Rapid identification and susceptibility testing results are of importance for the early appropriate therapy of bloodstream infections. The ePlex (GenMark Diagnostics) blood culture identification (BCID) panels are fully automated PCR-based assays designed to identify Gram-positive and Gram-negative bacteria, fungi, and bacterial resistance genes within 1.5 h from positive blood culture. Rapid identification and susceptibility testing results are of importance for the early appropriate therapy of bloodstream infections. The ePlex (GenMark Diagnostics) blood culture identification (BCID) panels are fully automated PCR-based assays designed to identify Gram-positive and Gram-negative bacteria, fungi, and bacterial resistance genes within 1.5 h from positive blood culture. Consecutive non-duplicate positive blood culture episodes were tested by the ePlex system prospectively. The choice of panel(s)  was defined by Gram-stained microscopy of blood culture-positive bottles . Results with the ePlex panels were compared to the identification results obtained by standard culture-based workflow. In total, 216 positive blood culture episodes were evaluable, yielding 263 identification results. The sensitivity/positive predictive value for detection by the ePlex panels of targeted cultured isolates were 97% and 99% for the Gram-positive panel and 99% and 96% for the Gram-negative panel, resulting in overall agreement rates of 96% and 94% for the Gram-positive and Gram-negative panel, respectively. All 26 samples with targeted resistance results were correctly detected by the ePlex panels. The ePlex panels provided highly accurate results and proved to be an excellent diagnostic tool for the rapid identification of pathogens causing bloodstream infections. The short time to results may be of added value for optimizing the clinical management of patients with sepsis. Bacteremia and severe sepsis are important causes of mortality in hospitalized patients, especially in those with increasing comorbidities and immunocompromised status . Delays The implementation of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in routine laboratory workflow has accelerated the identification of isolates, but this approach usually requires subculture, potentially generating delays. Direct identification by MALDI-TOF MS on bacterial pellets from blood culture broth has shown efficacy; however, the manual nature of bacterial isolation and the lack of FDA-cleared products for such in-house or commercial methods may impair their routine use . Recentlin vitro diagnostic medical devices); however, here we evaluated the analytical performance of the ePlex BCID panels in the RUO format compared to the routine testing results for the identification of pathogens that cause sepsis and their associated resistance determinants.The ePlex platform  relies on electrowetting technology to perform multiplexed nucleic acid extraction, amplification, and digestion, followed by the detection of analyte targets using eSensor technology. The ePlex research-use-only (RUO) blood culture identification (BCID) panels are fully automated PCR-based assays designed to identify 20 Gram-positive bacterial genera or species and 4 antimicrobial resistance genes (BCID-GP panel), 21 Gram-negative bacterial genera or species and 6 antimicrobial resistance genes (BCID-GN panel), and 16 fungal genera or species (BCID-FP panel) from positive blood culture. After this study was completed, the ePlex BCID panels have achieved CE-IVD marking  of adult patients sampled from January to October 2017 at CHU UCL Namur Mont-Godinne (CHUMG), a 370-bed tertiary university hospital comprising four intensive care units  and with a 120-bed surgical department covering subspecialties including cardiovascular thoracic surgery (lung transplantation), otorhinolaryngo-oncology, and neurosurgery. The CHUMG is part of a larger multisite institution (940 beds) resulting from the association of three hospitals serving a regional population  in the southern part of Belgium.The inclusion criteria for enrollment included nonduplicate positive blood culture episodes (one Gram-stained morphotype per septic episode per patient) with organisms visualized at Gram stain microscopy and presumptive clinically significant episodes based on the CDC definition of bloodstream infections  single . The resStreptococcus pneumoniae from the Streptococcus mitis/oralis group.The routine workflow for inclusion is summarized in Enterobacteriaceae species other than natural AmpC producers (Staphylococcus aureus (MRSA) among S. aureus strains (Enterobacteriaceae (AST-N236 cards) and by the disk diffusion method for Gram-negative nonfermenters and for all other bacteria using CLSI guidelines   by a chromogenic assay  for the detection of resistance to third-generation cephalosporin (3GCR), mostly mediated by extended-spectrum β-lactamase (ESBL) in roducers , and by  strains . Antimicidelines . The metidelines . VancomyCA, USA) .The ePlex BCID panels were performed by a microbiology laboratory technologist during daytime from 7 a.m. to 7 p.m. according to the manufacturers’ instructions. This occurred on the first positive blood culture bottle (SA or SN) from an episode of bacteremia within 1 h after Gram stain microscopy result. The choice of ePlex panel  was based on the Gram stain results. Briefly, 50 µl of the positive bottle broth was dispensed into the cartridge, which was loaded into the ePlex instrument, with results available after approximately 90 min of run time. The pathogens (species and genera) covered for identification and the resistance genes targeted by the three ePlex panels are listed in Table S1 in the supplemental material.Enterococcus and Gram negatives) on the isolate grown from solid culture but that did not generate a corresponding positive result by the ePlex system (FN) or as the detection of a resistance gene by the ePlex system that could not be detected on any of the growing colonies on agar plates (FP). The discrepancies were further investigated by performing subcultures on frozen discrepant blood culture broth aliquots for isolate recovery and by reviewing the conventional microbiological results of other blood culture samples collected during the same episode of sepsis. An FN ePlex result was eventually changed to true negative (TN) if the isolate identified in the first subculture was not recovered by the secondary subculture. An FP ePlex result was eventually changed to TP if the positive detection was subsequently recovered at secondary subculture or if it was found in at least one other blood culture bottle of the same blood culture episode. The sensitivity of each positive target by the ePlex system was calculated as TP/(TP + FN) and the specificity as TN/(TN + FP), TN being the number of samples tested negative for the target both by the ePlex panel and by culture. The overall sample agreement (both for identification and for detection of resistance determinants) was determined after resolution of discrepancies.The results from the ePlex system were compared to those obtained by standard procedures. For the identification of the pathogens, an ePlex result was categorized as true positive (TP) if confirmed on growing isolates, while a discrepancy was defined as growing organisms targeted on the ePlex panels which were not detected  or as a positive ePlex identification without the corresponding pathogen being detected or identified on culture . For the detection of the resistance determinants, a discrepancy was defined as the presence of the targeted resistance mechanism (PBP2a positivity for staphylococci and resistance-encoding genes detected for The distribution of positive blood culture episodes tested by the ePlex assays is presented in a flowchart in Listeria monocytogenes detection and one Lactobacillus detection were not confirmed by primary subculture of the samples. The BCID-GN panel correctly identified 119 Gram-negative and 7 Gram-positive (by pan-Gram-positive [Pan-GP] detections) isolates, while 4 Fusobacterium necrophorum, 1 Stenotrophomonas maltophilia, 1 Bacteroides fragilis, and 2 Pan-GP detections were not confirmed by primary subculture of the samples. The subsequent subcultures from frozen aliquots that yielded discrepant results did allow recovery of L. monocytogenes, S. maltophilia, and B. fragilis isolates that went unnoticed by initial culture. Results obtained by the ePlex thus were considered true positives for these three isolates. The other 7 positive detections by the ePlex panels not confirmed by secondary subcultures or cultured from the other BC+ bottles of the same patients were called false positives. On the other hand, 3 isolates also recovered in polymicrobial cultures with other organisms were missed by the ePlex . One S. epidermidis isolate missed by the BCID-GP panel was considered a contaminant in the setting of an S. aureus BSI. One Escherichia coli isolate was missed by the Pan-GN assay of the BCID-GP panel detecting an Enterococcus faecalis isolate. One Pseudomonas aeruginosa isolate was missed by the BCID-GN in a mixed BSI with E. coli. Regarding fungal organisms, all 6 Candida sp. isolates grown by culture and targeted by the ePlex were correctly identified by the BCID-FP panel. One isolate of Candida inconspicua not targeted by the BCID-FP panel was found in one episode of sepsis.The identification results of organisms targeted by the ePlex BCID panels are shown in Sixteen bacterial or fungal pathogens (6%) out of the 256 isolates growing from blood culture episodes were not targeted by the ePlex BCID panels. These organisms are listed in mecA signal, which was concordant with the primary subcultures in 19 samples growing a methicillin-resistant (MR) Staphylococcus isolate. Three MRSA isolates, confirmed by a positive PBP2a test result from colonies, were correctly detected as mecA-positive S. aureus by the ePlex, while the other 16 mecA-positive samples grew MR coagulase-negative staphylococci associated or not associated with methicillin-susceptible (MS) S. aureus. In one sample which detected S. aureus and mecA targets by the ePlex system, only MS S. aureus colonies were detected by primary routine culture. Subsequent subculture of this sample from a frozen aliquot yielded an MR S. epidermidis isolate in small proportion in addition to the predominant MS S. aureus colonies. One polymicrobial sample simultaneously containing MR S. aureus (MRSA) and a vancomycin-resistant E. faecium (VRE) isolate was correctly detected by the ePlex BCID-GP, giving mecA and vanA signals in addition to the detection of the two species. None of the 72 samples negative for Gram-positive resistance markers by the BCID-GP panel grew any methicillin-resistant Staphylococcus or vancomycin-resistant Enterococcus isolates. From the 122 BCID-GN valid results, four CTX-M type ESBL-producing E. coli and one Klebsiella oxytoca isolate  were also accurately detected by the ePlex system. Of the 117 samples negative for Gram-negative resistance markers by the BCID-GN panel, neither an acquired carbapenem resistance phenotype nor an acquired carbapenemase producer was found, while there was only one 3GCR E. coli isolate which carried an ESBL not targeted by the BCID-GN panel (TEM-52).The results of resistance genes detected by the ePlex BCID panels are presented in Following exclusion of invalid results and resolution of discrepant results, the performance of detection for each positive target of the ePlex panels compared to culture is detailed in Enterobacteriaceae (Candida) and the BCID-GN (pan-Gram-positive and pan-Candida) panels, consisting of targets from opposite panels and some of the most commonly found Candida species. These panels have coverage of at least 98%, 95%, and 93%, respectively, of the species encountered in BSI in Belgium  of polymicrobial infections among the 216 evaluable BC+ episodes. This could be explained in part by the higher proportion of BSI secondary to an intra-abdominal source (26%) in our hospital compared to the national data of BSI in Belgian hospitals  . This positive trend suggests great improvement in the robustness of the assay that would be foreseen in its upcoming CE-IVD format, which has <5% invalid rates according to the manufacturer’s guidelines.Regarding the identification of the bacterial and fungal pathogens associated with sepsis, complete agreement was observed for 234 isolates out