The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments. The biotechnological potential of marine bacteria has been exploited in several patented technological processes based on marine enzymes . MetagenOxygenases, which catalyze the addition of oxygen atoms into many organic compounds, show a remarkable enantio- and regio-selectivity and broad substrate specificity ,8,9,10. Type I Baeyer–Villiger Monooxygenases (BVMOs) belong to the Group B Flavin-dependent Monooxygenases (B-FDM), a diverse family of enzymes that also includes Flavoprotein Monooxygenases (FMO), N-hydroxylating Monooxygenases (NHMO) and YUCCA Monooxygenases  . BVMOs hCYP153 are soluble haem-containing monooxygenases, which catalyze the hydroxylation of medium-chain-length alkanes (from C6 to C11) at the terminal position to produce 1-alkanols. Applications of CYP153 enzymes include the conversion of terpene limonene into perillyl alcohol, a putative anticancer agent as well as the capability to hydroxylate piperidines, pyrrolidines and azetidines to useful pharmaceutical intermediates . MoreoveThe goal of this work was to identify Baeyer–Villiger and CYP153 Monooxygenases in a metagenomic dataset obtained from shotgun metagenomic sequencing of polar and subpolar coastal sediments , and to The dataset used in this work for the identification of sequences homologous to monooxygenase enzymes with biotechnological potential contains 23 assembled metagenomes of coastal sediments from four distant polar or subpolar environments . Both marine and brackish ecosystems exposed to oil-pollutants and cold temperatures are represented in this dataset . Microorr = 0.667, p = 0.01). This result suggests that the number of retrieved sequences could have been limited by the relatively low sequence depth (one lane of Illumina HiSeq 1500 per sample), which probably affected the assembly efficiency  searches . The numficiency . This isConsidering that some members of B-FDM different from BVMO share the same Pfam domain (PF00743) or have a high sequence homology against BVMOs sequences, the identified metagenomic sequences were further classified by phylogenetic analysis using reference sequences belonging to the different subgroups of B-FDM . The mosWith the aim of identifying novel features related to active sites or ligand specificities, a subset of divergent putative BVMO sequences was chosen for further analysis, based on the following selection criteria. First, clusters supported by bootstrap values higher than 50% and containing at least one full-length metagenomic sequence were identified in the phylogenetic tree  and steroid monooxygenase (STMO)  showed potential structural features different from the template structures . Lesser e (STMO) C. The 3De (STMO) D. In adde (STMO) E.Rhodococcus sp. HI-31 (pdb 3UCL), was used as the reference to establish the potentially productive orientations of the ligands for catalysis ; (ii) Port Värtahamnen, Stockholm, Baltic Sea, Sweden [SWE]; (iii) Ushuaia Bay, Tierra del Fuego Island, Argentina [ARG]; and (iv) Potter Cove, 25 de Mayo (King George) Island, Antarctica [ANT]. Details on sampling, DNA extraction and shotgun sequencing were previously reported . Briefly−5 were performed in IMG, using selected sequences from crystallized and/or biochemically characterized enzymes as query (For each enzyme family (BVMO and CYP153), sequences were obtained from the metagenomes using two complementary functional evidences . Firstlyas query . Duplicaas query . An addiReference sequences for each enzyme family were collected from the literature. Sequences coding for cytochrome P450 were obtained from a recent work published by Nie et al. . For BVMFor each metagenomic sequence, the HHpred web server was used to search for suitable templates for building high quality models . SelecteThree-dimensional structure files of ligand molecules were downloaded from the PubChem database , and com+ binding subdomains were considered as different interacting protein chains. The information to define subdomains was obtained from the crystal structure of the different Baeyer–Villiger Monooxygenase used as templates [Parameters associated with protein structure stability were calculated using PDBePISA server  . For calemplates . For theemplates . The obtemplates . The ideemplates .The methodological framework reported in this work allowed the selection of four sequences out of ~14 million PCSs as candidates for synthesis and heterologous expression, greatly aiding in the efficient and knowledge-based exploitation of a highly fragmented metagenomic dataset. These sequences include the putative BVMOs ANT05_100010021, NOR08_100070122 and ANT01_100026088, which were predicted to have a wide substrate range and novel substrate specificities. In addition, NOR08_100070122 and NOR08_100243532 could be regioselectives for abnormal lactones. On the other hand, the putative CYP153 ARG05_10097442 could present novel substrate specificity and regioselectivity. These putative enzymes could be used in the synthesis of a wide variety of lactones or 1-alkanols at room or low temperatures, where biooxidations may produce better results than the typical chemical oxidation with regard to performance and cost savings in energy consumption. These putative enzymes also could be applied to improve procedures aimed to overcome substrate and product inhibition, performing the reaction in a biphasic system by using organic solvents whose water miscibility can be varied by temperature . ConsideThe presented evidence suggests that the active sites of the selected putative monooxygenases would be larger and more accessible than those from previously biochemically characterized enzymes. It has been observed that low temperature adapted enzymes present larger catalytic cavities, more accessible to ligands, than mesophilic enzymes . A betteFinally, the methodology applied in this work can be used for mining genetic information with biotechnological potential from metagenomic datasets currently stored in public databases, as well as to complement approaches based on functional metagenomics or where only specific HMMs are constructed and applied in screening. This complement may strengthen the prediction aimed to rationale biocatalysts selection before biochemical characterization.


Determination of biomarkers may be useful in the surveillance of occupational exposure and workers’ health. The possibility of predicting development/clinical course of specific disorders or current disease, diagnosing in early steps, and health condition monitoring is a real necessity. Various agents present in the workplace environment (or their metabolites) can be measured in samples possessed from human body . On the other hand, inhalant exposure may induce specific or non-specific, local or systemic, acute or chronic biological response expressed by synthesis or releasing specific or non-specific substances/mediators that also can be determined in blood, nasal and bronchial lavage or sputum, tear fluid, exhaled breath, etc. The least is known about genetic markers which may predict individual susceptibility to develop some work-related disorders under the influence of occupational exposure. Due to common exposure to inhalant agents at workplace, researches on biomarkers that allow to inspect the impact of exposure to humans’ health are still needed. The authors of this article summarize the utility of biomarkers’ determination in work-related airway diseases in a recent clinical approach. Work-related respiratory diseases (WRRD) make up to 70% causes of all occupational disorder mortality among working population . In geneThis article presents reports on biomarkers in work-related airway diseases based on overview of available publications in the EBSCO and the MEDLINE online research databases. The search was limited to articles published mainly in the period of 2005–2016; however, a few previous source publications have been mentioned in references too. Biomarkers that can be determined in biological material samples collected directly from airways seem to be the most useful in diagnostic process and workers’ health monitoring, anywise blood investigation may be applicable too. The conception of “unified airway disease” (UAD) is worth noticing  because Investigation on biochemical and cellular composition of nasal lavage fluid (NLF), induced sputum (IS), or bronchoalveolar lavage (BAL) has provided the most reliable outcomes about local inflammatory process in respiratory tract. Additional good point is a non-invasive way of collecting these samples; however, procedures need to be carried out in referential medical centers with appropriate laboratory equipment and qualified personnel. Confirmation of eosinophilic inflammation in nasal secretion samples (smear or lavage) after the challenge with allergens has become the most useful diagnostic phenomenon in occupational rhinitis (OR) –7, 8•. DAlternating biomarker of eosinophilic inflammation in upper and lower airway is eosinophil cationic protein (ECP). Significantly elevated ECP levels have been measured in NLF collected from patients with OR and/or OA , 20, alsNO in clinical practice is valuable in airway diseases with eosinophilic inflammatory background, especially in asthma and EB. This is not a specific method for work-related respiratory disorders; however, the assessment carried out before and after work-shift or SIC brings information about possible escalation of local inflammatory process, especially if contraindications to induce sputum were certified or the procedure did not allow to obtain sufficient-quality samples [NO of 30–40% [NO in diagnosing OA and medical certification is still controversial and less valuable than assessment of cellular IS composition due to higher sensitivity to confounding factors  [The usefulness of measuring concentration of FE samples •, 23. Th samples . German  samples . An incrf 30–40% , 27 24 hsmoking) •. In exhsmoking) .Confirmation of non-specific bronchial hyper-responsiveness (NSBHR)-validated inhalant tests (with histamine/methacholine/mannitol/adenosine monophosphate) is indispensable part of objective diagnostic process of work-related asthma. Recording at least two measurements of NSBHR before and after the SIC may be helpful in predicting post-challenge asthmatic response in subsequent exposures  for the patients with OA who obtained a significant (more than