Several Latin American and Caribbean (LAC) countries have introduced pneumococcal conjugate vaccine (PCV-10 or PCV-13) in their routine national immunization programs.We aimed to summarize the evidence of PCV impact and effectiveness in children under 5 years old in the LAC Region.We conducted a systematic review of the literature on impact or effectiveness of PCVs on deaths or hospitalizations due to invasive pneumococcal disease (IPD), pneumonia, meningitis and sepsis. We searched Medline, WoS, Lilacs, Scopus, Central and gray literature published in any language from 2009 to January 2016. We included studies addressing the outcomes of interest in children in the target age group, and with the following designs: randomized trials, cohort or case-control, interrupted time series with at least three data points before and after the intervention, and before-after studies. Screening of citations, data extraction, and risk of bias assessment were conducted in duplicate by independent reviewers, according to the study protocol registered on PROSPERO. Descriptive analysis of the effectiveness measurements and sensitivity analysis were conducted. Effectiveness is reported as 1-OR or 1-RR for case control or cohort/clinical trials, and as percent change of disease incidence rates for before-after studies.We identified 1,085 citations, 892 from databases and 193 from other sources. Of these, 22 were further analyzed. Studies were from Brazil, Chile, Uruguay, Argentina, Peru and Nicaragua. Effectiveness ranged from 8.8–37.8% for hospitalizations due to X-ray confirmed pneumonia, 7.4–20.6% for clinical pneumonia, and 13.3–87.7% for meningitis hospitalizations, and 56–83.3% for IPD hospitalization, varying by age, outcome definition, type of vaccine and study design.Available evidence to date indicates significant impact of both PCV-10 and PCV-13 in the outcomes studied, with no evidence of the superiority of one vaccine over the other on pneumonia, IPD or meningitis hospitalization reduction in children under 5 years old. Streptococcus pneumoniae (S. pneumoniae or pneumococcus), which is considered the most common vaccine-preventable bacterial etiology of pneumonia, causing approximately 18% of cases in children globally [Pneumococcal diseases are infections caused by globally . In Latiglobally , 4.The World Health Organization (WHO) in 2012 recommended the introduction of pneumococcal conjugate vaccines (PCV) in childhood immunization programs with high priority to countries with mortality rate >50 deaths/1000 births in children under 5 years of age .Since 2009 countries in LAC Region have been among the first developing countries to introduce PCVs into their EPIs . As of MS. pneumonia [PCV-10 and PCV-13 were licensed mostly on the basis of comparative immunogenicity with PCV-7, and as such, studies on vaccine efficacy or effectiveness were not available at the time of its initial licensure . Notwithneumonia .The analysis of variation in the magnitude of the protective effect of PCV vaccines across study settings may be informative of the factors that influence their performance in immunization programs. This systematic review aims at summarizing the evidence of the impact and effectiveness of PCVs on hospitalization and mortality due to pneumonias, meningitis, and invasive pneumococcal disease (IPD) in children less than 5 years old in LAC.http://www.crd.york.ac.uk/PROSPERO/DisplayPDF.php?ID=CRD42016032693). (The study protocol was registered in PROSPERO under registration number CRD4206032693 (available at 032693). This study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. A systematic literature review was performed to identify all available data from published and unpublished studies conducted in Latin America and Caribbean, on the effects of PCV on hospitalization and mortality in children younger than 5 years of age. Details of the search terms and methods are presented in We included studies carried out in LAC countries made available (published or presented) between January 2009 and January 2016, without language limitation, with the following study designs: randomized trials, observational studies including cohort and case-control, and quasi-experimental studies including before-after and interrupted time series.To be eligible for review, studies had to target children aged less than five years of either sex. The study focused on the commercially available pneumococcal conjugate vaccines—PCV-10 and PCV-13—and considered any immunization schedule: 2 primary doses plus a booster (2+1) or 3 primary doses with or without a booster (3+1 or 3+0), with or without catch-up. The outcomes of interest were deaths or hospitalizations due to IPD, pneumonia, meningitis and sepsis. Secondary outcomes, such as serotype specific disease, adverse events, immunogenicity (antibody levels) and nasopharyngeal carriage were considered complementary information. All cause deaths and hospitalizations were not considered as study outcomes.The following exclusion criteria were considered: cross-sectional studies, case series and case reports as well as studies that only reported data before or after PCV introduction but not for both periods. Interrupted time series studies were included only if they presented data on a minimum of two data points before and after the intervention. For both before-after and time series studies, we excluded those in which only the number of cases were presented, without denominator information or incidence estimates presented. Studies specifically targeted at children with sickle cell disease, HIV-infection or conditions known to affect immune response were not eligible. Studies that considered only disease of selected serotypes, adverse events, immunogenicity (antibody levels), nasopharyngeal carriage, and all-cause mortality and hospitalization as primary outcomes; and studies assessing nosocomial infections were excluded.Citations were screened by two independent reviewers in a two-step approach. Titles and abstracts were first reviewed for duplication and inclusion criteria. Duplicates were excluded and full text of those papers meeting inclusion criteria were obtained for completion of screening on their eligibility. Screened articles were categorized as potentially eligible, unclear, or excluded. Citations on which eligibility reviewers disagreed were discussed or assessed by a third reviewer. Reasons for excluding studies were recorded.  Authors Data extraction was done independently by three reviewers, working in pairs, using abstraction forms developed specifically for this systematic review, by study design: cohort, case-control, before-after and time-series.As recently proposed to assess impact studies, to avoid multiple counting of reports from the same study, citations from the same study group on data originated from the same study protocol, population or information system were grouped for extraction, and reported as a single study .Data extracted included country, funding and ethical issues, study design, intervention details , study setting and period, data source, baseline information on study population, case definition and diagnostic criteria, data ascertainment methods, methods for data analysis, and main results including descriptive result and impact assessment results, corresponding confidence limits when available, and any results from sub-group analyses. Additional information on control and ascertainment of exposure for case-control studies and information on loss to follow-up for cohort studies were obtained.All studies were independently assessed for quality considering the items of structured quality scoring systems as checklists. Data elements of the Newcastle-Ottawa Scale (NOS)  were useDisagreements between reviewers were assessed and the major sources of divergence discussed until agreement was reached. When disagreement was not resolved, a third reviewer was used as an arbitrator.This paper presents data on effects of PCV on a variety of outcomes. As such, study results are presented by the following outcomes: pneumonia, meningitis, and invasive pneumococcal disease. As pneumonia studies included different case definitions, results were further grouped by the following categories: X-ray confirmed pneumonia (consolidated), and clinical pneumonia .A descriptive analysis of study characteristics including design, country, type and schedule of PCV introduced, data source, and endpoints considered was conducted. All results were stratified by age groups as presented by authors.For all studies, the main measure of interest was the vaccine effect in reducing the outcome of interest. In case control and cohort studies, this was reported as odds-ratio (OR) and relative risk (RR), and the resulting effectiveness was estimated as 1-OR or 1-RR. For time series studies, the effect was reported as either the % reduction in rates when modeling observed rates against predicted rates of disease, or resulting from a percent change in incidence rates when comparing the post and pre-vaccination periods.Stata Statistical Software: Release 10. College Station, TX: StataCorp LP.).In before-after studies, vaccine effects were reported as % change in rates . Studies reporting only number of cases were excluded from the analysis as it was not possible to determine the impact of the vaccine as a rate reduction. When possible, we calculated the percent change in rates using a systematic method in which all data points in the pre and post intervention period were considered, and the intervention year was excluded from the analysis. Percent change was calculated as ((pre PCV incidence rate–post PCV incidence rate)/pre PCV incidence) * 100. We conducted all analysis using Stata 10.0  and age group. Estimates of VE on hospitalization and mortality are presented separately. A posteriori, we defined the following age groups for which results are reported: <12m, 12-23m, 2-23m, 12-35m, 0-35m, 24-35m, 24-47m, 24-59m, <48m, and <60m. Further, combined results including all age groups < 2 years, the most important target age group for PCV, are presented.Sensitivity analysis was conducted to assess the impact of excluding studies which included pneumonia inpatients and outpatients combined, and selected outlier estimates from studies with significant potential for bias.A total of 1,085 references were identified and 33 were eligible for data extraction. They comprised 18 full reports and 15 abstracts and posters, reporting on 22 individual studies . Of the Of the 22 studies that met the inclusion criteria 5 were only available as poster/abstract. Studies were conducted in Argentina, Brazil, Chile, Nicaragua, Peru and Uruguay, with more than half of them being from Brazil. No study from the Caribbean countries was included. Most studies were published/presented in 2014–2016 (91%). PCV-10 was assessed in most of the studies (68%), and the predominant dosing schedules, regardless of vaccine type, were 3+1 and 2+1 (95%). A variety of endpoints, study designs and data sources were considered. Pneumonia was the most frequent endpoint of interest, and no study evaluating sepsis as an individual outcome was included in this review. Before-after was the most common study design, and surveillance the most common data source used in the