Hypoalbuminemia (HA) is a risk factor for the complications following pancreaticoduodenectomy (PD). This study aimed to explore the factors that affect HA following PD and evaluate the influence of HA on the short-term postoperative prognosis.Methods: Total 163 patients who underwent PD and met inclusion criteria were subdivided into two groups according to the status of HA. The relationships of postoperative albumin (ALB) level and exogenous ALB infusion with postoperative responses and complications were assessed by correlation analysis.Preoperative ALB ≥35.0 g/L and postoperative complication grade were factors influencing HA after PD. Correlation analysis demonstrated significant negative correlation of postoperative ALB level with white blood cell (WBC) count and neutrophil count. Postoperative exogenous ALB infusion positively correlated with blood urea nitrogen, creatinine, complication grade, postoperative intraperitoneal hemorrhage and pancreatic fistula. No significant differences were observed between the complications and30-day mortality rates with and without postoperative HA.HA after PD should not be considered as an indicator but rather a result of poor prognosis. WBCs, especially neutrophils, are involved in reducing postoperative ALB level. Infusion of exogenous ALB to maintain ALB > 30 g/L could not improve clinical outcomes. Pancreaticoduodenectomy (PD) is a complicated abdominal surgical procedure and postoperative mortality for PD has decreased to 3–5% with improvements in the surgical technique and perioperative management –3. HowevThe postoperative ALB level is affected by the recovery process, complications, and daily fluid therapy management strategy, such as the ratio of crystalloid/colloidal fluid. Therefore, this study aimed to explore the factors influencing postoperative HA and assess the relationship between ALB changes and complications after PD. In addition, we aimed to investigate the role of exogenous albumin in the prognosis of patients after PD.All subjects have given written informed consent and the study protocol was approved by Ethics Committee of Hunan Provincial People’s Hospital, The First Hospital Affiliated with Hunan Normal University . In this prospective study, 238 consecutive patients who had undergone PD during two-year period between January 2016 and January 2018 in our hospital were initially enrolled. Patients were excluded if they underwent: (1) pancreatogastrostomy reconstruction; (2) Braun anastomosis in digestive tract reconstruction; and (3) total laparoscopic PD and conversion to open procedure, to avoid the interference of additional procedures on patient prognosis. All patients were subdivided into two groups according to the postoperative status of HA: HA group and non-HA group. HA was defined by serum ALB level ≤ 30 g/L until the 14th day after operation independent of the supplementation with exogenous ALB.Routine blood tests, biochemical examination, measurement of carbohydrate antigen 19–9 (CA19–9), abdominal ultrasound, computed tomography (CT), magnetic resonance imaging (MRI)/magnetic resonance cholangiopancreatography (MRCP), and endoscopic ultrasound (EUS) were performed before operation. Only preoperatively latest examination results were adopted in this study. Routine blood tests, liver and kidney function tests, electrolyte levels, coagulation, and C-reactive protein (CRP) level, amylase levels in blood and drainage, and lipase detection were examined on the 1, 3, 5, 7, 10 and 14 days after operation. Postoperative abdominal ultrasound and/or CT were applied to evaluate ascites routinely.Classical PD was performed for all patients. Briefly, after kocherization of the pancreatic head and duodenum, the pancreas was divided anteriorly and to the left of the superior mesenteric vein and portal vein. En bloc removal of each specimen involved distal stomach resection at 10–40% and removal of all of the duodenum, gallbladder, and common bile duct (transected at the cystic duct confluence level) as well as approximately 10–20 cm of the proximal jejunum distal to the ligament of Treitz, with resection of the head, neck, and uncinate process of the pancreas. The scope of lymphadenectomy was dependent on the nature of the lesions and observations on operative exploration. Types of pancreaticojejunostomy reconstruction comprised of duct-to-mucosa end-to-side pancreaticojejunostomy with internal or external stent, invagination pancreaticojejunostomy with internal or external stent, or pancreaticojejunostomy without stent application. The stenting silicone tube could be guided externally through the jejunal loop  or cut short and left in the jejunal loop .Postoperative complications were graded according to the Clavien–Dindo criteria . PostopeAll patients received standard care for postoperative management. Somatostatin (3 mg/Q12h) was administered to all patients by transfusion with a micro-infusion pump through postoperative day 7 or extended according to postoperative complications. A gastric tube was routinely placed preoperatively and removed after the recovery of gastrointestinal function. The drainage tube was removed if there was no evidence of biliary fistula or pancreatic fistula. Total parenteral nutrition was used for patients with an inadequate diet and stopped when the patients could tolerate at least half of their oral diet. An exogenous ALB preparation  was adopted to maintain ALB level > 30 g/L. The endpoint was short-term analysis of early postoperative results.x2 test, Fisher’s test, or Wilcoxon rank sum test. The medians were compared by the Mann–Whitney U test. Binary logistic regression was used for postoperative univariate and multivariate analyses. Canonical correlation analysis was used to evaluate the relationship of postoperative ALB level and infused amount with indicators and complications. A standardized canonical coefficient (SCC) > 0.5 was considered relevant. All statistical analysis was performed using PASW Statistics software 18.0 . P < 0.05 was considered significant.Measurement data are depicted as median (minimum - maximum). Rates were compared by Pearson A total of 238 patients underwent PD between January 2016 and January 2018 in our hospital. Of these, 13 cases with pancreatogastrostomy reconstruction, 15 cases with Braun anastomosis in the digestive tract reconstruction, 43 cases with total laparoscopic PD, and 4 cases that required conversion to laparotomy were excluded. The remaining 163 patients were enrolled in the present study, including 35 cases of lower common bile duct cancer, 49 cases of duodenal papilla carcinoma, 49 cases of pancreatic head carcinoma, 17 cases of periampullary carcinoma, and 13 cases with an inflammatory mass at the head of the pancreas. On the postoperative pathology, 140 cases were confirmed to be adenocarcinoma, 2 cases were adenosquamous carcinoma, 5 cases were mucinous adenocarcinoma, 12 cases involved an inflammatory mass, 2 cases were cystadenocarcinoma, 1 case was sarcoma, and 1 case involved a retention cyst. The ratio of men to women was 1.3:1, and the average patient age was 57.8 years  cases had no complications. According to classification based on the Clavien– Dindo criteria, the percentages of all patients who experienced grade I to grade V complications were grade I 22.1% (36/163), grade II 11.0% (18/163), grade IIIa 4.3% (7/163), grade IIIb 3.7% (6/163), grade IVa 1.2% (2/163), grade IVb 0% (0/163), and grade V 3.1% (5/163), respectively. The 30-day in-hospital mortality was two cases in HA group and one case in non-HA group, and 60-day in-hospital mortality was three cases in HA group and two cases in non-HA group, showing no significant difference.The most common complication was ascites (37.4%), followed by infection (30.1%), pancreatic fistula (18.4%), and postoperative upper gastrointestinal tract hemorrhage (6.1%). Five patients presented with early intraperitoneal hemorrhage, of which two cases underwent reoperation and three received conservative treatment, two cases were cured after conservative treatment, and one died due to liver and kidney dysfunction. Nine cases experienced late intraperitoneal hemorrhage, of which three cases received laparotomy after hemostatic failure, two cases underwent interventional embolization treatment, and four cases were treated with double-catheter irrigation and local application of hemostatic drugs.The incidence of upper gastrointestinal hemorrhage was 6.1% (10/163), including one case that experienced early upper gastrointestinal bleeding within 12 h and was treated with hemostatic drugs and nine cases with late bleeding within 3 ~ 23 days postoperatively that resolved after conservative treatment. Five patients presented with biliary fistula (3.1%), including one case of both pancreatic fistula and late intraperitoneal hemorrhage that was treated by reoperation. The percentages of patients who experienced DGE of grades B and C were 1.8% (3/163) and 1.2% (2/163), respectively, and hyperbilirubinemia affected 6.13% (10/163) of the patients.Most complications were evaluated as grade I, including incision site infection and urinary tract infection. Intra-abdominal abscess formation (1.8%) was the most common type of infection among cases with grade II complications.Univariate analysis showed that the following five factors were associated with postoperative HA: preoperative TBIL ≥171.1 μmol/L, preoperative ALB ≥35.0 g/L, classification of complications, main pancreatic duct inner diameter > 0.3 cm, and intraoperative plasma transfusion (Table As shown in Table P = 0.186), ALB infusion frequency (P = 0.365), or postoperative 30, 60-day fatality rate  between the two groups. The classes of complications showed significant difference between the two groups (P = 0.020).No significant difference was observed in the rates of complications between the HA and non-HA groups Table . MoreoveIn this study, we analyzed the factors influencing HA after PD and the effects of HA on short-term prognosis. Multivariate analysis showed that preoperative ALB ≥35.0 g/L and the classification of postoperative complications significantly affected the occurrence of HA after PD, but the underlying mechanism is still elusive. We postulate that ALB level may indicate inflammation status in the body which may affect postoperative HA, but further studies are needed to confirm it.Correlation analysis showed that postoperative ALB level was only negatively correlated with WBC and neutrophil counts. The amount of exogenous ALB infused postoperatively was negatively correlated with TP but positively correlated with BUN, Cr, complications requiring surgical intervention, early intraperitoneal hemorrhage, late intraperitoneal hemorrhage and pancreatic fistula. Importantly, the incidences of complications and the 30-day mortality rates did not differ significantly between HA group and non-HA group.About 20–40% of patients present with HA after selected operations . In addiALB level after PD and infusion of exogenous ALB exhibited no relationship with HCT in this study, indicating that hemodilution was not the main reason for HA, consistent with previous report . The intThis study revealed that postoperative ALB level negatively correlated with postoperative inflammation indexes, such as WBC count, neutrophil absolute value, and CRP level. The correlation of CRP with postoperative ALB level was lower than that of the WBC and neutrophil counts, suggesting that WBC and neutrophils, especially neutrophils, have specific roles in the ALB decline after PD, probably via damage to the EG layer. We speculated that the surgery itself, as the initial stimulus of inflammation, led to an increase in the WBC count, especially in neutrophils. The release, activation and dynamic changes of different subgroups of neutrophils contributed to the damage and subsequent dysfunction of the EG layer. This may explain why infusion of exogenous ALB cannot reliably improve HA in some patients and even correcting ALB level > 30 g/L does not effectively prevent serious complications. Furthermore, such damage and functional loss of the EG layer may not be resolved while inflammation subsides during recovery, as reflected by a decrease in the neutrophil count, because serum ALB level slowly returns to the normal range over a relatively long time after the operation.Anastomotic fistula after PD, including bile leakage, pancreatic leakage, and intra-abdominal abscess formation, is the most serious complication that significantly increases postoperative mortality, and may become aggravated as late as the 8-12th day . This stBased on our results, the strategy for correcting HA after PD is to block or rapidly end the acute phase of the inflammatory response and protect or restore the EG layer, although the clinical benefit of this approach is questionable. In addition, careful attention to perioperative nutritional support can help patients pass through the acute stress reaction period, even though parenteral nutrition has been shown to seldom correct ALB concentration to normal level .There are several limitations in this study. First, PD procedures were performed by different physicians, and thus the amounts of fluid infused daily and the ratio of crystal and colloid varied. The impact of such differences on the occurrence of postoperative complications is difficult to evaluate. Second, exogenous ALB preparation used in this study was 20% concentration, and the impact of other concentrations of ALB on HA after PD remains unclear. Third, the variety of diagnoses may contribute to possible bias in our conclusion. Fourth, we only evaluated short-term outcomes of patients after PD and exogenous ALB infusion. Fifth, the sample size is not big enough. Further multi-center randomized prospective studies with long-term follow-up are needed.Our study provides further evidence that HA should not be considered as an indicator but rather a result of poor prognosis. WBC, especially neutrophils, exhibit unique role in reducing postoperative ALB level. Infusion of exogenous ALB to maintain ALB > 30 g/L has no clinical benefit for patients after PD.


