How has utilization of low-value carotid imaging for carotid bruits, preoperative evaluation, and syncope changed in the Veterans Health Administration system from 2007 to 2016?In this serial cross-sectional study of 809 071 carotid imaging examinations in the national Veterans Affairs Health system, there was a modest decrease in rates of imaging for carotid bruits that predated Choosing Wisely recommendations, and no significant trends in rates of preoperative carotid imaging or imaging for syncope.These findings suggest that to reduce low-value testing and utilization cascades, interventions targeting ordering clinicians are needed to augment the impact of public awareness campaigns. This serial cross-sectional study examines rates of low-value carotid imaging orders in the Veterans Health Administration before and after the release of the Choosing Wisely campaign. As part of the Choosing Wisely campaign, primary care, surgery, and neurology societies have identified carotid imaging ordered for screening, preoperative evaluation, and syncope as frequently low value.To determine the changes in overall and indication-specific rates of carotid imaging following Choosing Wisely recommendations.This serial cross-sectional study compared annual rates of carotid imaging before Choosing Wisely recommendations  and after  among adults receiving care in the Veterans Health Administration (VHA) national health system. Data analysis was performed from April 10, 2019, to November 27, 2019.Release of the Choosing Wisely recommendations.Annual rates of overall imaging, imaging ordered for stroke workup, imaging ordered for low-value indications . Indications were identified using a text lexicon algorithm based on electronic health record review of a stratified random sample of 1000 free-text imaging orders. The subsequent performance of carotid procedures within 6 months after carotid imaging was assessed.Between 2007 and 2016, 809 071 carotid imaging examinations were identified , of which 201 467 images (24.9%) were ordered for low-value indications , 257 369 (31.8%) for stroke workup, and 350 235 (43.3%) for other indications. Imaging for carotid bruits declined across the study period while there was no significant change in imaging for syncope or preoperative evaluation. Compared with the 6 years before, during the 4 years following Choosing Wisely recommendations, there was no change in the trend for syncope, a small decline in preoperative imaging , and a continued but less steep decline in imaging for carotid bruits . During the study period, 17 689 carotid procedures were identified, of which 3232 (18.3%) were preceded by carotid imaging ordered for low-value indications.These findings suggest that Choosing Wisely recommendations were not associated with a meaningful change in low-value carotid imaging in a national integrated health system. To reduce low-value testing and utilization cascades, interventions targeting ordering clinicians are needed to augment the impact of public awareness campaigns. In February 2013, the American Academy of Family Physicians17 recommended against screening for carotid artery stenosis in asymptomatic adults, building on prior recommendations against screening by the US Preventative Services Taskforce (USPSTF) released 2007 and updated in 2014.18 Also in 2013, the American Academy of Neurology19 recommended against carotid imaging for syncope without other neurologic symptoms, and the Society of Thoracic Surgeons20 recommended against preoperative carotid imaging prior to cardiac surgery in asymptomatic patients. To our knowledge, the proportion of imaging conducted for low-value indications, as opposed to evidence-based stroke workup, have not been described, and the associations of professional society recommendations with carotid imaging tests are unknown.One frequently identified low-value test is carotid imaging. The role of carotid imaging in the evaluation of symptomatic patients who have stroke or transient ischemia attacks (TIA) is well established, as a substantial proportion of ischemic strokes are attributable to carotid atherosclerosis.To understand the association of Choosing Wisely recommendations with carotid imaging tests, we examined national trends in carotid imaging in the Veterans Health Administration (VHA) health system over a 10-year period from 2007 to 2016. We conducted a time-series analysis comparing rates of carotid imaging before and after Choosing Wisely recommendations were released. To determine the cascade of utilization following low-value carotid imaging, we also examined the number of images ordered for low-value indications that were followed by a carotid revascularization procedure.STROBE) reporting guideline.We conducted a serial cross-sectional study of carotid imaging and interventions performed in the VHA health system between January 1, 2007, and December 31, 2016. Demographic, comorbidity, and imaging data were obtained from the VHA Clinical Data Warehouse. This study was approved by the University of California, San Francisco, institutional review board. This study was restricted to secondary data analysis; thus, the requirement for informed consent from participants was waived by the University of California, San Francisco. Data analysis was performed from April 10, 2019, to November 27, 2019. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  prior to a surgical procedure. This approach may misclassify testing performed for indications other than preoperative testing during the specified time period and may miss preoperative testing performed for a planned procedure that did not ultimately occur.26We sought to identify carotid images ordered for low-value indications  and those ordered for symptomatic indications  for which carotid imaging is a part of standard care. Prior studies of low-value testing have relied on diagnosis and procedure codes within administrative claims to identify indications.To overcome the limitations of prior approaches, we developed a text lexicon algorithm that searched documentation entered into Study Reason and Clinical History free-text fields by clinicians at the time of test ordering. The lexicon was developed iteratively through electronic health record review of a stratified random sample of 1000 carotid images , the immediate level change in 2013, and the difference in slopes between preintervention and postintervention trends are reported as annual images per 10 000 veterans with 95% CIs.We determined the annual rate of carotid imaging  overall, for low-value indications  and for stroke workup. Outcomes were inspected graphically and ordinary least-squares regressions were fit with Newey-West SEs to account for autocorrelation.P values were 2-sided, and statistical significance was set at .05.We lastly present the number and proportion of carotid images that were followed by a carotid procedure, overall and by image indication. Statistical analyses were conducted using Stata statistical software version 16 (StataCorp). During the study period, 809 071 carotid images were performed by the VHA radiology service. The mean age (SD) of patients undergoing imaging was 68.6 (10.1) years, and 776 632 patients were men (96.0%). Comorbidities were common, including hypertension (629 206 patients [77.8%]), coronary artery disease (303 214 patients [37.5%]), and prior stroke (125 266 patients [15.5%]), and 253 854 patients (33.6%) were current smokers .Nearly one-quarter of carotid images were ordered for low-value indications (201 467 images [24.9%]), while 257 369 images (31.8%) were performed for stroke workup, and 350 235 images (43.3%) were performed for other indications . Most imFor overall carotid imaging, there was no temporal trend before Choosing Wisely recommendations  and no immediate level change in the year of Choosing Wisely recommendation release . HoweverCarotid imaging for syncope demonstrated no significant temporal trends overall, before  or after Choosing Wisely release  .Imaging for carotid bruits declined across the study period B and wasThere was no significant change in preoperative carotid imaging B. PreopeP = .07).A total of 755 648 carotid images met criteria for carotid procedure analysis, of which 17 686 (2.3%) were followed by a carotid procedure within 6 months. Only 6555 carotid procedures (37.1%) were preceded by an imaging test ordered for stroke workup. The remaining procedures were preceded by an imaging test ordered for a low-value indication (3228 images [18.3%]) or other indications (7903 images [44.7%]) . The annThis serial cross-sectional study found that rates of overall carotid imaging in the national VHA health system remained stable between 2007 and 2016 and there was a small decline in low-value imaging driven by a decline in imaging for carotid bruits, which predated Choosing Wisely recommendations. We also observed a modest increase in imaging for syncope, despite Choosing Wisely recommendations, and a reversal of trends in preoperative imaging following Choosing Wisely recommendations, although the absolute magnitude of decline in imaging rates was small. These findings suggest that efforts to reduce low-value testing require more than public awareness campaigns.9 However, baseline rates of testing that are targeted by Choosing Wisely recommendations are frequently unknown, particularly for recommendations targeting specific indications that may not be identifiable from administrative data. Thus, our finding that more than 200 000 carotid images ordered in the VHA health system during the study were for low-value indications demonstrates that the potential impact of reducing low-value carotid imaging is substantial. Priorities for initiatives targeting overuse should also focus on testing with significant costs and risk for downstream patient harms.31 Our finding that more than 3000 carotid revascularization procedures were performed following carotid imaging testing for low-value indications suggests that successful efforts to reduce low-value imaging may result in both the reduction of the direct costs of imaging and the downstream utilization of invasive procedures with uncertain benefits.The first key to successful efforts to reduce low-value