In the present study, we investigated the efficacy of bladder hydrodistension combined with pentosan polysulfate (PPS) treatment in interstitial cystitis (IC)/bladder pain syndrome (BPS).In this study, 339 patients diagnosed with IC/BPS were categorized into two groups. The first group only received 300 mg/day PPS, while the second group received 300 mg/day PPS following bladder hydrodistension. The results were evaluated at the 3rd, 6th, and 12th months after the first dose using the interstitial cystitis symptom index (ICSI), international cystitis problem index (ICPI), visual analog scale (VAS), and female sexual function index (FSFI).PPS treatment started just after hydrodistension was significantly more effective than PPS treatment alone and combined treatment significantly reduced the rate of non-compliance such that, at the end of the 3rd month, 12.1% patients in Group-1 did not continue their treatment whereas only 1.9% of patients in Group-2 did not continue.The study results indicate that PPS treatment started just after hydrodistension yields significantly better results in terms of both symptom improvement and treatment compliance in patients with IC/BPS. Despite the advancements in medicine that have occurred at an unprecedented pace in the 21st century, there has not been a similar level of improvement in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS), causing continuing disappointment among both physicians and patients. Until 2002, the National Institute for Diabetes and Digestive and Kidney Disease (NIDDK) criteria were considered sufficient for the diagnosis.5Epidemiological studies report high prevalence rates of IC/BPS in the population, ranging between 2.7-6.5%.8Unfortunately, for a disease of such high prevalence that causes significant impairment in life quality, no definitive treatment has yet been established. Pentosan polysulfate sodium (PPS), intravesical dimethyl sulphoxide (DMSO), and many oral agents are used despite a lack of FDA approval , as well as hydrodistension, botulinum toxin injections, neuromodulation, and even cystectomy are methods currently used in treatment.9The only therapeutic agent currently approved for oral treatment of IC/BPS is PPS.In the present study, we compared the treatment success of oral PPS treatment alone and oral PPS treatment in combination with bladder hydrodistension for the purpose of enhancing treatment efficacy and treatment compliance in patients diagnosed with IC/BPS.The study was performed in a single center. We retrospectively reviewed 415 patients who were diagnosed with IC/BPS upon presenting to our center between November 2005 and July 2015. Since male lower urinary system pathophysiologies have different and more complicated presentations, the present study included only female patients in order to provide homogeneity in the study group. After applying study inclusion and exclusion criteria, the study included 339 patients. The inclusion criteria included female sex, patient aged 18 years old or older, complaints present for at least 6 months, no treatment received for IC during the last 6 months, no history of neurological disease, no history of previous pelvic surgery, no history of pelvic irradiation, not diagnosed with diabetes mellitus, no history of clinical urinary system infection within the last 1 month, and no pelvic organ prolapse. The exclusion criteria were consisted of patient younger than 18 years old, pregnancy or lactation, abnormal liver function tests, anticoagulant treatment, thrombocytopenia, hemophilia, history of gastrointestinal bleeding, and urolithiasis history.The diagnosis was based on medical history, physical examination, urinary culture antibiogram, cystoscopy and biopsy. The symptoms had to be present for at least 6 months. For every patient, scores from interstitial cystitis symptom index (ICSI), interstitial cystitis problem index (ICPI), and visual analog scale (VAS) for assessment of pain were recorded both before and after treatment for comparison. Sexually active individuals were assessed with the female sexual function index (FSFI).th, and 12th months after initiation of PPS treatment using the same assessment methods . The main purpose here was to avoid additional morbidity caused by repeating the same procedures in patients already diagnosed with biopsy and cystoscopy. In the first group, oral PPS treatment was initiated at 300 mg/day. In the second group, oral PPS treatment was initiated as early as possible after confirming the diagnosis with cystoscopy, hydrodistension, and biopsy. Patients were evaluated at the 3rd, 6 methods . The stuFor statistical analysis of the data, Pearson chi-square test, Cochran-Mantel test and Pairwise comparison tests were used, and p<0.05 was accepted as statistically significant.After applying the study inclusion and exclusion criteria to the 415 patients reviewed retrospectively, 76 were excluded from the study. Thus, the study included a total of 339 patients. According to the patient follow-up records, 50 patients in Group-1 (31.8%) and 21 patients in Group-2 (11.3%) did not show up for the control visits at the end of the first year. Mean patient age for the whole study group was 42.8 (21-74) years. For all patients, the diagnosis was made according to the definition of ICS.Mean duration of symptoms was 14 months (range 8-48 months). Of the 339 patients included in the study, 268 (79.05%) patients regularly attended all control visits until the end of the first year. Based on patients’ assessment results at the end of the first year, PPS treatment at 300 mg/day resulted in quite good improvement of symptoms in patients from both groups .Regarding comparison of the groups at the pre-treatment phase, it was observed that patients in Group-1, who had been diagnosed previously and had received various treatments before, but none except analgesics within the last 6 months, had milder symptoms in comparison to Group-2. Additionally, comparison at the end of the 3rd month of treatment showed that although patients in Group-2 had more severe symptoms, they responded very well to the treatment, and that treatment success was higher in comparison to Group-1.Another interesting finding was related to attendance to the control visits. Whereas 19 patients (12.1%) from Group-1 did not attend control visits at the end of the third month, this number was 2 (1.9%) in Group-2 (p<0.001). At the end of the sixth month, the number of patients who did not attend control visits was 13 (9.42%) in Group-1, and 4 (2.22%) in Group-2 (p<0.001). Comparison of the results at the end of the sixth month showed that both groups had similar treatment success rates.During the last six months of the follow-up period (starting from the 6th month until the 12th month), 18 (14.4%) patients from Group-1 and 15 patients (8.52%) from Group-2 did not attend control visits (p<0.001). Regarding evaluation of the last six months treatment period, it was observed that both groups showed progressive improvement, and that there was no statistically significant difference between the groups. In the present study, we investigated whether hydrodistension caused any change in the efficacy of PPS treatment in 339 IC/BPS patients diagnosed according to ICS criteria and confirmed with cystoscopy and biopsy. First, we observed that PPS 300 mg/day TID was very effective in treatment of IC/BPS. Literature data indicate that, compared to placebo, PPS treatment results in greater than 50% more improvement in patient symptoms.2O) and prolonged (>10 min.) hydrodistension has been shown to be more beneficial; however, it should be noted that it has higher complication rates , and therefore, must be performed with caution.Therefore, we used hydrodistension performed under general anesthesia for this purpose. AUA guidelines recommend hydrodistension as the third line treatment in BPSAt the end of the first three months, the proportion of patients who did not attend control visits was significantly higher in Group-1. This may be because these patients could not wait for optimal treatment efficacy to appear, and went on search for an alternative physician or treatment method. The number of patients who did not show up for follow-up was quite low in the group who received hydrodistension in combination with PPS treatment.Another study goal was to examine how IC/BPS affected female sexuality, and whether treatment could solve this problem. Based on evaluation of patients using the FSFI, it was observed that the majority of patients did not have a functional sexual life at all due to IC/BPS, and that no matter the level of improvement with treatment, patients continued an asexual life due to the pain they suffered during sexual intercourse.IC/BPS affects patients physically, but it can cause serious psychological, social, and sexual problems. PPS reduces the symptoms to nearly half, and is widely preferred due to the convenience of oral administration. The drug’s action is not immediately observed due to the nature of the disease, and it is a proven fact that its efficacy increases with continued use. Therefore, it would be appropriate to initiate PPS 300 mg/day treatment as early as possible after bladder hydrodistension under general anesthesia, as doing so would provide both acute symptom relief and increased patient compliance. Nevertheless, further studies and ideas related to combination of these two methods would aid in increasing the number of alternative treatment options.AS, FK conceived, designed and did statistical analysis & editing of manuscript.AS, FK did data collection and manuscript writing.AS, FK and CO did review and final approval of manuscript.