of the 263 identification results (89%), while 16 (6%) pathogens grown by culture were not targeted by the ePlex panels. Such a proportion of untargeted microorganisms was in line with the expected coverage based on the national BSI data  that were not visualized by microscopic examination. The inclusion of these Pan targets  could be of particular added clinical value, because they overcome the disadvantage of the system linking the choice of the panel to test and the Gram stain results, which requires skilled technologists who might not be available everywhere or at all times . The positivity of these Pan-GN and Pan-GP targets would trigger testing of the complementary ePlex panel on the same BC+ samples and may lead to adjusted therapy targeting the pathogens (bacteria or fungi) belonging to the other group that would not be covered if the treatment was based solely on microscopy. Other experiences did support the utility of multiplex molecular testing (containing targets of both Gram-stained groups) in better and faster recognition of polymicrobial BSI than microscopy .F. necrophorum [n = 4], Pan-GP [n = 2], and Lactobacillus [n = 1]). Although we did not attempt to perform additional molecular detection methods, these 7 positive detections called by the RUO software were analyzed by the manufacturer, indicating that all but one  would be true-negative results if the samples were assayed with the current version of released CE-IVD software . Updates to the thresholds of these RUO assays in the interpretative software could have avoided the occurrence of some false positives, but such modifications of thresholds were not available during the time of our evaluation and therefore could not be considered.Finally, positive detection on the ePlex could not be confirmed by primary culture or by secondary subculture of frozen aliquots in seven episodes , such as glycylcyclines or oxazolidinones. For Gram negatives, no carbapenemase-producing organisms causing BSI were found during the study period. The observation was in line with the epidemiological setting in our institution  and does not support the systematic use of rapid tests for the detection of carbapenemase in our routine workflow. In Enterobacteriaceae, all 5 CTX-M type ESBLs were detected by the ePlex system, but one ESBL family type (TEM-ESBL) not targeted by the BCID-GN panel was positive by BLT. Since the ePlex BCID-GN panel only targets selected resistance markers  among Gram negatives, phenotypical hydrolysis-based tests for the rapid detection of other resistance enzymes could be of complementary value , and no isolates carrying targeted resistance genes were missed by the ePlex system . Out of the 12 monomicrobial BC+ samples with ry value , and stary value , 20.F. necrophorum, Lactobacillus, and Pan-GP) showed perfect specificity. This excellent performance was in line with that achieved by other multiplex detection methods for BSI evaluated, with reported sensitivity/specificity ranging from 80% to 97%/91% to 100% for FilmArray BCID panel and 89% to 100%/93% to 100% for the two Verigene BC panels  to distinguish the S. anginosus group and E. faecium from E. faecalis is appreciated for the added information on the putative source of the BSI or for highlighting the intrinsic resistance of the identified species. For resistance marker detection, the 5 CTX-M ESBL producers would have been missed by FilmArray BCID, which targets only KPC resistance genes.After discrepant resolution, we found excellent performance of the ePlex BCID-GP and BCID-GN panels, with sample agreement, sensitivity, and positive predictive values of targeted species being found in 94% or more episodes for both panels. The vast majority of positive targets (32/35) had individual sensitivity of ≥95%, while all but 3 targets (C panels . Comparen = 7) of fungemia detections in our study prevents us from drawing conclusions about the performance of the BCID-FP panel for the detection of fungal pathogens associated with sepsis. However, one Candida inconspicua isolate identified by MALDI-TOF MS  compared to those obtained on subculture (5 h for the fastest-growing organisms) with expected substantial gain, especially in polymicrobial samples . The multiplex character of the ePlex also offers advantages for detecting polymicrobial infections in settings such as postoperative complicated abdominal infections. However, the main limit of our assessment is the observational nature of the study design without modification of our current routine workflow (microbiology workup and ePlex testing during the same daytime work hours). This prevented us from determining precisely the differences in time to results that could have been spared by the use of ePlex panels. We believe the increased speed for results could further be improved with the implementation of the system in a 24-h/7-day workflow, but this would need an appropriate antimicrobial stewardship program applied continuously to achieve significant clinical impact .In conclusion, we found that the ePlex BCID panels proved to achieve excellent accuracy and appeared very convenient for routine use  with min


The potentials of circulating tumour DNA (ctDNA) have been studied for non-invasive disease monitoring in patients with targetable mutations. However, the majority of cancer patients harbour no targetable mutations. A workflow including targeted next-generation sequencing (NGS) and droplet digital PCR (ddPCR) could be used for monitoring treatment in these patients. Thus, our aim was to evaluate the workflow for ctDNA monitoring in a cohort of non-small cell lung cancer patients.Forty patients were prospectively included. Plasma samples were collected prior to and during treatment. NGS (Ion AmpliSeq Colon and Lung Cancer panel v2) was performed on ctDNA from pre-treatment samples. The identified mutations were monitored by ddPCR in consecutively collected samples.TP53 (N=20) and KRAS (N=13). Treatment was discontinued due to non-response in 18 patients. In 16 of these, a simultaneous increase in ctDNA concentration was observed. A twofold ctDNA concentration increase confirmed in a second successive sample predicted non-response on the following imaging in 83% of patients (10/12).Mutations were detected in 21 patients. The most commonly mutated genes were ctDNA monitoring can be used for early detection of non-response in patients without targetable mutations, and therefore could supplement imaging data for treatment monitoring in this subset of patients. The potentials of circulating tumour DNA (ctDNA) for non-invasive disease monitoring have been intensively studied during the last decade. The primary investigated ctDNA source is plasma, and the small double-stranded DNA fragments are shed both actively and passively from tumour cells to the blood stream –3. Tumoua priori knowledge on the genetic constitution but often has lower detection limits and markedly shorter workflows than NGS making this approach useful for monitoring. The combination of these methods could present a workflow incorporable in the daily clinical routine that meet the needs associated with monitoring treatment in patients with unknown genetic constitution of their tumour.Recent advances in next-generation sequencing (NGS) and droplet digital PCR (ddPCR) have facilitated the detection as well as the quantification of ctDNA. NGS enables identification of multiple genetic alterations in each patient and is optimal for revealing the genetic constitution of the tumour. In contrast, ddPCR requires EGFR) and rearrangements of the anaplastic lymphoma kinase (ALK) during treatment with targeted therapies [EGFR mutations using ctDNA, studies have found correlation between decreasing ctDNA levels and tumour response on conventional imaging [EGFR mutations in ctDNA are cleared to an undetectable level may be predictive of response to EGFR-TKI therapy [EGFR and ALK wild type. These patients have no targetable mutations and no information on the genetic constitution of their tumours exists. In combination, these facts render monitoring of ctDNA less straightforward in this patient subset and could explain why information on the clinical utility of ctDNA analysis in these patients lacks. However, given the poor prognosis and lack of efficient second- and third-line treatment of this large patient subgroup, it is highly warranted to find a method that can lead to earlier detection of non-response and rapid discontinuation of ineffective treatment.In non-small cell lung cancer (NSCLC) patients, ctDNA monitoring has primarily been investigated in the patient subset with drug-targetable mutations present in tumour tissue. Thus, studies have focused on detecting and monitoring alterations in epidermal growth factor receptor  and KRAS (N=13). The median number of alterations identified in each patient was 2 (range 1-5). In the baseline sample, 93% (38/41) of the mutations were verified by ddPCR. We directly compared the allele frequencies obtained from sequencing and ddPCR and found median ratio of 0.95  of NGS compared to ddPCR, suggesting good quantitative agreement between the methods  and an ERBB4 mutation . Though, the ERBB4 mutation reappeared later in the treatment course (see Sequencing of the baseline sample succeeded in 36/40 patients (90%). At least one alteration was detected in 21 of the 36 patients (58%). In total, 41 alterations were identified with a median allele frequency of 2.5% (range: 1.0–71.1) , radiological progression was detected on the following imaging. Among these 10 patients, eight patients already had a twofold increase in ctDNA concentration in the first blood sample collected after only a median of 21 days (range 14-34) of treatment. The results of the ctDNA monitoring along with changes in tumour size measured on CT scans are illustrated for each of these ten individual patients in Figure P=0.0756)  was found. The tumour size was correlated to the ctDNA concentration found in the sample drawn at the time of the CT evaluation. If various mutations were identified in a patient, the mutation with the highest concentration at progression was used. The correlation approached but did not meet statistically significant  Figure .In this prospective study, we investigated whether a clinically relevant workflow including a targeted NGS panel and ddPCR could be utilised for treatment monitoring in a cohort of NSCLC patients with unknown genetic constitution of their tumour. We used a targeted 22-gene NGS panel including the most frequent mutations known in NSCLC which has proven useful for mutation detection in plasma ctDNA in previous studies , 24, 25.ALK [EGFR mutation-positive TKI-treated patients [EGFR and ALK wild-type patients there are very often more than one mutation with known functional impact [EGFR and ALK wild-type patients as we observed an increased ctDNA concentration from baseline to treatment discontinuation in 16 out of the 18 patients in whom discontinuation of treatment was due to radiological non-response. Interestingly, we found that a twofold ctDNA concentration increase confirmed in a second successive sample was an early indicator of progression in a substantial sub-set of the patients (10 out of 12 patients). In addition, an increase in the first available blood sample collected median 21 days after initiation of treatment predicted progression in 8 of these 12 patients. These findings suggest that a sustained increase in ctDNA could be a very important indicator of disease progression and could indicate that an expedited radiological evaluation should be performed.Several studies have been conducted in ALK  and, esppatients , 15, 17 l impact , and it Important issues to consider regarding the use of ctDNA analysis is the cost and time of the procedure and thereby the feasibility of transferring the method into the daily clinic. Results are required within a few days for optimal use of ctDNA analysis for treatment monitoring by clinicians. However, an important challenge with NGS has been the need for time-consuming bioinformatics. With the development of targeted NGS, where a panel of predefined clinically relevant genes is sequenced, the laboratory workflow has been markedly reduced, making incorporation into the daily clinical routine achievable. In combination with ddPCR, this set-up offers a relative inexpensive and fast approach with a workflow of only one to four days, which is