Total knee arthroplasty (TKA) is associated with significant perioperative blood loss and postoperative allogenic blood transfusion. Tranexamic acid (TXA) reversibly blocks lysine binding sites on plasminogen molecules and inhibits plasmin formation. Comparisons of the efficacy and safety of intra-articular and intravenous TXA in primary TKA have not previously been reported.A prospective randomized trial was conducted in 150 patients who underwent TKA, and these patients were randomized into three groups. Patients in Group A were injected by intra-articular TXA according to body weight (20 mg/kg). Patients in Group B received a standard dose of intra-articular TXA (2000 mg), and those in Group C were infused with TXA according to body weight (20 mg/kg) before tourniquet deflation and again 3 h later. Baseline characteristics and data collected at blood transfusion were compared. Differences among four time points  were carried out using ANOVA.p = 0.03). Group C had significantly less drain output than Group A after post hoc comparisons (p = 0.05), whereas no significant difference was observed between Group A and B (p = 0.09) or between Group B and C.The hemoglobin levels at postoperative day 5 were 10.6 g/dL for Group A, 10.6 g/dL for Group B, and 10.7 g/dL for Group C. The drain output was 399 ml for Group A, 314 ml for Group B, and 305 ml for Group C (The weight-adjusted dose of TXA administered intravenously significantly reduced the drain output but not the total blood loss when compared with the weight-adjusted dose of TXA administered intra-articularly. No significant difference was observed in the other parameters among the three groups.CRE-2013.644-T. Registered 1 March 2014.The Joint CUHK-NTEC CREC,  Total knee arthroplasty (TKA) is associated with significant perioperative blood loss, and frequently, allogenic blood transfusion is required. Such transfusion may lead to infection, delayed recovery, longer hospital stays, and increased mortality, while also increasing the costs . VariousTranexamic acid (TXA), a synthetic derivative of the amino acid lysine, is an antifibrinolytic agent that acts by reversibly blocking lysine binding sites on plasminogen and plasmin molecules, which then renders the plasmin unable to bind or degrade fibrin, thus preserving the fibrin clot and leading to hemostasis.Growing evidence suggests that TXA is effective in reducing perioperative blood loss, hemoglobin drop, and transfusion rates in primary TKA . HoweverThis is a single-institution, prospective, randomized trial conducted in a tertiary hospital in Hong Kong. Ethics approval was obtained from the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee (The Joint CUHK-NTEC CREC) . Informed consent was obtained from each subject. This study was in compliance with ICH-GCP and the Declaration of Helsinki. Inclusion criteria were all patients who were diagnosed with primary osteoarthritis of the knee and scheduled for elective primary TKA. Exclusion criteria included known allergy to TXA, diagnoses other than primary osteoarthritis, history of thromboembolic events , anticoagulant intake, and renal impairment with plasma creatinine level more than 200 μmol/l. Patients with preoperative hemoglobin levels of less than 12 g/dL were also excluded as low preoperative hemoglobin level is a known risk factor for postoperative transfusion. TKAs done with navigation guidance or patient-specific instrumentation were excluded to reduce possible confounding factors, as opening of the femoral intramedullary canal can also act as a source of bleeding.Randomization adopted random numbers generated by a computer. A dedicated experienced research nurse helped to collect the data. Missing data were minimized because of close monitoring by the research nurse. No crossover happened among the three groups.All patients underwent primary TKAs with general anesthesia or spinal anesthesia. A tourniquet was inflated at the start of the operation prior to skin incision. Medial parapatellar arthrotomy was employed. An intramedullary femoral guide and an extramedullary tibial guide were used. The femoral intramedullary canal was plugged with autologous bone before prosthesis implantation. Cemented posterior stabilized prostheses were implanted in all patients. The tourniquet was then released after the cement had hardened. Closure of the wound followed once hemostasis was achieved. One suction drain was inserted and clamped for 1 hour at the end of the operation. Mechanical prophylaxis for deep vein thrombosis with a sequential compression device was provided postoperatively until the patient began to ambulate.From March 2014 to February 2016, 256 patients underwent primary TKA, of which 106 patients were excluded because they met one or more than one of the exclusion criteria. At the end, 150 patients entered the study, and they were randomized into three groups. In the first group, intra-articular TXA according to patient’s body weight (20 mg/kg) was infused via the drain after wound closure (Group A (IA 20 mg/kg)). In the second group, a standard dose of intra-articular TXA (2000 mg) was infused via the drain after wound closure (Group B (IA 2 g)). In these two groups, the drains were clamped for an hour after TXA was given and then released. In the third group, intravenous TXA according to patient’s body weight (20 mg/kg) was infused before deflation of the tourniquet, followed by the same dosage 3 h later (Group C (IV 20 mg/kg)).Baseline demographics including age, sex, height, weight, and body mass index, were recorded. The preoperative hemoglobin and the postoperative hemoglobin levels at day 0, day 2, and day 5 were recorded. The drain output was recorded. The total blood loss was estimated by measuring the change in hemoglobin balance. Previous studies have suggested that, in abdominal and orthopedic surgeries, the blood volume does not change significantly from the preoperative value . Thus, tTransfusion triggers included hemoglobin level less than 8 g/dL, development of symptomatic anemia, or urine output less than 0.5 ml/kg/hour for three consecutive hours not responsive to fluid replacement therapy. Postoperative symptomatic deep vein thrombosis was assessed in terms of calf pain, calf swelling, postoperative persistent fever, or persistent tachycardia and was confirmed with ultrasound. All cases and assessments were done and reviewed by specialists from the local joint reconstruction team.p value ≤ 0.05 was considered statistically significant.Baseline demographics were summarized using mean standard deviation and range (minimum-maximum) where appropriate. One-way ANOVA and Fisher’s exact test were used to determine the difference in covariates in baseline characteristics, data collected at blood transfusion, and blood test results among the four data collection time points . Post hoc Bonferroni multiple adjustments were followed to look for significances under multiple comparisons. All statistical analyses were carried out using IBM SPSS version 25 . A two-sided No statistically significant difference was observed in the baseline demographics between the three groups, including age, sex, body weight, body height, and body mass index Table . Clinicap = 0.17). The hemoglobin levels at postoperative day 5 were 10.6 g/dL for Group A, 10.6 g/dL for Group B, and 10.7 g/dL for Group C (p = 0.76). The drain output was 399 ml for Group A, 314 ml for Group B, and 305 ml for Group C (p = 0.03). Group C had significantly less drain output than Group A in post hoc analysis (p = 0.05), whereas no significant difference was observed between Group A and B (p = 0.09) or between Group B and C (p = 1.00). The estimated blood loss by Lisander’s formula was 919 ml for Group A, 811 ml for Group B, and 790 ml for Group C (p = 0.07). Four patients required transfusion in Group A, one patient required transfusion in Group C, and no patient required transfusion in Group B (p = 0.13).The preoperative hemoglobin level was 13.7 g/dL for Group A, 13.4 g/dL for Group B, and 13.4 g/dL for Group C (p = 0.77). No pulmonary embolism was observed in any patients. One patient suffered from acute coronary syndrome in Group B. Another patient in Group B suffered from early infection, which required washout. One patient in Group C required arthrotomy and evacuation of a hematoma. No statistically significant differences were observed in the above complication among the three groups (Tables For the thromboembolic complication, one patient suffered from deep vein thrombosis in Group A, two patients suffered from deep vein thrombosis in Group B, and no patients experienced deep vein thrombosis in Group C (s Tables  and 2.TAs evidenced by multiple studies, TXA is known to be beneficial in reducing the blood loss and transfusion requirements associated with primary TKA , 9, 10. In our center, our statistics show no difference in the total blood loss, hemoglobin drop, or need for transfusion. That is, no definite results indicate whether topical or systemic administration is more beneficial in terms of decreased blood loss, hemoglobin drop, or need for transfusion. In another meta-analysis, two studies report on direct comparisons between topical and systemic administration. The results were inconclusive, with one study stating topical administration was superior in reducing blood loss, while the other study showed no difference .As for the comparison of various routes of administration of TXA, the most commonly compared would be the safety and efficacy between intravenous and intra-articular routes. Previous meta-analyses showed no increased risk of thromboembolic events when intravenous TXA was administered during arthroplasty compared with intra-articular administration . This fiIn this study, no difference was observed among the three groups, concurring with previous studies, regarding the weight-adjusted vs fixed dose or the route of administration, i.e., intra-articular vs intravenous, in terms of blood loss and hemoglobin drop.A decrease was noted in the total drain output in the intravenous group compared to the weight-adjusted group. However, the total drain output is only a bedside indicator of discharge from the wound, whereas the hemoglobin drop and the total blood loss would be a more genuine indicator of hemostasis. This difference in output may be attributable to the TXA that had been injected into the joint.With safety and efficacy bearing similar outcomes in all three groups, the option to choose a preferable way may lie with the cost and efficacy. A single injection is always preferred to repeated injections because it saves manpower and cost. Also, the surgeon would exercise more autonomy as the injection usually would be given intraoperatively, and no need would exist for a second dose if the first dose were administered by intra-articular injection.A fixed dose may also be more user-friendly compared to a weight-adjusted dose. Thus, the future regimen may lie in a single, fixed-dose, intra-articular administration.Current evidence suggests many different methods for the administration of tranexamic acid and a variable dosage. Our current study adds to the already large amount of evidence. However, our study, the first such study conducted in Hong Kong, had a simple regimen and few complications. The small sample size may obscure the potential positive results in the group. Ideally, recruitment of a larger sample size would be preferable. Estimation of the blood volume by calculation is another variable factor that may be a limitation. Measuring the actual blood volume, however, is unrealistic. By using Nadler’s formula and calculating all patient blood volumes with the same formula, we believe, however, that the margin of error has been minimized.No significant difference was observed in the total volume of blood loss, hemoglobin drop, or thromboembolic events across the three groups. The weight-adjusted dose of TXA administered intra-venously significantly reduces the drain output but not the total blood loss when compared with the weight-adjusted dose of TXA administered intra-articularly.