Supplemental Digital Content is available in the text Sepsis is the most common critical illness in the clinic, with a high incidence and mortality. Qingwen Baidu decoction (QWBDD) has been widely applied in the treatment of sepsis, however, there is no systematic review or meta-analysis of QWBDD in the treatment of sepsis. Hence, we provide a protocol of systematic review and meta-analysis to evaluate the efficacy and safety of QWBDD in the treatment of sepsis.The databases including Cochrane Library, PubMed, Embase, Web of Science, Cochrane Clinical Trial Database, World Health Organization International Clinical Trial Registration Platform, CNKI, CBM, VIP, and WanFang Database will be searched from the time when the respective databases were established to January 2019. All randomized controlled trials (RTCs) published in Chinese and English assessing QWBDD for sepsis will be included. Continuity data are expressed as mean difference (MD) or standard mean difference (SMD), and dichotomous data is expressed as relative risk. Analyses will be performed by using RevMan V.5.3.5 software.This study will provide high-quality synthesis of current evidence of QWBDD in the treatment of sepsis from the following aspects, including 28-day mortality, mean arterial pressure (MAP), blood lactate, procalcitonin (PCT), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), acute physiology and chronic health score (APACHE-II), intensive care unit stay, mean hospital stay, mechanical ventilation time, etc.Our systematic review will provide evidence for judging whether QWBDD is an effective intervention for sepsis.PROSPERO CRD 42019123078. Severe sepsis accounts for 10% of intensive care unit (ICU) inpatients and results in a 20% to 52% mortality rate, and the mortality rate of septic shocks could even be as high as 40% to 70%,–10 which is the leading cause of death secondary to coronary artery disease in European and American countries. With the aging of the population, the increase of drug-resistant pathogenic microorganisms, human medical activities, and immunosuppressed patients, the incidence of sepsis is on the rising. There are nearly 750,000 patients with severe sepsis and septic shock in the United States each year, and the annual medical cost of treating sepsis is as high as $16.7 billion. In China, the cost of treatment per day for every sepsis patient is as high as $502. Although the International Guidelines for Management of Sepsis and Septic Shock has been continuously updated and many new technologies for the treatment of sepsis have been emerged,,13,14 no effective methods have been found to reduce the incidence and mortality of sepsis.,16Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and still the leading cause of death in critically ill patients. Qingwen Baidu decoction (QWBDD) is comprised of 15 kinds of TCM . QWBDD has been used widely in clinical practice to treat sepsis with a certain effect by some low quality randomized controlled trials (RCTs) published in China.–21 However, there have been few systematic reviews regarding effectiveness and safety of QWBDD in the treatment of sepsis. Therefore, we provide a protocol of systematic review and meta-analysis to evaluate the effectiveness and safety of QWBDD for sepsis, in order to provide a stronger evidence-based medical basis for clinical application.Traditional Chinese medicine (TCM) is an important part of complementary and alternative medicine (CAM), which has been widely applied in clinical practice in China.22.12.1.1All the RCTs of QWBDD in the treatment of sepsis will be included, the language is limited to Chinese and English, regardless of whether blinding or allocation concealment was adopted. Non-randomized controlled trials, animal experiments, case or empirical reports, reviews, abstracts, and repeated publications will be excluded.2.1.2 There is no limitation to patient's age, sex, ethnicity, and severity of the disease. Patients with HIV infection, malignant tumors, connective tissue disease or immune system diseases will be excluded.Patients diagnosed as sepsis (older than 18 years) according to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) developed by Society of Critical Care Medicine and European Society of Intensive Care Medicine will be included.2.1.3 , and combined treatment of QWBDD and conventional treatment was used in the experimental group. The conventional treatment in the control group should be consistent with that of the experimental group. The dosage and route of administration of QWBDD are not limited. The course of treatment was at least 7 days and follow-up time was at least 28 days. However, there were no other TCM, acupuncture, acupoint sticking in both groups.The control group used the conventional treatment recommended in the guidelines2.1.42.1.4.1The mortality rate of 28 days.2.1.4.2Secondary outcomes will include central venous pressure (CVP), mean arterial pressure (MAP), urine volume, superior vena cava oxygen saturation (ScvO2), mixed venous oxygen saturation (SvO2), lactate clearance (LAC), blood lactate, procalcitonin (PCT), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), acute physiology and chronic health score (APACHE-II), ICU stay, mean hospital stay, mechanical ventilation time, adverse reactions.2.2We will search the following databases: The Cochrane Library, PubMed, Embase, Web of Science, Cochrane Clinical Trial Database, World Health Organization International Clinical Trial Registration Platform , China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), Chinese Science and Technology Periodical Database (VIP), and WanFang Database from the time when the respective databases were established to January 2019. The search terms include:QWBDD, sepsis, and RCTs. The English databases will be searched according to the keywords of each database combined with free words. The strategy for searching the PubMed will be shown as an example in Appendix A , and modified by using other databases.2.2.1We will search the references, related conference papers, or dissertations included in the study manually. In addition, relevant unpublished research results will be requested from other researchers or pharmaceutical manufacturers.2.32.3.1The articles will be imported to the Endnote  and duplicate studies will be excluded. Later, 2 authors will read the title and abstracts to exclude the studies clearly not meeting the inclusion criteria independently. Then, the 2 authors will read the full text of literature to determine whether to include them. Finally, results will be double checked for accuracy. Whenever there is a disagreement, it will be solved by discussion or consulting a third author. The process of studies selection and meta-analysis is presented in an adapted preferred reporting items for systematic review and meta-analysis (PRISMA) flow diagram Fig. .2.3.2The data extraction will be carried out independently by 2 authors via a standardized data abstraction form. The extracted content mainly includes: basic information of the study , basic characteristics , specific details of the intervention and treatment, outcome indicators and outcome measurement data, key factors of bias risk assessment . If there is a disagreement, it will also be solved by discussion or consulting a third author. The data will be double checked for accuracy.2.3.3The risk of bias of included literature will be assessed according to the bias risk assessment tool recommended by Cochrane Reviewer's Handbook V.5.3, including 7 domains: random assignment method, allocation concealment, blinding of patients, blinding of evaluator of outcome, incomplete outcome data addressed , selective reporting, other bias. The quality of included literature will be judged as “low risk,” “high risk,” and “unclear” from the above 7 aspects. If the opinions are inconsistent, an agreement will be reached through discussion or consulting a third author.2.3.4The dichotomous outcomes will be expressed by relative risk (RR) or odds ratio (OR) with 95% confidence interval (CI). For continuous outcomes, mean difference (MD) with 95% CI will be presented if the outcome measures of all studies are based on the same unit of measurement, otherwise, standardized mean difference (SMD) with 95% CI will be presented for analysis.2.3.5If the necessary information of the included study is unknown or lacking, we will contact the author via email or telephone to obtain it. If it is not available, the existing data will be processed by using data synthesis, and the impact of the missing data will be discussed.2.3.6I2 statistic. If P > .1, I2 <50%, there is no or low statistical heterogeneity. If P < .1, I2 ≥50%, statistical heterogeneity will be considered significant and further subgroup analysis or sensitivity analysis will be performed to find the source of heterogeneity.The heterogeneity will be assessed by chi-squared test and 2.3.7If there are >10 studies included, a funnel plot will be used to analyze whether there is a publication bias.2.3.8RevMan5.3 software  will be used to compute the data synthesis.P > 0.1, I2 <50%), a fixed effect model will be used for data synthesis. If there is a high statistical heterogeneity , further analysis will be conducted to find whether there is statistical or clinical heterogeneity, a random effects model will be applied after eliminating the impact of significant clinical heterogeneity. If there is a significant clinical heterogeneity, heterogeneity and inability to judge the source of heterogeneity, descriptive analysis will be used.If the heterogeneity is low or no statistical heterogeneity  software . The quality of evidence was divided into 4 levels: high, medium, low, and extremely low.3,23 Some clinical trials have shown that QWBDD may inhibit inflammatory reaction and regulating immunity–21 in patients with sepsis, however, the results were inconsistent, and so far no systematic review and meta-analysis of QWBDD in treatment of sepsis has been found, which has certain limitations on clinical guidance. Therefore, we intend to conduct a systematic review of QWBDD for sepsis in order to provide high-quality evidence of effects and safety of QWBDD for sepsis, and provide reference for scientific researchers and health policy makers. However, there may be some limitations in our reviews. Firstly, only studies published in Chinese and English will be included, which may increase the bias. Secondly, there may be a heterogeneity risk due to different nationalities, doses of herbs, age of the patient, and the small sample of the included study.Sepsis is the most common critical illness in the ICU, with high morbidity and high mortality. Modern medicine has made some progress in the methods and means of treating sepsis, however, the incidence and mortality of sepsis are still high, which is a worldwide problem.Conceptualization: Jingya Zhong, Hairong Cai.Data curation: Yonglian Huang.Funding acquisition: Ping Wang.Investigation: Dongjie Chen.Methodology: Yaxiu Huang.Project administration: Xiaohong Peng.Software: Shaoping Li.Supervision: Xiaohong Peng.Validation: Qifeng Cao.Writing – original draft: ping wang, Hefei Huang.