testing is identifying tests with a high rate of overuse.11 Similar to these early studies, we found no immediate declines in low-value testing following the Choosing Wisely recommendations release and additionally found no substantial differences in testing during a 3-year follow-up period. Second, prior national studies of low-value testing have largely relied on administrative claims, which lack the ability to directly identify indications for testing.23 To overcome this limitation, we examined the indications for imaging entered by clinicians at the time of test ordering, using a novel text lexicon search strategy that allowed us to characterize overall and indication-specific imaging trends. Notably, we found that more than 40% of images were ordered for indications other than stroke workup or for low-value indications. This finding is consistent with prior literature indicating that there are many reasons physicians order carotid imaging, most of which are of uncertain clinical significance.14Our findings build on earlier studies examining trends following Choosing Wisely recommendations and overcome 2 key limitations of prior studies. First, prior studies have largely been limited to studying the period immediately following the recommendations release, thus delayed uptake of recommendations may have not been captured.7 in the case of carotid imaging, low-value indications were targeted by multiple specialties that are likely to order imaging: the Academy of Family Physicians,17 American Academy of Neurology,19 and the Society of Thoracic Surgeons.20 However, carotid images may also be ordered by other clinicians who may not be aware of these recommendations, including primary care clinicians , acute care clinicians , and other surgical specialists. In the case of imaging for carotid bruits, we observed a decline in imaging over the period that predated the Choosing Wisely recommendations and may be associated with the 2007 USPSTF recommendations against carotid screening.18 Unlike Choosing Wisely lists, these recommendations are not specialty-specific and thus may have a wider impact.While Choosing Wisely lists may include testing largely ordered by different specialties,33 For imaging tests that may be ordered for different low-value indications, the target population for interventions may need to be adapted for each indication. Reducing low-value carotid imaging for acute conditions, such as syncope, likely requires clinician-focused interventions that span ambulatory, emergency department, and inpatient care settings. Whereas reducing low-value carotid screening tests requires both clinician- and patient-focused interventions, as patients may request low-value screening, driven by advertising from direct-to-consumer screening companies35 or receipt of prior testing.To achieve success in reducing low-value carotid imaging, targeted interventions are needed. Quality improvement initiatives, including clinical decision support tools, clinician education, and direct feedback to ordering clinicians, have demonstrated promise in single-center and health system studies of other low-value testing.36 would allow for collaborative decision-making and education on the value of certain tests and build on well-accepted and successful antibiotic stewardship program models.37Alternatively, interventions aimed at modifying ordering systems might address testing for multiple indications by either restricting the permissible indications for ordering carotid imaging or by providing clinical decision aids that inform clinicians of which indications may be of low value. Additionally, interventions may be more effective if they target the clinicians performing testing  in addition to the ordering clinicians. An imaging stewardship model in which ordering and performing clinicians discuss the merits of testing for specific patientsOur study has several limitations. First, our study took place in the national VHA health system, which serves a unique and primarily male patient population with a high comorbidity burden, thus overall imaging rates may not reflect the wider US population. Second, the free-text fields entered by ordering clinicians that we examined to identify indications for imaging were only available for carotid images performed by VHA radiology departments and not those performed by vascular laboratories. Vascular laboratories record study results in progress notes and do not include easily identifiable information on the indication for testing. Thus, our study is limited to this subset of images, which accounts for approximately three-quarters of all carotid images performed in the VHA health system. Third, our analysis of carotid procedures following imaging is likely conservative, as it is possible that after a carotid image, some patients may have received a carotid procedure outside the VHA health system owing to patient preference or service availability. Fourth, we were not able to determine the specialty of the ordering clinician. Fifth, although our study identified indications for testing using a newly validated text lexicon approach that provides improved specificity for identifying indications for testing compared with prior studies relying on administrative claims, this approach was found to have only modest sensitivity for preoperative testing.The findings of this serial cross-sectional study suggest that the release of Choosing Wisely recommendations from 3 professional societies were not associated with a substantial reduction in low-value carotid imaging in the national VHA health system. To reduce low-value testing and utilization cascades, direct interventions targeting ordering clinicians are needed to augment the impact of public awareness campaigns.


MYCN amplification, upstream TERT rearrangements or alternative lengthening of telomeres (ALT). The common defining feature of all three subgroups is altered telomere maintenance; MYCN amplification and upstream TERT rearrangements drive high levels of telomerase expression whereas ALT is a telomerase independent telomere maintenance mechanism. As all three telomere maintenance mechanisms are independently associated with poor outcomes, the development of strategies to selectively target either telomerase expressing or ALT cells holds great promise as a therapeutic approach that is applicable to the majority of children with aggressive disease.The majority of high-risk neuroblastomas can be divided into three distinct molecular subgroups defined by the presence of Here we summarise the biology of telomere maintenance and the molecular drivers of aggressive neuroblastoma before describing the most promising therapeutic strategies to target both telomerase expressing and ALT cancers. For telomerase-expressing neuroblastoma the most promising targeted agent to date is 6-thio-2′-deoxyguanosine, however clinical development of this agent is required. In osteosarcoma cell lines with ALT, selective sensitivity to ATR inhibition has been reported. However, we present data showing that in fact ALT neuroblastoma cells are more resistant to the clinical ATR inhibitor AZD6738 compared to other neuroblastoma subtypes. More recently a number of additional candidate compounds have been shown to show selectivity for ALT cancers, such as Tetra-Pt (bpy), a compound targeting the telomeric G-quadruplex and pifithrin-α, a putative p53 inhibitor. Further pre-clinical evaluation of these compounds in neuroblastoma models is warranted.In summary, telomere maintenance targeting strategies offer a significant opportunity to develop effective new therapies, applicable to a large proportion of children with high-risk neuroblastoma. In parallel to clinical development, more pre-clinical research specifically for neuroblastoma is urgently needed, if we are to improve survival for this common poor outcome tumour of childhood. Neuroblastoma is a common childhood malignancy arising from the sympathetic nervous system. It most commonly arises from the adrenal gland and presents with an abdominal mass, but has a heterogeneous clinical phenotype. Children aged less than 18 months often present with widespread metastases to the skin, liver and bone marrow (stage MS disease) .MYCN oncogene are classified as having clinical high-risk disease. High-risk neuroblastoma remains a major therapeutic challenge with survival rates of < 50% despite intensification of therapy [MYCN amplification, the molecular drivers of aggressive disease were unknown.Children aged > 18 months with stage M disease and/or amplification of the  therapy , 3. HoweMYCN amplification, rearrangements upstream to the telomerase reverse transcriptase (TERT) gene or alternative lengthening of telomeres (ALT) [In 2015 it was reported that aggressive neuroblastoma can be divided into 3 almost mutually exclusive subgroups with either es (ALT) , 5. Eaches (ALT) .Here we summarise TMMs in cancer, specifically focusing on the molecular alterations driving telomere maintenance in neuroblastoma, before describing potential novel therapeutic strategies to directly target TMMs for children with neuroblastoma.Telomeres are regions of repetitive nucleotide sequences (TTAGGG) located at the ends of chromosomes that protect chromosomes from DNA damage, unnecessary DNA repair and fusion with other chromosomes. In normal dividing cells, with each cell replication telomeres gradually shorten, until a critical level is reached, the Hayflick limit, after which cells undergo senescence . This grhTERC. hTR acts as a template for the synthesis of telomere DNA and is involved in the catalysis, localisation, and assembly of the telomerase holoenzyme [TERT gene/promoter or TERT promoter methylation.Telomerase, a functional ribonucleoprotein enzyme complex, maintains telomere length by adding telomeric DNA repeats at the 3′ ends of linear chromosomes. It is a reverse transcriptase that consists of a catalytic protein subunit TERT, and an essential RNA component known as human telomerase RNA (hTR), encoded by loenzyme , 11. Telloenzyme . In contloenzyme . This waATRX  in multiple malignancies, including neuroblastoma [ALT is defined as maintenance of telomeres in the absence of telomerase activity . It can blastoma , 