Pseudomonas aeruginosa infection. However, the indications in different guidelines on community-acquired pneumonia (CAP) are discordant. Our objectives were to assess the prevalence of antipseudomonal antibiotic prescriptions and to identify determinants of empirical antibiotic choices in the emergency department.Antipseudomonal antibiotics should be restricted to patients at risk of Observational, retrospective, one-year cohort study in hospitalized adults with pneumonia. Antibiotic choices and clinical and demographic data were recorded on a standardized form. Antibiotics with antipseudomonal activity were classified into two groups: a) β-lactam antipseudomonals (β-APS), including carbapenems, piperacillin / tazobactam or cefepime (in monotherapy or combination) and b) monotherapy with antipseudomonal quinolones.Pseudomonas aeruginosa being isolated in only nine (1.6%). Most (85%) prescriptions were compliant with SEPAR guidelines and 207 (37%) patients received antibiotics with antipseudomonal activity (14% β-APS and 23% levofloxacin). The use of β-APS was independently associated with ICU admission (OR 8.16 95% CI 3.69–18.06) and prior hospitalization (OR 6.76 95% CI 3.02–15.15), while levofloxacin was associated with negative pneumococcal urine antigen tests (OR 3.41 95% CI 1.70–6.85) but negatively associated with ICU admission (OR 0.26 95% CI 0.08–0.86). None of these factors were associated with P aeruginosa episodes. In univariate analysis, prior P aeruginosa infection/colonization , severe COPD , multilobar involvement  and prior antibiotic  were significantly associated with P aeruginosa episodes.Data were recorded from 549 adults with pneumonia, with Pseudomonas aeruginosa. The rationale for prescription was influenced by both severity-of-illness and pneumococcal urine antigen test (levofloxacin) and prior hospitalization and ICU admission (β-APS). However, these factors were not associated with P aeruginosa episodes. Only prior P aeruginosa infection/colonization and severe COPD seem to be reliable indicators in clinical practice.Antipseudomonal prescriptions were common, in spite of the very low incidence of  The clinical practice guidelines recommend that antimicrobial treatment for patients with community-pneumonia (CAP) should be prescribed empirically after evaluation of severity at presentation and pre-existing comorbid disease, and should be stratified based on prognostic risk scores –4. HowevP aeruginosa [Compliance of antibiotic treatment with the guidelines has been associated with improved outcomes, but mortality rates rise in patients at risk of multi-resistant microorganisms such as ruginosa , 9. Howeruginosa , 11, theP aeruginosa occurs in several distinct syndromes such as community-acquired pneumonia (CAP) and bacteremia in neutropenic hosts, and in intubated patients [P aeruginosa is controversial due to the different rates of prevalence reported in the literature [P aeruginosa isolates [P aeruginosa did not present any of these conditions [Pneumonia due to patients  In CAP, terature –18. Factisolates , 4, 19. nditions . Althougnditions .The aim of this study was to assess the prevalence of the use of antibiotics with antipseudomonal activity and the determinants of empirical choice, in patients attended for pneumonia in the emergency department. We hypothesizied was that antipseudomonal antibiotics might be prescribed more frequently than needed, and that determinants of prescription differ between quinolones and other choices.Retrospective, observational cohort study of all consecutive patients attended in the emergency department (ED) at Parc Tauli Hospital , between September 2010 and September 2011. The protocol was approved by the Sabadell Hospital Ethics Committee (2014/538).All patients ≥18 were evaluated for study inclusion. Patients were identified by primary ICD-9 codes for pneumonia  and by respiratory failure or sepsis, and had to meet the study definition of pneumonia prior to inclusion. Hospital admission was considered when the patient remained in the ED more than 24 h before discharge or was transferred to the ward or the ICU. The following cases were excluded: hospital-acquired pneumonia , witnessed aspiration pneumonia, solid organ or bone marrow transplantation, and alternative diagnoses .Pneumonia was defined as the presence of a new alveolar opacity on chest radiography plus one or more of the following: fever (temperature > 38 °C) or hypothermia (temperature < 35 °C), cough with or without sputum production, pleuritic chest pain and altered breath sounds on auscultation.Chronic obstructive pulmonary disease (COPD) was defined as a postbronchodilator FEV1/FVC ratio below 0.7 in accordance with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria in a patient with a smoking habit of more than 10 pack-years [ck-years .Immunocompromised group (ICP) included human immunodeficiency virus infection (HIV), neoplastic disease treated with chemotherapy or radiation therapy in the previous 3 months, hematological malignancy, anesplenia, and immunosuppressive therapy including chronic corticosteroid therapy  and non-steroid immunosuppressive therapy.Health Care-Associated Pneumonia (HCAP) was defined according to the criteria reported by the 2005 IDSA/ATS Guidelines [idelines . Episodeidelines .Shared Medical Record of Catalonia , and the Charlson index was calculated based on data stored in the hospital database [The following parameters were recorded in the ED: age, sex, smoking status, alcohol habits, and drug consumption, comorbidities, antibiotic treatment in the three months prior to ED visit, clinical symptoms, clinical signs, arterial blood gas measurements, chest radiograph findings, laboratory parameters, diagnostic procedures, and empiric antibiotic therapy. Previous functional status was evaluated by Barthel scale obtained through hospital databases and the database . All patInfluenza virus in epidemic periods). Likewise, pleural fluid was analyzed in case of pleural effusion. Samples were obtained for bacterial culture before starting antibiotic therapy in the ED. Likewise, urine samples for S pneumoniae and Legionella pneumophila antigen detection were routinely obtained before antibiotic therapy unless urgent antibiotic therapy was mandatory, and tested using the Binax NOW immunochromatography method . We considered the presence of positive urine antigens as evidence of a bacterial infection. Serological tests were not used routinely to detect atypical organisms. In patients in whom microbiological results were negative, or who did not undergo microbiological tests, the etiology was considered unknown. Bronchoscopic samples such as tracheobronchial aspirate or bronchoalveolar lavage from the lower airways were obtained in intubated patients.The hospital protocol included the performance of two blood cultures, respiratory secretions (when feasible) and nasopharyngeal swabs  was considered as any regimen that included an intravenous antipseudomonal antibiotic: carbapenems , antipseudomonal cephalosporin  or β-Lactam/ β-lactamase inhibitor (piperacillin–tazobactam) in monotherapy or in combination.Antipseudomonal quinolone was defined when levofloxacin was administered in monotherapy.Prior antibiotic therapy was defined as the exposure to antibiotics above 48 h during the 3 months prior to hospitalization.P. aeruginosa infection/colonization was defined as confirmed infection/colonization within the year prior to hospitalization, available from patients’ records.Prior S. pneumoniae were also considered.The SPSS statistical package version 21.0  was used for the statistical analysis. Results for categorical variables were expressed as absolute and relative frequencies, and continuous variables were expressed as mean values and standard deviation (SD). Differences in demographic and clinical characteristics between groups were assessed using the Chi-squared test and Fisher’s exact test for categorical variables. Means were compared using ANOVA or non-parametric tests when distribution was non-normal. Variables that might have influenced the treatment decision were selected for analysis: prior antibiotic exposure, prior hospitalization, nursing home residence, comorbidities such as COPD, bronchiectasis, diabetes mellitus, chronic liver disease, chronic heart failure, and criteria of immunosuppression. Likewise, episode severity, measured by the need for ICU admission, radiological extent and urine antigen detection of p < 0.05) were included in the model as covariates. The same analysis was run for levofloxacin as dependent variable. Hosmer–Lemeshow goodness-of-fit test was used for model calibration.Multivariate analysis using stepwise logistic regression was performed with prescription of empirical β-APS as a dependent variable. Variables showing a univariate association (Results were expressed as crude and adjusted odds ratios (OR) with 95% confidence intervals (95% CI). The level of significance was set at 0.05 (two-tailed).A total of 704 consecutive episodes of pneumonia in adults (669 patients) were recorded during the study period, 549 of whom required hospitalization. Pneumonia was classified as CAP in 295/549 (53.7%), as ICP in 125 (22.8%) and as HCAP in 129 (23.5%). The main demographic and clinical characteristics of hospitalized patients are shown in Table . The most frequently prescribed antibiotics in monotherapy were levofloxacin in 129 (42.3%) and amoxicillin-clavulanate in 108 (35.4%). Ceftriaxone plus azithromycin  was the most common combination. In 78 patients (14%) antipseudomonal β-lactam (β-APS) was prescribed in monotherapy or in combination. The empirical use of β-APS differed between groups and was significantly more frequent in ICP and HCAP .For initial treatment in the ED, 305 (55.6%) patients were prescribed one antibiotic and 244 (44.4%) were prescribed a combination regimen. Eighty-five percent of prescriptions complied with the SEPAR guidelines . Empirical antimicrobial regimens are summarized in Table 2p = 0.02). Streptococcus pneumoniae was the most frequently isolated pathogen : the pneumococcal urine antigen test was positive in 111/389 (28.5%) of patients evaluated. Other microorganisms had a lower frequency: Haemophilus influenzae 16 (2.9%), Moraxella catarrhalis 11 (2%) and Enterobacteriaceae 10 (1.8%). Pseudomonas aeruginosa was isolated in 9/549 (1.6%): three cases in the CAP group, two in the ICP group and four in the HCAP group. Moderate-severe COPD was present in five and nine patients respectively and a previous isolate of Pseudomonas aeruginosa was documented in two of them. Bacteremia occurred in only one patient, with chronic dialysis.We collected blood cultures in 340 episodes, and 145 valid respiratory specimens were retrieved. An etiological diagnosis was made in 184 