The observed diversity of intron number and presence/absence of maturases corroborated previously suggested correlation between the number of maturases in the pt genome and intron proliferation. Surprisingly, pt genomes of taxa belonging to Discoplastis and Lepocinclis encode two inverted repeat (IR) regions containing the rDNA operon, which are absent from the Euglenaceae. By mapping the presence/absence of IR region on the obtained phylogenomic tree, we reconstructed the most probable events in the evolution of IRs in the Euglenophyta. Our study highlights the dynamic nature of the Euglenophyta plastid genome, in particular with regards to the IR regions that underwent losses repeatedly.Photosynthetic euglenids (Euglenophyta) are a monophyletic group of unicellular eukaryotes characterized by the presence of plastids, which arose as the result of the secondary endosymbiosis. Many Euglenophyta plastid (pt) genomes have been characterized recently, but they represented mainly one family – Euglenaceae. Here, we report a comparative analysis of plastid genomes from eight representatives of the family Phacaceae. Newly sequenced plastid genomes share a number of features including synteny and gene content, except for genes  The plastids of both green algae and red algae were subsequently transferred to other eukaryotic lineages, which gave rise to secondary plastids. Green algal plastids were taken up by two groups of microbial eukaryotes (protists): euglenophytes and chlorarachniophytes1. Plastids from red algae were inherited to several algal lineages such as ochrophytes, haptophytes, and others. Although the origin and evolutionary history of plastids in various eukaryotic lineages are very different, almost all plastids contain their own genomes, which share some common features. Most sequenced plastid genomes are from land plants and green algae, but the number of other plastid genomes has increased substantially over the last few years. The vast majority of plastids have a similar structure, genes are dispersed among the inverted repeats (IR) and large and small single-copy (LSC and SSC)3 regions. The IR regions that contain genes for rRNAs and a variable number of tRNAs and proteins are broadly distributed in all primary and secondary plastids as well as in cyanobacterial genomes3. The IR has been suggested to play a role in the replication initiation4, genome stabilization5, and gene conservation6. Gene content varies greatly when we consider all photosynthetic eukaryotes, but within each of the evolutionary lineages, gene content and order are conserved features. In some plastid genomes, most notably those of euglenids and land plants, there are also self-splicing introns, which are thought to have invaded the plastid genome on multiple occasions8. Plastid-encoded group I introns, found in rRNA, tRNA or protein-coding genes, have been reported in apicomplexans, glaucophytes, stramenopiles and Viridiplantae3. Group II introns are found in the plastids of cryptophytes, euglenids, and Viridiplantae and usually exist within tRNA or protein-coding genes3. The most extreme examples of the group II intron derivatives are characteristic for plastid genomes of euglenids. These include “group III” introns reduced to 73–119 nucleotides, and “twintrons” which are group II introns nested within another group II intron11. Secondary origin of euglenid plastids and discovery of their unusual introns in plastid genomes triggered further studies on their plastid genomes evolution.Plastids derived from a single endosymbiotic event between a cyanobacterium and the common ancestor of the green algae (including land plants), red algae and glaucophytes - an event called primary endosymbiosisPyramimonas-related green alga12. Within photosynthetic euglenids, three evolutionary lineages are distinguished. A single mixotrophic species Rapaza viridis forms the most basal lineage13. Other photosynthetic euglenids are split into two groups: predominantly marine Eutreptiales and freshwater Euglenales. Euglenales are divided into two families: Phacaceae and Euglenaceae15. Genomic features of the secondary plastid of euglenids have been studied very intensively in the recent years as more plastid genomes have been sequenced. The first plastid genome of Euglena gracilis was sequenced more than two decades ago16 but the number of plastid genome sequences rapidly increased since 2012 resulting in 18 euglenid plastid genomes sequenced so far. Three Eutreptiales plastid genomes have been characterized18 and 15 from the Euglenales, including 14 of Euglenaceae19 and only one from Phacaceae20. The euglenid plastid genome has undergone dynamic changes, including genome reduction due to the gene loss or transfer to the nucleus12, the proliferation of introns21, and genome rearrangements. Intron proliferation has been proposed to be correlated with the number of maturases in the pt genome20. The majority of genes among previously sequenced euglenid pt genomes have had the same basic complement of protein-coding genes. However, there have been significant changes in the gene arrangement. More closely related taxa tend to have greater synteny21 than more divergent organisms, for which extensive rearrangement have been shown22.Photosynthetic euglenids (Euglenophyta) constitute a single subclade within euglenids. Their plastids, enclosed by three membranes, arose as the result of the secondary endosymbiosis between phagotrophic eukaryovorous euglenid and the Phacus, Lepocinclis, and Discoplastis. The family Phacaceae has been proposed quite recently based on phylogenetic relationships23 and morphological synapomorphy – the presence of numerous, small plastids without pyrenoids. The genus Phacus was erected in mid-XIX century24 to accommodate taxa from the genus Euglena that were rigid and did not undergo metaboly. The genus Lepocinclis was established a few years later by Perty25 to incorporate taxa from the genus Phacus that were not flattened. With the advent of molecular sequencing, the division of taxa between Phacus and Lepocinclis have been validated and a new genus Discoplastis was erected to accommodate several species formerly belonging to the genus Euglena and characterized by numerous small plastids without pyrenoids and strong metaboly of the cell26. Genus Phacus and Lepocinclis are species-rich and have been intensively studied on the species-level in the last decade, and several new species have been described31. In contrast, genus Discoplastis comprises only two species26.Phacaceae comprises three monophyletic genera: Lepocinclis, two taxa from Phacus, and one Discoplastis taxon. We sought to identify the main genomic changes that occurred in the investigated lineages. The examined genomes display considerable variability at all levels except gene content. We also present the phylogenomic trees inferred from the plastid genome sequences. Our results highlight the repeated losses of IR region during the evolution of Euglenophyta.Here, we report the structural features of the eight newly sequenced plastid genomes of the representatives of the family Phacaceae, including five taxa from Phacus orbicularis, has been sequenced20. When compared to the characteristics of Euglenaceae pt genomes32, they were very similar in their gene content. However, a few physical traits present within the Phacaceae were remarkable when compared to the rest of the Euglenales and the Euglenophyta.We investigated eight taxa representing all three genera encompassed in the family Phacaceae and found that the plastid genome experienced important alterations at the level of genome organization and intron prevalence during the evolution of the Phacaceae. Before our comparative analyses, relatively little was known about the extent of chloroplast genomic changes throughout Phacaceae and only one species, Eutreptia viridis contained the smallest genome among Euglenophyta at 65,513 bp17, followed by that of the fellow member of Eutreptiales – Eutreptiella gymnastica 12, leading to the assumption that there was an expansion in the genome size in the freshwater euglenids . Among members of the family Euglenaceae, Monomorphina aenigmatica11 was identified to contain both the smallest genome  and the smallest number of introns (53 + 1 unidentified ORF). Sequencing of the first representative of the Phacaceae family – Phacus orbicularis20 revealed that its pt genome  is smaller than any of the known Euglenaceae genomes and comparable in size with the pt genomes of Eutreptiella gymnastica. All the strains sequenced in this study possess relatively small pt genomes (below 91 Kb)  was identified as the smallest Euglenophyceae pt genome sequenced to date, even smaller than the genome of Eutreptiella gymnastica. That might suggest that the pt genome of P. inflexus underwent reduction, which could be explained by the loss of mat2 and mat5 and subsequently a smaller number of introns, along with the low average length of genes (531 bp) (Table Eutreptia viridis (587 bp).Previously, the pt genome of 33. The inferred relationships among genera of Euglenaceae are not well supported, and only those between closely related taxa are in accordance with multigene analyses on nuclear-encoded genes15. In contrast relationships among Phacaceae species were well resolved and highly supported. Phacus and Lepocinclis form sister clades with Discoplastis represented by D. spathirhyncha branching off first. That topology was previously recovered in phylogenetic analyses based on nuclear-encoded genes15.Before comparing the gene content and gene organization of the examined genomes, here we present the phylogenetic context required to interpret those results. Our chloroplast phylogenomic analyses were carried out using amino acid and