16–18.ATRX knockdown has been shown to induce ALT activity in cells of mesenchymal origin [ATRX depletion does not promote ALT activity in all cell types [A number of different non-canonical homologous recombination (HR) based mechanisms have been proposed to play a role in ALT telomere maintenance –22. Furtl origin . Howeverll types , 32 suggll types . FurtherDAXX (death domain associated protein) have also been shown to result in ALT due to a failure to localise ATRX to PML bodies [TP53) and retinoblastoma 1 (RB1) have also been associated with ALT [Genetic alterations in the histone chaperone and ATRX binding partner L bodies . In addiwith ALT  however In large-scale studies telomerase expression data is often used as a surrogate biomarker for telomerase activity , howeverThe identification of robust biomarkers of ALT has proved more challenging. The gold standard assay for ALT is to confirm the maintenance of telomeres, in the absence of telomerase activity through successive population doublings. However, this is only practical in cell lines, non quantitative, and requires long-term cell culture . AlthougPML bodies are ubiquitous throughout the genome and responsible for diverse functions including DNA repair. In ALT cancer cells PML bodies specifically co-localise to telomeric DNA, and are thought to facilitate HR , 26. ALTALT cells are also characterised by the presence of c-circles: single stranded, telomeric circular DNA strands which are thought to provide a template for ALT telomere synthesis . The c-cMYCN oncogene is found in almost 40% of clinical high-risk neuroblastomas [TERT gene, which induces transcriptional up-regulation of TERT by juxtaposing the TERT coding sequence with strong enhancer elements [ATRX [ATRX are associated with a distinct clinical phenotype including older age at diagnosis and a chronic progressive disease course [Amplification of the lastomas , and is lastomas . In an aelements . The thielements . Approxits [ATRX , 36. In e course .TP53 or RAS pathway associated gene is associated with an even worse prognosis [TP53 or RAS pathway mutations  in the absence of a concurrent TMM are not associated with worse survival and can spontaneously regress [Neuroblastoma harbouring a TMM is associated with a poor prognosis regardless of clinical stage , 44–46. TERT translocation or MYCN amplification [ATRX alteration the underlying drivers of ALT neuroblastoma are currently unknown, and somatic alterations in other genes, known to be associated with ALT in other malignancies are rarely found in neuroblastoma [Although the somatic alterations driving TMM’s in the majority of neuroblastoma are well defined, high telomerase expression can occur in the absence of either a fication . Also inblastoma . Numeroublastoma . Anotherblastoma . In keepblastoma .In summary, data is accumulating to support the hypothesis that TMMs are the common defining molecular feature of aggressive disease in the majority of children with clinical high-risk neuroblastoma. Therefore, both telomerase and ALT represent attractive targets for the development of novel therapeutic strategies with the potential to benefit a significant proportion of high-risk neuroblastoma patients. Here, we review the current pre-clinical and clinical research focused on targeting telomere maintenance with a specific focus on the potential relevance to patients with neuroblastoma.Imetelstat (GRN163L) is a competitive telomerase inhibitor with a complementary structure to the template region of the RNA component of telomerase that binds to and blocks the active site of the enzyme [e enzyme . Confirme enzyme –54.A phase I trial of imetelstat in 20 children with refractory or recurrent solid tumours demonstrated telomerase inhibition, which was sustained through to day 8 of cycle 1 of therapy . Two of BIBR1532 is a potent synthetic, non-nucleoside telomerase inhibitor [nhibitor . Howevernhibitor , and thiSodium metaarsenite (KML001) binds to telomeric sequences, displacing hTERT from the nucleus into the cytoplasm [ytoplasm  and has ytoplasm . In a phytoplasm .Telomestatin stabilises G-quadruplexes, which in turn, inhibits telomerase activity [activity . This coactivity  but is n6-thio-2′-deoxyguanosine (6-thio-dG) is a nucleoside analogue, which in telomerase active cells is recognised by telomerase and incorporated into telomeres resulting in telomere dysfunction [TERT activation. However, the response in MYCN amplified xenografts was mixed, likely reflective of the additional oncogenic pathways activated by MYCN [function . Pre-clifunction –66. Althfunction . 6-thio- by MYCN . This isXAV939 is an inhibitor of tankyrase, a positive regulator or telomerase. It has been shown to induce apoptosis in the telomerase expressing neuroblastoma cell line SH-SY5Y [ SH-SY5Y .It has been reported by Koneru et al. that ALT neuroblastoma cell lines are more resistant to topoisomerase inhibitors and that activation of ATM at ALT telomeres is associated with chemo-resistance. The ATM inhibitor AZD0156 was found to be synergistic with temozolomide and irinotecan therapy in both in vitro and in-vivo models of ALT neuroblastoma . AZD0156In 2015 Flynn et al. reported that the presence of ALT renders cells hypersensitive to ATR inhibition in osteosarcoma models . We haveMYCN amplified neuroblastoma cell lines show a significantly greater in vitro sensitivity to single agent AZD6738 than non-MYCN amplified neuroblastoma cell lines  is a cisplatin derivative that inhibits telomeric homologous recombination by targeting the telomeric G-quadruplex and has been shown to inhibit growth of ALT-cell xenograft tumours in mice [CX5461 is an RNA polymerase I inhibitor which has been shown to selectively kill ATRX mutant cells due to its effects on ribosomal RNA transcription. In-vitro sensitivity in ALT cancer cell lines has been demonstrated [pifithrin-α suppresses tumour growth in an ALT xenograft model [TP53 wild type [trabectedin, although the mechanism for this is not clear [Recent pre-clinical data focused on the therapeutic targeting of ALT in other cancer subtypes may give important insight into potential therapeutic vulnerabilities that can be exploited for ALT neuroblastoma Table : Tetra-P in mice . Thus fanstrated . It has ft model  althoughft model , callingild type . Finallyot clear .TERT alterations and MYCN amplification do co-exist in a small proportion of cases, there is no overlap between the telomerase expressing and ALT positive neuroblastoma [ATRX mutations and MYCN amplification are synthetically lethal in neuroblastoma [In some other paediatric malignancies, ALT (identified by the presence of ALT associated PML bodies) and telomerase activation have been shown to co-exist in the same tumour , 89. In blastoma . This isblastoma . Taken tblastoma , 5.ATRX LoF, it appears that ALT is a necessary adaption for cancer cell survival, and that reactivation of telomerase activity cannot overcome endogenous telomere dysfunction [Although data thus far indicates that subgroups of neuroblastoma are driven by either telomerase or ALT activation, it is highly likely that a selective pressure targeting one TMM will support the emergence of an alternative mechanism. In multiple other cancer subtypes it has been shown that long-term telomerase inhibition results in the emergence of features consistent with ALT –94. Convfunction .MYCN amplification and genetic alterations in ATRX are also associated with distinct patterns of wider transcriptional activation which drive malignant transformation [Finally although it has been shown that activation of a TMM is the key determinant of poor outcome in neuroblastoma, the key drivers of TMM’s in neuroblastoma; ormation , 95, 96.ormation , 98. TheMYCN amplified tumours, transcriptional up-regulation of TERT is only one of many oncogenic programmes up-regulated in MYCN amplified neuroblastoma cells.Despite evidence that telomere maintenance is a key driver of aggressive biology in neuroblastoma, clinical translation of novel therapeutics specifically targeting telomere maintenance remains extremely challenging. The only compound to make it into paediatric clinical trials so far, imetelstat is excessively toxic and pre-clinical data on other compounds targeting telomerase activity is extremely limited. The most promising telomerase targeting candidate to date is 6-thio-dG, however this agent is yet to make it into clinical trials. It must also be noted that in an aggressive malignancy such as neuroblastoma, rapid development of resistance to single-agent targeting of telomerase activation is highly likely and that combination therapies will be needed to overcome this. Also, in the case of For ALT driven cancers, there is preliminary data supporting specific roles of a handful of targeted therapeutic approaches but a dearth of robust evidence of pre-clinical efficacy specifically for neuroblastoma. The combination of ATM inhibition with chemotherapy is currently the most promising option for ALT neuroblastoma, although regimens combining cytotoxic chemotherapy agents and inhibition of master upstream regulators of DNA damage repair such as ATM are likely to be prone to significant toxicities.Despite these challenges, the development of effective new strategies for neuroblastoma by either selective targeting of telomerase or ALT offers great potential to treat the underlying drivers of aggressive disease biology, and is applicable to the greater proportion of neuroblastoma patients. Furthermore, the hypothesis that TMM targeting strategies may be particularly effective in neuroblastoma is supported by the fact that significantly fewer mutations are found in neuroblastoma in comparison to adult malignancies, which often arise due to an accumulation of oncogenic mutations over time .RAS and TP53 pathway genes [In addition, as recent data identifies an ‘ultra-high risk’ group of neuroblastoma patients with both telomere maintenance and mutations in ay genes , rationaAs our understanding of the molecular drivers of fatal neuroblastoma has significantly expanded in recent years, pre-clinical research efforts must now focus on translating this knowledge into effective, less toxic new therapies for children with neuroblastoma.