patients (33.5%): 50 (40%) in the ICP group, 103 (35%) in the CAP group and 31 (24%) in the HCAP group . This association was confirmed in the multivariate analysis .The main factors determining selection of antipseudomonal antibiotics are summarized in Table p < 0.0001). This factor was part of the HCAP definition and was present in up to 36% of the ICP population. In addition, an association was found between β-APS prescription and severe COPD and immunosuppression (ICP population), which are classical risk factors for P aeruginosa infection.Prior hospitalization was another significant determinant of β-APS use in community-acquired pneumonia , prior antibiotic  and severe COPD  presented significant associations. When prior P aeruginosa infection/colonization was included in the model, it was also associated with P aeruginosa pneumonia .Among the 381 episodes with blood cultures and/or respiratory samples available we tested the determinants of antibiotic use as risk factors for P aeruginosa coverage, this pathogen was documented in only 1.6% of episodes. Severity-of-illness, identified as the need for intensive care unit admission, and prior hospitalization were the main factors in the decision to prescribe β-APS. In contrast, the prescription of levofloxacin in monotherapy was mainly associated with the presence of negative S pneumoniae urine antigen and less severe disease.In this study we report the drivers of antibiotic prescription for pneumonia in the ED. Nearly 37% of hospitalized patients received antibiotics with potential antipseudomonal activity; although 14% of prescriptions were intended to provide Few studies carried out in the ED have focused on the determinants of antibiotic use in pneumonia, even though rates of inappropriate antibiotic treatment (for whatever reason) are known to be around 50% . ImproviS. pneumoniae strains with reduced betalactam susceptibility, Enterobacteriaceae and particularly atypical organisms, in agreement with the SEPAR guidelines [Therefore, although the indication for respiratory fluoroquinolones was to provide coverage against potential idelines , the potS pneumoniae etiology, but therapy is based on the result in fewer than 15% of cases [The specific determinants of the choice of levofloxacin over other options have not been studied in depth. In our study, apart from its being restricted to ward use, the most intriguing finding was its association with unilobar pneumonia, suggesting that radiological spread is interpreted as a sign of severity, as reported elsewhere , 29. Anoof cases . Althougof cases . Thus, aof cases .P aeruginosa, the clinical practice guidelines range substantially, from a non-specific recommendation [P aeruginosa is associated with increased mortality. For instance, in a multicentre study of 529 severely ill CAP admitted to the ICU, despite correct treatment according to IDSA guidelines in 15 (75%) of 20 cases of Pseudomonas aeruginosa infection, antimicrobial treatment at admission was inadequate. Chronic obstructive pulmonary disease, malignancy, previous antibiotic exposure, and radiographic findings demonstrating rapid spread of disease were associated with P aeruginosa pneumonia [In the case of the coverage of endation  to a recendation , 4. In gendation , 32–34. P aeruginosa as being more frequent in higher-risk groups than in low-risk groups, strengthening the argument that coverage of Gram-negative organisms is needed for these patients. However, in their study, only 5% of patients admitted to a respiratory ICU presented P aeruginosa.Few studies have evaluated aetiology according to severity-of-illness at ED presentation; Cilloniz and colleagues  identifiP aeruginosa.In our study, antipseudomonal coverage was provided in 22/59 of cases admitted to ICU, but in fact none of these patients were infected by In severe episodes of CAP requiring vasopressors or mechanical ventilation, beta-lactam/macrolide combinations were preferred in view of the evidence of reduced mortality due to the immunomodulatory effects of macrolides , 37. Morp < 0.0001).In our study, site of care was a more decisive factor than high PSI; 35.6% of ICU patients received β-APS compared with 11.4% of ward-admitted patients  COPD was the main comorbidity, but immunosuppression was present in only two patients.Other factors related to the use of β-APS were immunosuppression and severity of COPD, which are well-known risk factors for P aeruginosa coverage used in the ED were not useful in predicting P aeruginosa infection in our study. However, prior P aeruginosa infection/colonization together with severe COPD seem to be good predictors, although multivariate analysis was not carried out due to the low number of P aeruginosa episodes. These results are in agreement with Restrepo et al [Determinants of po et al  and withpo et al  recommenThe most consistent risk factor for PA is prior colonization. Unfortunately, this information is not always available at the ED of a referral center because not all CAP patients have prior isolates. In any case, our data suggest that potential prior PA respiratory colonization reported in computerized clinical records should be checked in the ED.The main limitation of this study is its single-centre retrospective design. Furthermore, the generalizability of the findings is limited as this is a single-centre cohort from 2010 to 2011 that may no longer reflect current clinical practices or pathogen distribution; for example, moxifloxacin is not approved for systemic use in Spain, a circumstance that may limit the generalization of our findings. However, the single-centre design allowed us to avoid confounding factors such as differences in protocols or bias in patient selection.Pseudomonas aeruginosa. Levofloxacin was prescribed to patients with low severity and negative pneumococcal urine antigen tests. Antibiotic prescription intended to provide P aeruginosa coverage was guided mainly by severity-of-illness, prior hospitalization and, less frequently, by specific risk factors. However, these factors were not associated with P aeruginosa episodes. Only prior P aeruginosa infection/colonization and severe COPD seem to be reliable in clinical practice.Antipseudomonal prescription was common, in spite of the very low incidence of


Thalassemia is a group of hereditary diseases characterized by a common recessive monogenic hematological disorder, presenting a significant public health concern in the developing countries. Recent studies have identified the renal effects of thalassemia syndrome. Chronic hypoxia, long-term anemia, iron overload, and iron chelators are the major causes of renal tubular dysfunction and glomerular filtration abnormalities, while glomerulonephritis is not considered a major cause of abnormal urinalysis.We report a case of a 38-year-old female patient with immunoglobulin A (IgA) nephropathy accompanied by anemia who was misdiagnosed initially, but was diagnosed with alpha-thalassemia after gene tests. We administered a combination of oral prednisolone, leflunomide, and angiotensin receptor blockers as well as folic acid and mecobalamin. During the follow-up, her proteinuria was significantly reduced, and her anemia was improved.The possibility of occurrence of thalassemia should be considered in IgA nephropathy complicated with refractory anemia, especially in high-incidence areas of the disease. Thalassemia refers to a group of hereditary hemolytic anemias, wherein mutations or deletions of the globin gene lead to various degrees of inhibition in α or β globin synthesis. The clinical manifestations are correlated with the severity of the disease. The manifestations primarily include anemia, jaundice, and hepatosplenomegaly. Population mobility and migration have allowed the disease to become increasingly common. The prevalence of α-thalassemia and β-thalassemia in China was 7.88 and 2.21%, respectively and the diseases were mainly distributed in the southern China [A 38-year-old female was admitted to our center with a complaint of abnormal urine tests. She was diagnosed with chronic nephritis syndrome 10 years ago, but did not receive appropriate medical care during this period. The patient also complained of menorrhagia with unexplained anemia. Physical examination revealed no positive signs such as bilateral lower limb edema or hepatosplenomegaly. Urinalysis revealed proteinuria (2+), blood (3+), and more than 75% the dysmorphic red blood corpuscles. The protein content was 0.71 g in 24-h urine analysis. The urinary N-acetyl-β-D-glucosaminidase was 11.14 U/g·Cr, and urinary α1 microglobulin was 12.6 mg/L. In additional renal function tests, creatinine clearance rate was 96.1 ml/min, glomerular filtration rate (GFR) was 93.1 ml/min; and serum creatinine was 71 μmol/L. The albumin content was 34.5 g/L, and the results of thyroid function tests were within the normal ranges. Tests for anti-neutrophil cytoplasmic antibodies, anti-nuclear antibodies and anti-double-stranded DNA antibodies were negative. No viral markers of hepatitis were detectable, and all the antibodies were negative except the surface antibodies. Levels of serum IgA and complement component C3 were also normal. Ferritin level was 44.1 ng/mL. Complete blood tests revealed microcytic hypochromic anemia. Hemoglobin (Hb) was 84 g/L, mean corpuscular volume was 69 fL, and mean corpuscular Hb was 22 pg. Ultrasonography results of kidney were normal. Results of renal biopsy showed that 15 glomeruli were detected by light microscopy and one of them exhibited fibrocellular crescents. Renal tubules displayed localized atrophy, and inflammatory cell infiltration as well as fibrosis in the interstitium. Arteries and arterioles were unremarkable. Immunofluorescent analysis revealed diffuse IgA (3+) and C3 (2+) staining were present in several mesangial areas. Based on these results, the patient was diagnosed with IgAN accompanied by iron deficiency anemia. We treated the patient with a combination of oral prednisolone, leflunomide, and angiotensin receptor blockers (ARBs). In addition, polysaccharide-iron complex was also administered. A complete remission of proteinuria was observed, and hematuria was also relieved. However, Hb was constantly under the normal reference range, which could not be explained by the iron deficiency anemia. In 2016, the patient was diagnosed with α-thalassemia based on the results of our genetic tests showing the deletion of --SEA gene. Serum HbF and HbA2 were elevated to 2.47 and 3.04%, respectively. During the 3-year follow-up, a significant improvement in anemia was observed following the treatment consisting of a small dose of ARB with folic acid and mecobalamin administration. Laboratory tests in March 2019 showed that Hb levels were elevated to 157 g/L. Urine occult blood was 2+, 24-h