two-fold) increase in NSBHR , 30•. MeNO) was found also among female hairdressers exposed to high concentrations of ammonia in comparison to unexposed controls [In comparison to asthmatic workers, neutrophilic inflammation in IS has been observed among patients who suffered from COPD , 44. Whacontrols . German controls . These fDetermination of skin and serum-specific immunoglobulin E (sIgE) antibodies demonstrates the sensitization to allergens; however, commercially available standardized tests are prepared only for a few occupational allergens , 49. DetDetection of antigen-specific IgG (sIgG) antibodies is helpful in searching the causative agents of occupational hypersensitivity pneumonitis (OHP) , 62•, buSimilar to IS samples, evaluation of ECP levels in serum has been considered as the exponent of systemic eosinophilic inflammation in workers with OA , 71; howAirborne iron may be responsible for siderosis and lung cancer among welders , 75, andSusceptibility to develop some work-related respiratory disorders has been investigated through identification of “candidate” gene researches. It has been found that the HLA-DPB11(E69) allele is closely associated with sensitivity to airborne beryllium, and inheritance of this gene increases the risk of development of chronic beryllium disease even thirty-fold in exposed workers . VariousAt present, most of the well-known and reliable biomarkers are related to work-related airway disease with allergic background. Numerous researches have been carried out on occupational asthma and resulted in indicating the cellular assessment of induced sputum as the most useful in diagnostic process. A comprehensive overview of current knowledge on biomarkers’ utility in diagnosis of occupational asthma was described elsewhere •. Biomol


Psoriasis is a chronic inflammatory systemic disease with a prevalence of 2–3%. Overwhelming evidence show an epidemiological association between psoriasis, cardiovascular disease and atherosclerosis. Cardiovascular disease is the most frequent cause of death in patients with severe psoriasis. Several cardiovascular disease classical risk factors are also increased in psoriasis but the psoriasis-associated risk persists after adjusting for other risk factors.Investigation has focused on finding explanations for these epidemiological data. Several studies have demonstrated significant lipid metabolism and HDL composition and function alterations in psoriatic patients. Altered HDL function is clearly one of the mechanisms involved, as these particles are of the utmost importance in atherosclerosis defense. Recent data indicate that biologic therapy can reverse both structural and functional HDL alterations in psoriasis, reinforcing their therapeutic potential. Psoriasis is an immunomediated disease classified by some authors as autoimmune , 2, althCardiovascular disease is associated with a 6-year shorter life expectancy in patients with severe psoriasis .Atherosclerosis is the pathological process underlying cardiovascular disease (CVD) and mortality. All phases of atherosclerosis are related to inflammatory events, in a strikingly similar fashion to the inflammatory events observed in psoriasis . FurtherHigh-density lipoproteins (HDL) are of pivotal importance in atherosclerosis. These are extremely complex particles with a unique capacity for milieu adaptation, constantly changing both its chemical composition and its tri-dimensional structure . HDL hasThis review discusses psoriasis-associated alterations in HDL structure and function that may play a role in atherogenesis and vascular disease associated with psoriasis. We searched ISI Web of Science, Medline and PubMed for psoriasis, atherosclerosis, cardiovascular disease, HDL, dysfunctional HDL and anti-HDL antibodies. We started by searching for each term separately and afterwards an advanced search was carried out by combining “psoriasis” with each search term in keywords, abstracts and/or titles. The references cited in the selected articles were also analysed. After a comprehensive review, duplications were excluded and the final selection was based on relevance and date.Lipid metabolism in patients with psoriasis has been a subject of study for more than 50 years. Numerous studies show decreased levels of HDL and/or increased levels of low-density lipoproteins (LDL) and of very-low density lipoproteins (VLDL) and triglycerides (TG) –18. HoweThe published studies are heterogeneous because they include patients with different disease durations and systemic treatments, factors that may per se influence the lipid metabolism. Based on the results of 200 patients investigated at the onset of skin disease showing higher cholesterol concentrations in VLDL and HDL, Mallbris et al.  suggesteLipoproteins form a complex lipid transport system. The smallest lipoproteins in molecular size are HDL and they contain the highest proportion of apolipoproteins to lipids. Apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) are major HDL associated proteins. They are secreted into plasma by the liver and the intestine. The highly dynamic and flexible conformation of the apoA-I molecule provides a possible explanation for how apoA-I determines HDL subclass structure and heterogeneity , 24, 25.HDL takes up cholesterol from cell membranes and other lipoproteins and transports it from the periphery to the liver for excretion during the process of reverse cholesterol transport (RCT). Active export of excess cholesterol to HDL is mediated by the ATP-binding cassette transporters ABCA1 and ABCG1/G4. RCT is a process of major biological importance and is believed to be an important mechanism to explain how HDL might prevent cardiovascular disease.Furthermore, HDL has several anti-inflammatory and antioxidant functions that may also be as important as RCT in protecting against atherosclerosis Table . NoticeaApolipoprotein E (apoE) is another important component of the HDL particle . It is aApoE polymorphisms modulate susceptibility to many diseases, namely neurodegenerative disorders and atherosclerosis, as well as psoriasis , 32. In Whilst most studies have been focused on the HDL protein components, the HDL lipids represent 50–80% of the total HDL mass and have been shown to affect particle stability, cholesterol efflux from macrophages and cholesterol elimination . It is nHDL has known immunomodulating properties related both to the innate and the adaptive immune system .HDL modulates several immune cell functions, mainly by modifying the lipid rafts’ cholesterol content. Thereby it modulates dendritic cells (DC), monocytes, macrophages, T cells and B cells . ImportaIn chronic inflammatory diseases however, the antioxidative and anti-inflammatory properties of HDL become impaired . ProteomImpaired cellular efflux of lipids to HDL is another pro-inflammatory mechanism that is associated with activation of intracellular STAT3 signalling and enhanced vascular inflammation .These phenomena are important in chronic inflammatory diseases like rheumatoid arthritis and systemic lupus erythematosus (SLE), which are also associated with increased risk of cardiovascular disease. Chronic inflammation induced alterations in lipid metabolism certainly contribute to the higher atherosclerotic risk in these patients.In patients with psoriasis, not only lipoprotein levels can be altered, but also their composition and function may be significantly different from controls. This is an important observation because the composition and function of HDL are actually considered more important than the quantity of HDL itself.Mehta et al.  found a HDL isolated from 15 psoriatic patients  revealedA recent study demonstrated that psoriasis associated HDL particle characteristics and function were also altered in 44 paediatric patients with psoriasis. These patients showed reduced cholesterol efflux capacity compared to age-matched controls, before and after adjusting for confounding variables, suggesting that reverse cholesterol transport defects start early in life even with low levels of chronic inflammation .A cross-sectional study designed to characterize the biologic activities of plasma lipoproteins, involving 25 patients with psoriasis and 25 controls, showed lower plasma levels of HDL and reduced PON-1 activity. PON-1 activity was found to negatively correlate to disease severity, measured by PASI. Other HDL properties were also altered, namely a) protection against LDL oxidation, b) inhibition of TNF-α induced monocyte adherence to endothelial cells, c) prevention of oxidized low-density lipoprotein (ox-LDL)-induced monocyte migration and d) protection of endothelial cells from TNF-α induced apoptosis .Reduced PON-1 activity was also demonstrated by Usta  in a cohIn chronic inflammatory conditions, protein structural modification and appearance of neo-epitopes may lead to autoantibody production and further HDL dysfunction. Elevated titres of autoantibodies against HDL (aHDL) and apoA-I have been demonstrated in diseases associated with an increased cardiovascular risk . These aIt should be emphasized that published studies are heterogeneous regarding both clinical and laboratorial data. Patient selection includes different types of psoriasis (e.g. with or without arthritis) in various clinical stages with varying degrees of inflammation or remission and under different systemic therapies. Laboratory methodology is also variable; measuring HDL-cholesterol is not equivalent to measuring HDL particle number. NMR spectroscopy and ion mobility can be used to quantify HDL particles, however, these methods give different estimates of HDL particle concentration and size. Another problem is that all methods used to assess HDL subclasses measure only static concentrations with no assessment of the dynamic processes regulating HDL subclasses . DespiteInflammatory processes occurring chronically in psoriasis have an impact on HDL in many different ways resulting in proteomic and lipid composition alterations and consequently altered HDL function. These alterations help to explain the epidemiological evidence associating psoriasis to atherosclerosis and CVD, though further investigation on this subject is necessary. Importantly, anti-psoriatic therapy reverses these alterations. A better understanding of these mechanisms of disease will contribute to the development of novel biomarkers and of new therapeutic strategies.