reported studies . A totalThe variety of study designs and methods used in the studies made it inappropriate to conduct a meta-analysis.Thirteen of the studies included in this review evaluated PCV effectiveness on pneumonia . Among tHospitalization rates for pneumonia in 13 studies varied widely , mainly by outcome (inpatients only vs. inpatients and outpatients combined), and age subgroups .All included studies on pneumonia hospitalization and deaths reported 35 effectiveness estimates in different age subgroups. When we consider all the above reports on PCV-10 and PCV-13 effectiveness (VE), regardless of outcome, age group, data source and study methodology, VE point estimates varied from a lower 7.4% to 84.6%. Some of the estimates had very wide confidence intervals , whereasWhen results are stratified by the different pneumonia case definitions, regardless of age subgroup, PCV-10 and PCV-13 effectiveness estimates for clinical pneumonia varied from 7.4% to 49.3%, whereas for consolidated (X-ray confirmed) pneumonia VE ranged from 8.8% to 84.6%.In sensitivity analysis, disregarding the outlier estimate reported by Hortal , VE for When only effectiveness estimates from the studies included in sensitivity analyses were stratified by vaccine type, for clinical pneumonia outcome, VE varied from 7.4 to 20.6% among the various age subgroups. Those results concern studies conducted in sites using PCV-10 (n = 4) as studies from sites using PCV-13 did not fulfill the criteria above for sensitivity analysis . When X-ray confirmed pneumonia (consolidated) outcome was considered, estimates varied from 11.9% to 25.3% for PCV-10 (n = 6), and from 8.8 to 37.8% for PCV-13 (n = 2). Estimates for both PCV vaccines in either pneumonia outcome fell under overlapping 95% confidence limits .Given the great diversity of age subgroups in the reports ; Fig 2, VE for pneumonia caused by vaccine-type serotypes  were notAll three studies that assessed PCV impact on pneumonia mortality –24 showeAnalysis of effectiveness for combinations of dosing schedules and catch-up doses was not performed, due to the existing methodological variation among studies, already referred to.Five studies addressed PCV effectiveness against pneumococcal meningitis, all of which were conducted in Brazil, where PCV-10 is used. They comprised four before-after , 41, 42,Considering all reports on PCV-10 effectiveness regardless of age group, VE point estimates varied from 13.3% to 87.7% . The higMost studies reported effectiveness against pneumococcal meningitis of all serotypes for <12m, 12-23m, and younger than 2 years. Two studies reported on VE for children < 12 months of age, ranging from 36.6%  to 62.8%As showed in Two studies addressed pneumococcal meningitis mortality , 20 and Estimates of serotype-specific PCV-10 effectiveness against meningitis were reported by some authors, mainly as proportion of cases before and after vaccine introduction. In addition, most of such studies reported on a small number of cases with serotype data. In Paraná State, Brazil, Hirose et al.  reportedFour studies addressed invasive pneumococcal disease (IPD) hospitalizations , 44, 45,Reported VE against all-type IPD was generally high regardless of age, ranging from -14.7% to 66.0% , Fig 4. Both interrupted time series studies conducted in Chile  and BrazEstimates of PCV-10 effectiveness against IPD caused by individual serotypes were reported by some authors, but were mostly based on the comparison of the number of cases when comparing pre- and post-vaccine periods. A study in Chile  reportedTwo studies conducted in Brazil , 32 estiAndrade et al.  by meansS. pneumoniae, which enhanced specificity of case definition. However, the authors acknowledged that case ascertainment took place in a small number of hospitals with the laboratory capacity for pneumococcal identification. Moreover, case detection of IPD via surveillance system resulted in over-representation of meningitis, despite being the least common invasive syndrome. Selection of controls is a major challenge and there is no “ideal” control group for case-control studies. Neighborhood controls appeared a reasonable and efficient approach in that study conducted in a developing country [An inventory of potential for bias was detailed for all studies included in the review  Tables.  country , 49. A s country , ancilla country  was nestBefore-after and ITS were the most common study designs in this review (11 studies) Tables , 3 and 4Selection bias was an issue rarely addressed in the studies even in those based on passive surveillance, hospital and laboratory surveillance, or sentinel surveillance systems. Only one of them acknowledged the changed approach to diagnosis engendered by PCV implementation .Vaccination coverage was presented in ten studies , 32, 44,To our knowledge this is the first systematic review of the impact and effectiveness of the 2 current commercially available pneumococcal conjugate vaccines (PCV-10 and PCV-13) in LAC countries. The thorough review of the literature allowed assessment of PCV impact and effectiveness on the most relevant clinical syndromes of pneumococcal disease, which lead to hospitalization and mortality in children under 5 years old. In this review, pneumonia was the most frequently targeted clinical presentation by the studies, which showed high incidence rates. Studies in this review showed that PCV had both direct and indirect effects on the three clinical syndromes most relevant for hospitalization, in all different age-groups, schedules, countries, and study designs, except for very few instances in untargeted age groups . A positThe studies selected in our review were carried out in countries using the vaccination schedule 2+1, except Brazil which used 3+1 and Nicaragua, 3+0. Brazil We selected pneumonia and IPD hospitalizations and deaths as study outcomes, which are relevant in terms of disease burden and severity, and for which there are available data sources for impact assessment. These are the most commonly measured disease outcomes in countries with PCV impact studies .A variety of case definitions and endpoints for pneumonia assessment were used in the included studies. It is known that the sensitivity of pneumonia diagnosis and the estimated effectiveness of PCV on pneumonias vary according to the endpoint and case definition considered . This afFour of the 12 studies that evaluated pneumonia hospitalizations considered combined endpoint of pneumonia inpatients and outpatients , 29–31. Effectiveness estimates were consistently high for meningitis and IPD when compared to other outcomes, reaching 56.8–83.8%. Other authors have found similar findings, as reported by a recent systematic review on the impact of PCV in pediatric older children in low and middle income countries . This isSeveral studies reported on serotype-specific PCV effectiveness, and some authors acknowledged the small number of type-specific IPD cases and low representativeness of reported cases as an important limitation to demonstrating type specific VE , 45. As Our findings indicate higher VE for all study outcomes in selected age groups (ie 12–23 months). This is likely a result of the fact that children in this age group had the opportunity to have completed the vaccination schedule recently. Nonetheless, as the overall disease burden is higher in younger children, impact as total burden of disease averted was most significant in younger age groups (ie <12 months), as reported in a global review of pneumococcal disease burden by age and region .S. pneumoniae achieved with high vaccine coverage [Issues related to study design were a major concern in this review. Most studies study analyzed secondary data, and all but two studies of pneumonia had a before-after or an interrupted time-series (ITS) approach. The assessment of vaccination through ecologic study design using aggregate data, such as ITS and before-after studies, provide impact measures that combined direct effects, related to individual protection from immune response, and indirect effects including non-vaccinated subjects who benefitted from reduced circulation of coverage . On the coverage . As othecoverage , severalAs reported in the literature , our resThis study has several limitations that are worth mentioning. While the strength of this analysis is to provide a wealth of information on the heterogeneity of the vaccine impact and effectiveness as well as on the methodological quality of the studies, there are some limitations. It was not possible to perform a meta-analysis which could allow us to estimate a common measure since we found an important heterogeneity on study designs, end points, and age group stratification within studies included in this analysis. Only studies from six countries were included in the final analysis. No studies from the Caribbean countries met the inclusion criteria for this study. The small number of countries investigated could affect the comprehensive understanding about the vaccine impact and effectiveness in Latin American countries.Finally, potential publication bias, resulting in under publication of studies with negative results has to be considered when interpreting these results. We believe that the extensive literature search strategy and sources in our study contributed to reduce publication bias. Nonetheless, it is unclear to what extent and impact that selective publication of favorable results may have had in this review. Five of the 22 studies were funded by the industry, and 6 others had co-authors with some link to vaccine manufacturers. Nine studies were sponsored by governmental and/or international organizations and two did not disclose sponsorship. As shown in previous research , sponsorNo studies in this review have compared the effectiveness of PCV-10 and PCV-13 directly, and thus, only indirect comparisons were possible. Considering the outcomes studied and the available evidence to date, we found no evidence of the superiority of one vaccine over the other with regards to impact and effectiveness on hospitalization reduction in children under 5 years old. Considering the inclusion criteria established in this study there is insufficient evidence so far to compare the impact and effectiveness of both vaccines with regards to mortality. Studies directly comparing the effect of PCV-10 and PCV-13 in developed countries have demonstrated similar effectiveness with different schedules on pneumonia and IPD hospitalizations , 68.Currently PCV is one of the most expensive vaccines recommended by PAHO and WHO, accounting 75% of the total vaccine cost of immunizing a child in the majority of LAC countries . It is cS1 Appendix(PDF)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The aim of this study is to observe the effect of Peony-Glycyrrhiza Decoction (PGD) on hyperprolactinemia in women with schizophrenia induced by Amisulpride.n = 20) and PGD groups (n = 21). Maintaining the original Amisulpride dose, the two groups were given placebo and PGD, respectively. The levels of Prolactin (PRL) and other hormones were measured on the initial day and at weeks 4 and 8 after treatment. Changes of clinical symptoms in patients with hyperprolactinemia were observed. The PANSS scores were recorded to