 imagery to monitoring powdery mildew in winter wheat. The performance of this approach was evaluated from two aspects, for one thing, by compared with traditional single-date based methods, the accuracy of the proposed approach had increased by 7.7%, for another, we also compared this method with the CART, BPNN, and KNN, which suggested that the multi-temporal VIs-based KNN method has an excellent performance in monitoring powdery mildew in winter wheat at the regional scale, with the overall accuracy of 84.6%. This approach provides an evidence for using the satellite observations on guiding field disease prevention and management. Our future work would focus on: (1) the detection and classification of multiple diseases at the field scale, and (2) the relationship analysis between early growth information of wheat  and disease evolution.


Thy-1 was discovered over 50 years ago, and in that time investigators from a broad variety of fields have described numerous and heterogeneous biological functions of Thy-1 in multiple contexts. As an outwardly facing cell surface molecule, it is well positioned to receive extracellular signals; previously reviewed studies have confirmed an important role in cell-cell and cell-matrix adhesion, cell migration, and regulation of outside-in signaling. More recent studies reviewed here expand the repertoire of Thy-1 effects on signaling pathways, and reveal novel roles in mechanotransduction, cellular differentiation, viral entry, and extracellular vesicle binding and internalization. All of these studies contribute to understanding Thy-1 as a context-dependent integrator of a diverse range of extracellular information, and provide impetus for further studies, some of which are suggested here. More than 50 years after its original description as a lymphocyte marker , Thy-1 . AccordiThe effects of Thy-1 on cell signaling have been intriguing. An excellent recent review by a pioneer in the field details the signaling effects of Thy-1 relative to its abundance and location on cell surfaces . Thy-1 d-/- mice fail to resolve fibrosis following bleomycin-induced lung injury, associated with the persistence of apoptosis-resistant myofibroblasts. A study in hepatocellular carcinoma cells showed that the presence of Thy-1, which is a cancer stem cell marker in these tumors, is correlated with enhanced Notch signaling 90, has long been recognized and broadly utilized as a marker of hematopoietic and mesenchymal stem cells. Until recently, however, any mechanistic role in the stem cell phenotype has not been appreciated. Reprogramming of differentiated fibroblasts into induced pluripotent stem cells (iPSCs) using a limited number of transcription factors is one of the major milestones in 21st century biology . A study-/- mice were found to have decreased osteoblastic differentiation and increased adipocyte differentiation compared to those from WT mice, consistent with findings of increased adiposity and decreased bone density in Thy-1-/- mice , such as exosomes, larger microvesicles, and apoptotic bodies. We had previously demonstrated release of Thy-1 from fibroblasts in response to inflammatory stimuli, and antibody studies indicated that Thy-1 was likely released in a membrane-bound fraction . SubsequHow do these seemingly disparate new findings inform our current view of Thy-1? The last half century has produced a substantial body of work on this molecule, during which its roles in a large number of biological fields have become apparent. What insights can be gained from what is known about Thy-1, and what are important questions for future studies? First, Thy-1 is a primitive molecule, conserved to the level of chordates , and it Many unanswered questions remain. Little is known, for example, about how the mode of anchorage of Thy-1  affects its function. Is it merely by regulating its proximity to integrins and other surface molecules, or is the lipid moiety of Thy-1 itself able to interact with other molecules to affect signaling? How important are other post-translational modifications, most notably glycosylation, in some of the seemingly discordant effects of Thy-1 in different contexts? Many of the downstream effects of Thy-1 involve cell phenotype and “identity.” These are likely regulated at the level of transcriptional control and epigenetic modifications. Does Thy-1 directly affect nuclear machinery, or are all its effects secondary to its effects on cytoskeletal rearrangements and signaling cascades? With improved understanding of mechanisms comes the ability to target specific aspects of Thy-1 function. However, the translational potential of manipulating Thy-1 remain unexplored. Perhaps the next half century will see important breakthroughs as a result of additional careful study of this enigmatic integrator of extracellular information.JSH reviewed the relevant literature and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Squalene epoxidase  is a key rate-limiting enzyme in cholesterol biosynthesis. Anil Padyana and colleagues report the long awaited structure of human squalene epoxidase (SQLE). They solved the crystal structure of the catalytic domain of human SQLE alone and in complex with two similar pharmacological inhibitors and elucidate their mechanism of action. SQLE is the target of fungicides and of increasing interest in human health and disease, particularly as a new anti-cancer target. Indeed, in a companion paper, Christopher Mahoney and colleagues performed an inhibitor screen with cancer cell lines and identified SQLE as an unique vulnerability in a subset of neuroendocrine tumours, where SQLE inhibition caused a toxic accumulation of the substrate squalene. The SQLE structure will facilitate the development of improved inhibitors. Here, we comment on these two studies in the wider context of the field and discuss possible future directions. A case in point is SQLE, which catalyzes a second rate-limiting step downstream of HMGCR and has been relatively neglected until now.3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) catalyzes the initial rate-limiting step during cholesterol biosynthesis and is the target of the highly successful statin class of cholesterol-lowering drugs. However, relatively little is known about the 20 or so other enzymes involved in cholesterol synthesis2. More recently, human SQLE has been gaining prominence as a bona fide oncogene and target in cancer therapy5.And yet this enzyme is becoming a hot topic. The yeast homologue ERG1 is a target of antifungals like terbinafine. The human enzyme has a long history of being a potential cholesterol-lowering target, and natural products in common foods and beverages, such as garlic, red wine and green tea, may lower blood cholesterol levels by inhibiting SQLES)-oxidosqualene 8.The molecular interactions of the SQLE enzyme-substrate complex must be exquisitely precise to rigidly control the regio- and stereochemistry of the epoxidation reaction9.Work over the past two decades has characterized several key structural features of SQLE. The catalytic domain Fig.  containsAlthough substrate requirements, cofactors, and inhibitors have been investigated for SQLEs of various origins, no structural model has been available so far. Moreover, the domains responsible for enzymatic activity and inhibitor interactions are not well understood. Therefore, a structure of human SQLE has been long awaited.10. They determined the crystal structures of the FAD-bound human SQLE alone and with two potent inhibitors NB-598 and Cmpd-4”. Despite interspersed primary sequence, the N-terminal-truncated protein contains three distinct domains, the FAD-binding domain, the substrate-binding domain, and a C-terminal helical membrane-binding domain ,22(S),23-dioxidosqualene6 -oxidosqualene can be flipped and reinserted into the substrate binding pocket, hydrocarbon head-first and that the same regio- and stereo-specificity applies. The significance of this second epoxidation is that it represents the entry-point for diverting the product 2,3(S),22(S),23-dioxidosqualene into a shunt in the biosynthesis pathway. This shunt pathway is most active when SQLE activity is high and the activity of the subsequent enzyme lanosterol synthase is low. The final product 24(S),25-epoxycholesterol appears to serve as a measure of cholesterol synthesis and to protect against surges in the production of this potentially cytotoxic molecule. In addition, endogenous 24(S),25-epoxycholesterol is a natural ligand for the liver X receptors which induce expression of cholesterol efflux-related genes as well as promoting dopaminergic neurogenesis13.SQLE can act again to introduce a second epoxide to produce 2,3-oxidosqualene. However, no well-diffracting crystals of the SQLE•FAD complex with either of the substrates could be obtained. Squalene is a large hydrophobic molecule without a polar group to anchor it to the aqueous interface, and so may reside in the midplane of the lipid bilayer14. But how is squalene extracted from the membrane to be acted on by SQLE? Presumably, supernatant protein factor (perhaps in concert with anionic phospholipids) transfers squalene from the membrane midplane to the active site of SQLE, but it is not known how the catalytic domain of SQLE associates with the membranes of the endoplasmic reticulum. The hydrophobic regions at the C-terminus are probably membrane associated perhaps via two putative transmembrane domains (Fig. Given the authors’ pharmaceutical background, their focus on inhibitors is understandable. From a more basic biology viewpoint, more structural information may be required to elucidate the actions of SQLE on its native substrates squalene and 2,34. Terbinafine shares the tertiary amine motif of the two inhibitors tested but has a naphthalene moiety which is bulkier than the corresponding moieties in either NB-598 or Cmpd-4”. Accordingly, Padyana and co-workers10 found terbinafine to be a far less potent inhibitor, with an IC50 of 7.7 μM compared to 63 and 69 nM for NB-598 or Cmpd-4”, respectively. The idea that terbinafine is a weak partial inhibitor against human SQLE fits neatly with species differences in the residues lining the binding