There are few data available about childhood tuberculosis (TB) in rural hospitals in low-income countries. We assessed differences in epidemiological characteristics and treatment outcomes in children with TB aged 0–4 versus 5–14 years in rural Ethiopia.For this retrospective cross-sectional study, we analyzed childhood TB registers from a rural Ethiopian hospital. We collected data on the number of cases, type of TB, and treatment outcomes using standard definitions. By means of binary and logistic regression analyses, data were compared from 1998 to 2015 in children aged under 5 versus those aged 5–14 years.n = 862) had pulmonary TB (PTB), which was more common in younger children  than in older ones . Most cases of PTB  were smear negative, including virtually all  younger children and most older ones . The most common types of extrapulmonary TB (EPTB) were TB adenitis  and bone TB . Children under five showed a lower prevalence of adenitis TB , bone TB , and abdominal TB . Most diagnoses were new cases of TB . Overall, 63.5% (n = 814) of the children successfully completed treatment . In total, 16.3% (n = 209) transferred to another center . Thirteen percent of patients (n = 167) were lost to follow-up . Fifty-two (4.1%) children died (no age differences). Being aged 5–14 years was independently associated with successful treatment outcomes .We included 1282 TB patients: 583 (45.5%) were under 5 years old, and 699 (54.5%) were aged 5–14 years. More than half  contains supplementary material, which is available to authorized users. Tuberculosis (TB) remains an important challenge for global health. The World Health Organization (WHO) estimated that in 2017 there were about 10 million incident cases of TB worldwide, and 10% of these were in children under 15 years old . The burManagement of childhood TB poses challenges to low- and middle-income countries. Morbidity and mortality are high enough to make this one of the three primary infectious diseases related to poverty –6. In 20A 2018 WHO report ranked Ethiopia 14th worldwide with regard to the global TB burden; 117,705 cases were reported in 2017, with an estimated incidence rate of 164 cases per 100,000 population per year. Of the reported cases in 2017, about 20,000 were in children under 15 years [Mycobacterium tuberculosis. Disease manifestation patterns show clear associations with age at the time of primary infection. Immune-compromised children also carry an increased risk for TB [Given the natural history of TB, age remains the most important variable influencing the risk of disease progression following a primary infection with k for TB , so it sk for TB .Although several studies have been published about childhood TB in Ethiopia, few have taken place in a rural environment. Moreover, they do not generally compare age groups in terms of epidemiology, disease manifestations, diagnosis or outcome –12, evenEthiopia’s National Tuberculosis and Leprosy Control Programme (NTLCP), launched in 1992, helped standardize the registry of TB cases following WHO guidelines , 14. ThiThis is a retrospective cross-sectional study from January 1998 to December 2015. We examined patient records in children aged less than 15 years old and diagnosed with TB at Gambo General Hospital, collecting data in line with the NTLCP reporting guidelines and WHO standards , 14.Gambo General Hospital is a private rural general hospital with 135 beds. Since 2003, the hospital has had an inpatient feeding center with around 15–20 beds for treating severe acute malnutrition. It is 18 km from Arsi Negele, the capital of West Arsi Province; 45 km from Kuyera Hospital ; and 250 km south of Addis Ababa. West Arsi Province has a population of 1.9 million, and Gambo General Hospital serves a population of about 95,000 people, living mostly in a rural setting and subsisting from agriculture and livestock.In a study performed from July 2013 to June 2014 in the Hetosa District of Oromia Region, Ethiopia, the incidence of smear-positive pulmonary TB (PTB) was 214 / 100,000 persons per year . A recenPatients with a clinical suspicion of TB underwent a diagnostic study before starting treatment. If symptoms were consistent with PTB, patients provided three sputum samples, usually from spontaneous sputum, although gastric aspirates were taken in a few of the younger children. Two positive smears indicated smear-positive PTB. Patients with three negative smears received treatment with antibiotics, and if the symptoms continued, they were examined by chest X-ray. The following abnormalities on chest X-ray were suggestive of TB :(1) enlarged hilar lymph nodes and opacification in the lung tissue,(2) miliary mottling in lung tissue, and (3) cavitation (common with older children) or pleural or pericardial effusion. In case of respiratory symptoms, patients with suggestive chest X-rays whose condition did not improve with antibiotics were considered to have smear-negative PTB. Miliary tuberculosis was classified as pulmonary TB because of the lesions in the lungs , 14.Extrapulmonary TB (EPTB) was generally diagnosed clinically. The investigations used to diagnose the common forms of EPTB were: (1) for peripheral lymph nodes, lymph node fine needle aspiration; (2) for TB meningitis, lumbar puncture; (3) for pleural effusion, chest X-ray plus pleural tap for biochemical analysis, cell count and culture; (4) for abdominal TB, abdominal ultrasound and ascites fluid analysis; (5) for osteoarticular TB, X-ray or joint tap, (6) for pericardial TB, ultrasound and pericardial tap. Tuberculous pleural effusion, without radiographic abnormalities in the lungs, was considered EPTB , 14. ChiPatients with both PTB and EPTB were classified as having PTB; these cases were not included in both categories. The only exception was TB meningitis: if patients diagnosed with TB meningitis by lumbar puncture had an abnormal chest X-ray, they were considered as having TB meningitis. Bacteriological confirmation by culture was not undertaken. The Xpert MTB/RIF assay had not yet been implemented during the study period.A TB diagnosis resulted in three potential management approaches: 1) hospital admission (seriously ill PTB or EPTB patients), 2) registration and ambulatory treatment at the TB clinic, or 3) transfer to the patient’s local TB clinic.Clinicians completed case notification forms for all registered patients according to NTLCP reporting guidelines , 14, colThe treatment scheme changed during the period of study, as shown in Additional file P values of less than 0.05 were considered statistically significant.All collected data were double-checked for completeness, entered into Excel, and exported to SPSS version 22.0 for analysis. We report variables of interest as frequencies or means. We performed the chi-square test and Fisher’s exact test to compare variables in univariate analysis, expressing results as crude odds ratios (ORc) with their 95% CIs. We then fit a logistic regression model to identify predictors of treatment outcome based on the adjusted odds ratio (ORa) and 95% CI. As the study was retrospective in nature, informed consent was not required from patients’ parents or legal guardians; however, all records were anonymized prior to analysis. The approved Local Research and Publication Committee of the Gambo General Hospital and the Health Unit and Ethical Review Committee of the Ethiopian Catholic Secretary approved this waiver as well as the protocol and the study (permit number: GH/MSMHF/710).From 1998 to 2015, 3534 patients were diagnosed with TB, including 1282 (36.3%) children: 583 of them (45.5%) were aged 0 to 4, and 699 (54.5%) were 5 to 14 years old. Figure p < 0.001). Most (87.5%) cases of PTB were smear negative; this result was more common in children under five (99.6%) than in older ones  (Table Diagnostic results showed that PTB was the main type of TB (67.2%), and it was more common in younger children (82.5%) than in older ones (54.5%) p < 0.00. Most (8p < 0.001), bone TB  and abdominal TB . TB meningitis was slightly more common in the younger age group (2.6% versus 1.6%).TB adenitis was the most common type of EPTB (54.5%), followed by bone TB (20%), abdominal TB (11.7%) and TB meningitis (6.2%). Children under five showed a lower prevalence of adenitis TB . Indeed, no TB cases in the under-five group showed failure or previous treatment, compared to eight and five cases, respectively, in older children .Most patients were registered with new cases of TB, especially younger children . Only 2.3% of the patients tested had a positive result. Determination of HIV status increased dramatically following implementation of the NTLCP in 2005 .HIV status was tested in 31.2% of the children   were admitted, with a predominance of young children among those hospitalized (p < 0.001); 209 (16.3%) transferred to another center ; 167 (13%) were lost to follow-up ; and 52 (4.1%) died  had successful treatment .Table More than a third (36.3%) of the total recorded cases of TB in our study population occurred in children—a higher proportion than reported elsewhere in Ethiopia , 11, 17,Given the scant data available on childhood TB (and the dominance of high-income settings where studies exist), in 2007 WHO called for additional research to characterize the global epidemiology of the disease [Nearly half (45.5%) of the total childhood TB cases were in children under five, which is consistent with results from a previous study in Tanzania  but highChildhood TB is mostly due to recent transmission, so its burden provides a good proxy measure for TB prevalence in the community . TherefoThere has been a decrease in prevalence of tuberculosis in Ethiopia over the past 15 years [Most cases 98.2%) were new TB rather than previously treated TB. This proportion was slightly higher than in other studies of childhood TB (92%) [.2% were PTB was the most common type of TB in our children, which is consistent with the literature . In our M. tuberculosis in a greater percentage of cases in this group [Using other sample types, such as gastric aspirate, results in a higher diagnostic yield than spontaneous sputum in children. The Xpert MTB/RIF assay in sputum and gastric aspirates may identify is group –27. Thusis group , 27.EPTB made up a third of the childhood TB cases, which is a lower proportion than in the studies by Hailu et al.  and TilaOn the other hand, there was a slightly higher proportion of TB meningitis in children under five ; it is well known that this type of TB is more frequent in children under three .Abdominal TB appeared less frequently in younger children (4.9%) compared to older ones (13.8%). Although this variant is rare in children, most reported cases are in low- and middle-income countries with a considerable TB burden. Consistent with our results, most cases of abdominal TB are in patients aged over five , 29. DiaOverall TB/HIV co-infection in our series showed a prevalence of 2.3%, much lower than elsewhere in Ethiopia, where it ranges from 10 to 28.2% , 30–34; In our study, 39.8% of the children were admitted to hospital. Some patients were hospitalized for reasons other than TB, such as severe