Cerebral edema is a devastating complication of DKA which is extremely rare in adults but is the leading cause of diabetes-related death in the pediatric population. Newly diagnosed diabetes, younger age, first episode of DKA, severity of DKA at presentation, and administration of bicarbonate are predictive of cerebral edema in DKA. We present a case of a young adult with DKA as the presenting symptom of diabetes, whose clinical course was complicated by renal failure, refractory shock, and cerebral edema. This case addresses the controversy surrounding bicarbonate therapy in DKA and its possible role in the development of a rare fatal complication of DKA. Diabetic ketoacidosis (DKA) is a serious acute complication of diabetes mellitus. The combination of insulin deficiency and an increase in counterregulatory hormones results in severe metabolic derangements. Cerebral edema (CE) is a devastating complication of DKA that is well-described in the pediatric population but exceptionally rare in adults. We present a case of a morbidly obese young adult with no known history of diabetes who presented with DKA and developed multiorgan failure and cerebral edema.2), he had no known chronic medical problems. He was on no medications and denied exposure to drugs, alcohol, or hazardous chemicals. He had multiple first-degree relatives with diabetes mellitus, but it is unknown if they had type 1 or type 2 diabetes mellitus. On initial examination, he was afebrile, tachypneic, tachycardic, and hypertensive, with normal oxygen saturation on room air. He was alert and responsive (GCS 13) but noted to be dehydrated and in severe respiratory distress. His cardiopulmonary examination was unremarkable except for a regular pulse rate of 100/min. His initial random blood glucose of 694 mg/dL, bicarbonate of 5 mmol/L, arterial pH of 6.86 and calculated anion gap of 43 satisfied all the diagnostic criteria for DKA. He was oliguric with serum creatinine 1.37 mg/dL, potassium 3.5 mmol/L, and serum sodium (corrected for hyperglycemia) 138 mmol/L. His white blood cell count was 19,500/mm3. The arterial blood gas panel was consistent with primary metabolic acidosis with appropriate respiratory response, which was attributable to ketoacidosis and lactic acidosis (venous lactic acid was 4.3 mmol/L). Urinalysis revealed glucosuria, proteinuria (100 mg/dL), and ketonuria and was not suggestive of infection. Chest X-ray showed crowding of bronchovascular structures  and a streaky left retrocardiac opacity suggestive of atelectasis. He was given intravenous fluid boluses , empiric antibiotic coverage with piperacillin-tazobactam, intravenous potassium chloride (40 mEq), and intravenous calcium gluconate. Intravenous insulin infusion was started at the rate of 8 units/hour and thereafter titrated per the hospital's diabetic ketoacidosis protocol. Concurrently, another 80 mEq of KCl was given intravenously. Repeat serum potassium was 3.0 mmol/L, and the insulin infusion was paused per protocol to prevent further hypokalemia. Despite receiving an additional 30 mmol potassium phosphate intravenously and 140 mEq potassium chloride enterally, the hypokalemia worsened (serum potassium 2.7 mmol/L). Due to lack of improvement in severe acidosis, he was given 100 mEq and then another 150 mEq of sodium bicarbonate intravenously, and an infusion of 100 mEq sodium bicarbonate in 0.45% sodium chloride was begun at the rate of 250 mL/hr. He became hypothermic (bladder temperature 35.0 degrees Celsius) and hypotensive (96/27 mmHg) and was started on vasopressor support with norepinephrine. He remained severely acidotic  with worsening hypoxemia and hypercarbia. He was intubated and mechanically ventilated using assist control-volume control mode, with a tidal volume of 600 mL and a minimum set respiratory rate of 25/min. A chest X-ray revealed interval increase of interstitial markings, a new left retrocardiac opacity, and patchy perihilar and bibasilar opacities, suggesting pulmonary congestion and possible developing pneumonia. He went into anuric renal failure, and continuous venovenous hemofiltration was initiated but could not be maintained due to profound hypotension and subsequent clotting of the circuit. At this stage, the patient was on norepinephrine, vasopressin, and phenylephrine infusions to maintain his mean arterial pressure over 65 mmHg. It was then noted that he was not responsive to painful stimuli despite not being on any sedation for several hours. Pupils were unequal and nonreactive to light. An emergent noncontrast computed tomography of the head revealed diffuse cerebral edema with transtentorial and cerebellar tonsillar herniation, for which a dose of mannitol was administered. Due to his poor prognosis, the next of kin elected to withdraw life-sustaining measures, and the patient passed away. Posthumously, laboratory results came back with HbA1C of 12.9%, and antibodies to islet cell, glutamic acid decarboxylase, and insulin were negative, suggesting the patient had underlying type 2 diabetes mellitus.A young Caucasian male in the third decade of life presented to the emergency room with confusion and tachypnea. He had a 2-week history of generalized weakness and a 2-day history of nausea, vomiting, diarrhea, polyuria, and polydipsia. Apart from morbid obesity  is a serious and potentially life-threatening complication of diabetes mellitus. Although typically associated with insulin-deficient states like type 1 diabetes, it may also occur in type 2 diabetes in conditions of extreme physiologic stress or in a variant of type 2 diabetes called ketosis-prone diabetes . In the Cerebral edema (CE) is a devastating complication of DKA. It is well-described in the pediatric population—it occurs in 0.2-1% of children with DKA and is the leading cause of diabetes-related death in this age group –9. The rAlthough the precise pathogenesis of CE in DKA remains poorly understood, a widely accepted theory blames aggressive intravenous rehydration, use of hypotonic fluids, rapid correction of hyperglycemia, and fluctuating oncotic pressures, all together causing an osmotic shift of fluid into the intracellular space resulting in cell swelling , 20. On Another school of thought implicates the effects of cerebral reperfusion, neuroinflammation, and disruption of the blood-brain barrier in the development of CE in DKA , 19. Somβ-hydroxybutyrate levels [In general, severe acidemia causes impaired myocardial contractility and decreased peripheral vascular resistance . Howevere levels . This she levels . Additioe levels .Total body potassium is low upon initial presentation in DKA, due to osmotic diuresis, poor oral intake, and gastrointestinal losses. However, the serum potassium level is often normal or high, due to the extracellular shift of potassium in the setting of insulin deficiency and acidosis , 29. A sCurrent guidelines from the American Diabetes Association recommend that adults with pH lower than 6.9 should receive an isotonic solution composed of 100 mmol sodium bicarbonate in 400 mL sterile water with 20 mEq KCl, given at a rate of 200 mL per hour for two hours until the venous pH > 7.0. If the pH is still lower than 7.0 after this is infused, they recommend repeating this infusion every two hours until pH reaches 7.0 . The exi


To evaluate an innovative open necrosectomy strategy with continuous positive drainage and prophylactic diverting loop ileostomy for the management of late infected pancreatic necrosis (LIPN).n = 23), open necrosectomy with colonic segment resection (ON+CSR) group (n = 8) and open necrosectomy with prophylactic diverting loop ileostomy (ON+PDLI) group (n = 11). Continuous positive drainage (CPD) via double-lumen irrigation–suction tube (DLIST) was performed in ON+PDLI group. The primary endpoints were duration of organ failure after surgery, postoperative complication, the rate of re-surgery and mortality. The secondary endpoints were duration of hospitalization, cost, time interval between open surgery and total enteral nutrition (TEN).Consecutive patients were divided into open necrosectomy (ON) group (p = 0.11). The length of stay in the ON+PDLI group was shorter than the ON group (p = 0.001). The hospitalization cost in the ON+PDLI group was less than the ON group (p = 0.0052).The recovery time of organ function in ON+PDLI group was shorter than that in other two groups. Colonic complications occurred in 13 patients (56.5%) in the ON group and 3 patients (27.3%) in the ON+PDLI group (ON+PDLI can avoid the intestinal dysfunction, re-ileostomy, the resection of innocent colon and reduce the intraoperative trauma. Despite being of colonic complications before or during operation, CPD + PDLI may show superior effectiveness, safety, and convenience in LIPN. Severe acute pancreatitis (SAP) is a serious disease involving multiple disciplines and systems. A certain proportion of patients with SAP would develop infected pancreatic necrosis (IPN) in the later course of the disease . IPN is Therefore, in current study, we aimed to evaluate the effectiveness, safety, and convenience of CPD + PDLI in LIPN compared to the other two open necrosectomy approaches, no matter with or without colonic complications before or during operation.From January 2012 to February 2017, all the consecutive patients admitted to our center with a diagnosis of IPN were registered in an internal database and screened for potential enrollment. Patients who were performed open pancreatic necrosectomy and diagnosed with IPN during the study period were collected. The data were assembled and analyzed retrospectively. Informed consent was exempted because this retrospective study was harmless to the patients and contained no personal data. The study was approved by the Institutional Review Board of Jinling hospital.The inclusion criteria for the study were: (1) patients diagnosis with IPN according to the presence of gas bubbles within pancreatic necrosis on contrast-enhanced CT scan or a positive bacterial culture obtained by fine-needle aspiration, first