assess the psychotic symptoms. A total of 41 female schizophrenia patients receiving Amisulpride were randomly divided into placebo (p < 0.01), and level of Estradiol in the PGD group increased significantly at week 8 (p < 0.05). There were no differences in PANSS scores and biochemical indexes between two groups at weeks 4 and 8. Compared with placebo group, level of PRL decreased while Progesterone increased remarkably in the PGD group at weeks 4 and 8 ( PGD can improve symptoms of hyperprolactinemia and hormone levels in women with schizophrenia caused by Amisulpride, without affecting their mental symptoms and biochemical indexes. Schizophrenia, a severe mental illness, with high prevalence rate and disability rate, has been characterized by hallucinations, delusional perception, delusions of control, thought disorders, and social function withdrawal . The useThe majority of antipsychotic medications in clinic, such as Haloperidol, Chlorpromazine, Risperidone, Amisulpride, Sulpiride, and Olanzapine, will bring hyperprolactinemia . Some stPRL is a polypeptide hormone, synthesized and secreted by luteotroph in the anterior pituitary, and its main function is to regulate reproduction, lactation, and puerpera behavior . HyperprHyperprolactinemia may cause short-term menstrual disorders, distending pain of the breast, lactation, and sexual dysfunction. If it lasts for a long time, the effect to reproductive development, bone density, and cardiovascular system will come into being . TherefoIn recent years, the intervention of Chinese herbs in hyperprolactinemia caused by antipsychotic drugs has been studied. The results showed that Peony-Glycyrrhiza Decoction (PGD) could increase the binding capacity of dopamine receptor, inhibit the secretion of PRL , and sigIn this study, female participants aged 18–40 years with diagnosis of paranoid schizophrenia ) were screened. Majority of return visits were conducted in outpatient clinics; only few were finished in inpatient department. Patients in stable physical, neurological, and endocrinological condition with normal laboratory values  were enrolled to the study. Clinical inclusion criteria included (1) taking Amisulpride monotherapy for at least 3 months; (2) stable mental state, stable treatment for at least 3 months, and PANSS scores less than 60 ; (4) at n = 20) and PGD groups (n = 21). The baseline characteristics of patients were tested before this study.This is a preliminary study. The double-blind, randomized, placebo-controlled trial, lasting for 2 months, was conducted between January 2014 and February 2017 in the Department of Psychiatry of Brains Hospital of Hunan Province. A total of 41 patients were included in this study. Patients were randomly divided into placebo   in the it-test. p values less than 0.05 were considered as significant and those less than 0.01 were significant remarkably.The randomization code was not broken until all data were collected. Statistical analysis was performed using SPSS 24.0. For continuous variables, descriptive statistics  were calculated, while for discrete variables number of patients and percentages are given. Shapiro-Wilk test was used to test normality of distribution, and variables that did not follow normal distribution were transformed for best normality. Means, standard deviations, and confidence intervals are reported for nontransformed variables, and results of tests are reported for transformed or nontransformed variables. The difference between proportions was analyzed by Fisher's exact test, while intergroup differences were assessed by The two groups of patients were given placebo and PGD, respectively, lasting for 8 weeks. In the end, 34 of them have completed this study. In the placebo group, 5 patients were lost (25%), among them 2 patients had to quit because of severe akathisia and 3 patients due to amenorrhea after 4 weeks, while in the PGD group only 2 patients were lost (9.52%) for extrapyramidal side effect after 4 weeks. It was a double-blind study. The baseline characteristics of patients are shown in p = 0.00) and week 8 . Levels of Estradiol have no statistical significance at week 4  but have statistical significance at week 8 . Levels of Progesterone have statistical significance at week 4  and week 8 . Levels of FSH have no statistical significance at week 4  and week 8 . Levels of LH have no statistical significance at week 4  and week 8 .Figures 2The biochemical indexes of patients at week 8 in the study are shown in X2 = 1.11, p > 0.05).After 8 weeks' treatment, the incidence of adverse reactions in TESS  was 3/15 in the placebo group and 5/19 in the PGD group, respectively. The difference between these two groups has no statistical significance (p = 0.10), week 4 , and week 8 . At week 8, there was no improvement of hyperprolactinemia-related symptoms in the placebo group. On the contrary, 15 patients in the PGD group improved hyperprolactinemia-related symptoms in different aspects, among them, 2 cases in amenorrhea, 4 cases in abnormal menstruation, 3 cases in galactorrhea, 4 cases in reduced libido, and 2 cases in sexlessness.PANSS scores  have no A great number of studies have shown that western medicine, such as Bromocriptine, Aripiprazole, and other drugs, will do antipsychotic-induced hyperprolactinemia more harm than good , 24. SidThe PGD contained the following: 60 g Paeoniae Radix Alba and 30 g Glycyrrhiza Radix Et Rhizoma. According to previous studies on the treatment of Qi-deficiency and blood-stasis syndrome, the ratio of Paeoniae Radix Alba to Glycyrrhiza Radix Et Rhizoma in PGD should be 1 : 1 . HoweverConsidering that the high dose of PGD may increase the side effects to patients, our study detected the liver and kidney function, blood lipids, blood glucose, and thyroid hormone, combined with the TESS to assess the safety of the medication. Fortunately, the parameters above did not change too much after 8 weeks' treatment in PGD group, and the TESS did not show overt side effects. Although some extrapyramidal reactions appeared, they were controlled easily by using trihexyphenidyl hydrochloride. The improvement of corresponding symptoms by traditional Chinese medicine has given the patients confidence to finish this study, resulting in such low expulsion rate.From all above, when western medicine cannot relieve Amisulpride-induced hyperprolactinemia, the PGD may work. This traditional Chinese decoction is simple and effective, with low side effect, which is worth being popularized in clinic. Further studies should be carried out to investigate the mechanism of how does it affect hyperprolactinemia.


The cultivation stage of wheat and yellow peas, and the electricity used during the manufacturing stage of food production, were the hotspots in the life cycle. The nutritional and greenhouse gas (GHG) data were combined as the nutrition carbon footprint score (NCFS) (NBS/g CO2 per serving), a novel indicator that reflects product-level nutritional quality per unit environmental impact. Results showed that yellow pea flour increased the NCFS by 15% for pan bread, 90% for breakfast cereal, and 35% for pasta. The results and framework of this study are relevant for food industry, consumers, as well as global and national policy-makers evaluating the effect of dietary change and food reformulation on nutritional and climate change targets.Incorporating low cost pulses, such as yellow peas, that are rich in nutrients and low in fertilizer requirements, into daily food items, can improve the nutritional and sustainability profile of national diets. This paper systematically characterized the effect of using Canadian grown whole yellow pea and refined wheat flours on nutritional density and carbon footprint in cereal-based food products. Canada-specific production data and the levels of 27 macro- and micronutrients were used to calculate the carbon footprint and nutrient balance score (NBS), respectively, for traditional and reformulated pan bread, breakfast cereal, and pasta. Results showed that partial replacement of refined wheat flour with yellow pea flour increased the NBS of pan bread, breakfast cereal, and pasta by 11%, 70%, and 18%, and decreased the life cycle carbon footprint (kg CO The global food demand is projected to increase by 70% by 2050 . AccordiPulses are a subgroup of legumes that are solely harvested as a dry grain . From anAs a significant source of macro- and micronutrients, including protein, fiber, folate, iron, potassium, and magnesium, pulses are included in dietary guidelines and cultural dietary patterns across regions as nutrient-dense foods . ObservaCanada is the world’s largest producer of yellow peas and, relative to production, dry yellow peas remain the most underutilized pulse in North America. Moreover, yellow peas are the lowest cost pulse available. Therefore, their utilization as a flour can be a cost-effective method for bolstering the nutritional density and sustainability profile of staple processed food products commonly consumed in North America, or other regions.Life cycle assessment (LCA) is a comprehensive framework used to assess the environmental impact of food products from the extraction of raw material through to disposal ,12,13,14This study assessed the nutritional and greenhouse gas outcomes of cereal-based food products, such as pan bread, breakfast cereal, and pasta, that have been reformulated with Canadian-grown yellow pea flour as a replacement for refined wheat flour. Unlike previous studies that focused on single nutritive outcomes, such as caloric or protein levels in food ,17,18,19Given that one or more of pan bread, breakfast cereal, and pasta represent important sources of nutrition in various regions , they weThe type of refined wheat flour used by each product formulation varied in protein levels and reflects the type of wheat flour typically used to produce corresponding foods . CalciumThe nutrient composition of raw ingredients is summarized in Whole yellow pea flour is obtained from milling dry whole yellow peas . The nutIn Canada, the Reference Amount represents a regulated serving of food and was used in this study as the serving sizes for the traditional and reformulated food products. The Reference Amounts are 75 g for pan bread, 30 g for breakfast cereal , and 85 g for pasta (dry) .The nutritional quality of traditional and reformulated foods was determined using the nutrient balance concept (NBC) proposed by Fern et al.  and nutrkQI is the QI of an individual nutrient food k, 2000 kcal represents the total daily energy intake to which nutrition labelling is based in Canada [kE is the number of calories per serving of food k : 85 g [a relative to DV is represented by q,ja/q,jDV. qN is the number of qualifying nutrients (q) considered (qN = 27). If the QI value is >1, the food item is considered nutrient dense; if the QI value is <1, the food item is considered to be energy dense [The QI is defined as the mean of the ratio of qualifying nutrients contained in 2000 kcal of a given food relative to their daily values (DV) across qualifying nutrients (Equation (1)).n Canada , and Ek y): 85 g ). The amThe DVs for qualifying nutrients are summarized in d  in a food product relative to their daily maximal reference values (MRV). It is calculated as the average of the sum of ratios of disqualifying nutrients to their MRV (Equation (2)) in 2000 kcal food [kDI is the disqualifying index for food k. Again, 2000 kcal represents the total daily energy intake, and kE is the energy content of a serving of pan bread (75 g), breakfast cereal (30 g), or pasta (85 g). The level of each disqualifying component a per MRV within food k is represented by d,ja/d,jMRV. dN is the number if disqualifying components in food k. MRVs for disqualifying nutrients are summarized in dN = 5. If the DI value is > 1, the food item is deemed “compromised” because it contains disqualifying nutrients in values higher than the MRV relative to the energy content of the food item [The DI represents the levels of 5 public health-sensitive nutrients cal food .