pocket. Moreover, the crystal structure of the human enzyme proved useful for rationalizing why mutations of residues lining the binding pocket render certain fungal strains resistant to terbinafine treatment. Therefore, the SQLE structure will stimulate the development of better inhibitors that are more potent and selective against either the human or fungal versions of this enzyme.Terbinafine, a common antifungal agent, has been proposed as a new treatment strategy for human cancers5 investigated NB-598 sensitivity in numerous cancer cell-lines of neuroendocrine origin, including from small cell lung cancers. Using multiple approaches, they concluded that NB-598 sensitivity was due to toxicity caused by squalene accumulation rather than to insufficient cholesterol being synthesized. The enhanced toxicity relied on lipid droplets serving as storage for excess squalene, since inhibition of lipid droplet formation made even insensitive cells highly vulnerable to NB-598. The link between SQLE, squalene and lipid droplets clearly warrants further research, particularly in an oncogenic context.Mahoney et al.Therefore, the crystal structure of the catalytic domain of human SQLE is certainly a boon to the growing throng of researchers interested in this fascinating enzyme vital in human health and disease. Indeed, the accompanying chemical biology study provides more compelling evidence that SQLE has a prominent role in certain cancers.


Depression is a very common condition that impairs functioning and is often untreated. More than 60% of the treatments for depressive disorder are administered in primary care settings by care providers who lack the time and expertise to treat depression. To address this issue, we developed Ascend, a therapist-supported, mobile phone–delivered 8-week intervention administered at the Meru Health Online Clinic in Finland.We conducted two pilot studies to examine the feasibility of the Ascend intervention, specifically, dropout rates, daily practice, weekly group chat use, and changes in depression symptoms. We also explored whether daily practice and weekly group chat use were associated with changes in depression symptoms.A total of 117 Finnish adults with elevated depressive symptoms enrolled in Ascend, a program that included daily cognitive behavioral and mindfulness meditation exercises delivered through a mobile phone app, anonymous group chat with other users, and chat/phone access to a licensed therapist. Eight weekly themes were delivered in a fixed, sequential format. Depression symptoms were measured at baseline, every second week during the intervention, immediately after the intervention, and 4 weeks after completion of the intervention. Data were analyzed using intent-to-treat repeated-measures analysis of variance and linear regression models.P<.001). We found that both more daily practice and chat group use predicted the occurrence of fewer depressive symptoms at 4 weeks postintervention .For studies 1 and 2, we observed dropout rates of 27% and 15%, respectively, decreasing daily practice and group chat use, and decreased depression symptoms from baseline to immediately and 4 weeks after the intervention (This therapist-supported, mobile phone–delivered treatment for depression is feasible and associated with reduced depression symptoms. Design features that enhance daily practice and group chat use are areas of future investigation. Validation of these results using a controlled study design is needed to establish the evidence base for the Ascend intervention. Depression is one of the most common mental disorders, impacting more than 300 million people worldwide according to the World Health Organization (WHO) . In addiThere are several effective psychological ,4 and phOnline interventions for mental disorders, especially depression and anxiety, have gained increasing popularity during the last decade. These online interventions can be delivered in many different formats including psychoeducation, self-help programs, chat support groups, interactive health coaching, and professional-led online therapy . Online There is a growing body of evidence for the beneficial effect of online interventions for depression. A recent meta-analysis  of contrg=.22) and a moderate reduction compared to an inactive control group (g=.56). This study also found no statistical difference between the effect of mobile phone only and mobile phone plus human/computerized components for the treatment of depression. Furthermore, apps focused on mental health produced a greater reduction in depression symptoms than apps focused on cognitive training. Interestingly, the integration of specific features, including mood monitoring, CBT, and mindfulness, did not alter the observed effects sizes. In addition, a more conservative subanalysis of studies (n=16) that used intention-to-treat analyses or reported complete outcome data showed a moderate effect of these treatment methods on depression symptoms (g=.40). This meta-analysis suggests that mobile phone apps may be a potentially effective tool to introduce self-management skill