drainage and/or operation ; (2) patThe exclusion criteria were: (1) pregnant patients; (2) patients received chemotherapy for malignancy or auto-immune diseases; (3) patients received abdominal surgery before IPN and was present due to abdominal compartment syndrome (ACS), perforation of a visceral organ, bleeding during the current episode of AP; (4) patients received ON for IPN before admitting to our institute during the current episode of AP; (5) IPN was caused due to trauma (6) the treatment strategy was not completed for nonmedical reasons.Initial medical treatment and minimally invasive step-up approach were carried out for every patient before and after IPN confirmation according to the international recommendations , 19.For each participant, the following variables were collected, including age, sex, etiology, and body mass index (BMI), time interval between AP onset to operation. Baseline characteristics such as acute physiology and chronic health evaluation (APACHE) II score, sequential organ failure assessment (SOFA) score, laboratory data including C-reactive protein (CRP), procalcitonin (PCT), Interleukin-6 (IL-6) and white blood cells (WBC) were also collected and assessed within 24 h before surgery, at the third and seventh day after surgery.Our primary endpoints were duration of organ failure after surgery, postoperative complication, the rate of re-surgery and mortality. Secondary endpoints were duration of hospitalization, cost, time interval between open surgery to total enteral nutrition (TEN). The main postoperative complications included colonic complication, hemorrhage, pancreatic fistula. The colonic complications included colonic suspected imminent or overt ischemia/perforation, stenosis, hemorrhage, colonic fistula, pseudo-obstruction.Organ functions were evaluated in cardiovascular, renal and respiratory systems. The criteria for cardiovascular, renal and respiratory failure were defined based on international consensus , 20, carGastrointestinal fistula was defined as the discharge of the gastric contents, bowel from drain or surgical wound. Intra-abdominal bleeding was defined as peritoneal/retroperitoneal bleeding that required surgical, radiologic or endoscopic treatment. Gastrointestinal bleeding was defined as that the blood loss from the mouth to the rectum was more than 500 ml/24 h.Percutaneous or endoscopic transgastric drainage was firstly performed. The preferred route was through the left retroperitoneum, thereby facilitating minimally invasive retroperitoneal necrosectomy at a later stage. If there was no clinical improvement after 72 h and if the position of the drain was inadequate or other fluid collections could be drained, a second drainage procedure would be performed. If this was not possible, or if there was no clinical improvement after an additional 72 h, then, video-assisted retroperitoneal débridement with postoperative lavage, was performed.Three ON strategies were used in the management of LIPN. Among them, continuous positive drainage via a large-bore double-lumen irrigation-suction tube is one of the key techniques. The significance of positive drainage system has been confirmed by several studies , 13, 14.Additionally, jejunostomy was performed for enteral feeding and the abdomen was closed. The first group patients without suspected imminent or overt ischemia/perforation before or during operation were performed ON alone. The second group patients associated with suspected imminent or overt ischemia/perforation before or during operation underwent colonic segmental resection (CSR) with proximal ostomy, as well as ON. The third group patients, in addition to ON, underwent PDLI, despite suspected imminent or overt ischemia/perforation before or during operation. According to the different surgical procedures, they were divided into three groups, ON, ON+CSR, and ON+PDLI.P < 0.05 was considered as statistically significant.SPSS software  was applied for statistical analysis. Measurement data was presented as median and interquartile range (IQR). Kruskal-Wallis was performed to compare variance among three groups and Bonferroni correction was used to compare two groups. For categorical variables, chi-squared test was performed to compare the constituent ratio among the three groups. Fisher’s exact test was performed between two groups. From January 2012 to December 2017, a total of 234 patients with IPN were admitted to our hospital, of which 65 patients were performed with open surgery. Forty-two patients were treated with ON for LIPN after failed minimally invasive approach were enrolled in the retrospective cohort study. The other 23 patients were excluded as the following reasons. Five patients underwent laparostomy for ACS, 5 patients underwent laparotomy for abdominal hemorrhage, 1 patient was trauma-related, 7 patients received exploratory laparostomy in other hospital and 5 patients with incomplete clinical data. . Meanwhile, at the 3rd day, PCT in the ON+PDLI group was also lower than the ON group (p < 0.05). At the 7th day, CRP in the ON+PDLI group was significantly lower than that in the ON group (p = 0.002). . ]. Our results showed systematic condition was improved in all groups after treatment. However, it was significant in the ON+PDLI group than in the ON+CSR at the 3th day after surgery (p < 0.05). . Fig. a APACHE 5). Fig. b.Fig. 4p = 0.11). In the ON group, 7 cases underwent re-operative ileostomy or colostomy due to colonic complication. Comparing to the ON+PDLI group, the rate of re-surgery in ON group was significantly higher (P = 0.04). As for the other major complications including pancreatic fistula, hemorrhage, and death showed no significant differences in the three groups. In addition, in the ON+CSR group, 5/8 patients were colonic necrosis/perforation, 3/8 patients were pericolitis and fat necrosis, which were confirmed by histological examination.In ON group, 13 patients (56.5%) had colonic complication, including 2 patients with colonic fistula, 4 patients with colonic stenosis, 2 patients with colonic fistula, 2 patients with stenosis, 3 patients with pseudo-obstruction, 1 patients with duodenocolonic fistula and 1 patients with biliary-colon fistula. The complication rate decreased to 27.3% (3/11 patients) in ON+PDLI groups, consisting of colonic fistula in 1 case, colonic stenosis in 1 case and colonic fistula with stenosis in 1 case. The incidence rate of colonic complication in ON+PDLI group was lower than that in the ON, but there was no significant difference (p = 0.004). . , 17 (14–20) and 9 (8–11) days in ON, ON+CSR and ON+PDLI groups respectively. A remarkable decrease of postoperative duration of circulatory failure existed in ON+PDLI group compared with ON+CSR group (4). Fig. a Meanwhiy). Fig. b There wp = 0.0011) and ON+PDLI groups (p = 0.0069).The median time from AP onset to open surgery was 25 (19–35), 44 (34–54), and 22 (15–34) days in ON, ON+CSR and ON+PDLI groups respectively. The median days in ON+CSR group were significantly longer than that of in the ON (p = 0.0052)   days] were similar to that of the ON+CSR group [95.5 (72.5–129.8) days], while the days in ON+PDLI group were significantly shorter than the ON groups Fig. a. Higher52) Fig. b. Additily) Fig. c. (Tablely) Fig. .Fig. 6CSevere acute pancreatitis (SAP) is a serious disease involving multiple disciplines and systems. IPN is a complication of SAP at its end stage, with a high mortality rate 14–69%) due to multiple organ failure, despite advances in critical care and surgical technique . IPN is 9% due toSeveral researches recommend a low threshold for colonic resection due to suspected imminent or overt ischemia/perforation by outside inspection during surgery. But 12.5–50% patients who were performed with aggressive surgical approach suffered innocent colonic resection without ischemia/perforation , 16. JusSustained colonic complications may prolong parenteral nutritional support, which is attended by potential poor prognosis . Many reIn the current study, WBC, CPR, PCT and IL-6 were chosen as indicators of the infection and inflammatory response –36. The Patients with LIPN who underwent ON could benefit from CPD + PDLI approach as follows. Continuous positive drainage via a large-bore double-lumen irrigation-suction tube is one of the key techniques to ensure adequate drainage after operation. Early enteral nutrition can be started with PLDI. According to the clinical manifestations, we can adjust the strategy of enteral nutrition timely and safely, which can implement total enteral nutrition as early as possible by keeping colon out of circuit. This is important to protect the gut barrier, reduce bacterial translocation, and even decrease the morbidity and mortality by reducing the septic complications. PDLI can manage the diversion proctocolitis by antegrade succus entericus reinfusion, which may also prevent the colonic complications such as diarrhea, obstruction, missing colon fistula and stenosis after performing closure of the stoma , 39. PDLThere were also some limitations in this study. First, it was a retrospective cohort study with a small sample size. Large samples randomized controlled study should be needed for in-depth investigation in the future. Second, intraoperative bleeding patients were excluded, and the effect of PDLI in these patients has not been evaluated. Furthermore, complications such as post-operative bleeding cannot be avoided, and there is no significant reduction in post-operative mortality. Finally, the time to pulmonary, circulatory and kidney failure were not evaluated in this study. Therefore, whether patients would have developed these outcomes after discharge was still unknown. Further study was needed to evaluate these outcomes.Our study suggests that CPD has initially solved the problem of inadequate drainage after ON. Though it may be impossible to control for all the operative and surgical variation that inherently exists in the management of LIPN, our study may provide a new strategy for treatment of LIPN.