(2)DIk=t cereal  g, or pakNBS is the nutrient balance for food k. q,kQI is the qualifying index for each nutrient q in food qk. N = 27 is the number of nutrients identified as qualifying ingredients. As outlined by Fern et al. [q,kQI for a given nutrient q is >1, the QI is truncated to 1, based on the rationale that the requirement for a specific qualifying nutrient is already met. If QI is ≤1, it remains unchanged. An NBS of 100% is achieved if a food satisfies 100% of the daily dietary requirement for every qualifying nutrient in a 2000 kcal diet. Conversely, a value of 0 implies that none of the qualifying nutrients are contained in the diet [The third metric, the NBS, was calculated using the average QI of all 27 qualifying nutrients (Equation (3)):n et al. , when cathe diet .It is well known that the agricultural inputs, processes, field operations, and outputs, such as crop yields and emissions, vary widely across the globe, even for a given particular crop. Most of the existing LCA inventory databases provide geographically generic or EU (European Union)-based crop inventory data. Utilizing such data makes the calculated environmental impact a proxy and an estimate. To achieve a more accurate life cycle footprint, regional  datasets are required. Therefore, in this study, where possible, crop production, milling, processing, and manufacturing data corresponding to Western Canadian conditions were used. When not available, existing LCA inventory databases provided geographically generic or EU-based crop inventory data.2 equivalents (eq). Three supply chain stages for pan bread, pasta, and breakfast cereal were considered for the LCA component of this study: (a) Crop cultivation (yellow peas and wheat), (b) Milling (converting grains into flour) and, (c) Product manufacturing . GHG emissions from packaging, retail, product utilization, and post-consumer recycling were not considered, as these stages are highly site-dependent, and outside the scope of this study. It was assumed that, regardless of the formulation, the emissions from these stages would be largely unchanged across traditional and reformulated foods. Furthermore, within the LCA, these stages often contribute marginally to the total carbon footprint of the foods relative to the production, processing, and manufacturing stages [The carbon footprint of the traditional and reformulated pan bread, pasta, and breakfast cereal was assessed as COg stages .2 eq/kg N; cereals: 3.48 kg CO2 eq/kg N; peas and cereals: 1.62 kg CO2 eq/kg P2O5) were based on emission factors from the DataSmart Life Cycle Inventory [2 eq/ha; cereals: 39 kg CO2 eq/ha), as well as the production and use of fuel for field operations   was derived from studies [2: 2.7 kg/L; CH4: 0.00038 kg/L; N2O: 0.000069 kg/L) [2 eq associated with field operations. Results demonstrated that GHG emissions associated with the production of dry peas in Western Canada was 0.188 kg CO2 eq/kg peas (range: 0.134–0.243 kg CO2 eq/kg) [2 eq/kg cereals (range: 0.259–0.404 kg CO2 eq/kg) and used as a proxy for GHG emissions from wheat grown in Western Canada.Greenhouse gas emissions corresponding to the cultivation of yellow peas and wheat in Western Canada were from a recent systematic review . Brieflynventory . Seed apnventory  and MacWnventory , respect studies ,44,45,46 studies  and combThe grain, energy, and water requirements for milling 1 kg of yellow peas and wheat into flour is presented in Data from Guidice et al.  showed tFor Canadian yellow peas, it was assumed that 1.14 kg whole yellow peas  and 0.20A detailed breakdown of the natural gas, electricity, and water required during the manufacturing stage of food production is provided in 2 eq. per kg ingredient) and emissions from the production of non-flour ingredients  were considered during the manufacturing stage of this analysis  (The impact assessment method IPCC (2013) for Global Warming Potential (20 years) was used in the present LCA. Carbon emission factors from Ecoinvent   and the eq/unit) .2 eq per serving) (Equation (4)).A combined nutrition carbon footprint score (NCFS), as an indicator of product level nutrient density per unit environmental impact, was determined by dividing the NBS by the carbon footprint of the food product ) was relatively unchanged when foods were reformulated with whole yellow pea flour. Overall, pan bread had the highest NBS, QI, and DI values for both the traditional and reformulated scenarios.As expected, across all three reformulated food products, as the proportion of the yellow pea flour in the total flour increased from 0 to 100%, the NBS increased linearly . For eac2 eq/kg, respectively. Overall, reformulation with whole yellow pea flour decreased total GHG emissions by 4% for pan bread, 11% for breakfast cereal, and 13% for pasta. Contrary to emissions data standardized per kg produced, when the carbon footprint per serving size was considered, traditionally formulated pasta represented the food with the largest carbon footprint at 65.20 g CO2 eq/85 g (dry) serving (2 eq/75 g serving) and the breakfast cereal (32.99 g CO2 eq/30 g serving) per serving were similar.The carbon footprint of products reformulated with yellow pea flour is lower than the traditional products containing wheat flour only . The mea serving . Carbon 2 eq/kg) and pasta (0.518 kg CO2 eq/kg), the cultivation of wheat represented 60% and 85% of the total greenhouse gas emissions. However, for traditional breakfast cereal, the high energy requirements during manufacturing stage was the largest contributor to its total carbon footprint at 51% (0.498 kg CO2 eq/kg), followed by cultivation of wheat (45%). Given that the carbon footprint for the cultivation of yellow pea (0.188 kg CO2 eq/kg) is approximately half the carbon footprint of Canadian wheat from a cereal-cereal and oilseed-cereal rotation (0.33 kg CO2 eq/kg) [The contribution of each life cycle stage to the calculated carbon footprint of foods are presented in 2 eq for pan bread, 0.020 kg CO2 eq for breakfast cereal, and 0.026 kg CO2 eq for pasta. Similarly, on a per serving basis, for each 10% increase in whole yellow flour, carbon emissions were decreased by 0.847 g CO2 eq (pan bread), 0.665 g CO2 eq , and 2.784 g CO2 eq (pasta) ( (pasta) .2 eq/serving) were combined, the NCFS (Equation (4)) was highest for the pan bread  followed by breakfast cereals , and pasta  in both traditional (0% pea flour) and reformulated scenarios (2 eq.) than pasta (65.20 g CO2 eq.), its NBS (30%) was lower than that of pasta (43%), which moderated the effects of reformulation on the NCFS. Overall, reformulating with yellow pea flour increased the NCFS by 15%, 90%, and 35% for pan bread, breakfast cereals, and pasta, respectively. 2 eq/serving for pan bread, 0.13 NBS/g CO2 eq/serving for breakfast cereal, and 0.09 NBS/g CO2 eq/serving for pasta. In addition, when whole yellow pea flour represented 100% of the flour in the formulation, the NCFS was increased to 2.65 (+78%) for the pan bread, 2.19 (+141%) for the breakfast cereal, and 1.58 (+136%) for the pasta, compared to the traditional formulations.When the NBS and carbon footprint data  and reformulated pasta (56.85 g CO2 eq/serving) displaced breakfast cereal  as the largest producer of GHG emissions . However, Espinoza-Orias et al. [2 eq/kg. The carbon footprint from LCA for breakfast cereals manufactured in Europe was 1.85 kg CO2 eq [2 eq/kg). Nette et al. [2 eq/kg, which is, again, much higher than levels in this study. Differences between the present analysis and previous LCA can be attributed to different crop yields, agricultural practices, and electricity mixes between regions.The calculated carbon footprints of the three products in the present study compare well with the previous LCAs. For example, Braschkat et al.  demonstrs et al.  showed tg CO2 eq , and is e et al.  demonstrA lower carbon footprint in Canadian production of wheat is attributed to widespread no-till farm practices that lowers on-farm GHG emissions  . Western2 eq/kg. The variability was due to the variety of bread and processing requirements across bread types, and suggests the need to use localized datasets as a means to accurately evaluate the product-specific carbon footprints. Rather than site-generic or globally averaged emission factors from different databases ,52,55, tIt is increasingly clear that all stakeholders in the global food supply chain, be it on the supply side  or the demand side (consumers), should act to minimize impacts on the environment . CombiniThe present study has limitations and weaknesses that must be taken into account while interpreting the data. First, knowledge gaps exist around the milling and manufacturing stages of food production, depending upon the technology used and which also varies widely between and within countries, and industry stakeholders. Therefore, the published literature was relied upon to provide some of the inputs and carbon emission coefficients  for the 6, iron, zinc, riboflavin, or magnesium [Second, it is important to emphasize that unfortified refined wheat flour was used to model the effects of traditional and reformulated foods on nutritional and carbon emissions. Thus, as discussed previously, the present study presents a worst-case scenario for the NBS in traditional and reformulated scenarios. In Canada, it is mandatory that all food products produced with refined wheat flour use wheat flour that is fortified with thiamine, riboflavin, niacin, folic acid, and iron ,26. In aagnesium . TherefoThird, the NBS does not account for bioavailability of nutrients or protein quality. The latter refers to the ability for dietary protein to be used by humans for metabolic work . TypicalFinally, the present analysis did not consider the impact of other environmental categories of concern, such as water use , land usResults from this study complement other studies that evaluate the potential of dietary supplements , biofortInclusion of higher amounts of pulses in food products could bring substantial nutritional and environmental advantages, in terms of lower GHG emissions, and a more nutritionally balanced diet. Simultaneous assessment of the effects of food production and reformulation on nutrient density and environmental impact can be used by national and global policy-makers to set quantitative targets , evaluate alternative strategies to achieve them  while, at the same time, monitor the impact on the nutritional performance of the food systems.