nucleotide data sets that included representatives of all genera of Euglenophyta . The amino acid data set was generated using 57 protein-coding genes , whereas the nucleotide data set contained two rRNA genes . The obtained phylogenetic tree  making up the standard rRNA operon were present in the pt genome of Discoplastis, and all analyzed Lepocinclis  but absent from the representatives of Phacus. Initial assemblies of the plastid genomes of Discoplastis and Lepocinclis were not circularized and the resulting contigs differed in the coverage, suggesting duplication. The contigs were joined via PCR reactions and additional PCR reactions were used to extend the pt genome to the reported genomic sequences. The resulting assembly confirmed that the circularization was impossible due to the presence of an inverted repeat of the ribosomal operon. The arrangement of the plastid genomes in Discoplastis and Lepocinclis was very similar, IRs are separated by a very short SSC, which doesn’t contain any genes, only tandem repeats.The most prominent structural difference observed among analyzed pt genomes is the presence/absence of inverted repeat (IR) regions encoding the rRNA operon. Two identical IR copies containing all five genes . Only the operons of two species of Eutreptiales, Eutreptiella gymnastica12, and Etl. pomquetensis18, resemble the operons and their strand orientation in prasinophytes36, whereas one of the copies was most probably lost during the divergence of Euglenales. Although all known pt genomes of Euglenales lack one copy of IR, in E. gracilis and Stromobomonas acuminata37 they have been shown to contain tandemly repeated copies of the rRNA operon38.The plastid genomes of land plants and green algae often contain two copies of an IR encoding the rRNA operonEutreptia, however we have to take into account that only one representative of that genus was analyzed and its sequence is incomplete17. The second loss occurred during the evolution of the genus Phacus, as none of the three analyzed species carried two copies of IR. Finally, the third loss most probably appeared in the common ancestor of all taxa belonging to the family Euglenaceae, which were lacking the quadripartite arrangement of the plastid genome. That scenario is more likely than the creation of an IR de novo from an IR-less plastid genome39. However, some of the Euglenaceae pt genomes are incomplete, and the number of losses might increase with the completion of those genomes. Specifically, pt genome of Colacium vesiculosum and Strombomonas acuminata have traits of the possible remnant of the IR; they possess an additional piece of 16S rRNA or 23S rRNA, respectively37.By mapping the presence/absence of IR on the obtained phylogenomic tree, we reconstructed the most probable events in the evolution of IRs in the Euglenophyta Fig. . Our res40. It has been shown that the substitution rates are several times lower for the IR relative to the single-copy (SC) regions among several angiosperms41 suggesting a major impact of the IR on the rate of plastome sequence evolution. The omnipresence of the quadripartite structure of plastid genome suggest its important role, however loss of the IR regions, although rare, is known from land plants42 and several lineages of the green algae, such as prasinophyceans43, trebouxiophyceans40, and streptophytes44. The molecular mechanisms underlying these events remains unclear, although several hypotheses have been proposed based on studies on plants and green algae39. It might be a consequence of repeated events of IR contraction, the complete excision of one of the IR sequence or through the differential elimination of the gene sequences from one IR copy. Among the plastid genomes of the Phacaceae, we didn’t observe any intermediate types of IR copies, which might suggest the step-wise loss of one of the IRs. In contrast, in Etl. gymnastica two copies of the IR differ, which might indicate that one of them is on its way to be lost12. More plastid genomes sequences form the early branching lineages of Euglenophyta is needed to support either of the aforementioned hypotheses.The plastid genomes of almost all land plants and algae carry two identical copies of a large IR sequenceDiscoplastis and Lepocinclis possess two copies. We did not identify any traces of 5S rDNA in two species, namely L. ovum and L. playfairiana, and only the core of 5S rDNA (27 nucleotides long) in L. steinii. The 5S rRNA was also not identified in Etl. gymnastica pt genome12 and in some green algae, like Pyramimonas parkeae and Pycnococcus provasolii35. However, the absence of 5S rRNA in plastid genomes, in particular among protists, have been previously shown to be the result of difficulties with identification, not the lack of 5S rRNA in those genomes45.The rRNA operon was present in all investigated strains and roaA, which was absent in P. inflexus and D. spathirhyncha, and tRNA L-(UAG) which was absent in L. ovum. The number of genes encoding proteins and tRNAs was also consistent with other Euglenophyceae taxa21. In several taxa, roaA (P. pleuronectes) and atpF  began with alternative start codons, which was also previously observed for other taxa21.The majority of the protein-coding genes and tRNAs were shared among all investigated taxa, except mat5 gene has patchy distribution along the tree of photosynthetic euglenids, and it was present in two out of seven analyzed species (both strains of L. tripteris and P. pleuronectes)  Fig. . Bennettmat2 was absent in two out of the three analyzed Phacus species (P. inflexus and P. orbicularis) and one Lepocinclis species (L. tripteris)  in this family. Discoplastis and all analyzed Lepocinclis strains contained two maturases and average (50–59) to a high (79–95) number of introns (Table P. pleuronectes (104 introns) 17. The observation of the greater number of introns in strains with more maturases seems to be consistent but doesn’t explain the mechanism of intron proliferation.It was proposed that proliferation of introns might be related to the number of maturases. Eutreptiales, with one maturase, has the lowest number of introns and Euglenacae with two or three maturases, tend to have more introns introns 04 in thi introns 04 in thipsbC, which carries an intron-encoded maturase ycf13, is considered the ancestral intron because it is the only homologous intron in euglenophyte cpDNAs46. Surprisingly, intron1 in psbC of both strains of L. tripteris was not homologous with E. gracilis psbC intron2, though L. tripteris psbC intron1 still contained ycf13.An intron of psbF and psbD genes in E. gracilis16 are unique to that species and absent not only in all Euglenaceae22 but also in Phacaceae20 (Table psbC (intron1/Egra.2) (Table 20. Our data contradicted the suggestion of petB twintron (intron1) as a synapomorphy for all Euglenophyta21 as we identified the petB twintron in taxa representing all genera of Phacaceae except L. ovum and P. pleuronectes   were very similar, and ~90% of nucleotide sites were identical when compared across the entire genomes. This was a much higher identity than was observed in the two strains of Euglena viridis, which were only 70.5% identical21. The identity was even higher within coding space – 95.3%, which was also higher than between two Euglena viridis strains (91.5%). The pt genomes of the L. tripteris strains were also very similar in size but a comparison between the two genomes revealed that the area between tRNAs-S(GCU) and Y(GUA) were different , Germany); Lepocinclis ovum, SAG 1244-8; Lepocinclis playfairiana, MI102 (strain isolated at Michigan State University); Lepocinclis steinii, UTEX 523 ; Lepocinclis tripteris, UTEX 1331; Lepocinclis tripteris, (MI101); Phacus inflexus, ACOI 1336 ; Phacus pleuronectes SAG 1261-3b. Cultures were maintained as described in21. Lepocinclis playfairiana and L. tripteris (MI) cells were identified in field samples collected from the East Lansing, MI, USA area and brought into culture in the following manner: single cells were picked from field samples using a sterile Pasteur pipette under a Leica MZ16 dissecting microscope  and transferred through a series of sterile drops of growth media, modified AF-6 medium51 with 150 mL L−1 of Soil-Water Medium , to ensure the presence of only one cell. The isolated cells were then placed into one well of a 96-well plate containing sterile growth media, one cell per well, and allowed to divide for ~2 weeks. Following this, well contents were moved into 12 mL culture tubes that contained sterile growth media and were allowed to grow for another ~2 weeks. Finally, the culture tubes were subsampled and viewed with a Zeiss Axioscope 2 plus microscope  to ensure that they were uni-algal and verify the identity of the culture. These two cultures were also maintained as described previously21.The following eight cultures were used in this research: L. ovum, L. playfairiana, L. steinii, L. tripteris (UTEX), L. tripteris (MI), P. inflexus, and P. pleuronectes were concentrated, washed, and had their DNA extracted with following protocols52, with the following alterations: Percoll  was substituted for Centricoll  in the P. inflexus process; L. ovum, L. playfairiana, L. steinii, and P. pleuronectes cultures were not run through the gradient step. DNA of D. spathirhyncha was extracted using phenol/chloroform, and DNA separation, using a cesium chloride gradient, was performed as described before17. All DNA was sequenced with Illumina paired-end reads at the Michigan State University Research Technology Support Facility: D. spathirhyncha with HiSeq 2 × 100 bp reads; all other taxa with MiSeq 2 × 150 bp reads. Phacus inflexus raw sequence data were assembled into contigs with the ‘De Novo Assemble…’ program in Geneious Pro version 6.8.1  