Schizochytrium sp., AM) and micro-minerals  on the immune and antioxidant status, and the expression of hepatic genes involved in the regulation of antioxidants, inflammatory cytokines, lipid metabolism, and organ growth of largemouth bass  fed high-and low-fishmeal (FM) diets. For this purpose, two sets of six iso-nitrogenous (42% crude protein) and iso-lipidic (12% lipid) diets, such as high (35%) and low (10%) FM diets were formulated. Within each FM level, AM was used to replace 50% or 100% of fish oil (FO), or without AM (FO control) and supplemented with either OM or IM . Diets were fed to juvenile LMB  to near satiation twice daily. The results indicated that FO replacement by dietary AM did not change the levels of most biochemical , antioxidants , and immune (IgM and lysozyme) parameters in LMB, except ALP and CAT. MM affected only hepatic GSH, with lower values in fish fed the OM diets. FM influenced the levels of ALP, AMY, GLOB, IgM, and MDA (P < 0.05). A three-way interactive effect (P = 0.016) was found on IgM only, with lower levels in fish fed diet 12 . Subsequently, the relative expressions of hepatic antioxidants (Cu/Zn-SOD and GPx-4), inflammatory cytokines (TNF-α and TGF-β1), lipid metabolism (FASN and CYP7A1), and organ growth (IGF-I) related genes were affected by the dietary treatments, with interactions being present in Cu/Zn-SOD, TNF-α, TGF-β1, FASN and IGF-I. Overall, dietary AM could be used as an alternative to FO in low-FM diets without compromising the health of LMB, especially when it is supplemented with MM.A 12-week factorial experiment was conducted to investigate the interactive effects of dietary algal meal ( Micro-algae, such as Schizochytrium sp., and other thraustochytrids are recognized as prominent, sustainable, and alternative sources of oils rich in long-chain n-3 polyunsaturated fatty acid (n-3 LC-PUFA) since fisheries providing food-grade fishmeal (FM) and fish oil (FO) have already reached their limit of sustainability4. Individually or in combination, micro-minerals (MM) including iron (Fe), zinc (Zn), manganese (Mn), copper (Cu) and selenium (Se) are involved in diverse physiological functions such as facilitating cofactors of several enzymes, antioxidant defense, reduction of inflammation, protein synthesis, and so on7. Thus, it is generally recommended to supplement MM into plant-based fish feeds, due to the above essential roles of MM, and their low levels in plant feed ingredients.Algae have been recognized as an alternative source of fatty acids  for use in aquafeeds10. Liver is one of the main organs involved in accumulation of fats, such as PUFAs, which are essential for membrane function in fish11. However, a large content of these lipids implies a high risk of oxidative stress, because they are the major targets for reactive oxygen species (ROS)13. To maintain the endogenous ROS at relatively low levels and to attenuate the damage related to the high reactivity of ROS, fish are equipped with a variety of enzymatic and non-enzymatic antioxidant scavenging systems14. In liver, lipid metabolism can directly induce body inflammatory responses15, including pro- and anti-inflammatory responses . Simultaneously, organ growth is under endocrine control, specifically through the growth hormone (GH) - insulin-like growth factor (IGF) axis16.In fish nutrition studies, many parameters related to biochemical, immune, antioxidants, and inflammatory cytokine status have been used as vital indicators of fish healthMicropterus salmoides) is a freshwater carnivorous teleost native to North America, which has been cultured in the United States since the 1890s17. Recently, LMB production has increased in the United States. It has been introduced to China and is becoming one of the most commercially valuable fish there18. Due to its carnivorous feeding behavior, formulated feeds for LMB fundamentally rely on FM and FO as critical dietary components. The evaluation of alternative, sustainably produced ingredients for the optimization of cost-effective and nutritious feeds for LMB could improve the profitability of aquaculture operations and encourage the expansion of LMB production in the United States and beyond. There are few reports on the health/ immunonutrition studies for LMB29 and to the best of our knowledge, there is no report about the potential interactive effects of FM, algal meal (AM), and MM  on the growth, health, and signaling molecules involved in antioxidant and inflammatory cytokines responses or lipid metabolism and organ growth of LMB. In an earlier study we conducted, the complete replacement of FO by dietary AM supplemented with MM did not negatively affect the growth and feed utilization of LMB, even at low FM levels . Therefore, we designed this study to investigate the interactive effects of dietary FM, AM and MM (OM vs IM) including Fe, Zn, Mn, Cu and Se, on the biochemical, immune, antioxidant and inflammatory cytokines status of juvenile LMB fed high- and low-FM diets. In this study, nutrigenomics was used to investigate the relationship between the nutrients  and target genes involved in antioxidant and inflammatory responses and in lipid metabolism and organ growth.Largemouth bass , 12% lipid, and an estimated 14.64 kJ/g digestible energy (DE). The first set of diets contained 35% FM as the primary protein source (these are considered the high-FM diets) while the second set of diets contained 10% FM (these are considered the low-FM diets). Within each FM level, dietary Diet preparation was carried out as follows: solid ingredients were mixed for 30 min using a Hobart mixer  followed by the addition of FO and water until the appropriate consistency for pelleting was achieved. The resulting moist diets were passed through a pelletizer with a 3.2 mm die to form pellet strands and air-dried for 24 hours to a moisture content <10%. Diet strands were broken up and sieved using a standard testing sieve , sealed in vacuum-sealed plastic bags, and stored in a freezer (−20 °C) until used. A subsample of each diet was collected for proximate and test-mineral composition analyses , and all the experimental protocols were approved by the Alliance. Juvenile LMB were obtained from F&L Anderson Fish Farm, Lonoke, AR, USA, and transported to the aquatic animal nutrition laboratory (AANL), School of Aquaculture and Aquatic Sciences, KSU, Frankfort, KY, USA; and were acclimatized to the experimental conditions for three weeks. During the acclimatization period, fish were hand-fed to apparent satiation with a 45% CP and 15% crude fat commercial feed .The use of experimental fish was under scientific research protocols of Kentucky State University (KSU) and complied with all relevant local and/or international animal welfare laws, guidelines and policiesThe experiment was conducted in a closed recirculating freshwater aquaculture system (RAS) at AANL. At the commencement of the feeding trial, fish were hand-graded and stocked in each of thirty-six 110-L glass tanks at a stocking density of 15 fish /tank. Three tanks were randomly assigned to each experimental diet. Fish in each tank were hand-fed with their respective diet to apparent satiation twice daily (at 08:00 and 16:00 hours) for 12 weeks. All water quality parameters were maintained within the acceptable ranges for LMB, including temperature (25.27 ± 0.5 °C), pH (7.56 ± 0.03), dissolved oxygen (6.19 ± 0.04 mg/l), total ammonia nitrogen (0.44 ± 0.02 mg/l), nitrite nitrogen (0.28 ± 0.01 mg/l) and salinity (1.61 ± 0.03ppt). A 12-h photoperiod was provided by artificial lighting controlled by a timer.g at 4 °C for 10 min. The supernatant was removed and stored at −80 °C for plasma biochemistry and immune parameters assays. Liver samples of the anaesthetized fish were quickly removed and stored at −80 °C, such as 9 samples per treatment for hepatic peroxide content, and antioxidants enzymes activity and 6 samples per treatment for gene expression assays.At the end of the 12-week feeding trial, sampling was conducted after a 24-h fasting. Three representative fish from each tank (9 fish per treatment) were anaesthetized with 100 mg/l of tricaine methanesulfonate (MS-222). Blood samples were obtained from the caudal vein using heparinized syringes and transferred to 2.0 ml tubes. Blood samples were centrifuged at 3,000 × 10. Plasma immune parameters, such as lysozyme31 and immunoglobulin M (IgM)32, were measured using commercial kits following manufacturer’s protocols . Hepatic peroxide (MDA) and antioxidants, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH), were analyzed by spectrophotometric measurements using commercial kits , according to Kumar et al.4 and Habte-Tsion et al.9.Plasma biochemical parameters were analyzed using a comprehensive diagnostic profile kit from VetScan Analyzer . The following parameters were analyzed: alkaline phosphatase (ALP), amylase (AMY), globulin (GLOB), albumin (ALB), and total protein (TP)33. Briefly, total RNA was isolated from LMB liver samples using a TRIzol® reagent . To avoid genomic contamination, the extracted RNA was treated with RNase-Free DNase . The quality and quantity of the isolated RNA were assessed using a NanoDrop™ OneC spectrophotometer . Complementary DNA (cDNA) was synthesized using the PrimeScriptTM RT reagent kit, following the manufacturer’s instructions . Specific primers for most target genes  were designed using online resources according to the partial cDNA sequences of the target genes for Micropterus salmoides transcriptome analysis, using the published sequences of LMB or adopted from published articles  was reacted with 10.0 µL SYBR Premix Ex Taq II (2X), 0.8 µL forward primer (10 µM), 0.8 µL reverse primer (10 µM), 0.4 µL ROXTM reference dye or dye II (50X), and 6.0 µL RNase-free distilled water in a final reaction volume of 20 µL. The real-time PCR was carried out in a StepOnePlusTM Real-Time PCR System . The thermocycling conditions for the target genes were initiated with the denaturation step at 95 °C for 30 s followed by forty cycles of 95 °C for 5 s, 60 °C for 34 s, and 95 °C for the 30 s, 95 °C for 3 s, and 60 °C for 30 s. A melting curve analysis was performed (over a range of 50–95 °C) to verify that a single PCR product was produced. Threshold cycle number (CT) for each sample was determined using Applied Biosystems software and was related to the concentration of the target genes. The housekeeping gene for M. salmoides23 (β-actin) was used to normalize the expression levels of the target genes. After verifying that the primers were amplified with 100% efficiency, the expression results were analyzed using the 2−ΔΔCt method34.Real-time qPCR was used to determine mRNA levels for the target genes and performed according to standard protocols of the manufacturersAll data were validated for normality and homogeneity of variances by Shapiro-Wilk and Levene’s tests, respectively. A three-way analysis of variance (ANOVA) was performed to detect the interactive effects of dietary FM (high and low), AM , and MM source (OM and IM). Statistical significance was considered as P < 0.05. If significant interactions were identified, the Tukey’s honest significant difference (HSD) test was used for mean separation. In the absence of significant interactions (additive model), the means of any significant factor with more than two levels were separated using