During the follow-up, 456incident cases of type 2 diabetes occurred. When comparing extreme quartiles, intake oftotal polyphenol was inversely associated with the risk of type 2 diabetes ; 1 sd increase was associated with a reduced risk of diabetes . Among the main classes of polyphenols, flavonoids, phenolicacids, and stilbenes were independent contributors to this association. Both subclasses ofphenolic acids were associated with decreased risk of type 2 diabetes, whereas amongsubclasses of flavonoids, high intake of flavanols, flavanones, flavones and anthocyaninswas significantly associated with decreased risk of type 2 diabetes. Total dietarypolyphenols and some classes of dietary polyphenols were associated with lower risk oftype 2 diabetes.This study aimed to test the association between dietary content of total and individualclasses of polyphenols and incident cases of type 2 diabetes in Polish adultsparticipating to the Health, Alcohol and Psychosocial factors In Eastern Europe study. Atbaseline, diet by 148-item FFQ and health information were collected from 5806participants free of diabetes. Self-reported incident type 2 diabetes was ascertained at2–4-year follow-up visit. OR and 95 % CI of type 2 diabetes comparing the variouscategories of polyphenol intake to the lowest one (reference category) and as 1 Among the most attractive hypotheses potentially explaining the benefitsassociated with high consumption of fruits and vegetables, polyphenols content is one of thebest candidates as determinant of health. In fact, besides the known macro- and micronutrientscontained in fruit and vegetables, polyphenols are highly representative in beverages such ascoffee, tea and some alcoholic beverages that have demonstrated to have a positive impact onhuman health. Polyphenolic compounds are molecules highly contained in the aforementioned foodand plant-derived beverages divided into four main classes on the basis of their chemicalstructure, including flavonoids, phenolic acids, stilbenes, and lignans, and ‘other’polyphenols including various type of compounds. Overall, polyphenols comprise thousands of different molecules have beenclassified and described. Even though, the main challenges of studying these compounds rely onthe identification of their potential effects in humans. In vitro studiesdemonstrate consistently antioxidant and anti-inflammatory effects at cellular and tissuelevel, but questions remain regarding their effective relevance for human health, as exposurein normal diet occur at lower concentrations than in laboratory setting.Plant-derived dietary pattern have been suggested to exert protection against metabolicdisorders, such as type 2 diabetes. However, available data on type 2 diabetes prevalence and incidence is focusedonly on flavonoids. Only recently research has expanded to other polyphenol groups, such asphenolic acids and individual subgroups but data are overall scarce. Moreover, as polyphenolconsumption varies with a great extent with the dietary habits of a population, it is ofinterest to collect information from various countries in order to provide stronger evidenceof their potential effects. In this study, we aimed to test the association between dietarycontent of total and individual classes of polyphenols and self-reported incident cases oftype 2 diabetes in Polish adults participating to the Health, Alcohol and Psychosocial factorsin Eastern Europe (HAPIEE) study.Although causality is best supported by randomised trials, observational epidemiologicalstudies also contribute to causal inference and are of great interest to evaluate the ‘realworld’ consumption of polyphenols and their potential association with health outcomes.Regarding metabolic disorders, type 2 diabetes has been one of the most studied outcomeevaluated in epidemiological studies. A relatively recent meta-analysis showed that increasedintake of flavonoids, especially flavonols, may decrease the risk of type 2 diabetes. Information on methods used are reported in detail elsewhere. The present study was conducted on a subcohort of the Polish arm of theHAPIEE cohort (a random sample of 10 728 adults aged 45–69 years recruited in 2002–2005(response ratio of 59 %) in the urban area of Krakow, Poland) which was free of diabetesor impaired fasting glucose (fasting plasma glucose from 6·1 to 6·9 mmol/l) at baseline(n 9420). Study participants completed a structured questionnaire andunderwent to a clinical examination during the baseline data collection (wave I) and atfollow-up visit (wave II). Participants were followed for a median follow-up period of 4years (range 3·2–5·4 years). For the purpose of this study, only individuals free ofdiabetes at baseline who attended the last follow-up visit in 2006–2008 were included inthe analysis . Sample included in theanalysis did not substantially differ in background characteristics or variables ofinterest concerning dietary habits. All participants provided written informed consentbefore joining the study.The HAPIEE study is a multicenter prospective cohort study investigating the role ofbiological, dietary, lifestyle and environmental factors in cardiovascular and otherchronic diseases. The FFQ consisted of 148 food and drink items accompanied by acountry-specific instruction manual that included photographs to facilitate the estimationof portion sizes. Participants were asked how often, on average, they had consumed thatamount of the item during the last 3 months, with nine responses ranging from ‘never orless than once per month’ to ‘six or more times per day’. Participants were also asked toinclude additional foods and frequency of consumption by manual entry.Dietary data were collected by using a FFQ previously validated,8. Twww.phenol-explorer.eu). The process of estimation of polyphenol intake has been described in detailselsewhere. In brief, food items of the FFQ containing more food components wereseparated according to their ingredients and foods that contained no polyphenols wereexcluded from the analysis. The average food consumption was calculated (in g or ml) byfollowing the standard portion sizes used in the study and then converted in 24-h intake.An advanced search was carried out in the Phenol-Explorer database to retrieve meancontent values for all polyphenols contained in the foods obtained and individualpolyphenol intake from each food was calculated by multiplying the content of eachpolyphenol by the daily consumption of each food. Total polyphenol intake was calculatedas the sum of all individual polyphenol intake from all food sources encountered accordingto this process. In this study we investigated exposure to total polyphenols and theirmain classes phenolic acids, flavonoids, stilbenes and lignans; the main subclasses ofphenolic acids, including hydroxybenzoic acids and hydroxycinnamic acids; the mainsubclasses of flavonoids, including flavanols, flavonols, flavanones, flavones,anthocyanins, and isoflavones; and ‘other’ polyphenols, including alkylmethoxyphenols,alkylphenols, curcuminoids, furanocoumarins, hydroxybenzaldehydes, hydroxybenzoketones,hydroxycinnamaldehydes, hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols,naphtoquinones, phenolic terpenes and tyrosols.Data on the polyphenol content in foods were obtained from the Phenol-Explorer database and categorised in low, moderately and high activity level. Individualswere categorised according their smoking status as non-smoker and current smoker. Alcoholconsumption was categorised as (i) non-drinkers (ii) drinkers; alcohol intake wasconsidered as a continuous variable (g/d).. BMI was calculated according to the formula weight (kg)/height(m2).Physical examination included measurement of height, weight, waist circumference andblood pressure using standard proceduresParticipants were considered to have type 2 diabetes at baseline if they had elevatedglucose concentrations (plasma glucose concentrations of ≥7 mmol/l) or treatment withhypoglycaemia medications  within the last 2 weeks.Among individuals free of diabetes at baseline, cases of new type 2 diabetes were definedas those participants self-reporting of being professionally diagnosed with type 2 ortaking hypoglycaemia medications within the last two weeks at follow-up.χ2 test was used for comparisons of categorical variables, the Kruskal–Wallistest was used for continuous variables because these variables did not fit a normaldistribution.We categorised individual flavonoids by dividing intake into quartiles and providing meanintakes for each category of exposure (expressed as glycosides and esters).Characteristics of the study cohort were described by quartiles of total polyphenolconsumption. Sex-specific analyses were conducted. Descriptive presentation relied oncross tabulations. Continuous variables are presented as means and standard deviations,categorical variables as counts and percentages. Variables were examined for normality(Kolmogorov test). The sdincrease intake). Tests for linear trends were also performed by assigning the medians ofeach quartile as scores. Variables included in the multivariable model were age, totalenergy intake, BMI, physical activity, educational status, smoking status, alcoholconsumption, alcohol intake, fibre and menopausal status (women only). As Spearmancorrelation test showed significant correlation between main classes of polyphenols andsubclasses of flavonoids (data not shown), we did not include in the model all mainclasses of polyphenols/flavonoids. Graphical representation using restricted cubic splinesanalysis for total polyphenols was applied. When examining the association with totalpolyphenols, a sensitivity analysis was performed by including one at the time the majorfood sources of polyphenols based on our previous publication, to test whether the level of association was driven by one individual foodcomponent. Statistical significance was accepted at P<0·05. Allstatistical analyses were performed with SPSS for Windows 21.0 (SPSS Inc.).The association between baseline consumption of polyphenols and incident type 2 diabeteswas assessed by logistic regression analyses. Age- and energy-adjusted, andmultivariable-adjusted models were performed: OR and 95 % CI of having type 2 diabeteswere calculated for polyphenols considered as both categorical  and continuous exposure . The sensitivity analysis by adjusting for major food sources of polyphenolsshowed no differences with previous results (data not shown). Among the main classes ofpolyphenols, flavonoids, phenolic acids and stilbenes were contributors to this association.When considering individual subclass contributors among phenolic acids, both hydroxybenzoicacids and hydroxycynnamic acids showed an inverse relation with type 2 diabetes in a lineardose–response manner . These results are consistent with previous findings of the cross-sectionalanalysis of baseline data from the same cohort in which we found that the same classes offlavonoids were associated with lower odds of having impaired glucose metabolism. Such findings may explain the results of previous observations showing betteroutcomes related with plant-based dietary patterns in the HAPIEE cohort(–). Recent prospective studies reported certain benefits for higher intakes ofanthocyanins, flavanols and flavonols, despite contrasting results have been also published. Findings from meta-analyses regarding the potential benefits of flavonoids ontype 2 diabetes and glycaemic control also provided evidence of association between some flavonoid classes anddecreased risk of type 2 diabetes. From a mechanistic point of view, flavonoids may reducebiological pathways related to the development of type 2 diabetes by improving endothelialfunction, which has been correlated with insulin resistance. Moreover, experimental studies in vitro and invivo demonstrated that flavonoids interact with molecular targets and affectsignalling pathways resulting in improvement of glycaemia and suppression ofgluconeogenesis(–). Despite we found some null results regarding the flavonol class, which hasbeen previously related with health benefits associated with tea consumption, some limitations of our study, including reverse causation and geneticsrelated to high tea consumption may explain our unexpected findings.Flavonoids are mainly contained in fruit, vegetable and tea, which have been associatedwith benefits toward metabolic disorders. We previously showed in the HAPIEE cohort the association between coffee andmetabolic