acute malnutrition, and were only then diagnosed with TB. Hospitalization was more frequent in the under-five group.TB programs in sub-Saharan Africa rarely assess treatment outcomes in children . The WHOIndeed, the transfer rate was relatively high in our series , 9, 11 bIn our series, the proportion of patients who successfully completed treatment was lower in children under 5 than in those aged 5–14 (56.6% versus 69.2%), but the transfer rate was higher (19.4% versus 13.7%). The rate of treatment loss to follow-up was 13%—higher than in other studies in Ethiopia with more urban and periurban settings , 9 and iWith regard to the mortality rate, the 4.1% we observed is within the range reported in other studies of childhood TB in Ethiopia , 11, 33.Young children, particularly those under two years of age, are at higher risk of dying from TB because their immune system is still immature , 27, 42.In terms of treatment outcome predictors, age group was the only variable that was independently associated with treatment success. This outcome was significantly higher in 5–14-year-olds compared to the under-5 group. Other studies have reported similar findings , 18, 43.In contrast, other series have reported significant associations between treatment failure and HIV co-infection, unknown serostatus and malnutrition , 39, 44.The findings of this study must be interpreted in light of its limitations. First, it is common to find both PTB and EPTB in the same pediatric patient, but in this study the variable was dichotomized (EPTB only in the absence of PTB), following WHO definitions. In children, this classification can obscure very important information about serious cases of TB, for instance if the child is diagnosed as having PTB but also has TB meningitis, which could be much more life threatening. Secondly, we were unable to collect sociological data and only limited data about HIV infection, which might have affected the risk of a poor outcome. Finally, Gambo General Hospital is a private hospital, and patients were often referred from other health facilities for diagnosis and admission but then transferred to their local health center following the intensive treatment phase. Thus, we might have underestimated treatment failure and mortality rates. Additional limitations are the retrospective and hospital-based nature of the study, which could cause bias. Moreover, there also seem to be recent changes in health system in the area, which could depress estimates of prevalence.Data collection on childhood TB in rural health facilities is important for understanding the epidemiology of this disease. Because spontaneous sputum smear has a very low diagnostic yield in children in low- and middle-income countries (and particularly in children under five), alternative diagnostic methods, such as Xpert MTB/RIF in gastric aspirates, become more important. In this rural setting in Ethiopia, children under five usually present with new smear-negative PTB. They have less EPTB than children aged 5 to 14 but more TB meningitis. Younger children are admitted more frequently and show lower rates of successful treatment.Additional file 1:Annex 1. Treatment regimen according to type of tuberculosis following the Tuberculosis and Leprosy Prevention and Control Programme in Ethiopia. (DOCX 88 kb)


Vibrio spp. is the major infection-producing marine bacteria in commercially important bivalve Paphia undulata. The host resistance is the major determining factor for the development of pathogenesis. To explore defense mechanisms, researchers have focused primarily on the study of differential expression of individual or specific groups of host immune genes during pathogen-challenge.V. alginolyticus and virulent V. parahaemolyticus to mark the possible molecular mechanisms of pathogenesis. Comparison of the differentially expressed genes between the two groups of Vibrio-infected clams revealed that the number of down-regulate genes in V. parahaemolyticus injected clams (1433) were significantly higher than the other group (169). Based on Gene Ontology classification, a large proportion of these down-regulate genes were found to be associated with cellular and molecular mechanisms for pathogen recognition, and immunity development thereby explaining the low survival rate for the V. parahaemolyticus-treated clams and suggesting a higher virulence of this bacterium towards the surf clams. Quantitative real-time PCR of 24 candidate genes related to immunity involving the JAK-STAT signaling pathway, complementary cascade, cytokine signaling pathway, oxidative stress, phagocytosis and apoptosis down regulated under V. parahaemolyticus infection, indicating compromised host defense. Furthermore, we could demonstrate a central role of JAK-STAT pathway in bacterial clearance. dsRNA mediated depletion of a clam STAT homolog gene results in dramatic increase in the infection by V. alginolyticus, a mildly pathogenic strain under control conditions.We compared the expression profile in the surf clams infected with avirulent Vibrio species with a differential pathogenicity to P. undulate and downstream molecular analysis could enlighten on the probable molecular mechanisms of the Vibrio pathogenesis and the virulence of V. parahaemolyticus in surf clams, which also benefits to develop new strategies for disease control in surf calm aquaculture.The difference in gene expression profiles in surf clams treated with two  Vibrio and the protozoa Perkinsus have been identified as the major disease-producing pathogens affecting the development and survival of clams and diminishing the meat quality and thereby the price of the products [Bivalves are one of the earliest yet ubiquitous group of aquatic invertebrates with an estimated 10,000–20,000 living species. They are both economically and ecologically important with respect to food source, biomass and effects on communities. However, there is a steady progressive decline in the production of the bivalves following mass mortality among the farmed species due to marine microbial infections [products .The host resistance is the major determining factor for the development of pathogenesis. The defense mechanism in mollusks mainly relies on the effectors of innate immunity, which is mediated by circulating competent cells- referred to as hemocytes, and highly diversified humoral antimicrobial factors. Both these cellular and humoral components work in a synergistic way to initiate the recognition, segregation and ultimately elimination of pathogens and other non-self entities , 5. The To explore defense mechanisms, researchers hitherto have focused primarily on the study of differential expression of individual or specific groups of host immune genes during pathogen-challenge. Recent application of high-throughput next generation sequencing technologies involving direct sequencing of transcripts (RNA-seq) are providing extensive information about host-microbe interactions at the transcriptional level including global gene expression and novel gene discovery –12. The Vibrio-infections remains largely unexplored. There are only two previous studies on the expression analyses of defense-related genes in surf clams (Mesodesma donacium) during Vibrio spp. (V. anguillarum)-challenge [Vibrio-infection in surf calms, we utilized Illumina RNA-seq to score gene expression changes in P. undulate infected with two Vibrio pathogens- V. parahaemolyticus and V. alginolyticus. Of these two strains, V. parahaemolyticus was found to be more virulent than V. alginolyticus, as evidenced by the survival rate of P. undulate post pathogenic injection. Thus the comparison of the transcriptome of P. undulate infected with these two Vibrio strains could help us identify specific immune genes contributing to host resistance and molecular mechanism underlying the pathogenesis of marine molusks.The surf clams are the bivalves supporting the largest proportion of the shellfishery market in China. In spite of it economic importance, the underlying molecular mechanism of surf clam defense towards hallenge , 17. In Vibrio species, V. parahaemolyticus and V. alginolyticus towards surf clam Paphia undulate, the survival rate of the infected clams were measures at 24 h, 36 h, 48 h, 60 h and 72 h post-injection. A clear difference in the survivality was observed between clams infected with V. parahaemolyticus (VP) and the ones infected with V. alginolyticus (VA) in comparison to the controls (C)  Fig. . The surVibrio mediated infection of surf clam P. undulate, high-throughput RNA-seq based transcriptomic analysis was performed. cDNA libraries were prepared for the V. parahaemolyticus and V. alginolyticus infected clams  and were sequenced using Illumina platform. All three libraries were assembled into annotated 74,433 sequences, which were used for references sequence for quantification analysis. The total mapped reads were 14,651,562, 13,544,017 and 14,529,523 for VP, VA and C groups respectively  analysis showed that the DEGs were clustered into distinct groups . The values were mostly clustered between 0.5 and 1 while very few remain scattered between the ranges 2 to 4 for both the dataset. Therefore, qRT-PCR data supported the sequencing results and provided data about the suitability of using the Illumina sequencing approach for de novo assembly of the surf clam hemocytes transcriptome without a reference genome.Repression of large number of immune genes in is Table . Strikinis Table . Additiois Table . The patVibrio infection in surf clams, we utilized JAK-STAT pathway inhibitors- methotrexate and ruxolitinib. We investigated infection induced apoptosis in JAK/STAT pathway inhibitor treated surf clams. Suppression of JAK/STAT pathway induced apoptosis in VA infected clams, and the observed apoptotic index was significantly higher than DMSO treated controls , inhibitor of apoptosis (IAP) and tumor necrosis factor (TNF) in unigene0039277 depleted clams. GSTs are proteins which help in cellular detoxification by conjugating the toxins with glutathione (GSH), increase their solubility and thereby aiding to the easy removal of the toxins from the cells , 21. On Vibrio infection in P. undulate, and presumable the virulent strain V. parahaemolyticus establishes infection by suppressing this gene by utilizing a yet unknown mechanism. The suppression of this gene is presumably a key step in the establishment of the virulent strain V. parahaemolyticus.Next, we investigated the effect of unigene0039277 on the survival of surf clams under VA and VP infection. dsGFP injected clams were used as controls and uninfected dsGFP clam showed a near 100% survival rate at about 70 h post infection - signal transducers and activators of transcription (STAT) pathway is a pleiotropic signal transduction cascade that transduce a multitude of signals critical for cellular homeostatis, development and immunity [In our study, 24 candidate genes were selected for qRT-PCR analyses based on their implication in the host-immune response towards immunity . Mechaniimmunity . The sigimmunity . As a reimmunity , 30–32. immunity . In addiDrosophila, mice and humans [Drosophila