Exopolysaccharides (EPSs) are biological polymers secreted by microorganisms including Lactic acid bacteria (LAB) to cope with harsh environmental conditions. EPSs are one of the main components involved in the formation of extracellular biofilm matrix to protect microorganisms from adverse factors such as temperature, pH, antibiotics, host immune defenses, etc.. In this review, we discuss EPS biosynthesis; the role of EPSs in LAB stress tolerance; the impact of environmental stresses on EPS production and on the expression of genes involved in EPS synthesis. The evaluation results indicated that environmental stresses can alter EPS biosynthesis in LAB. For further studies, environmental stresses may be used to generate a new EPS type with high biological activity for industrial applications. The unique structural features have made bacterial EPSs of particular interest in the fields of chemistry, medicine and food industry Streptococcus, Lactococcus, Pediococcus, Lactobacillus, Leuconostoc and Weissellale are often used to produce EPSs Among EPS producing bacteria, Lactic acid bacteria (LAB) have has grasped the attention of researchers thank to their strong ability to produce EPSs. The LAB strains as Previously, there have been several reviews to describe the stress response in LAB -Why are EPSs related to stress resistance?-What type of stress to apply?-Is it possible to control EPS biosynthesis using environmental stress?Therefore, in this review, we will discuss EPS synthesis; the physiological functions of EPSs as well as the impact of environmental stresses on EPS production and the expression of genes involved in EPS biosynthesis in LAB. This assessment will clarify the relationship between environmental stresses and changes in LAB EPS synthesis. It also suggests that environmental stress can improve the productivity of EPSs from LAB and produce customized EPSs with desired functionality.2.eps gene cluster and housekeeping genes. These gene products can be categorized into four groups (or modules) basing on their functions: polysaccharide assembly machinery , phosphoregulatory system managing polysaccharide assembly , glycosyltranferases and sugar nucleotide biosynthetic pathways. Genes encoding acetyl- and pyruvyl transferase involved in the chemical decoration of EPSs also present in the cluster  or a translocase moves the lipid carrier-repeating unit complex from the inner surface of cytoplasm membrane to periplasmic. Then, a polymerase (encoded by wzy or cpsH) catalyzes the coupling of repeating units Secondly, the synthesis process of repeating units begins by attaching the first sugar nucleotide to the isoprenoid lipid carrier, undecaprenyl phosphate, which is attached to the cytoplasmic membrane of the cell, and being catalyzed by priming glycosyltransferase. This is followed by the sequential addition of sugar nucleotides to form repeating units and glycosyltransferases encoded by gense in the EPSs are synthesized to serve various functions in the bacteria. One of these is to ensure bacteria survive under stress conditions. The function of EPSs in LAB's stress resistance is discussed in the next section.3.EPSs are the important structural component of LAB cell wall To adapt to environmental stresses, LAB can alter their cell surface by producing more EPSs As discussed, LAB enhance EPSs synthesis to create a physical barrier on the cell surface which separates the cell from stress. Especially, in low pH conditions, this EPS layer restricts the access of exogenous acids to bacterial cells due to the anions bound to EPSs as phosphate groups Latilactobacillus sakeiIn the case of osmotic stress, a sudden increase in osmotic pressure made by stress results in water movement from the inside to the outside of cell, causing a detrimental loss of cell turgor pressure and changing intracellular solute concentration, which ultimately can seriously affect cell viability L. mesenteroides by 10 times under oxidative stress and influence promoting the aerobic growth of oxygen-sensitive strains such as Lactobacillus and BifidobacteriumA lot of bacteria respond to carbon dioxide stress by producing EPSs to create a barrier for slowing the diffusion of toxic substance into cells, which in this case would be carbon dioxide Lactiplantibacillus plantarum is related to better tolerance to low pH The ability to protect cells from environmental stresses depends on EPS-phenotype. Terms such as ‘ropy’, ‘mucoid’, and ‘slime’ have been used to describe the different EPS producing phenotypes of LAB 4.Together with the cell protection function, the positive advantages of EPSs are highlighted through the essential contribution to the human health such as prebiotic, anticoagulant, antioxidant, anti-inflammatory, antiviral, cholesterol lowering effects and even anticancer activity L. plantarum 47FE and Lactiplantibacillus pentosus 68FE, respectively, exhibit strong anticoagulant and fibrinolytic activity LAB's EPSs have been showed an essential functional role in blood coagulation prevention. The strong anticoagulant activity of EPSs in sulphate derivatives has been demonstrated. Heparin Cofactor II is a potent inhibitor of thrombin in the coagulation pathway and the sulphated EPSs provides an acidic medium condittion to facilitate the inhibitory effect of Heparin Cofactor II on thrombin L. plantarum can stimulate probiotic bacteria growth. It is low-digested by artificial gastric juice and show to put non-probiotic bacteria off growing that Enterobacteriaceae is a representative instance In vitro EPSs produced by Weissella cibaria, Weissella confusa, L. plantarum and Pediococcus pentosaceus could be used as a prebiotic ingredient in the food industry to modulate gut microbiota towards health benefits Prebiotic effects were also observed at LAB's EPSs in vitro assay, EPSs produced by L. plantarum BR2 show cholesterol lowering properties (45%) in vitro experiments, several hypotheses to explain the cholesterol lowering mechanism of EPSs have been proposed including bile removal, anabolism and cholesterol conversion, co-precipitation effects, etc...Another health promoting functions of EPSs produced by LAB are cholesterol lowering effects Lactobacillus gasseri FR4 have a good free radical activity, while hydroxyl and superoxide radical capture activities are dependent on EPS concentration in-vivo conditions, LAB'S EPSs have been shown to increase the activity of hepatic superoxide effutase, serum catalase, and glutathione S-transferase simutaneously reducing serum malondialdehydes and monoamine oxide activity. These are excellent antioxidant and anti-aging evidence created by EPSs Free radicals usually cause serious health problems. Therefore, EPSs are such an important natural antioxidant to prevent the free radicals. LAB's EPSs also exhibit high antioxidant activity. One evidence showed that EPSs from In recent decades, the immunomodulatory potential of EPSs has received a lots of scientific consideration. Many in-vitro studies have demontrated that EPSs produced by different LAB species have the immunomodulatory ability L. plantarum 70810 can significantly inhibit the proliferation of tumor cells such as HepG-2, BGC-823, especially HT-29 In vitro evaluation of anticancer properties of Lactobacillus acidophilus EPSs in colon cancer cell lines demontrated that they were able to inhibit the expression of genes involved in angiogenesis and tumor survival Levilactobacillus brevis MSR10 are used to synthesize the silver nanoparticles (AgNPs). According to the results obtained, these AgNPs not only had high antimicrobial and antioxidant capabilities but also significantly reduced the percentage of live HT-29 cells Cancer is one of the health problems getting a lot of attention today and it is usually treated via chemotherapy method. However, chemotherapy can cause some unexpected effects which can range from minor to severe and life-threatening L. plantarum LRCC5310 were able to resist human rotavirus in vitro Many recent researches have been conducted to show the antiviral bioavailability of EPSs and they are considered to be an immune stimulant affect in a number of ways in the immune system, contributing to the protection of human cells against certain viruses 5.Under environmental stresses, LAB have different adaptation mechanisms which involve the accumulation of compatible solutes and energy storage compounds; regulation of energy production pathways, as well as the modulation of cell envelope, i.e., membrane, cell wall, surface layers, and EPSs 5.1.et al. demonstrated that EPS production in Lactobacillus lactis subsp. cremoris LC 330 is stimulated by nitrogen limitation Lactobacillus delbrueckii ssp. bulgaricus was recorded increased EPS production in additional nitrogen-enriched The composition of nutrients is one of the factors which affects the growth and metabolism of cells Lactobacillus confusus TISTR 1498 Lactobacillus strains  yield the highest EPSs when growing on fermentation medium comprising 20% sucrose as carbon source Fructilactobacillus sanfranciscensis LTH2590 rose by increasing the sucrose concentration in the medium and reached about 40 g/L at sucrose concentration of 160 g/L Leuconostoc mesenteroides NRRL B-1299, culture medium with sucrose concentration over 5 g/L caused more dextran production Streptococcus thermophilus (W22) and L. delbrueckii subsp. bulgaricus  was stimulated by high glucose concentration L. casei and Lacticaseibacillus rhamnosus, although the growth is apparently decreased Excessive sugar presence in the culture medium also increases EPS production in LAB. The possible explanations for the increased EPS synthesis under stress of high sugar concentration are osmosis, unlimited supply of sugar building blocks and high energy availability Bifidobacterium longum JBL05, increased proportionally to carbon dioxide concentration in the range of 0–20% L. casei growing in carbon dioxide-rich environment was surrounded by a membrane like EPS component B. longum declined growth and EPS accumulation B. longum varies under different stress conditions. Although oxidative stress reduces the accumulation of EPSs in B. longum, it increases EPS production in B. scardovii and B. adolescentis. One evidence has shown an increase in EPS production and the cell surface hydrophobicity of B. scardovii and B. adolescentis under oxidative stress In some LAB strains, carbon dioxide can used as a carbon source for growth because it is a substrate in carbamoyl phosphate synthesis and other metabolic reactions in LAB 5.2.L. rhamnosus GG, while it enhanced biofilm formation in Limosilactobacillus reuteri strains Lactobacillus helveticus ATCC 15807 under controlled pH of 6.2 was lower than that observed at pH 4.5 Ligilactobacillus salivarius UCO_979C-2, adapted variant strain, after 24 h at pH 2.6 was 690 mg/L, compared to native L. salivarius UCO_979C-1 strain that was only 450 mg/L at pH 6.4 EPS production in LAB may be stimulated by various physical stresses as a cellular defense response, which could also enhance the formation of biofilms et al., the EPS synthesis of L. confusus TISTR 1498 did not depend on biomass and stress of high NaCl concentration could enhance EPS production in solid state fermentation Leuconostoc mesenteroides/pseudomesenteroides 406 achieved maximum EPS yield in the presence of 5% NaCl L. helveticus ATCC 15807 The negative effects of osmotic stress on cells may be limited because of the presence of EPSs. Therefore, the presence of substances caused high osmotic pressures, such as NaCl, can stimulate EPS synthesis on the cell wall. As previously described by Seesuriyachan et al. demonstrated that sub-lethal thermal stress increases EPS production and improves the viability of B. bifidumExcessive temperature causes protein denaturation, nucleic acid and membrane damage 5.3.Saccharomyces cerevisiae. Lactobacillus kefiranofaciens JCM 6985 enhanced the production of kefiran, an exopolysaccharide, in co-culture with S. cerevisiae IFO 0216 L. rhamnosus strains was also increased by 39–42% and a higher level of EPS operon expression was observed for L. rhamnosus RW-9595M in co-culture L. paracasei co-cultured with Saccharomyces cerevisiae resulted in the overexpression of gene (coding for polyprenyl glycosylphosphotransferase) involved in EPS production Saccharomyces cerevisiae is induced by direct and physical contact with components on the surface of yeast cell In biotechnology, co-culture has been shown to make microorganisms more resistant to environmental changes and can perform more complex metabolic activities through the culture combination of various strains esp genes under environmental stresses. Details are presented in section 6.In general, the biosynthesis of EPS can be altered either up or down under different stress conditions. These changes may be related to expression level of genes involved in EPS synthesis. To clarify this hypothesis, we have discussed the expression of 6.Bacteria respond to stresses by activating various regulatory mechanisms including activities involved in metabolisms, cell envelope and gene expression, giving them the potentiality to adapt to extreme environmental conditions  [108]. Cgtf01207 gene, encoding for a priming glycosyltransferase related to EPS synthesis, was observed in B. animalis subsp. Lactis after exposure to stress of acid, bile salts and osmosis epsNMLKJ genes in Streptococus thermophilus ASCC 1275 increased. However, the expression of genes involved in the synthesis of sugar nucleotides such as dTDP-rhamnose and UDP-GlcNAc reduced epsNMLKJ cluster, but the expression of eps1C and eps1D genes increase while that of eps2C and eps2D decrease gtf gene encoding for enzyme which produces beta-glucan (membrane-linked glycosyltransferase enzyme) in Lacticaseibacillus paracasei caused 60 times higher heat tolerance, 20 times higher acid tolerance compare to control strain Xanthomonas campestris pv. L. rhamnosus HN001 during heat stress. In contrast, the heterological expression levels of glyceraldehyd-3-phosphate dehydrogenase and phosphoglycerate kinase, related to EPS biosynthesis of Xanthomonas axonopodis pv. Glycines) The correlation between stress and the expression of genes involved in EPS biosynthesis has been documented in LAB. Increasing expression of 7.eps cluster and the impact of environmental stresses can alter the expression of these genes resulting in increased EPS production. Accordingly, environmental stress may be considered as a major factor to control LAB's EPS biosynthesis. The impact of different stresses on EPS synthesis is summarized in L. helveticus ATCC 15807 is stimulated by stress at low pH but inhibited by sodium chloride stress. Conversely, a specific stress may stimulate EPS production in one species but inhibit it in another (In order to survive under environmental stress conditions, LAB react by synthesizing EPSs to form a protective barrier around the cells. This EPS synthesis is catalyzed by enzymes encoded by genes in the  another .The EPSs produced by LAB can be the key ingredients showing promising functional roles for various utilities in food, medicine, etc. However, low EPS productivity could be a problem limiting commercial applications of these EPSs. Currently, EPS production improvement studies often focus on optimizing culture mediums, using genetic engineering, using cheap fermentation substrates, and environmental stress L. reuteri Mh-001 was demonstrated to relate to their anti-inflammatory activity, in particular galactose content enhances EPS anti-inflammatory effects on the macrophages In addition, the biological activities of EPSs such as prebiotic, anti-oxidant, anti-inflammatory, ... are related to the monosaccharide compositions of EPSs. It has been proved that EPSs with distinct monosaccharide compositions vary in their therapeutic effects


Aim: High-frequency longitudinal tracking of inflammation using dried blood microsamples provides a new window for personalized monitoring of infections, chronic inflammatory disease and clinical trials of anti-inflammatory drugs. Results/methodology: Using 1662 dried blood spot samples collected by 16 subjects over periods of weeks to years, we studied the behavior of 12 acute phase response and related proteins in inflammation events correlated with infection, vaccination, surgery, intense exercise and Crohn's disease. Proteins were measured using SISCAPA mass spectrometry and normalized to constant plasma volume using low-variance proteins, generating high precision within-person biomarker trajectories with well-characterized personal baselines. Discussion/conclusion: The results shed new light on the dynamic regulation of APR responses, offering a new approach to visualization of multidimensional inflammation trajectories. In this paper, we explore the longitudinal behavior of inflammation-related acute phase response (APR) proteins in a unique collection of dried blood spot (DBS) samples. These included 1522 samples collected longitudinally by eight individuals over periods of up to 9 years (with extended periods of daily sampling) and 140 samples collected by eight elite athletes at multiple time points per day during 1 week of Olympic training using a Sportomics approach [Inflammation is central to the understanding of human health and disease. Underlying the classical definition , inflammation involves a complex balance between protective and destructive processes implemented by a varied cast of proteins under active regulatory control. In clinical contexts, these complex processes are usually summarized by measuring only a single blood biomarker: CRP , a key cMany therapeutics that reduce inflammation are in use or under development. These usually act either directly  or indirectly (by treating causes of inflammation). Inflammation signaling is generally targeted by biologics, including a wide and rapidly growing range of molecules such as the anti-TNF alpha antibodies infliximab  and adalCan a single biomarker adequately summarize such complex mechanisms and outcomes? The current use of CRP as a biomarker implicitly assumes that inflammation can be considered a fixed response  to many in vivo  to changes in one or more biomarkers of effect . However, since frequent longitudinal blood sample collection by venipuncture is generally impractical outside a medical environment , longitLongitudinal sample collection can be radically improved by using a less intrusive means of blood collection: fingerprick capillary blood dried on filter paper (‘dried blood spots’: DBS). DBS have been used as clinical samples since the pioneering studies of Guthrie  on inborHere we report results that illustrate the complex and informative behavior of APR proteins (and related proteins) in tracking inflammation due to multiple causes. The data demonstrate the potential for major improvements in diagnostic test interpretation using personalized baseline values and the feasibility of personalized multiparameter biomarker response models useful in clinical trials.A unique set of 1522 long-term longitudinal capillary blood DBS samples (sample set I) was self-collected at home between 2008 and 2017 by 8 participants using lancet finger-pricks  and dried on Whatman 903 Protein Saver DBS cards . The DBSSupplementary Table 1).A panel of proteins of known clinical significance was measured using SISCAPA-MRM mass spectrometry . These iSamples were analyzed in four campaigns at different times, measuring slightly different but largely overlapping sets of measured protein targets . These ywww.tableau.com) and joined with Excel tables defining sample characteristics . Data analysis and visualization were performed in Tableau Desktop .PAR data were assembled in Tableau Prep Builder (Supplementary Table 1).To minimize the effect of variations in plasma volume between DBS from the same individual , we refined and applied a method developed previously  to normaAfter this subject-specific normalization, subject-specific baselines were computed for each protein as the average level in the half of samples with lowest CRP . Fold-changes were computed relative to these baselines and so focus on relative responses in the individual rather than absolute scale of change.Supplementary Figure 1A shows DBS protein levels before and after normalization in 250 serial samples collected by subject S-04 over almost 5 years.Amounts of selected APR and related proteins were measured using an automated, multiplexed SISCAPA-MS protocol ,39 and nSupplementary Figure 2).In addition to maintaining stable levels over time, two of the normalizing proteins (Hx and IgM) exhibit large quantitative baseline differences between individuals. As a result, each subject's sample set, in many cases including hundreds of samples collected over many years, forms a tight cluster on a plot of Hx versus IgM, almost completely separated from the other subjects A. In genSupplementary Figure 1B, extreme inflammatory events such as major infections affect the concentrations of most plasma proteins to some degree, including those used here for normalization. With the present approach, normalization bias due to inflammation is mitigated due to the fact that while IgM varies little, Alb shows a weak negative APR and Hx shows a weak positive APR, so that the combined index is to a great extent balanced with respect to inflammation.While the selected normalizing proteins exhibit striking longitudinal stability, we nevertheless observed two significant sources of variation in these proteins within subjects. Supplementary Figure 1B), with fold increases relative to baseline ranging from 0.036 (LPSBP) as great as the fold increase in CRP to 0.002 (Hx).The largest perturbations observed in levels of most proteins occurred during inflammatory events that were noted by the study subjects and were coincident with increases in the acute phase reactants CRP C and SAASupplementary Table 3). On average, the ratio of assay CV (from replicate standards) to average subject baseline CV  was 0.52, satisfying the criterion (<0.6) allowing statistically meaningful analysis of within-subject biological variation [Supplementary Table 2).Given the significant fraction of samples that showed evidence of an inflammatory response, we defined personal ‘normal’ baseline levels for each biomarker protein based on the subset of samples with CRP below a cutoff equal to the personal median CRP value across all of a subject's samples; in other words, selecting the half of each subject's samples with lowest CRP values. For each protein, the average value in these low-inflammation baseline samples was taken as the subject's personal baseline. The standard deviation in these baseline samples allowed calculation of a personal baseline CV. The average baseline CV calculated over all subjects, samples and proteins was 12.3%  compared with 38.0%  for combined baseline and nonbaseline  samples . Inflammatory responses  surgical procedure and subsequent recovery period of 97 days during which daily (or more frequent) samples were collected C. FollowA combination of rapid APR protein changes superimposed on a slow declining trajectory were observed in subject S-10, who has Crohn's disease and has diligently explored dietary and other measures to control symptoms. Over a period of ∼2.5 years, CRP and SAA levels were substantially elevated on numerous occasions B. OveralThe most rapid APR protein changes observed were the result of intense physical training, in this case using dried venous blood samples from elite Brazilian beach volleyball athletes during a week of Olympic training. Four samples were collected on each training day: Differences in timing between earlier and later-peaking inflammation markers suggest an improved method for visualizing an inflammatory response trajectory as loops in two dimensions, starting at baseline, through injury and returning to baseline . Figure The same approach can be applied B to visuvia DBS, a broad panel of acute phase proteins and precise, multiplexed wide-dynamic-range quantitation using SISCAPA-MS. This picture captures details of inflammation processes on multiple time scales, reveals complex relationships among inflammation-related proteins and extends the measurable dynamic range downward to cover biology-driven fluctuations in APR baselines (‘microinflammation’). The results move beyond what can be observed through sporadic measurements of a single marker such as CRP and offer the potential for major