doable in a daily clinical setting.EGFR patients was treated with erlotinib while the current recommendations advice chemotherapy or immunotherapy in first- and second-line. However, non-targetable mutations were used for the monitoring leaving the treatment extraneous, and our results are comparable with studies of patients receiving other treatment regimens [This cohort of wild-type regimens , 27.EGFR mutation (L858R). Concentrations of ctDNA were measured at initiation of therapy and again after 8 weeks of treatment, and in line with us, they found a correlation between the ctDNA concentration change and tumour response in the 10 patients with detectable ctDNA. This indicates that our findings are applicable to other treatment regimens.In a recent study by Cabel et al. , the preA correlation approaching but not meeting statistically significance between tumour size and ctDNA concentration was observed. This correlation has been observed in several other studies –12, and EGFR mutations detected in ctDNA by ddPCR at three time-points within one day (N=22) [This study is the first of its kind and strengthened by the prospective nature. We evaluated a homogeneous patient cohort receiving the same treatment and with complete clinical data. Yet, the study has some limitations to consider. The number of patients was limited and our results are primarily hypothesis generating. Moreover, the biological variation of ctDNA is not well studied and could potentially influence our results. A recent study found no significant alterations in y (N=22) . HoweverAdvanced-stage NSCLC patients were prospectively enrolled in a single-centre study at the Department of Oncology, Aarhus University Hospital, Denmark from April 2013 until August 2015. Details on the study design have been published previously . In shorPatients were treated with erlotinib in a palliative setting, and treatment was continued until radiological or clinical progression, unacceptable toxicity or death. A baseline computed tomography (CT) scan of the chest and abdomen was performed on all patients before erlotinib start. An evaluation CT scan was performed after 9-11 weeks of treatment or earlier on clinical indication. During the treatment course, additional CT scans were performed every 12 weeks. Neuroimaging with magnetic resonance imaging (MRI) was performed on clinical indication. Radiological response was quantified as percentage change in sum of longest diameter of target lesions according to Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 criteria . Tumour EGFR mutation testing was performed in all patients with adenocarcinoma . During the inclusion period, ALK rearrangement testing was incorporated as part of the routine diagnostic work-up in adenocarcinoma patients and was performed using IHC (screening) and Fluorescence in situ hybridization (verification).As part of the routine diagnostic work-up, In this present study, patients were considered eligible for inclusion if the following criteria were met: EGFR wild-type tumour, at least two blood samples available, a response evaluation CT or MRI scan had been performed and no more than three weeks between the last blood sample and the radiological evaluation. Inclusion of patients is illustrated in Blood samples collected from anonymous blood donors were used for investigating limit of detection (LoD) of the ddPCR. These were collected from the blood bank at Aarhus University Hospital.B2M) gene by ddPCR as described in Ten millilitres of blood were collected in an EDTA-containing tube at each blood sampling. Within 2 hours of withdrawal, samples were centrifuged (1400 g for 15 min). Plasma was isolated and subsequently frozen at -80°C until further analysis. Cell-free DNA (cfDNA) was extracted from a range of 1-2 mL of patient plasma and from 4-5 mL of donor plasma using the QIAamp Circulating Nucleic Acid kit  according to the manufacturer’s protocol. Elution of cfDNA was performed in 100 μL elution buffer supplied with the kit. The amount of cfDNA was quantified by measuring the beta-2-microglobulin , a CE-IVD-marked version of the Ion Ampliseq Colon and Lung Cancer Research Panel v2 (referred to as the Colon and Lung panel), was used to prepare sequencing libraries from cfDNA (1.1-10 ng). Sample preparation was performed using the Ion Chef™ Instrument, and sequencing was conducted on the Ion Personal Genome Machine® (PGM™) System . Each Ion 316™ v2 BC chip was loaded with eight samples. If samples did not meet the criteria of mean depth ≥ 2000, they were disqualified. Variants were called if they were exonic, previously observed, reported to COSMIC, and if the allele frequency ≥ 1% . Benign The performances of each of the 92 amplicons were evaluated by investigating the mean coverage over the 36 patient samples successfully sequenced. Five amplicons generally performed poorly and did not meet the defined mean coverage . These were excluded from further analyses. The majority of the sequence data has been previously published [The ddPCR reactions were performed using the QX200™ AutoDG™ Droplet Digital™ PCR System . Conditions for ddPCR reactions are described in th august 2017). The estimate of median OS was calculated using the Kaplan–Meier method and the median follow-up time by the inverse Kaplan–Meier method. All tests were two-sided, and P-values less than 0.05 were considered to be statistically significant. Statistical analyses and graphic artwork were performed using STATA version 13 (Stata Corporation).To investigate the correlation between measurements obtained from NGS and ddPCR, llinear regression was performed. Agreement between methods was investigated using Bland-Altman plots. Log-transformed data was back-transformed to achieve median ratios. Spearman’s correlation was used to evaluate the correlation between ctDNA concentration and tumour size. Overall survival (OS) was determined as the time from start of erlotinib treatment until death of any course or last follow-up date (14In this study, we demonstrated that quantitative ctDNA monitoring by targeted NGS and ddPCR could be used for early detection of non-response in patients with unknown genetic constitution of their tumour. Particularly, a twofold ctDNA concentration gain in two successive blood samples was found to be an early indicator of progression. If these data are validated, ctDNA analysis could in the future supplement imaging data acquired by CT scans for treatment monitoring in this patient subset. While the prognosis is generally poor for this patient subgroup, a non-invasive method that could improve identification of non-responding patients early after treatment initiation could lead to earlier discontinuation of ineffective treatment. This will markedly reduce the risk of unnecessary toxicity and increase the chance of receiving other potentially effective treatments before worsening of performance status.


With the advancement of antiretroviral therapy and improved life expectancy, women living with HIV/AIDS are enjoying a better sexual life. Yet, the consistent utilization of contraceptive in such patients is highly recommended. There is paucity of data regarding contraceptive use among HIV-positive and negative women in Ethiopia. The present study aimed at examining the use of contraceptives among HIV-positive and HIV-negative women in Ethiopia.p-value of 0.05 was deemed significant with corresponding 95% confidence intervals.A comparative cross-sectional study was conducted among HIV-positive and HIV-negative women attending family planning Clinic of Gondar university referral hospital between January 2016 and August 2017. Descriptive statistics were used to present categorical data and Pearson’s chi-square test was done to examine differences in the utilization of contraceptives between HIV-positive and HIV-negative women. Kaplan Meier test was also carried out to determine the incidence of unintended pregnancy. A p < 0.05]. Unlike HIV-negative women (2.9%), HIV-positive (28.4%) women reported a higher rate of intrauterine device use. Male condom was used more commonly in HIV-infected women (26.7%) as compared to HIV negative (3.9%) women .A total of 894 participants consisting of 314 HIV-positive and 580 HIV-negative women were included in the study. The rate of previous unintended pregnancy was 280 (31.3%) in HIV-negative women and 115 (12.9%) in HIV-infected women. Women who routinely utilized contraceptives were more likely to avoid unintended pregnancy [log rank: 2.89, Intrauterine contraceptive device was reported to be the most commonly used contraceptive method in HIV patients. Further, unintended pregnancy was relatively common in women with low contraceptive practice. The use of dual contraceptives should be advocated for HIV-positive women so as to protect unintended pregnancy and curtail the transmission of HIV. Globally, 36.7 million people were living with HIV/AIDS at the end of 2016 and 50% of all adults with HIV infection were women .. However, previous studies reported low rate of contraceptive use in the same set-up. This discrepancy might be due to the changing attitude of clients towards family planning and the decline of risk of HIV transmission through interventions at antenatal care units and HIV follow-up clinics. Comparative study has been obtained from Addis Ababa, Ethiopia, in which about three-fourth of HIV infected women and nearly two-third of HIV non-infected women utilize contraceptive . ContracOur study revealed that, HIV positive patients usually utilized Intrauterine contraceptive device (IUCD) as compared to HIV negative clients who instead preferred hormonal contraceptives.. This might be due to the fact that hormonal contraceptives are expected to have untoward possible pharmacokinetic interactions between antiretroviral drugs which directs the preference to intrauterine device and condom use for HIV- positive clients on HAART . In addiThe utilization of dual contraceptive methods found to be frequently observed among HIV-positive patients. None of the respondents reported dual contraceptive use in the HIV non-infected group. The parallel application of condom use along with other methods is warranted to reduce the risk of unwanted pregnancy, transmission of HIV to a non-infected couple, to minimize transmission of drug resistant virus to an individual with HIV infection , 20. AltThis study also illustrated that women who had unintended pregnancy used contraceptive methods occasionally. Even though Ethiopia has a promising achievement on contraceptive coverage, it is still difficult to control unintended pregnancies. This might be due to the lack persistence with the modern contraception. In addition, the low literacy and the high level of unemployment complicate the odds of unintended pregnancy in developing countries.In general, the present study provided a comparative evidence regarding the pattern of contraceptive use among HIV-infected and non-infected women. However, its findings are only interpreted in the light of the following limitations. First of all the study is limited to single center and qualitative data on the reasons to choose a certain type of contraceptive is not included. Therefore, we recommend a multicenter study including large number of subjects and mixed type of study design to incorporate qualitative aspects. Further, the present study also highlighted that significant number of patients were not on HARRT. The reason for low consumption of the HARRT should be investigated.Intrauterine contraceptive device was reported to be the most commonly used contraceptive method in HIV patients as compared to HIV negative counter parts. Male condom use was found to be higher among HIV positive patients as compared to HIV negative women. Nearly, one-half of HIV negative clients use injectable contraceptive methods as compared to around a quarter in the HIV positive women. Further, unintended pregnancy was relatively common in women with low contraceptive practice. The use of dual contraceptives condom use in particular, should be advocated for HIV-positive women so as to protect unintended pregnancy, HIV transmission to partner, other strain and sub type HIV infections. Education and Communication campaigns should be strengthened in the community about reproductive health services in general and family planning services in particular. Access to quality contraceptive methods should be scaled up in community.


