Scientific Reports 10.1038/s41598-019-45974-7, published online 02 July 2019Correction to: This Article contains errors. The colours in Figure 6B are inverted; the correct Figure 6 appears below as Figure In addition, the total phospholipid content is not provided. The text in the Results and Discussion subsection ‘Physicochemical stability characterization of the liposomes’,“The liposomes, i.e., empty liposome, PLGba, and RGD-targeted PLGba, had a mean particle size of about 100 nm with narrow particle size distribution (polydispersity index or PDI of <0.150), suitable for the objective of the current study, which is the exclusive particle accumulation in tumor environment based on the EPR effect. The small particle size, morphology, and to some degree the narrow particle size distribution were confirmed as depicted by the TEM graph of PLGba.”should read:“The liposomes, i.e., empty liposome, PLGba, and RGD-targeted PLGba, had a mean particle size of about 100 nm with narrow particle size distribution (polydispersity index or PDI of <0.150), suitable for the objective of the current study, which is the exclusive particle accumulation in tumor environment based on the EPR effect. Moreover, the total phospholipid concentration of the liposomes was 56.6 ±1.8 mM which agreed with the expected phospholipid concentration (60 mM). The small particle size, morphology, and to some degree the narrow particle size distribution were confirmed as depicted by the TEM graph of PLGba.”Finally, information on extruder type and extruding times is incomplete. The text in the Materials and Methods subsection ‘Liposome Preparation’,“Subsequently, the mixture was passed through polycarbonate membranes of 0.4, 0.2, 0.1, and 0.05 μm pore size .”should read:“Subsequently, the mixture was extruded  by passing 11 times through polycarbonate membranes of 0.4, 0.2, 0.1, and 0.05 μm pore size .”


The study of tumor markers (TM) in pleural effusion (PE) was not extensive.TM in PE and serum were analyzed to determine whether TM was expressed in intrathoracic and extrathoracic tissues. To further verify the correlations between serum TM and tumor size, TNM stage of lung adenocarcinoma.P > .05). Serum CEA, serum CA125, serum CA15‐3 were positively correlated with tumor size, T stage, N stage, M stage (P < .05). Serum CA19‐9 was not significantly correlated with tumor size and T stage (P > .05), but was positively correlated with N stage and M stage (P < .05). The levels of PE CEA, PE CA125, PE CA15‐3 were higher than those of serum CEA, serum CA125, serum CA15‐3 . The level of PE AFP was lower than that of serum AFP (P < .05). The level of PE CA19‐9 was not significantly different from that of serum CA19‐9 (P > .05). The positive rates of PE CEA and PE CA125 were higher than those of serum CEA and serum CA125 (P < .05). The positive rates of PE AFP, PE CA15‐3, PE CA19‐9 were not significantly different from those of serum AFP, serum CA15‐3, serum CA19‐9 (P > .05).PE CEA, PE CA125, PE CA15‐3 were moderately positively correlated with serum CEA, serum CA125, serum CA15‐3, respectively . However, PE AFP and PE CA19‐9 were very strongly positively correlated with serum AFP and serum CA19‐9, respectively .Serum AFP was not correlated with tumor size, T stage, N stage, and M stage (The expression characteristics of TM in PE and serum supported the correlations between serum TM and tumor size, TNM stage of lung adenocarcinoma. This study found that although several TM expressed differently in different degrees in patients with lung adenocarcinoma, their expression may be in primary or metastatic lesions, or both. These expression characteristics were helpful for the diagnosis and metastasis judgment of lung adenocarcinoma. Immunohistochemistry was the golden standard for the detection of TM expression, and there were few reports on this aspect in pathology. In addition, using PE to study TM of lung adenocarcinoma may be more useful than using serum. The pathogenesis of PE was tumor invading the pleura , or inflammation invading the pleura. Both of these conditions can affect the production of PE by parietal pleura or the absorption of PE by visceral pleura, resulting in increased PE. The formation mechanism of PE in lung cancer may be both.The expression products of lung tumors first entered PE. PE was diluted after entering the blood. Therefore, the expression products of lung tumors first appeared in PE, followed by blood. Blood and PE were in circulation. Similarly, the expression products from extrathoracic tissues first entered the blood, followed by PE. It was assumed that a certain TM was expressed only in the primary tissue of lung adenocarcinoma, but not in any metastatic tissues. The level and positive rate of this TM in PE was significantly higher than that in serum.In our previous study,22.1This study was approved by the ethics committee of Guangxi Medical University Cancer Hospital with an Ethics approval number of LW2018011. This was a retrospective study that reviewed patients' TM indicators. The patients in this study consisted of two parts. The first part were 335 cases of lung adenocarcinoma patients, which were hospitalized from October 2014 to August 2017. Included patients were those who met the following criteria: (a) The pathological diagnosis was lung adenocarcinoma; (b) No antineoplastic treatment; (c) Blood samples were collected on the second day of admission to test TM; (d) There were definite tumor size and TNM stage; (e) No other malignant tumors. The second part consisted of 84 cases of lung adenocarcinoma patients who were hospitalized from October 2015 to July 2018. Included patients were those who met the following criteria: (a) The pathological diagnosis was lung adenocarcinoma; (b) PE and serum were drawn on the same day to test TM; (c) No other malignant tumors; (d) PE came from the side of lung adenocarcinoma. All cases were staged according to the 2009 seventh edition2.2The tests were completed on the day of blood drawn and PE drawn to avoid attenuation. All assays were performed according to instrument and reagent specifications. CEA, AFP, CA125, CA15‐3, and CA19‐9 were measured using ARCHITECTi2000SR automatic electrochemical luminescence instrument and matching reagents . TM levels above the following values were considered abnormal: CEA, 5ng/ml; AFP 30ng/ml; CA125, 35ng/ml; CA15‐3, 30ng/ml; CA19‐9, 37ng/ml.2.3Chest CT scans were performed using a Siemens somatom sensation 64‐slice CT or a GE Discovery CT 750 HD. Scanning scheme: Ask the patients to hold their breath for a scan after inhaling. Using CARE DOSE technology, 120 kV, pitch 1.000, layer thickness 5 mm; lung window , mediastinum window , layer thickness 1 mm, interval 1 mm. Films were read by two experienced radiologists.2.4P < .05 (two tailed). Based on the normality test (skew and kurtosis coefficients), TM levels did not obey normal distribution. The levels between groups were compared by nonparametric rank sum test, Mann‐Whitney U test. The positive rates between groups were compared by Chi‐square test (χ2). The correlation was analyzed by Kendall's tau‐b correlation analysis. The levels of TM may be influenced by tumor size, T stage, N stage, M stage, and other factors, so, the correlation coefficients between tumor size, TNM stage, and TM levels were low. The correlation analysis in part 1 was based on P < .05, not the correlation coefficient. In part 2, the correlation coefficients were still used to determine the correlation. Correlation coefficient grading: 0.8‐1.0 Very strong correlation, 0.6‐0.8 strong correlation, 0.4‐0.6 moderate correlation, 0.2‐0.4 weak correlation, 0.0‐0.2 very weak correlation or no correlation.Data were statistically analyzed using IBM SPSS21.0 software. The significance level was 33.11 83 (24.8%), T2 157 (46.9%), T3 58 (17.3%), T4 37 (11%); N0 107 (31.9%), N1 41 (12.2%), N2 114 (34%), N3 73 (21.8%); M0 203 (60.6%), M1 132 (39.4%).Table 1 7 (8.3%), T2 10 (11.9%), T3 9 (10.7%), T4 36(42.9%), Tx 22 (26.2%); N0 5 (6%), N1 4 (4.8%), N2 22 (26.2%), N3 31 (36.9%), Nx 22 (26.2%); M1 84 (100%).Part 2 was the information of 84 cases of lung adenocarcinoma all in stage IV and had been treated with anti‐tumor agents. In this cohort, there were 51 (60.7%) males and 33 females (39.3%). The average age was 56.6 years (30‐75 years old). Some patients did not have detailed TNM stage data, T3.2r = 0.146; r = 0.218; r = 0.349; r = 0.358, all P < .05). CA125 was positively correlated with tumor size, T stage, N stage, M stage . CA15‐3 was positively correlated with tumor size, T stage, N stage, M stage . CA19‐9 was not significantly correlated with tumor size and T stage . CA19‐9 was positively correlated with N stage and M stage . There were no correlations between AFP and tumor size, T stage, N stage, M stage .Table 3.3Table z = −4.397; z = −7.735; z = −2.000, all P < .05). The level of PE AFP was lower than that of serum AFP . The level of PE CA19‐9 was not significantly different from that of serum CA19‐9 .In both PE and serum, the lowest values of CEA, AFP, CA125, CA15‐3, and CA19‐9 were lower than the upper line of normal value, the maximum values of CEA, CA125, CA15‐3, and CA19‐9 were higher than the upper line of normal value, the maximum value of AFP was lower than the upper line of normal value. The median of CEA, CA125, and CA15‐3 were higher than the upper line of normal value, the median of AFP and CA19‐9 were lower than the upper line of normal value. The levels of PE CEA, PE CA125, and PE CA15‐3 were higher than those of serum CEA, serum CA125, serum CA15‐3, respectively .The positive rates were ranked as CA125>CEA>CA15‐3>CA19‐9>AFP in both PE and serum. The positive rates of PE CEA and PE CA125 were higher than those of serum CEA, serum CA125, respectively. The positive rates of PE AFP, PE CA15‐3, and PE CA19‐9 were not significantly different from those of serum AFP, serum CA15‐3, serum CA19‐9, respectively . PE AFP, PE CA19‐9 were very strongly positively correlated with serum AFP and serum CA19‐9, respectively . Figure PE CEA, PE CA125, PE CA15‐3 were moderately positively correlated with serum CEA, serum CA125, serum CA15‐3, respectively (4Exclude patients without tumor size data, only 335 cases of lung adenocarcinoma were studied in part 1. It was noteworthy that the conclusions were consistent with the previous 424 cases of lung adenocarcinoma.AFP was a TM of hepatocellular carcinoma and was irrelevant to lung adenocarcinoma.CEA was the antibody to be tumor‐cell specific. CEA was widely recognized as a TM with high sensitivity and specificity for lung cancer,CA15‐3 was commonly used in diagnosis and prognosis of breast cancer.Some studies suggested that the level of PE CA19‐9 in malignant PE and benign PE were similar,5The expression characteristics of TM in PE and serum supported the correlations between serum TM and tumor size, TNM stage of lung adenocarcinoma. Serum CEA, serum CA125, and serum CA15‐3 were positively correlated with tumor size, T stage, N stage, M stage of lung adenocarcinoma. Serum CA19‐9 was positively correlated with N stage, and M stage of lung adenocarcinoma, but not correlated with tumor size and T stage. Serum AFP was not correlated with tumor size, T stage, N stage, and M stage of lung adenocarcinoma.The authors have no conflicts of interest to declare.ZQC, YW and MF conceived and designed the project. ZQC and YW collected the clinical specimens and data. ZQC, YW and MF performed the statistical analysis and wrote the manuscript. ZQC, YW and MF contributed to the writing and critical reading of the paper. All authors read and approved the final manuscript.This study was approved by the ethics committee of the Guangxi Medical University Cancer Hospital with an ethics approval number of LW2018011.Not applicable.