as previously described21. All other raw sequence data were assembled into contigs with the ‘De Novo Assembly’ program in CLC Genomics Workbench version 5.5.1  as previously described21. The number of contigs and their lengths, number of reads per contig, and average coverage per contig are provided in the Supplementary Table Cultures of 21. The primers designed to confirm the presence of IRs are listed in the Supplementary Table 53, RNAmmer54, or by manual aligning, we used MFannot tool55 and confirmed its annotation by homology searches with 5S rRNA Database56. The search for potential twintrons was performed as previously described21, with the exception that instead of a manual search, a Python script was created (Supplementary File https://github.com/ankarn/groupIII_twintrons) to find the 3′ conserved motif for Group III twintrons9 within the homologous external introns. Genome maps were drawn using OGDRAW57. Newly generated organelle genomes were deposited in GenBank , sequence cleanup (as necessary), PCR primer creation, annotation, arrangement, and genome map creation were as performed as previously described58. Mauve performs syntenic comparisons between multiple genomes and displays these syntenic regions graphically. In the Mauve alignment the repeat regions of rRNA were not included because Mauve will not align repeat regions which have multiple matches in both genomes.Synteny between the pt genomes of Phacaceae was determined and visualized with progressive Mauve 2.3.159, trimmed with BMGE ver. 1.1260, and concatenated with SequenceMatrix ver. 1.861, leaving 11,499 reliably aligned amino acid (aa) positions and 3,959 reliably aligned nucleotide positions (rRNA). The protein data set was assembled from protein-coding genes: atpA, B, E, F, H, I, ccsA, petB, G, psaA, B, C, I, J, psbA, B, C, D, E, F, H, I, J, K, L, N, T, rbcL, rpl2, 5, 12, 14, 16, 20, 22, 23, 32, 36, rpoB, C1, C2, rps2, 3, 4, 7, 8, 9, 11, 12, 14, 18, 19, tufA, ycf4, 9, 12, 65. We excluded from the dataset sequences from the strains of the same species (E. gracilis var. bacillaris KP686076 and E. viridis KP686075). We excluded also Colacium vesiculosum (JN674636), which has been previously shown to have the unstable position at phylogenomic trees, and its elimination doesn’t lead to different topology but could reinforce the support for the relationships within the tree33. As an outgroup, we used three species representing Chlorophyta, previously shown to be the close relatives of Euglenophyta33. For Maximum Likelihood (ML) analyses each protein-coding gene was divided into a separate partition, and one rRNA partition was applied, resulting in 58 partitions. We determined the best choice of model for each partition with ModelFinder62 implemented in IQ-TREE ver. 1.6.163. ML analyses were performed with IQ-TREE ver. 1.6.163, using a partitioned analysis for multigene alignments under the recommended models64 and 1,000 ultrafast bootstrap65.The 57 orthologous protein sequences and two rRNA nucleotide sequences encoded by 26 analyzed plastid genomes (Supplementary Table Supplementary Information


Tofacitinib is a new oral Janus kinase inhibitor that has shown promising clinical benefit in various rheumatologic diseases. However, many concerns related to the development of malignancies have been reported with its use.  A 43-year-old female patient received tofacitinib for refractory rheumatoid arthritis (RA). Two years after 5 mg bid daily dosing, the patient developed chronic myelogenous leukemia (CML), for which she received imatinib and tofacitinib was discontinued. She then remained in remission for rheumatoid arthritis and within the expected milestone outcome for her CML.  This is the first reported case of CML after the use of tofacitinib. This event is of particular interest knowing the possible benefits tofacitinib carries in the treatment of CML demonstrated in a few studies. Tofacitinib is an oral Janus kinase (JAK) inhibitor that was approved in 2012 for moderate to severe active rheumatoid arthritis (RA), in 2017 for active psoriatic arthritis, and recently in 2018 for induction and maintenance of moderate to severe ulcerative colitis [The immune-modulator effect of tofacitinib in such a relatively new mechanism of action has raised a concern about adverse events, particularly malignancies , 3.Herein, we report a case of a patient who developed CML after receiving tofacitinib for rheumatoid arthritis.A 43-year-old female patient with Hashimoto's thyroiditis was diagnosed with seropositive RA  in April 2013 when she presented with bilateral wrist, PIP, elbow, shoulder, knee, and ankle synovitis. She was started on prednisone taper at 20 mg/day and methotrexate 10 mg once a week optimized in increments up to 25 mg/week, with calcium, vitamin D, and folic acid supplements. Despite this therapy and despite significant improvement, there was recurrent, intermittent low-grade synovitis in both wrists. In mid-2016, X-rays revealed early erosions in the left ulnar styloid and capitate as well as the bases of the left second and third metacarpal bones. It was then decided, in January 2016, to start her on targeted therapy with tofacitinib 5 mg bid. The patient had rapid favorable response, and within 3 months, she was in clinical remission. The dose of methotrexate was progressively tapered down and stopped by the end of December 2016. The patient then remained in remission. Unfortunately, a routine laboratory test on June 23, 2016, showed leukocytosis of 27,500. The hemoglobin count was 11.3, and the platelet count was 610,000. A repeat CBC 5 days later confirmed leukocytosis with a WBC of 32,300 , hemoglobin count of 10.6, and platelet count of 703,000. Tofacitinib was discontinued, and the patient was referred to hematology/oncology.Initial blood work was positive for t BCR-ABL (p210 b3a2) on FISH analysis. Bone marrow aspirate and biopsy showed myeloid and megakaryocytic hyperplasia with 2% blasts and a myeloid-to-erythroid ratio of 10 : 1. The karyotype showed t detected in all the examined cells. The flow cytometry showed that 5-6% of flow events express early myeloid markers.The patient was diagnosed with chronic myelogenous leukemia (CML), and she was started on imatinib 400 mg. Two weeks later, blood tests showed a WBC of 18,200, platelet count of 514,000 and hemoglobin count of 9.9 that later further dropped to 6.5 with significant fatigue. Therefore, imatinib was decreased to 300 mg/day. Three months later, the patient remained in clinical remission for rheumatoid arthritis with hematological remission with WBC 4,700, Hg 12.5, and platelets 291,000. Her repeat quantitative PCR for BCR-ABL was 0.86%.Various malignancies have been reported after the use of tofacitinib in previous case reports and meta-analyses . HoweverFor instance, in the rheumatoid arthritis clinical development program, the incidence rate of malignancies in patients treated with tofacitinib, excluding nonmelanoma skin cancer (NMSC), was reported to be 0.85/100 patient-year (py) (107 out of 5671 patients) in a total tofacitinib exposure of 12,644 py. These include both solid cancers  and hematologic malignancies (lymphoma and T-cell CLL) . Cohen eOn the contrary, the data on cancer development in psoriatic arthritis patients treated with tofacitinib are more unclear. For instance, two out of three phase 3 clinical trials reported malignancies as adverse events. In OPAL Broaden, 3 patients developed solid tumors (excluding NMSC) out of 107 patients 2.8%) taking continuous 5 mg bid tofacitinib for up to 12 months . In the % taking Likewise, in ulcerative colitis, three phase 3 clinical trials  evaluated the safety and efficacy of tofacitinib in induction and maintenance. No malignancies (excluding NMSC) were reported in any of these trials, but an ongoing LTE study revealed 8 cases giving an overall incidence rate of 0.48/100 py  , 10.In addition, Xie and Ma reported a case of AML 2 years after the use of tofacitinib for refractory ulcerative colitis , despiteNatural killer (NK) cells, in addition to other cytokine transduction signaling (such as interferon types I and II), have been shown to have a major inhibitory role in tumorigenesis . TofacitFrom another perspective, various studies have investigated the role of the JAK/STAT pathway in the pathophysiology of CML , 19; BCRTo our knowledge, this is the first reported case of CML that occurred 2 years after tofacitinib use at 5 mg bid in a patient with rheumatoid arthritis. It is difficult to tell whether our patient developed a de novo CML or she already had acquired the BCR-ABL translocation during her treatment for rheumatoid arthritis which was possibly “silenced” by inhibiting the JAK3 pathway with tofacitinib. Moreover, a solid cause-effect relationship between the use of tofacitinib and the development of CML cannot be established. However, by discontinuing tofacitinib and treating her with imatinib, the patient has been so far in remission for her rheumatoid arthritis and within the expected milestone outcome for her CML.Whether imatinib contributed to the maintenance of her rheumatoid arthritis in remission or not is not known. An extensive mutational workup and studying the effect of reintroduction of tofacitinib in case of relapse of either rheumatoid arthritis or CML would be interesting to perform in this case.Despite the potential benefits of tofacitinib in the treatment of CML, we illustrated a unique case of CML that developed after its use for RA. It may represent a potential side effect that may be added to the list of malignancies associated with this drug. In contrast, the response of both RA and CML to imatinib shed the light on the interrelation of pathways between RA and CML and may be the subject of many interesting studies.