proteinuria was reduced to 0.08 g, and serum creatinine and albumin were normal.Thalassemia is a common inherited disease of hemoglobin synthesis worldwide. Patients with thalassemia have a wide spectrum of clinical phenotypes depending on their respective genotype characteristics. Some patients with thalassemia may not have any clinical symptoms, leading to misdiagnosis. Patients with thalassemia major, the most severe type, usually require regular blood transfusions for survival and some patients even die in utero or shortly after birth. The mortality of thalassemia is considerable, particularly among children .Although clinical manifestations of thalassemia can be accompanied by multisystem complications including cardiopulmonary dysfunction and endocrine organ disease, kidney involvement has received less attention. Previous clinical studies have identified thalassemia-associated proximal tubular dysfunction and abnormalities in GFR , which hThe pathogenesis of kidney dysfunction among patients with thalassemia may involve chronic hypoxia that causes proximal tubular cells dysfunction and interstitial fibrosis with consequent renal damage. Anemia induces renal hemodynamic changes in the presence of other risk factors , 7. NickThalassemia associated with IgAN is relatively rare. In 1994, Harada  reportedICs are considered important pathogenic factors of IgAN. Factors affecting the deposition of ICs in glomeruli are mainly the one associated with the ability of the body to clear ICs. The elimination of ICs in human circulation is mostly accomplished by erythrocytes through immune adhesion and phagocytosis. When erythrocyte immune function is impaired or the complement-dependent clearance mechanism is affected by immune deficiency, the activity of complement receptor type 1 on erythrocyte membrane is decreased. Its ability of recognizing, adhering to, and clearing ICs is also decreased . TherefoThe present report is the first one describing IgAN associated with α-thalassemia. The occurrence of IgAN in this case may be related to the impaired clearance of circulating ICs caused by thalassemia. However, the possibility of a concomitant existence of these two diseases could not be excluded, as both are common diseases in China. The patient has been followed up for more than 7 years and her condition is relatively stable under the current treatment. While treating with patients having urinary abnormalities combined with anemia, especially in cases wherein the renal function is insufficient, thalassemia is more likely to be misdiagnosed or missed, even in high incidence areas of the disease.


Asterella wallichiana and Plagiochasma apendiculatum growing in natural habitats. Observations revealed Ni-excess induced negative impacts on abundance, frequency of occurrence of reproductive organs, population viability and morphological traits, spore viability and physiological attributes of both the liverworts. Results led us conclude that P. appendiculatum survived better with the lowest impact on its life cycle events than A. wallichiana under Ni excess in natural habitats. Our findings collectively provide insights into the previously unknown mechanisms of Ni-induced responses in liverworts with respect to phenological attributes, as well as demonstrate the potential of P. appendiculatum to survive better in Ni excess habitats.Bryophytes are potent metal absorbers, thriving well on heavy metal (HM)-polluted soils. Mechanisms controlling uptake, compartmentalization and impacts of HMs on bryophytes life cycle are largely unknown. The current study is an effort to decipher mechanisms of nickel (Ni) excess-induced effects on the phenological events of two bryophytes,  Nickel (Ni) is a non-biodegradable heavy metal (HM), which poses environmental pollution threats, damaging biosphere and human health worldwide5. Ni is found in natural soils at trace concentrations [< 0.005–100 parts per million (ppm)], and finds its way into environment through anthropogenic activities like metal smelting, municipal sludge, industrial effluents, fertilizers and pesticides6. Ni toxicity is manifested at numerous levels, leading to inhibition of mitosis7, compromised plant growth, altered photosynthesis and plant water status, inhibition of Calvin cycle enzyme activities8, repression of nitrogen metabolism9 and generation of oxidative stress, as well as blockade of adequate absorption of other essential metals8. Most common symptoms of Ni phytotoxicity include chlorosis, necrosis, stunting of shoots and roots, and decrease in leaf size6. Although it is phytotoxic, Ni forms an important constituent of urease, the sole enzyme known to maintain Ni in stoichiometric proportion in higher plants8.Uncontrolled urbanization and human interference have tremendously contributed toward releasing toxic elements into the surrounding ecosystems, generating genotoxic stress that causes species extinction and depletion of biodiversity11. Reports have indicated that reduced pollen load, delayed or early dehiscence of anthers and disruptive changes in post-fertilization of eggs in higher plants are associated with HM uptake and translocation14. However, translocation of HMs and their impacts on reproductive abilities of lower plants like bryophytes are currently unknown. So far, a few scientific researches conducted in lower plants like algae16 and bryophytes19 have provided very shallow information.Translocation of HMs from roots to leaves and reproductive organs, and their cumulative effects on the development of reproductive organs in higher plants have been recently studied20. Bryophyte acts as a sink for various HMs, accumulating higher concentrations of metal cations than those found in the associated substrates. The high metal-accumulating capacity of the bryophytes can be attributed to their higher surface-to-volume ratio and frequent absence of the cuticle21. HM-induced physiological responses and impacts on the phenological events of these tiny plants are least understood. Among bryophytes, liverworts represent mostly mesic organisms that grow on humus, exposed surfaces of rocks and deserts, and constitute a significant component of vegetation in the temperate biomes19. Lack of a significant cuticle and well-developed conducting system has made bryophytes potent metal absorbers, bioindicators and biomonitoring agents22.In general, bryophytes are the simplest and most primitive group of land plants. They are divided into three divisions namely liverworts, hornworts and mosses, which differ from each other in terms of morphological features of gametangia and sporophytesAsterella wallichiana (Lehm. & Lindenb.) Grolle23 and Plagiochasma apendiculatum Lehm. & Lindenb.25 are natives of western Himalaya, and represent a group of commonly distributed dioecious liverwort species of the North-Western Himalayas and Western Ghats of India26. A few studies on these liverworts have been conducted, mainly for evaluation of HM-induced changes in antioxidant systems of A. wallichiana27 and P. appendiculatum28. Largely, the impacts of HMs on the life cycle (reproductive abilities) of these tiny plants remain least understood, and have so far been ignored. The present study constitutes an effort to bridge the gap of missing information of HM-induced phenological changes in bryophytes. We explored the morphological and physiological, as well as reproductive responses of liverworts toward tolerance to Ni excess on natural sites. Our findings demonstrate profound effects of Ni excess on the life cycle of these bryophyte species and enable us to discover previously unknown mechanisms of Ni responses in these liverworts in a comparative manner.−1) and the Ni excess group NS (125 ± 5.16 mg kg−1). In the CS, the macronutrients together with their determined concentrations were organic carbon , phosphorus , organic matter (9145 ± 89.98 mg kg−1) and nitrogen  (Table −1), zinc , iron  and manganese   concentration, namely the control group CS (10.25 ± 0.79 mg kgsum) and Nemerow pollution index (PINemerow). PIsum and PINemerow showed approximately 10- and 3-times higher scores, respectively, in NS than CS. According to the soil classification based on PINemerow index to assess overall degree of soil pollution, the examined NS were placed in the class IV showing moderate level of soil pollution, whereas the CS were placed in the class II with warning limit of soil pollution  were noted in the gametophytic thalli of A. wallichiana and P. appendiculatum, respectively, in the NS relative to that of the CS under natural conditions   of reproductively active archegonia and antheridia in both the liverworts compared to CS  carried out on the young gametophytic thalli of  CS Fig. a,b. Howetum Fig. a,b. Furttum Fig. c,d. Impotum Fig. a–d.FigurA. wallichiana and P. appendiculatum, respectively, recorded in NS compared to CS  and 0.9 (P. appendiculatum) as compared with their respective value in CS  content was recorded in young gametophytic thalli of A. wallichiana and P. appendiculatum, respectively, grown in NS compared with CS  and P. appendiculatum (20%) in NS, when compared with CS , with 24% and 10% reduction in CS Table . Ni toleCS Table . RhizoidA. wallichiana and P. appendiculatum, respectively, in NS Compared to CS  and glutathione (GSH). About 41% and 12% increase in ascorbic acid (ASA) content was recorded in CS Table . GlutathCS Table .A. wallichiana and P. appendiculatum in both NS and CS, approximately 32% and 21% decrease in frequency of occurrence (FOC) of antheridia  was noted for A. wallichiana and P. appendiculatum, respectively  was noted for A. wallichiana and P. appendiculatum, respectively, relative to that of CS. While, FOC of normal archegonia encounter (NArcE) of A. wallichiana and P. appendiculatum showed reduction by 25% and 13%, respectively, in NS compared to CS  in both  CS Fig. a–d. Grey32. Results of current study revealed differential responses in terms of urease activity in young gametophytic thalli of A. wallichiana and P. appendiculatum grown in NS and CS  is a key enzyme regulating the hydrolysis of urea into ammonia and bicarbonate. Understanding the role of urease in facilitating Ni uptake and assimilation into urease could be predicted via determining its activity.1.5 is a6. Soils excess with heavy metals (HMs) like Ni often result in poor plant growth and development34. Plants grown in Ni-excess soils often show reductions in species richness, abundance and plant growth, and increase in membrane damage, and perturbation in physiological mechanisms like photosynthesis and reproductive potential. Current study showed similar impacts of Ni excess on growth, abundance and other physiological and reproductive attributes of both Asterella wallichiana and Plagiochasma appendiculatum growing in natural habitats.Nickel (Ni) phytotoxicity has been proven to be more disastrous than its deficiency in plants10. Present study, in natural habitats  has shown that A. wallichiana is endowed with higher Ni-uptake potential than P. appendiculatum. In general, soil pH has been associated with mobility of metal ions in soil solution and in the rhizosphere zone of a plant18. Bryophytes, being hyper-accumulator of metal ions like Cu, Zn, Ni and Pb, uses several mechanisms to reduce metal uptake and/or adsorb metal ions on the surface alone18. In the current study, the higher accumulation and availability of more Ni2+ ions in NS, A. wallichiana and P. appendiculatum could be linked to decrease in pH value from 8.3 in CS to 7.7 in Ni-excess NS  concentrations and low C/N values in the humus have also been advocated as factors causing decline in abundance and species richness of bryophytes36. In current study, NS (n = 5) showed reduced abundance of A. wallichiana and P. appendiculatum compared with CS (n = 5)  Fig. a–c. ThisCS Table .Nemerow, are widely used to evaluate the threat level of a particular element in soil37. These indices revealed strong contamination of Ni metal in the NS soils compared with CS soils among the three heavy metals , as only Ni scored higher values of PI, Cf and PINemerow in the NS soils than in CS soils 40. Additionally, our population viability analysis (PVA) of the data obtained under field conditions also indicated negative impact of Ni excess on the number of reproductively active archegonia and antheridia of both the liverworts, with higher negative Ni effect being observed on A. wallichiana than P. appendiculatum  proved that exposure of moss to Ni could reduce the canopy size41. Reductions in rhizoids surface area (RSA) of A. wallichiana and P. appendiculatum thalli in NS could be a morphological adaptation of these liverworts to reduce the areas exposed to Ni excess  has been shown to improve Ti potential by adsorption of metal ions on the surface of rhizoids18. Such that, reduced values of Ti for A. wallichiana in NS could be attributed to more reduction in RSA in comparison with P. appendiculatum compared to CS  were shown to translocate from soil into floral organs, such as pistil, anther and nectary12. This HM translocation was found to negatively impact pollen viability, pollen removal and deposition, thereby affecting the overall plant fitness in C. pepo12. Similarly, HM translocation and accumulation have been shown to impact pollen germination and pollen tube length in tobacco plants43. Parallel to these observations, current study also pointed negative impact of Ni excess on the reproductive behavior of A. wallichiana and P. appendiculatum. Accumulation of Ni ions in the sporophytes and gametophytes of both the liverworts were observed  in A. wallichiana than in P. appendiculatum compared to CS  contents48. Ni-induced negative impacts on the photosynthetic pigments were more evident in A. wallichiana as compared to P. appendiculatum in NS  and ascorbic acid (ASA), play vital role in tolerance to HMsA. wallichiana and P. appendiculatum has helped to understand mechanisms operative in liverworts for the management of Ni-induced oxidative stress under field conditions. Findings are of immense significance in establishing the mechanistic pathway of Ni-induced damage on the life cycles of the liverworts  Grolle and Plagiochasma apendiculatum Lehm. & Lindenb. were carried out fortnightly over one year (from January 2017 to December 2017).The area selected for the present study was district Reasi of the Union territory of Jammu and Kashmir, India which is geographically situated between 33° 4′ 58.1016′′ N latitude and 74° 49′ 59.9268′′ E longitude with an area spanning over 151,701 hectares. Collections of A. wallichiana and P. appendiculatum were collected and analyzed for the presence of various macro- and micronutrients. Estimation of macronutrients in soil samples was performed by the methods of Subbiah and Asija52 for nitrogen (N2) and Olsen et al.53 for phosphorus (P). Determination of micronutrients  was estimated by atomic absorption spectroscopy method using standard procedures provided with the instrument . Soil pollution indices, including single pollution index (PI), contamination factor (Cf), sum of contamination (PIsum) and Nemerow pollution index (PINemerow), were calculated for Cu, Zn and Ni using the standard procedures described in Qingjie et al.54 and Kowalska et al.37. These indices were used to classify soils into different classes depending upon the degree of the contamination of a specific HM. For instance, soils with PI < 1, < 2, < 3, < 5 and > 5 indicated absent, low, moderate, strong and very strong soil pollution. For the Cf, soils were classified as follows: Cf < 1 = low contamination, Cf between 1 and 3 = moderate contamination, Cf between 3 and 6 = considerable contamination, Cf > 6 = very high contamination. For PIsum, geometric mean of PI of each HM present in the soil was taken. For PINemerow, the following criterion was applied for the soil classification: Class I , II , III , IV  and V . Class I, II, III, IV and V refers to clean, warning limit, slight pollution, moderate pollution and heavy pollution, respectively.Soils inhabited by n = 5, having minimum Ni concentration) and NS  sites were washed with tap water followed by distilled water to remove soil particles and other vegetation. To determine the Ni uptake potential of liverworts collected from the field, about 300 mg of oven dried samples of A. wallichiana and P. appendiculatum collected from CS and NS were placed in the muffle furnace (300–400 °C for 5 h) to ash, which was then digested using wet-digestion procedure in a mixture of HNO3 and HClO4  as described elsewhere. The concentrations of Ni (in mg kg−1 tissue) were determined using Atomic Absorption Spectrometry  following manufacturer instructions.The young gametophytic stages of both the liverworts collected from the natural sites designated as CS  were collected from the CS (n = 5) an NS (n = 5) in district Reasi and brought to the laboratory in polyethylene bags. At this stage, samples were divided into two parts for short-term physiological and biochemical, and long-term morphological analyses:Fresh samples of A. wallichiana and P. appendiculatum collected from NS and CS were used for physiological and biochemical parameters such as malondialdehye content , H2O2 content (µmol g-1 fresh weight) and relative electric conductance (REC%)55.(i) Short-term analyses: For short-term physiological analyses, gametophytic stages of A. wallichiana and P. appendiculatum growing on NS and CS. These sites were visited every week for a period of 1–2 months to observe the development of young gametophyte into mature gametophytes bearing antheridia and archegonia, and development of sporophyte.(ii) Long-term analyses were carried on the young gametophytic thalli of A. wallichiana and P. appendiculatum, a comprehensive population viability analysis (PVA) was carried using the Vortex ver. 10.3.156. The impact of Ni excess on the frequency distribution and number of the reproductively active archegonia and antheridia of the A. wallichiana and P. appendiculatum was determined for the young gametophytic thalli of the two liverworts growing in CS and NS. Vortex was also used to determine the quasi-extinction risk imposed by Ni using several input parameters, such as initial population size , mortality (number of gametophytes perished under Ni excess in NS), catastrophe (Ni excess considered as a catastrophe reducing number of reproductively active antheridia and archegonia in NS compared to CS) and reproductive potential of the antheridia and archegonia of both the liverworts.For measuring the impact of Ni excess on the survival and normal functioning of the reproductive structures (antheridia and archegonia) of 2 patch of area under study) were recorded. Plants were photographed in the field using a digital camera . Each sample was divided into two parts; one part was kept for the preparation of herbarium and another for the identification of thalli using gametophytic and sporophytic characters.For field conditions, ecological attributes of the habitats of two liverworts, including mean temperature and mean relative humidity  staining procedure was used58. In brief, A. wallichiana and P. appendiculatum gametophyte and sporpophytic stages collected from CS and NS were thoroughly washed with double distilled water. Later, tissues were air dried and then placed in petri plates containing dimethyl glyoxime (DMG) solution for 10 min, followed by washing with distilled water to remove any surface retention of DMG. Accumulation of Ni2+ ions upon DMG staining was observed as pink color.To visualize NiA. wallichiana and P. appendiculatum were cut manually. Sections were mounted in glycerin before photomicrography using a NIKON ECLIPSE E400  camera.The male and female mature gametophyte developmental attributes like archegonia and antheridia numbers were counted and dry biomass was measured. Sporophyte viability was determined by staining the spores with 2,3,5-triphenyltetrazolium chloride (TTC). For anatomical studies, ventral section of thallus stained with DMG for CS and NS of both the A. wallichiana and P. appendiculatum was done using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) based fluorescence microscopy59. Briefly, the thalli were placed on glass Petri plate containing Ni solution  for 10 min and distilled water . They were then allowed to float on a 60 µM H2DCFDA solution prepared in buffer  for 10 min in dark. After a brief wash with buffer, the thalli were observed using Leica DM1000 fluorescence microscope under GFP filter having 470/40 nm bandpass excitation and emission of 525 nm fluorescence microscopy. The whole thalli were imaged, and the grey values were calculated by drawing ROI (region of interest) bearing similar area for all the samples. The average intensities were used to calculate the ROS concentration.Reactive oxygen species (ROS) detection in young gametophytic thalli of 29.Estimations of ascorbic acid (ASA), glutathione (GSH) and proline (PL) were done as described in Choudhary et al.A. wallichiana and P. appendiculatum collected from the NS and CS following the method of Kandeler and Gerber32.Among the Ni-specific metalloenzymes, urease enzyme activity was estimated in P ≤ 0.05. Principal component analysis (PCA) was carried using meV software version 4.060.Otherwise stated, for each experiment, five biological repetitions were designed, and the resulting data were expressed as mean values ± standard errors. In entire experiments, each biological repetition had three technical repeats. One-way analysis of variance (ANOVA) was carried out, and data were presented at a significance of Supplementary Information.