Microalgae are widely be used in carbon sequestration, food supplements, natural pigments, polyunsaturated fatty acids, biofuel applications, and wastewater treatment. However, the difficulties incurred in algae cell separation and harvesting, and the exorbitant cost required to overcome these challenges, are the primary limitations to large-scale industrial application of microalgae technology.Euglena gracilis. Our results demonstrate that flocculation can be induced by increasing the medium pH to 8.5; however, most of the algae cells were broken by increasing the pH > 10. Magnesium phosphate, calcium phosphate, and their derivatives precipitation jointly led to flocculation, although calcium phosphate and its derivatives precipitation had a greater effect.Herein, we explore the potential of inducing flocculation by adjusting the pH for pre-concentrating E. gracilis under a pilot-scale culture system. Moreover, it also maintained the microalgae cells’ integrity, chlorophyll production, and increased paramylon production. These findings provide a theoretical basis for reducing the cost of large-scale E. gracilis harvesting; as well as provide a reference for harvesting other microalgae.This study demonstrates that pH treatment-induced flocculation is efficient and feasible for the pre-concentration of  E. gracilis by alkalescent pH 8.5 treatment.1.It is efficient to pre-concentrate microalga2.Magnesium phosphate, calcium phosphate and their derivatives precipitation jointly led to flocculation, although calcium phosphate and its derivatives precipitation had a greater effect.E. gracilis cell integrity, chlorophyll production , and improves the paramylon production.3.This flocculation method maintains the Microalgae exhibit a high biomass production and growth rate and do not compete with crops, as algae can grow on non-arable land . MicroalEuglena gracilis, which is characterized by the absence of a cell wall, produces a wide variety of bioactive compounds  with tremendous potential for metabolic engineering and commercialization , PEM + PO43−  and the control  at the CPV. Dissolved Ca2+ and Mg2+ ion quantification was performed using inductively coupled plasma optical emission spectroscopy (ICP-OES) . The specific steps were performed according to Trevelin et al. [To further investigate the relative importance of CaThe pilot-scale (60-L column photobioreactors) culture FE was determined by alkalescent pH 8.5 treatment at 0.5, 1, 1.5, 2, and 12 h. Next, the algae biomass DW was analyzed before and after flocculation, while chlorophyll production and paramylon production were analyzed at 2 and 12 h.Chlorophyll production determination was conducted as described by Price , using tg for 5 min, the supernatant was removed. 1.5 mL of 1% SDS solution was added to the tube, after which the contents were transferred into a 1.5-mL centrifuge tube and heated in a water bath at 85 °C for 2 h. Again, the supernatant was removed after the centrifuge tube was centrifuged at 2000g for 5 min. The precipitate was washed and centrifuged in 1 mL deionized water, then oven-dried at 70 °C to a constant weight. The obtained precipitate was paramylon. The paramylon production (g/L) was calculated as shown in Eq.  were considered significantly different, p < 0.05 statistically different, and p values more than 0.05 (p > 0.05) not significant. Standard errors were included in all the graphs.Each growth curve, pH value, flocculation efficiency test, ion concentration, paramylon production, and chlorophyll content test were performed in triplicate and the average was reported. All data were statistically analyzed by Student’s Additional file 1: Fig. S1. The flocculation efficiency of E. gracilis cells and the change in sedimentation under varying pH treatments. Table S1. Concentrations of the main ions (> 1 mg/L ) in the PEM culture medium.


Trueperella pyogenes, separated from cow milk with mastitis, were determined.Lactoferricin (Lfcin) is an antimicrobial activity center of lactoferrin, produced by hydrolysis from the N-terminal of lactoferrin. It was hypothesized that the intramolecular disulfide bond in Lfcin could affect its antibacterial function through influencing its molecular structure. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with an intramolecular disulfate bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. The antibacterial activity of the peptides against 2O, similar ratios of secondary structures with bLfcin and bLfcin C36G under ionic conditions, and close percentages of secondary structure with bLfcin under hydrophobic conditions. The synthetic peptides exhibited strong antimicrobial activity against T. pyogenes isolates, T. pyogenes ATCC 19,411, and E. coli ATCC 25,922. The antimicrobial activities of the three peptides were greater against T. pyogenes than against E. coli, and bLfcin DB exhibited higher antibacterial activity compared with its derivatives.The secondary structure of bLfcin DB showed more β-turn and less random coil than the other peptides in HThe intramolecular disulfide bond could change the molecular structure of bLfcin under alternative ionic strengths and hydrophobic effects, and the formation of the disulfide bond is beneficial to executing the antibacterial function of bLfcin. The resistance of pathogenic bacteria to conventional antimicrobial agents has become an increasingly serious threat to human public health , 2. ThisS. aureus and E. coli  was calculated using the following formula:CD spectra of samples were acquired on a spectropolarimeter  . Samplesθ is the measured ellipticity in mdeg, c is the concentration in mg/mL, l is the cuvette pathlength in cm, and MRW is the mean residue weight of the peptide. MRW is defined aswhere MW is the molecular weight of the peptide in daltons, and n is the number of residues in the peptide.where T. pyogenes was separated from cow milk with mastitis. Mastitis milk samples of cows were obtained from commercial dairy herds located at Gansu province in China. After delivered to laboratory, 10 µl of milk sample was inoculated onto a trypticase soy agar (TSA) plate containing 5% fetal bovine serum or a MacConkey agar  and cultivated at 37 °C for 36 to 48 h. Colonies with the dark pink to red colours were further confirmed by the Vitek system . A total of 5 T. pyogenes isolates were identified as described previously [2.To evaluate the effect of the disulfide bond on the antibacterial activity of bLfcin, eviously , and theT. pyogenes isolates were carried out by ADD. The reference strains T. pyogenes ATCC 19,411 and E. coli ATCC 25,922 were used as controls. Briefly, an inoculum aliquot /mL) was placed on Mueller-Hinton agar (MHA) . The ADD assays were carried out at only a one concentration for each peptide (400 µg/disc), followed by the incubation for 48 h at 37 °C. As a positive control, 1.25 µg/mL ciprofloxacin was used for all tested strains. Sterile water was used as a growth control. Each of these tests was performed three times.The susceptibility assays of three T. pyogenes, and there were no published MIC breakpoints for AMP in the CLSI criteria or the literature, the susceptibility assays of the isolated strains were carried out by broth dilution methods that have been reported previously [T. pyogenes ATCC 19,411 and E. coli ATCC 25,922 were selected as control groups. The MIC and MBC were determined using the following microdilution assay. In brief, the bacterial strains were incubated at 200 rpm for 12 to 15 h, at 37 °C, in a Mueller Hinton broth (MHB)  until an optical density of 0.15 to 0.30 (620 nm) was obtained. Then, 75 µL of 2 × AABSA solution (0.02 µL/mL acetic acid and 0.4 mg/mL BSA) containing peptide was added to each well on the first column of a 96-well microtiter plate. The peptide serial dilution was performed by the 2 × AABSA solution from the first column to the tenth column. Then, the peptide solution was mixed with 75 µL of the inoculum (1 × 106 CFU/mL) in each cell. The concentrations of peptide serial dilution were halved, and the final serial concentrations were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 µg/mL, respectively. At the same time, the concentration of the bacteria reached 5 × 105 CFU/mL, in line with previous finding [Because there was no CLSI method available for the determination of MICs for eviously . The str finding . The sol6 CFU/mL in MHB using a nephelometer. The peptide solutions at 2-fold MBC of bLfcin were added to isopyknic bacterial medium in culture tubes. Bacteria reached a final cell density of 5.0 × 105 CFU/mL, and the peptide concentrations reached MBC of bLfcin. The bacteria and peptide mixtures were incubated for time periods up to 120 min at 37 °C. Aliquots of cultures were taken at 0, 15, 30, 60, 90, and 120 min post-inoculation. Serial 10-fold dilutions of each aliquot was prepared in basal medium and plated in triplicate onto suitable agar media. Viable bacterial counts were enumerated by a standard dilution and plating method. Time-kill curves were constructed by plotting log10CFU/mL against the time of incubation. Killing assays were performed in triplicates and the results were averaged.The test strains were cultured to reach exponential growth phase at 37 °C in MHB. The strains were then adjusted to achieve cell density of 1.0 × 10