To date, adult behavioral and neurochemical alterations have been mainly investigated in ENU2 mice, whereas there are no clear indications about the onset of these deficiencies. Here we investigated and report, for the first time, a comprehensive behavioral and neurochemical assay of the developing ENU2 mice. Overall, our findings demonstrate that ENU2 mice are significantly smaller than WT until pnd 24, present a significant delay in the acquisition of tested developmental reflexes, impaired communicative, motor and social skills, and have early reduced biogenic amine levels in several brain areas. Our results extend the understanding of behavioral and cerebral abnormalities in PKU mice, providing instruments to an early preclinical evaluation of the effects of new treatments.Phenylketonuria (PKU) is one of the most common human inborn errors of metabolism, caused by phenylalanine hydroxylase deficiency, leading to high phenylalanine and low tyrosine levels in blood and brain causing profound cognitive disability, if untreated. Since 1960, population is screened for hyperphenylalaninemia shortly after birth and submitted to early treatment in order to prevent the major manifestations of the disease. However, the dietetic regimen  is difficult to maintain, and despite the recommendation to a strict and lifelong compliance, up to 60% of adolescents partially or totally abandons the treatment. The development and the study of new treatments continue to be sought, taking advantage of preclinical models, the most used of which is the PAH Phenylketonuria  is the most common metabolic inherited disease caused by a deficiency in phenylalanine (Phe) metabolism, resulting in high Phe and low tyrosine levels .have abandoned the treatment, partially or totally.Since 1960, newborn screening and early treatment (a Phe-free diet) have considerably improved outcomes, such that early treated children can lead nearly normal lives. Treatment consists in a low-protein diet supplemented with generally unpleasant Phe-free mixture of amino acids and nutrients that must be consumed in large amount. Although this is still the best therapeutic option, the diet involves major alterations of the lifestyle, and patients and their families experience difficulty in maintaining compliance to dietetic regimen. Moreover, dietary therapy can be associated with deficiencies of several nutrients, some of which may be harmful to brain development, and consolidated evidence indicates that even early-treated PKU patients show cognitive deficits caused by mildly elevated blood Phe levels , 5, 6, 7The pressure created by these difficulties has renewed the interest in identifying different approaches. During past years, treatment strategies alternative to the diet  have been proposed and submitted to preclinical studies , 10, 11 enu2 (ENU2), the genetic murine model of PKU. The chemically induced genetic mutation [The development and the study of new treatments take advantage of preclinical models, the most used of which is the PAHmutation  in the cmutation , 27, 28.To be efficacious, PKU treatment must start as soon as possible in the early days of life in order to prevent neurodevelopmental perturbations .rd week of postnatal life as a critical window of brain amine availability in ENU2 mice [Previously, we identified the 3NU2 mice  demonstrNU2 mice . This tiNU2 mice , 32, 33.However, to the best of knowledge, no data are available on the behavioral phenotype of ENU2 mice during early postnatal life, as studies focused on adult animals , 28, 34.In order to investigate early behavioral abnormalities in ENU2 mice, we conducted a large set of behavioral phenotyping assay in both males and females of healthy and mutant mice. In particular, we evaluated body measurements, communicative behavior, developmental milestones acquisition, neonatal social recognition, and motor skills development.Results showed that ENU2 mice are significant smaller than WT, have a different pattern of ultrasonic vocalizations and have a significant delay on the acquisition of several developmental reflexes, never reaching controls’ score in most of them. ENU2 mice also result to have impaired motor and social skills: significant differences were indeed found compared to WT in velocity and distance moved, and in Homing Test ENU2 spend less time in maternal bedding area.s behavioral alterations; the biogenic amine levels were found to be reduced in several brain structures in ENU2 pups, with serotonin (5-HT) being the most affected one, as already shown in adult mice.Neurotransmitters level deficits supportOur results extend the understanding of brain and behavioral abnormalities in ENU2 mice, providing instruments to evaluate early the effects of new treatments.All experiments were conducted per the Italian Law (D. Lgs. 26/14) and the European Communities Council Directive of November 24, 1986 (86/609/EEC) regulating the use of animals for research.All experiments of this study were approved by the ethics committee of the Italian Ministry of Health and therefore conducted under license/approval ID #: 10/2011-B, according with Italian regulations on the use of animals for research (D. Lgs. 26/14) and NIH guidelines on animal care. Heterozygous multiparous females (+/-) were mated with heterozygous males to obtain -/- ENU2 or +/+ Wild type (BTBR) offspring. Ten days after breeding females were individually housed; delivery day was considered as pnd 0. Nine litters were used, and those made of less than 6 and more than 10 pups were excluded from the experiment. In order to investigate gender-related effects and reduce variability, as well as based on our previous experience maximum 10 animals for sex  were used for all experiments. All pups were equally manipulated but only the score obtained by two male and female pups per litter were randomly chosen to be included in the statistics, in order to be sure that all the litters were equally represented. The experimenters were unaware of genotypes until pnd 10–12, when ENU2 mice become recognizable from the fur.Before the beginning of the tests (pnd 3) mice were marked through distal phalanx removal, in order to be recognizable during the whole testing period. For genotyping, tail biopsy was performed; DNA was prepared using the Easy DNA Kit , and the PAH mutation on exon 7 was detected by PCR amplification and digestion thought restriction enzyme BsmAI .Behavioral assessment begun on pnd 4 and finished on pnd 18; animals were then weaned on pnd 28. When possible every tested pup underwent the complete test battery, in order to be able to follow its development.On pnd 14 six behaviorally naïve male and female pups per strain were sacrificed through decapitation and the brains collected, immediately frozen into liquid nitrogen and stored at -80° until the biochemical analysis.During mating and breeding mice were provided with food and water ad libitum and were on a 12:12-h dark:light cycle (lights on from 7 AM to 7 PM) and everything was made to minimize animals discomfort.Body weights and lengths of pups were measured to investigate offspring growth on pnd 5, 8, 11, 14, 17, 21, 24 and 28 . Data weThe ultrasound emission is the main communicative behavior pups display to elicit maternal care. Abnormalities in the typical emission pattern might underlie damaged mother-pup bound, anxiety problems and development impairment. From pnd 4 to 14 to evaluate ultrasounds, after an hour acclimatization in the testing room, pups were firstly separated from the mother and after 5 min individually placed in a clean beaker covered with clean sawdust and the emissions recorded for five minutes. To prevent cooling, pups were maintained on a hot plate (36°) and after recording they were returned to the home cage. No more than 5–6 pups per litter were tested .The registration apparatus, shown in The curve of the number of USVs emitted was analyzed by two-way ANOVA for repeated measures  followed by post-hoc Duncan’s test for multiple comparisons. The analysis of the USVs features on pnd 6 was performed by one-way ANOVA to evaluate the genotype effect .From pnd 5 to pnd 17 sensorimotor reflexes such as righting reflex, negative geotaxis, screen test, vertical screen test, auditory startle, cliff avoidance, vibrissae stimulation reflex, visual placing response, were recorded , 39.The parameters analyzed were the latency to act out the behavior (righting reflex and negative geotaxis) or the score obtained on a four level scale If no response was present within 30 seconds, the animal was given 0 points, or 30” (cut off time) in the latency evaluation.Development of reflexes was analyzed by two-way ANOVA for repeated measures  followed by post-hoc Duncan’s test for multiple comparisons.Pups were tested on pnd 10. After an hour habituation in the testing room, mothers were removed from the home cages and placed on a hot plate (36°) to prevent offspring cooling. The test started after 5 min separation.The testing apparatus was made of a central compartment and 2 lateral arms , one covered with home cage bedding, the other with clean sawdust. Pups were individually placed in the empty center and, after 1 minute of habituation, they had to choose between the two arms. Time spent into the arm containing the nesting litter, percentage of maternal arm choices, latency to reach maternal and zone crossings were scored , 39, 40.A video-based EthoVision system  was used to record and analyze the data. Statistical analysis was performed by one-way ANOVA to evaluate the effect of the genotype on the parameters .Animals underwent the Open Field test on pnd 18 to evaluate motor skills development.we used the EthoVision system  was used.Pups were individually tested for five minutes , 41, 42.Statistical analysis was performed by one-way ANOVA  to evaluate the effect of genotype on the parameters.Punches were obtained as previously described . Frozen The punches were stored in liquid nitrogen until the day of the analysis.of for the effect of genotype on the single structure and compound was performed by one-way ANOVA.Frozen samples were weighed and homogenized in 0.05 M HClO4. The homogenates were centrifuged at 14000 rpm for 20 min at 4°C. Tissue levels of 5-HT, DA, NE and their metabolites 5-HIAA, DOPAC, HVA and MHPG were assessed using HPLC system . The HPLSince no significant difference between male and female was found in all the tests, the results are shown as sex-balanced groups. = 8,149; p<0.001; post hoc analysis p < .05 and .001 for ENU2 to WT at pnd 18, 21 and 24;  = 28,165; p<0.001; post hoc analysis p < .001 and .05 for ENU2 to WT at pnd 21, 24 and 28; Body growth was measured from pnd 5 to 28 . StatistENU2 pups are shorter and weight less than WT, and these differences become significant at the beginning of the weaning, when maternal nourishing is reduced. = 2,898; p<0,05; post hoc analysis p < .001 for ENU2 to WT at pnd 6 and 8): ENU2 pups emit significant fewer calls and never reach the peak present in WT calls, although a curve of emissions is still present.Measurements of ultrasonic emissions in separated pups  = 14,788; p<0,001), the calls emitted have a higher mean peak frequency  = 7,971; p<0,01) and a lower mean peak amplitude  = 11,063; p<0,01), and, even if the difference in the mean duration of the calls is not significant, the total amount of time spent calling is significantly lower  = 13,539; p<0,001).One-way ANOVA revealed that at pnd 6 ENU2 mice emit less calls in comparison with WT  = 7,783; p<0.001; post hoc analysis p < .001 and .05 for ENU2 to WT at 5, 8 and pnd 11)., that is = 2,676; p<0.05; post hoc analysis p < .05 for ENU2 to WT at pnd 14), that is = 45,504; p < .001) but no significant interaction with age., the abi, i.e. th = 5,777; p < .05) and age effect  = 23,177; p < .001)., measure = 20,330; p<0.001; post hoc analysis p < .01 and .05 at pnd 8 and 17)., i.e. ab = 65,192; p<0.001; post hoc analysis p < .05 and .001 pnd 11 and 17). has been = 6,042; p<0.001; post hoc analysis p < .05 and .001 at pnd 14 and 17). measuresAltogether, analyzed data showed that ENU2 mice have impairments in almost all tested skills, showing a motor and sensory developmental delay. = 