Young adults with depression and evidence of inflammation may represent a high-risk group for cardiometabolic disorders, but studies of cardiometabolic risk in this population are scarce. We aimed to examine: (1) the prevalence of low-grade inflammation in young-adults with depression; (2) cross-sectional and longitudinal associations between cardiometabolic risk factors and depression with or without evidence of inflammation.The ALSPAC birth cohort participants were assessed for depression and serum high-sensitivity C-Reactive Protein (CRP) levels at age 18, alongside cardiometabolic measures  at age 18 years, and body mass index at ages 9, 13 and 18 years. Low-grade inflammation was defined as CRP>3 mg/L. Multinomial regression was used to examine associations of cardiometabolic markers with depression cases with and without evidence of inflammation. Sensitivity analyses were conducted to examine for interactions between depression, inflammation and cardiometabolic traits.Out of 2932 participants, 215 met ICD-10 criteria for depressive episode at age 18 years; 23 (10.7 %) had CRP>3 mg/L and 57 (26.5 %) had CRP 1−3 mg/L. Depressive episode with raised CRP (>3 mg/L) was associated with higher triglycerides , higher BMI  and insulin insensitivity , and longitudinally with higher BMI at ages 9  and 13 . There was evidence for interaction between BMI and CRP for the risk of depression at age 18  and between CRP and depressive symptoms for the risk of increased BMI at age 18 .A notable proportion of young adults with depression have evidence of inflammation. These individuals are at increased risk of cardiometabolic disorders. Management of cardiometabolic risk in depressed individuals with evidence of inflammation should form part of routine clinical practice. Co-morbidity of depression with cardiovascular diseases (CVD) , type 2 Based on data from the Avon Longitudinal Study of Parents and Children (ALSPAC), a general population-representative prospective birth cohort from the UK, we have examined the prevalence of low-grade inflammation (serum hsCRP levels >3 mg/L) in participants meeting the ICD-10 criteria for current depressive episode at age 18 years. We have compared cardiometabolic risk between depression with and without evidence of inflammation using a range of established cardiometabolic risk factors at age 18 years and body mass index (BMI) in childhood at ages 9 and 13 years. We hypothesized that depression with evidence of inflammation would be associated with greater cardiometabolic risk both cross-sectionally and longitudinally.22.1http://www.bristol.ac.uk/alspac). The study website contains details of all available data through a fully searchable “data dictionary” . Parents completed regular postal questionnaires about all aspects of their child’s health and development from birth. Since age 7, the children attended an annual assessment clinic during which they participated in various face-to-face interviews and physical tests.The Avon Longitudinal Study of Parents And Children (ALSPAC) birth cohort initially recruited 14,541 pregnant women resident in county Avon, a geographically defined region in southwest of England, with expected dates of delivery 1 st April 1991 to 31 st December 1992, resulting in 14,062 live births . DetaileThe current study is based on all participants with a measure of the outcome and exposures, after removing participants with CRP > 10 , to minithe ALSPAC Law and Ethics Committee, and Local Research Ethics Committees. All participants provided informed consent.2.22.2.1In total, 3287 participants provided a blood sample. Samples were frozen at −80 °C. The measurements were assayed in 2008 after a median of 7.5 years in storage. There was no evidence of freeze-thaw cycles during storage period. CRP was measured by automated particle-enhanced immunoturbidimetric assay . All assay coefficients of variation were <5%. The minimum detection limit for CRP was 0.03 mg/L. Twenty-nine participants (0.6 % of the sample) were below this limit and were assigned values of 0.01 (n = 16) and 0.02 (n = 13); they were also included in the analysis. In the total sample, CRP values ranged from 0.01–176.10 mg/L (32 subjects had CRP levels >10 mg/L). Participants with CRP > 10 mg/L (n = 32) were excluded due to the risk of confounding by acute inflammatory state (e.g. infection). We defined evidence of low-grade inflammation as serum CRP>3 mg/L, and no inflammation as CRP<3 mg/L, as is most commonly used in the literature to define low-grade inflammation . In addi2.2.2The computerised version of the Clinical Interview Schedule Revised (CIS-R) was self-administered by cohort participants in assessment clinics at mean age 17.8 years (SD = 0.38). The CIS-R is a widely used, standardized tool for measuring common mental disorders in large community samples . The CIS2.32.3.12.3.1.12) for Insulin Resistance  and fasting insulin (FI). Fasting samples were taken at 0900 after a 10 -h fast (water only). Blood samples were immediately spun, frozen and stored at −80 °C and measurements were assayed within 3–9 months of the samples being taken with no previous freeze-thaw cycles. FPG and FI were measured by an ultrasensitive ELISA  automated microparticle enzyme immunoassay. Its sensitivity was 0.07 mU/L, and inter- and intra-assay coefficients of variation were <6%. Insulin resistance was calculated as a continuous measure from FPG and FI by using the computerized, updated version of the Homeostatic Model Assessment , low-density lipoprotein (LDL) and triglycerides (TG). Fasting samples were taken at 0900 after a 10 -h fast (water only). Plasma lipid concentrations were measured by modification of the standard Lipid Research Clinics Protocol by using enzymatic reagents for lipid determination. LDL concentration was determined via the Friedwald equation ).2.3.1.3Self-reported data on smoking, alcohol and drug use were collected using a questionnaire. The number of cigarettes/roll ups smoked per day was coded as a binary variable if participant reported smoking ≥1 cigarette per day. Alcohol use was coded as a binary variable if participants reported consuming drinks containing alcohol twice a week or more frequently.2.3.2BMI was calculated as weight (kilograms) divided by height (metre squared) and was measured at various timepoints in a relatively large sample during childhood.2.4We adjusted cross-sectional analyses for sex, ethnicity, paternal social class, physical activity , smoking , alcohol use , and BMI . For our longitudinal analysis, we adjusted for sex, ethnicity and paternal social class. We excluded adjustment variables where they were the exposure of interest, since they were already apparent in the statistical model. See Supplementary Data for further information.2.5Exposures that were non-normally distributed were natural log-transformed . Resultant variables were standardized (Z-transformed), so the statistical estimations represent the increase in risk of depression (with or without raised CRP) per SD increase in exposure. We completed tests for multi-collinearity of exposures/confounders in a linear regression model. The variance inflation factor for all covariates was between 1.01–1.11, suggesting minimal multi-collinearity. Adjustments were added using the enter method of multiple regression. All statistical analysis was done using IBM SPSS 24.0.2.5.1We calculated the proportion of currently depressed participants with “low”, “medium” and “high” inflammation at age 18 years, compared with non-depressed participants. Chi-square tests were used to examine for differences between currently depressed and non-depressed participants in CRP levels, and for associations between severity of depression and CRP levels.2.5.2We used multinomial regression to calculate odds ratios (ORs) and 95 % confidence intervals (C.I.) for the cross-sectional and longitudinal associations between cardiometabolic risk factors and depression with or without evidence of raised CRP. ‘No depression’ was the reference category. We performed likelihood ratio tests to examine whether the estimates for groups were significantly different from each other. Multinomial regression models were adjusted for potential confounders as described above. ORs represent the increase in risk of depression with/without evidence of raised CRP per standard deviation (SD) increase in exposure variable, compared to participants with no depressive episode.2.5.3We performed a sensitivity analysis to examine for any cross-sectional association or interaction between CRP (continuous data) and the cardiometabolic factors described above, on risk of current depression at age 18 years. We performed adjustments as described above. For each cardiometabolic risk factor, we included CRP in the logistic regression model alongside an interaction term (CRP*cardiometabolic risk factor). ORs represent the increase in risk of depression per standard deviation (SD) increase in exposure or interaction variable, compared to participants with no depressive episode.2.5.4We performed a sensitivity analysis to examine for any cross-sectional association of CRP (continuous data) and interaction between CRP and depressive symptom score (continuous data), for the outcomes of cardiometabolic traits (continuous data). Regression models were adjusted as described above. For each continuous cardiometabolic outcome, we included CRP and depressive symptom score in a linear regression model alongside an interaction term (CRP*depressive symptom score). Beta coefficients and 95 % C.I’s represent estimates for the SD increase in continuous cardiometabolic trait per SD increase in exposure or interaction variable.2.5.5p-values for adjusted regression models for cross-sectional analyses were corrected for multiple testing using the Holm-Bonferroni method , and increased levels of CRP with increasing severity of depression  (= 0.089) .Table 1C3.2p-value for likelihood ratio test <0.001) , triglycerides  and fasting insulin  were associated with current depression with raised CRP, after adjusting for potential confounders. The likelihood ratio test showed that these effect estimates were significantly different between the two depressed groups  and without , but the association was stronger for cases with raised CRP (Holm-corrected  <0.001) . BMI  and 13  years was associated with depression with raised CRP at age 18 years See .Table 5L3.4We found trend evidence for an interaction between CRP and BMI  and between CRP and triglycerides  on risk of current depression. See Supplementary 3.52 , fasting insulin , HDL  and triglycerides . However, there was no evidence for interaction between CRP and depressive symptoms scores in relation to these outcomes.We found trend level evidence for an interaction between CRP and depressive symptom score for the outcome of BMI . CRP was associated with other cardiometabolic outcomes after controlling for potential confounders: HOMA4Using a general population-based birth cohort study, we report that a notable proportion of young adults with current depression have evidence of raised CRP. In our sample, approximately one in ten people meeting ICD-10 criteria for current depressive episode at age 18 had CRP >3 mg/L, while about a quarter had CRP 1−3 mg/L. To our knowledge, this is one of the first studies of the prevalence of low-grade inflammation in young adults enrolled in a population-representative birth cohort. The meta-analytic prevalence of inflammation defined as CRP>3 mg/L is about 27 % , and in We report that participants with a current depressive episode and raised CRP were more likely to be female and have increased cardiometabolic risk factors at age 18 years, and increased BMI in childhood assessed at ages 9 and 13. Our findings may indicate that depression with evidence of inflammation may be more common in females. However, in our sample there were only three male participants who had depression and evidence of inflammation. Therefore, the possibility of type I error due to low statistical power because of small sample size cannot be ruled out. In future, studies with larger samples of depressed cases are required.Cardiometabolic risk factors were associated with cases of current depression with, but not without, evidence of inflammation. These findings are consistent with the idea that inflammation could be a common mechanism for comorbid depression and cardiometabolic disorders. Comorbidity between depression and cardiometabolic disorders is well known , and is Previous research which considered depression as a single entity, including our own work from the ALSPAC cohort, reported that associations between current depression and cardiometabolic risk factors could be explained by sociodemographic and lifestyle factors including sex, ethnicity, social class and physical activity . In the 2, fasting insulin, triglycerides, and HDL) after adjustments for potential confounders including depressive symptoms. HOMA2 is a reliable measure of insulin resistance ,  for this research project. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. GMK acknowledges funding support from the MQ: Transforming Mental Health , the Wellcome Trust , the Medical Research Council , and the BMA Foundation J Moulton Grant (2019). The UK Medical Research Council and Wellcome (Grant ref: 102215/2/13/2) and the University of Bristol provide core support for ALSPAC. This publication is the work of the authors and the authors will serve as guarantors for the contents of this paper. A comprehensive list of grants funding is available on the ALSPAC website: http://www.bristol.ac.uk/alspac/external/documents/grantacknowledgements.pdf/. This research was specifically funded by The Wellcome Trust (Grant no. 08426812/Z/07/Z) (Awardee: Dr Golam M Khandaker).BIP is funded by Based on existing ALSPAC cohort data, this specific study design and analysis were conceived by GMK. Analysis was conducted by BO and BIP. The manuscript was prepared by BIP. GMK and PBJ provided oversight and revised the manuscript.Data was obtained following a successful application to the ALSPAC executive committee.None.