Oreochromis niloticus and Nile perch Lates niloticus from Lake Victoria, complemented with recent data on Cu, Zn, Cd, and Pb. This also is the first report on Cr, Ni, As, and Ag levels in invertebrates: the shrimp Caridina nilotica, gastropod Pila ovata, and bivalve Mutela bourguignati. The study was conducted at five sites in the Kenyan part of Lake Victoria: four sites in Winam Gulf influenced by various anthropogenic pressures, including a site near Kisumu City, and one in the main lake, with lesser direct anthropogenic influence. Apart from Cu and Ag, which were highest in O. niloticus liver, the invertebrates had higher levels of trace elements than fish. Contamination of the gulf with trace elements was best mirrored by the invertebrates, whose mobility is limited; they accumulated Cr, Cd, Ag, and Pb corresponding to the levels in the surface sediment. The accumulation of trace elements in fish species and their bioindicative potential corresponded to their habitats and feeding behaviour. The tissue contents of most trace elements were higher in the inshore-dwelling, omnivorous O. niloticus compared to the pelagic, piscivorous L. niloticus. Cu (465 ± 689 mg/kg dw) and Ag (3.45 ± 1.49 mg/kg dw) in the liver of O. niloticus were up to 10 and 119 times higher than in L. niloticus, respectively. Oreochromis niloticus therefore has bioindicative potential for Cu and Ag contamination. Both the invertebrates and fish showed positive correlations between Cu and Ag concentrations, indicating similar source and/or uptake route. The target hazard quotients (THQ) show that there is no human health risk associated with the consumption of these fish. However, the levels of Zn, Cd, and Pb in P. ovata surpassed maximum food safety limits and are hence potentially unsafe for human consumption.This is the first comprehensive report on the accumulation of Cr, Ni, As, and Ag in the fish species Nile tilapia The online version of this article (10.1007/s00244-020-00715-0) contains supplementary material, which is available to authorized users. Trace elements, which include heavy metals and metalloids, occur naturally in the environment , the gastropod Pila ovata (Ampullaridae), the bivalve Mutela bourguignati (Iridinidae), and commercial fish: O. niloticus (Cichlidae) and L. niloticus (Laditae). Caridina nilotica is the only shrimp species known in Lake Victoria   of Kenya’s total aquatic food production. Moreover, 35% of the lake’s total fish production is landed in Kenya for internal and external markets, of which Nile perch L. niloticus accounts for 84.7% of total fish exports to Europe, Asia, United States, and Central America  in the invertebrates 2 with a mean depth of 40 m and maximum depth of 79 m  drains through Rwanda, Burundi, Tanzania, and Uganda, while the main river outlet is the Nile , Uganda (43%), and Tanzania 51%), is the world’s largest tropical lake and the second largest freshwater lake in the world, covering a total of 69,000 kmile Crul . The Ken%, is theThe study was performed in five selected sites in the Kenyan part of Lake Victoria both in Winam Gulf and part of the main lake as indicated in Fig. Caridina nilotica was obtained from AB, KM, and RS using seine nets. The samples were transferred into clean plastic bags, put on ice, and were transported protected from light to Maseno University laboratory in Kisumu City, where they were rinsed with Millipore water. In order to achieve sufficient material for analyses, 15–20 specimens were pooled into 15-ml polypropylene (PP) tubes and oven-dried for 48 h to constant weight at 90 °C. In total, 72 sample were obtained: 26 from AB, 24 from KM, and 22 from RS. Mollusc specimens of P. ovata (≈ 40-mm shell height) and M. bourguignati (≈ 60-mm shell length) were handpicked (wearing nitrile gloves) from the littoral areas of the study sites. The specimens were transported alive to the laboratory in aerated containers with lake water. For P. ovata; 10 specimens from AB, 17 from KM, 7 from MN, and 15 from RS were taken, whereas for M. bourguignati 8 specimens were obtained from MN and RS each. The specimens were kept in aerated lake water for 48 h for depuration  and 1 ml of 30% H2O2 using a microwave MARS XPRESS system (CEM Corporation). The digested samples were transferred into 15-ml volumetric flasks and brought up to volume using milliQ water. The leachates were filtered through 0.2-µm PTFE pre-syringe filters (VWR). Reference samples comprising 0.2 g (dry weight) of fish protein (DORM-3 and DOLT-5) obtained from the National Research Council Canada (NRCC) were digested and diluted in the same manner as described above. Trace element  concentrations were determined using total x-ray reflection fluorescence spectrometry  and, when necessary, graphite furnace atomic absorption spectrometry (GF-AAS) using a PinAAcle 900Z (Pelkin Elmer).Dried tissue samples were homogenized with a mortar and pestle, and approximately 0.2 g was digested in 9 ml of 34% HNOU tests (for pairwise comparisons) where applicable. For the ANOVA post hoc test, Tukey’s HSD was applied for pairwise mean comparisons where homogeneity of variance was established, whereas the Games Howell test was applied for unequal sample sizes and variances  of fish.Statistical analyses were done using IBM SPSS 21. The Shapiro–Wilk Test, which is very sensitive to detecting deviations from normality (Mohd and Yap The bioaccumulation factors (BAFs) were determined according to the formula:In which the variables x and y stand for matrices that are compared to each other, such as sediment, invertebrate, and fish tissue. Element concentrations in the sediment were based on data from Outa et al. .O. niloticus and L. niloticus in the region. THQ is the ratio between the potential exposure to a substance and the reference dose (level at which no adverse effects are expected) (USEPA The body trace element burden (mg/kg dw) of mollusks and fish muscle obtained in our study were converted to fresh weight by applying a conversion factor of 0.2 based on an estimated water content of 80%  USEPA . THQ vald) USEPA . For cald) USEPA , which wd) USEPA  is as foThe variables in the equation are defined as follows: EF is the exposure frequency (350 days/year); ED is the exposure duration (26 years); IRF is the average fish consumption per day. The per capita fish consumption in Kenya is 4.5 kg per year FAO , which tL. niloticus, we treated all fish for this study as one sample, whereas values for O. niloticus were calculated per site. With the exception of Cu and Ag, which were highest in O. niloticus liver, the invertebrates had the highest concentrations of the trace elements measured in this study. The levels of most trace elements were higher in the liver than in the muscle of fish , which we did not observe in P. ovata foot or in M. bourguignati. Caridina nilotica from the less disturbed RS site had lower concentrations than samples from the AB and KM sites . Similarly, P. ovata viscera from RS had lower concentrations compared with AB and KM . This agrees with the results for Cr in sediments from these sampling sites  from the Mekong River Delta, Vietnam. This is even lower than the mean Cr concentration recorded in C. nilotica from the RS site (1.19 ± 1.0 mg/kg dw). Regarding human consumption, most international regulations do not provide maximum values for Cr. One exception is China, where a maximum level of 2 mg/kg ww is allowed in mollusks  = 2.263, p = 0.688 and X2(4) = 2.073, p = 0.722, respectively). In fish, Cr is predominantly taken up through the gills and distributed via the blood to the tissues, with little or no bioconcentration at environmentally relevant concentrations , which are higher than in the fish species from our study.Chromium concentrations in fish liver were significantly higher than in muscle in all samples, and there was no significant difference between the two fish species Table . Comparions Reid . As noteons Reid , the levons Reid , who recl et al.  reportedC. nilotica, P. ovata, or M. bourguignati. In the current study, the variations in concentrations in crustacean and mollusc samples from the study sites (Table Pila ovata viscera had the highest concentrations (9.63–36.1 mg/kg dw), which were at least four times higher than in the other invertebrate tissues. Compared with other studies, lower Ni levels have been reported in mollusks: 0.02–1.54 mg/kg dw in Pomacea caniliculata from the Mae Klong River, Thailand . This agrees with Coetzee et al.  from Lake Naivasha, Kenya. In our study, the value was below the LOD (0.040 mg/kg dw) in muscle tissue of O. niloticus in three of the sampling sites  but was detected in the rest of the samples, albeit at low levels. In the rather unpolluted Vaal Dam in South Africa, similar levels were reported from cyprinid fish , indicating that translocation to various organs, including the foot is not restricted. Pila ovata accumulated 10–18 times more Cu than M. bourguignati. This finding concurs with Gundacker (C. nilotica), 4.40 (P. ovata viscera), and 3.96 (P. ovata foot). Comparing Cu levels with other studies, Abdennour et al. . In mollusks, lower levels were recorded in snails and bivalves