The aim of the present study is to evaluate the expression of microRNA (miRNA) in peri-implant soft tissue and to correlate epigenetic information with the clinical outcomes of the implants up to the five-year follow-up. Seven patients have been rehabilitated with fixed screw-retained bridges each supported by implants. Peri-implant bone resorption and soft tissue health parameters have been recorded over time with a five-year follow-up. Mini-invasive samples of soft peri-implant tissue have been taken three months after implant insertion. miRNA have been extracted from cells of the soft tissue samples to evaluate gene-expression at the implant sites by microarray analysis. The epigenomic data obtained by microarray technology has been statistically analyzed by dedicated software and compared with measured clinical parameters. Specific miRNA expression profiles predictive of specific clinical outcomes were found. In particular, some specific miRNA signatures appeared to be “protective” from bone resorption despite the presence of plaque accumulation. miRNA may be predictors of dental implant clinical outcomes and may be used as biomarkers for diagnostic and prognostic purposes in the field of implant dentistry.  Currently dental implants are widely used for fixed rehabilitation of partially or completely edentulous patients and demonstrate predictable outcomes. However, the biological mechanisms of possible complications and implant failure are not clear and are debated in the dental scientific community.In particular, the specific endogenous characteristic of the host  may strongly affect the success of the rehabilitation. Modern innovative technologies using molecular biomarkers may help in identifying individual susceptibility.In a previous paper  we provimiRNAs are small noncoding RNAs (ncRNAs) of approximately 22 nucleotides responsible for specific regulation of gene expression in a post-transcriptional manner. They are the main regulator of gene transcription and bear relevance in predicting clinical outcomes. Indeed, only less than 5% of expressed genes producing messenger RNA is really translated into proteins while microRNAs are fully functionally active in cell cytoplasm ,4.A major problem is currently represented by the lack of a predictive marker to personalize risk after peri-implant surgery. Indeed, some authors have suggested peri-implant soft tissue biotype as a risk indicator of peri-implants tissue disease ,6. HowevMolecular biomarkers are already extensively used in medicine to accurately classify each patient according to their real risk of developing complications. This approach, referred to as personalized or “theranostic” medicine, has been already well developed in oncology but is still under development in dental science. Molecular predictive biomarkers should reflect the pathogenic process modulating clinical outcome in the target tissue of the diseases. Furthermore, the molecular alteration investigated may occur years before the appearance of the related clinical consequence thus opening the possibility of preventing adverse clinical outcomes before their onset.At this regard, miRNAs are currently identified as predictive biomarkers for degenerative diseases, because they do not undergo a post-transcriptional selection, being themselves the controllers of gene transcription. Consequently, compared to genomic or transcriptomic biomarkers, miRNA expression has by far a higher probability of being related with clinical variables representing a new tool for predictive medicine.On the other side, several factors, including the implant surface , might aThe role of miRNA in implant dentistry has been established. In a clinical trial, we demonstrated that miRNA expressed by peri-implant tissues are related with the clinical outcome . HoweverThe aim of the present study is to examine the predictivity of miRNA profiling to categorize patients according to risk categories of developing more or less probably adverse consequences of oral implants on a long-term basis.In particular, the expression of micro-RNA (miRNA) in peri-implant soft tissue will be correlated with the clinical outcomes of dental implants recorded up to the five-year follow-up.Between January 2013 and July 2014, 7 patients  who were referred to the Division of Implant and Prosthetic Dentistry of the Surgical Sciences Department (DISC) of the University of Genoa  and that required the insertion of two implants into an edentulous area were recruited if they met the following criteria: desire to be treated with fixed prostheses supported by dental implants and good general health without any contraindications for undergoing oral surgery and the related prosthodontic protocols.Exclusion criteria included: an uncontrolled medical condition such as diabetes mellitus, immune suppression, intravenous bisphosphonate medication, oro-facial cancer, chemotherapy or head and neck radiotherapy, infarct during the preceding 6 months, heavy smokers (≥20 cigarettes/day). Healing time since tooth extraction in the intended implant sites had to be longer than 1 year. Post-extractive implants and implant sites that required a bone graft were excluded.The present report was conducted in accordance with the Helsinki Declaration and was approved by the local Scientific Ethical Committee of the University of Genoa. Patients were instructed in detail on the planned clinical procedure and signed an informed consent before being scheduled in the experimental protocol.Each patient of the sample received two adjacent implants: Osseotite implants  with a dual acid-etched (DAE) surface in the apical portion and a machined coronal part (hybrid implants), and Full Osseotite implants (Biomet 3i) completely DAE (DAE implants).All the implants had identical macro- and micro-structure, external hexagon connection, a 4 mm diameter, a 10–13 mm length (depending on available bone). Implants were placed with a single-stage delayed loading approach and were assigned specific codes for blinding. The healing abutments remained in situ up to the 4-month follow-up appointment when impressions of the implants were taken to fabricate the fixed prostheses. No bone nor soft tissue grafting procedures were performed at the implant sites. Patients were accurately instructed on the hygienic procedures to be followed and were prescribed adequate dietary guidelines in order to ensure optimal recovery.Screw-retained prostheses, provided with a metal framework and composite resin veneering material, were delivered 5–6 months (mean 24 weeks) after surgery . Spontaneous bleeding, probing depth (PD), bleeding on probing (BoP), plaque index (PI), and suppuration were recorded at 3 and 6 weeks and at 3-, 6- and 12-months post-surgery and then at the five-year follow-up appointment. At the five-year appointment the fixed prostheses were unscrewed in order to record the parodontal parameters. PD was recorded in four points for each implant  using a non-metallic probe. BoP was simultaneously evaluated in four points for each implant . PI was measured at four points for each implant using an erythrosine gel .Technical and biological complications were also recorded.Intraoral radiographs were taken at the time of implant placement and at 3-, 6- and 12-months post-surgery and then at the five-year follow-up appointment. The implant–abutment interface was used as the reference point for interproximal bone level measurements over time. Bone resorption (BR) over the duration of the study was assessed from these reference points to the most coronal bone at the mesial and distal aspects of each implant.t-test.The above-mentioned clinical parameters recorded at the five-year follow-up appointment were statistically analyzed and differences between DAE and hybrid implants were evaluated by Student’s After 3 months from surgery, a sample of soft tissue containing both epithelium and connective tissue was collected: a mini-invasive sample of soft tissue (diameter < 3 mm) surrounding each implant was taken using a surgical blade to be histologically analyzed by miRNA microarray at the Department of Health Sciences (DISSAL) of Genoa University.MicroRNA microarray analysis is an innovative technology, which allows the evaluations of thousands of genes in a single experiment. MicroRNAs (miRNA) have been extracted from cells of the soft tissue samples to evaluate gene-expression at the implant sites by microarray analysis.miRNA expression data was evaluated in the peri-implant tissue as previously reported . The expThe results of miRNA microarray have been compared with clinical parameters measured over time for five years since miRNA analysis. The previously published paper reported correlation between miRNA data and one-year outcomes (T0). The present paper reports the correlation between miRNA data and clinical outcomes recorded at the five-year follow-up appointment (T1).mean bone resorption (BR): normal/increased mean probing depth (PD): normal/increased mean plaque index (PI): low/high mean bleeding on probing (BOP): yes/no All clinical continuous variables recorded at the five-year follow-up have been turned into categorical ones depending on the following criteria arbitrarily chosen by the authors:On the base of these parameters, several categories of implants  have been determined. In addition, implants with the contemporary presence of BOP, altered PD and bone resorption were considered affected by peri-implantitis (PITI).Implant sites were also divided in two categories according to the periodontal biotype: thin (TH) and thick (TK). To evaluate the biotype, a periodontal probe was placed into the facial aspect of the peri-implant mucosa. The peri-implant biotype was categorized as thin if the outline of the underlying periodontal probe could be seen through the gingiva and thick if the probe could not be seen .Relationship between miRNA and clinical events were tested by Genespring software . During the five-year follow-up, there were no dropouts and all the patients attended the five-year recall appointment. No implant failure occurred resulting in an implant cumulative survival rate (CSR) of 100%. No technical complications were detected, resulting in a prosthodontic CSR of 100%. All the patients anecdotally reported to be satisfied with their implant rehabilitation.Five patients presented a thin periodontal biotype and two a thick periodontal biotype.Mean bone resorption at the five-year follow-up appointment was 1.98 mm : 1.8 mm and 2.2 mm for implants with a machined coronal portion and DAE implants respectively. No patient presented spontaneous bleeding or suppuration. No statistically significant differences were found between hybrid implants and DAE implants for the clinical variables recorded at the five-year follow-up. Mean and median values of the other soft tissue health parameters are reported in An overall view of the miRNA alterations as related to clinical outcomes occurring in the five years after surgery was performed using scatter plot  and volcFor each clinical variable, the predictor miRNAs were identified by volcano plot analysis .BR: 48 miRNA were significantly  dysregulHierarchical cluster analyses were used to classify patients according to their miRNA expression profile as related to BR in the five years following implant surgery. Implants with a normal miRNA expression level  are located in the left part of the cluster, while implants with a decreased miRNA expression (blue) are located in the right part of the cluster. A severe alteration of miRNA expression at three months occurs in patients with BR ≥ 2 mm at the five-year follow-up appointment a. These PD: 29 miRNA were significantly . They plBOP: 66 miRNA were significantly . They plPITI: 10 miRNA were significantly . They plThe number of altered miRNAs as related to BR, peri-implant probing depth (PD) and BOP recorded at the five-year follow-up was dramatically increased (T1) as compared to T0 . These fp-value = 0.0986; Fischer’s exact p-value = 0.2308). Conversely, miRNA expression as focused on the cluster of PITI miRNA predictors  or thin (high-risk). The ability of periodontal biotype to predict the clinical outcome of dental implants in the following five years was tested by chi square analysis . Of the tors see  was ableIn fact, PCA of miRNA expression at T0  distinguThe miRNA pattern was compared in patients undergoing plaque accumulation (high PI) between those with or without BR. The aim of this approach was to identify miRNA protecting peri-implant tissue from the adverse effect of plaque accumulation. Obtained results are reported in Both scatter plot and volcano plot identified differences between these two groups of patients. The miRNAs that protected peri-implant tissue from BR despite the presence of