Tukey HSD test. All statistical analyses were performed using Statistical Analysis System software . Results are expressed as mean ± standard error (SE).Plasma biochemical  and immune (IgM and lysozyme) parameters are presented in Table A three-way interaction (P = 0.016) of FM, AM and MM was detected for IgM, with its higher concentration in the plasma of LMB fed diet 6  compared to those fed diet 12 , while no significant differences were found among other treatments. Plasma lysozyme was not significantly affected by dietary treatments.Hepatic peroxide and antioxidant parameters including MDA, SOD, GPx and GSH, were not significantly affected by replacement of dietary FO with AM and no interactions were found Table . SignifiThe relative expressions of antioxidants genes in LMB fed the experimental diets are shown in Figs. Figures The relative expressions of genes related to hepatic lipid metabolism are shown in Figs. Figure 35. In this study, we did not find any significant three-way interactive effects of FM, AM, and MM on any analyzed plasma biochemistry parameters in LMB. Simultaneously, the replacement of dietary FO by AM did not influence most of the plasma biochemistry in LMB, except ALP. Similarly, no significant effects of dietary Schizochytrium sp., supplemented with either inorganic or organic MM  were observed on blood plasma chemistry of Atlantic salmon7. Our recently published study revealed that dietary Schizochytrium meal could replace dietary FO up to 75% and play an imperative role as a source of essential fatty acids in shrimp (Litopenaeus vannamei) diets without compromising growth and health4. However, in the present study, there were significant differences in blood plasma ALP levels between treatments with 50% and 100% dietary FO replacements by AM, with ALP levels higher in fish fed AM50, but these were not significantly different compared to the AM0 treatments. Nonetheless, the highest level of plasma ALP in our study (87.0 U/L) is below the normal ranges that reported in Atlantic salmon (647–988 U/L)36. In humans, the level of ALP in a healthy adult range from 20–140 U/L; however, children tend to have significantly higher levels of ALP than adults because their bones are still growing37. The presence of ALP activity in plasma is directly related to the release of ALP enzymes from cells to the extracellular fluids and elevated ALP activity may occur when there is cell growth, tissue necrosis, or leakage of preformed ALP39. Therefore, in this study, the higher levels of plasma ALP in LMB fed AM50 could be related to the cell/ bone growth of juvenile LMB. Nevertheless, further study is needed to understand the specific mechanisms of the effects of dietary AM on plasma ALP in fish.In aquatic animals, blood parameters are considered as important indicators for physiological conditions and health status in response to dietary treatments40. Lysozyme is a ubiquitous bacteriolytic enzyme that is part of the nonspecific defense mechanisms in most animals41. In this study, FO replacement by dietary AM did not affect the levels of the immune parameters such as IgM and lysozyme, in the plasma of LMB. However, LMB fed diet 6  displayed significantly higher plasma IgM in contrast to those fed diet 12 , but not among other treatments. We also found interactive effects of dietary FM, AM and MM on the IgM content in LMB. Our findings suggest that dietary AM could maintain immune defense system/ health of LMB, even when replacing 100% of dietary FO, especially when supplemented with MM. This is in agreement with a study on Atlantic salmon that reported whole Schizochytrium sp.-based microalgae can provide a good alternative to the depleting marine resources of n-3 LC-PUFA without detrimental health effects42, even more pronounced for salmon fed OM7. Nevertheless, this is the first study to investigate the interactive effects of three factors  in fish, therefore further study is needed.In teleost fish, producing immunoglobulins is a specific immune response after being stimulated by antigen and the IgM class is the predominant immunoglobulin in most fish species11, which implies a high risk of oxidative stress since these lipids are major targets for ROS13. The antioxidant defense system helps fish to maintain endogenous ROS at relatively low levels and to attenuate the oxidative damage induced by the high reactivity of ROS14. An increase in free radicals causes overproduction of MDA, which is one of the final products of lipid peroxidation in the cells. Thus, the MDA level is commonly known as a marker of oxidative stress43. Nogueira et al.44 reported that a decrease in MDA content is associated with an increase in antioxidant enzymes in the defense system, which indicates MDA is a toxic substance that is caused by free radicals. The key antioxidants in the antioxidant defense system include SOD, GPx, CAT, and GSH13. In fish, antioxidant enzymes activities correlate with nutritional factors45. In the present study, we did not find significant differences between the control (FO control) and AM treatments in the hepatic MDA, SOD, GPx and GSH levels of LMB, except higher CAT activity in LMB fed AM-diets. The significantly higher MDA content observed in the liver of LMB fed high-FM compared to low-FM diets might be related to the MDA content of dietary FM, which was not quantified in this study. Whereas IM supplementation supported significantly higher GSH in comparison to OM (1.37 ± 0.01 vs 1.35 ± 0.00), such narrow differences suggest limited practical significance.Fish hepatic tissue has large quantities of PUFAs46. In this study, there were no significant differences in the level of hepatic Cu/Zn-SOD mRNA among treatments, except the down-regulation in the high-FM treatment (with AM0 and OM). A three-way interactive effect (P = 0.030) of dietary FM, AM, and MM was found on Cu/Zn-SOD mRNA level. The gene expression of hepatic GPx-4 was significantly affected by dietary AM only, with its lowest level in LMB fed AM100 diets. The antioxidants mRNA levels showed similar trends to the activities of antioxidant enzymes in this study. The regulation of hepatic antioxidant genes expression in LMB fed various dietary treatments in this study could be related to the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway that play a vital role in the regulation of antioxidant enzymes gene transcriptions in fish9. Furthermore, though we did not find significant effect of MM source as a single factor (organic and inorganic) on the antioxidant defense of LMB, the interactive effects of FM, AM and MM on the Cu/Zn-SOD mRNA level in this study suggest that the MM  could be involved in the antioxidant defense of LMB fed low-FM diets. Other studies reported that dietary MM supplemented in OM forms were more effective than IM forms in the antioxidant defense of other fish species47. The possible reason for the different findings of these studies could be due to differences in experimental design, fish species, minerals source and other factors.The activities of antioxidant enzymes in fish are closely related to their corresponding gene expression levels48. In this study, interactive effects of dietary FM and AM were found on the relative gene expression of hepatic TNF-α and TGF-β1. Concurrently, an inverse trend was observed between the relative gene expressions of TNF-α and TGF-β1, with dietary AM  down-regulating TNF-α mRNA and up-regulating TGF-β1 in fish fed low-FM diets; although not significant for the former. The regulation of these genes by dietary FM and AM could be mediated by the signaling molecules of nuclear transcription factor-κB (NF-κB)49 and target of rapamycin (TOR)8, although this needs further elucidation.TNF-α is pro-inflammatory cytokines, whereas TGF-β1 is anti-inflammatoryde novo synthesis of fatty acids50. CYP7A1 is involved in cholesterol metabolism or elimination51 through catalyzing the first and rate-limiting step in the classic pathway of bile acid synthesis52. In this study, AM up-regulated the mRNA levels of hepatic FASN in LMB fed low-FM diets. Interactive effects of dietary FM and AM were also detected on the relative gene expression of hepatic FASN, while the expression of CYP7A1 mRNA was regulated by dietary AM only, with its augmented levels in LMB fed AM100 diets. A previous study reported that the mechanism of bile acids regulating the CYP7A1 expression is mainly through the farnesoid X receptor signaling (FXR)/ small heterodimer partner (SHP) pathway (FXR/SHP pathway) in the liver53. Our study proved that dietary AM could play an important role in lipid metabolism and cholesterol elimination in the liver of LMB through de novo synthesis of fatty acids and bile acids. Nevertheless, to our knowledge, this is the first report regarding the effect of dietary AM on FASN and CYP7A1 mRNA levels in the hepatic cells of fish, and the specific mechanisms behind the regulation of de novo synthesis of fatty acids and bile acids (FASN and CYP7A1 augmentation), cholesterol elimination, and promotion of dietary lipids metabolism in LMB needs further investigation.FASN is a key enzyme for the 55. In this study, the relative expression of hepatic IGF-I was regulated by the dietary treatments. Specifically, IGF-I was significantly augmented in LMB fed the high-FM diet (with AM0 and OM) but was not different from those fed the low FM and AM50 diets. Interestingly, interactive effects of FM, AM, and MM were found on the relative expression of IGF-I mRNA. Thus, the relative expression of this signaling molecule could supply some important insights in understanding the underlying mechanisms for liver growth and, ultimately, the growth of LMB fed low FM diets supplemented with AM and MM.Furthermore, in previous studies, the relative expression levels of the GH-IGF system were effectively used in the evaluation of organ growth and, ultimately, fish growth in response to different dietary treatments and alternative feed ingredientsThe results of this study indicated that dietary AM could completely replace FO (even in low-FM diets) and improve/ maintain the immune, antioxidant, anti-inflammatory, and lipid metabolism capacity of LMB. Similarly, the MM  could be involved in the antioxidant defense of LMB fed low-FM diets. Indeed, dietary AM can be used as a good alternative to FO without detrimental impacts on the health of LMB, especially when it is supplemented with MM. Moreover, the relative expressions of hepatic Cu/Zn-SOD, GPx-4, TNF-α, TGF-β1, FASN, CYP7A1, and IGF-I mRNA were regulated by the dietary replacements, with interactive effects on the mRNA levels of Cu/Zn-SOD, TNF-α, TGF-β1, FASN, and IGF-I. These signaling molecules could aid in the understanding of the mechanisms behind antioxidant, inflammatory, and lipid metabolism status and organ growth of LMB in responses to dietary FM, AM, and MM. Furthermore, despite no significant effects of MM source as a single factor were identified for any of the response parameters reported herein, we believe that the observed interactive effects among FM, AM, and MM found contribute relevant information to the literature. Overall, our findings provide new insights for future studies on fish nutrition, particularly those geared towards the optimization of nutritionally balanced, cost-effective and environmentally friendly commercial feeds for LMB and other farmed species.