melanogaster [Beauveria bassiana in insects [The JAK/STAT pathway is one of the major signaling cascades required for providing resistance against pathogenic attack, both in vertebrates and invertebrates. Although the pathway was initially discovered while researching antiviral mechanisms of interferons, it was later shown to play significant role against other pathogens as well . Anti-vid humans , 33. Losnogaster , 35. Parnogaster , 37. JAKnogaster . There anogaster ; however insects , 39. Add insects . Encapsu insects . Thus, iIn comparison to the wealth of information available for JAK-STAT pathway and its biological function in vertebrates and insects, relatively few studies have investigated the same in ecologically and economically important marine organisms. Although there are reports about the involvement of JAK-STAT pathway in anti-pathogen defense in various aquaculture organisms including shrimps and crabs , 42. In P. undulate. Knockdown of a particular putative STAT gene increased the virulence of V. alginolyticus, a bacterium generally perceived to be mildly pathogenic to P. undulate. Thus, we assume that VP, which is virulent to P. undulate, functions by suppressing the STAT homolog (unigene0039277). Interestingly, STAT3-dependent signal transduction has also been reported to be involved in immune response in whiteleg shrimp, Litopenaeus vannamei during Vibrio spp. infection. Increased mortality in Vibrio-infected Litopenaeus vannamei was reported due to inhibition of JAK/STAT pathway by cell death regulatory genes indicating that the latter was necessary for anti-bacterial defense [Vibrio infection in surf clam remains to be elucidated. Interestingly, knockdown of the STAT homolog, unigene0039277, in clams resulted in repression of several other key immune genes like GST, IAP and TNF inspiring the idea that surf clam STAT indeed might be a key gene that regulate other immune genes in a systematic fashion. A thorough analysis of unigene0039277 knockdown clam is required to address these questions in the future.In this study, JAK/SAT pathway plays a key role in bacterial clearance as evidenced by increased infection under inhibitor treatment in surf clams  defense . HoweverV. alginolyticus. The expression of some of these genes remained low at a level almost comparable to the controls, in case of V. parahaemolyticus infection.The ability of a host organism to overcome a pathogenic attack depends on how successfully it can mount a defense in response to pathogenic attack. On the other hand, a pathogenic establishment requires overcoming or bypassing this host defense . PathogeVibrio strain. The variation of expression of a large number of genes detected by RNA sequencing in our study provides a rich resource for studying the novel genes involved during Vibrio-challenge in surf clams. Comparative genomic analysis of P. undulate infected with two different Vibrio strains helped us demostrate key role of JAK/STAT pathway in clam defense and pathogen clearance. A specific putative STAT gene (unigene0039277) was found to provide enhanched resistance against Vibrio attack, and the suppression of this gene is presumed to be an important step in the establishment of infection by the virulent strain V. parahaemolyticus. However, the exact mechanism of repression of unigene0039277 or other immune genes remains unclear. Our findings might lead to the identification of therapeutic targets for infection minimization further helping us maintain ecological and economic stability of the surf clams.To the best of our knowledge, this is the first study on the investigation of transcriptome profile of the surf clams during the challenge by virulent Tetraselmis suecica and Isochrysis galbana.Surf clams used in this study were obtained from the local Fish Market of Xiamen, Fujian Province, China, and maintained at 18 °C - 22 °C in tanks with recirculating seawater for a 2 week prior to bacterial infection. Prior to the experiments, the clams were acclimatized to the aquarium conditions for 2 week. The clams were fed twice daily with the marine algae, V. alginolyticus (1 × 107 CFU/ml) or V. parahaemolyticus (1 × 107 CFU/ml) . Clams injected with 100 μl of phosphate buffered saline (PBS) were taken as controls [The clams were injected in the muscles with 100 μl of either controls . Then thVibrio infected groups of clams 24 h post pathogenic injection, using a 0.5 mm diameter (25G) disposable needle. In total, 300 clams representing five biological replicates of bacterial challenge  were used for the expression analysis and stored at − 80 °C for RNA isolation. For RNA isolation, hemolymph was pooled from 20 individuals from each group. The experiment was repeated in triplicates. Briefly, the hemolymph was centrifuged at 3000 g for 10 min at 4 °C. The pellet was suspended in 250 μl of TRIzol (Invitrogen). Total RNA was isolated from each sample following the manufacturer’s protocols; followed by RNA purification with the RNeasy Mini kit (Qiagen) after treating with RNAase-free DNase I (NEB). The concentration and purity of isolated RNA was measured at 260 nm/280 nm (A260/A280) using spectrophotometer (Thermo Scientific). The RNA integrity was examined on Agilent 2100 Bioanalyser RNA Nano Chip  and by running on agarose gel electrophoresis.Hemolymph was withdrawn from the adductor muscle of the control and the two V. parahaemolyticus-infected groups of clams and to compare it with the control and V. alginolyticus-infected group, quantitative gene expression analysis for all the three groups of clams was performed using the Illumina RNA-seq technology on HiSeq 2000 platform [To characterize the immune response among platform , 48.The extracted mRNA was converted to cDNA using SuperScript II reverse transcriptase (Invitrogen). cDNA was ligated to Illumina TruSeq RNA multiplex adaptor sequences using the TruSeq RNA sample prep kit (Illumina). Size-selected cDNA fragments of 200 bp (±25 bp) excised from a 2% agarose gel were amplified using Illumina PCR primers for paired-end reads (Illumina), and 15 cycles of the PCR programme comprising 98 °C for 30s, 98 °C for 10s, 65 °C for 30s and 72 °C for 30s, followed by an extension step of 5 min at 72 °C. After quantified by TBS380, two RNAseq libraries were sequenced in single lane on an Illumina Hiseq Xten sequencer  for 2 × 150 bp paired-end reads.http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). Initial removal of low-quality reads and multiplex index adaptor sequences (Illumina) was performed with edgeR [Quality of reads was visualized with FastQC  were assembled and mapped to the reference transcriptome of surf clam using edgeR; allowing only 1 or 2 bp mismatch [http://www.ncbi.nlm.nih.gov/sra/) under the accession numbers PRJNA560440.The clean reads with quality scores greater than 20/30 (Qmismatch . For funhttp://www.geneontology.org) using Amigo (http://amigo.geneontology.org/) software. GO based functional analysis gave us a broad overview of the ontology content with respect to biological processes, cellular components and molecular functions [For GO analysis, the DEGs obtained from Illumina platform were mapped to terms in GO database version GO slim . The qRT-PCR reactions were performed in triplicate on a Light Cycler 480 platform (Roche) in a volume of 20 μl using 1 μL of diluted cDNA (1:10), 0.4 μL of each of the forward and reverse primers (10 mM) and 10 mL of 2× Master Mix  with the cycling conditions as follows: 95 °C for 5 min, followed by 40 cycles of 94 °C for 5 s, 60 °C for 31 s . Beta ac7 CFU/ml of either V. alginolyticus or V. parahaemolyticus. Clams treated with dimethyl sulfoxide (DMSO) were used as control. Then the clams were returned to the tanks and maintained at 25 °C until sampling. The hemolymph was withdrawn from their adductor muscles after 24 h and plated onto TCBS media and incubated for 12 h at 37 °C and were observed for the growth of Vibrio spp. The positive colonies formed on the plates were counted and compared with control.The clams were injected with 5 mg/kg and 20 mg/kg of methotrexate (Sigma) and ruxolitinib (Sigma) respectively in their adductor muscles; followed by infection with 100 μl of 1 × 105 hemocytes were collected for apoptotic analysis, and total of 10,000 cells were obtained to detect in the flow cytometry. Finally, the result was conducted by FlowJo software.For apoptotic index, the percentage of apoptotic hemocytes were analyzed in a flow cytometry  using fluorescence intensity based (photometric) assay targeting the hallmark of apoptosis  assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation) . ApproxiVibrio spp. as described before. Apoptosis indices and survival rate of the clams were measured. The relative expression of inflammatory molecules including glutathione-S-transferase (GST), apoptosis inhibitors (IAP) and tumor necrosis factor (TNF) were measured using qRT-PCR of mRNA isolated from these clams. The primers used to synthesize dsRNA are given in Table dsRNA synthesis was performed using the MEGAscript T7 Transcription Kit  following the protocol. 50 μg of dsRNA was microinjected into the adductor muscles of the oyster to knockdown the expression of target genes. Then the clams were infected with http://www.r-project.org) [P-values≤0.05 was considered statistically significant. The correlation statistics was performed and Pearson correlation coefficient (R) was calculated at P value< 0.0001.One way ANOVA was used to analyze the mean differences among samples. Pair-wise t-test was then applied to compare gene expression levels between the two surf clam populations. All statistical analyses (avo functions and pairwise t-test functions)) were conducted in the R computation environment (ect.org) . P-value