Breakfast is considered to be one of the most important meals of the day. Its omission has been reported to be associated with increased disease risk, such as obesity, diabetes, and coronary heart disease, as well as unhealthy lifestyle and lower dietary quality. Using data from the National Adult Nutrition Survey (NANS)—a food consumption survey conducted among 1500 Irish men and women over 18 years of age, residing in the Republic of Ireland at the time the survey was conducted—we aimed to characterize breakfast regularity, identify dietary patterns associated with regular breakfast consumption, and assess the nutritional quality of such dietary patterns, using the nutrient-rich food index score NRF9.3. We determined two breakfast regularity categories and assessed dietary quality, by means of adherence to the principal component analysis derived dietary patterns and the NRF9.3 dietary index. Regular breakfast consumers were identified as those who consumed breakfast 3–4 times out of the 4 days of the collection period; such consumers comprised the majority of the population (94.4%). They had the highest adherence to healthier dietary patterns, namely, the “vegetarian” (odds ratio (OR): 2.59: 95% Confidence Interval (CI): 1.40, 4.77), “fish and vegetables” , and “breakfast cereals”  dietary patterns. Breakfast significantly contributed to the daily micronutrient intake by providing, on average, 24% of dietary fiber, 32% of iron, 30% of calcium, 32% of folate, and 37% of riboflavin. The importance of regular breakfast consumption on those who skip breakfast should be highlighted, in order to improve compliance with nutritional recommendations and adherence to a healthy lifestyle. Breakfast is often referred to as the most important meal of the day and its consumption has been associated with an overall healthy dietary pattern, as measured by adherence to the Mediterranean-type diet  or the HNANS , is a cross-sectional food consumption survey, conducted among adult Irish participants. Details of the study design and methodology have been previously described . Data onn = 14) were excluded and the final sample for analysis consisted of 1486 participants. Missing values on weight (n = 87) were excluded from the NRF9.3 index computation. The demographic information collected included: Sex , age (years), marital status (married/widow/single/divorced), and education level . Dietary data were collected by means of a 4-day food diary, in which participants were requested to record and weigh any food or beverage consumed over 4 days and provide information, regarding the type of food and meal consumed, its preparation method, manufacturer, and the time of ingestion. Weighed Intake Software Program WISP version 3.0  was used to analyze nutrient intake data. A total of 2552 food codes (including 233 supplements) was categorized into 19 food groups for data analysis. Lifestyle variables examined included: Smoking status (currently smoke/used to smoke/never smoked), physical activity /home activity (MET h/w)) and time spent watching television (h/w). Analysis was conducted for breakfast consumers only; therefore, those participants who skipped breakfast for all 4 days of the food dairy  were self-reported by participants. Therefore, in order to be consistent with definitions previously published in the literature, we verified that our breakfast variable was aligned with the one provided by Timlin et al. : “The fiTo determine the regularity of breakfast consumption, the number of days in which breakfast was consumed, during the 4-day collection period, were classified as follows: 4/4—breakfast consumed on 4 out of 4 days; 3/4—breakfast consumed on 3 out of 4 days; 2/4—breakfast consumed on 2 out of 4 days; and 1/4—breakfast consumed 1 out of 4 days. Regular and irregular breakfast consumers were defined as consuming breakfast 3–4 out of 4 days and 1–2 out of 4 days, respectively. The overall food-based dietary patterns followed by participants of the study were identified by principal component analysis (PCA). This method was used to identify common underlying dimensions (factors) of food consumption, by deriving factor loadings for each pre-defined food group. Factors were subsequently rotated, using a varimax procedure, to maintain uncorrelated factors. Analysis of eigenvalues, scree plot, and the interpretability of the factor solution were used to support a final decision on retaining a 7-factor solution. A factor score for each participant was calculated by summing the daily intake of each food group and weighting it by their factor loadings. Further, the factor score of each of the 7 dietary patterns was split, according to the tertiles—which were also used in the analysis—whereby the highest tertile indicated highest adherence to a specific dietary pattern. To assess how breakfast regularity related to the type of meal consumed during the remainder of the day, we used a generic meal coding system, previously applied to the NANS dataset. The methodology has been described in detail elsewhere ,16. The To assess the overall nutritional quality of the dietary patterns derived, we used the Nutrient-rich food index NRF9.3 , which ip values < 0.05 (two-sided) found were considered statistically significant. All statistical analyses were performed, using the IBM SPSS Statistics for Windows, version 24 .An ANOVA test for continuous variables and a chi-square test for categorical variables were used to compare the data between the categories of breakfast consumers. Characteristics of the study population were presented as counts, percentages for categorical variables, and mean and SD for continuous variables. Non-adjusted logistic regression models were used to assess the association between adherence to PCA-derived dietary patterns (3rd tertile vs. 1st tertile) and the regularity of breakfast consumption. Additionally, to assess the relationship between overall dietary quality, measured by the nutritional index NRF 9.3, and adherence to dietary patterns identified by PCA, the Pearson correlation coefficient was used. The Of the 1486 participants in the study, 94% consumed breakfast on at least 3 days out of the 4-day collection period and were considered regular breakfast consumers, as shown in Participants of all age groups were equally represented, with slightly fewer participants being over 50 and 64 years of age . A total of 46% of the cohort reported a higher level of education, 58% reported being married, and 52% reported to never have smoked. Nearly 40% of the participants preferred to skip a light meal and the majority of the population (93%) consumed a main meal, containing protein- and carbohydrate-based food groups. With respect to breakfast regularity, significant differences were observed according to gender, age, marital status, smoking habits, time spent watching television, and the type of light meal consumed during the day, between irregular and regular breakfast consumers. Approximately half (52%) of the regular breakfast consumers were females. Younger adults (18–35 year) were more likely to be irregular breakfast consumers, while the older population (50 years of age and older) were most likely to consume breakfast regularly. No difference was observed in the education level of regular and irregular breakfast consumers. Single participants and those who currently smoke were most likely to be irregular breakfast consumers. Though no difference was observed between regular and irregular breakfast consumers, in terms of the level of recreational and at-home physical activity, those who consumed breakfast regularly spent significantly less time watching television. Irregular breakfast consumers were more likely to skip the consumption of a light meal, while regular breakfast consumers were more likely to consume a sandwich as a light meal. The assessment of overall dietary quality showed that regular breakfast consumers, compared to irregular breakfast consumers, had a significantly higher NRF 9.3 score. p < 0.05) lower intakes of dietary fiber, iron, calcium, folate, B2, and vitamin D, as well as higher intake of sodium and fat at breakfast, as shown in Overall, it was observed that breakfast provided 19.9% of total daily energy intake, as well as 24.4% of dietary fiber, 31.6% of iron, 29.8% of calcium, 32.0% of folate, and 36.5% of B2 intakes. Compared to regular breakfast consumers, irregular breakfast consumers had significantly (p = 0.006), as shown in A total of four dietary patterns were most likely to be followed by regular breakfast consumers: the “Sandwich”, “Breakfast cereal”, “Fish and vegetables”, and “Vegetarian” dietary patterns. The “Sandwich” pattern was characterized by higher intakes of breads and rolls, butter, oils, and cheese. According to the NRF9.3, greater adherence to this dietary pattern was associated with the lowest nutritional quality. The “breakfast cereal” pattern was characterized by higher intakes of breakfast cereals, dairy, nuts, and seeds. Higher adherence to the “Vegetarian” dietary pattern was correlated with increased consumption of nuts, seeds, herbs, soups, fruits, vegetables, and nutritional supplements. The “Fish and vegetables” pattern was strongly correlated with higher intakes of fish and vegetables, as