disorders, including type 2 diabetes. Meta-analyses of observational studies conducted on coffee reportedsignificant decreased risk of type 2 diabetes and general metabolic disorders. Together with their antioxidant properties, CGA have been found to exertdirect effects in regulating glucose metabolism. CGA have been hypothesised to exert effects on glucose metabolism throughtheir specific competitive inhibition of the glucose-6-phosphate translocase, which in turninhibit gluconeogenesis, and activation of AMP-activated protein kinase, a sensor andregulator of cellular energy balance that may lead to suppression of hepatic glucoseproduction and fatty acid synthesis.In this study, hydroxybenzoic and hydroxycinnamic acids, the main subclasses of phenolicacids, were both inversely associated with the risk of type 2 diabetes. Among the moststudied hydroxycinnmic acids, chlorogenic acids (CGA) which are contained in coffee, havebeen reported to exert beneficial effects towards cardiovascular risk factors and metabolicdisorders. A recent meta-analysis on resveratrol treatment as an adjunct topharmacological management in type 2 diabetes mellitus reported a significant reduction offasting glucose, insulin, glycated Hb (HbA1c) and insulin resistance levels in participantswith type 2 diabetes but did not affect glycaemic measures in non-diabetic individuals. We reported that high total stilbenes intake was related with decreasedincidence of type 2 diabetes, thus confirming earlier findings derived from clinicalsetting. The main effects of stilbenes in human health are relative to their role incellular defense against oxidative stress through the nuclear factor-erythroid-2-relatedfactor-2 (Nrf2) and the potential roles of SQSTM1/p62 protein in Nrf2/Keap1 signalling andautophagy. Moreover, modulation of expression of visfatin, sirtuin-1 andGLUT (2 and 4) may improve glucose metabolism by suppressing oxidative stress and increasingpotential to internalise glucose by extrahepatic tissues. Besides the antioxidant effects, stilbenes have also been suggested toregulate circulating leptin levels and improve insulin signalling, both implicated in type 2diabetes risk. However, the mechanisms of action for the hypothesised effects are by farcomplex and the knowledge on them is still evolving.Studies on stilbenes have been mostly focused on the effects of resveratrol oncardio-metabolic health. However, other epidemiological studies reported contrasting results and analyses of large European and US cohorts showed no association of soyafood intake, isoflavones and lignans and risk of type 2 diabetes. Consumption of soya foods in our cohort was very low and main food sources ofphyto-oestrogens in non-Asian population are generally legumes, whole wheat products andseeds. Clinical trials on flaxseeds and lignans and glycaemic control showed similarcontrasting results. A meta-analysis of twenty-four intervention studies on soy and isoflavoneintake and glycaemic control showed no significant effect on fasting glucose and insulin ofeight trials with isoflavone extract and six studies with isolated soya protein, butreduction in fasting glucose concentrations in nine studies that used whole soya foods orsoya diets . Among other issues related to the contrasting results on phytoestrogens hasbeen pointed out the large inter-individual variation in plasma concentrations ofisoflavones, which may limit the use of intake data for risk assessment. Phyto-oestrogens have been hypothesised to ameliorate glucose metabolism andprevent type 2 diabetes by decreasing the activity of key enzymes related to blood glucoseand HbA1c levels  and activating the PPAR gene expression pathway. However, given the aforementioned issues, further studies are needed toprovide stronger evidence of the biological rationale.In the present study, no significant results were found for phyto-oestrogens, such asisoflavones and lignans. In our previous cross-sectional analysis, we found some positiveassociation between impaired glucose metabolism and intake of isoflavones, which have a weakoestrogenic action. These findings are substantially in contrast with other recent studiesshowing that urinary excretion of isoflavones and lignans or markers of consumption wereassociated with lower risk of type 2 diabetes. Nevertheless, it could contribute to an underestimation of the associationsfound because of the higher no-participation in diabetics. Third, as information on diet wascollected at baseline only, we were unable to test whether dietary habits had changed duringfollow-up. However, during 4 years it is unlikely that there was a major change in dietaryhabits of the population. We also acknowledge that our assumption on the induction periodbetween nutritional exposure to polyphenols and T2D might have been too short. In any case,the FFQ usually captures the habitual diet and may represent a proxy for long-term dietaryexposures. The potentially unrealistic assumed induction period remains a limitation of ourstudy, and it more probably would have produced and underestimation of the association.Fourth, although sensitivity of diabetes self-reports is high (about 70 %), there is a small proportion of individuals that are unaware of their disease.If we consider that diabetics could have dropped the study to a larger extent thannon-diabetics, this causes that the ascertained number of cases would be under-estimated,thus reducing the statistical power of the analyses. However, given the prospective designof the study, it is unlikely that this under-recording of cases would be differentialaccording to the exposure, thus affecting the precision, but not the validity of theresults. However, such issues are common to all previous studies using the same methodologyand as polyphenol exposure was ascertained before diagnosis of disease, misclassificationwould tend to bias estimates toward the null and underestimate true associations. Fifth,some polyphenol-rich foods, such as herbs and spices, were not included in FFQ, which mightlead to underestimation of the exposure. Nevertheless we don’t expect that contribution ofpolyphenols from spices and herbs in the total polyphenols intake is high enough tomaterially affect the results. Fifth, use of table content databases would have inevitablyled to some misclassification of polyphenol intake. Sixth, consumption of some polyphenolclasses  in ‘normal’ diets can be negligible, andconsequently difficult to be accurately estimated. Despite we found significant results forsuch polyphenol groups, it should be considered that amount consumed was very low. Seventh,despite we adjusted the analyses for a number of potential confounders, other untestedvariables (i.e. family history of diabetes) may contribute to residual confounding.Other limitations should be taken into account when considering results from this study.First, the observational design of the study can only partially provide evidence ofcausation. Second, due to relatively low response rate in the follow-up examination, numberof participants included to the analysis was substantially lower than screened at baseline.This influenced the representativeness of the sample negatively, but there is evidence thatlow participation rate affect less the relations studiedIn conclusion, higher intake of a variety of polyphenols may decrease the risk of type 2diabetes. Besides the fact that results from this study may not be generalisable to otherpopulations, they provide further evidence that potential association between polyphenolsintake and cardiovascular risk factors may exist. Our study added to the current knowledgefurther information regarding other polyphenol classes potentially explaining theaforementioned contrasting results. Limitation of previous studies investigating individualclasses of polyphenols may have led to underestimation of their effects by not taking intoaccount possible interactions. Further studies with experimental design are needed toestablish the effects that can specifically attributed to each polyphenol class in order toidentify specific polyphenol-rich foods that may contribute to the prevention ofcardiovascular risk factors.


Renal staghorn stones are challenging for urologists to ensure maximum stone clearance and minimal morbidity. Percutaneous nephrolithotomy (PCNL) has become the gold standard treatment for renal staghorn stones. To assess the safety and efficacy of a novel integrated rigid and flexible percutaneous nephroscope(Rigi-flex nephroscope) in PCNL for renal staghorn stones.We present our initial experience with this new technique.From March to July 2016, a prospective analysis of 3 patients with staghorn stones treated with Rigi-flex nephroscope in PCNLunder totally ultrasound guidance by paravertebral block (PVB) anesthesia was done. PCNL was performed with the rigid section of a 13-Fr Rigi-flex nephroscope firstly and the stones were disintegrated into fragments by holmium laser.Then the stones were removed by active flushout, followed by a search for residual stones in other inaccessible calyces with the flexible section. Finally, the residual stones were disintegrated into small fractions by holmium laser in situ or repositioned with a set of disposable retrieval baskets to pelvic or other accessible areas. The whole procedure was accomplished via only one nephrostomy tract. The operating time, stone-free rates (SFR), postoperative hemoglobin drop, complications, length of hospitalization, were recorded.The operation time were 89, 62 and 45 min, respectively, the postoperative hemoglobin drop was 1, 0.8 and 0.9 mg/dl, respectively.The postoperative Kidney-Ureter-Bladder (KUB) radiograph of the three patients showed no residual fragment >3 mm. No patients needed blood transfusion and suffered significant complications. The length of hospitalization was 9, 6 and 4 days, respectively. No patient needed multiple tracts PCNL or staged auxiliary measures one month after the operation.The application of Rigi-flex nephroscope in PCNL under ultrasound guidance for staghorn stones has its unique advantages as monotherapy with increasing procedural stone free rate (SFR) via single nephrostomy tract, hence there is less morbidity as it does not require additional tracts dilation and staged auxiliary procedures combination. However, SFR should also be evaluated at a longer follow-up, particularly for staghorn stone, further large-scale multicenter prospective clinical trial are needed to verify its feasibility. Percutaneous nephrolithotomy (PCNL) has become the gold standard treatment for large renal stones and currently is recommended for staghorn stones, as it has stone-free rate three times higher than extracorporeal shock wave lithotripsy (ESWL), along with lower morbidity, shorter length of hospital stayand operating time as well as faster return to work than open stone extraction surgery –3.RetrogMost of staghorn stones were approached with PCNL primarily in accordance with existing techniques, but the large stone burden volume and scattered distribution in various parts of the pelvocalyceal system are challenging for most urologists to ensure maximum stone clearance and minimal morbidity.As a rigid endoscope, conventional nephroscope or semi-rigid ureteroscope can not access the renal calyces situated at an acute angle with the calyx of entry, which may increase needs of multiple tracts PCNL or staged auxiliary measures. Creation of multiple tracts to maneuver into various parts of the pelvocalyceal system, for staghorn stones or migrated stone fragments, increases potential risks of access-related morbidity of the procedure . Staged It is necessary to explore a new concept and definition of PCNL.So we proposed that PCNL is redefined beyond a surgical technique as a new requirement for the operation procedure:P-Patient oriented, C-Cost efficient, N-New features, L-Less invasive. The new concept of “PCNL” indicates that: the therapeutic schedule including the selection of surgical technique and instruments shall be individualized based on cost-efficacy and safety. The operation shall be completed by novel less-invasive and high-efficacy instruments. Surgeon’s expertise, experience and skills, as well as the new instruments are of upmost impotantance in the new “PCNL” for precise treatment of complex nephrolithiasis.Based on the new concept of “PCNL”, we proposed “integrated rigid and flexible Percutaneous Nephrolithotomy(Rigi-flex PCNL)” via a novel integrated rigid and flexible