plaque deposits are reported in miRNA are biomarkers of several diseases and in recent years, some studies have begun to focus on the regulatory role of miRNAs in the inflammatory response ,11.miRNA are fundamental in controlling cell function and also influence osteoinductive pathways and inflammation in several ways. On the other side, miRNA expression is influenced by many factors and some authors have suggested that different implant surfaces may condition miRNA expression ,13. However, studies investigating the role of miRNA in implant dentistry are still scarce. Some in vitro studies are present ,14 and sA recent review  found onOur previous study suggested that miRNAs play an important role in conditioning clinical outcomes after implant insertion . SpecifiThe present study with a five-year follow-up confirmed the potential role of miRNA in patients’ susceptibility to peri-implant disease and the potential role of miRNAs as predictors of clinical outcomes of dental implants. In fact, miRNA expression at three months of healing was predictive of the clinical variables recorded at five years of follow-up.The miRNA mostly involved in the prediction of clinical outcomes was miR-548. Indeed, this miRNA predicted PD and BOP . This miThe definition and etiology of peri-implant disease are controversial . In partIndeed, a specific miRNA expression profile was found in patients with normal BR at five years, despite the presence of plaque accumulation , and miRNA expression analysis was far more accurate than periodontal biotype in predicting PITI occurrence in the five years after implant surgery . The present investigation shed light on possible biomarkers that may be predictable of dental implants clinical outcomes. The variables that can compromise implant therapy are numerous  and the miRNA can be considered a useful tool for only evaluating the “predisposition” of the peri-implant tissues to a stable osseointegration process. The integrated findings derived from clinical and laboratory analysis helped with understanding the biological mechanisms affecting peri-implant tissue healing and maintenance. This will help clinicians in the choice of a better treatment plan for a predictable success of dental implant rehabilitations. In fact, specific miRNA expression profiles are predictive of specific clinical outcomes in implant dentistry and may be used as diagnostic and prognostic tools, thus affecting therapeutic strategies. These findings can be tools for a more personalized medicine approach in the treatment of edentulous patients using dental implants.Moreover, the possible identification of “protective” miRNA also discloses the possibility of using miRNAs in future studies as therapeutic agents in implant dentistry. miRNAs might be used as possible drugs or coatings for implant surfaces, in order to improve healing and maintenance of peri-implant tissues.The results of the present clinical trial suggest that miRNA may be predictors of dental implants clinical outcomes and may be used as biomarkers for diagnostic and prognostic purposes in the field of implant dentistry.


The regenerative medicine field has been revolutionized by the direct conversion of one cell type to another by ectopic expression of lineage-specific transcription factors. The direct reprogramming of fibroblasts to induced cardiac myocytes (iCMs) by core cardiac transcription factors  both in vitro and in vivo has paved the way in cardiac regeneration and repair. Several independent research groups have successfully reported the direct reprogramming of fibroblasts in injured myocardium to cardiac myocytes employing a variety of approaches that rely on transcription factors, small molecules, and micro RNAs (miRNAs). Recently, this technology has been considered for local repair of the pacemaker and the cardiac conduction system. To address this, we will first discuss the direct reprograming advancements in the setting of working myocardium regeneration, and then elaborate on how this technology can be applied to repair the cardiac pacemaker and the conduction system. The regenerative medicine field has been revolutionized by the direct conversion of one cell type to another by exogenous of over expression of lineage-specific transcription factors (TFs). The direct reprogramming field finds it origin in the ground-breaking work by Yamanaka and colleagues who demonstrated that pluripotent stem cells (PSCs) can be generated by forced overexpression of a combination of transcription factors  in somatic cells, such as fibroblasts . After tIf’) and the associated so called ‘Membrane-clock’, and the ‘Ca2+-clock’ mechanism that was later integrated with the former in the coupled-clock concept . Al. Al+ iCMSimilar to Ieda et al., S. Protze et al. screened for triplet combinations of 10 candidate factors in comparison with GMT to generate iCMs from Myh6-tdTomato reporter mice using mouse embryonic fibroblasts (MEFs) and neonatal CFs. The read-out for reprogramming efficiency was activation of a broader range of cardiac-specific genes. This screen yielded Tbx5, Mef2c and Myocardin (TMM) as the most potent combination for iCM generation with a higher activation of multiple cardiac genes . However, this combination did not result in the generation of beating CMs . AnotherThe Olson group additionally screened for protein kinases that can boost cardiac reprogramming by altering the relevant intracellular signaling pathways. Their protein kinase library results showed that TTF reprogramming was accelerated by addition of Akt1 to GMTH (GMTHA). The mechanism of action is not entirely understood but Akt1 seems to enhance fibroblast reprogramming through pathways involving IGF1, P13K, mTORC1 and Fox3a signaling . The samIn the context of reprogramming, small molecules are typically defined as chemical compounds that have the potential to alter cellular signaling pathways. This can be achieved by their exogenous in vitro or in vivo application (for a certain duration of time) to cells that are in the process of reprogramming. Advantages of the use of small molecules include their ease of application and strict temporal control, which make them highly effective in optimizing the process of TF-mediated reprogramming. Moreover, there is emerging knowledge on guiding the differentiation processes of PSCs and cardiac progenitor cell-derived cells. This has illustrated the usefulness of small molecules in modulating signaling pathways that are important for cardiomyocyte development and maturation ,28,29. HIn an effort to further identify highly effective small molecules and/or potential novel pathways Mohamed et al. employed a high throughput screen using a library of 5500 chemical compounds. This screen was ultimately narrowed down to two potent small molecules, the previously identified TGFβ inhibitor SB431542 and the Wnt signaling inhibitor XAV939. GMT overexpression combined with a cocktail of these two inhibitors (GMTc) accelerated in vitro reprogramming as compared to GMT alone. Beating cells were now observed as early as eight days post transduction (as compared to 6–8 weeks with GMT) and the reprogramming efficiency was increased by 8-fold. In vivo administration of GMTc in a mouse model of MI increased the ejection fraction (EF) one week after its application, as compared to GMT alone (EF GMTc ~45% vs. EF GMT ~35%) and persisted throughout the 12-week study (EF GMTc ~40% vs. EF GMT ~35%). Although this screen failed to identify novel pathways, it provided a platform for further high-throughput testing and emphasized the robustness of the TGFβ and Wnt signaling pathways .Myh6 and Nppa) revealed demethylation of CpGs. These results clearly indicate enrichment in cardiac genes and suppression of fibroblast specific genes . Ad. Ad2+ osThe group of Singh and colleagues showed that addition of Hand2 and myocardin or MiR-590 to the GMT cocktail reprogrammed porcine and human fibroblasts into iCMs. Approximately 5% of the iCMs exhibited cTnT expression together with MYH6 and TNNT2 upregulation . The mecFinally, Fu et al. used human H9ESC derived fibroblasts (containing a α-MHC-driven mCherry reporter), neonatal and skin fibroblasts for additional iCM optimization studies. They reported effective reprogramming for a five-factor combination containing GMT, ESRRG and MESP-1. Interestingly, addition of MYOCD and ZFMP2 further enhanced reprogramming efficiency. This seven-factor combination resulted in cTNT expression in approximately 5% of iCMs. Global gene expression data of cTNT+ iCMs showed a shift from fibroblasts to CM profile . The samThe progress made by direct reprogramming of fibroblasts using a combination of core cardiac TFs, small molecules, and miRNAs encourages the use of this technology to regenerate the pacemaker and the CCS. Previous findings indicate that GMT-based reprogramming generates mostly ventricular-like CMs , while GThe TFs Shox2, Isl1, Tbx3, and Tbx18 play a crucial role in SAN formation during embryonic development . A prereThe above-mentioned studies provide us with experimental evidence on how forced expression of specific TFs in CMs drives them towards pacemaker-like phenotypes. This knowledge may also be applied to directly reprogram fibroblasts towards pacemaker-like cells. A first attempt in this direction has been reported by Nam and colleagues. To optimize their approach, they used the Hcn4-GFP reporter mouse line (with high GFP expression in the SAN) to screen a pool of 20 TFs . With thCre/+; NICD (Notch intracellular domain) transgenic mice, Notch-activated hearts exhibited an enlarged AVN region. Histological and electrophysiological examination revealed the presence of additional accessory conduction pathways between the atria and ventricles leading to ventricular pre-excitation [Cre/+; NICD) drives CM transdifferentiation towards a CCS phenotype [In addition to efforts towards the generation of pacemaker cells, attempts have been made to induce other pacemaker and CCS phenotypes by direct reprogramming. Notch signaling has been implied to be crucial in CCS development. Rentschler and colleagues have used a loss-of-function and gain-of-function approach to demonstrate the crucial role of Notch signaling in AV canal (AVC) formation during CCS formation in mouse . Mlc2vCrcitation . In a suhenotype . Gene exhenotype . These thenotype . Moreovehenotype . Moreovehenotype . Taken tSeveral viral vector systems have been used for the delivery of lineage-specific factors to support direct reprogramming in vitro and in vivo. When these vectors are considered for repair of the pacemaker and the CCS, they should ideally (i) target specific components of the CCS, (ii) have absent-to-minimal pathogenicity, (iii) be none or minimal immunogenic, (iv) harbor limited species-specificity , and (v) have a high transduction efficiency. There have been significant advancements in developing viral vectors that can efficiently deliver gene of interest into target cells. Adenoviral vectors have been a popular delivery method for temporary gene expression in the heart and have successfully been used to reprogram embryonic fibroblasts towards pluripotent stem cells . Adeno ADirect reprogramming holds substantial promise for the regeneration of myocardium and restoration of cardiac pacemaker and CCS function . To achiThe embryonic development of SAN and CCS clearly involves highly regulated and precisely controlled stage-specific TF expression, signaling molecules and molecular pathways ,60,61. TA relatively un-explored aspect in direct reprogramming is the translation to large animal testing. Given the reasonable efficacy of Sendai-GMT-based reprogramming, it would be useful to learn more about how effective such a strategy is in a clinically relevant large animal model. With regard to large animal testing of direct reprogramming approaches aimed at regeneration of the pacemaker and the CCS, the recent Tbx18 study  has provThere are several potential target patient populations being considered for initial clinical studies. An obvious target cohort includes patients with acute MI as this has been the setting where direct reprogramming has been most extensively studied. With regard to dysfunction of the pacemaker and the CCS, demand pacing of the ventricle is expected to become feasible with currently available technology. Similar to other gene therapy-based approaches to biological pacing ,73,74, tCurrently available experimental data shows significant promise for the general concept of direct in vivo reprogramming of fibroblasts. However, current methods need further refinement aiming for higher reprogramming efficiencies and more pure phenotypes to better recapitulate cardiac function in settings of myocardial repair and regeneration of the pacemaker and CCS.