from less contaminated freshwater bodies. For instance, in the Ipoba Stream, Nigeria, Ezemonye et al.  from the polluted Fankou stream in southern China. In fish, Cu is predominantly taken up from the diet through the intestines and directly from the water across the gill epithelia . The levels in O. niloticus liver were 4–10 times higher than in the liver of L. niloticus. The omnivorous inshore-dwelling O. niloticus is probably exposed to more Cu uptake through the diet and from contact with the contaminated water and surface sediment. Cu in fish tissue generally shows weak or negative correlations with fish size . Nonetheless, the Cu content in O. niloticus liver had high positive correlations with TL  at KM, where the Cu levels were highest in surface sediment. This agrees with the findings of Çoğun et al.  from our study sites, however, were higher than in the same species from Lake Nasser (67.1–102 mg/kg dw) (Mohamed T. zillii (123 ± 148 mg/kg dw) from Lake Naivasha  and 7.89 (M. bourguignati).The body concentration of Zn, an essential element, is regulated to relatively constant levels in invertebrates . The mean concentrations in M. bourguignati and P. ovata viscera from our study sites (383–1361 mg/kg dw) are higher than in molluscs from other areas in Africa. For instance, mean Zn concentrations in the viscera of P. ovata from various sites in the Ikpoba River in Nigeria ranged from 23.6 to 45.2 mg/kg dw , which received effluent from a Zn/Pb mine. There, mean Zn concentrations in P. canaliculata ranged from 3000 to 6400 mg/kg. Regarding the risk for consumption by humans, the mean levels of Zn in P. ovata viscera at AB (1350 ± 433 mg/kg dw), KM (893 ± 769 mg/kg dw), and RS (1361 ± 1132 mg/kg dw) and M. bourguignati at MN (829 ± 460 mg/kg dw) surpassed various national food safety limits: Australian Legal Requirements for Food Safety (750 mg/kg dw), Ministry of Public Health, Thailand (667 mg/kg dw), and Brazilian Ministry of Health (250 mg/kg dw)  and O. niloticus . Elsewhere, mean Zn values of 12.1 mg/kg and 10.3 mg/kg were recorded in the muscle tissue of O. niloticus and L. niloticus, respectively, from Lake Nasser (Mohamed O. leucostictus (604 ± 82 mg/kg dw) from Lake Naivasha  from Lake Turkana, Kenya  from our study were higher than the mean concentrations in O. leucostictus (73.1 mg/kg dw)  and in L. niloticus (37.8 mg/kg dw) from Lake Nasser  were on the same magnitude with the findings of Tu et al.  were lower than in the bivalve Unio pictorum from Lake Maggiore, Italy  given by the China Food and Drug Administration (CFDA O. niloticus specimens from RS (0.036 ± 0.01 mg/kg dw), and the mean concentration in L. niloticus was (0.043 ± 0.01 mg/kg). Our results agree in part with the findings of Machiwa  = 688.030, p < 0.001, F  = 9.425, p < 0.0001 and F  = 13.286, p < 0.0001). As indicated in Fig. C. nilotica and P. ovata are therefore potential indicators for Ag contamination. Like with Cu and As, the levels in the foot and viscera of P. ovata did not differ significantly . The concentrations in invertebrates were significantly positively correlated with Cu levels . Our results therefore point to the potential common sources or intake paths and sequestration for Cu and Ag in the invertebrates. There is a paucity of data on the accumulation of Ag in invertebrates from natural waters. Experimental results, however, demonstrated that under Ag exposure some mollusks can accumulate high levels of Ag in their tissues. For instance, in an experiment where sediment was spiked with AgNO3 to 100 mg/kg up to 75 mg/kg dw, Ag was recorded in the hepatopancreas of the viviparid snail Bellamya aeruginosa  = 37.232, p < 0.0001), with the highest concentrations in fish at the site impacted by Kisumu City . This positive correlation is strongest at low concentrations of Ag (up to 1.5 mg/kg dw), after which the levels of Cu do not correspond with the rise in Ag levels . Ni uptake may involve proton-coupled divalent metal transporters with binding sites similar to those for Cu and sequestration into Ni-induced metallothioneins  but are in the same range as O. niloticus liver samples from AB, KK, MN, and RS . Mutela bourguignati from RS also had lower levels compared with MN . Similarly, we recorded a spatial variation in Cd contents in P. ovata viscera  = 10.8, p < 0.001), with the samples from RS having significantly lower levels than elsewhere in the gulf ; this points to a common source and/or intake path for these elements. Most of our findings on Cd levels in the invertebrates were not consistent with data from other studies. Mwamburi   surpassed the 1.0 mg/kg ww safety standard . These higher levels in O. niloticus may result more from exposure and uptake via the gills from the habitat, namely contaminated shallow inshore areas. Cd has a long biological half-life in vertebrates . In other studies, Otachi et al. . Similarly, the concentrations in P. ovata viscera varied significantly between the study sites  = 4.087, p = 0.012), with the highest values at KM and the lowest at RS  that we recorded in C. nilotica. Likewise, concentrations in M. bourguignati (0.73 ± 0.97 and 1.41 ± 0.68 mg/kg dw) and in P. ovata viscera from our study sites (0.48–15.5 mg/kg dw) were lower than the mean in Sphaerium sp. (31.8 mg/kg dw) from Lake Victoria  in Fankou stream, which received effluent from a Zn/Pb mine in southern China. Regarding the implications for human consumption, the Pb content of P. ovata viscera (3.09 ± 6.0 mg/kg ww) from the KM sampling site, surpassed the maximum limits of 1 mg/kg ww . Similarly, Pb was positively correlated with Zn in O. niloticus liver  and Cu in L. niloticus muscle tissue . Fish size did not correlate to Pb concentrations in liver and muscle in our study, which agrees with the findings of Szarek-Gwiazda and Amirowicz Below is the link to the electronic supplementary material.


The OM in the surface sediment of ZJB was a mixture of terrestrial and marine sources, and was dominated by marine OM (54.9% ± 15.2%). Compared to the central ZJB, the channel and coastal ZJB areas had higher δ13C and δ15N values, higher TOC and TN concentrations, and lower TOC/TN ratios, indicating higher primary productivity and higher percentages of marine OM in the latter two subregions. Mariculture activities, sewage inputs, and dredging were responsible for these phenomena. Clear seasonal variations in OM were observed in ZJB. The average proportions of terrestrial OM in summer increased by 10.2% in the ZJB channel and 26.0% in the coastal ZJB area compared with those in spring. Heavy rainfall brought a large amount of terrestrial OM into the channel and coastal ZJB areas, leading to the increase of the terrestrial OM fraction in these two subregions in summer. In summary, anthropogenic influences had a significant influence on the spatial and seasonal variations of sedimentary OM in ZJB.Elemental  and total nitrogen (TN)) and stable carbon and nitrogen isotope compositions (δ Organic matter (OM) in sediment is a mixture of components from different sources. The concentration of total organic carbon (TOC) is used as a primary proxy to describe the abundance of OM . Under t13C and δ15N, respectively) and total organic carbon to total nitrogen (TOC/TN) ratio  with a population of approximately 7.3 million. There are many sewage outfalls in ZJB [13C and δ15N values in the surface sediments of ZJB during spring and summer. Spatial and seasonal variations in OM signatures were determined and used to evaluate the effects of anthropogenic activities and environmental changes on the SOM. The results of this study will help to address the gaps in the understanding of the effects of anthropogenic activities and seasonal environmental variations  on the SOM of subtropical bays.Zhanjiang Bay (ZJB) is a semi-enclosed bay located in the northwestern coastal region of the South China Sea . It coves in ZJB . Zhanjias in ZJB . Over res in ZJB ,43. ZJB s in ZJB . These as in ZJB . Howevers in ZJB ,46,47,48ZJB is located in the southern part of the Chinese mainland and is linked to the South China Sea by a narrow inlet that is 1.9 km in length . The watSampling was carried out in ZJB during two cruises in April (spring) and August (summer) in 2017. Fifteen stations, covering most of the bay , were se2O2 to remove OM, and with 1 M HCl to remove carbonates. The pretreated samples were then washed 3 times with deionized water. The solids were dispersed with 10 mL of 0.05 M (NaPO3)6 and then analyzed for grain size (between 0.02 and 2000 μm) using a Malvern Mastersizer 2000 laser diffractometer. The percentages of the clay (<4 μm), silt (4–64 μm), and sand (>64 μm) fractions were determined.For grain-size analysis, a small portion of each sediment sample was pretreated with 30% H13C, the sediment samples were treated with a 1 M HCl solution to remove carbonates. The samples were then washed with deionized water, obtaining a neutral condition before being dried at 60 °C [13C analyses. TN and δ15N were analyzed without pretreatment with HCl. The elemental and isotopic analyses were conducted at the Third Institute of Oceanography, Ministry of Natural Resources, China. Concentrations of the TOC and TN, as well as the δ13C and δ15N