Dimethoate (DM) is an organophosphorus (OP) pesticide with wide use in the pest control. Its persistence in crops and soils could possibly cause adverse health consequences in humans as well as other non-target species. Since molecular studies confirming potential genotoxicity of DM have not been previously reported, the acute in vivo toxicological impact was evaluated in Wistar rats. Significant micronuclei induction and metaphase chromosome abnormalities in bone marrow cells exposed to three different DM doses  at multiple treatment durations  indicated positive dose response relationship, confirming its genotoxic and cytotoxic potential. Significant mitotic index decrease was seen in dosed animals compared to vehicle control. The study used peripheral blood comet assay, indicating DM-mediated damage to DNA at all exposure levels in a time responsive manner. These assays were found to be an effective, precise, and fast technique with applied value in biomonitoring studies. Cell cycle and apoptosis along with mitochondrial membrane potential (MMP) in flow cytometric analyses confirmed DM exposure decreased MMP, affected the cell cycle, and inflicted DNA damage, which led to cellular apoptosis of leukocytes culminating into immunotoxic effects. The in silico experiments consequently augmented that DM showed acceptable binding energy value for Cyclin A2, suggesting that it could inhibit the cell cycle progression by inhibiting cyclin A2. Also, the environmental changes brought about by humans are a potential threat to life. Due to intense usage of pesticides presently, it is crucial to evaluate the genotoxicity induced by chemicals including the organophosphates. This is an alarm and hence, is emphasized by various agencies and investigators ,2,3. Dim in rats . In rece in rats . Even so in rats . Various in rats  and leuk in rats . Its gen in rats , the neg in rats  were acc in rats . However in rats ,16. Impo in rats . They alIt is enlightening to consider reports on various other organophosphates analyzed for genotoxicity by in vivo studies ,20. ChroGenotoxicity is related to the potential for compound to modify genetic components at sub-toxic concentrations, directing to its diffusion during cell division . A singlAn effort is made to compare the DNA damage owing to acute toxicity and unravel a mechanistic basis of cellular apoptosis. To the best of our knowledge, toxicity reports on DM necessitate mutagenicity assessment in mammalian test systems, especially when acute toxicity reports are controversial and molecular events leading to such cytogenetic changes are lacking. The current study is thus aimed at elucidating a dose response relationship by assessing (i) the cytotoxicity and genotoxicity of DM in rat bone marrow cells via micronucleus test (MNT), chromosomal aberration (CA) assay, and mitotic index (MI) profile; (ii) DNA strand break by comet assay; (iii) cell cycle alteration to assess apoptotic induction; and (iv) cellular stress due to impaired mitochondrial membrane potential, MMP (ΔΨm) in white blood cells (WBCs) of treated rats, and (v) Molecular docking study of DM with cell cycle regulatory protein.2-EDTA, Trizma base, Ethidium bromide (EtBr), propiodium iodide (PI), RNAse, Rhodamine 123 (Rh123), electrophoresis reagents, sodium bicarbonate, phosphate buffered saline (PBS), and tris buffered saline were products of Sigma Aldrich . FACS Lysing Solution was a product of Becton Dickinson . Bromophenol blue, Fetal bovine serum (FBS), and Colchicine were obtained from Hi-Media Pvt. Ltd. . Cyclophosphamide—CPA (Cyclocel 500) was obtained from Celon Laboratories . May—Gruenwald and Giemsa stain were procured from Merck (India).The test compound DM (99% purity), low melting point agarose (LMPA), normal melting agarose (NMA), NaRattus norvegicus  were acquired from Indian Institute of Integrative Medicine (IIIM-CSIR) , quarantined, acclimatized and sacrificed as per standard practice conforming to the University Ethical Regulations. After acclimatization, rats were divided into five groups by stratified randomization with five animals per group. Three groups were exposed to DM while two additional groups served as normal and positive controls. This grouping was maintained for all three sub-lethal doses for acute time periods of 24, 48 and 72 h.The experimental animals—50 obtained for DM was 200 mg/kg-bw, used in the current study [50 = 20 mg/kg-bw) to medium (20% of LD50 = 40 mg/kg-bw) and high dose (30% of LD50 = 60 mg/kg-bw). Cyclophosphamide  and water (vehicle control) were undertaken for comparative study. Distilled water was used to prepare dosing solutions of test agent and injected via intraperitoneal (i.p.) injection [As per the method of Musch, the LDnt study . The thrnjection . At 24, The cytogenetic assays were conducted in compliance with OECD guideline .MN assay followed the protocol of Schmid . ImmediaMethod of Preston et al. was used for preparing the metaphase chromosomes . An aqueDetection of DNA strand break was performed according to protocol of Singh et al. with some modifications . BrieflyAlkaline conditions (pH > 13) were used to perform the electrophoresis. After lysis, slides were subjected in electrophoretic buffer at 4 °C for 40 min to achieve the DNA denaturation that allow the DNA unwinding and the expression of alkali labile site. Electrophoresis was then carried out at 0.74 V/cm for 40 min  at 4 °C by raising or lowering buffer. Thereafter, neutralization buffer  were employed to wash the slides. Slides were stained by 80 μL of 1× Ethidium Bromide and excess stain was removed by dipping in chilled distilled water. Slides were examined at 40× magnification under fluorescence microscope coupled with a charge coupled device camera (Olympus). WBCs were isolated by following the protocol of Lone et al. . Blood wx-axis, PI fluorescence) versus cell counts (y-axis) is demonstrated in the FL2 histograms.Post completion of exposures, PBS was used to wash the harvested cells and fixed in cold ethanol (70%) for 48 h. Cells were digested by RNase (400 μg/mL) at 37 °C for 45 min. After incubating the cells with Propidium iodide (10 μg/mL), examined instantly on flow cytometer FACSAriaII . A total of 10,000 events were obtained for cell cycle analysis . DNA conProtocol of Majeed and colleagues was employed for flow cytometric measurements of change in Ψm . WBCs wehttp://pubchem.ncbi.nlm.nih.gov) in SDF format (2D-strucuture data) and converted into pdb file, and further were subjected to energy minimization and ligand preparation using Discovery studio suit. 3D structure of cyclin A-2 was acquired from Protein Data Bank (https://www.rcsb.org/) (PDB ID: 4II5). Molecular docking was performed by Autodock 4.2 software using default parameters.The structure of compounds was fetched from PubChem database (CID3082 and CID2907)  followed by post hoc Tukey multiple comparisons using Statistical Package for Social Sciences (SPSS) Version 20.0. Results were considered significantly different if the P-value was 0.05, unless otherwise stated.Compound related mortality did not occur during the study as the doses selected were below the median lethal dose. Animals in experimental groups were comparable to control without any lethality. A reduction in mean body weights of exposed individuals was found in high dose group at all time periods of study; however, these decreases were statistically insignificant. Although the data is not shown, it was generally observed that the mean body weights of control and DM exposed animals were reasonably similar during the study period.p < 0.01; p < 0.05) were observed at all DM concentrations, where increases up to 5.6 ± 0.7above the normal control value of 0.27 ± 0.04 were noted. Dose dependent increases in MNPCE frequency were seen while time dependent decreases within a particular group were evident. Highly significant (p < 0.001) enhancement in MNPCEs at all exposure durations in the CPA control, reflected the expected outcome of a positive mutagen.Representative PCE (blue–purple), NCE (pink) and MNPCE are shown in p < 0.05) decrease up to 0.711 were noted at two higher concentrations. The decrease in the cytotoxicity indicator reflected a dose and time responsive relationship. The animals exposed to the positive control agent, CPA, resulted in a significant reduction (p < 0.01) in the P/N ratio at all time interval.p < 0.01) was observed at the highest concentration 24 h post treatment while damaging effects on chromosomes were less intense at the other two durations of each concentration. However, as the time elapsed, a decrease in the total aberration mean was quite obvious in each treatment group. This reflects anticipated elimination of altered cells caused by DM toxicity. Exposure to a positive mutagen led to highly significant enhancement (p < 0.001) in CA frequency, providing experimental proof-of-concept. The study additionally investigated the mitotic index (MI) to describe cell proliferation. A significant dose dependent decrease (p < 0.05) in the MI  compared to normal control  dose-dependent increase in terms of mean tail length, measured 24 h posttest agent exposure (Comet parameters scored as the extent of DNA damage measured by (i) Tail Length and (ii) Olive Tail Moment / OTM for each dose and duration group are presented in exposure . The taiexposure a. Expectexposure  in OTM a1 exhibit the cells percentage that have undergone apoptosis. The effect of multiple DM doses and durations on cell cycle phase distribution is summarized in p < 0.001) in the apoptotic phase from 79.2 ± 2.32 at the lowest, to 83.53 ± 5.37 at the highest DM dose was noted. Within a group, a time dependent decline in the apoptotic cell count was quite conspicuous for all treatments. Hence, DM-induced apoptosis was significant in leukocytes due to dosing, with highest sub-G1 counts at 24 h.The alteration in cell cycle progression obtained from a single animal as function of DM treatment has been shown in i) values were projected to be 259.67 and 54.6 µM, respectively. “Van der Waals”, “Hydrogen bond” and “Desolvation” energy components for DM, and CPA interaction with cyclin A2-CAS were −5.15 and −6.56 kcal/mol, respectively. Additionally, “Electrostatic Energy” components for “DM-Cyclin A2”, and “CPA-Cyclin A2” interactions were found to be −0.16, and −0.13 kcal/mol, respectively. Ligplot analysis reveals that there is a strong hydrophobic interaction and hydrogen bonding in “DM-Cyclin A2”, and “CPA-Cyclin A2” interactions  of cyclin A2 was observed to interact with DM through 12 amino acid residues, namely His233, Asn237, Asp240, Pro309, Thr310, Val311, Leu341, Tyr347, Leu348, Tyr350, Pro352 and Ile355 ; with CPractions b.A characteristic flow cytometric image obtained from a single experiment depicting Rh 123 fluorescence due to DM intoxication is shown in At 48 h exposure period , DM showRattus norvegicus. Until now, limited acute genotoxicity studies on DM are available; and most