improvements in diagnostic medicine and for monitoring subject responses in clinical trials.Here we present a high-resolution multi-dimensional molecular picture of inflammation events and personal baseline biomarker levels in 16 individuals, combining dense longitudinal sampling While previous studies have examined many of these proteins alone, or occasionally in small groups, this study is unique in applying a broad set of APR markers across many indications at high frequency. Key to this broader coverage is selection of a group of APR proteins that vary widely in response kinetics, magnitudes and function. Each inflammation-associated protein biomarker shows a different characteristic time course of expression during an inflammation event. APR proteins, which are generally produced in the liver, exhibit a protein-specific early phase increase, likely governed by mRNA transcription and expression triggered by a short-lived cytokine signal. Among the APR proteins, LPSBP typically rises most rapidly, followed in order by SAA, CRP, A1AG, FibG, Hx, MBL, Hp and C3. MPO, which in the context of whole blood serves as a surrogate for the neutrophil count, typically shows a spike just before or coincident with LPSBP, reflecting the fact that pre-existing neutrophils can be released from the bone marrow immediately upon cytokine signaling, without a time-lag required for new protein synthesis. APR proteins decline following an order  that is generally consistent with previously measured half-lives of CRP, LPSBP, A1AG and C3 of 26, 32, 104 and 84 h, respectively . ProteinIn addition to different expression times, the APR proteins also differ widely in the amplitude of changes during inflammation events. Proteins exhibiting fast responses typically show very large changes  while those showing smaller, though still statistically significant changes , typically show slow responses. Changes in Alb and IgM generally involve decreases from baseline during major infections .versus small scale inflammation events. The APR panel used here thus achieves its overall design goal: to include proteins responding differently in time, magnitude, polarity and functional responses to inflammatory signals.The data also demonstrate directly, for the first time, that the quantitative relationships between strongly responding APR proteins such as CRP, SAA and LPSBP are nonlinear; in other words, producing different relative change magnitudes in large Supplementary Figures 1–4); the impressive tight clustering of subjects' samples in Underlying these observations is an implicit assumption that proteins  are themselves stable in DBS samples over extended time periods . While the present study was not designed to test stability directly, several results presented here provide compelling evidence of long-term stability: the general constancy of individual subject baselines for most proteins across many years  which allows inflammation events to be visualized in two dimensions as shown in Our long-term objective, however, is to create a true multi-dimensional, personalized longitudinal inflammation model including additional APR components and related features such as neutrophil bursts (measured here as released MPO) preceding APR and adaptive immune responses (measured here as IgM) following some infections. Such a model will incorporate the temporal and magnitude relationships among the biomarkers (including the nonlinear regulatory effects first reported here), fitting multi-protein data from observed events to generate important derived parameters. Some of the required methods may be adapted from classical pharmacodynamic (PD) modeling , in whicA number of novel observations emerged from examination of our extended longitudinal sample series. First is the unexpectedly large number of identifiable inflammation events, most of which were apparently due to infections in apparently normal subjects. A surprisingly high proportion (12–45%) of each subject's samples showed indications of inflammation, whether defined as CRP levels >twofold above personal baselines, or inclusion in the 57 discernable APR events. Similar proportions are obtained when considering only those samples collected on a near-daily basis (about 1/3 of the samples), in which the timing of collection should be less susceptible to subjects' selection bias. Such high event frequencies, if confirmed in a larger subject population, suggest that unrecognized short-term “sub-clinical” inflammatory events are likely to occur frequently during investigational studies and drug trials, and perhaps at even higher frequency in unwell individuals (of which the Crohn's disease subject is an example). Such changes may impact behavior of drugs not directly related to inflammation, for example, due to the well-known binding of many small molecules to the APR proteins Alb and A1AG .The frequency of small inflammation events also suggests that randomly-timed single CRP measurements  may overestimate a patient's true baseline level if collected during an inflammatory episode , and thus bias predictions of cardiovascular disease risk ,51. ConvCharacterizing personal baseline inflammation proved to be more challenging (and interesting) than anticipated. In the half of each individual's samples with the lowest CRP (presumably the samples showing the lowest levels of inflammation), we observed persistent correlations between CRP, SAA and LPSBP, indicating that a significant amount of the observed baseline variation represents low-level, but real, biological response fluctuations (‘microinflammation’), rather than analytical ‘noise’. As a result, the spectrum of inflammatory responses measurable using APR biomarkers appears to extend smoothly from ±10%  to 1000-fold, a total dynamic range of over 10,000. This observation suggests the possibility of correlating detectable subclinical inflammatory triggers with a variety of contextual data including diet, environmental exposures, recurring infections and chronic inflammatory disease.The 57 discrete inflammation events we cataloged included events driven by a variety of causes, both ‘planned’  and ‘unplanned’ . Given the small number of subjects covered, these observations represent individual case reports and require follow-up in well-designed studies of larger cohorts to confirm the generality of our observations.Total hip replacement surgery in subject S-04 represented a ‘planned’ inflammatory intervention, whose known start time and 2-h duration allowed precise timing of samples with respect to the inflammatory stimulus A. Post-sSupplementary Figure 4), after which the levels subsided to pre-existing baseline levels. The production of such large amounts of IgM after specific infection events is consistent with the expected timeframe for an adaptive immune response to a pathogen, and may provide an opportunity to identify endogenous human monoclonal antibodies from DBS samples with potential therapeutic value as anti-infectives. In contrast, MPO, which serves in whole blood as a surrogate for neutrophil count, was frequently increased at the earliest stage of infection followed by spikes every 2 or 3 days , consistent with periodic release from and regeneration of neutrophil pools in the bone marrow [A series of infection events generated the largest APR protein changes observed here, seven of which resulted in SAA levels ≥100-fold above personal baselines A. These e marrow .In a single case of Crohn's disease, we observed a higher frequency of APR events than in the other subjects, including eight subject-identified ‘Crohn's attacks’, three ‘colds’ and one instance of food poisoning. Interestingly, three of the Crohn's attacks occurred at times of minimal APR levels, while the others coincided with APR spikes of 2- to tenfold above baseline levels. The brief timecourse of the attacks highlights the necessity for frequent sampling as a basis for selecting an optimal sampling strategy for any given condition: a weekly sampling schedule would have completely missed most of the events B & C. OfIntense exercise with accompanying heat stress and strong sun, in this case occurring during Olympic training for beach volleyball, was included here to assess the potential magnitude of very short-term physical effects on APR responses. The most significant changes observed were large decreases in Hp  and incTaken together, these results demonstrate that DBS collected under field conditions and stored for periods up to 9 years can be analyzed by SISCAPA-MRM to deliver high-precision biomarker measurements spanning a very wide dynamic range.unpublished data).However, we note several limitations of the present study. While the sample numbers analyzed here exceed those in all but a handful of biomarker studies, the number of subjects is modest, with only five individuals contributing more than 100 longitudinal samples each. Future studies will require larger numbers of subjects in order to generalize conclusions about the frequency, scale and time courses of inflammation events, to link this data with specific causes and outcomes, and to determine optimal sampling frequencies for specific applications. While not strictly necessary in studies such as ours that refer biomarkers to personal baselines, we also look forward to reporting calibration of the individual biomarkers against validated clinical assays, and rigorous comparison of dried capillary blood with liquid venous serum  was collected from 16 individuals over periods up to 9 years, including daily collection for extended periods.A panel of nine positive and three negative APR proteins was measured by SISCAPA-LC-MRM mass spectrometry.Spot-to-spot volume variation in dried blood spots was reduced substantially by normalization of a three-protein panel, providing total workflow assay CVs on replicate DBS samples of 2.5–6%.A total of 57 discrete inflammation events were observed related to major infections, vaccination, surgery, extreme exercise and Crohn's disease.APR proteins were differentially regulated during these events, with unique nonsynchronous time courses and nonlinear regulatory relationships that require PD-like modeling and interpretation more advanced than currently used ratios.CRP, SAA and LPSBP remained correlated in the subset of samples with lowest CRP, indicating that very low level ‘microinflammation’ phenomena can be measured and extending the dynamic range of measurable APR to >10,000.Multiplexed measurement of a broad panel of acute phase response (APR) proteins, including CRP, at frequent timepoints using DBS microsamples provides improved insights into the magnitude and dynamics of inflammation processes.The approach provides personal baselines for each protein, allowing improved diagnostic performance.Clinical trials of anti-inflammatory biologics, antibiotics and cancer therapeutics can benefit from improved multi-marker pharmacodynamic models of inflammation.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