Non-alcoholic fatty liver disease (NAFLD) is a public health problem lacking an approved pharmacological treatment. Omega-3 fatty acids have shown to reverse NAFLD. Chia is a seed rich in α-linolenic acid (ALA), antioxidants, and fiber; therefore, it could be useful to treat NAFLD.In a single arm experimental design study, the effect of 25 g/day of milled chia was assessed in 25 patients with NAFLD. After two weeks of dietary stabilization  and eight weeks of a chia-supplemented isocaloric diet, liver:spleen attenuation index and visceral abdominal fat (VAF) were measured by computed tomography. Lipids, lipoproteins, free fatty acids (FFA), and ALA plasma concentrations were also determined.P < 0.05 for all).Dietary chia supplementation induced an increase in plasma ALA concentration (75%) and dietary fiber (55%) consumption. After chia supplementation, VAF (9%), body weight (1.4%), total cholesterol (2.5%), non-high density lipoprotein cholesterol (3.2%), and circulating FFA (8%) decreased. Furthermore, NAFLD regressed in 52% of the treated patients (The results of the present study show that 25 g/day of milled chia ameliorates NAFLD. Chia is an accessible vegetal source of omega-3 fatty acids, antioxidants, and fiber, which could have the potential to prevent metabolic abnormalities in NAFLD patients. Considering that there is no pharmacological treatment approved for NAFLD, the findings of the present study suggest that a chia-supplemented diet could be an innovative alternative to control this disease.https://clinicaltrials.gov/show/NCT03942822 Parallel to the obesity epidemic, non-alcoholic fatty liver disease (NAFLD) prevalence has markedly increased during the last years . Recent Although no drug has been specifically approved for the treatment of NAFLD, recent studies indicate that dietary supplementation with marine-origin omega-3 fatty acids (eicosapentaenoic acid [EPA]/docosahexaenoic acid [DHA]) and the Mediterranean-style diet are useful to treat NAFLD , 5. EPA Salvia hispanica) is the richest vegetal source of omega 3-fatty acids, antioxidants, and fiber [Therapies that focus on the use of functional foods, rich in a variety of phytochemicals, mono/polyunsaturated fatty acids, antioxidants, minerals, and fiber, have shown antioxidant, anti-inflammatory, and lipid-lowering effects , 8, whicnd fiber . Althougnd fiber , 11, itsInstituto Nacional de Cardiología Ignacio Chávez in Mexico City, Mexico  study, performed at the ico Fig. . EligiblInstituto Nacional de Cardiología Ignacio Chavez (No. 16–980), and was retrospectively registered at clinical trials.gov (NCT03942822).The experimental protocol was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki, it was approved by the Research and Ethics Committee of the To know the current eating habits, including total energy, macronutrients and fiber intake, 24-h dietary recalls were applied on the first visit by a trained nutritionist. As previously recommended, two weekdays and one day of the weekend were considered for the recalls . To stan2). Waist circumference was measured with a non-stretch tape . The measurement was made in duplicate, with the patient without clothes around the waist, at the midway between the lowest rib and the iliac crest, measures were averaged; however, a third measurement was taken if they differed more than 0.5 cm.Anthropometric measurements were obtained according to the International Society for the Advancement of Kinanthropometry (ISAK) regulations, by a certified nutritionist. Weight and height of the patients were recorded before 8:00 a.m. in fasting conditions; participants were required to remove shoes and all outer cloths and heavy pocket items. A digital scale , and a SECA 220 wall stadiometer  were used, with an accuracy of 0.1 kg and 0.1 cm, respectively. Body mass index (BMI) was calculated as weight (kg)/height . Glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and uric acid (UA) concentrations were determined using direct standard enzymatic colorimetric methods , in a COBAS c311 analyzer. Low-density lipoprotein cholesterol (LDL-C) concentration was estimated using the DeLong formula . The repLiver enzyme values were considered high when ALT, AST, or GGT were ≥ percentile 75 . These cut-off values were obtained from 101 men and 180 women in the GEA study, without obesity and normal lipid, glucose, and blood pressure values.v/v) mixture, by vigorous vortexing for 1 min, according to the Folch’s method [2SO4 (2%). Fatty acid methyl esters were extracted three times with 2 mL of n-hexane, which was separated and dried under nitrogen. The dry residue was dissolved in 50 μL of n-hexane, and 1 μL was analyzed in a Shimadzu GC-8A gas chromatograph equipped with a flame ionization detector  and an SP2330 capillary column of 25 m length and 0.25 mm internal diameter . Fatty-acid peaks were identified by using the Supelco 37 component FAME Mix . A plasma control sample was run in each extraction assay; ALA inter-assay coefficient variation was lower than 13%.To 100 μL of plasma, 50 μL of a solution of heptadecanoic acid  (1 mg/mL) in chloroform was added as an internal standard. All solvents and chemicals were analytical grade from J. T Baker . Plasma lipids were subsequently extracted three times with a chloroform:methanol (1:2 s method . After cv/v), containing 0.002% of butylated hydroxytoluene (BHT) as antioxidant, in an ice bed overnight. At the end of this period, 1 mL of NaCl solution (0.8%) was added and vortexed for 1 min; the solvent was decanted and collected. This step was repeated twice. The organic layer was dehydrated with anhydride sodium sulfate, filtered and evaporated under a gentle stream of nitrogen. Fatty acids of the residue were transesterified to their corresponding methyl ester and analyzed by gas chromatography as described above.The chia seeds were milled and analyzed for fatty acid content according to the AOAC  procedur2). Subcutaneous abdominal fat (SAF) was calculated by subtracting the VAF from the TAF area.Computed tomography is a validated method for measuring VAF  and evalP values < 0.05 were considered statistically significant. All the analyses were performed using SPSS for Windows .Data are presented as mean ± standard deviation, median (interquartile range) or prevalence. Comparisons were made by paired Student’s t, Wilcoxon matched-pairs signed-rank or Chi-squared tests, as appropriate. The study included 25 NAFLD patients; general characteristics are shown in Table n = 16), the improvement in FFA (mmol/L) was greater . Regarding liver function tests, there were no differences in the median concentrations , with consequent BMI and waist circumference reduction Table , panel AThe present study evaluated the effects on NAFLD of an isocaloric diet supplemented with 25 g/day of milled chia. To the best of our knowledge, the present study shows for the first time that milled chia reversed NAFLD in 52% of the treated patients. This was accompanied by decreases in BMI, waist circumference, and VAF reduction, particularly in obese subjects. These results suggest that milled chia intake could be an accessible therapeutic alternative for the control and prevention of NAFLD, and its main causes of morbidity and mortality (T2DM and CAD).Intervention studies with chia in humans are scarce and vary in the way of seed administration , duration of the intervention (weeks or months), type of basal diet , and characteristics of the included patients  , 20–23; In line with previous reports , 22, a sPreviously, it had been suggested that a high omega-6:omega-3 index could be implicated in the etiopathogenesis of NAFLD , 26. EvaTherapeutic use of omega-3 fatty acids for the management of NAFLD has gained interest, due to its potential antioxidant effect, among other benefits, since oxidation is part of the pathophysiology of fatty liver disease . The benNAFLD has a multifactorial origin and a certain genetic susceptibility has been reported , 3. CompThe effect of chia on glucose and lipid levels has been less studied. Nieman et al. did not observe improvements in these parameters when analyzing normolipidemic women . HoweverThe main strength of the present study was the tomographic analysis of abdominal fat, which allowed assessing chia’s effect beyond body weight and its effect on fat depots with a greater pathophysiological transcendence. Based on previous studies, an adequate dose of the milled seed was administered, which allowed for a greater bioavailability of its nutrients and maximal increment in ALA; besides, adherence to treatment was higher than 90%, and this was verified by quantifying ALA in plasma.The most important limitation was the lack of a control group; however, previous studies agree about the difficulty of having an adequate placebo. In a placebo-controlled chia study, authors analyzed the blinded design in participants, finding that 60% of subjects on the placebo arm acknowledged being on placebo treatment . On the The results of the present study show that 25 g/day of milled chia ameliorates NAFLD. Due to its composition, chia could be an accessible vegetal source of omega-3 fatty acids, antioxidants, and fiber. These phytochemicals have the potential to prevent the appearance of metabolic abnormalities, advanced stages of NAFLD, and development of T2DM and CAD in NAFLD patients. Functional foods are suggested as part of innovative therapies for the treatment of cardiometabolic diseases. Although findings of the present study expand this information, more studies are needed to consolidate these nutriments as therapeutical alternatives.