14,135; p<0,05); also the percentage of entries in the nest area was significantly different  = 5,309; p < .001). Significant genotype effect was also found in latency of entries in the maternal area  = 6,516; p < .05), likewise significant was the difference in the entries in the non-maternal area  = 13,147; p < .01) shown after the analysis of the apparatus zone crossing.A significant genotype effect was found on the percentage of the choice of the area containing maternal litter sawdust . ANOVA d = 9,250; p<0,01) and distance moved  = 9,213; p<0,01) parameters, showing that at pnd 18 ENU2 are significantly slower and move less than healthy control mice.Locomotor activity of 18-day-old pups in an empty Open Field was lower in ENU2 mice compared to WT . In partCenter time in the apparatus did not differ between groups.mpFC; Nucleus Accumbens, NAC; Caudate Putamen, CP; Hippocampus, HIPP; Amygdala, AMY) are reported in Results from analysis of neurotransmission in different brain areas  and metabolites  ±ES.Data are indicated as mean concentrations of biogenic amines , the levels of 5-HT and of its metabolite 5-HIAA were early and significantly reduced in ENU2 mice regardless of the brain areas.In a murine model of a metabolic inherited disease it is essential to analyze behavioral and neurochemical phenotyping during the early developmental period in order to identify the exact onset of behavioral symptoms and brain alterations, and then to indicate the best time for early intervention and what parameters to keep an eye on during preclinical evaluation of experimental treatments. To date, although behavioral and neurochemical alterations have been extensively demonstrated in adult mice , 43, this we decided to go deeper into the characterization. The analysis of different parameters of the emissions at pnd 6 revealed that ENU2 pups emit less call, and even if the mean duration of the calls did not differ between strains, the overall time spent calling is reduced in ENU2; the calls are also “softer”, as indicate by the Amplitude and are more frequent indicating also qualitative differences in the emissions. These data add another aspect to the behavioral profile of EN2 mice, being communication damaged in both quantitative and qualitative features.Mice emit and perceive Ultrasound Vocalizations (USVs) to communicate; in particular USVs are emitted by pups when taken out from the nest and separated from their mother, to induce maternal retrieval in the nest. Therefore, USVs are commonly used in preclinical studies to measure communication deficits in murine models of neurodevelopmental disorders. We registered USVs from pnd 4 until pnd 14 to study the whole curve of emissions, and we found that ENU2 pups never reach the peak present in WT calls. Since pnd 6 represents the peak of emissionMoreover, in every developmental milestones ENU2 mice showed a difference in comparison to WT in terms of reduced performance, in at least one time point. Generally, ENU2 reached fully developed reflexes later than healthy littermates, as showed by increased latency or reduced scores obtained in many of them.Homing Test allows investigating complex abilities, such as sensory and motor skills, odors recognition and preference for social inputs. In the Homing Test, 10-day-old ENU2 pups showed longer latency to reach, less time spent in and lower percentage of entries in the area containing nesting odor, in comparison to WT. Although ENU2 pups present reduced locomotor activity, in the Homing Test they carry out a number of zone crossings comparable to healthy littermates. Therefore, data suggest slower development of social olfactory and related cognitive abilities as compared to control mice.In addition, distance moved and velocity during the Open Field Test showed that 18-day-old ENU2 pups had lower general activity than age-matched WT. These data are in agreement with several previous studies showing reduced locomotor activity in adult animal model of PKU , 35, 43 According to behavioral data, neurochemical analysis revealed early abnormalities in 14-day-old ENU2 pups. ENU2mouse brain is early characterized by relevant cerebral deficits, with particularly dramatic deficiency in serotonergic metabolism, characterized by more than 50% of level reduction in all investigated brain areas. With respect to catecholamine reduction, the presence and the seriousness of effects depend on the observed cerebral structure.The brain amine reduction observed here is consistent with clinical and preclinical neurochemical studies showing reduced aminergic metabolism in brain  and ceres the face validity of the genetic murine model of PKU. Furthermore, these early neurobehavioral alterations make possible, in the future, to evaluate precociously the effects of alternative treatments, monitoring early stages of postnatal life in live hyperphenylalaninemic mice.Present study identifies early behavioral and neurochemical alterations in the best murine model of PKU, providing a comprehensive assay of the several deficits in young ENU2 mice, as well as the parameters to keep an eye on during preclinical evaluation of alternative treatments. Our data showing early behavioral and neurochemical characterization of ENU2 strengthen


Supplemental Digital Content is Available in the Text.An 8-week treatment with 1800 mg of Gralise decreased the severity of postmastectomy pain syndrome and the degree of sleep impairment and catastrophizing behavior. Chronic pain is a common and debilitating complication following breast surgery. One of the most challenging for treatment is the neuropathic pain condition, postmastectomy pain syndrome (PMPS). Gabapentin is a pharmacotherapy for neuropathic pain disorders; however, its once-daily, gastroretentive formulation, Gralise, has not been evaluated in PMPS.To evaluate the safety and effectiveness of Gralise in patients with moderate-to-severe PMPS.The primary effectiveness endpoint was a change in the worst pain intensity score from baseline to completion of 8 weeks of Gralise therapy. The secondary endpoints included the change in mood, coping behavior, sleep, and function. Sensitivity to experimental stimuli was tested before and after treatment via quantitative sensory testing. The incidence and type of adverse event were used to evaluate the safety and tolerability of Gralise.Twenty-one patients with confirmed moderate-to-severe PMPS were enrolled. Nineteen of 21 (90.5%) patients completed the 8-week treatment with Gralise. A significant positive change was found in pain intensity, pain impact, and sleep. There was no change in sensory testing scores. Of total, 63.16% of patients reported reduction in present pain, 78.95% in average pain, 89.47% in worst pain, and 84.21% in overall pain severity at posttreatment visit. No significant adverse effects were noted in the study.Variation in type of breast surgery, small sample size, lack of placebo control.There was a significant improvement in pain and sleep, and Gralise was well tolerated in patients with PMPS. Further investigation is warranted. Food and Drug Administration (FDA) for patients with postherpetic neuralgia.11Gralise is a once-daily, gastroretentive formulation of gabapentin that gradually releases gabapentin over a 24-hour period to the optimal site of absorption, the proximal small intestine. Among other advantages, this gradual release reduces the chance of saturating intestinal uptake3 no studies have been conducted using Gralise, and its safety and efficacy profiles have not been investigated in breast cancer survivors with chronic pain. Therefore, the purpose of this study was to determine whether Gralise has a potential as a safe, tolerable, and effective treatment for PMPS and how it affects pain-related traits. In the large observational epidemiological study, we identified patients who underwent breast surgery and reported moderate-to-severe pain at 6 months or later postoperatively.6 We hypothesized that Gralise would reduce their pain intensity, increase sensitivity thresholds for painful stimulation, and improve mood, sleep, coping behavior, and overall pain impact scores. This study aimed to inform future randomized clinical trials with Gralise on phenotyping strategies for comprehensive evaluation of PMPS and related traits.Despite some positive evidence of PMPS relief with gabapentin,27 After the 2-week period, patients received a stable dose of 1800 mg/d for an additional 8 weeks. At the end of the 8 weeks, Gralise was gradually discontinued over 1 week , and stored according to the manufacture's guidelines. At the first visit, subjects were dispensed a 2-week titration package, 8-week maintenance package, and 1-week discontinuing package supply of Gralise. Treatment was initiated at a dose of 300 mg/d and increased over a 2-week period to a total daily dose of 1800 mg/d. As recommended by manufacturers and previously described, 1800 mg/d is the clinically effective regimen.6 In Belfer's original study, these patients were screened for postmastectomy pain at least 6 months following their surgery, and the average postoperative screening time was 38.3 months.6 Patients' pain quality was described using 3 instruments, The Brief Pain Inventory, The Short Form McGill Pain Questionnaire and the Breast Cancer Pain Questionnaire (BCPQ), as well as the pain burden index, as described previously, were calculated for each subject.6 Twenty-one patients amongst the 611 were selected to participate in this study based on the presence and scale of current postmastectomy pain, presence of neuropathic pain symptoms, overall health, history of pain management, and availability for this trial. These patients had all reported moderate-to-severe pain  at the time of their initial screening and were included in this study if they reported current pain level ≥4/10 at follow-up, to ensure that their pain had not decreased spontaneously over the several year span. Medical charts were reviewed for creatinine levels, history of kidney disease, and list of pain medications. Full demographic, clinical, and medical data from eRecords were available for each patient.The subject cohort was selected from Belfer's database of over 600 postmastectomy patients for whom pain and related psychosocial traits were previously obtained and described.28 Patients were required to have pain level ≥4/10 at present time, which was first determined via phone interview and then confirmed at visit 1. Patients were excluded from this study if they were pregnant or breastfeeding, had any known allergy to gabapentin, had a history of gabapentin dose-limiting tolerance or lack of efficacy at doses of ≥1200 mg/d or pregabalin at doses of >300 mg/d, had any current pain management (such as treatment with duloxetine or opioids) and creatinine clearance <60 mL/L.Women 18 years or older who underwent total or partial unilateral mastectomy due to a breast cancer diagnosis and suffered from moderate-to-severe PMPS were eligible for the study. Moderate-to-severe PMPS was defined as shooting, pressing, burning, or pricking pain in the remaining breast, chest wall, armpit, and/or arm, persisted from the time of breast surgery and sometimes accompanied by tingling and/or numbness (as per BCPQ descriptors). Pain intensity score cutoff was ≥4/10 on the NRS, which was reported as a clinically significant level affecting patients' daily activities and quality of life.http://links.lww.com/PR9/A4). At the first clinic visit to the Pain Sensory Lab at the Magee Women's Hospital, confirmation of eligibility was determined via a neuropathic pain assessment by a chronic pain expert .23 All patients recruited to this study were confirmed as having PMPS with both specific pain descriptions as per BCPQ and specific sensory tests . Serum creatinine levels were obtained through local laboratories. Signed informed consent was attained before study procedures from each subject. Once the quantitative sensory testing (QST) session and study questionnaires were completed during the first visit, the patient was given a Gralise supply and scheduled for a future second visit. The clinical coordinator contacted the study subject the following day to confirm that