This study aimed to understand the ability of fixed-bearing posterior cruciate ligament (PCL)-retaining implants to maintain functionality of the PCL in vivo. To achieve this, elongation of the PCL was examined in six subjects with good clinical and functional outcomes using 3D kinematics reconstructed from video-fluoroscopy, together with multibody modelling of the knee. Here, length-change patterns of the ligament bundles were tracked throughout complete cycles of level walking and stair descent. Throughout both activities, elongation of the anterolateral bundle exhibited a flexion-dependent pattern with more stretching during swing than stance phase . The posteromedial bundle remained shorter than its reference length (defined at heel strike of the level gait cycle) during both activities. Compared with loading patterns of the healthy ligament, postoperative elongation patterns indicate a slackening of the ligament at early flexion followed by peak ligament lengths at considerably smaller flexion angles. The reported data provide a novel insight into in vivo PCL function during activities of daily living that has not been captured previously. The findings support previous investigations reporting difficulties in achieving a balanced tension in the retained PCL. Posterior cruciate retaining total knee arthroplasty (PCR-TKA) has been advocated as a surgical advancement to better restore joint function. The retained posterior cruciate ligament (PCL) is thought to assist femoral rollback with knee flexion, which can consequently increase the moment arm of the knee extensor muscles and increase the knee range of motion (ROM) ,2,3,4. HA comparison of PCL function in healthy vs. PCR-TKA knees reported in the literature indicates that PCR-TKAs may subject the PCL to abnormal loading. Our previous systematic review and meta-analysis of the literature was able to extract the average loading patterns of the natural PCL, and reported that the ligament is increasingly stretched as the knee flexes, before peaking at 90°, after which it shortens . HoweverMost studies reporting PCL loading after PCR-TKA examined cadaveric specimens using a variety of different assessment techniques . As suchFluoroscopic imaging and image processing now enable the relative kinematics of the tibial and femoral implant components to be measured during restricted movements ,21,22. BSix subjects  with a unilateral PCR-TKA , each with a neutral mechanical alignment were recruited to take part in this study. Subjects had good postoperative functional and clinical outcomes  and were measured at least 1 year after surgery (mean time ± SD: 4.2 ± 3.5 years). Each subject provided written, informed consent before participation in this study, which was approved by the local ethics commission (EK 2011-N-6).A moving fluoroscope ,27 was uA previously developed multibody knee model  was scalThe model represented the anterolateral (AL) and posteromedial (PM) bundles of the PCL using 10 one-dimensional elements (per bundle) connecting their origin and insertion sites. For each activity trial, the fluoroscopically measured 6 DoF tibiofemoral kinematics over time were prescribed to the model tibiofemoral joint. The length of each ligament fibre throughout the entire activity cycle was calculated by the standard analysis tools available within OpenSim, and normalised to their reference length, which was considered to be each fibre’s own length at heel strike of the level walking cycle . For theFor each ligament bundle, intra-subject variabilities of the ligament elongation patterns were assessed by calculating the SDs between the data obtained for the five trials captured from each subject for every degree of knee flexion. The mean SDs were then calculated by averaging the intra-subject SDs achieved at each degree of knee flexion. Finally, the inter-subject variability was determined at each degree of knee flexion by calculating the average SD across all trials and all subjects. A repeated measures ANOVA based on statistical parametric mapping (SPM ) was useTo assess sensitivity of the ligament elongation patterns to variations in implant kinematics, the 3D poses of the implant components were perturbed around the baseline kinematics captured by the moving fluoroscope during a single gait cycle. Here, the anterio–posterior (AP), medio–lateral (ML) and proximal–distal (PD) translations were varied for each frame within ± 5 mm, while the flexion–extension (FE), varus–valgus (VV), and internal–external (IE) rotations were perturbed within ± 5° . The perturbed kinematics were then used to drive the subject-specific multibody model and the corresponding elongation patterns were calculated. In addition, change of the maximum ligament elongation due to variation in each implant kinematic parameter was calculated and used to estimate the range of possible errors that might occur from inaccuracies in the employed image-based technique.The intra-subject PCL elongation patterns remained largely consistent between the five repetitions captured in each subject during each of the studied activities . The maximum length-change of the AL bundle during stance phase was 5.4 ± 2.5% observed at 67°, whereas the corresponding value for the swing phase was 11.1 ± 3.6% at 58°. At this 67° angle, the PM bundle was around 8% shorter than its reference length during the stance phase, while at the same flexion angle during the swing phase, it had almost recovered to its reference length.Throughout the stance and swing phases of stair descent, the flexion dependent ligament elongation patterns exhibited similar trends. Interestingly, however, both bundles were longer during the swing than the stance phase , and thip < 0.02 for AL and PM bundles).At low flexion angles (less than around 40°), the SPM showed no statistically significant differences in the elongation patterns of either PCL bundle between level walking and stair descent . HoweverIn general, ligament elongation patterns were partially affected by changes in each of the implant kinematic parameters . AmongstDespite the evolution of PCL retaining knee replacement implant designs, it remains unknown whether the natural function of the postoperative ligament is restored, including natural PCL kinematics, throughout activities of daily living. In our study, elongation patterns of the AL and PM bundles of the PCL in six subjects with a fixed bearing PCR-TKA implant were quantified throughout complete cycles of level walking and stair descent. Here, the implant kinematic data, measured using a moving fluoroscope, were used to prescribe the motion of multibody knee models with the PCL bundles represented by a series of one-dimensional elements. Throughout both studied activities, the AL-PCL exhibited more lengthening during the swing than the stance phase. The PM-PCL remained shorter than its reference length (defined as its length at heel strike during level gait) throughout both level walking and stair descent. These data, together with the differences observed between the swing and stance phases, indicate that PCL function in PCR-TKA is critically governed by the loading conditions. As a result, the reported data throughout complete cycles of walking and stair descent, including their loaded and unloaded phases, provide novel insights into in vivo PCL function in activities of daily living. Specifically, our study reveals more elongated PCL bundles during swing than the stance phase, which has not been previously captured, and which suggests that assessment of PCL functionality should be performed throughout complete activity cycles.Correct intraoperative tensioning of the PCL is consistently emphasised in the literature in order to achieve a successful PCR-TKA ,34,35. IImportantly, this study found significant differences in PCL elongation between the swing and stance phases of walking and stair descent. Our results indicate greater lengthening of the AL-PCL during swing than the stance phase. This is once again likely to result from the anterior femoral translation during the swing phase that was reported in the same subjects . These kOur comprehensive literature review demonstrated large variability in the reported PCL loading patterns after PCR-TKA . A decreImplant geometry can balance the PCL contributions to femoral roll-back and thereby impact postoperative PCL tension ,41,42. TThe intra- and inter-subject variabilities observed in the elongation patterns obtained in the current study correspond well with variations in the kinematic patterns reported in the same subjects . Here, tThere are some other limitations that need to be considered when interpreting the results presented in this study. First, since subject-specific medical images were not available to reconstruct the knee geometry and segment ligament attachment sites, a generic model was scaled to subjects’ anthropometry to represent subject-specific knee models. Moreover, despite delivering highly accurate in-plane kinematics, single-plane fluoroscopy is known to have a limited accuracy in capturing out-of-plane kinematics. The sensitivity analysis performed in this study  indicateThis study revealed similar trends to the PCL elongation patterns in a group of PCR-TKA subjects to those previously observed in healthy subjects. Here, we additionally show clear differences in the PCL strains under different activities and loading conditions, as well as between the different PCL bundles. However, the differences observed between PCL elongation patterns captured post-TKA and those of the healthy PCL reported in the literature support previous investigations reporting difficulties in achieving healthy tension balance in PCR-TKA.


One anastomosis gastric bypass- minigastric bypass (OAGB-MGB) is an emerging bariatric surgery that is being endorsed by surgeons worldwide. Internal herniation is a rare and dreaded complication after malabsorptive bariatric procedures, which necessitates early diagnosis and intervention.We describe a 29-year-old male with chylous ascites caused by an internal hernia 8 months following laparoscopic one anastomosis gastric bypass. An abdominal CT showed enlargement of lymph nodes at the mesentery, with a moderate amount of liquid in the abdomen and pelvis. An emergent exploratory laparoscopic surgery demonstrated an internal hernia at the Petersen’s space with a moderate quantity of chylous ascites. The patient made an uneventful recovery after surgery.Internal herniation can occur after OAGB-MGB and in extremely rare cases lead to chylous ascites. To our knowledge, this is the first reported case of chylous ascites following one anastomosis gastric bypass. One-anastomosis gastric bypass- minigastric bypass (OAGB-MGB) is an emerging bariatric surgery technique currently endorsed by surgeons worldwide , 2. SimiEarly complication rates of OAGB-MGB range from 0.5 to 3.1% . This, h2; weight loss 53 kg). On physical examination, the patient’s vital signs were within normal range. The patient had no fever. Abdominal auscultation revealed normal bowel sounds. Upon abdominal palpation, the patient complained of diffuse tenderness. His lab results, including lactate, amylase and lipase were all within normal limits.We report a 29-year-old male, otherwise healthy, who presented to our emergency department, complaining of severe diffuse abdominal pain that had developed over the last 24 h, and had become progressively worse with time. The patient did not report any constipation, diarrhea, or distention despite having nausea and vomiting. He had undergone laparoscopic OAGB-MGB in a different medical center 8 months prior to his admission (Body mass index (BMI) prior to the surgery: 42 kg/mA computed tomography scan of the abdomen and the pelvis following oral and intravenous contrast administration demonstrated no evidence of intestinal obstruction or free air. Enlarged lymph nodes at the root of the mesentery were observed, with a moderate amount of free liquid of unknown origin in the abdomen and pelvis Fig. . The aboParallel to dramatic increases worldwide in obesity prevalence, surgical procedures aimed to induce and maintain weight loss have increased considerably over recent years. Both RYGB and OAGΒ-MGB are common, well-tolerated and effective operations for morbid obesity. Both surgeries carry similar rates of complications and reoperations, with leaks and hemorrhages being the most common due to the gastrointestinal anastomoses .An internal herniation is a dangerous late complication that may occur after both RYGB and OAGB-MGB procedures  and is characterized by symptoms of abdominal obstruction . CliniciChylous ascites in a bariatric surgery setting generally occurs by a loop passing through the Petersen’s defect, which causes direct compression of the mesenteric vessels and fluid extravasation. In previous case reports of patients with chylous ascites following RYGB, the complication was resolved by the release of the internal hernia compressing the mesenteric lymphatic vessels . SimilarClosure of the mesenteric defect at the time of the primary surgery has been shown to reduce the occurrence of internal hernia following RYGB procedures . AlthougIn conclusion, internal hernia is a dangerous complication following OAGB, which in extremely rare cases can lead to chylous ascites. Surgical intervention is required to release the hernia and relieve the compression of the lymphatic vessels. Closure of the potential spaces that can lead to internal herniation should be evaluated in OAGB.