nephroscope (Rigi-flex nephroscope)  for accessing all calyces of the pelvocalyceal system through only one nephrostomy tract, with the objective of assessing the feasibility and safety of this new technique, especially in increasing procedural SFR. The seamless switching of Rigi-flex nephroscope between rigid mode and flexible mode is straightforward. Technical parameters and pictures of Rigi-flex nephroscope are shown in Table No reference to a similar study or use of a similar instrument could be found during a thorough literature search. To the best of our knowledge, this is the first clinical study using this endoscope anywhere in the world.Our study was a small-scale clinical observational trial, which used the Rigi-flex nephroscope to evaluate the efficacy and safety of PCNL for patients with staghorn stones. The study was approved by the ethics committees of our hospital. All patients gave written informed consent for the Rigi-flex PCNL and the use of their information in our research, according to the Helsinki II declaration.From March to July 2016, three patients  with staghorn stones were admitted in our department. Eligible patients were 18 years of age or older with staghorn stones that required PCNL.The demographic characteristics are reported in Table In addition to routine preoperative examination, urine culture and sensitivity was also tested. Preoperative Kidney-ureter-bladder (KUB) radiograph and abdominal non-contrast computed tomography (NCCT) were used to proved staghorn stones and delineate kidney  and adjacent viscera.All patients with urinary tract infection were treated with culture specific antibiotic therapy until repeat urine culture was negative. All other patients with negative urine culture received empiric antibiotic therapy for three days before operation.After total paravertebral block (PVB) anesthesia was accomplished.An externalized ureteric stent was placed into ureter retrogradely with cystoscope in lithotomy position for retrograde saline injection if intraoperative artificial hydronephrosis is needed, and a 18-Fr Foley catheter was remained in the bladder.Then, the patients were repositioned to prone position with a pillow under the abdomen for establishment of percutaneous nephrostomy tract under totally ultrasound guidance with 3.5-MHz convex abdominal ultrasound probe . The percutaneous renal puncture was finished by an 18G access needle with echogenic tip . First, the surgeons observed the kidney  and adjacent viscera in lower paravertebral region by ultrasound, and then selected an optimum percutaneous puncture spot  usually through the posterior middle calyx, which led straight to maximum stone burden, and kept the adjacent viscera out of the preset needle access. Two-steps precise puncture method was performed for good visualization of entire needle from preset spot into target cylax:First, the echogenic tip of the acccess needle was moved from skin into the perinephric fat tissue where the direction of the needle can be adjusted accompanying respiratory movement.Second, the echogenic tip of needle was inserted into target cylax fornix quickly.If percutaneous renal puncture was accomplished successfully with the flow-out of clear and transparent urine from needle, a hydrophilic 0.035-inch J-tip coaxial guidewire was placed into the collecting system through access needle (ultrasound can detect the J-tip in collecting system). After 1 cm skin surrounding the access needle was incised, tract dilatation was serially performed by 10 to 22-Fr fascia dilators  through the guidewire and a 22-Fr working access sheath was kept in the collecting system.A 13-Fr Rigi-flex nephroscope was advanced into pelvocalyceal system in its rigid section through working access sheath along the longitudinal axial direction of nephrostomy access. The stones were disintegrated into fragments by holmium laser fiber . A 500-μm laser fiber was used for rigid mode with power setting 3.0/20 Hz.The stones were actively flushed out with transportion of stone fragments by continuous irrigation backflow through access sheath alongside the nephroscope or by filling of collecting system with high pressure and quickly removing the nephroscope resulting in immediate inversion of irrigation water-flow with spillage-like removal of stones via the access sheath , 8.All vThe operations were terminated when no residual fragments could be detected with the help of Rigi-flex nephroscope and ultrasound screening.A double J-tip ureteric stent  was inserted into ureter antegradly and a 20-Fr nephrostomy tube was placed in each patient.All patients were initially evaluated with KUB in postoperative 1st day.Nephrostomy tubes were removed in postoperative 2nd day.The patients were reevaluated with KUB about one month post-operation and double J-tip ureteric stents were removed. NCCT was performed when the stone status were sub-optimally evaluated with KUB.Residual fragment <3 mm were defined as clinically insignificant residual fragments (CIRF). Larger stones >3 mm were defined as residual stones. Patients who were complete stone free or had only CIRF were considered to have a successful surgery.Stone free rate (SFR), postoperative hemoglobin drop and length of hospitalization was recorded. All complications occurring within one month post-operation were recorded according to the modified Clavien Classification system..Theperioperative data and postoperative complications were reported in Table The first description of percutaneous renal stone extraction was reported by Fernstrom and Johansson in 1976 .2, staghorn and partial staghorn calculi. American Urological Association (AUA) recommends that all newly diagnosed staghorn stones should be actively treated, because untreated staghorn stones have a tendency to destroy the kidney and cause life-threatening urosepsis [The indications for PCNL include stone factors , patient factors , and failure of other treatment modalities (ESWL and flexible ureteroscopy). The accepted indications for PCNL are stones larger than 20 mmrosepsis .As compared to ESWL as well as RIRS, althrough PCNL with the highest SFR after one-stage single treatment in case of large or multiple renal stones , 13.HoweOf the 1466 patients with staghorn stones undergoing PCNL in the CROES database, the SFRs was only 56.9% . IncreasTo the best of our knowledge, this is the first clinical study to use this novel type device-integrated rigid and flexible percutaneous nephroscope in PCNL under totally ultrasound guidance by total PVB anesthesia.As compared with traditional PCNL, Rigi-flex PCNL has its unique advantages:Rigi-flex percutaneous nephroscope can find almost all stones in various calyces with single nephrostomy tract through intraoperative seamless switching of Rigi-flex nephroscope between rigid mode and flexible mode. Medical expenses and access-related morbidity were reduced as it does not require multiple tracts and staged procedures with less instruments. In our initial experience, three patients with staghorn stones underwent Rigi-flex PCNL and were almost completely stone free. The postoperative complications were fewer and minor. The length of hospitalization was slightly longer than before due to treatment of postoperative fever with urosepsis and hematuria. It is a promising tool that has the potential to reduce the morbidity of PCNL in cases of multiple or staghorn stones and improve stone clearance rates.However, there were certain limitations in our study:This is the initial experience of a new technique from a single center.Since the number of patients included in this study was small, the results were not statistically significant.The SFR should be evaluated in a longer follow-up, particularly for staghorn stones.Further large-scale multicenter prospective clinical trials are needed to verify the cost efficacy and safety of the device.The application of Rigi-flex nephroscope in PCNL under ultrasound guidance for staghorn stones has its unique advantages as monotherapy with increasing procedural stone free rate (SFR) via single nephrostomy tract, hence there is less morbidity as it does not require additional tracts dilation and staged auxiliary procedures combination. Given the limited case number, further large-scale multicenter prospective clinical trials are still required.


Erythropoiesis stimulating agents (ESAs) were proposed to enhance survival of renal tissues through direct effects via activation of EPO receptors on renal cells resulting in reduced cell apoptosis, or indirect effects via increased oxygen delivery due to increased numbers of Hb containing red blood cells. Thus through several mechanisms there may be benefit of ESA administration on kidney disease progression and kidney function in renal patients. However conflicting ESA reno-protection outcomes have been reported in both pre-clinical animal studies and human clinical trials. To better understand the potential beneficial effects of ESAs on renal-patients, meta-analyses of clinical trials is needed.Literature searches and manual searches of references lists from published studies were performed. Controlled trials that included ESA treatment on renal patients with relevant renal endpoints were selected.Thirty two ESA controlled trials in 3 categories of intervention were identified. These included 7 trials with patients who had a high likelihood of AKI, 7 trials with kidney transplant patients and 18 anemia correction trials with chronic kidney disease  patients. There was a trend toward improvement in renal outcomes in the ESA treated arm of AKI and transplant trials, but none reached statistical significance. In 12 of the anemia correction trials, meta-analyses showed no difference in renal outcomes with the anemia correction but both arms received some ESA treatment making it difficult to assess effects of ESA treatment alone. However, in 6 trials the low Hb arm received no ESAs and meta-analysis also showed no difference in renal outcomes, consistent with no benefit of ESA/ Hb increase.Most ESA trials were small with modest event rates. While trends tended to favor the ESA treatment arm, these meta-analyses showed no reduction of incidence of AKI, no reduction in DGF or improvement in 1-year graft survival after renal transplantation and no significant delay in progression of CKD. These results do not support significant clinical reno-protection by ESAs. Erythropoietin (EPO) is a circulating hormone produced by the kidney, that stimulates erythropoiesis by binding and activating the EPO receptors (EPOR) on erythroid progenitor cells . SubjectChronic anemia can result in organ damage affecting the cardiovascular system, kidneys, and the central nervous system –6 thus aClinical interventions to see if there is a relationship between ESAs and renal outcomes included short-term prophylactic ESA treatment where there was a high likelihood of acute kidney injury (AKI), e.g., patients undergoing coronary artery bypass grafting (CABG) surgery. In another modality, ESA treatment at the time of surgery might mitigate the ischemic damage and delayed graft function (DGF) that occurs during the perioperative period following kidney transplant. DGF increases the risk of acute rejection, impaired graft function, and reduces long term patient and graft survival. In a third modality, treatment of CKD patients to correct anemia associated with renal failure presumes that ESA treatment might delay or prevent renal disease progression through direct anti-apoptotic effects on renal cells or indirect effects of anemia correction, eg improved oxygen delivery.Most of the trials examining the effect of ESAs on renal patients were small, outcomes were not robust or they varied across studies. Therefore, results from individual trials were inconclusive, but meta-analyses of results from those clinical trials may allow more definitive conclusions. We reasoned further that meta-analysis of multiple modalities would add additional value. The three modalities above were selected for meta-analysis because they examined direct and/or indirect effects of ESAs on renal disease progression or renal function. We report here that meta-analyses show no significant beneficial effects in any of the modalities, suggesting that ESAs have little reno-protective benefits, at least with the patient populations examined and clinical designs employed.We wished to assess the effect of ESAs on kidneys by analyzing data from human clinical trials where ESAs might mitigate effects