Handball is a strenuous body-contact team sport that places high loads on the knee joint. Anterior cruciate ligament (ACL) tear is one of the most devastating injuries that any handball player can suffer, and female athletes are at particular risk due to their intrinsic anatomical, hormonal, neuromuscular and biomechanical characteristics. The purpose of this study was to analyze the horizontal jumping biomechanics of female elite handball players with or without previous ACL reconstruction.Twenty-one female participants (6 with previous ACL reconstruction and 15 uninjured controls) were recruited. Two horizontal hopping tasks were evaluated using inertial sensor unit (ISU)-based technology to assess jumping biomechanics through a direct mechanics-based approach.P < 0.05) reduction in the unilateral triple hop for distance compared with the healthy controls. Furthermore, during the initial propulsive phase of the unilateral cross-over hop, the control participants generated significantly (P < 0.05) higher force values in the mediolateral direction (the X axis) with their dominant limb compared with the ACL-reconstructed (ACL-R) limb of previously injured participants.The athletes with previous ACL reconstruction demonstrated a significant (Three-dimensional horizontal jumping biomechanics analyses using ISU-based technologies could provide clinicians with more accurate information regarding the horizontal jumping biomechanical patterns among elite handball female athletes. Furthermore, several mechanical alterations could still be observed among those players who had undergone previous ACL reconstruction, even when several years have passed since the original ACL injury. Handball is a body-contact team sport that elicits high-intensity maneuvers such as abrupt changes in direction, velocity and sudden single leg landings , 2. The The reported incidences of ACL injury for male and female handball athletes are approximately 0.24 and 0.86 injuries per 1000 h of exposure, respectively . TherefoThe clinical relevance of ACL injury does not rest solely on the ligament disruption itself; the functional implications of concomitant associated knee injuries for the athletes’ function can play an important role in the clinical prognosis of the athlete after injury . AdditioThis ambiguity may expose the athlete to both higher risk of graft rupture and a new injury of the healthy contralateral knee . Thus, tFunctional performance evaluations have traditionally been highlighted as a key point in relation to decisions regarding resuming play after ACL injury , 13–16. Several biomechanical and neuromuscular impairments at the trunk, hip and knee joint levels have been widely reported in the literature as a result of motion analysis and inverse mechanics procedures during the abovementioned and other sport-specific tasks –21. UnfoIn relation to handball, Myklebust et al.  observedThe aim of this study was to examine the biomechanical differences in horizontal jumping between elite female handball players with previous ACL reconstruction who had returned to their previous sport activity, and level-, sex-, and age-matched pairs of control counterparts. The hypothesis of the present research was that the ACL-R players would present lasting biomechanical alterations in terms of greater supported three-axis peak forces during single-limb horizontal jumping maneuvers compared with their control counterparts, despite have continued with elite competition for several years after the original ACL injury.A descriptive case series study design was selected. The examinations were conducted at the athlete’s habitual training court. The jumping task battery included the unilateral triple hop for distance (UTHD) and the unilateral triple cross-over hop for distance (COHD). These tests have been established as reliable methods for evaluating lower limb function in relation to ACL injury in previous investigations , 30, 31.Twenty-one female elite handball players competing in their highest national division league and European championships were recruited. The sample comprised 6 athletes who had undergone ACL reconstruction, two of them bilaterally , and 15 uninjured controls . Among the athletes with bilateral reconstructions, both limbs were recorded as ACL-R limbs. The average and standard deviation of the data collection time since surgical reconstruction was 6.0 ± 3.5 years. For the control group, athletes who had sustained a previous lower limb injury lasting more than 6 weeks were excluded to avoid jumping pattern bias due to potential functional alterations resulting from severe lower extremity injury. The participants and coaches were informed in detail about the experimental procedures and the possible risks and benefits of the project, which was approved by the Ethical Committee of the Public University of Navarra and performed according to the Declaration of Helsinki.An inertial orientation tracker  was attached over the L3-L4 region of the subject’s lumbar spine and provided data on kinematic and kinetic variables such as accelerations, orientations and velocity at a sampling rate of 100 Hz. A technical explanation describing the inertial sensor-derived variables has been previously provided . Before starting the measurement, the inertial sensor unit is calibrated and the sensor axes are aligned with anatomical directions. The acceleration signal consists of gravitational and inertial components. The inertial sensor unit registers gravity as a static vertical component, in addition to the dynamic acceleration caused by changes in velocity during locomotion. The gravity component must be subtracted to estimate the dynamic acceleration. The 3D orientation data provide the position of the inertial unit with respect to the gravitational vector, allowing the calculation of the inertial component for each axis. The gravitational constant was estimated by leaving the inertial sensor unit still on a flat surface for 2 seconds. In previous studies –28, bodyThe data reported by the sensor was analyzed using direct mechanics-based procedures that considered the subject as a mechanical system and estimated the movement and actuation of forces through the center of mass displacement –39. As pZ- velocity signal was used to distinguish the boundaries between the different phases of both tasks and were considered positive when the subject moved upwards (corresponding to the propulsive phases of the three consecutive jumps) and negative when subject moved downwards (corresponding to the pre-loading and landing phases). The different phases of the jumping task have been described succinctly in previous studies [Briefly, in order to facilitate the biomechanical analysis of the jump, the task was divided into separate phases. The identified phases were based on the results obtained from the vertical velocity curve recordings (Z-axis) through a self-customized computer application implemented with MatLab 7.11 . The  studies –29  calculation was defined as the ratio between the jumping performance (cm) and the sum of the peak ground reaction forces supported at the centre of mass level (N). The amount of the sum of three-dimensional forces would penalize or benefit the ratio in the horizontal jumping task. The ME, aims to determine to what extent the supported peak ground reaction forces are during the absorptive phases, in relation to the distance reached during the maneuver. Supporting greater peak ground reaction forces during the absorptive phases, could lead to a more harmful mechanical overload which could increase the injury risk.Descriptive statistics  were calculated for all the collected variables .p < 0.05. “SPSS® statistical software  was used for the abovementioned statistical calculations.Afterwards, descriptive statistics for the selected variable groups  were applied. After normal distribution of the data and variances equality were checked through the Shapiro-Wilk and Levene tests respectively, a 2 X 2 (group by limb) multivariate analysis of variance (ANOVA) was performed to analyse interaction levels between factors. The dominant limb of the control group was matched to the involved limb of the ACL-R group and the non-dominant limb was matched to the non-involved limb of the ACLR group . Thus, ip value) significance, emphasizing that the magnitude of an effect would be more relevant than any statistically significant effect especially in the clinical practice or when treating elite athlete’s data [Apart from that, intra and inter-group differences were analysed using magnitude-based inferences (MBI). This statistical method was chosen in order to highlight the practical significance over the statistical  lighter and smaller than their non-ACL-R counterparts. No significant interaction effects were found between factors for UTHD and COHD tests. Therefore, the results are delimitated to the description of the main effects observed.After the data processing, the number of analysed limbs in both control and ACL-R group was the following: 8 ACL-R reconstructed limbs and 4 ACL-R in both UTHD and COHD maneuvers; 13 dominant and non-dominant limbs in the UTHD and 14 dominant and non-dominant limbs in the COHD of the control group. The ACL-R players were significantly (p < 0.05). Indeed a non statistical trend although a large effect size was find in realtion to a gretater X mediolateral force production during the first hop in controls in comparison to ACL- reconstructed players.  when executing this horizontally oriented jumping task (Table p = 0.010). The Bonferroni post-hoc analysis revealed that the dominant limbs of the controls displayed significantly greater X-medial-lateral axis forces than the injured limbs of the ACL-R group (p < 0.05). No significant differences were found for the remaining analyzed variables  Table . Howeverles Fig. .The ACL-R limbs of the cases demonstrated a trend towards lower mechanical efficiency ratios  when executing this side-to-side and horizontally oriented jumping task.For more information, available complementary material is included about the 3-axial forces results for the UTHD  players. In this sense, it seems that male handball professional players are able to recover their lower limb full performance capacities without lasting biomechanical alterations that can be in contrast observed among their female counterparts. Although evidence exists referring no sex influence in relation to increased risk for ACL graft failure among sportspeople,  may be, Traditionally, lower limb functional evaluations have been carried out in order to determine the athlete capacities with regard to return to sport participation. tIndeed, jumping biomechanical have also been performed in relation to injury risk factor identification showing huge correlation between poor unilateral limb performance values and knee dynamic instability . In thisThus, despite knee joint moment description is not possible when analysing a jumping task through a direct mechanics approach, by placing an ISU on the L3-L4 level, clinicians by using this jumping biomechanical analysis method, could look for jumping aberrant patterns identification that have been previously linked to a greater knee joint injury risk due to excessive mechanical overload during high demand athletic tasks.In this way, it is possible that ACL-R female athletes, could have developed lasting movement pattern adaptations during single-limb actions in the attempt to improve lower limb stiffness through movement pattern reprogramming at the central nervous system level , 46. ThiThe identification of lasting functional and biomechanical jumping alterations several years after the injury in both the present and