The easypod connect observational study (ECOS) assessed treatment adherence among paediatric patients receiving growth hormone (GH) via the easypod electronic injection device.ECOS was an open-label, observational, longitudinal study conducted in 24 countries between 2010 and 2016, enrolling children treated with GH.The primary endpoint was the rate of treatment adherence during 5 years of follow-up. Impact of adherence on growth outcomes was assessed using Spearman’s product–moment correlations.n = 409), 75.5% after 4 years (n = 143) and 70.2% after 5 years (n = 43). Statistically significant correlations between adherence and 1-year change in height standard deviation score  and height velocity (P < 0.001) were observed.Overall, 1190 patients had easypod data available for ≥3 months; most patients had GH deficiency (75%); 606 of these patients were GH naïve at baseline. Over the first year of monitoring, the median rate of adherence was 93.7% among patients overall and >93.0% in GH-naïve patients, irrespective of the treatment indication. Clinically meaningful improvements in growth rates were observed after 1 year of treatment across all GH indications. Adherence decreased with increasing treatment duration, but the overall median adherence rate remained high after 3 years of follow-up: 87.2% (ECOS produced accurate, real-time adherence data in a large population of GH-treated children over 5 years of follow-up. Using the easypod connect system, physicians can potentially identify patients with inadequate adherence and poor response to treatment, enabling them to take appropriate action to help them maximise the benefits of GH treatment. Recombinant human growth hormone (GH) is approved for use in the treatment of children with various aetiologies, including growth hormone deficiency (GHD), Turner syndrome (TS) and born small for gestational age (SGA) with no catch-up growth , 2, 3. TDetection of poor adherence to GH therapy can be difficult because patients or carers may be reluctant to admit to it and may overestimate reported rates . Even thThe easypod device is currently the only electronic GH injection device available; it is approved for use in more than 40 countries so far, including the European Union and the United States. It was awarded the gold medal at the 2007 Medical Design Excellence Awards  and receThe easypod connect observational study (ECOS) was a 5-year investigation to assess treatment adherence among paediatric patients receiving GH via the easypod device. Secondary objectives included investigation of any correlation between adherence, socioeconomic factors and changes in long-term growth outcomes.ECOS was an open-label, observational, longitudinal study conducted in 24 countries  between November 2010 and February 2016. Adherence data in Spain were not collected via easypod connect and, therefore, Spanish results were not included in this analysis; interim results for Spain have been published separately .®, Merck KGaA, Darmstadt, Germany). Eligible patients from each of the participating countries were enrolled in the database and attended one baseline visit followed by 1–4 visits per year, according to local routine clinical practice. Planned duration of follow-up was at least every 6 months for up to 5 years. Owing to the observational nature of the study, all diagnoses and treatment decisions were at the discretion of the investigating physician, following standard endocrine practice.Patients included in the study were aged 2–18 years or >18 years without fusion of growth plates and were receiving GH via the easypod device  guidelines and applicable national legal and regulatory requirements. Written informed consent was obtained from patients (or their parent/guardian) prior to study enrolment. The study was approved by local ethical committees for the centres in each country and the details for these may be found in the online Adherence data were primarily derived from injections recorded in the easypod device, while baseline and outcome measures were obtained by physician data entry into clinical report forms. A protocol amendment was made after the start of the study to capture GH-naïve status. GH naïve status in a patient was defined as any patient who had not been treated with any GH formulation before starting treatment with easypod.The primary endpoint was the rate of treatment adherence derived from the easypod device, defined as the percentage of prescribed injections that were recorded as being administered. Variables assessed with respect to adherence in this study included prior exposure to GH preparations, the indication for GH treatment, age at easypod start Suspected serious adverse events (SAEs) that occurred during the study were collected and reported directly to the sponsor’s Global Drug Safety Officer. Adverse events were coded according to the current Medical Dictionary for Regulatory Authorities (MedDRA) version in the sponsor’s Global Safety Database (ARIS) at the time of SAE reporting .This population-based observational study aimed to generate hypotheses relating to influencers of adherence and, therefore, was not limited in terms of sample size. The primary analysis population was the easypod adherence data analysis set (DAS), comprising patients who had a recorded treatment start date, no gaps >1 week of recorded injections, height measurements available at both treatment start and 1 year (±3 months) and who had at least 3 months of data from the easypod device. Data were analysed for these patients overall, and for the subgroup of GH-naïve patients.Adherence rates were calculated on a cumulative basis, by duration of easypod use and for individual treatment periods (from the beginning of treatment to the last complete week with adherence data available for each patient). Because of the single-arm, observational nature of the study, the statistical analysis was descriptive, with summary statistics for primary and secondary endpoints. Height standard deviation score (SDS) was calculated using World Health Organization reference data  and heigA total of 2420 patients were enrolled in ECOS, of whom 1203 had sufficient data for inclusion in the study, and 1190 patients had easypod data for ≥3 months and comprised the easypod adherence DAS; 606 of these patients were GH naïve at baseline. The main indication for GH treatment was GHD (75% of patients); the remaining patients had SGA (17%), TS (7%) or ‘other’ . A summary of baseline demographic characteristics and auxological data is presented in Adherence data were available for at least 1 year for >98% of patients in the easypod adherence DAS. Over the first year of monitoring, the median rate of adherence was 93.7% among patients in both the easypod adherence DAS and the subgroup of GH-naïve patients. Median adherence rates were >93.0% in patients in each of the indication groups, including GHD (93.4%), SGA (95.0%) and TS (93.2%). In the easypod adherence GH-naïve subgroup, adherence was >90.0% for patients with GHD, irrespective of the cause: idiopathic isolated GHD (92.3%), organic GHD of congenital origin (91.1%) or tumour origin (96.4%).n = 409), 75.5% after 4 years of follow-up (n = 143) and 70.2% after 5 years of follow-up (n = 43). The proportion of patients with an adherence rate of ≥80%  across each of the indication groups; median overall HV over the first year of treatment was 8.2  cm/year and HV SDS was 2.11 , indicating a positive growth response . The groAmong GH-naïve patients, the greatest change in height SDS was observed in those with organic GHD of congenital origin  and HV SDS 2.63 ) followed by idiopathic isolated GHD  and HV SDS 2.54 ; P < 0.001) for change in height, 0.13 (P < 0.001) for change in height SDS, 0.14 (P < 0.001) for HV and 0.08 (P = 0.013) for HV SDS, indicating a positive correlation between adherence rate and growth response. Positive correlation values were also generally observed in each of the indication groups, although these only reached statistical significance for the GHD patients.Spearman’s product–moment correlations between adherence rate and growth measures  were 0.1Equivalent growth outcome and correlation data were also obtained from the GH-naïve subgroup . Growth P < 0.001) and 0.11 (P < 0.01) for the correlations between adherence rate and change in height SDS and adherence rate and HV SDS, respectively. With regard to the GH-naïve subgroup, Spearman’s product–moment correlations between adherence rate and measures of growth response were generally slightly lower than those for the overall group and consistently failed to reach statistical significance across the different measures.For each indication, positive correlations were seen in the easypod adherence DAS for patients with GHD, which constituted the majority of the assessed population: the Spearman’s product–moment values were 0.14 , the overall mean (±s.d.) duration of treatment was 935.2 (±456.5) days, with a mean GH dose of 0.03 (±0.01) mg/kg/day administered at study entry (data available for n = 1185). In the GH-naïve subgroup (n = 606), the overall mean duration of treatment was 964.6 (±464.5) days and mean dose at study start was 0.03 (±0.02) mg/kg/day.For patients included in the easypod adherence DAS  in 11 patients were considered to be related to Saizen  and 63 SThe global 5-year ECOS is the first large-scale study to provide an objective assessment of adherence and effects on growth outcomes during GH treatment with the easypod device in paediatric patients with growth disorders. Median adherence rates were high (94%) over the first year of study treatment, and only gradually decreased with longer duration of follow-up. An adherence rate of ≥80% was maintained by the majority of patients over 3 years of treatment and over each individual treatment period. The associations between high adherence rates and positive growth outcomes were statistically significant in the overall adherence DAS population.n = 824), the recorded dose history showed adherence of 87.5%, with significantly higher adherence in treatment-naïve patients than in treatment-experienced patients (n = 75), a median adherence rate of 96.0% was recorded over a period of 343 days . There were no clear differences in adherence rates according to age (<6 years or ≥6 years), gender, pubertal stage or parents’ employment status. Further investigations of potential confounders are needed to more accurately quantify the observed trends.The limitations of this study include its non-interventional nature, which is associated with a high level of missing data, high inter-patient variability, inclusion of both GH treatment-naïve and non-naïve patients and the absence of detailed recording of actions performed by healthcare providers and carers when poor adherence and/or poor response to treatment was recorded. However, these limitations occur in all surveillance studies , whereasNo other large-scale patient registries have provided comparable insights into patient adherence to GH treatment; this is because of the lack of devices comparable to easypod for enabling assessment of real-time adherence . Other sECOS has provided accurate, robust and real-time adherence data in a large population of patients receiving GH via easypod. The study showed that through using easypod and easypod connect, physicians can identify patients with inadequate adherence, which will enable them to take appropriate action to help maximise the benefits of GH treatment. Poor adherence to treatment is an issue for the management of most chronic diseases, and early detection to enable improved adherence in patients receiving GH is crucial to achieve normal adult height. The study also confirmed the value of using an e-Health platform to monitor adherence to obtain better outcomes. Statistically significant associations were found between adherence and growth outcomes, supporting the monitoring of adherence. No new safety signal was identified in ECOS, and the benefit–risk balance for GH via easypod remains favourable.E K and G S are employees of Merck KGaA, Darmstadt, Germany. P S W D and L O have received honoraria and research grants from Merck KGaA, Darmstadt, Germany.This study was sponsored by Merck KGaA, Darmstadt, Germany. Medical writing assistance was provided by David Candlish, inScience Communications, Tattenhall, UK, and sponsored by Merck KGaA, Darmstadt, Germany.E K, G S, L O and P S W D contributed to the data analysis, review and interpretation and writing/revisions/approval of drafts and final manuscript. L O also provided biostatistical services.