the first dose was taken. For the next 10 weeks, the patient was called weekly, and pain and adverse event (AE) data were collected via structured phone interview. If patients did not self-report drug-related side effects, patients were asked about the presence of AEs commonly observed with gabapentin treatment. During the second visit , the subject completed the same surveys and underwent the same QST procedures as the first visit (baseline). A follow-up telephone call addressing any AEs was conducted at week 12 (7 days after treatment completion). The duration of the subject's participation in the study was 15 weeks (after eligibility determination), which included the 3-week washout period, 2-week titration-up period, 8-week treatment period, 1 week titration-down period, and 1 week follow-up period.Patients were contacted by the clinical coordinator and invited to participate in the study. There were a total of 2 clinic visits and 10 telephone visits . The Brief Pain Inventory (BPI)38 was used to characterize pain. The location, intensity (measured by the NRS), duration, frequency and quality of pain, and pain-related functional limitations were recorded. Primary endpoint was a change in the worst pain level over a 24-hour period. Anxiety, symptoms of depression, and sleep disturbances were assessed using short-form instruments from the National Institutes of Health roadmap initiative, Patient-Reported Outcome Measurement Information System (PROMIS).12 These instruments have been calibrated on more than 20,000 subjects and have been extensively validated in studies comparing results with established scales.30 Catastrophic thinking associated with pain was quantified via the Pain Catastrophizing Scale.40 This scale has been validated in patients with chronic pain.An extensive phenotyping of pain and comorbid traits was achieved through validated questionnaires and QST  with a flat round transducer (probe area 0.785 cm2) applied bilaterally at the trapezius muscle and on the dorsal aspect of the proximal forearm over the extensor muscle compartment. Pressure was increased at a steady rate of approximately 1 kg/s until the subject perceived the pressure first as painful (indicating the patient's threshold) and then unbearable (suprathreshold sensitivity or tolerance), and the pressure in kilograms was recorded.Sensitivity to painful stimulation was assessed by measuring both mechanical (pressure) and thermal (cold pressor) pain thresholds and tolerance, as described previously.Cold pain sensitivity was determined via immersion of the right hand in a circulating cold water bath maintained at 4°C for 30 seconds. This was repeated 3 times, with 2 minutes in between immersions. The third immersion lasted until the subject reached the pain tolerance (or a 3-minute maximum), and the time in seconds was recorded. The intensity of cold pain was rated using the NRS at the midpoint of and 30 seconds after each test. All procedures were performed by the clinical coordinator and a trained clinical volunteer under Belfer's supervision, and the questionnaires were self-administered under the supervision of the study staff. All data were entered into a web-based database in real time.The mean change in patient reported worst pain level measured over 24 hours with NRS (via the BPI instrument) from baseline (visit 1) to week 10 .The mean change in patient reported levels of present and average pain, mood, coping behavior, sleep, and function, as well as in pain sensitivity scores  from baseline to week 10.t tests in IBM SPSS Statistics 22.0. Differences between baseline and posttreatment values of >30% for psychosocial variables and pain sensitivity scores, and a difference of 1.5 to 2 points on the NRS for pain intensity were considered clinically significant. A probability of <0.05 was considered statistically significant. This proof-of-concept pilot study aimed to collect the data for power and sample size calculations for future larger-scale clinical trials.All continuous variables were normally distributed, and the mean values and SDs were calculated. To assess the change in PMPS and PMPS-related outcomes from baseline to the end of treatment, the pretreatment and posttreatment mean scores were compared using paired sample P = 0.0001). The subjects' average pain intensity showed a decrease of pain at a mean level of 4.6 (SD = 1.9) before treatment started to 2.2 (SD = 1.8) on the NRS at the end of 8 weeks of treatment. The mean difference of subjects' average pain intensity ratings between baseline and the end of the study was 2.4 (SD = 2.7) (P = 0.001). Of the 19 subjects who completed the treatment, 12 (63.2%) reported significant reduction in present pain , 15 (79.0%) reported significantly lower average pain , 15 (79.0%) reported significantly lower worst pain , and 16 (84.2%) reported significantly lower overall pain severity  at posttreatment visit  and included in Table R2 = 0.56).All study participants were women of Caucasian origin. The mean age of the 21 patients was 55.2 (SD = 6.5) years old at the baseline. The mean body mass index was 27.5 (SD = 4.9). Table P = 0.002) compared with their survey answers pretreatment, 38.89% of patients reported feeling less stressed, and 61.1% of patients reported having greater feelings of control. There was also a significant improvement in sleep variables after Gralise treatment compared with baseline. Of total, 61.9% of patients reported significantly improved sleep demonstrated by a decrease in restless sleep, a decrease in difficulty sleeping, and a decrease in trouble staying asleep , while 27.8% of patients reported better sleep quality at the end of Gralise treatment, demonstrated by feeling more satisfied with sleep, feeling that sleep was more refreshing, and feeling that they got enough sleep. Posttreatment catastrophizing scores were also significantly lower . However, Gralise was not noted to have a significant effect on anxiety or depressive symptoms .Additional objectives for this study were to assess if there were any changes in the impact of pain on daily activities, mood, coping behavior, and sleep patterns. Compared with baseline, 73.7% of patients reported significantly lower impact of their pain on daily functioning at the posttreatment visit  and right arms (P = 0.3). Likewise, there was no difference in pressure threshold (P = 0.51) or tolerance on the left arm (P = 0.3) and right arm (P = 0.59) between pretreatment and posttreatment measurements. There was a trend towards higher tolerance  to ice-cold water when comparing baseline to posttreatment scores (P = 0.09). All the psychological factor data are displayed and summarized in supplemental Figure 2s (available at http://links.lww.com/PR9/A4) and Table The secondary effectiveness endpoint used QST to assess if Gralise therapy improved patients' sensitivity to mechanical and thermal experimental stimulation. There was no difference between pretreatment and posttreatment scores in patients' average pressure thresholds on both the left P = 0. and righhttp://links.lww.com/PR9/A4). Patients were categorized as having marked, mild or unchanged/worse pain relief and also none, not significant, or significant side effects. None of the patients in the study had the significant side effects of diarrhea, vomiting, nausea, or weight gain that are reported in previous gabapentin trials, suggesting better tolerability of Gralise compared with gabapentin.The safety and tolerability were evaluated by the incidence of AEs reported while on Gralise therapy. No severe AEs were noted. Of the total 21 patients enrolled in the study, only 1 patient (61 years old) withdrew from the study on week 2 due to AEs . There were a total of 5 AEs: fatigue, dizziness, dry mouth, sleeplessness, and itchiness. All AEs were mild and did not require treatment. Fourteen patients reported at least 1 of these events. Of total, 42.9% (9/21) of the patients reported fatigue, 33.3% (7/21) reported dizziness in the morning, 4.8% (1/21) reported dry mouth, and 9.5% (2/21) reported sleeplessness. Among the 14 patients with the before mentioned side effects, 7 patients reported 2 or more. All side effects, with the exception of itchiness, subsided by week 7 of the study. One patient (4.5%) reported itchiness that lasted from week 3 to week 12. At the end of the 12 weeks, patients were asked to report their general impression of their degree of pain relief; these results were compared simultaneously with patient reports of side effects from the Gralise therapy -δ site of voltage-dependent calcium channels and modulates the influx of calcium, with a resulting reduction in excitatory neurotransmitter release. Gabapentin  has been used for the management of pain related to postherpetic neuralgiaam), when patients are sleeping. Consequently, patients report a lower rate of dizziness and sedation relative to immediate release of gabapentin and pregabalin.19 Indeed, fatigue and dizziness observed in our study had mild symptoms, resolved spontaneously by week 7 of treatment, and were not accompanied by other AEs commonly observed in gabapentin trials, such as diarrhea, vomiting, nausea, or weight gain. Therefore, this pilot study indicates better tolerability of Gralise compared with nongastroretentive gabapentin that should be confirmed in follow-up studies.Gastroretentive gabapentin, Gralise, is a unique extended-release, once-daily formulation of gabapentin that provides both efficacy and increased tolerability. It was developed using AcuForm technology, a polymer-based drug delivery system that retains the tablet in the stomach and upper gastrointestinal tract for a sustained period. When administered with a meal, the tablet swells and remains in the stomach for up to 15 hours, releasing the drug gradually for absorption by the small intestine. The starting dose is typically 300 mg/d once daily and increased over 2 weeks to a target dose of 1800 mg/d. When administered with an evening meal, the peak dose occurs in the early morning  with a threshold score for inclusion in the study41 would facilitate the recruitment of patients with a neuropathic component to their postmastectomy pain and further enrich the study population. Finally, evaluation of long-term effects of Gralise (beyond 8 weeks of treatment) in the future prospective studies will help to determine its ultimate value for pain management in patients with persistent and severe PMPS.Breast cancer is the most common cancer in women worldwide. Partial or total mastectomy is the most common treatment choice in patients with breast cancer. Furthermore, with recent advances in breast cancer risk assessment and prediction, bilateral prophylactic mastectomy has become the most common risk-reducing surgery in high-risk women. Therefore, the rates of preventive mastectomy are on the rise.This study, for the first time, found that an 8-week treatment with 1800 mg of Gralise significantly decreased the severity of PMPS and improved patients' sleep and catastrophizing traits associated with PMPS. The positive findings from this study suggest that Gralise may expand treatment options for PMPS; however, the possibility that pain reduction experienced by patients is, in part, due to a placebo response cannot be ruled out. Future studies would require a placebo control and use of a noninferiority trial with gabapentin. Data from this study may lead to large-scale trials on the efficacy of Gralise in comparison with other pain medicine for chronic neuropathic pain relief and functional improvement in breast cancer survivors.The authors have no conflicts of interest to declare.This study was supported by the Investigator-Initiated Research grant , internal funds of the Department of Anesthesiology, and the Clinical and Translational Science Institute (CTSI) at the University of Pittsburgh, Pittsburgh, PA.Appendix Ahttp://links.lww.com/PR9/A4.Supplemental Digital Content associated with this article can be found online at