of them are inconclusive or unacceptable due to questionable validity of the results or due to a design or reporting deficiency. Only limited numbers of reports are acceptable [The conflicting Dimethoate toxicity reports and mutagenicity assays, with serious lack of research on genotoxic effects of Dimethoate in mammalian models, prompted this investigation on ceptable ,36. IARCceptable . Also, aceptable . The durceptable ,40. ThusThe rodent erythrocyte micronucleus and chromosomal aberration assay are markers of early biological effects of carcinogens. Hence, International regulatory agencies recognize MN and CA tests as most successful and reliable assays for genotoxic carcinogens ,42. The “S” to “M” phase of cell cycle further corroborated by our cell cycle experiments. These findings confirmed that the test doses induced cytotoxic effect, with recovery and repairing ability in mitosis at later exposure times. Also, DM demonstrated direct interaction with DNA leading to damage of genetic material evident by various structural chromosomal aberrations such as break, deletions, gap, rings and acentric fragments. These aberrations caused by improperly repaired DNA breaks confirms the clastogenic potential of Dimethoate. It is also crucial to note that when cells with few chromosomal abnormalities persists for longer, their further division could be associated with mutations that could be passed on to the next generation through clonal expansion, hence capable of manifesting the damage in the genome, thus the study holds promising results from heritable genetics view point too. Information regarding DNA damages obtained by cytogenetic methods were quite promising. Hence, DNA damage at the individual cell level was measured using comet assay—a potentially useful in vivo genotoxicity test on account of its rapidity, simplicity to perform, higher sensitivity and specificity [Nevertheless, May-Gruenwald/Giemsa staining in MN assay could not distinguish between micronuclei-containing fragments or whole chromosomes, hence chromosome aberration assay was performed further. The rate of bone-marrow proliferation (expressed in terms of MI) was significantly inhibited in DM-treated groups relative to control. The decrease in MI  indicates a slower cells progression from cificity ,44. Our cificity . Jagetiacificity . The enhcificity . The inccificity , which mcificity . The sticificity .2/M cell cycle arrest at either duration or doses of DM, suggesting the DNA repair process was insufficient to repair the DNA damage incurred. Consequently, transition of cells from G2/M phase to the sub-G1 or apoptotic phase was evident in a dose response manner. The appearance of a Sub-G1 peak, characteristic of apoptosis, supports this shift. Our next aim was to unravel the probable target of Dimethoate regulating apoptosis in the present case. Therefore, further docking experiment were performed, in which free energy of binding determine the strength of interaction between a ligand and target protein [Apoptosis, a distinctive pathological process, may lead to disease if it is mis-regulated . Therefo protein ,53. The  protein ,55. Acco protein . TherefoAlthough flow cytometry-based assays are associated with limitations regarding detection of lysed or damaged cells , the majThe MMP decrease due to DM treatment reflects DM as an uncoupler of oxidative phosphorylation leading to decreased mitochondrial membrane potential in a concentration dependent manner. It could thus be believed that at initial duration, i.e., 24 h all DM doses is more toxic to mitochondrial oxidative phosphorylation. Loss of MMP is indicative of mitochondrial membrane leakage and permeability, and release of cell death mediators triggering the final apoptotic damage. This is one of the many crucial pathways for apoptosis of the cells of immune system as confirmed by various researchers ,62. ThusWe also interpret the overall findings of this study focused on DM-mediated DNA damage through the biochemical approach. The incurred DNA damage could be due to the interaction of DM or its metabolite with cellular DNA, due to reactive oxygen species (ROS) production leading to strand breaks. We already established role of ROS in our earlier research on DM acute effects leading to oxidative stress . A compaThe most important application of the findings of this study is for safeguarding the human population. Most pesticides have confirmed carcinogenic effects in humans via a variety of mechanisms including genotoxicity, chromosomal aberrations, immunotoxicity, oxidative stress etc. . OrganopCytogenetic endpoints indicate that DM administration in mammalian model has cytotoxic and genotoxic effect confirmed by micronuclei formation, production of structural chromosome aberrations, bone marrow suppression and increased DNA damage indices in the comet assay. Molecular analysis confirmed the potential of Dimethoate to induce cytotoxicity as well as DNA toxicity, highlighted by appearance of the SubG1 apoptotic peak in the cell cycle and ΔΨm dysfunction in treated rats. In silico analysis augments cell cycle alteration by this mutagen could possibly be by the inhibition of cyclin A2. All together these data substantiate the sensitivity of the experimental protocol followed in the detection of DNA damaging effects. We conclude that DM exposure lead to cytogenetic damage, and its genotoxic and immunotoxic potential is confirmed from the present endeavor.


APP indicated fibrosis stage independent from hepatitis activity grade. A cut-off value = 4.349 diagnosed cirrhosis with area under ROC more than 0.8. Multivariate analysis revealed that smaller APP independently contributed to HCC prevalence and overall mortality. The results were validated in another 707 patients with HCV infection. In conclusion, APP was not confounded by age or hepatitis activity grade contrary to Fibrosis-4 index. APP is as simple as physicians can calculate it by pen calculation. The product serves physicians in managing patients with chronic liver disease.Fibrosis-4 index, a conventional biomarker for liver fibrosis stage, is confounded by age and hepatitis activity grade. The current retrospective multicenter study aimed to formulate the novel indices of liver fibrosis by mathematically combining items of peripheral blood examination and to evaluate ability of prognosis prediction. After a novel index was established in a training cohort, the index was tested in a validation cohort. Briefly, a total of 426 patients were enrolled in a training cohort. Albumin and platelet most strongly correlated to fibrosis stage among blood examination. Albumin platelet product (APP) = Albumin × platelet/1000 could differentiate the four stages of liver fibrosis ( One half of the liver disease-related deaths attributes to cirrhosis, the most progressed status of liver fibrosis. Evaluation of liver fibrosis stage enables a physician to predict and prevent patients from several complications of cirrhosis including esophagogastric varix, ascites, and hepatic encephalopathy expected in the future2. Hepatocellular carcinoma (HCC), the sixth leading malignancy and the third common cause of cancer death3, also typically complicates patients with cirrhosis4.Liver disease brings the world approximately 2 million of annual deathsLiver biopsy examination used to be the gold standard for staging liver fibrosis.5. The most inexpensive and simple modality should be several indices combining some items from complete blood count and liver function test, as represented by Fibrosis-4 index (FIB-4)7.However, noninvasive strategies to estimate fibrosis stage have already been replacing it9. Furthermore, the index is difficult to calculate without ready-made online calculators. In the current retrospective multicenter study, we formulated a novel index of the liver fibrosis stage and prognosis by mathematically combining two items of peripheral blood examination in a training cohort, and validated their clinical significance in a validation cohort.However, FIB-4 index has been reported to be confounded by age and hepatitis activity gradeAccording to diagram in Supplementary Fig. To determine items of an equation, linear trend through fibrosis staging was evaluated for age, platelet, total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil), and gamma-glutamyl transpeptidase (γGTP). Among them, age and T-Bil linearly increased, and platelet and albumin decreased Table . R squarDifferences in median values between two fibrosis stages were analyzed for four indices using Steel–Dwass test Fig. . The resReceiver operating characteristic (ROC) analysis was performed to assess the ability to distinguish advanced fibrosis (F3–4) from nonadvanced fibrosis F0–2) and cirrhosis (F4) from noncirrhotic stages (F0–3). As shown in Fig.  and cirrThe AUROC of the indices to detect cirrhosis differentially from noncirrhotic stages resulted greater than 0.8 in APP (a), Bilirubin platelet quotient (c) and Three math (d) as shown in Fig. The greatest AUROC was presented by APP among four indices. The second was Three math. Based on the analyses above, APP and Three math were extracted as candidates of liver fibrosis staging.ROC analysis revealed, as shown in Supplementary Fig. 9. Influence of hepatitis activity on Albumin platelet product was evaluated based on HCV-specific patients in the training cohort. As shown in Supplementary Table Indices of liver fibrosis have been reported to fluctuate according to hepatitis activity gradingIn 93 patients with stage 2, APP in grade 0–1 patients did not differ from that in grade 2 patients .To evaluate the diagnostic ability of liver fibrosis staging, the APP was calculated for each fibrosis stage in the validation cohort. As shown in Supplementary Fig. ROC analysis revealed that the AUROCs of the APP for distinguishing advanced fibrosis from nonadvanced fibrosis (a) and cirrhosis from noncirrhotic status (b) were greater than 0.8 (c), as shown in Fig. The clinical impact of the APP on HCC-free survival and overall survival was confirmed using Kaplan–Meier analysis in the validation cohort through 15 years observation  APP is able to diagnose fibrosis stage, especially, advanced liver fibrosis and cirrhosis without confounding by age or hepatitis activity grade. (2) Smaller APP independently correlates with greater HCC prevalence and mortality.12. Thus, a cut-off value of FIB-4 is proposed for each age group8. However, APP is not confounded by age because the index is not based on age.Including age in its equation, FIB-4 is reported to overestimate fibrosis stage in senior patientsfloribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP) fluctuated in an identical fibrosis stage according to activity grade9. APP might be more suitable for fibrosis staging than other indices.Fibrosis staging by APP equals in accuracy to FIB-4. Diagnostic ability of APP was lined by stability of it against hepatitis activity grading. We previously reported