values, were determined using an elemental analyzer integrated with an isotope ratio mass spectrometer . Replicate analysis of one sample (n = 5) gave a relative standard deviation less than 0.8% for TOC and less than 0.7% for TN. The TOC/TN ratios presented here are the molar ratios of TOC to TN content. The δ13C and δ15N values are reported relative to the Vienna PeeDee Belemnite standard (V-PDB) and atmospheric nitrogen, respectively. Acetanilide was used as a working standard (δ13C = −29.53‰ and δ15N = 1.18‰). The instrument analytical precision was ± 0.2‰ for δ13C and ±0.25‰ for δ15N.The frozen sediment samples were freeze-dried, ground, and homogenized with a pestle and mortar, then passed through a mesh sieve (150 μm in pore size) prior to analysis of the elemental concentrations and stable isotope ratios. For analysis of TOC and δat 60 °C ,50, homoGrain size compositions can significantly influence the behavior of sedimentary parameters. The ternary diagram in r > 0.56, P < 0.05; r > 0.94, P < 0.001; The TOC in sediment represents OM that escapes remineralization . It is wBased on the data from the two seasons, the TOC was highest in the channel (0.82% ± 0.37%), intermediate in the coastal bay (0.55% ± 0.38%), and lowest in the central bay (0.49% ± 0.19%) . The TN 13C and δ15N in the surface sediments of ZJB in spring and summer. In spring, the values of δ13C ranged from −25.0‰ to −22.2‰, with a mean of −23.4‰ ± 0.8‰. In summer, they ranged from −25.7‰ to −21.8‰, with a mean of −23.7 ± 1.0‰. Overall, the spatial distributions and the values of δ13C in the two seasons were rather similar , intermediate in the coastal bay (−23.4‰ ± 0.9‰), and lowest in the central bay (−24.3‰ ± 0.9‰). Here, δ15N showed a similar distribution pattern, with the highest value in the channel (7.3‰ ± 0.9‰), an intermediate value in the coastal bay (6.4‰ ± 1.2‰), and the lowest value in the central bay (5.7‰ ± 1.4‰) , an intermediate average of TOC/TN in the coastal bay (13.3 ± 9.4), and the lowest average of TOC/TN in the channel (10.1 ± 3.0)  . The val1 ± 3.0) . Additio13C values also indicated an increase in marine-derived OM in the channel sediment, which is discussed later.TOC/TN ratios have often been used for tracing OM sources in sediment samples ,59,60. I13C  may also alter the TOC/TN ratios in SOM . For the3 pathway have δ13C values ranging from −30‰ to −24‰ [4 pathway have δ13C values ranging from −16‰ to −10‰ [13C values ranging from −22‰ to −18‰ [3 plants has depleted δ13C values compared to marine OM. Diagenetic effects on the δ13C signature of OM have been shown to be small [13C is a perfect tool for distinguishing different OM sources [Terrestrial plants with a C to −24‰ , while t to −10‰ . Marine  to −18‰ . In genebe small ,79. Ther sources ,66, and  sources ,19,66.4 plants are uncommon in the area surrounding ZJB, where the dominant cultivated plant is rice (a C3 plant) and the natural ecosystem is a subtropical forest. Therefore, the contribution of C4 plants to the SOM in ZJB can be neglected [13C values in the surface sediments of ZJB ranged from −25.7‰ to −21.8‰ , intermediate in the coastal bay (−23.4‰ ± 0.9‰), and lowest in the central bay (−24.3‰ ± 0.9‰), suggesting that the channel and the coastal bay were more influenced by marine phytoplankton than the central bay. According to previous studies, δ13C values of SOM in coastal sediments generally increase in a seaward direction [13C in this study was influenced by human activities. Relatively high δ13C values have also been seen in the coastal area of northern Shandong Peninsula [irection ,77. The eninsula . The chaeninsula ,58,82. Meninsula , which ceninsula ,37. In teninsula ,85,86,8713C values in the channel and coastal bay in spring, the δ13C values in these two subregions in summer were relatively negative  generally showed greater depleted values and less seasonal variability than particulate OM obtained in the same area by Cai [The δa by Cai  , while the δ15N values of terrestrial OM usually range from −10‰ to 10‰ (mean 2‰) [15N values than terrestrial OM [15N values of these two sources overlap to some extent. Also, the δ15N values of OM could potentially be modified by selective diagenesis during sinking and after sedimentation [15N does not discriminate as effectively as δ13C between terrestrial and marine OM sources. However, compared to δ13C, δ15N can be effectively used to distinguish between sewage inputs and other sources because of their different δ15N values [15N values (10‰–22‰) [15N values (~10‰) in their tissue [15N values in the sediment. Aquaculture could also lead to elevated δ15N values in the sediment (δ15N > 7‰) [13C in identifying sewage inputs is limited, because the δ13C values of sewage effluents (which vary from −26‰ to −22‰) overlap with the values of terrestrial and marine sources [The δs of SOM ,36,89. Tmean 2‰) . Althougtrial OM ,91, the entation . TherefoN values . Nitroge10‰–22‰) ,94. Marir tissue . The set15N values for the complete set of samples in ZJB ranged from 2.8‰ to 9.0‰  was significantly higher than in summer (4.8‰ ± 1.4‰)  . The decplankton . In wintton [15N ,97. In senomenon ,91.15N and TOC/TN [13C [13C in either spring or summer  and marine OM (fmar) in the sediments of ZJB can be assessed using the δ13C-based two end-member mixing model [13Cter and δ13Cmar are the δ13C values of the terrestrial and marine OM end-members, respectively. Due to the lack of δ13C values for the terrestrial and marine end-members for the SOM in ZJB, we chose the δ13C value of C3 plants (average: −27‰) as the terrestrial end-member [13C value of phytoplankton (−20.8‰), collected from the northern South China Sea, was chosen [Diagenesis can affect the chemical and isotopic compositions of OM, especially in the case of δd TOC/TN ,66. Howe/TN [13C . In ZJB,r summer . Addition spring . Therefong model ,40. The d-member ,76. For s chosen .13C-based two end-member mixing model are displayed in mar: 54.9% ± 15.2%). In spring, the calculated fter ranged from 22.1% to 68.2%, with a mean of 42.8% ± 13.7%. In summer, it varied from 16.4% to 78.4%, with a mean of 47.5% ± 16.8%. The relatively higher average fter value in the summer could probably be related to the high rainfall in this season, which could bring large amounts of terrestrial OM into ZJB [ter value of the different subregions in different seasons confirmed this conclusion, which is discussed below.The results from the δinto ZJB . The aveter and fmar values also showed spatial and seasonal variations, as summarized in ter value were observed in the channel and coastal bay .This study investigated the spatial and seasonal variations of OM in the surface sediments of ZJB. TOC, TN, TOC/TN, δ13C, and δ15N in the surface sediments of ZJB. Compared to the central bay, relatively higher δ13C and δ15N values, relatively lower TOC/TN values, and relatively higher TOC and TN concentrations were found in the sediments in the channel and coastal bay. This indicated elevated primary productivity and relatively high marine OM in these areas. Nutrients from nearby sewage outlets, rapid urbanization, mariculture activities, and heavily fertilized farming in the surrounding area were found to be responsible for these phenomena. Dredging caused subaerial exposure of the surface sediment and affected OM preservation in the central bay. Lower δ13C and δ15N values and higher TOC/TN ratios, together with the lower TOC and TN concentrations in the central bay sediments, indicated the low-efficiency storage of marine OM in this area.Anthropogenic activities had a significant influence on the distribution patterns of TOC, TN, TOC/TN, δ13C values being higher in spring in the channel and coastal bay, and the TOC/TN ratios being higher in spring in the coastal and central bays. Additionally, the δ15N values were higher in spring in the channel and central bay. Higher temperatures were responsible for the low concentrations of TOC and TN in the channel and coastal bay in summer. Heavy summer rainfall increased the input of terrestrial OM in the channel and coastal bay, resulting in a decrease in δ13C values in this season. Relatively low TOC/TN ratios in summer in the coastal and central bays may be related to the accumulation of fresh OM in this season when primary production was high. High rainfall in summer was also responsible for low δ15N values in this season in the channel, while the low δ15N values in summer in the central bay may be related to the changing sources of nitrogen used by phytoplankton.Seasonal variations in SOM parameters were observed in ZJB, with the TOC and TN concentrations and δThe results of this study indicate that large amounts of terrestrial OM can be brought to coastal sediment areas during summer when rainfall is high. The decomposition of SOM can consume a large amount of oxygen in water. This indicates that high rainfall may aggravate the risk of hypoxia for the bottom water in coastal areas through increasing terrestrial input. Therefore, measures should be taken to reduce the input of terrestrial OM during high rainfall. More investigations should be conducted to study the influence of terrestrial OM during high rainfall periods for the protection of coastal environments.