training for adults with depression. However, we need to better understand the potential mechanisms  and pretreatment patient features  that predict outcome and help us refine mobile phone–delivered interventions for depression.More recently, the increased use and flexibility of mobile phones has presented a more promising route to extend the reach of clinical interventions. A recent meta-analysis involving 3414 participants across 18 RCTs of mobile phone–based interventions for depression  showed aTo address the public health need for more accessible interventions for depression, we developed a new mobile phone–delivered intervention that includes self-help modules, support from a personal remote therapist, and chat group access to an anonymous patient peer-group support.The goal of this pilot study was to examine the feasibility of a newly developed mobile phone–delivered, therapist-supported 8-week intervention for depression in self-referred depressed individuals. We examined dropout, number of days of practice, and weekly use of an online group chat. For symptom reduction, we examined intervention-related changes in depression symptoms and whether daily practice and weekly group chat use predicted a reduction in depression symptoms. We proposed three hypotheses. First, we expected a dropout of ≤30% among all participants, daily practice on at least half of the days , and weekly group chat use for at least half (4/8) of the weekly group chat sessions. We also examined the pattern of change in daily practice and weekly group chat over the 8-week intervention. Second, we expected the Ascend program to be associated with significant decreases in depression symptoms. In study 1, we used the Beck Depression Inventory-II (BDI-II)  to measuFor studies 1 and 2, participants were recruited from March to September 2017 in Finland via online Facebook advertisements for the Meru Health Online Clinic, a national remote health care provider approved by the Finnish National Supervisory Authority for Welfare and Health . Meru Health operates under HIPAA ; legislation and all protected health information is stored in a HIPAA-compliant cloud storage hosted by a company called Datica . All datParticipants were offered a free online intervention for depression that included mobile phone–delivered content, anonymous weekly chat group, and private phone/chat access to therapists. All participants were required to have a mobile phone. To enter the study, participants had to demonstrate some depression symptoms based on the BDI-II (>9) in study 1 and at least mild depression symptoms based on the PHQ-9 (>4) in study 2. We increased the depression symptom cut-off in study 2 to match the validated score of PHQ-9 for screening mild depression. Other inclusion criteria included the perceived ability of the participant by both the participant and therapist to commit to an 8-week online intervention with a minimum of 20 minutes of practice 6 days per week. The exclusion criteria included previous suicide attempts, severe suicidal ideation with a specific plan, severe self-harm, active substance abuse, and a history of psychotic disorder.We informed participants that the goal of the study was to examine an online intervention for depression, delivered through a mobile phone app. Participants provided informed consent through the Ascend mobile phone app for their anonymous data to be used for further intervention refinement and research purposes. Participants were not compensated for their participation. They enrolled in the program at no cost. Institutional review board exemption was granted by Pearl IRB  for analyses of previously collected and deidentified data.We provided participants a link to download the Meru Health Ascend app on their mobile phone to access the intervention content. Participants used a unique identification number to record depression symptoms at baseline, during the intervention, immediately after the intervention, and 4 weeks after the intervention. We trained participants on how to access an anonymous chat group and converse with the study therapist via direct one-to-one chat messaging or, in few exceptions, phone calls.As part of the standard treatment procedure at the Meru Health Online Clinic, Ascend study therapists conducted phone interviews to examine inclusion and exclusion criteria before enrollment and determine participant suitability for the online intervention. After the program, the study therapist spoke to participants via phone calls to assess the participant’s experiences and address any further needs. In addition, participants had an opportunity to speak directly with the therapist via phone calls during the program. This occurred in only a few cases when a participant had a specific concern or question that could not be properly communicated by chat messaging with the study therapist.The Ascend program consists of 8 modules delivered in a fixed order through a mobile phone app over an 8-week period, including practices derived from mindfulness-based stress reduction , mindfulThe Meru Health therapists provided ongoing