that FIB-4, APRI, enhanced liver fibrosis score (ELF score) and Wisteria FIB-4 consists of four items, age, AST, platelet, and square root of ALT. Calculating FIB-4 using an electronic calculator is not easy in general practice because the square root of ALT locates as one of Denominators in the equation. Therefore, customized calculators for FIB-4 have been open in the internet. The equation of APRI is simpler compared to FIB-4. However, diagnostic abilities of APRI for liver fibrosis were not competitive to that of FIB-4 in the current cohort.In summary, the data probed that APP was as reliable as FIB-4 in liver fibrosis staging. In addition, the equation of APP is as simple as APRI because APP is calculated by two items alone. Physicians in office can calculate APP by an electronic calculator or even by pen calculation.The limitations of the current study might lie in the fact that (1) because the current observation focused on patients with HCV infection patients, eligibility of APP in patients with other etiologies should be evaluated in further studies. (2) Although the influence of antiviral therapy on patients with HCV was evaluated in terms of HCC-free survival and overall survival, the prognostic impacts of nucleos(t)ide analogues for patients with HBV, ursodeoxycholic acid for patients with PBC, and steroidal agents for patients with AIH were not considered in the analyses. (3) In the training cohort, the baseline year at which liver biopsy was performed varied among some decades. Thus, the baseline year was quite variable, probably confounding the prognosis of patients in the training cohort. The validation cohort may have a much smaller risk of such confounding. (4) Finally, a prospective study is necessary to exclude any other potential biases originating from the retrospective nature of this study.In conclusion, APP indicates liver fibrosis stage and prognosis in Japanese patients with chronic liver diseases, predominantly with HCV infection. The diagnostic accuracy of APP to differentiate fibrosis stage was competitive to that of FIB-4, and free from confounding by age or hepatitis activity. Furthermore, smaller APP independently contributes to HCC prevalence and mortality. APP enables physicians to manage patients with chronic liver diseases.The current retrospective study investigated novel indices for liver fibrosis and prognosis consisting of two or three blood exams. Clinical information and pathological stage of liver fibrosis were available to readers of the novel index, but not available to the liver pathologists. Baseline complete blood count, biochemical test, and coagulation function test were performed within a week before performing the liver biopsy.10. To control any potential biases in a training cohort, clinical significance of a novel index was evaluated in a validation cohort. Sample size was validated based on post-hoc power analysis. The study was performed according to STARD and STROBE statement14.After a novel index was established in a training cohort, the index was tested in a validation cohort. Diagnostic potential for liver fibrosis stage was described by area under ROC curve, sensitivity, specificity and positive likelihood ratio. Prognosis prediction by a novel index was presented using two endpoints, prevalence of HCC and overall deaths. Multivariate analysis was performed to process potential confounders within allowable number of parameters ruled by number of events15. Informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was approved by the Institutional Review Board at Kagawa University, Faculty of Medicine (Heisei-30-151)To select candidate blood exam items for a novel index, linear trend through fibrosis stage was evaluated for them. Considering R squares, two or three items were mathematically combined as a simple product or ratio.Japanese patients with HCV and HBV infection, primary biliary cirrhosis definite and probable autoimmune hepatitis patients who underwent percutaneous liver biopsy examinations for a clinical condition between 1986 and 2019 in Kagawa University Hospital, were consecutively enrolled. Patients who had hepatocellular carcinoma when the liver biopsy examinations were performed were excluded.9/l) × √ALT (U/l))16. APRI was calculated using the following equation: 100 × (AST (U/l)/upper limit of normal AST values (U/l))/(Plt (109/l)17.The following clinical data were extracted from the participants’ medical records: age, gender, platelet count, AST, ALT, γGTP, T-Bil, total protein and albumin in blood examinations. T-Bil (mg/dl) was converted to T-Bil (µmol/l) according to the equation: T-Bil (mg/dl) × 17.1. FIB-4, a conventional liver fibrosis index, was calculated using the following equation: age × AST (U/l)/ as follows: stage 1, portal or central fibrosis; stage 2, some septa; stage 3, many septa; stage 4, cirrhosis. The METAVIR grading system was used to assess hepatic inflammatory activity22. Pathological stage of PBC was evaluated using the Scheuer classification  by experienced pathologists who specialized in liver pathology24. Staging and grading were performed by experienced pathologists who specialized in liver pathology.For HCV, HBV, and AIH samples of liver biopsies, the extent of fibrosis was assessed using a modified METAVIR score , followed by the Steel–Dwass post hoc test, was used to assess significant differences in terms of fibrosis stages . Categorical variables were analyzed using Fisher’s exact test. Cut-off values in ROC analysis were determined using Youden index25. P < 0.05 was considered statistically significant.Continuous variables were presented as median and interquartile ranges. Mann–Whitney 27, at Kagawa University.For the training cohort, statistical analyses above were performed using GraphPad Prism 6  and EZR , a graphical user interface for R software For the validation cohort, the abovementioned statistical analyses were performed using SPSS statistical software version 26.0 , JMP 14 , and EZR at Nagasaki Medical Center.Supplementary Information 1.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.


This retrospective clinical study investigates the frequency of biological and technical complications in patients rehabilitated by natural root-retained overdentures (RODs) with cast post-and-cores (root caps) wearing precision attachments and analyses factors influencing complication rates .Patients formerly treated with RODs were invited for a cost-free clinical visit to evaluate their intraoral status. Furthermore, they were interviewed and patient records were screened for complications occurring since denture delivery. Statistical models include descriptive analyses, Fisher’s exact test, odds ratios, and a multivariate regression model.p = 0.0029), in subjects cleaning their dentures less than twice a day (p < 0.001), in subjects regularly using CHX-containing products (p = 0.036) and in subjects with a plaque index > 40% (p < 0.001).A total of 114 patients wearing 128 RODs with a total of 280 abutment teeth were evaluated (mean service time: 7.9 years). Technical complications occurred in 68.8% of the RODs, with matrix loosening being the most frequent complication (50.1%). Biological complications occurred in 53.9% of all RODs, with the presence of denture stomatitis being the most common biological complication (38.3%). The presence of denture stomatitis was significantly higher in the maxilla relative to the mandible (Root cap-retained overdentures with precision attachments are a viable treatment option in partially dentate subjects, even over long-term periods. However, high complication rates should be expected.Establishing good oral hygiene is a decisive factor in preventing complications in RODs. Furthermore, CHX-containing products may not be recommended for routine domestic use. Due to the continuous development of oral care and strategies and preventative measures, the frequency of caries and periodontitis has decreased over the past several decades. Consequently, the number of people suffering from total edentulism is also decreasing, whereas the number of people with residual teeth is rising . HoweverOne strategy to reestablish a more favorable crown-root ratio is to shorten the clinical crown. Subsequently, teeth with shortened crowns can serve as abutments in a root-supported overdenture (ROD) . OverdenTo protect roots or tooth surfaces that are exposed to the oral cavity, ROD abutment teeth can either be covered by direct filling materials (e.g. resin composite)  or by caIt has been shown that RODs require extensive maintenance to prevent them from technical (e.g. chipping fractures or loss of retention) , 20 and/The current clinical retrospective study was approved by the ethics committee of Bern, Switzerland (KEK-BE 268/15). All participants provided their written informed consent.The electronic accounting database of the Department of Reconstructive Dentistry and Gerodontology, School of Dental Medicine, University of Bern, was searched to identify subjects receiving an ROD with cast copings wearing precision attachments between 2002 and 2016 in either the pre- or post-graduate clinics. All identified subjects were contacted by a letter explaining the purpose of the study and were invited for a cost-free clinical examination. After three further attempts by telephone, non-responding subjects were marked unavailable and excluded from the study.Clinical examinations were performed between October 2016 and August 2017 by two post-graduate clinicians who had not been involved in the treatment of any of the included patients. To calibrate inter-examiner reliability, the first five clinical examinations were executed together.First, study participants completed a general and oral dental history, as well as a dental hygiene questionnaire. Their dental status, including the Decayed, Missing, Filled Teeth index (DMFT), stomatological status, O’Leary plaque index and prosthetic condition, was recorded on standardized forms. Furthermore, the dentures were examined in terms of the ROD design (open vs. closed design) and the type of precision attachment. Additionally, the participants were asked about and the patient records were screened for any biological or technical complications that had occurred since ROD delivery.The primary outcome parameter was to analyse the frequency of any biological or technical complications since ROD delivery. Biological complications included abutment tooth loss, the presence of denture stomatitis, recurrent caries and abutment tooth fractures. Denture stomatitis was diagnosed by visual inspection and comprised all three types according to the Newton classification: multiple pin-point hyperaemic lesions (type 1), diffuse erythema confined to the mucosa contacting the denture (type 2) and inflammatory papillary hyperplasia (type 3) . Caries Technical complications included decementation of the gold coping, coping or post-fractures, loss of retention, loss of the