Calendula officinalis L. and their mutant lines obtained though chemical mutagenesis using diethyl sulphate (DES)  and dimethyl sulphate (DMS) . The studied cultivars displayed different sensitivity to DMS and DES mutagens. More M1 plants with morphological changes were observed in C. officinalis cv. ‘Zolotoe more’ than in cv. ‘Rajskij sad’. DMS and DES at low concentrations had positive effects on main agro-metrical traits in both cultivars including plant height, inflorescence diameter and number of inflorescences per plant. Dose-dependent increase in number of various meiotic abnormalities was revealed in both mutant lines. Comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotype stability in M1 and M2 plants. Seed treatments with DMS and DES at certain concentrations resulted in higher yields of inflorescences in M1 plants compared to the control. In M2 generation, dose-dependent reduction in the yields of inflorescences was observed. Our findings demonstrate that DMS and DES at low concentrations have great potential in calendula mutation breeding.The morphological, meiotic and chromosomal variability were studied in two cultivars of  Calendula officinalis L. (Asteraceae), commonly known as calendula or pot marigold, is an annual aromatic herb with yellow or golden-orange flowers. This species is widely used in pharmaceutical industry, medicine, food and cosmetology2. The chemical composition of C. officinalis is well-determined. The flowers, leaves and stems contain flavonoids, xanthophylls, carotenoids, essential oils, coumarins (scopoletin) and water-soluble polysaccharides4. The raw material contains triterpenoid saponins 2–10% (oleanolic acid glycosides), triterpene alcohols  and steroids. The seeds are rich in fatty acids (about 20%) including calendic acid (about 60%). It was shown that conjugated fatty acids are active substances for the treatment of obesity and also they can protect against cancer7. The flowers of C. officinalis contain rutin which has antioxidant, anti-inflammatory and anticarcinogenic activity8.A valuable medicinal plant, C. officinalis, and also unstable active ingredients, e.g., carotinoids, were revealed9. Besides, calendula productivity was found to correlate with seed types and also a number of seed rows in inflorescences10. It is therefore important to direct research efforts towards the development of new promising C. officinalis cultivars with the desirable morpho-agronomic traits and/or biochemical profile.At the same time, polymorphism in active chemical compounds of the raw material was observed in 20. As an example, seed treatment with low concentrations of sodium azide and diethyl sulphate was found to influence on seed germination percentage, plant height, leaf area, fresh plant weight, flowering date, inflorescence diameter and gas-exchange measurements in calendula plants of M1 and M2 generations. Also, these chemical mutagens had significant effects on total soluble proteins, acid phosphatase, and catalase activity fractions of the calendula leaves21.Currently, for obtaining new plant cultivars with valuable economic traits, a mutagenesis approach is used alongside with transgenic crops and recombinant DNA technology. Particularly, chemical mutagenesis is an effective and simple method for obtaining valuable starting material for plant breeding as chemical mutagens  induce a high frequency of non-lethal point DNA mutations and generate novel genetic diversity in various crops22. However, in C. officinalis, a comprehensive study of meiotic changes resulting from chemical mutagenesis has not been performed yet. Besides, currently available cytogenetic information regarding this species is rather scarce and mostly obtained by simple chromosome staining. It was shown, for example, that a karyotype of C. officinalis was composed by metacentric and submetacentric chromosomes which were small in size (1.5–3.5 µm)25. This is, probably, the reason why different karyotype formulas23 and chromosome numbers: 2n = 3226 and 2n = 2827, were described for this species. In one study, a more detailed FISH-based analysis of calendula karyotype was performed that revealed two large chromosome pairs bearing 45S rDNA loci and one pair with 5S rDNA loci27. However, chromosome variability in calendula mutant lines has not been investigated yet, although a comparative molecular cytogenetic analysis of C. officinalis cultivars and mutant lines could provide important information on possible karyotypic reorganization induced by chemical mutagens.Moreover, the quality and quantity of meiotic aberrations are considered to be reliable indicators of sub-lethal effects (doses) of chemical mutagens as well as an effective monitoring system for successful plant mutation breedingC. officinalis cultivars (cv. ‘Zolotoe more’ and cv. ‘Rajskij sad’) and their mutant lines (M1 and M2 generations) obtained though chemical mutagenesis using diethyl sulphate (DES) and dimethyl sulphate (DMS) mutagens.In the present study, we investigated the morphological, meiotic and chromosomal variability in two The original calendula cultivars differed in their main agro-metrical traits. Plants of cv. ‘Zolotoe more’ were considerably lower, had less inflorescence diameters and golden-yellow (with a brown center part) semi-double flowers whereas plants of cv. ‘Rajskij sad’ had larger inflorescences with bright double orange flowers Fig. .Figure 1C. officinalis cultivars, seed treatments with DMS and DES induced a number of morphological changes including plant dwarf, presence of several anthodiums in one inflorescence, polymorphism and fasciation of inflorescences, polypetalous flowers with an increased inflorescence diameter, etc.  compared to cv. ‘Rajskij sad’ (1841 kg/ha in 2014 and 1853 kg/ha in 2015). The effects of different concentrations of both mutagens on calendula productivity were also different. In cv. ‘Zolotoe more’, the treatments with DMS and DES did not result in a higher yield of inflorescences in M1 plants compared to the control. In M2 generation, some reduction in the yields was observed. In cv. ‘Rajskij sad’, the treatments with 0.08% DMS (in both M1 and M2 plants) and 0.025% DES (in M1 plants) resulted in a significant increase in the yields  at diakinesis and metaphase I . In particular, chromosome associations  were detected Fig. . At anapC. officinalis cultivars and mutant lines contained 2n = 32 metacentric and submetacentric chromosomes including two satellite chromosomes pairs (1 and 9) with karyotype formula K = 2(4 m + 10 sm + 2 smst). The chromosome sizes ranged from 3.5 to 5.0 µm  and chromosome idiograms of ted Fig. .In M2 plants of cv. ‘Zolotoe more’, the minor 45S rDNA sites observed on chromosomes 5 and 10 were polymorphic. After the treatment with 0.025% DES and 0.04% DMS, the pattern of chromosomal distribution of 45S and 5S rDNA sites was similar to that revealed in the control plants (cv. ‘Zolotoe more’). At higher mutagen concentrations , the minor 45S rDNA sites were not visualized. In M2 plants of cv. ‘Rajskij sad’, the patterns of chromosomal distribution of 45S and 5S rDNA loci were mostly similar to the control plants. The exception was the treatment with 0.04% DMS. In this case, the minor 45S rDNA locus was not visualized.C. officinalis karyotypes, any chromosomal rearrangements were not revealed.In the studied C. officinalis including plant height, number of inflorescences, inflorescence diameter, etc. Currently, an optimal plant height is a desirable agronomic trait that contributes to a high yield. Besides, the variation in calendula plant height is one of the initial associated traits in response to a mutagen treatment even at low concentrations28. In this study, DMS and DES treatments resulted in plant height changes in both C. officinalis cultivars, and our results are consistent with previous findings on other flowering plants including Salvia splendens29, Euonymus japonicus30 and Amaranthus31. At the same time, the observed variations in this important agronomic trait were dose-dependent, and those changes were different between the examined calendula cultivars. Considered all, the mean value of the plant height was less in M1 and M2 plants of cv. ‘Zolotoe more’ compared to the original cultivar (control). However, in M1 and M2 plants of cv. ‘Rajskij sad’, this parameter was more than in the control. These differences are probably related to different levels of plasticity in plants of the studied cultivars, and in some cases, mutagen-induced damages of the plant cells could be repaired at the initial ontogenesis stages10. In general, both examined mutagens induced the increase (in different degrees) in number of inflorescences, inflorescence diameter, shoots, and doubleness in flowers, and these findings agreed with the earlier reported data on DMS and DES mutagenic effects32. It is important to note, however, that the weather environment factors during growing period should also be considered in making an assessment of these mutagenic effects33.In the present study, DMS and DES mutagens stimulated the diversity in morphological characters of 34. Deviations from the normal bivalent conjugation could be displayed as univalent and multivalent formation at metaphase I stage36. In the studied calendula lines, alongside with normal meiotic segregation patterns, we observed various chromosomal associations at metaphase I . The univalent formation induced by mutagens was supposed to be a result of changes in the structure of chromosomes followed by the reduction of chiasma frequency due to restriction of pairing to homologs37. The appearance of the multivalent associations is considered to be caused by mutagen-induced chromosome breaks followed by the reciprocal translocation joining38. Alternatively, these associations can be a result of chromosome mismatches and breakages which lead to translocations and inversions43.Many chemical mutagens were shown to influence the plant genome and cause the meiotic disorders manifested themselves as typical chromosome aberrations  as well as mass fragmentation, nondisjunction, chromosome stickiness and other abnormalitiesNigella sativa44.Chromosome nondisjunction, occurred at anaphase I, is considered to be a serious meiotic abnormality which resulted in chromosome loss as well as unequal distribution of genetic material. Most cells of the studied here specimens had normal chromosome disjunction (16:16) at anaphase I and anaphase II, however, cells with abnormalities  were also revealed. These disorders could be related to the paracentric inversions as previously described in tomatoes and 20. At the same time, zygotes with chromosome mutations  were shown not always to produce viable seeds45. Considering that karyotypes in M2 plants did not differ in chromosome number and morphology from the original calendula plants, our findings indicates that DMS and DES at low concentrations are rather effective mutagens for calendula breeding, which had positive effects on vegetative parameters without dramatic consequences for the calendula genome.Thus, in the present study, the analysis of the chromosome behaviour during meiosis revealed an increased number of microsporocytes with various meiotic disorders in calendula mutant lines if compared with the control. Moreover, a high level of phenotypic variability observed in the examined mutant lines could also be related to mutagen-induced chromosome mutations. An estimation of cytological abnormalities and their magnitude during mitosis or meiosis is believed to be a key component to determine the effect of a mutagen and also species sensitivity to sublethal doses in mutation breeding experimentsC. officinalis chromosomes. As a result, the karyotype formula of C. officinalis reported here differed from that described earlier23 by a ratio of metacentric and submetacentric chromosomes. Moreover, we have identified two satellite chromosomes in the karyotypes and also specified the chromosome number of C. officinalis as both numbers 2n = 3226 and 2n = 2827 have been described before.Basically, the results of karyotype study in the calendula cultivars agreed with earlier reported data which were mainly based on simple monochrome staining. At the same time, the application of DNA intercalator 9-AMA allowed us to obtain longer by half chromosomes in metaphase plates and also specify the morphology of 48. In plant species with small chromosomes, chromosome banding patterns are usually rather poor which makes it difficult to identify precisely each chromosome in a karyotype50. Typically, in small-sized chromosomes, large heterochromatic bands located in the pericentromeric regions, small telomeric bands and few small intercalary bands are revealed54. The application of DNA intercalator 9-AMA allowed us to increase the resolution of chromosome DAPI-banding patterns, improve accuracy of physical mapping of rRNA genes, and then use these markers for analysis of calendula chromosomes. As a result, all chromosomes in karyotypes of C. officinalis original cultivars and mutant lines were identified for the first time. The results of FISH-based analysis of chromosomal distribution of major 45S and 5S rDNA in the original C. officinalis cultivars agreed with the currently available data27. Moreover, we detected minor 45S rDNA loci on chromosome 5 (both cultivars) and on one homolog of chromosome 10 (cv. ‘Zolotoe more’). These additional 45S rDNA loci were probably related to intraspecific genomic polymorphism.DAPI-banding was shown to reveal AT-rich heterochromatic regionsIn M2 plants, the analysis of chromosomal distribution of 45S and 5S rDNA sites revealed dose-dependent differences in localization of these minor 45S rDNA sites on chromosomes 5 and 10. Moreover, the plants of cv. ‘Zolotoe more’ were more sensitive to these mutagenic effects than cv. ‘Rajskij sad’.55. Both DMS and DES mutagens were successfully applied for improving the vital characters of the floricultural crops including calendula21. Our results show that DMS and DES had positive effects on valuable agro-morphological traits in C. officinalis, and therefore these mutagens have great potential for improvements in the viability, yield and quality of calendula. Particularly, seed treatments with 0.08% DMS and 0.025% DES resulted in higher yields of inflorescences in M1 and M2 plants of cv. ‘Rajskij sad’ compared to the control. In cv. ‘Zolotoe more’, slight increase in the yields of inflorescences in M1 plants and dose-dependent reduction of this parameter in M2 plants were observed. Therefore, these chemical mutagens can stimulate useful traits in calendula genotypes, and this is very important for developing new promising cultivars in order to meet market demands. Currently, induced mutagenesis has become widespread in plant mutation breeding to solve a very wide range of problems, and particularly, this method is successfully used for developing improved crops within the Asteraceae family including Dendranthema grandiflora