Few studies have examined the relationship between retinal microvascular abnormalities and chronic kidney disease (CKD). This study aims to examine the association between retinal vessel diameters and CKD in the rural China in order to provide the scientific basis for the early detection and diagnosis for CKD.Participants and data were extracted from the Handan Eye Study, a population-based cross-sectional study performed from 2006 to 2007. The central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) were summarized by the average arteriolar and venular caliber of each eye. The estimated glomerular filtration rate (eGFR) and a urinary albumin to creatinine ratio (ACR) were recorded. Multivariate logistic regression models were used to determine any associations between CRAE, CRVE, arteriole-to-venule ratio (AVR), retinopathy, and CKD in the recruited participants.P = 0.037) and CKD  after adjustment for potential confounding variables. Retinopathy was associated with the occurrence of albuminuria  and CKD . In participants with diabetes, the ORs for the 1st and 4th quartiles of CRAE and CRVE were 2.292  and 2.113 , respectively. Among the participants with hypertension, retinopathy was also observed to be associated with CKD .CKD was found in was 17.3% (892/5158) of this population with a 0.9% (48/5545) rate of reduced renal function and 16.7% (922/5538) rate of albuminuria. Retinopathy was present in 9.6% (571/5925) of participants. Compared to the 4th quartile of AVR, the first group was found to have a higher risk of albuminuria (odds ratio [OR] = 1.261, 95% confidence interval [95%CI]: 1.015–1.567, The parameters of retinal vessel diameter may be a useful index evaluating the occurrence and development of CKD. Therefore, early detection, diagnosis, and treatment are the cornerstones of CKD management.Chronic kidney disease (CKD) has become a major public health problem worldwide and has been associated with premature morbidity and mortality.7 and microvascular alterations in renal circulation in CKD have been documented in animal models.8 Thus, microvascular disease has been suggested to play a major role in the etiology and evolution of CKD.Some epidemiological studies have shown a relationship between CKD retinal microvascular abnormalities. Risk factors for microvascular damage, such as diabetes and hypertension, have been strongly associated with CKD,11 Since rural population accounts for the vast majority of China's total populace, exploring the relationship between retinal microvascular abnormalities and CKD in this population is of great significance.However, epidemiological studies are generally focused on urban and economically developed areas, while little is known about rural areas where lifestyle, level of education, cultural customs, nutrition, and environmental factors are very different.This study examines the association between retinal vessel diameters and CKD in rural China in order to provide a scientific basis for the early detection and diagnosis for CKD.9 Residents of Yongnian County in Handan City, Hebei Province aged 6–30 years were randomly selected using a stratified, clustered, and multistaged sampling technique. Of the 458 villages in this region, 13 were randomly selected and stratified by geographic landform (plains or hillside). The protocol for this study was reviewed and approved by the Ethics Committee of the Beijing Tongren Hospital in accordance with the guidelines of the Helsinki Declaration, and written informed consent was obtained from all participants. Participants with missing information on serum creatinine, urinary albumin, or unclear retinal photography were excluded.All patient data were extracted from the Handan Eye Study, a population-based cross-sectional study performed from 2006 to 2007. A detailed explanation of this study and its methodology was published by Liang et al in 2009.9 Albuminuria was measured by immunoturbidimetric methods , and urinary creatinine was measured by the Jaffé kinetic method.The information collected from participants included sociodemographic status , past medical history , and lifestyle information (including assessment of smoking and drinking). Physical examinations were performed to measure height, weight, and blood pressure. Blood samples were obtained and fasting blood glucose, lipid levels, urea nitrogen, urea albumin, and creatinine were measured.9 Retinal vessel diameters from digital retinal images were measured using a computer-based program by trained graders who were blinded to the status of each participant. The central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) were summarized using the average arteriolar and venular caliber of each eye. This data were obtained by measuring all arterioles and venules coursing through a zone between 0.5 and 1 disc diameters away from the margin of the optic disc.10The procedures for retinal photography and the grading of retinal microvascular signs have been described in a precursor study.Retinopathy was considered present if any characteristic lesion, as deﬁned by the ETDRS severity scale, was present including microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading, and/or new vessels.2) or albuminuria evaluated by urinary albumin to creatinine ratio .11 The eGFR was calculated using a modified version of the Modification of Diet in Renal Disease equation developed by the Peking University First Hospital, based on data from Chinese patients with CKD.12CKD was deﬁned as the presence of either reduced renal function as evaluated by estimated glomerular filtration rate  and participants were classified into either an overweight/obesity group (≥24.0 kg/m2) or a normal/underweight group (<24.0 kg/m2). Level of education was based on number of years of schooling and classified into 2 groups, ≥9 and <9 years. The arteriole-to-venule ratio (AVR) was calculated as the ratio of CRAE to CRVE. The parameters of CRAE, CRVE, and AVR were categorized into quartiles.Body mass index (BMI) was calculated (kg/mPopulation characteristics were described using means and standard deviations (SDs) or frequencies and proportions. The characteristics of the different quartiles of CRAE, CRVE, and AVR were also summarized and tested with one-way analysis of variance or the Chi-square test where appropriate. Multivariate logistic regression models were used to determine if associations existed between CRAE, CRVE, AVR, and/or retinopathy and CKD in the recruited population. Separate analyses were also performed for participants with diabetes, hypertension, and obesity. The effects of potential confounding variables and bias were controlled by adjusting the age, sex, BMI, education, hypertension, diabetes, smoking, drinking, total cholesterol, triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels.P value of <0.05 was considered to be statistically significant. All analyses were performed using SAS software, version 9.1.3 .Odds ratios (ORs) and their 95% confidence intervals (95%CIs) were estimated using the maximum likelihood method. A 2-sided A total of 5925 participants were recruited into the study. Retinal imaging and retinal vessel diameter measurements were obtained for all participants. eGFR data were available for 5158 participants and albuminuria data for 5538.P < 0.05). Total cholesterol, triglycerides, and LDL levels were also found to be higher than in the general population (P < 0.05).In this population, CKD had a prevalence of 17.3% (892/5158), reduced renal function in 0.9% (48/5545), albuminuria in 16.7% (922/5538), and retinopathy in 9.6% (571/5925) of participants. Basic characteristics of the participants are shown in Table P < 0.05, Table P < 0.05, Table P < 0.05, Table The differences between the CRAE quartiles were statistically significant for age, BMI, hypertension, diabetes, smoking, total cholesterol, LDL levels, and HDL levels (P = 0.037) and CKD  when adjusted by age, sex, incidence of smoking, alcohol consumption, BMI, level of education, and total cholesterol, triglycerides, and LDL, and HDL levels. Retinopathy was also associated with the occurrence of albuminuria  and CKD .The associations between CRAE, CRVE, AVR, and CKD were analyzed with multivariate logistic regression models Table . CompareP = 0.032) compared with 2.113  in the 4th quartile (Table P = 0.047), as shown in Table In participants with diabetes, the ORs for CKD in the first CRAE and CRVE quartile 2.292 , retinopathy was related to a decrease in eGFR and albuminuria.7 In our analysis, we found a weaker association between retinopathy and CKD compared to the above studies.According to the Atherosclerosis Risk in Communities Study, people with retinopathy were more likely to suffer from CKD  after adjustment for potential confounding factors.18 Adjustment for all potential confounding factors is extremely important in order to determine the association between retinopathy and CKD, and further studies may be warranted.In the Chronic Renal Insufficiency Cohort study, presence and severity of retinopathy at baseline were strongly associated with the risk of subsequent progression to end-stage renal disease and reductions in eGFR in univariate analyses. However, this association was not statistically significant after adjustment for initial eGFR and 24-hour proteinuria.19 On the other hand, in the Atherosclerosis Risk in Communities Study, patients with retinal arteriolar/venular narrowing were more likely to suffer from CKD compared to the general population. In the SiMES, only retinal arteriolar narrowing was related to increased albuminuria, while no difference was found for retinal venular changes.7In this study, we did not find an association between quartiles of CRAE and CRVE and CKD considering the whole sample or just hypertensive participants. The presence of retinal arteriolar abnormalities was also not associated with deteriorating renal function in the Cardiovascular Health Study.20 who reported that no significant associations could be found between AVR and eGFR/microalbuminuria in hypertensive patients. However, our results could be explained by the specificity of this population.Interestingly, we found that the likelihood of suffering from CKD increased with decrease in retinal diameter. Therefore, AVR may act as a marker for the early stages of CKD. However, this association could not be observed once the patients were stratified based on hypertension and diabetes diagnoses. This confirms the results obtained by Masaidi et al23 For example, the SiMES showed that retinal arteriolar narrowing was associated with CKD.19 We did not find statistically significant associations between these variables in the general population, but we did find that retinal arterial or venular narrowing increases as CKD progresses in patients with diabetes. Previous studies have also reported that type 2 diabetic patients with proliferative diabetic retinopathy are more likely to present with renal involvement.23 Therefore, all patients with proliferative diabetic retinopathy or with retinal arterial or venular narrowing should undergo an evaluation of renal function, including urinary albumin measurements.The association between CKD/albuminuria and retinopathy has been evaluated innumerousstudies.24 reported that the prevalence of CKD was 14.3% in 13 provinces in China – not including Hebei province analyzed herein – with 2.8% of the population having reduced renal function and 7.3% having albuminuria. Xue et al25 sampled residents of Guangxi Province and reported that the prevalence of albuminuria and reduced renal function were 12.5% and 0.4%, respectively – the overall CKD prevalence was 15.3%. Compared to these studies, we have found similar prevalence of CKD but a higher rate of albuminuria which may be related to lifestyle differences.Zhang et alThe main limitation of this study is its cross-sectional nature which limits determination of causal inferences. Secondly, albuminuria was measured only once using urinary ACR, which may overestimate or underestimate the real prevalence of albuminuria. Additionally, a selection bias may have occurred due to exclusion of eligible participants with incomplete records. The results from this population-based data from rural China suggest that retinal vessel diameter is associated with CKD, especially in the diabetic population, and may serve as a useful index for evaluating the occurrence and development of CKD.