of ischemia or disease progression. This necessitated comprehensive searches and identification and analysis of controlled trials with renal patients where ESAs were used to protect kidneys from ischemia or to slow renal disease progression. All trials that had relevant renal endpoints were selected and analyzed, and data was extracted from those that might test the hypothesis.Literature searches were performed using OVIDSP (Wolters Kluwer companies) to access MEDLINE and other databases including Current contents, Embase and BIOSYS previews, using search terms for ESAs  in combination with anemia terms , kidney or kidney injury , and terms describing possible beneficial outcome . Searches of the Clinicaltrials.gov and the Cochran database websites were performed using ESA terms combined with anemia, renal, kidney and transplant, to further identify potential papers of interest. A manual search of the reference lists in papers, review articles and other meta-analyses identified additional papers.Papers considered for inclusion described human clinical data with ESA treatment and renal endpoints. Papers were rejected if they were not controlled trials, were case reports, described only preclinical data, or lacked the relevant renal endpoints. Papers with ESA treatment of renal patients on dialysis were omitted because renal disease progression was not applicable. The final list included controlled clinical trials that utilized ESAs in transplantation, AKI, and for anemia correction in predialysis CKD patients.The data was recovered by SE and reviewed by ZE. Recovered data included the study characteristics, study location, length of study, ESA treatment, nature of the comparator arm, number of subjects in each arm, time intervals and definitions of renal endpoints. Results were grouped according to study type . For trials involving AKI, data collected for meta-analysis was the number of patients with AKI and number of patients with renal recovery following AKI. Other endpoints recovered from those trials were any creatinine-based or enzymatic markers that were measures of renal function or renal injury. With kidney transplant studies the measures recovered for meta-analysis were incidence of DGF within the first week post-surgery and graft loss/survival over a 1 year period. Other data collected were any creatinine-based data, incidence of proteinuria, and enzymatic-based markers of renal injury. The meta-analysis endpoint in anemia correction trials was incidence of progression to renal replacement therapy  at any time during the study. Other data recovered were, estimated glomerular filtration rate (eGFR), serum creatinine (sCr), and their rate of change over time, and incidence of proteinuria. All the trial information and secondary measures are summarized in Tables Data extracted to assess trial quality (bias) included randomization, concealment of allocation, masking of patients and clinicians, documentation of dropouts and withdrawals, and whether analysis was by intention-to-treat.2 statistic was not equal to zero. Risk ratios (RR) and 95% confidence intervals were calculated to compare results for patients treated with ESA with the control group. Heterogeneity or inconsistency across studies was assessed using Cochrane’s Q  and the I2 statistic. The p-value for the z-test comparing treatment groups was also determined.Data were summarized using Comprehensive Meta-Analysis Software (V2) . A random-effects model was used because it assumes treatment effects are not identical in all studies. However, results of analyses using a fixed-effects model, which assumes that the treatment effect is the same in each study and that differences in results are due only to chance, are also provided when the IThe titles of papers from the searches were reviewed, and abstracts examined. Papers with potential relevance to ESAs, human clinical trials and tissue protection were recovered. This process resulted in 4056 papers. The selection and rejection process for these papers is shown in Fig. Trial quality  was evaluated utilizing Jadad  and CochNine trials were identified , 24–29 tThe endpoint tested in the meta-analysis was the number of patients that developed AKI within 2–7 days . Four of the trials were performed by overlapping members of the same study groups , 27, 29.2 = 60%), 3 trials showed benefit in the ESA arm, while the other 4 were neutral, or favored the control arm. This heterogeneity is further apparent when other renal endpoints were examined (Table p = 0.054) the ESA group and in the 7th, creatinine-based markers favored the ESA group. In 3 trials there was no difference in eGFR between groups, while in another trial, eGFR was improved in the ESA arm. Overall the secondary outcome analyses using non-creatinine-based renal biomarkers did not demonstrated significant reno-protection by ESAs. In 3 trials urine or plasma NGAL or serum cystatin C) were the same in both groups; in the 4th, urinary NGAL was lower in the ESA arm, although the significance of this difference is uncertain.Overall 107 of 367 29%) of the subjects developed AKI in the ESA groups, with 133 of 357 (37%) in the control groups .A meta-analysis with 450 subjects utilizing the DGF endpoint , is shown in Fig. 2 = 0%). Excluding the retrospective study [Meta-analysis of long term graft loss over 1 year in four trials showed similar outcomes Fig. . Fifteenve study  reduced In the 7 trials, additional renal outcomes were reported that showed no differences between ESA and no-ESA groups Table . These iCKD patients are often anemic, and ESA treatment to increase and maintain Hb levels is long-term. Therefore, analysis of ESA anemia correction clinical trials is a potentially useful method to assess the effect of Hb increases, and oxygen delivery to renal tissues, on renal disease progression.In the 19 anemia correction trials identified, CKD patients were typically divided into 2 groups; those remaining at their starting Hb (control) and those where ESAs were used to target a higher Hb. ESAs in the 19 trials , 38–55 wPatients that progressed to RRT included those that began dialysis or received a transplant. In one trial a patient withdrew because of sepsis and AKI . This evThe remaining 18 anemia correction trials had a combined total of 8020 subjects; 3964 in the treatment arm (higher Hb) and 4056 in the comparator (low Hb control) arm. Trials were of varying size; 3 had over 600 subjects. The initial and achieved Hbs in the 2 groups are shown in Table 2 = 25.0%). This lack of effect on disease progression is supported in 18 trials by other assessments of change in renal function, including proteinuria, or creatinine based markers where there were no significant differences reported between groups  of subjects in the treatment arm and 786 (19.3%) in the control arm, progressed to RRT  there were differences in patient selection, treatment regimen and outcome definition. Finally, the meta-analyses were based on aggregated, not individual patient level data, which precluded adjustments for confounding factors such as age and comorbidities.In contrast to some preclinical studies demonstrating reno-protection by ESAs in animals, anemia correction, prophylaxis or post-injury intervention with ESAs provided no significant clinical reno-protection in humans. This suggests that ESAs may not have robust, nor reproducible direct, or indirect, benefits on renal function.


Nutrient needs, including those of the essential nutrient choline, are a population wide distribution. Adequate Intake (AI) recommendations for dietary choline  are grouped to account for the recognized unique needs associated with age, biological sex, and reproductive status . Established and emerging evidence supports the notion that common genetic variants are additional factors that substantially influence nutrient requirements. This review summarizes the genetic factors that influence choline requirements and metabolism in conditions of nutrient deprivation, as well as conditions of nutrient adequacy, across biological sexes and reproductive states. Overall, consistent and strong associative evidence demonstrates that common genetic variants in choline and folate pathway enzymes impact the metabolic handling of choline and the risk of nutrient inadequacy across varied dietary contexts. The studies characterized in this review also highlight the substantial promise of incorporating common genetic variants into choline intake recommendations to more precisely target the unique nutrient needs of these subgroups within the broader population. Additional studies are warranted to facilitate the translation of this evidence to nutrigenetics-based dietary approaches. N-methyltransferase (PEMT) pathway  and women (n = 31 completed) were confined to metabolic wards and given identical diets . After ten days of calibration at 550 mg choline/day, the subjects were restricted to <50 mg of choline per day until they developed signs of organ dysfunction, indicating choline deficiency, or for a maximum of 42 days. While consuming the choline deficient diets, subjects were randomized to treatment with a folic acid supplement vs. consumption of only dietary folates. Subjects were considered to have choline deficiency-associated organ dysfunction if they displayed a greater than: five-fold increase of serum creatine phosphokinase (CPK) activity; a 1.5-fold increase in serum activity of aspartate transaminase, alanine transaminase, γ-glutamyl transpeptidase, or lactic dehydrogenase; and/or a 28% increase in liver fat content (assessed by magnetic resonance imaging) that was resolved after repletion of dietary choline [The seminal study by Zeisel and colleagues which demonstrated that dietary choline is an essential nutrient for humans was published in 1991 and consisted of a highly controlled metabolic ward trial conducted in a cohort of healthy, young men . All sub choline . While tS-adenosylmethionine (SAM), the universal methyl donor. To test this hypothesis, the odds of developing choline deficiency were calculated based on the presence or absence of common single nucleotide polymorphisms (SNPs) in the genes for nine choline metabolizing enzymes [MTHFD1) was associated with a seven-fold increased odds of developing organ dysfunction in individuals randomized to no supplemental folic acid; in premenopausal women who were carriers of this variant, the odds of developing organ dysfunction reached a striking 15-fold increase. No significant effects were noted for carriers of common variants in methylenetetrahydrofolate reductase (MTHFR) or the reduced folate carrier (RFC). For variants in choline metabolism, a polymorphism in the promoter region of PEMT  increased the odds of developing organ dysfunction 25-fold, whereas an A→C substitution (rs9001) in choline dehydrogenase (CHDH), the enzyme which converts choline to betaine, yielded a protective effect. The lack of significant effects among other tested genotypes  on choline-deficiency associated organ dysfunction may be due to a true lack of functional significance or to the limited statistical power. For example, the sample in this study only included four homozygous individuals for each MTHFR SNP. The functional significance of variants within these genes requires further study.Zeisel and colleagues hypothesized that common genetic variants impairing choline and folate metabolism would account for this variation in susceptibility to choline deficiency. Folate and choline metabolism are tightly linked due to their common participation in one carbon metabolism and ability to donate methyl groups to generate  enzymes ,7  in the promoter region of the al women . Furtheris needs . In a cois needs .The highpulation  and theipulation  on susceMTHFR 677C→T (rs1801133) 677CC or TT genotypes while consuming varied choline intakes [9) for the last three weeks. While consuming the study diets, all participants displayed normal liver enzyme values, suggesting that 300 mg choline/day was sufficient in this population to prevent organ dysfunction. However, all treatment groups exhibited decreases in serum folate and an increase in plasma homocysteine, suggesting that the folate RDA is insufficient for men of Mexican descent; this effect was most pronounced in homozygous carriers of the MTHFR 677T allele, who exhibited a 170% increase in plasma homocysteine relative to an 18% increase in participants homozygous for the C allele . The plasma homocysteine response in the 677TT men