individual support as needed and curation of the group chat during the Ascend intervention. The interaction took place primarily via chat messaging and, in a few instances, phone calls. Therapists included one medical doctor and two master’s-level clinical psychologists, each of whom had training in mindfulness-based stress reduction or mindfulness-based cognitive therapy. The patients could reach out to their therapist whenever they wanted. However, more than 50% of the communication was initiated by the therapist, not the patient. The therapist would normally check in with the patient via chat at least 2-3 times/week to see how the patient was doing. On an average, the therapist spent 20 minutes on each patient per week, including time spent chatting with the patient and reviewing the patient’s data and progress.Study therapists used a Professional Dashboard , which is a secure web-based tool to monitor participant progress and chat with participants. The mobile phone app hosted all the weekly program content, including text, video, audio, and graphics. The intervention included video lessons every week, audio-guided mindfulness-meditation practices, visual graphics that illustrated cognitive-behavioral principles, and journaling prompts.The mobile phone app consisted of the following components: a “Me” screen to access the daily practices and learning materials; a “Program” screen to view the structure of the whole Ascend program, identify where on the timeline one is currently located, and access already completed modules and practices; a “Group” screen to view other anonymous participants’ written reflections on different practices and lessons ; a “Notifications” screen to track therapists’ messages and newly available lessons or practices; and an “Other” screen to view the Emergency Plan, Privacy Policy, Terms of Use, and a single button to contact the therapist via chat messaging.We used multiple measures to assess the feasibility of the Ascend intervention. We measured dropout from the intervention, operationally defined as less than 4 weeks of active participation during the 8-week intervention. We also measured completion of mobile phone–delivered practices (number of days during the 8 weeks) and participation in the once-weekly chat group (a binary yes/no measure). As stated above, we shifted from the BDI-II in study 1 to the PHQ-9 in study 2 to measure depression symptoms, because it became clear that the latter self-report measure is more commonly used across a wide variety of health care settings.Depressive symptoms were measured using the BDI-II, a scale containing 21 items rated 0-3 points in terms of intensity, with total scores ranging from 0 to 63. BDI-II scores of 14-19 points suggest mild depression; 20-28 points suggest moderate depression, and 29-63 points suggest severe depression. To enter the Ascend intervention, the participant had to score a minimum of 10 points at baseline. The BDI-II has demonstrated high internal consistency  in outpatient samples .The PHQ-9 is the 9-item depression scale extracted from the full PHQ. Because each of the 9 items can be scored from 0  to 3 (nearly every day), the PHQ-9 score can range from 0 to 27 points. PHQ-9 scores of 5-9 points suggest mild depression, 10-14 points suggest moderate depression, 15-19 points suggest moderately severe depression, and 20-27 points suggest severe depression. To enter the Ascend program, the participant had to score a minimum of 5 points at baseline. Prior large-scale studies have shown that the PHQ-9 has excellent internal reliability, with Cronbach alpha of 0.89 in primary care settings, and excellent test-retest reliability .To assess the patient-therapist interaction, we used a single-item question: “How valuable has the therapist interaction been for you?” This rating ranged from 1 to 5, with higher values indicating greater value. The patient-therapist interaction was assessed at weeks 1, 3, and 6 and after the intervention.We computed descriptive statistics for different indicators of intervention engagement. We plotted the number of days per week of practice during the intervention and the number of weeks of chat group use and used a repeated-measures analysis of variance (ANOVA) with Huynh-Feldt correction for autocorrelation of adjacent time points to examine whether there was significant change over time in the number of days of practice completed and online chat group use.