Tzvelev, Artemisia pallens Bess. and Helianthus annuus L.59.The optimal conditions for pre-sowing seed treatments with chemical mutagens was shown to result in improved vegetative growth and flowering characteristics, increased yields and stress tolerance in comparison to normal plantsC. officinalis cultivars displayed a different sensitivity to DMS and DES mutagens. This could be indicative of genetic polymorphism among C. officinalis cultivars, which correlates with variability in the cultivar characteristics. Therefore, a careful testing of these mutagens at different concentrations is required for successive realization of plant breeding programs.Thus, our findings demonstrated that DMS and DES at certain concentrations had positive effects on morphological and yield attributes of calendula which could be useful for further breeding. At the same time, the studied C. officinalis cv. ‘Zolotoe more’  and cv. ‘Rajskij sad’  were treated with aqueous solutions of DMS (0.04% and 0.08%) and DES (0.025% and 0.05%) for 18 h. The control seeds were treated with water. The types of mutagens, their concentrations and treatment time were optimized based on our previous studies10 as well as early described data on application of chemical mutagens in breeding of horticultural crops32. After the mutagen treatment, the seeds were washed in water and sowed into the greenhouse for obtaining seedlings (M1 generation). At the stage of 4–5 true leaves, the seedlings were planted in the field in crop geometry of 60 cm × 30 cm according to the earlier described approach60. Plant vegetative parameters were measured at the stage of flower maturity of the plants. Seeds from the individual M1 plants were harvested and then used both for cytogenetic assays and for cultivation of M2 plants in the next growing season. At early May 2015, these seeds were sowed into the greenhouse and then grown in the field in the same way as M1 plants. The productivity (yield of inflorescences) of calendula plants was determined as a total sum of ten-round harvesting of inflorescences during the flowering period according to the earlier described approach60. Statistical data analysis was performed using standard functions of Microsoft Excel 2013.The calendula plants were grown in the Botanical garden of the All-Russian Institute of medicinal and aromatic plants  during two successive experimental seasons (summer 2014 and summer 2015). At late April 2014, seeds of 62. After the pre-treatment, the root tips were fixed in ethanol:acetic acid (3:1) fixative for 48 h at room temperature. Before squashing, the roots were transferred into 1% acetocarmine solution in 45% acetic acid for 15 min. The cover slips were removed after freezing in liquid nitrogen. The slides were dehydrated in 96% ethanol and stored at −20 °C until use.For FISH assays, the modified technique of chromosome spread preparation from root tips was applied. The seeds were germinated in Petri dishes on the moist filter paper at room temperature. Root tips (of 0.5–1 cm) were excised and stored for 16–20 h in ice-cold water with 1 µg/mL of 9-aminoacridine (9-AMA)  to inhibit chromosome condensation process and accumulate prometaphase chromosomes63. After freezing in liquid nitrogen, the cover glasses were removed, and the slides were stored in 96% ethanol at −20 °C until use.For meiotic chromosome preparation, young floral buds (prefoliation) were fixed in ethanol:acetic acid (3:1) fixative for 30 min at 4 °C and then chromosome spreads were prepared as previously described64. This DNA probe was labelled directly with SpectrumAqua .pTa71 - a 9-kb-long sequence of common wheat encoding 18S, 5.8S, and 26S rRNA genes including spacers65. This DNA probe was labelled directly with SpectrumRed fluorochrome .pTa794 - a 420-bp-long sequence of wheat containing the 5S rRNA gene and intergenic spacerThe following probes were used for FISH:et al.51. After overnight hybridization, the slides were washed as described previously66.FISH procedure was carried out according to Muravenko After FISH procedure, chromosome slides were stained with 0.1 μg/ml DAPI   dissolved in Vectashield medium .52.The slides were examined using an Olympus BX-61 epifluorescence microscope . Images were captured with monochrome charge-coupled device camera . Then they were processed with Adobe Photoshop 10.0 software . At least 15 metaphase plates were investigated for each specimen. For meiosis analysis, at least 200 cells (10 plants) of each sample were analyzed. The meiotic chromosome preparations were analyzed as described previously


Intravenous crystalloid solutions are administered commonly for critically ill patients. We performed this meta-analysis of randomized trials with trial sequential analysis (TSA) to evaluate effects of chloride content of intravenous crystalloid solutions on clinical outcomes among critically ill adult patients.Electronic databases were searched up to June 1, 2018, for randomized trials of use of balanced crystalloids versus 0.9% saline solutions in critically ill adult patients. The outcome variables included mortality, renal outcomes, serum content alterations and organ function. Subgroup analysis was conducted according to patient settings, types or volume of crystalloid fluid, or among sepsis versus non-sepsis, TBI versus non-TBI or subpopulations by the categories of baseline kidney function. Random errors were evaluated by trial sequential analysis.p = 0.06). The use of balanced crystalloid solutions was associated with longer RRT-free days , less risk of increase in serum concentrations of chloride  and sodium , less risk of decline in serum base deficit , longer ventilator-free days  and vasopressor-free days . Subgroup analysis showed that balanced crystalloid solutions were associated with a reduced in-hospital mortality rate among septic patients  and non-traumatic brain injury patients , while the TSA results indicated a larger sample size is still in need.Eight studies with 19,301 patients were analyzed. A trend of in-hospital survival benefit with no statistical difference could be observed with balanced crystalloids compared with 0.9% saline , https://www.crd.york.ac.uk/prospero/#recordDetailsThe online version of this article (10.1186/s13613-019-0506-y) contains supplementary material, which is available to authorized users. Intravenous crystalloid solutions are administered commonly in critical care unit, especially for patients with need of resuscitation. Crystalloid solutions as the fluid of choice for initial resuscitation and subsequent intravascular volume replacement in patients with sepsis and septic shock were recommended in guidelines for management of sepsis and septic shock published in 2016 [0.9% saline solutions have been the most commonly administered intravenous fluid. Since first developed by adding the buffer lactate to Ringer’s solution in the 19th century, balanced crystalloid solutions such as  have been preferable alternative for relatively physiological concentrations of ions –5. Data A recent large pragmatic trial suggested balanced crystalloid fluid administration in critically ill adults compared with use of 0.9% saline could decrease incidence of a major adverse kidney event within 30 days  rather tThere is no requirement for ethical approval, and patient consent for this meta-analysis is based on previous published studies. The protocol for this meta-analysis was registered on PROSPERO: International prospective register of systematic reviews (CRD42018102661).We conducted a search for randomized trials in the following databases until June 2018: Medline, EMBASE, Cochrane  database, Elsevier, Web of Science and ClinicalTrials.gov. The details of search strategy were as the meta-analysis by Kawano-Dourado used , shown iThe titles and abstracts were screened to determine whether a study should be included by two reviewers (MX and FL) independently. Then, the full texts were reviewed according to the inclusion and exclusion criteria. Any discrepancies were resolved by a consensus on the inclusion or exclusion of a study after a discussion with a third reviewer.Type of study: randomized controlled trials and cluster randomized trials.Population: Acutely ill adult patients in the ICU or surgical adult patients transferred to ICU in the perioperative period.Intervention: balanced crystalloid fluid characterized by a near-physiological chloride concentration , given intravenously for resuscitation or maintenance.Control: 0.9% saline solution works as control group with relatively high chloride content for fluid resuscitation or maintenance.Outcomes: The primary outcome was in-hospital mortality. The secondary outcomes were renal outcomes including new RRT use, stage 2 or higher AKI development according to kidney disease: improving global outcomes (KDIGO) criterion  after enWe included trials with the following features:If they made the comparisons between crystalloids versus colloids, hypertonic saline versus balanced solutions, or among solutions with different colloid components, to minimize the confounding factors and make sure the difference between the experimental and control groups involved a buffer in the solution .If they were involved with the patients receiving renal transplantation.We excluded trials with the following features:All available data including characteristics of the selected studies, details of the population enrolled, details of the intervention including type of crystalloid fluid, volume and duration and details of the predefined primary outcome were independently extracted by two investigators (MX and FL). Disagreements between the two investigators were resolved by a consensus after discussing with a third reviewer (XZ).We summarized the evidence by evaluating design, quality, consistency, precision, directness and possible publication bias of the included studies in accordance with the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system  .I2 test were used to assess the statistical heterogeneity and inconsistency in RevMan 5.3 [p value < 0.10 was predefined as the statistically significant heterogeneity in the M–H Chi-square test, while I2 index was used to assess heterogeneity in the meta-analysis. Higgins and colleagues proposed 25%, 50% and 75% of I2 values would mean low, medium and high heterogeneity, respectively [Data were conducted by RevMan 5.3 software according to the Cochrane Handbook. Mantel–Haenszel (M–H) Chi-square test and the vMan 5.3 . A p valectively . Both thectively .Predefined subgroup analysis was conducted comparing patients setting (ICU versus transferred to ICU in perioperative period), crystalloid fluid type  and fluid volume (mean or median volume above versus < 5 L). To investigate the potential sources of heterogeneity, we performed post hoc analyses according to specific patients. Patients were classified into sepsis and non-sepsis, TBI and non-TBI or subpopulations according to the categories of baseline kidney function by Semler study in 2018 : normal Sensitivity analyses were used to estimate the effect of chloride content of intravenous fluid on clinical outcomes of in-hospital, 30-day and 60-day mortality, development of stage 2 or higher AKI, new RRT use, RRT-free days. It was conducted by sequentially omitting a single study each time, to identify the potential influence of an individual study by STATA .α = 0.05 and β = 0.10 (power 90%) and a required diversity-adjusted information size based on the intervention effect suggested by the included trials using a random-effects model, which aimed to assess the effects of chloride content of intravenous crystalloid fluid on in-hospital mortality of critically ill patients [To prevent the risk of random error from being increased by repeated updates, a two-sided trial sequential analysis  was performed with patients –19.The flow diagram shows the study selection process in Fig. A total of 19,301 patients reporting outcomes and analyzed were exposed to crystalloid solutions of different chloride contents as outlined in Table We assessed each included trial by the mode of randomization, allocation concealment, level of blinding and loss to follow up  among all in-hospital mortality analyses  and 10.9% (1045/9557) in groups of balanced crystalloids and 0.9% saline, respectively. Pooled estimates of included studies indicated a trend toward survival benefit with no significant difference in patients receiving balanced crystalloid solutions versus 0.9% saline with an RR of 0.92  failed to reach the traditional boundary (p = 0.05), the trial sequential monitoring boundary as well as the estimated information size boundary  [I2 = 0%, p = 0.24). Among them, three studies  with 423 patients provided detailed indications for new RRT use, which included oliguria, acidemia, blood urea nitrogen over 70 mg/dL, serum creatinine over 3.39 mg/dL and organ edema. The data showed no difference between groups of patients treated with balanced crystalloid and 0.9% saline solutions  are summarized in Table  = 0.11) , 10, 25.p = 0.06, Additional file p = 0.03, Additional file p = 0.31 for interaction).Two studies of Selmer in 2016 and 2018 introduced the composite outcome of MAKE30, which is defined as the occurrence of any of the following: in-hospital mortality, new RRT use or a 200% increase in serum creatinine from admission to discharge at 30 days after ICU admission , 25. Thep < 0.001; I2 = 0%) and sodium  compared to 0.9% saline. There was a significant decline in serum base deficit  with 0.9% saline compared to balanced crystalloid fluid infusion. There was no significant difference in alterations of serum pH value and bicarbonate concentration between balanced crystalloids and 0.9% saline groups.Three studies , 21, 23 p < 0.001; I2 = 0%) and vasopressor-free days  indicates high risk of bias; yellow (?) indicates unclear risk; and green (+) indicates low risk of bias. Figure S2. GRADE profile for assessing quality of evidence. Figure S3. Forest plots for indications of new RRT use after enrollment. Figure S4. Forest plots for MAKE30 in predefined subgroups. a Sepsis and non-sepsis subgroups. b Subgroups according to categories of baseline renal function. MAKE30 is for major adverse kidney events within 30 days. Figure S5. Forest plots for alterations’ of serum content among critically ill patients. Figure S6. Forest plots for organ support. a MV use of critically ill patients. b Ventilator-free day of critically ill patients. c Vasopressor-free days of critically ill patients. MV is for mechanic ventilation. Figure S7. Funnel plots. a Funnel plots for in-hospital mortality. b Funnel plots for 30-day mortality. c Funnel plots for 60-day mortality. d Funnel plots for development of stage 2 of higher AKI of critically ill patients. e Funnel plots for new RRT use of critically ill patients. f Funnel plots of for RRT-free days of critically ill patients. AKI is for acute kidney injury according to KDIGO criterion; RRT is for renal replacement therapy.