previous research –27, coulSome potential limitations could be observed in the present study. Given the uniqueness of the analyzed population, which was limited to an exclusive cohort of female professional handball athletes, the results should be interpreted with caution and in relation to this sport level, discipline and sex. Additionally, there was a lack of standardization of the postoperative rehabilitation protocols and the graft type used for the ligament repair among the ACL-R athletes. The heterogeneity of the rehabilitation process may have biased the long-term outcome in terms of physical activity level and sport-specific performance. However, previous studies have reported that no differences exist between reconstructions using different graft types in relation to long-term function of the knee . FurtherIn conclusion, elite female handball players with previous ACL reconstruction demonstrated a attenuated jumping capacity in the THD test. Indeed, they also displayed lower X-medial-lateral axis peak force generation, especially during the first propulsive phase of the CTHD. This fact could be interpreted as a protective effect against the lower limb collapse. As main clinical implication, ISU systems can aid the implementation of real-time simple biomechanical jumping examinations by sports medicine professionals in clinical settings to reduce the residual uncertainty that often arises during the ACL rehabilitation process regarding the return to sports. However, due to the uniqueness of the analyzed cohort the present results must be considered with caution and restricted to the intrinsic characteristics of these top level female handball players.Additional file 1: Appendix A. Technical explanation of ISU technology-derived analysis [Appendix B. UTHD manoeuvre phases breakdown in terms of centre of mass force orientation values. Descriptive statistics and effect size calculations. Appendix C. UCOHD manoeuvre phases breakdown in terms of centre of mass force orientation values. Descriptive statistics and effect size calculations.analysis . Appendi


Background: Progressive multifocal leukoencephalopathy (PML) is an opportunistic infection with JC-virus (JCV), a papova-virus, affecting mostly oligodendrocytes and the white matter of the central nervous system. Progressive Multifocal Leukoencephalopathy (PML) almost exclusively occurs in immunocompromised patients based on different underlying conditions of severe cellular immunodeficiency such as HIV/AIDS, secondary to neoplastic and autoimmune diseases, or during immunosuppressive therapy.Case presentation: We present the case of an otherwise healthy and immunocompetent patient without immunosuppressive therapy who was admitted with hemianopsia to the right side, sensory aphasia and changes of behavior. Magnet resonance imaging (MRI) and laboratory testing confirmed the diagnosis of PML, although functional tests did not show any evidence for cellular immunodeficiency. Extensive immunological tests did not reveal an apparent immunodeficiency. During symptomatic therapy the patient developed seizures which were assumed to be caused by a spontaneous immune reconstitution inflammatory syndrome (IRIS) demonstrated by MRI. We added a high dose of intravenous corticosteroids to the antiepileptic treatment and seizures ended shortly thereafter. However, the impairments of vision, behavior and language persisted.Conclusions: Our case report highlights that an apparently immunocompetent patient can develop PML and IRIS spontaneously. Therefore, MRI should be applied immediately whenever a rapid progression of PML symptoms occurs as treatment of IRIS with corticosteroids can result in a marked clinical improvement. Progressive Multifocal Leukoencephalopathy (PML) is an opportunistic infection of white and gray matter cells caused by JCV-reactivation within the brain almost exclusively occurring in immunocompromised patients. Hence, PML has been explained as an opportunistic infection secondary to underlying predominantly cellular forms of immunodeficiencies  , 2. JCV We present a case of PML in a patient with no detectable immunodeficiency at time of PML diagnosis. As far as neuroimaging by cMRI suggested a subsequent development of an IRIS, a corticosteroid-treatment was initiated resulting in a marked benefit of the PML.A 68-year-old male patient was transferred to our neurological department after he had been admitted to another hospital twice before. The first admission had taken place 3 months before in July 2018 due to visual impairments (hemianopsia to the right) and uncommon behavior. The MRI had shown a T2-weighted (T2w)-FLAIR hyperintensity in the left temporooccipital lobe that had been interpreted as an ischemic stroke. The results of cardiovascular investigations had been normal and the patient had been discharged. In August 2018 he had been admitted again with a progression of symptoms consisting of severe sensory aphasia, psychomotoric deficits and progressive visual impairments. The MRI had displayed a massive enlargement of parietooccipital lesions in both hemispheres predominantly affecting the left white matter . The polThe patient was then transferred to our department for further diagnostics. His general physician and relatives revealed that the medical history contained no record of frequent or severe infections. He had never received any kind of immunosuppressive treatment. In 1999 the patient had suffered from a myocardial infarction. The medical history was unremarkable apart from arterial hypertension and a helicobacter-positive gastritis. In 2017 he had got a viral infection with mild influenza-like-symptoms. Neither the patient nor his family knew of any recent vaccinations.Upon admission to our neurologic department he had developed a right sided homonymous hemianopsia, severe sensory aphasia and disorientation. Our CSF analysis showed a pleocytosis (22 cells/μl) and an elevated protein concentration (0.72 g/l). The cell-distribution was mostly lymphocytic, but monocytes and granulocytes were found as well. CSF specific oligoclonal bands (type 2) were present and there was an intrathecal production of IgG and IgM. The JCV-PCR (1E3 c/ml) from CSF and the JCV-antibody-index (24.000 AU/ml serum) were positive. All additional tests were negative  in the CSF by PCR, as well as tickborn encephalitis (TBE), enterovirus, and cytomegalovirus serologically; CSF-antibody-indices for HSV, EBV, VZV and CMV). All investigated autoimmune encephalitis antibodies , -Zic4) were negative in serum and CSF.18F-Fluorodeoxyglucose (18F-FDG) 60 min p.i. to search for a malignancy or an inflammatory disease. The PET scan showed prominent mediastinal lymph nodes with moderately high metabolism, however, it did not reveal any profound hypermetabolic lesions. 18F-FDG uptake in the brain was further evaluated using statistical parametric mapping (SPM) for comparison with a reference group. The cerebral glucose metabolism was lowered bilaterally in parietotemporal and occipital areas with a clear predominance on the left side. The metabolism in the frontal cortex, basal ganglia, and cerebellar regions appeared to be normal , elevated rheumatoid factor (60.5 IU/ml), Cardiolipin-IgM-antibodies (14 MPL-U/ml), and antinuclear antibodies  of unspecific speckled pattern in the immunofluorescence and without specificity against ds-DNA or extractable nuclear antigens. An abdominal sonography showed a splenomegaly and prominent lymph nodes but no sign of neoplasia. We performed a whole-body PET/CT (including the brain) with 296 MBq e normal .A biopsy of the prominent mediastinal lymph nodes identified in the PET investigation showed no signs of malignancy or granuloma. Additionally ACE and the soluble IL-2-receptor in the blood were normal, so sarcoidosis seemed unlikely. The HLA-DR-expression and phenotyping of lymphocytes showed normal results . In addition to extensive phenotyping of peripheral blood mononuclear cells (PBMC) even the functional analyses by proliferation assays of PBMC by plant lectins , tuberculin (PPD) and anti-CD3, spontaneous and antibody-dependent NK-cell activity (ADCC) were within normal range.During inpatient treatment the patient developed a cough and had impaired breathing. The tracheal exudate was positive for HSV using PCR so we started a therapy with intravenous aciclovir which improved the respiratory symptoms markedly. In September 2018 a progression of the patient's symptoms with focal and generalized seizures occurred. We started a medication with levetiracetam and had to add valproate and lacosamide to the antiepileptic treatment due to recurring seizures. The MRI showed a further expansion of the lesions with a new peripheral contrast enhancement  as a sigAfter nearly 2 months the patient was transferred to a rehabilitation facility. Visual impairments, disorientation and a sensory aphasia were still persistent. During the following weeks the patient developed severe dysphagia until oral intake of food and fluids was no longer possible. The patient died 2 months after the transfer to a nursery home due to a respiratory infection and pulmonary insufficiency.18F-FDG-PET/CT scan. HIV-results were repeatedly negative. A chronic autoimmune disease was also not detected and a biopsy of mediastinal lymph nodes showed no sign of sarcoidosis. Blood tests revealed a normal distribution and function of PBMC, especially the amount of CD4+-cells was normal. However, we cannot exclude a stage of transient immunodeficiency before PML developed, e.g., secondary to an intercurrent viral infection which had presented “flu-like” and clinically self-limiting in 2017. However, an episode of symptomatic tracheobronchitis with HSV-reactivation shown in the BAL in 09/2018 might indicate persisting immunodefiency, although we did not find further evidence for a capable immunological impairment at the same time. Hence, a hypothetical explanation of the course of PML in the presented case may be a (a) transient cellular immunodeficiency potentially due to the suspected viral infection in 2017 as the predisposition for development of PML and JCV-reactivation; (b) clinical development of PML as a demasking IRIS since summer 2018 during spontaneous immunoreconstitution; (c) further progression of PML-IRIS after antiviral treatment of HSV-tracheobronchitis in 09/2018 and (d) clinical improvement by corticosteroid-pulse subsequently. The HSV reactivation in the respiratory tract would be in accordance to this hypothetical course because it is known to be caused by immunodeficiency and as well by IRIS (The presented case shows evidence for the rare development of severe PML and spontaneous IRIS in a patient with no apparent immunodeficiency. A neoplastic disease could not be detected by abdominal ultrasound, thoracic X-ray and a whole-body 18F-FDG PET/CT might indicate mild infiltration by inflammatory cells (Few comparable cases with PML in apparently immunocompetent patients have been published –15 but ary cells  or even It has been demonstrated that JCV-genome can be found in brain tissue of patients suffering from other neurological diseases than PML and even in neurologically healthy individuals . HoweverAll authors gave their consent for publication.Please contact corresponding author for data requests. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.Written informed consent was obtained from the patient and his legal guardian for the participation as well as the publication of the case report and any accompanying images. A copy of the written consent is available upon request from the corresponding author.LK wrote the manuscript and was involved in the diagnostic and therapeutic clinical progress. PR contributed MR-images and legends , 3. RS aThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gasoline- and diesel-fueled engines are ubiquitous sources of air pollution in urban environments. They emit both primary particulate matter and precursor gases that react to form secondary particulate matter in the atmosphere. In this work, we updated the organic aerosol module and organic emissions inventory of a three-dimensional chemical transport model, the Community Multiscale Air Quality Model (CMAQ), using recent, experimentally derived inputs and parameterizations for mobile sources. The updated model included a revised volatile organic compound (VOC) speciation for mobile sources and secondary organic aerosol (SOA) formation from unspeciated intermediate volatility organic compounds (IVOCs). The updated model was used to simulate air quality in southern California during May and June 2010, when the California Research at the Nexus of Air Quality and Climate Change  study was conducted. Compared to the Traditional version of CMAQ, which is commonly used for regulatory applications, the updated model did not significantly alter the predicted organic aerosol (OA) mass concentrations but did substantially improve predictions of OA sources and composition , as well as ambient IVOC concentrations. The updated model, despite substantial differences in emissions and chemistry, performed similar to a recently released research version of CMAQ  that did Organic aerosol (OA) is a major component of atmospheric fine particulate matter . Source 12, single-ring aromatics, isoprene, and mono- and sesquiterpenes. Research and regulatory efforts have historically focused on emissions of primary organic aerosol (POA), but recently the attention has shifted to secondary organic aerosol (SOA) since SOA appears to dominate OA mass concentrations even in urban areas . Typical12+) that are difficult to speciate using traditional gas chromatography–mass spectrometry (GC-MS) techniques due to the very large number of constitutional isomers and/or polarity of partially oxidized species (12 and single-ring aromatics). Recently, a series of experiments investigated the VOC emissions and SOA formation from gasoline vehicles, diesel vehicles, and small off-road engines recruited from the California in-use fleet , b. May  species . Jathar In this work, we used an updated version of CMAQ to simulate ambient OA from gasoline and diesel sources in southern California. The updates included new mobile source emissions profiles for VOCs based on  and emisIn this section, we provide a brief overview of CMAQ followed by more detailed descriptions of the OA model and emissions.http://www.epa.gov/ttnchie1/net/2011inventory.html) and biogenic emissions were estimated using the Biogenic Emission Inventory (BEIS) version 3.14 model . Details about the application of this version to CalNex can be found in 14 model . Gridded14 model . The firThe OA model used here builds on the volatility basis set (VBS) implementation in CMAQ  and is rIn the VBS-IVOC model, we extended the work of C*): non-volatile and logarithmically distributed bins from 100 to 103 μg m−3 at 298 K. The gas–particle partitioning of semi-volatile organic compounds in each basis set is assumed to be in equilibrium and to form a quasi-ideal solution with all of the OA.The VBS version of CMAQ includes four distinct volatility basis sets to separately track different classes of OA: anthropogenic POA, anthropogenic SOA, biogenic SOA, and biomass burning POA . The VBSIn the VBS-IVOC model, we used emission inventories developed by We (in the VBS-IVOC model) and In the VBS-IVOC model, we used new VOC speciation profiles for tailpipe emissions from gasoline and diesel sources . These sC* bins of 103 and 104 μg m−3  were reclassified as IVOCs, consistent with the parameterization of For gasoline and diesel sources, we estimated the IVOC emissions in the VBS-IVOC model based on the gas-phase carbon-balance analysis of Following To illustrate the effects of these changes, The magnitude of the POA emissions was identical between all three models with the exception that some of the POA emissions were reclassified as IVOCs in the VBS-IVOC model as described earlier. The BTEX emissions were identical between the Traditional and VBS models, but lower in the VBS-IVOC model because we renormalized the NMOG emissions to account for IVOCs. The Traditional model did not include IVOC emissions. The IVOC emissions in the VBS-IVOC model were a factor of 4 higher for gasoline sources than in the VBS model of x yields; there was no NOx dependence in the SOA yield from sesquiterpenes and IVOCs. Emissions profiles for VOCs, IVOCs, and their SOA yields, specific to gasoline and diesel tailpipe emissions, are presented in SOA production from VOCs was simulated using the parameterizations of IVOC emissions from gasoline and diesel sources were represented separately using two (one for gasoline and one for diesel) gas-phase species in the chemical mechanism (CB05-TUCL) and the parameterizations of −11 cm3 molecules−1 s−1. These aging reactions formed products with a vapor pressure reduced by 1 order of magnitude. Biogenic SOA was not aged based on previous findings that aging reactions over-predicted OA concentrations in rural areas  during the simulation period.−3 with POA accounting for slightly more than half of the OA in source regions such as downtown Los Angeles (a “source” region was defined as one with high anthropogenic emissions of species such as POA) and SOA dominating in non-source regions and off the coast.Gasoline sources were predicted to contribute ~ 35 % of the inland OA, while diesel sources contributed less than 3 % (for details see Sect. 4). The predicted gasoline OA exhibited a slightly different spatial pattern than total OA, with higher downwind concentrations near Riverside than those near central Los Angeles, reflecting the importance of atmospheric production of SOA. As expected, biogenic SOA was more important outside of the urban areas contributing 5 % of total OA in urban areas versus 10–20 % in non-urban areas. Other OA contributed slightly more than half of all OA in the urban areas. Other OA was dominated by cooking POA, biomass burning POA, and other anthropogenic SOA  and the CalNex Pasadena ground sites. Predictions from the VBS-IVOC model were slightly lower than the filter-based measurements at the CSN sites, similar to other studies . The fraOA mass concentrations are only one measure for evaluating model performance. Given the myriad sources of and complexity in SOA production, a model can predict the right absolute OA concentration for the wrong reason. Therefore, it was important to evaluate the model against OA composition. Before discussing the normalized composition predicted by the VBS-IVOC model, we briefly describe the findings from In Although predictions from the VBS-IVOC model were much better than the Traditional model for the POA–SOA split and the fractional source contribution/composition of OA, in A novel aspect of the VBS models (VBS and VBS-IVOC) is that they track IVOCs, an important class of SOA precursors . Campaig−3) from the VBS-IVOC model were 35 % lower when compared to the hydrocarbon IVOCs concentrations measured by −3). In contrast predictions from the VBS model were a factor of 4 lower than the measurements, which highlights the improved representation of IVOCs in the VBS-IVOC model; the Traditional model predicted essentially no IVOCs. The under-prediction of VBS-IVOC could partly be a result of the inability of the model with a 4 km horizontal resolution to capture the location-specific concentrations at Pasadena. The model–measurement comparison suggests that the VBS-IVOC model reasonably simulated the emissions, transport, and chemistry of IVOCs from mobile sources. Furthermore, the VBS-IVOC model predicted that the majority of the hydrocarbon IVOCs originated from gasoline sources. Coincidentally, the predicted IVOC sum for other anthropogenic sources and biomass burning (4.3 μg m−3) compared well with the measured oxygenated IVOCs (4.1 μg m−3). Given the uncertainty in the model emissions of IVOCs for non-mobile sources (POAx1.5), the comparison with oxygenated IVOCs needs to be explored in future work.Predicted gasoline and diesel IVOC concentrations (3.9 μg m−3) with very little SOA (0.2–0.4 μg m−3), while the OA predicted by the VBS-IVOC model had equal proportions of POA and SOA.We compared predictions from the VBS-IVOC model to OA predictions from Although the VBS and VBS-IVOC models contain very different representations of mobile source emissions, these emissions contributed, on average, to slightly more than one-third of the total OA in southern California (see Sect. 4). Therefore, the updates used in the VBS-IVOC model had a limited influence in affecting the overall OA burden. Strict regulations have dramatically reduced emissions from motor vehicles over the past three decades, which has both improved air quality and increased the relative importance of other sources to OA . For exaThe similarity between predictions from the VBS and VBS-IVOC models was also due to the importance of aging reactions. Both models used the same aging scheme applied to POA and SOA vapors  VOC and IVOC SOA precursors from gasoline and diesel sources and (ii) experimentally constrained parameterizations for SOA production from IVOCs. Predictions of OA mass concentrations from the updated model (VBS-IVOC) slightly under-predicted daily averaged, filter-based measurements at CSN sites in California during May and June 2010  but were a factor of 3 lower than aerosol mass spectrometer-based measurements made at Pasadena as part of the CalNex campaign. The Pasadena site may have been influenced by local sources and transport not captured by the model at a 4 km resolution. We recommend future modeling studies to be performed at higher resolution.When compared to a Traditional model of OA in CMAQ that includes a non-volatile treatment of POA and no SOA from IVOCs, the VBS-IVOC model produced different spatial patterns of OA with lower (~ 50 %) concentrations in source regions but higher (~ 20–40 %) concentrations away from the sources. The VBS-IVOC model in comparison to the Traditional model improved predictions of the sources and composition of OA. These findings are consistent with previous comparisons between the Traditional and VBS models and highlight the importance of the use of an OA model that includes semi-volatile and reactive POA and SOA formation from IVOCs.*C and O : C of the OA and fragmentation reactions can be increasingly important at longer timescales (Predictions of OA from the VBS-IVOC model were similar to those from a recently released research version of CMAQ (VBS) that included semi-volatile POA and SOA formation from IVOCs . The premescales . Furthermescales ; future For the first time, we compared model predictions to ambient measurements of IVOCs. The new VBS-IVOC model better predicted the ambient IVOC concentrations compared to the Traditional and VBS models. This suggests that the updated model reasonably simulated the emissions, transport and chemistry of IVOCs from mobile sources. However, the model representation of IVOCs from non-mobile sources remains poorly constrained and needs to be explored through future emissions, laboratory and modeling studies.Finally, the VBS-IVOC model predicted that mobile sources accounted for 30–40 % of the OA in southern California, with half of the OA being SOA. The diurnal variation of OA in Pasadena supports the hypothesis that substantial OA is produced through photochemical reactions vs. primary emissions . GasolinSupp