η2p) using SPSS, version 25 . We also computed a depression-change score from the baseline to the 4-week follow-up to be used as the dependent variable in subsequent linear regressions.We used an intent-to-treat analysis that included all participants regardless of whether they completed or dropped out of the intervention. We used the more conservative method of last observation carried forward to insert missing data immediately postintervention and during the 4 weeks of follow-up. We implemented repeated-measures ANOVA and reported effect size as partial eta-squared  from week 1 (mean 4.57 days [SD 1.72]) to week 8 (mean 1.52 [SD 1.83]) of the intervention . Participants reported a high and consistent level of the participant-therapist interaction across four time points during the 8-week intervention (mean 4.12).Dropout from the 8-week Ascend intervention was observed in 6 of the 22 participants (27%). For mobile phone–delivered exercises during the Ascend intervention, participants completed daily practices on 42% of the 56 days . A repeated-measures ANOVA, with Huynh-Feldt correction for autocorrelation of adjacent time points, showed that the mean number of days per week of practice decreased significantly  as well as from before to 4 weeks after the intervention .Using a linear regression controlling for baseline depression symptoms, we found that greater number of days of practice significantly predicted a reduction in depressive symptoms from the baseline to 4 weeks postintervention  in a linear trajectory from week 1 (mean 5.04 days [SD 1.71]) to week 8 (mean 2.63 [SD 2.31]) of the intervention . Participants reported high and consistent levels of participant-therapist interaction across four time points during the 8-week intervention (mean=4.13).Dropout was noted in 14 of the 95 participants (15%). Participants completed the mobile phone–delivered exercises during the Ascend intervention on 55% of the days . A repeated-measures ANOVA, with Huynh-Feldt correction for autocorrelation of adjacent time points, showed that the mean number of days per week of practice decreased significantly  as well as from before to 4 weeks after the intervention  versus 30% dropout from computer-delivered CBT . For comDaily practice of mobile phone–delivered exercises decreased linearly over the course of the 8-week Ascend intervention in both pilot studies. Given that the amount of daily practice significantly predicted a reduction in depression symptoms in each of the two pilot studies, this finding is disconcerting and strongly suggests that further effort is needed to investigate methods to support sustained practice, as it may lead to greater clinical improvement. This may involve introducing more novel types of practices or different modes of delivery of practices to maintain the participant’s attention and sustained engagement. Ongoing measurement of engagement during the intervention and tailoring the delivery to individual participants may help increase sustained practice. This could involve the use of individual participant feedback after each practice to direct what type of practice and modality is to be used when delivering subsequent practices. A brief motivational interview by the therapist may also identify and overcome obstacles to engagement. This is a component of internet interventions that would benefit from further research. Sustained engagement may likely increase the effect of the Ascend intervention and possibly reduce the probability of depression relapses.Group chat, often referred to as synchronous text-based dialogue, is an increasingly important component of online mental health interventions. In our studies, the number of weeks of chat group use decreased significantly over the 8-week intervention. Like daily practice, group chat use was a significant predictor of a reduction in depressive symptoms. A recent review  found thg=.43 to .54) for reduction of depression symptoms across the two pilot studies. Our findings are in line with the results from a recent meta-analysis [g=.40), CBT-based interventions (g=.53), and interventions that included mindfulness practices (g=.49). Importantly, subanalyses in this meta-analysis that focused on diagnosed mood disordered samples showed that mobile phone–delivered interventions were most effective for patients with self-reported mild-to-moderate levels of depression symptoms (g=.52). This is important because individuals with mild-to-moderate depression symptoms are most likely to have the capacity to use mobile phone and other online interventions.The Ascend intervention was associated with a reduction in depression symptoms across two pilot studies. However, because we did not incorporate a comparison control group, we cannot rule out regression to the mean as a reason for the reduced depression symptoms. Nonetheless, our intent-to-treat analysis revealed a moderate effect  and pharmacological interventions as well as a control group without an active intervention. Although this study provides initial evidence of a reduction in depression symptoms, future studies should include diagnostic assessment of participants. In addition to changes in clinical symptoms, it will be important to measure putative mediators of the Ascend intervention related to mindfulness meditation  and CBT . Future studies might consider determining whether the amount  or type of therapist-patient interaction  predicts intervention outcome.The overall finding from this pilot study is that the 8-week online Ascend intervention was associated with reduced depression symptoms. However, more rigorous controlled trials that compare the Ascend intervention with both other online mental health interventions and gold-standard clinical interventions for major depression are needed.