well as lower intakes of meat, showing a moderate positive linear relationship with NRF 9.3. The “Western” pattern was characterized by higher intakes of grains, pasta, beverages, meat, and eggs, and had the lowest probability to be followed by regular breakfast consumers (odds ratio (OR): 0.45: 95% CI: 0.26, 0.79; Regular breakfast consumption was associated with overall higher dietary quality, in our study. Participants who consumed breakfast regularly had the highest adherence to the healthiest dietary patterns, namely, the “Vegetarian”, “Fish and vegetables,” and “Breakfast cereals” ones. In contrast to irregular breakfast consumption, regular breakfast consumption, as observed in the NANS, contributed significantly to the total micronutrients profile of participents. It provided, on average, as much as 24% of dietary fiber, 32% of iron, 30% of calcium, 32% of folate, and 37% of riboflavin to the daily micronutrient intakes of participents.In our study, examination of the different types of dietary patterns, followed by participants who consumed breakfast on most of the days of the survey, showed that they adhered to a healthier diet, as indicated by the types of foods consumed. This also included higher intakes of dairy, breakfast cereals, nuts, seeds, fruits and vegetables, fish, and nutritional supplements, as well as lower intakes of red meat and sugar confectionery. These findings are in line with the results of Hopkins et al. , who fouA number of studies have investigated whether regular breakfast consumption is associated with a lower prevalence of obesity. For example, young adults in the US National Health and Nutrition Examination Survey (NHANES), who consumed ready-to-eat cereals for breakfast, were 31% less likely to be overweight or obese and 39% less likely to have abdominal obesity, compared to those who skipped breakfast . In a reOur study has some limitations and strengths worth considering. The participants of the NANS were predominantly white Irish, which might imply specific dietary patterns. As such, the results presented here might not be generalizable to other populations. Further research in a more heterogeneous population is needed to confirm our findings. The analyses applied in our study were cross-sectional in nature; therefore, it could be of interest for future studies to investigate whether the dietary patterns followed by irregular and regular breakfast consumers are constant over time. It is also worth considering that, currently, there is no universal definition of “breakfast consumption.” Therefore, suggestions for future direction should include the validation of our definiton of breakfast regularity and investigation into the relationship between regular and irregular breakfast intake and chronic disease progression. Our study has several strengths. The cohort included a large, nationally-representative sample of Irish men and women, as well as a thorough examination of diet, which were both unique features of the NANS. Very few studies have collected data capturing dietary intake, during specific eating occasions. This feature allowed us to categorize the regularity of breakfast intakes and to study the relationship, not only of food-based dietary patterns, but, more importantly, of the meals consumed throughout the day. In conclusion, our study suggests that regular breakfast intake is associated with overall higher dietary quality, lower prevalence of smoking, and decreased television watching time. Therefore, our study could serve as an indicator of healthy dietary and lifestyle behavior. Based on the findings obtained, it could be suggested that health professionals highlight the importance of regular breakfast consumption to those who skip breakfast. This simple message could improve compliance with nutritional recommendations and adherence to a healthy lifestyle.


Timely and effective dissemination of the latest clinical evidence to health care providers is essential for translating biomedical research into routine patient care. Online platforms offer unique opportunities for dissemination of medical knowledge.In this study, we report the profiles of health care providers participating in the New York State HIV-HCV-STD Clinical Education Initiative online program and their evaluations of the online continuing professional development courses.We compiled professional and personal background information of the clinicians who completed at least one online course. We collected their self-reported program evaluation data with regard to the course content, format, knowledge increase, and impact on clinical practice.usefulness/relevance, 91.08%; easy comprehension, 89.09%; knowledgeable trainer, 92.00%; appropriate format, 84.35%; knowledge increase, 48.52%; intention to use knowledge, 85.26%; and plan to change practice, 21.98%). Comparison with the reference data indicated that the online program successfully reached out to the primary care communities. Both the younger generation and the senior health care providers were attracted to the online program. High-quality multimedia resources, flexibility of access, ease of use, and provision of continuing professional development credits contributed to the initial success of this online clinical education program.We recorded a total of 4363 completions of 88 online courses by 1976 unique clinicians during a 12-month study period. The clinicians’ background was diverse in terms of demographics, education levels, professional disciplines, practice years, employment settings, caseloads, and clinical services. The evaluation of online courses was very positive (We have successfully characterized a diverse group of clinicians participating in a statewide online continuing professional development program. The evaluation has shown effective use of online resources to disseminate clinical evidence on HIV, hepatitis C virus, and sexually transmitted disease to primary care clinicians. Timely and effective dissemination of the latest clinical evidence to health care providers is essential for translating biomedical research into routine patient care . DissemiSince 2008, the New York State Clinical Education Initiative (CEI) program has developed hundreds of multimedia learning modules, online continuing medical education (CME) and continuing nursing education (CNE) courses, interactive case simulation tools, and other online resources -10. ThesPrevious research found that the traditional methods for dissemination of clinical evidence to health care providers were mostly ineffective . LimitatEarly initiatives to use information technology for dissemination research generated mixed results ,16. WithThe HIV pandemic has been a serious threat to global public health for decades. According to the Joint United Nations Programme on HIV/AIDS, there were 1.8 million new diagnoses, 1 million deaths, and 36.7 million people living with HIV worldwide in 2016 . In the In recent years, HCV also has increasingly become a major concern of public health. CDC estimated 33,900 acute HCV cases in 2015 and 3.5 million people with chronic HCV in the United States . HIV andHIV research has advanced very rapidly since the 1990s, with many completed and ongoing clinical trials on treatment, prevention, and behavioral intervention . More thThe CEI program  is sponsTo leverage the latest development in information technologies and to explore the opportunities offered by the widely used digital platforms, we initiated the CEI online program in 2008. We collaborated with domain experts across the nation and developed a large repository of online resources, including hundreds of multimedia learning modules, online CME/CNE courses (the focus of this study), and guideline-driven interactive case simulation tools -10,34. Wstrongly agree, agree, neutral, disagree, and strongly disagree), and the responses were further grouped into positive (strongly agree and agree) and nonpositive  responses for data analyses. For item (5), we used five discrete levels to indicate a clinician’s knowledge on a specific training topic  before and after the training, and then calculated the difference (at least one level increase vs no increase) for data analyses. In addition to these structured questions for course evaluation, clinicians can provide free-text comments on their experiences of the CEI online training program. Once the evaluation is completed, the CME/CNE credits associated with an online course are immediately awarded. At any time, the clinician student can review and print the CME/CNE certificate. Partial screenshots of the CEI Student Portal to collect course evaluation and student profile data are shown in To compile the clinicians’ profiles and their evaluations of the CEI online CME/CNE courses, we queried the CEI Student Portal and obtained data of: (1) all clinicians who successfully completed at least one online CME/CNE course from November 2014 through October 2015; and (2) their evaluations of all completed online CME/CNE courses during this period. The CEI Student Portal is a centralized system to manage clinician students, online courses, and program evaluations. A clinician user can sign up for the student portal at any time. As part of the registration, clinicians need to provide their personal  and