exhibited high variation and further modeling of this response variable suggested that serum folate, plasma riboflavin, choline intake and the PEMT 5465G→A SNP (rs7946) collectively accounted for >80% of the variation [MTHFR 677CC carriers; the decrease in DNA methylation associated with compromised folate status was greatest in the 300mg choline intake group relative to the 1100 and 2200 mg groups.Work in the Zeisel lab has been formative in establishing nutrient-nutrient  and nutrient-genotype interactions that influence the risk and onset of organ dysfunction in individuals deprived of dietary choline. The Zeisel lab’s work, coupled with earlier evidence from animal nutrition, suggested a strong interdependence between dietary choline and dietary folate needs; however, the impact of folate status and genetic variants in folate metabolism on choline needs outside of the context of frank choline deficiency required further exploration. To address this question, the Caudill laboratory undertook a controlled feeding study to assess the adequacy of the folate RDA in Mexican American men with the  intakes . The T a→T rs18013 677CC oariation . Additioariation , suggestariation . Signifi9/PC-d9 enrichment ratio among men with the MTHFR 677TT genotype consuming 1100 mg choline/day relative to the 550 mg choline/day group (p = 0.03) [MTHFR 677TT genotype displayed a greater urinary betaine to choline enrichment ratio (p = 0.04) and higher urinary d3-sarcosine enrichment. Collectively, these results suggest a greater turnover of choline to betaine and use of betaine as a methyl donor among 677TT men, as compared to 677CC men. Notably, the enrichment ratios for d3-PC and d-9 PC were not significantly different, indicating that greater use of betaine as a methyl donor did not affect the balance of PC derived from either the CDP-choline or PEMT pathways, a question of potential functional significance given their distinct fatty acid profiles.The use of an isotope tracer is an important aspect of this study, as it allowed the Caudill lab to assess choline’s partitioning and flux through metabolic pathways. While observing static measures, such as metabolite pool size, can be informative, many metabolites are subject to homeostatic maintenance which can mask the impact of nutrient-nutrient and nutrient-genotype interactions on choline dynamics. Indeed, null associations between genotypes and static measures may lead investigators to question the functional importance of a specific polymorphism, while the impact of the variant may occur at the level of partitioning, recycling or metabolic flux. In the feeding study noted above, Mexican men consuming 550 and 1100 mg choline/day achieved similar unlabeled plasma betaine and PC concentrations; however, isotope data revealed a greater betaine-d = 0.03) . FurtherMTHFR C677T genotypes  consumed 135 μg/day of dietary folate equivalents for 7 weeks, and were subsequently randomized to 400 or 800 dietary folate equivalents for an additional 7 weeks; choline intakes through this period were ~350 mg/day. Folate restriction led to decreases in plasma PC and sphingomyelin, with strong trends for this reduction in women with the 677CC or CT genotype, suggesting that the 677TT genotype plays a compensatory role during conditions of low folate intakes by shunting choline towards the CDP-choline pathway to maintain PC pools. A follow-up analysis of this study examined the effects of additional variants  on plasma homocysteine and PC levels [PEMT rs7946 variant AA genotype and the MTHFD1 variant AA genotype exhibited higher concentrations of plasma homocysteine during folate restriction. Additionally, women with the PEMT rs12325817 variant CC genotype exhibited an attenuated decline in circulating plasma PC. These results run counter to the hypothesis that PEMT impairing SNPs would compromise PC pools and reduce S-adenosylmethionine consumption and subsequent homocysteine levels. However, loss-of-function SNPs in PEMT may lead to a compensatory upregulation of the CDP-choline pathway, as seen in mice lacking PEMT [Additional controlled feeding studies have been undertaken by the Caudill lab to assess the impact of nutrient-genotype interactions during folate inadequacy with fixed choline intakes. In a study by Abratte et al. , MexicanC levels . Women wing PEMT , therebyn = 26), non-pregnant (n = 21), and lactating (n = 28) women consumed identical diets and a total of 480 or 930 mg choline/day for 10–12 weeks along with ~400 dietary folate equivalents, and 600 μg as supplemental folic acid . After week six, 22% of choline was provided as deuterium labeled choline chloride-(trimethyl-d9), a metabolic tracer that enables tracking of dietary choline throughout different metabolic pathways and compartments such as blood, urine, and breast milk. This allows for elucidation of the effects of various factors  on the metabolic flux and partitioning of dietary choline [MTHFR rs1801133, methionine synthase (MTR) rs1805087 (wildtype), methionine synthase reductase (MTRR) rs1801394, and MTHFD1 rs2236225, were all found to influence choline dynamics, frequently through interactions with reproductive state and choline intake [MTHFR rs1801133 and MTR rs1805087 wildtype) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, these findings indicated that loss-of-function variants in folate metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent PEMT-derived PC synthesis, and suggested that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.Early trials provided clear evidence that common genetic variants in folate and choline metabolism exhibit reproducible effects of a relevant magnitude during conditions of nutrient inadequacy. Most individuals in the U.S. population however, consume choline intakes well above those used in deficiency studies, and folate intakes are very high. Whether genetic variants are impactful during conditions of nutrient adequacy remains an active area of investigation. To address this research question, the Caudill lab has undertaken controlled feeding studies providing recommended levels of nutrient intakes in women across the reproductive life cycle . Pregnan choline ,24. With choline ,24 assesCHKA rs10791957 risk A allele was associated with a decreased use of dietary choline for PEMT-derived PC synthesis relative to CDP-derived PC synthesis, and decreased use of choline-derived methionine for PEMT-derived PC synthesis. Similar phenotypes were found for carriers of the PEMT rs4646343 T allele, the PEMT rs7946 T allele, and the CHDH rs12676 C allele. Differences in the use of choline as a methyl donor were also identified with greater use of choline-derived betaine as a methyl donor among women with the FMO3 rs2266782 G allele, and non-pregnant women with the CHDH rs12676 A allele. Furthermore, women with the SLC44A1 rs7873987 C allele and rs3199966 G allele exhibited a greater use of betaine for methionine synthesis in the higher choline intake group as compared to the lower intake group, suggesting that these genotypes readily utilize additional dietary choline for methyl donation. The partitioning of dietary choline between betaine and CDP-derived PC was also influenced by CHDH rs9001 and BHMT rs3733890. Overall, many of these SNPs appear to be associated with a differential need for dietary choline at adequate intakes.Effects on choline dynamics were also detected for SNPs in choline enzymes  Figure . For exaCollectively, the results of this work establish an effect of common genetic variants on choline metabolism among individuals consuming nutrient-adequate diets. Numerous hypotheses can be generated from this data based on the identification of alterations in metabolic partitioning and flux. Further work identifying functional outcomes associated with these nutrient-genotype metabolic interactions will be paramount to translating these metabolic differences to genotype-specific dietary choline recommendations. Common outcomes such as decreased partitioning of choline towards the oxidative pathway  and alterations in PEMT-derived PC may well have functional consequences. For example, PEMT plays a critical role in exporting lipids from the liver to the bloodstream, and generates a PC molecule that is enriched in long chain omega 3 fatty acids such as docosahexaenoic acid . AlteratPEMT and MTHFD1 genes, appear to substantially modify choline needs during conditions of deficiency; furthermore, these and numerous other common variants throughout choline and folate metabolism alter the metabolic flux and partitioning of dietary choline during conditions of choline adequacy across reproductive stages.Complex, lifecycle-dependent, nutrient-nutrient, nutrient-reproductive state, and nutrient-genotype interactions clearly impact dietary choline needs, modify the risk of choline deficiency and alter nutrient metabolism. Notable common variants, such as those found in the A major goal of identifying genetic factors that influence choline needs is to inform dietary recommendations. Carriers of common genetic variants represent subgroups within the population whose risk of inadequacy is altered relative to the population mean; the studies characterized in this review highlight the substantial promise that the incorporation of genetic variants into dietary planning and recommendations may improve the assessment of nutrient inadequacy. However, at present, the translation of the results from these trials to nutrigenetics-based dietary approaches remains limited and substantial challenges remain for the field.MTHFR C677T genotypes and numerous health outcomes [There is a need for well-powered trials that recruit individuals within genotypes, randomize to varied choline intakes, and assess causal impacts of these variants on functional outcomes. This need extends beyond just choline to the entire field of nutrigenetics. Randomized controlled trials have thus far been invaluable in the assessment of dietary choline needs and will be essential for the field of nutrigenetics and precision nutrition to advance; however, additional lines of evidence from observational analyses will also be key in assessing dose-response relationships, nutrient-nutrient interactions, the impact of co-morbidities, and long-term health outcomes. Currently, epidemiological analyses linking choline and other one-carbon nutrients to health outcomes are limited, and commonly do not consider the impact of genotypes, thus averaging across groups that may have distinct effects, and limiting the detection of functional health consequences associated with dietary choline intake. Given the impracticality of long-term dietary studies and concerns regarding exposure assessment and confounding in observational studies, future epidemiological analyses which utilize genetic variants as pseudo-randomized instrumental variables  may provide useful data for making causal inferences about the impacts of genetic alterations in nutrient metabolism. Several of these analyses exist already for the outcomes ,28. If gPEMT SNPs that render PEMT insensitive to the high estrogen milieu of pregnancy, choline needs are dramatically higher during the latter half of pregnancy. Furthermore, choline intakes have been implicated in the risk for several chronic diseases, including non-alcoholic fatty liver disease (protective) and cardiovascular disease  [The current nutrient recommendations largely focus on outcomes related to the prevention of nutrient deficiency. Indeed, the choline AI was established to prevent elevated liver enzymes. However, numerous lines of evidence suggest that choline intakes may impact other outcomes as well. For example, dietary choline intakes higher than the current AI consumed during pregnancy may improve infant cognition ,30 and pimental) ,26. As tIn summary, there is consistent and strong associative evidence demonstrating that common genetic variants throughout choline and folate metabolism impact the metabolic handling of choline and the risk of nutrient inadequacy across varied dietary contexts. Additional evidence from large, well-powered, randomized controlled trials are needed to further define an estimated average requirement (EAR) for choline intakes across the lifespan, and clarify the utility of genetic variants in the assessment of nutrient inadequacy for both groups and individuals. Future dietary assessment and planning approaches that incorporate common genetic variants into this risk assessment hold substantial promise.