A. cruentus) and “Zobor” (A.hypochondriacus x A. hybridus) were evaluated and compared to nonirradiated controls Ficha (A. cruentus L.) and K-433 (A. hypochondriacus x A. hybridus) and commercial varieties, Aztec (A. cruentus L.), Plainsman and Koniz (A. hypochondriacus x A. hybridus). Mutant varieties, “Pribina” and “Zobor”, showed superior 1000-seed weight performance compared to all investigated amaranth samples. The change in quantitative seed trait was accompanied by significantly higher oil and squalene content compared to commercial varieties. Moreover, significantly higher content of essential linoleic acid was detected in mutant variety “Zobor”. The present findings suggest that seeds of irradiation-derived varieties have high nutritional potential and can be used as a supplementary crop in the human diet.Grain amaranth is known as an alternative crop with exclusive nutritional value and health benefits. The purpose of this study was to investigate the effect of gamma irradiation on quantitative and qualitative amaranth seed traits, including 1000-seed weight, amino acids, fatty acids content, oil, and squalene yield. Two Slovak mutant varieties “Pribina” ( Amaranth is a multipurpose crop that can be used as food and animal feed, as well as an ornamental plant . AmarantThe lipid content in amaranth seed is up to three times higher compared to buckwheat and common cereals . The fatAmaranth is also considered a good source of insoluble fiber and has high vitamin and mineral content, such as riboflavin, niacin, ascorbic acid, calcium, and magnesium ,21. AmarOne of the tools for improving seed traits of important crops is mutation breeding, which has been successfully utilized for variety development and generating polygenic variability ,23. Our Amaranthus cruentus) and “Zobor” (Amaranhtus hypochondriacus × Amaranthus hybridus), in their nonirradiated control genotype Ficha (A. cruentus) and hybrid K-433 (Amaranhtus hypochondriacus × Amaranthus hybridus), and in the preferred commercial varieties Aztec (Amaranthus cruentus), Plainsman and Koniz (Amaranhtus hypochondriacus × Amaranthus hybridus). We evaluated 1000-seed weight as an important yield parameter and determined the content of important seed nutritional properties, such as amino acids, total oil, fatty acids, and squalene.Hence, we aimed at the comparison of selected quantitative and qualitative seed traits in two gamma-ray derived Slovak varieties “Pribina” , Plainsman (0.68–0.73 g), and Koniz (0.68–0.71 g).Among the tested amaranth samples, the lowest 1000-seed weight was found in commercial varieties Koniz and Plainsman, especially when harcested in 2018 (0.68 g for both). Similar results of 1000-seed weight in similar conditions, i.e., Central European conditions, have been previously reported ,34,35.Amaranthus cruentus and Amaranthus hypochondriacus. Authors reported that 1000-seed weight ranged from 0.62 to 0.93 g and was influenced by genotype. Other authors [Gimplinger et al.  analyzed authors  reportedAmaranthus cruentus accessions from 0.33 to 0.76 g. Our results showed up to 67% higher 1000-seed weights compared to this report.Rivelli et al.  investigMoreover, we observed an effect of the growing season on this tested yield parameter . The latp ≤ 0.05) between mutant varieties “Pribina” and “Zobor” and their nonirradiated counterparts Ficha and K-433, a long-term increase in 1000-seed weight of mutant varieties was observed. Considering that high yield is influenced by seed parameters, such as 1000-seed weight, it is one of the crucial breeding targets for the improvement of yield. As both mutant varieties had higher 1000-seed weight, they are very important for the market.As a result of mutagenesis treatment, demonstrated by significant differences  content in seeds of mutant varieties “Pribina” and “Zobor”, controls Ficha and hybrid K-433, and commercial varieties Plainsman and Koniz during three consecutive growing seasons (2016–2018) were performed. p ≤ 0.05) content of summed AA was found in seeds of the commercial variety Plainsman (92.76 g/16 g N), followed by mutant variety “Zobor” (90.75 g/16 g N) in 2017 and 2018, respectively  and aspartic acid (6.55 ± 0.137.53 ± 1.20 g/16 g N) from NEAA and arginine (6.16 ± 0.34–7.66 ± 1.29 g/16 g N) from EAA . These raranthus ,41. The Leguminosae than to cereals, except for sulphur-containing AA present in higher amounts in amaranth than in Leguminosae [Amaranthus cruentus grains have a similar AA composition to Chenopodium quinoa but the concentration of essential leucine, lysine, and phenylalanine is higher. Hovewer, the AA content depends on the amaranth species and cultivar [The advantage of amaranth seeds over cereal grains is a relatively high protein content. The quality protein is determined by balanced AA composition, especially with a significant amount of EAA, like lysine and suplhur-containing methionine. Generally, the AA profile of amaranth seeds is more similar to uminosae ,42. Palouminosae  presentecultivar .Amaranthus hypochondriacus whole flour. Shukla et al. [The lysine content of amaranth samples analyzed herein was, on average 4.33 g/16 g N , with tha et al.  reporteda et al. ,45. Matta et al.  analyzeda et al. .As reported by Kugbe et al. , irradiap ≤ 0.05) content of essential isoleucine (2.09 ± 0.04 g/16 g N) and valine (2.30 ± 0.05 g/16 g N), and nonessential tyrosine (2.75 ± 0.30 g/16 g N) ompared to nonirradiated control K-433  during the monitored period (p ≤ 0.05) content of both EAA and NEAA in mutant variety “Zobor” over the hybrid K-433 in 2016 and 2018  and NEAA (55.15 g/16 g N) in 2017. Hovewer, the lowest content of summed EAA (24.91 g/ 16 g N), as well as NEAA (39.80 g/16 g N), was also detected in this variety in season 2018, indicating the instability of Plainsman in this analyzed trait. Although variety “Zobor” exhibited relatively unstable performance in 2017, except for methionine and cysteine, the content of all tested AA in “Zobor” seeds was the higher than all other analyzed samples . Thus, vAmaranthus hypochondriacus x Amaranthus hybridus) as Plainsman and “Zobor” showing some degree of instability. Shukla’s findings [The most consistent AA performance was observed for hybrids K-433 and Koniz . Interesfindings  suggest Amaranth seeds generally have a higher lipid content than common cereals, but it is less in comparison to oilseeds.p ≤ 0.05) oil content was detected in mutant varieties “Zobor” (5.55 ± 0.39%) and “Pribina” (5.42 ± 0.32%), followed by genotype Ficha (5.41 ± 0.56%), and hybrid K-433 (5.41 ± 0.23%) over commercially known Aztec (4.86 ± 0.51%), Koniz (4.82 ± 0.40%), and Plainsman (4.57 ± 0.29%).The crude oil content in the analyzed amaranth seeds  ranged fAmaranthus hybridus, 4.58% in Amaranthus hypochondriacus, and 3.21% oil in Amaranthus cruentus seeds, whilst Zhang et al. [Amaranthus dubius and Amaranthus tricolor seeds . According to the reports, oil content was influenced by amaranth species, cultivar, agrotechnical practices, and growing location. This is also supported by the findings of other authors [Amaranthus cruentus and Amaranthus hypochondriacus from 6.39 to 8.20%, whereas in another study oil levels ranged from 1.09 to 5.00% in Amaranthus cruentus and from 3.03 to 5.97% in Amaranthus hypochondriacus [He and Corke  reportedg et al.  determin authors ,56. Theyndriacus .Squalene (SQ) is one of the unsaturated hydrocarbons of triterpene that is synthesized in plants, animals, fungi, and bacteria as a precursor for the synthesis of hormones, vitamins, and sterols . SqualenMutant variety “Pribina” showed the highest SQ value (6.94 ± 0.70%), while the lowest content was detected in variety Aztec  oil and SQ yield compared to commercial varieties, Plainsman, Aztec, and Koniz.We can conclude that the seeds of the mutant varieties “Pribina” and “Zobor”, and genotype Ficha, can be considered an exclusively rich plant source of SQ in comparison to other investigations ,55,62,63Oil extracted from amaranth grain  is attrp ≤ 0.05) only for myristic, oleic, and linoleic acid content in the analyzed amaranth seeds  showed the lowest content of oleic acid (23.80%) but the highest content of linoleic acid (40.70%).Mutant variety “Zobor” exhibited a significantly lower content of oleic acid, which may be caused by the negative correlation between linoleic and both oleic and palmitic acids ,55. In tAmaranthus hypochondriacus (31.50%) and in Amaranthus cruentus (31.30%). The content of another saturated FA, stearic acid, ranges according to different species and different authors reported yields from 0.1% in Amaranhus asplundii to 4.1% in Amaranthus dubius [The content of saturated palmitic acid varied between 19.67% (Pribina) and 20.14% (’Zobor’), which is in accordance with most published data ,62,65. Hs dubius ,56. The Amaranthus cruentus and Amaranthus hypochondriacus, respectively.The values of α-linolenic acid in the analyzed amaranth seeds ranged from 0.69 to 0.75%. Similar data were previously obtained by Jahaniaval et al.  and LeónThe saturated FA comprised on average 24.03% of the total FA and the unsaturated FA 75.00%, leading to an average saturated/unsaturated ratio of 0.32. Similar values were determined by other authors ,55,56.Our results confirmed previous reports, suggesting amaranth oil as a good source of unsaturated FA with high levels of linoleic acid suitable for human nutrition .Amaranthus cruentus L.  and Amaranthus hypochondriacus x Amaranthus hybridus . Slovak mutant varieties “Pribina” and “Zobor” were bred at the home institute—Institute of Plant Genetics and Biotechnology by mutation breeding [Two grain amaranth species were evaluated in the present study: breeding ,38. Briebreeding  in the JAmaranth plants were cultivated in three consecutive years (2016–2018) at the locality of Nitra , with a mean annual precipitation of 600 mm and a mean annual temperature of 9.5 °C. The soil type at the experimental field in Nitra is a Haplic Luvisol with pH of 7.4. Weather conditions  in the vegetation season of all three years are shown in Seeds were manually sowed in May and plants were harvested in September in each year. The seeds were hand-threshed from dried panicles. During the growing season, no fertilizers or sprays were applied.The weight of 1000 seeds was calculated as an average value of 10 independent measurements for each sample.2O2 and 85 % HCOOH. 