professional  background information. When taking an online CME/CNE course, a clinician must complete watching the multimedia materials and pass a short exam to test his/her knowledge related to the topics covered by the course. After completion of each online CME/CNE course, the clinician student must provide an evaluation with self-reported data on: (1) usefulness/relevance of the information presented in the course; (2) ease of comprehension; (3) knowledge of the trainer; (4) appropriateness of the online training format; (5) knowledge level on the course topic before and after the training; (6) intention to utilize the learned knowledge; and (7) plan to change practice after the training. Here, for items (1), (2), (3), and (6), we used a five-point Likert-scale measure  were female, 1103 (55.82%) were white, 492 (24.90%) were African American, 128 (6.48%) were Asian, and 436 (22.06%) were Hispanic. In terms of the clinicians’ education level, 308 (15.59%) held a doctoral degree and 486 (24.60%) held a master’s degree. The details of the clinicians’ demographics and education levels are shown in The top categories of the recorded primary professional discipline were physician , nurse , case/care manager , nurse practitioner , and social worker . Regarding the practice years in the primary professional discipline, 124 (6.28%) indicated more than 30 years of experience, 224 (11.34%) between 21 and 30 years, 402 (20.34%) between 11 and 20 years, and 1182 (59.82%) 10 years or less. For clinicians’ employment setting, the top categories recorded were community health center , hospital/hospital clinic , state/local health department/clinic , private/group practice , managed care organization , and professional organization . Among these, 1257 (63.61%) were in urban settings, 439 (22.22%) were in the suburbs, and 280 (14.17%) were in rural areas. Details of the clinicians’ primary professional disciplines, years in practice, and employment settings are provided in For the caseload, 171 (8.65%) clinicians reported seeing more than 100 HIV patients per month, 82 (4.15%) reported seeing more than 100 HCV patients per month, and 108 (5.47%) reported seeing more than 100 STD patients per month. The caseload category of 1-10 patients per month recorded the largest number of clinicians . The top clinical services provided for HIV patients were risk reduction intervention , case management , and HIV screening/testing . The top services for HCV patients were risk reduction intervention , HCV screening/testing , and HCV treatment . The most popular services for STD patients were risk reduction intervention , STD screening/testing , and STD treatment . The details of the clinicians’ caseloads and clinical services are provided in Regarding the number of online CME/CNE courses taken by the health care providers, 1454 (73.58%) clinicians completed only one course, 182 (9.21%) completed two, and the remaining 340 (17.21%) clinicians completed three or more. Excluding a CEI staff who worked as a system tester, the largest recorded number of online CME/CNE courses completed by a single clinician was 47, and the average number of courses taken was 2.17.HIV/AIDS Confidentiality Law Overview, with 1379 completions; (2) Street Drugs & HIV, with 195 completions; and (3) STD-HIV Inter-Relationship, with 158 completions. A complete list of the top 20 most popular online CME/CNE courses is shown in As a measurement of course popularity, we recorded a list of courses with the most numbers of completions. The top three on the list were: (1) In terms of course evaluation, the overall responses from the clinicians were very positive. With the 4363 completed courses, 3974 responses (91.08%) indicated that the information presented was useful and relevant, 3887 (89.09%) indicated that the course was easy to comprehend, 4014 (92.00%) indicated that the trainer was knowledgeable, and 3680 (84.35%) indicated that the online training format was appropriate. With regard to the impact of the online training, 2117 of the 4363 responses (48.52%) indicated at least one level increase in knowledge, 3720 (85.26%) indicated intention to use the learned knowledge, and 959 (21.98%) indicated a plan to change clinical practice. Excluding those who did not provide direct patient services at the time, 39.43% (959/2432) of those who responded planned to change their clinical practice after the training. A summary of the course evaluation data is shown in A total of 288 comments from the clinicians were collected during the study period. Review and analysis of these comments excluded 156 invalid or meaningless responses. For the remaining 132 comments, we further categorized them into five themes: information/content, format, trainer, technical issues, and general positive feedback, with 4 belonging to two separate themes. Among these, 39 indicated that the clinicians learned a lot from the presentation and found it very informative. For example, a comment under this theme was “the information is useful for updating agency policy,” indicating that the clinician planned to utilize the learned information in their facility. The 53 general positive feedbacks such as “I like it” and “excellent program” only expressed clinicians’ overall positive experiences but without details. Additional positive comments were recorded on knowledgeable trainer (n=3) and appropriate online format (n=1). We recorded 25 comments about technical issues encountered by the clinicians, for example, “video was cut off at 40 minutes’ mid-sentence.” Other negative comments included challenging materials or other information issues (n=4), problem with the trainer (n=5), and request for improvement of the format (n=6).In this study, for the first time, we reported a detailed profile of health care professionals participating in a statewide online HIV-HCV-STD knowledge dissemination program. Although there were limited reference data, we had a few interesting findings. First, compared to the data of general health care workforce in New York State , the proTo relieve the potential concern on privacy, we did not collect CEI participant’s date of birth. Instead, we used years of practice as an alternative measure, which we believed could roughly estimate a health care provider’s age in most cases. The data showed that a majority (60%) of the CEI audience had 10 years or less of experience in practice. This result was consistent with previous finding that an online program tended to attract younger clinicians . In addiSimilar to a previous pilot study , we recoWith regard to caseload, our data indicated a wide range of distribution, from 0 to 100+ HIV, HCV, and STD patients per month. The largest category of the participating clinicians had 1-10 patients per month, a typical scenario in the primary care setting. It is interesting to note that 4%-9% of the clinicians had 100+ patients per month, indicating that the CEI online program also attracted HIV, HCV, and STD specialists, as suggested by anecdotal evidence. In addition, 39%-54% of the clinicians had no patients at the time—they likely were those: (1) in the HIV, HCV, and STD clinical care and public health teams but with no direct interactions with patients; or (2) new practitioners preparing to engage in HIV, HCV, and STD patient care.According to the Accreditation Council for Continuing Medical Education 2015 Annual Report, there were close to 37,000 enduring internet material activities that provided more than 74,000 hours of instruction to 4.8 million physicians and over 6.8 million other learners . NeverthThe data from a recent national survey called for a central repository for listing educational opportunities and tracking continuing education credits . NonetheThis study has a few limitations. First, we focused only on a specific category of online resource, multimedia CME/CNE course, while many other types of resources such as case simulation, InfoButton, and clinical decision support could also be leveraged for dissemination of clinical evidence ,43,45. AIn conclusion, we have successfully characterized the profiles of clinicians participating in the New York State CEI online CME/CNE program for dissemination of the latest HIV, HCV, and STD clinical evidence. We have identified a diverse group of health care providers in terms of the demographics, education levels, professional disciplines, practice years, employment settings, caseloads, and clinical services. The participating clinicians’ evaluation of the CEI online CME/CNE courses was very positive with regard to the content, format, and knowledge increase. A significant portion of the participating clinicians planned to adopt the learned knowledge and skills in their practice. These initial set of evaluation data have demonstrated the effectiveness of using online resources for dissemination of HIV, HCV, and STD clinical evidence to primary care clinicians. Future research is required: (1) to assess the effectiveness of knowledge dissemination with multiple types of online resources and using direct measures on health care processes and patient outcomes; (2) to examine the comparative effectiveness with control groups; and (3) to analyze the co-variates through development of a predictive model for effective online training.