Physical activity (PA) is associated with changes in body composition that affect insulin sensitivity and leptin levels. Few studies have assessed the effect of lifestyle interventions on changes in objectively measured PA levels in obese children. To evaluate the effects of a multidisciplinary lifestyle intervention on anthropometric indices, biochemical parameters and accelerometer measured PA in abdominal obese children.A randomized control trial was performed in 106 children and adolescents with abdominal obesity. Participants were randomly assigned to usual or intensive care group for 8-week. PA was measured by accelerometry over four days including, at least, two weekdays in all participants. Both groups were encouraged to accumulate an extra time of 200 min per week in their PA.At baseline, 75% of subjects do not fulfill the WHO recommendation of being more than 60 min/day on moderate-to-vigorous PA (MVPA). The intensive care group achieved a significant reduction in anthropometric indexes compared to the usual care but no significant change was found in biochemical or PA parameters. Both groups achieved a significant reduction in light PA. Interestingly, intensive care participants significantly increased MVPA in 5.5 min/day. Moreover, an inverse association between changes in MVPA and leptin levels was found.The two lifestyle intervention reduced anthropometric indexes and lowered light PA in abdominal obese children. No significant differences were observed between intensive care and usual care in regard to PA. Intensive care participants significantly increase physical activity (MVPA) and, changes in MVPA were inversely associated with changes in leptin levels after the intervention.ClinicalTrials.gov, Identifier: NCT03147261. Registered 10 May 2017. Retrospectively registered.The online version of this article (10.1186/s12887-019-1468-9) contains supplementary material, which is available to authorized users. Dyslipidemia, hypertension, insulin resistance or type 2 diabetes, are the main alterations that derive from obesity and contribute to aggravate cardiometabolic risk in pediatric populations . Obese cSedentary lifestyle, the fourth leading cause of global mortality, is becoming more frequent in pediatric populations , 6. “GloIntervention Grupo Estudio Navarro de Obesidad Infantil) study is a randomized control trial (NCT03147261) conducted in Pamplona, Navarra (Spain). It is a 2-year family-based lifestyle intervention program for children with abdominal obesity. Seven to sixteen year-old children were recruited from the Endocrinology Pediatric Units of the University of Navarra Clinic, Navarra’s Hospital Complex and Primary Health Care Centers in Pamplona. General inclusion criteria for enrollment was waist circumference above the 90th percentile, according to national data [The IGENOI , as described in other trials with pediatric populations . One hunThe primary endpoint of the IGENOI study was to assess the effectiveness of the lifestyle interventions based on BMI-SDS values. Changes in BMI-SDS are the main outcome since successful interventions should decrease BMI-SDS near 0.5 units in order to reduce cardiometabolic risk . Taking The IGENOI study is a family-based lifestyle program carried out by a multidisciplinary team  in a clinical setting. It consists of a two-year program that comprises an 8-week phase with individual and group sessions and a follow-up period of 22 months. Our research group has different weight loss interventions where we have observed that in a short time period (8 to 10 weeks) there are substantial changes in weight loss and other metabolic parameters . In thisParticipants were randomly assigned to the usual or intensive care group with a ratio of 1:3. The randomization was performed using a computer-generate randomization. The intensive care group was advised to follow a fully-day meal plan during the intensive phase. This diet consists on a moderate hypocaloric Mediterranean diet to not to interfere with children’s growth. Energy restriction (10 to 40%) was calculated according to obesity degree and physical activity levels, as described elsewhere , 25. TheIntensive care participants and their parents received six 30 min-sessions lead by the dietitian during the 8-week period in order to monitor the accomplishment of the diet. One parallel group sessions was organized for 1) intensive care participants and 2) their parents or legal tutors. During the group sessions parents were told their role in the intervention and the obesity related comorbidities, while children were taught about different topics such as energy balance, portion sizes, groups of foods, the importance of the breakfast and physical activity . On the 2), and these values were converted into standard deviations (BMI-SDS) using age and sex-specific cut-off points according to Spanish reference growth charts [Anthropometric measurements  were evaluated by trained personnel following standard procedures. Body mass index (BMI) was calculated as weight divided by squared height  and the presence of the team .Venous blood samples were obtained after an overnight fast. Glucose, insulin and lipid profiles were determined by standard autoanalyzer techniques. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin values. Leptin levels were measured by ELISA . All the measurements were taken at baseline and after the 8 week period.Physical activity and sedentary time were objectively assessed using triaxial accelerometry over four days, including, at least, two weekdays in all participants. Participants and parents were instructed on wearing the accelerometer  around the non-dominant waist all the time, including sleep time, and removing it just for water-related activities (bathing or showering). The monitors were initialized using 60-s epochs, as described elsewhere .Accelerometry data were analyzed using ActiLife 6.0 software . Continuous 24-h accelerometer data were recorded from weekdays and weekend days and were analyzed separately. Total PA was obtained by weighting 5 times weekdays plus two times weekend days by two and dividing the result by seven, as previously reported . Data weSleep was assessed using the accelerometer Actigraph wGT3X-BT, and the data was analysed using the Sadeh algorithm derived from fundamental research performed by Avi Sadeh et al.  .This alp value < 0.05 was considered as statistically significant. Variables were described using mean ± SD. Student’s t test was used for the comparison between groups (two independent groups test) and for the comparison within group (paired test) before and after the intervention. The change in each variable was calculated as the difference between post- and pre- intervention values for each subject.All statistical analyses were two-tailed and a Analyses of covariance (ANCOVA) were performed to assess the changes in anthropometric, biochemical and PA variables between usual care and intensive care groups after the adjustment for potential confounders: the studied variable at baseline, baseline BMI-SDS, sex and Tanner stage." Stata 12.0  for Windows was used in all the analyses.Furthermore, we fitted a multivariable adjusted linear regression to examine the association of changes in leptin with changes in metabolic parameters and PA intensity.The study population includes 106 children with obesity (BMI-SDS 2.89 ± 1.05) (37.7% boys), with waist circumference over the sex and age-specific 90th percentile and a mean age of 11.31 ± 2.47 years old. At baseline, participants spent, on average, 997.9 (101.6) min/day on sedentary activities, including sleep time, 43.9 (23.4) min/day in MVPA activities, and 394.2 (95.9) min/day on light PA.Most abdominal obese children (74.5%) do not fulfilled the WHO recommendations of being more than 60 min/day on MVPA at baseline. Participants were more active during weekdays compared to weekend days  had significantly higher glucose levels compared with the intensive care group (n = 79) (p = 0.016). We did not find significant differences between groups regarding age, sex or Tanner stage.As expected, due to the randomization, participants in the two groups were similar for most of the clinical parameters at baseline, except for blood glucose levels Table . The usup = 0.029) and hip circumference (p = 0.019) was significantly higher in the intensive care group compared to the usual care group after adjusting for potential confounders (Table The decrease in BMI-SDS (e p = 0.09 was sigp = 0.004), insulin (p = 0.010), and leptin (p < 0.001) levels were observed in the intensive care group. Meanwhile, subjects in the usual care group did also significantly decrease glucose (p < 0.001) and leptin (p < 0.001) levels.Regarding biochemical parameters, no differences were found between the two lifestyle interventions. A reduction in glucose (p = 0.024) in the intensive care group . Moreover, changes in leptin were also associated with changes in anthropometric and metabolic parameters  . Both stEvidence shows that PA levels are different between normal-weight and obese subjects. Besides, PA levels are higher in children than in adolescents and in boys than in girls, with accelerometer-measured MVPA ranging from 49.1 to 85.1 min/day in normal-weight children and adolescents , 30–32.We addressed the achievement of WHO recommendations on MVPA levels in our population. Only 25% of participants accumulated more than 60 min/day of MVPA at baseline. When considering the day of the week, 32% of the participants achieved the recommendations during the weekdays but only 16% of them did it at weekends. The evidence regarding this point is controversial. Some authors recommend the promotion of PA during the weekdays because children have PA lessons at the school , 34, whiIn the literature we found several lifestyle intervention studies that examined both adiposity indexes (i.e. BMI-SDS) and objectively measured PA levels , 16, 18 To our knowledge, this is the first intervention program that achieved a significantly increase in MVPA  in intensive care subjects. We did not found differences in PA between intervention groups. Intensive care subjects were under a moderate hypocaloric Mediterranean diet and achieved a successful weight loss (Δ BMI-SDS = − 0.51) that was higher than usual care group. It has been described that the combination of PA and Mediterranean diet might provide greater health benefits that those acquired separately in a recent meta-analysis . In addiLeptin is a peptide hormone secreted by adipose tissue which plays a central role in regulating human energy homeostasis , 25. LepThe strengths of our study include:  the longIn conclusion, the two lifestyle interventions were successful, since a reduction in anthropometric indexes and light PA in abdominal obese children was achieved. No significant differences were observed between intensive care and usual care groups in regard to PA. Intensive care participants following a hypocaloric Mediterranean diet significantly increased MVPA. Changes in MVPA were inversely associated with changes in leptin levels after the intervention.Additional file 1:Table S1. Physical activity characteristics measured by accelerometry before lifestyle intervention in children with abdominal obesity (N = 106). (DOCX 13 kb)Additional file 2:Table S2. Baseline characteristics in participants with abdominal obesity divided by type of intervention. (DOCX 14 kb)