1:9) was determined in an automatic amino acid analyzer AAA 400  and results were analyzed using CHROMuLAN software. Seeds and leaves were homogenized for both procedures, and a 0.5 g sample was used. Hydrolysis was carried out at 110 °C while refluxing under a stream of nitrogen for 23 h.Essential amino acids: lysine (Lys), histidine (His), arginine (Arg), threonine (Thr), valine , isoleucyne (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe) and nonessential amino acids: aspartic acid (Asp), glutamic acid (Glu), serine (Ser), proline (Pro), glysine (Gly), alanine (Ala), tyrosine (Tyr), and cysteine (Cys) were analyzed in amaranth seeds from all amaranth samples (in duplicate) except variety Aztec. The content of amino acids after acid hydrolysis (6M HCl) and sulphur amino acids after oxidation hydrolysis . Extractions were performed in an ASE350 apparatus (Dionex). Twelve grams of seed powder were mixed with 1.1 g of Celite powder and added to a 22 mL extraction cell. The extraction solvent was 100% n-hexane, the extractuion emperature was 70 °C, the static time was 10 min, and 3 extraction cycles were performed. After extraction, the solvent was evaporated using a rotavapor apparatus (Büchi). The temperature of the water bath was set at 60 °C and the vacuum was set at 500 mbar. After evaporation, the flasks were left to stand open overnight for any residual solvent to be removed, after which the mass of oil was recorded. The amount of squalene in the oil was determined by gas chromatography  using a capillary MXT-5 column  equipped with a SSL injector (inlet temperature 350 °C) and FID detector  under a temperature gradient . A constant helium flow of 7.0 mL/min was applied.Samples for GC analysis were prepared by mixing 0.1 g of a 10 m% oil solution in n-heptane with 0.04 g of a 1 m% tetradecane solution in n-heptane  and diluting to 1 g with n-heptane. To perform quantitative analysis of squalene a calibration was performed using a squalene standard (Acros Organics). Analysis results were evaluated using Chromeleon 6 software.The gas chromatography  equipped with capillary column DB-23  and FID detector  was used for amaranth oil metyl esters fatty acids (FAME) profile analysis . Tempera2). Isolation between each amaranth variety was secured by a corn plot (1 m wide).The experimental field design was a block in a split plot arrangement with four replications. For each experimental variant, the plot size was 2.0 m × 1.5 m . Analysis of variance (ANOVA), Tukey HSD multiple comparison test, and Fisher LSD test were employed to identify significant differences among the different processes at The mutant varieties analyzed in this report exhibited stable and consistently superior performance of 1000-seed weight over nonirradiated controls and commercial varieties during a three year period. The content of all tested amino acids in mutant variety “Zobor” seed was the highest from all analyzed amaranth samples, except for methionine and cysteine. In addition, “Zobor” seeds had a significantly higher content of essential linoleic acid. Furthermore, both mutagenesis-derived varieties “Pribina” and “Zobor” had significantly higher oil and squalene yield compared to the three tested commercial varieties: Aztec, Plainsman, and Koniz. Thus, these two Slovak varieties can be used by farmers and producers as promising new germplasms generated by induced mutagenesis.


To explore the relationship between body mass index (BMI) and clinicopathological characteristics in patients with papillary thyroid carcinoma (PTC).2), overweight recombination (24.0 ≤ BMI < 28.0 kg/m2) and obesity group (BMI ≥ 28.0 kg/m2). The clinicopathological characteristics of PTC in patients with different BMIs group were compared.The clinical data of 1,579 patients with PTC, admitted to our hospital from May 2016 to March 2017, were retrospectively analyzed. According to the different BMI of patients, it can be divided into underweight recombination (BMI < 18.5 kg/m), normal body recombination , advanced T stage (T III/IV), and advanced tumor-node-metastasis stage (TNM III/IV) in the overweight group were higher, with OR (odds ratio) = 1.99(1.41–2.81), OR = 2.01(1.43–2.84), OR = 2.94(1.42–6.07), respectively, relative to the normal weight group. The risk for ETE and T III/IV stage in the obese group were higher, with OR = 1.82(1.23–2.71) and OR = 1.82(1.23–2.70), respectively, relative to the normal weight group.BMI is associated with the invasiveness of PTC. There is a higher risk for ETE and TNM III/IV stage among patients with PTC in the overweight group and for ETE among patients with PTC in the obese group. The incidence of thyroid cancer has been increasing in recent years worldwide. Thyroid cancer in women has become the fifth most common malignant tumor in the United States . ThyroidA total of 1,702 patients with PTC  who received surgical treatment in Tianjin Medical University Cancer Institute and Hospital from May 2016 to March 2017 were considered. After excluding patients with histories of thyroid surgery, antithyroid drug consumption, and thyroxine administration before surgery, 1,579 subjects were eligible for analysis in this study.  Each participant signed an informed consent form, which was uploaded in supplementary materials. This study was approved by the Ethics Committee of the Tianjin Medical University Cancer Institute and Hospital. Ethics Committee reference number is Ek2018117.2, underweight; 18.5 ≤ BMI < 24.0 kg/m2, normal weight; 24.0 ≤ BMI< 28.0 kg/m2, overweight; and BMI ≥ 28.0 kg/m2, obese)  levels, combined with postoperative pathological features, including tumor size (maximum diameter of the tumor), lymph node metastasis, multifocality, and the extrathyroidal extension (ETE) and TNM stage based on the eighth edition of the Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC). We reviewed the height and the weight of the patient during admission, calculated BMI according to the Chinese obesity classification standard  . SubsequLogistic regression analysis was used to analyze the relationship between BMI and the clinical pathological features of thyroid cancer. The odds ratio (OR) and 95% confidence interval were used. The adverse clinicopathological features analyzed included multifocality (number of lesions ≥ 2), tumor size ≥ 1 cm, ETE, lymph node metastasis, high T stage (stage III + IV), and high TNM stage (stage III + IV). Logistic regression  was used to analyze the relationship between BMI and the adverse clinicopathological features of PTC. Similarly, logistic regression analysis (adjusting for age and TSH) was used to analyze the relationship between BMI and adverse clinicopathological features of PTC in men and women. For those older than ≥55 years and <55 years, logistic regression analysis (adjusting for gender and TSH) of the relationship between BMI and adverse clinicopathological features of PTC was performed.The Chi-square test was used to analyze whether there were differences in gender, age, level of TSH, number of tumors, tumor size, ETE, lymph node metastasis, T stage, and TNM stage among different BMI groups.P < 0.05.Statistical analysis was performed using SAS V9.3 software  with a statistical significance noted at 2 with mean BMI 25.52 ± 3.79 kg/m2. A total of 704 patients (44.6%) had lymphatic metastasis, 228 (14.4%) had ETE and 565 (35.5%) had multifocal tumors. With regards to the T stage, 1,322 (83.7%) patients were in the T1 stage and 257 patients (16.3%) were in the T3/4 stage. With regards to the TNM stage, 1,515 (95.9%) patients were in stage I and II, and 64 patients (4.1%) were in stage III and IV  years, a median age of 46 years, 1,129 patients (71.5%) aged <55 years, and 450 (28.5%) aged ≥55 years. BMI ranged from 16.00 to 48.33 kg/mI and IV .χ2 = 80.28, P < 0.0001) and age  between different BMI groups. BMI is associated with invasion of the envelope , T stage , and TNM stage (P = 0.0002) in the pathological features of the tumor (There are differences in the distribution of gender (he tumor .P < 0.0001), high T stage , and TNM III/IV stage  than patients with a normal weight. Subjects in the obese group also had a greater risk of ETE  and high T stage  than normal weight subjects. Whether in the overweight or obese group, BMI has no correlation with lymph node metastasis.We further explored the risk of more aggressive clinicopathological features according to BMI . MultiplP = 0.0002), high T stage , and TNM III/IV tumors ; the obese group had a greater risk of ETE , high T stage , and TNM III/IV tumors   . In maleP = 0.009, ORs = 2.18(1.22–3.89), P = 0.008, and ORs = 2.42(1.15–5.13), P = 0.02, respectively. ETE  and high T stage  were each more frequent in the obese group than in the normal weight group , ht group .P = 0.01, ORs = 1.80 (1.16–2.78), P = 0.008 respectively. ETE , high T stage  and multifocality  were each more frequent in the obese group than in the normal weight group , ht group .Thyroid cancer is the most common malignant tumor in the endocrine system. Its incidence has increased year by year in the past 20 years. In 2012, the number of new cases of thyroid cancer in China accounted for 15.6% of the global number of new cases, and the number of deaths accounted for 13.8%2. PTC is the most common histological type of thyroid cancer, accounting for about 80% of its incidence . In receP = 0.02) and the risk of ETE  increased significantly in overweight group, while tumor size, lymph node metastasis, and multifocal tumors were not significantly associated with BMI; the risk of ETE  in the obese group increased with BMI. At present, the relationship between obesity and the pathological features of PTC remains controversial. At present, molecular mechanisms related to obesity and tumors indicate that obesity can promote tumor invasion and metastasis through a variety of obesity-related factors and metabolic pathways . AdiponeIn summary, obesity is closely related to the risk of PTC and the invasiveness of tumors. Controlling body weight through regular exercise and a reasonable diet and reducing obesity should be important prevention and treatment methods for patients with papillary thyroid cancer and high-risk groups.10.7717/peerj.9675/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9675/supp-2Supplemental Information 2Click here for additional data file.