Radiologic severity may predict adverse outcomes after lower respiratory tract infection (LRI). However, few studies have quantified radiologic severity of LRIs. We sought to evaluate whether a semi-quantitative scoring tool, the Radiologic Severity Index (RSI), predicted mortality after parainfluenza virus (PIV)-associated LRI.We conducted a retrospective review of consecutively-enrolled adult patients with hematologic malignancy or hematopoietic stem cell transplantation and with PIV detected in nasal wash who subsequently developed radiologically-confirmed LRI. We measured RSI (range 0–72) in each chest radiograph during the first 30 days after LRI diagnosis. We used extended Cox proportional hazards models to identify factors associated with mortality after onset of LRI with all-cause mortality as our failure event.After adjustment for patient characteristics, each 1-point increase in RSI was associated with an increased hazard of death . Baseline RSI was not predictive of death, but both peak RSI and the change from baseline to peak RSI (delta-RSI) predicted mortality . A delta-RSI of ≥19.5 was 89% sensitive and 91% specific in predicting 30-day mortality.We conclude that the RSI offers precise, informative and reliable assessments of LRI severity. Progression of RSI predicts 30-day mortality after LRI, but baseline RSI does not. Our results were derived from a cohort of patients with PIV-associated LRI, but can be applied in validated in other populations of patients with LRI. The use of mortality as a primary endpoint in clinical trials of antimicrobials has been hotly debated due to difficulty in the determination of attribution , 2. The Systematic quantification of radiological severity could allow for a valid and reliable score that can serve as a biomarker of mortality. While thoracic computed tomography (CT) and chest X-ray (CXR) are important clinical tools for evaluating pneumonia , few stuTo explore the value of quantitative assessments of radiologic severity, we sought to determine whether radiologic progression of pneumonia as quantified by a systematic scoring tool, the Radiologic Severity Index (RSI) , would pWe conducted a retrospective chart review of a previously-established cohort of consecutively-enrolled adult patients with hematologic malignancies or those who had undergone hematopoietic stem cell transplantation (HSCT) who developed symptomatic PIV upper respiratory tract infection (URI) between October 1, 2002 and November 30, 2007 . PIV wasPIV-associated LRI was defined as the detection of PIV from nasal wash with symptoms of a clinical pneumonia syndrome  in accordance with guidelines . We chosAll clinical and oncologic data were collected prospectively from a database of viral infections used in infection control. Data collected included demographic information , underlying malignancy, cancer status , PIV detection in BAL (if performed), presence of BAL co-pathogens, cytotoxic chemotherapy, and administration of antiviral therapy with ribavirin and/or intravenous immunoglobulin therapy. For patients who underwent HSCT, we collected type of HSCT)  prior to LRI, and corticosteroid administration within 30 days of infection.We applied a semi-quantitative scoring tool for LRI severity initially developed in patients with H7N9 influenza LRI  to our sTwo senior thoracic radiologists  reviewed the RSI literature with two pulmonary investigators  and scored sample CXRs and CTs from a training set to establish a consistent rule-based system for scoring images. This training set consisted of patients without PIV-associated lower respiratory tract infection (LRI) but with a broad range of severity of infiltrates. After training, the radiologists independently scored all CXR and CT images performed within 60 days of LRI in sequential order, and we used the mean of the two RSI scores for analyses. The radiologists were blinded to the primary outcome of death and to each other’s scoring. We defined baseline RSI as the RSI score on the day of LRI onset. We defined peak RSI as the highest RSI score on CXR or CT after LRI onset and delta-RSI as the change from baseline to peak RSI.2 method to determine which model was best [2 estimates in Cox models are often low due to censored data and do not reflect the robustness of models; however, this technique can compare different models.We used chi-square or Fisher’s exact test to analyze categorical data. We used a Student’s t-test or Wilcoxon rank sum to analyze continuous data. We used an extended Cox proportional hazards model to analyze the factors associated with time to death after onset of LRI . We incowas best . GeneralWe used univariate logistic regression models with baseline, peak and delta-RSI as separate predictors of 30-day mortality. We used Bland-Altman plots , paired In our primary extended Cox model, we incorporated 241 measurable RSI-CXR scores. Non-survivors underwent more radiologic tests within the study period . In univariate Cox regression models, each 1-point increase in RSI-CXR measured as a time-varying covariate was associated with a 13% increase in the hazard for death . The proportional hazards assumption was met. AML was associated with mortality in our univariate Cox regression model . After adjusting for age, presence of AML and gender, only the progression of RSI remained in the multivariate model and predicted mortality  [In separate univariate Cox models using all RSI-CXR and RSI-CT scores for 14-, 30- or 60-day outcomes, RSI remained predictive of mortality . In secondary univariate analyses, extended Cox models using the number of involved zones predicted mortality , but RSI was better at predicting mortality than the number of involved zones (RSI: R = 0.04) .Using univariate logistic regression models, we found that longitudinal assessments of RSI were informative, whereas baseline assessments were not. Mean delta-RSI scores were highly predictive of mortality , as were peak RSI scores  . HoweverReliability between readers was excellent for all RSI scores (ICC: 0.99). We present the Radiologic Severity Index (RSI), a systematic radiologic scoring tool for LRI severity previously used in H1N1 influenza LRI , and tesFew studies have attempted to systematically quantify LRI severity radiologically. Grieser et al. developed a semi-quantitative CT-scoring tool for the acute respiratory distress syndrome after H1N1 influenza LRI in 23 patients . This toOur study of RSI adds to the existing literature by showing that progression of RSI on longitudinal assessment predicts mortality after PIV-associated LRI. Moreover, delta-RSI had excellent discriminatory ability to predict 30-day mortality, while baseline RSI scores were not predictive of mortality after PIV-associated LRI. This demonstrates that it is the change in radiologic severity that is informative. Commonly used clinical risk assessment tools for community acquired pneumonia (CAP), such as the CURB-65, were primarily developed to predict outcomes using baseline data and therefore cannot serve as outcome measures of pneumonia –32. In aOur findings build on those of other investigators. A substantial proportion of patients have persistent infiltrates weeks after LRI onset. Mittl et al. found that in 81 patients hospitalized with CAP, 33% had delayed resolution of pneumonia , but thiIn addition, precise quantification of radiographic results likely improved our ability to detect associations of radiologic severity with mortality. Others have found that multilobar pneumonia, as determined on qualitative chest radiograph interpretation, is associated with death –41, treaThe high reliability of RSI among readers improves precision in the assessment of radiologic severity. In studies using qualitative interpretations of radiologic severity, inter-observer variability between readers of chest radiographs may have impacted the ability to find an association between radiologic severity and mortality. For example, El Solh et al found that when determining whether chest radiographs had consolidation, interstitial infiltrates, or a mixed pattern, the kappa statistic between two radiologists was 0.6.  Taylor eOthers have highlighted the discrepancy between CXR and CT when diagnosing LRI, with discordance between imaging modalities in the diagnosis of LRI in 27–48% of patients , 44, 45.Not all patients in this study had proven PIV LRI, which is defined as detection of PIV from a lower respiratory tract sample and has been well established as a criterion , 47, 48.Our study must be interpreted in the context of certain limitations. Since we tested RSI only in a cohort of patients with PIV-associated LRI, further validation is necessary in other cohorts with LRI such as CAP or RSV-associated LRI. The study was retrospective in nature, and measurements were not obtained systematically. Measurement bias may exist because sicker patients were likely to have more assessments, and patients who died may not have had radiologic assessment at peak severity. However, the lack of follow-up studies in healthier patients would likely bias our results towards the null hypothesis, since we do not capture resolution of pneumonia. Furthermore, the lack of radiologic studies in patients who died before follow-up studies could be obtained would also bias our results towards the null hypothesis, because we would miss the progression of severe pneumonia. Our method of imputation ensured that our model likely captured progression of clinical disease, since radiology is typically performed due to changes in clinical status and not performed when a patient’s clinical status is stable . RadioloIn summary, we show that progression of radiologic infiltrates as assessed by the RSI is predictive of mortality in a high-risk cohort of patients with LRI. Longitudinal assessments of RSI were predictive of mortality, while baseline assessments were not. RSI was superior to previous methods to quantify LRI severity due to improved precision and reliability. Substantial discrepancies exist between CT and CXR assessments of LRI severity. RSI was tested in a cohort of patients with PIV-associated LRI, and future studies are needed to validate RSI in other LRI populations and to correlate RSI with other important outcomes such as health care utilization and cost. RSI is a promising tool that offers granular, reliable, precise and informative assessments of LRI severity and is well suited for consideration as part of a composite endpoint of clinical treatment failure in therapeutic trials of pneumonia.S1 DiscussionThis section offers a brief discussion on the utility of RSI depending upon a study population’s baseline risk of death and based on the type of statistical analysis employed.(DOCX)Click here for additional data file.S2 DiscussionThis section offers a brief discussion on pitfalls of data sampling in retrospective and prospective cohort studies, with suggestions on optimal data sampling techniques for potential prospective studies using RSI as a biomarker of radiological pneumonia severity.(DOCX)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.