denture matrix and denture base or denture teeth fractures.Furthermore, the influence of smoking habits (yes vs. no), dental/prosthetic status in the opposing jaw, recall frequency (< 6 months vs. 1–2 years vs. never), the adjuvant use of chlorhexidine (CHX) containing mouthwash or gel , the attachment type , the number of abutment teeth (≤ 3 vs. > 3) at the day of ROD delivery, the ROD design (open vs. closed), denture cleaning and tooth brushing habits (< 2 vs. ≥ 2/day), denture wearing habits (day only vs. day and night), the denture fabrication setting (pre- vs. post-graduate clinic), the plaque index (≤ 40% vs. > 40%) and the denture location (maxilla vs. mandible), on the frequency of complications was analysed.p values and odds ratios were adjusted for the number of abutment teeth per ROD with a logistic regression analysis. Furthermore, ORs, the Youden index and Fisher’s exact test were applied to analyse the influence of ROD age on the complication frequency. Regarding the influence of the type of attachment , an additional analysis (Fisher test) with the total number of abutment teeth as a reference was applied. The Mann-Whitney U test was used to compare the distribution of the metric variables  in RODs with and without complications. Finally, a multivariate analysis with a logistic regression was applied, identifying factors that increase the overall complication risk , as well as the risk for developing denture stomatitis. All statistical tests were two-sided with a significance level of 5%. The analyses were done with the software Stata/IC 14.2 for Windows.For descriptive data analysis, the relative amounts of biological and technical complications, using the total number of RODs as the reference, were calculated. Fisher’s exact test was used to test for independence of complications and categorical factors. Furthermore, odds ratios (OR) and 95% confidence intervals (CI) were calculated, comparing the complication risk in respective groups (e.g. smokers vs. non-smokers). The A total of 511 former patients treated with RODs could initially be identified from the clinic files. The mean age of those 511 patients was 76.1 ± 11.4 years, wearing RODs with a mean number of 2.5 ± 1.2 abutments. Of those, 246 subjects could not be contacted, and 150 declined the invitation, resulting in a total of 115 subjects with 130 RODs willing to attend a cost-free clinical evaluation. One patient with two RODs was excluded from the study because the original number of abutment teeth could not be identified from the patient file or the ROD itself, resulting in a final study sample of 114 participants with a mean age of 70 years (min-max: 42.9–88.4 years) wearing 128 RODs originally retained by 280 abutment teeth. The mean number of abutments per denture was 2.2 ± 0.9. The mean exposure time of the RODs was 7.9 years (min-max: 3.0–14.9 years). A detailed overview of the patient and OD characteristics is given in Tables p = 0.051). The mean age of RODs with and without biological complications was 8.5 ± 3.2 years and 7.3 ± 3.5 years, respectively (OR: 1.11). According to the Youden-index, an ROD age of 8 years is the cut-off point: overall biological complications  and tooth loss  were more frequent in RODs older than 8 years. No influence of the ROD age on other types of biological complications could be demonstrated.Of the RODs, 46.1% were free of biological complications. At least one biological complication occurred in 53.9% of the RODs. Five types of biological complications could be identified. The most frequent complication per denture was the presence of denture stomatitis (38.3%), followed by the loss of abutment teeth (14.8%), abutment tooth caries (8.6%), fracture of abutment teeth (7.8%) and apical lesions on abutment teeth (3.9%). Table p = 0.029, OR: 2.32) and in subjects cleaning their RODs less than twice a day . The presence of denture stomatitis was also influenced by using CHX-containing products (p = 0.036). The lowest frequency of denture stomatitis was found in subjects never using CHX-containing products. Stomatitis tended to occur more frequently in subjects wearing dentures with a closed design; however, this observation was not statistically significant in the participant cohort . An overview of the factors influencing the presence of denture stomatitis is given in Table p = 0.016) (Table The presence of denture stomatitis was significantly higher in the maxilla (U test confirmed that the plaque index was significantly higher in RODs with abutment tooth caries and denture stomatitis (Table p < 0.001) and abutment tooth caries (p = 0.037) were significantly more frequent in denture sites with a plaque index > 40%. No further factors influencing the frequency of biological complications could be identified.The Mann-Whitney is Table . Stomatip = 0.453) nor of any of the various types of technical complications was significantly influenced by ROD age. The mean age of the RODs with and without technical complications was 8.1 ± 3.3 years and 7.6 ± 3.5 years, respectively (OR: 1.04).Of all RODs, 31.2% had no technical complications; at least one technical complication occurred in 68.8% of the dentures. Retention loss of the matrices was the most frequently observed complication (50.1%), followed by denture base fractures (19.5%), decementation of the root caps (14.1%), loss of the matrix housing (13.3%) and post-fractures (3.1%). Details on the number of technical complications are given in Table p = 0.032). Furthermore, post-fractures  were significantly more frequent in RODs worn only during daytime. Post-fractures (n = 4) only occurred in RODs with 1 or 2 abutment teeth. However, a higher number of abutment teeth (> 3) correlated significantly with a higher risk of denture base fractures . The plaque index was significantly higher in dentures that lost matrix retention (p = 0.006) (Table Post-fractures were significantly more frequent in mandibular RODs (p < 0.001), using CHX-containing products occasionally (p = 0.006) or daily (p = 0.015) compared with never, and in maxillary RODs (p = 0.016). At least one biological or technical complication occurred in 80.5% of all dentures, which means that only 19.5% of the dentures were completely complication-free. Complications were more frequently observed among RODs made and delivered in the pre-graduate clinic (p = 0.023). No further factors influencing the overall complication rate could be identified.The multivariate logistic regression analysis demonstrated a significantly higher risk for denture stomatitis in subjects brushing their teeth less than twice a day , were clinically evaluated. Furthermore, complications could be retraced from the patient files, as most of the patients were followed up in the Department of Reconstructive Dentistry, University of Bern, after denture delivery. Although RODs are commonly used in Switzerland as an alternative to clasp-retained RPDs, the shortcomings of the extensive tooth preparation and the elective endodontic treatment of the abutment teeth should be considered. Consequently, RODs might better constitute an alternative to implant overdentures.The overall complication rate of 80.5% (at least one complication per denture) in the present study was high compared with numbers given in similar studies . To corrIt is very concerning that denture stomatitis was found in approximately one-third of all denture sites. In the scientific literature, even higher frequencies of stomatitis are reported among removable denture wearers . It seemThe significantly higher frequency of denture stomatitis in subjects regularly using CHX-containing products is a very interesting finding, especially since most of the current literature encourages the use of CHX due to its anti-candidal effect . A higheAbutment tooth and post-fractures were more frequently observed in mandibular compared to maxillary RODs. Additionally, post-fractures only occurred in RODs with fewer than 3 abutment teeth. As fractures are generally regarded to be a direct consequence of overloading, it is not surprising that a low number of abutment teeth results in high forces on these teeth  and, consequently, an increased fracture risk . AccordiIn all cases of root cap decementation, caps could be recemented. In four RODs, a post-fracture of the recemented root cap or other root caps in the same jaw occurred. This may be related to an overloading of the root caps if they were not cemented in the exact same position. Therefore, in cases of root cap decementation, it might be beneficial to remove the matrices and housings from the ROD, and to repolymerize them into the denture intraorally, thereby preventing misfit-induced overloading. Misfit resulting from tooth migration during the night, when RODs are not worn, could also explain the beneficial outcomes in terms of post-fractures and decementation in day-and-night ROD wearers. However, many negative factors, such as increased plaque levels, higher frequencies of gingival inflammation, caries, periodontitis and pneumonia (in elderly subjects), are associated with day-and-night denture wearing –43. TherThe major limitation of the current study is its retrospective design and the high dropout rate. Although patients were asked to provide information on any complication since denture delivery, and all patient records were checked, it cannot be ruled out that the actual number of complications was higher than the reported number. Some patients were followed up in a private practice setting and came back only to participate in the current study, and some patient records could not be located. Furthermore, it would have been interesting to include Kaplan-Meier curves, estimating the likelihood of complications over time. But for many complications, the exact time of occurrence could not be verified, so it was decided not to include such analyses. Additionally, the current study did not analyse periodontal parameters or their influence on the complication frequency.Nevertheless, the current study provides several key insights on complications in RODs, which have, to the best of the authors’ knowledge, not been described so far.Root cap-retained overdentures with precision attachments are a viable treatment option in partially dentate subjects, demonstrating satisfactory clinical outcomes, even over long-term periods. However, frequent technical and biological complications may be expected. The frequency of biological complications is influenced by ROD age, with 8 years being the cut-off point. An open denture design, as well as well-established oral hygiene, can prevent biological complications. Day-and-night denture wearing may reduce the frequency of root cap decementation and post-fractures, but should only be recommended in subjects with excellent oral and denture hygiene routines. A minimum of three abutments seems beneficial for preventing post-fractures. CHX-containing products may foster denture stomatitis if used on a daily basis.