This study was to present meta-analysis findings across selected clinical trials for the effect of health information technologies (HITs) on glycemic control among patients with type 2 diabetes.HITs may be promising in diabetes management. However, findings on effect size of glycated hemoglobin level (HbA1c) yielded from HITs varied across previous studies. This is likely due to heterogeneity in sample size, adherence to standard quantitative method, and/or searching criteria .We systematically searched Medline, Cumulative Index of Nursing and Allied Health Literature (CINAHL), and the Cochrane Library for peer-reviewed randomized control trials that studied the effect of HITs on HbA1c reduction. We also used Google Scholar and a hand search to identify additional studies. Thirty-four studies (40 estimates) met the criteria and were included in the analysis. Overall, introduction of HITs to standard diabetes treatment resulted in a statistically and clinically reduced HbA1c. The bias adjusted HbA1c reduction due to the combined HIT interventions was − 0.56 [Hedges’ g = − 0.56 ]. The reduction was significant across each of the four types of HIT intervention under review, with mobile phone-based approaches generating the largest effects [Hedges’ g was − 0.67 ]. HITs can be an effective tool for glycemic control among patients with type 2 diabetes. Future studies should examine long-term effects of HITs and explore factors that influence their effectiveness.The online version of this article (10.1007/s11892-018-1105-2) contains supplementary material, which is available to authorized users. Diabetes is now a world epidemic. It affected 8.8% 415 million) adult population (age 20–79 years) in 2015 worldwide and the prevalence will rise to 10.4% (642 million) by 2040 (10.4%) 5 million, 4. HoweTo improve diabetes management, development of innovative self-care strategies is warranted. Advances in health information technologies (HITs) have introduced approaches that support effective and affordable health care delivery and patient education. Technologies in mobile, computer, e-mail, and internet approaches have shown evidence in enhancing chronic disease management , 7, suggThe benefits of HITs in diabetes management seems well illustrated in theory—they have the potential to empower patients and support a transition from a role in which the patient is the passive recipient of care services to an active role in which the patient is informed, has choices, and is involved in the decision-making process ••. They We systematically searched Medline for eligible articles from 1946 to December 2017, using combinations of the following MeSH (M) and textword (TW) search terms: (1) Diabetes Mellitus Type 1 (M), Diabetes Mellitus Type 2 (M), diabetes (TW), diabetes mellitus (M), prediabetic state (M), prediabetes (TW), (2) telemedicine (M), mHealth (TW), cell phone (M), cell phone$ (TW), mobile phone$ (TW), telehealth (TW), eHealth (TW), internet (M), ambulatory monitoring (M), and wearable$ (TW). Similar searches were conducted in Cumulative Index of Nursing and Allied Health Literature (CINAHL), and the Cochrane Library. We also used Google Scholar to identify additional studies not listed in the abovementioned databases. We also performed supplementary searches using references lists of eligible articles and relevant systematic review and other review articles we encountered.Articles were deemed eligible if they were peer-reviewed RCTs containing methodology and results sections that studied the effect of HITs including mobile phone-based, web-based, short message/text and other technologies on HbA1c among patients with T2D. Articles were excluded if they only included patients with type 1 diabetes, involved continuous glucose monitors, were feasibility trials, or were not written in English.A multistage screening process was used. Search results were first pooled and duplicates were removed. Next, article abstracts were screened for apparent relevance. Then the article texts were reviewed to confirm eligibility status. Articles extracted from reference lists underwent an identical process.Following the screening process, data from eligible articles were extracted independently by two researchers. A coding manual was used to maintain reliable practices. The coding manual specified study characteristics , intervention characteristics , and clinical outcome (HbA1c). For the clinical outcome, additional data were extracted concerning the intervention’s treatment effect compatible with meta-analysis. Discrepancies were unanimously resolved before final data entry.The Cochrane Collaboration Risk of Bias Assessment tool was used to appraise the quality of each article by two reviewers. Six domains of bias  are included in the tool and risk scored as low, high, or unclear . For eacN test [We appraised the risk of selective reporting or publication bias by visual inspection of funnel plot symmetry of the standard error of each trial plotted against its estimated effect. This assumes that trials with larger sample sizes were less subject to publication bias. We further assessed the publication bias using a fail-safe N test .I2 statistics [P < 0.05 or the I2 statistics was ≥ 50% [RCTs containing methodology and results sections that studied the effect of mobile or potentially mobile HIT on type 2 diabetes were eligible for meta-analysis inclusion. We calculated a raw effect size measure of HIT on HbA1c (%) across 40 point estimates in 34 studies  changes from baseline to follow up time between intervention and control groups). We used Comprehensive Meta-analysis Version 3 statistical software to calculate two similar effect size measures modified in two ways , 42. Firatistics . We consas ≥ 50% . In addias ≥ 50% . It showas ≥ 50% . Reportias ≥ 50% .Although the effects of HIT on HbA1c is a mix of both HITs and standard diabetes care, including medication adherence and lifestyle modifications, in 16 of the reviewed trials, this effect was not clearly distinguished . For this reason, we repeated the overall synthesis analysis using data from the remainder 18 studies (53%) that compared outcome between a combined HITs and standard care intervention group and standard care alone control group.Figure n = 29, 85%) received standard care and/or consultation from health care professionals [n = 23, 68%) have incorporated education components in their interventions including self-care and monitoring, life style modifications and/or medication administration and adjustment [Study characteristics are presented in Supplemental Table ssionals , 79, 80 ssionals , 76, 78.ssionals . One stussionals . Three sssionals , 71, 74.ssionals , 78. Eigssionals , 79, 80.ssionals , 76, 77.ssionals , 74, 75.justment –76, 80. justment , 76–79. The risk of bias assessment of the studies is shown in Supplemental Fig. N test indicates that an additional 2426 studies would have to be added before the loss of statistical significance would occur. This suggests that publication bias may not be a serious issue in our analysis. Moreover, the trim-and-fill method [A funnel plot  in 25 out of 40 estimates. The mean reduction in HbA1c after intervention between intervention group and control group was − 0.65 (result not shown in table or figure). The standardized mean reduction in HbA1C resulting from HITs was − 0.57  with values ranging from − 2.10 to − 0.01   and 6.5 to 9.9% (eAG ranges from 7.8 to 13.2 mmol/L) in intervention and control groups, respectively. We found mean HbA1C reductions due to HITs across all studies. Reductions in HbA1C levels were statistically significant   with values ranging from − 2.96 to − 0.01  were statistically significant. We found an overall adjusted effect size (Hedges’g) of 0.56 . This effect size is larger than what was reported in previous review studies , 37, 82,Our analysis detects a significant clinical impact of HITs in reducing dysglycemia. Evidence suggests that every 1% decrease in HbA1c over a ten-year period is associated with a risk reduction of 21% for diabetes-related death and 37% of microvascular complications . This reIn our study, a significant reduction in HbA1c was observed with the addition of all types of HIT interventions reviewed, including the use of mobile phone-based applications, web-based application, SMS/Text, and others. The effect size, however, varied among these groups. Among all technology features under review, we found mobile phone-based and SMS/Text approaches to be particularly effective in improving glycemic profile. These findings appeared consistent with findings of two previous systematic reviews. One review focused on the effect of telemedicine on HbA1c concluded that text messaging may be especially effective mechanism for linking providers and patients with diabetes; therefore, they hold great promise to influence diabetes care positively . The othThe heterogeneity observed in the study is potentially explained by the wide variation of interventions included. Among trials under review, interventions ranged from simple messages providing diabetes management suggestions for patients  to more One limitation of the study is that more than 80% of the trials included did not provide clear information on blinding to participants and personnel on outcome measurement. However, evidence suggests that lack of blinding is unlikely to influence an objectively assessed outcome such as HbA1c . AnotherFurther, our study did not include papers published in non-English language nor trial registry data, which can be another source of publication bias. Otherwise, broad inclusion/exclusion criteria were used to increase the likelihood of capturing relevant studies, including a hand search of reference lists of eligible articles, relevant systematic reviews, and narrative reviews. Moreover, while results from our risk-of-bias assessment helps to highlight the quality of the reviewed trials, unobserved confounding may also influence findings. Lastly, long-term evaluation of HITs’ effect on HbA1c warrant further research. Previous evidence showed that antidiabetic drugs achieved their maximal effect in lowering HbA1c by 3 to 6 months . Most ofOur meta-analysis shows that overall, HITs have favorable impact on glycemic control of patients with type 2 diabetes. On average, mobile-based interventions produce larger effects compared to other forms of approaches under review. Our findings have critical clinical implication since HbA1c is the strongest predictors of disease progression and development of microvascular and macrovascular complications in patients with type 2 diabetes . This reSupplementary Figure 1(DOCX 15 kb)Supplementary Figure 2(PPTX 41 kb)Supplementary Table 1(XLSX 19 kb)