Streptococcus disease at an eldercare facility, in which 5 persons died, was biphasic. Although targeted chemoprophylaxis contained the initial outbreak, a second phase of the outbreak occurred after infection control processes ended. To retrospectively investigate the genomic epidemiology of the biphasic outbreak, we used whole-genome sequencing and multiple bioinformatics approaches. Analysis of isolates from the outbreak and isolates prospectively collected during the outbreak response indicated a single S. pyogenesemm81 clone among residents and staff members. Outbreak isolates differed from nonoutbreak emm81 isolates by harboring an integrative conjugative genomic element that contained the macrolide resistance determinant erm(TR). This study shows how retrospective high-resolution genomic investigations identified rapid spread of a closed-facilty clonal outbreak that was controlled, but not readily cleared, by infection control management procedures.A 3-month outbreak of invasive group A  Streptococcus pyogenes, or group A Streptococcus (GAS), is a gram-positive, human-adapted opportunistic bacterial pathogen. GAS causes a wide variety of clinical manifestations, from relatively benign self-limiting infections of the nasopharynx or skin to life-threatening invasive infections such as bacteremia, pneumonia, and necrotizing fasciitis  was hospitalized in early June 2014. The initial 3 case-patients were admitted within 24 hours of each other, and GAS was isolated from blood or tissue cultures from all 3 case-patients. During the first phase, 6 case-patients with confirmed GAS infection were hospitalized; 5 died of presumed sepsis. Outbreak investigations and control measures were subsequently implemented and included screening staff members and residents by collecting throat swab samples and providing targeted chemoprophylaxis for residents and staff members who were in direct contact with case-patients. These interventions continued until early July 2014; however, in late July, the outbreak recurred and continued until mid August 2014. In this second phase, a staff member was hospitalized with severe pharyngitis, after which 5 residents were hospitalized with soft tissue infections, septicemia, or both.According to disk-diffusion testing, all GAS isolates collected during the first phase were susceptible to penicillin but resistant to erythromycin. Accordingly, a suspected case-patient was defined as any resident or staff member from the facility who was unwell from early June 2014 through mid-November 2014, and a confirmed case-patient was any person from the facility from whom erythromycin-resistant GAS was cultured from blood, throat, or skin samples. The definition of a suspected case-patient was kept intentionally broad because of the wide-ranging symptoms among initial case-patients. A carrier was defined as any asymptomatic person from the facility from whom erythromycin-resistant GAS was isolated from a skin or throat swab sample.emm typing at ESR according to previously described methods .After the initial 3 cases were confirmed, throat swab samples were collected from all residents and from facility staff members who worked in nursing, kitchen, or waste collection. Swab samples were also collected from any skin lesion on staff members or residents. Hospital staff working in the wards where residents had been admitted were also asked to consent to collection of throat swab samples. The outbreak isolates were compared with nonoutbreak isolates for contextual purposes. Nonoutbreak isolates were defined as clinical isolates submitted to the Institute of Environmental Science and Research (ESR) in New Zealand from across New Zealand during 2002–2014. Although including only contemporaneous nonoutbreak isolates would have been ideal , New Zealand’s small population and the fact that GAS infections are not notifiable in New Zealand meant that we had to select nonoutbreak isolates over a broader time frame. All outbreak and nonoutbreak isolates underwent initial https://www.illumina.com). To enable fine-mapping of the outbreak, we completely sequenced a representative emm81 outbreak isolate, DMG1800716, by using Pacific Biosciences long-read technology (https://www.pacb.com). To validate the consensus assembly of the reference genome, we used Illumina short reads. We used Prokka (https://blast.ncbi.nlm.nih.gov/Blast.cgi) comparisons of the 55 emm81 genomes relative to DMG1800716 by using the BLAST Ring Image Generator (https://www.ncbi.nlm.nih.gov/sra) under BioProject PRJNA494270.We performed genome sequencing and assembly for outbreak and nonoutbreak isolates. All isolates underwent Illumina whole-genome sequencing  were detected by swabbing of unwell persons with suspected cases, and the other 3 confirmed cases were identified by prospective sampling of all 75 residents and 30 hospital staff members . Prospective swabbing also identified 1 resident as a carrier (erythromycin-resistant GAS was isolated from the resident’s throat).The average case-patient age was 79.5 years. Residents exhibited a variety of signs and symptoms . One staff member was hospitalized with severe pharyngitis; the other staff members were treated at home for minor skin infections. During the first phase of the outbreak, a characteristic feature was the rapidity with which case-patient conditions deteriorated; 1 died within a few hours of symptom onset. Five confirmed case-patients, all residents, died of streptococcal sepsis during the first phase. During the second phase of the outbreak, no deaths were reported. Five persons had suspected cases  during the outbreak but were excluded from this analysis because erythromycin-resistant GAS was not isolated.During our investigation, we obtained 18 erythromycin-resistant GAS isolates, which were cultured from a variety of body sites including blood, throat, and soft tissue . For 2 rAfter the initial 3 cases were confirmed and the outbreak was recognized, public health staff members initiated targeted chemoprophylaxis. A 10-day course of penicillin or amoxicillin was given to all staff members, any resident who was unwell or had been in contact with a case-patient, and any resident from whom GAS was isolated. The 4 staff members with outbreak strain infections stayed away from work until they had completed their course of antimicrobial therapy, their clinical signs of infections had resolved, and a throat swab sample culture was negative. Outbreak control measures initially continued for 1 month after the last case in the first outbreak phase was identified.When the outbreak recurred, additional surveillance and environmental control measures were initiated and continued for 3 months after the last case of the second phase was identified. Other additional control measures included educating staff and residents about hand hygiene, monitoring the temperature of any resident with a skin lesion, cleaning all furniture and upholstery with diluted bleach where possible, replacing all toothbrushes, using disposable wound dressing trays rather than trolleys, inspecting the hands of staff members for skin lesions daily, and instructing the hospital to collect blood and throat swab samples for culture from any residents admitted from this eldercare facility and to place them in a single room. Items used communally by residents and staff  were cleaned with diluted bleach after meals or each use.emm81.0 gene allele. Only 5 contemporaneous nonoutbreak emm81 isolates were collected in New Zealand during 2014; the remaining 32 nonoutbreak isolates were collected during 2002–2013. Core-genome comparisons of the 18 outbreak strains with the 37 nonoutbreak emm81 isolates showed that the outbreak isolates were highly clonal and formed a separate clade in the emm81 phylogeny ; all other nonoutbreak isolates sampled during the outbreak period, which were all from other geographic regions, were ICE negative . ICE-SpDMG1800716 also harbored the abortive infection operon, AbiE operon, which is associated with bacteriophage resistance and stabilization of extrachromosomal elements (rum) gene  gene , panel Cemm81 GAS clone that was resistant to erythromycin and exhibited inducible clindamycin resistance. emm81 GAS is one of the most common M-types that causes invasive disease in New Zealand (emm81 as a global GAS strain is highlighted by its inclusion in the experimental 30-valent M-protein vaccine (emm typing, would not have had the discriminatory power to differentiate the outbreak isolates from other emm81 isolates that were already in New Zealand. Along with other recent reports of GAS outbreaks of a single emm type (Through our clinical and genomic epidemiologic analyses, we determined that a fatal GAS outbreak in an eldercare facility was associated with a single emm81 isolates identified that the outbreak clone had acquired a macrolide resistance determinant within a putative integrative and conjugative element, ICE-SpDMG1800716. Outbreaks of GAS disease have previously been linked to the acquisition of mobile genetic elements, such as an ongoing polyclonal emm12 and emm1 scarlet fever outbreak in Hong Kong and mainland China associated with horizontal acquisition of multidrug resistance and a superantigen-encoding prophage (,Comparative analyses of the outbreak clone with 37 nonoutbreak SpDMG1800716 within the genomes of the outbreak isolates.In addition to harboring macrolide-resistance genes, the integrative conjugative element in the outbreak isolates also contained the abortive infection protein AbiE, which may have contributed to the relative fitness of the outbreak isolates. AbiE may confer bacteriophage resistance and has been shown to stabilize extrachromosomal elements such as plasmids (GAS carriage among healthcare workers in this and other outbreaks serves as a reminder that staff member sampling is integral to GAS outbreak investigations (As was the case for other reported outbreaks in long-term care facilities (In summary, our data further highlight the potential for invasive GAS to cause rapid and fatal outbreaks, particularly in closed communities such as eldercare facilities. Invasive GAS disease is not notifiable in New Zealand, nor is there mandatory surveillance for invasive GAS infections. The incidence of invasive GAS infections in New Zealand and elsewhere is particularly high among those >75 years of age (Streptococcus disease in nursing care facility, New Zealand.Additional methods and results for study of biphasic outbreak of invasive group A Streptococcus disease in nursing care facility, New Zealand. Additional results for study of biphasic outbreak of invasive group A