The aim of the presented study was to evaluate the differences between selected biochemical markers in infants with moderate or severe hypoxic-ischemic encephalopathy (HIE) and their impact on patient prognosis.A total of 57 cooled newborns were divided into groups according to Sarnat staging of HIE . The differences between groups were evaluated depending on the mode of delivery, pregnancy and labor complications, gestational age at birth, birth weight, and Apgar score at 1.3 and 5 min. The differences in biochemical biomarkers of HIE  as well as biomarkers of hepatic injury , alanine transaminase (ALT), prothrombin time (PT), and activated partial thromboplastin time (APTT)), kidney failure , myocardial injury (troponin T (TnT)), levels of fibrinogen, and platelet counts were also examined. Univariate Kaplan-Meier method was used for survival analyses.The biomarker levels in severe HIE newborns compared with moderate were as follows: pH (7.10 vs. 6.99), serum lactate (22.50 vs. 17.00 mg/dL), AST (109.50 vs. 270.55 IU/L), ALT (27.30 vs. 108.05 IU/L), PT (17.00 vs. 44.20 s), APTT (47.75 vs. 47.90 s), TnT (0.22 vs. 0.85 ng/mL), creatinine (0.68 vs. 1.15 mg/dL), urea (44.55 vs. 73.30 mg/dL), and fibrinogen (1.65 vs. 1.90 mg/dL). Survival analyses showed significantly reduced survival for severe HIE infants (75%) vs. moderate HIE (100%).In conclusion, the severity of HIE can be evaluated based on selected markers; however, their levels do not correspond with future prognosis of newborns. Neonatal encephalopathy (NE) is a term used to describe a neurological syndrome caused by a disorder of the brain. Clinical manifestations of NE include features of altered level of consciousness, respiratory depression, seizures, and abnormal muscle tone and reflexes . NE occuHIE is associated with increased risk of neonatal mortality; 15–20% of neonates die due to complications of perinatal asphyxia . MoreoveEvaluation of the severity of HIE is a crucial determinant in predicting the future outcomes for children with HIE. Physical examination combined with neurological assessment, performed according to Sarnat staging , gives uMeta-analyses of previous studies showed that therapeutic hypothermia (TH) reduces mortality and neurodevelopmental disability in groups of term or late preterm newborns with suspicion of peripartum asphyxia . Before Taking into consideration that early evaluation of severity of HIE, as well as early detection of potential multiorgan injury caused by asphyxia in diagnosed neonates, can be helpful in the prognosis of newborns’ outcomes; the aim of the presented study was to assess whether the differences between selected biochemical markers in infants with moderate or severe HIE affect the future patient’s prognosis.A retrospective study of 57 neonatal patients with diagnosed HIE was conducted in the Department of Neonatology at Poznan University of Medical Sciences between 2009 and 2017. Therapeutic hypothermia was introduced into the standard clinical practice for infants born ≥ 36 weeks of gestation with suspected perinatal asphyxia within 6 h after birth. Before the application of TH, newborns with presumed HIE were evaluated in accordance with specific eligibility criteria. All patients enrolled in the study met both treatment criteria A and B. Criterion A included at least one of the following conditions: (1) acidosis of arterial blood or the blood obtained from the umbilical cord  or base excess (BE) ≥ 16 mmol/l; (2) Apgar score ≤ 5 at 10 min after birth; and (3) resuscitation ≥ 10 min after birth. Criterion B included patients with moderate-to-severe HIE according to Sarnat staging, especially with a state of impaired consciousness (lethargy or coma), and with at least one additional feature such as decreased muscle tone, abnormal stimulus response , and clinically proven seizures. Before induction of the cooling procedure, the patient’s brain activity was assessed for at least 20 min using an amplitude-integrated electroencephalogram (aEEG). All of the aEEG records presented features of moderate or severe brain dysfunction and seizures. Moderately abnormal aEEGs were defined as visible changes in the activity band (discontinuous background pattern—lower margin of the band < 5 μV). The presence of burst suppression, low continuous voltage, or flat tracing in recorded activity band was classified as patterns of severely abnormal aEEG .Patients were cooled with a selective head cooling Cool-Cap system (14 neonates) and a whole-body cooling device . After application of therapeutic hypothermia, neonates were cooled for 72 h. Selective head cooling (SHC) and whole-body cooling (WBC) require a continuous body  temperature measurement. During SHC, the core body temperature was reduced to 34.5 °C. In a group of patients cooled with WBC, core temperature was maintained at 33.0–33.5 °C. After that, the patients were slowly rewarmed (0.5 °C/h) to 36.5 °C.Newborns were divided into groups according to Sarnat staging of HIE . The differences between groups were evaluated depending on the mode of delivery, pregnancy and labor complications, gestational age at birth, birth weight, and Apgar score at 1.3 and 5 min. The differences in biochemical plasma biomarkers of HIE were also evaluated. The comparison of metabolic acidosis parameters (pH and base excess) was performed on fetal umbilical cord arterial blood samples obtained immediately after birth. The measurements of pH, base excess, and level of serum lactate were carried out on two additional specimens of blood obtained from umbilical vessels after 1 h and at latest after 4 h after birth. The levels of plasma biomarkers of hepatic injury (aspartate transaminase (AST), alanine transaminase (ALT), prothrombin time (PT), and activated partial thromboplastin time (APTT)), kidney failure , myocardial injury , levels of fibrinogen, and platelet counts were also examined. All blood samples were collected in the first 24 h after birth.U test, Student’s t test, or Cochran-Cox test.Statistical analysis was performed using Statistica. Descriptive statistics are presented as percentages for categorical variables, mean and standard deviation for continuous, normally distributed variables, and median with range for continuous variables with asymmetry of distribution. Normality of the distributions was checked by Shapiro-Wilk test. The prevalence of variables was assessed by the chi square test, the Fisher exact test, or Fisher-Freeman test. Continuous variables were compared using the Mann-Whitney A total of 57 neonatal patients were enrolled in the study. Table p = 0.027) and results of base excess in material obtained from two samples . Statistically significant results were also observed in the levels of hepatic injury biomarkers—AST  vs. B, 270.55 IU/L (range 66.10–3175.00), p = 0.0004), ALT  vs. B, 108.05 IU/L (range 16.60–1195.30), p = 0.0008), PT  vs. B, 24.40 s (range 14.50–320.00), p = 0.0004), APTT  vs. B: 47.90 s (range 30.30–400.00), p = 0.302); myocardial injury biomarker—TnT  vs. B, 0.85 ng/mL (range 0.05–10.00), p < 0.0001); and kidney failure markers—creatinine  vs. B, 1.15 mg/dL (range 0.63–3.16), p < 0.0001) and urea  vs. B, 73.30 mg/dL (range 32.20–137.90), p = 0.002). Also, different levels of fibrinogen were noted  vs. B, 1.90 mg/dL (range 0.30–6.90), p = 0.274) and thrombocytopenia  was observed in 8/27 cases of moderate HIE and in 20/29 cases of severe HIE (p = 0.003).The neurological examination provided information about clinical staging of HIE. Patients were divided into groups according to Sarnat staging: 27 out of 57 (47.4%) met the criteria of a group HIE - A moderate; the rest of them 30 out of 57 (52.6%) were classified as cases of severe HIE - B. Statistically significant differences between the groups of moderate and severe HIE were noted (Table p = 0.100826), TnT (p = 0.218603), AST (p = 0.957588), ALT (p = 0.285924), creatinine (p = 0.794386), or urea (p = 0.306928) had no effect on the probability of survival of patients with HIE  compared with that of patients with moderate HIE (group A). While the survival probability for newborns with moderate HIE is 100%, for children with severe HIE, it is only 75% , however higher levels of serum lactate in the severe HIE infants compared with the moderate in second specimen taken at latest after 4 h after birth (p = 0.027). This is in accordance with studies of Helijic et al. [In the presented study, besides neurological examination, serum lactate was used to predict the severity and outcome of neonatal HIE. Interestingly, we noted no significant differences between tested groups in first specimen taken in the first hour of life . Moreover, greater differences between moderate and severe HIE babies (two times more vs. four times more) were observed in the case of ALT, which is considered to be a more specific marker for hepatic injury than AST [p = 0.0004), which confirmed the observations obtained in another cohort study [Ischemic hepatic injury occurs in about 40–60% of the diagnosed infants with HIE . ALT andthan AST . The resthan AST , 31, 32,rt study . In turnrt study .p = 0.002 and p < 0.001, respectively). These findings mean that kidney biomarkers, such as urea or creatinine concentrations, correlated with the degree of HIE. Similarly, a rising trend in the concentrations of blood urea and creatinine as HIE staging of neonates progressed was also observed by El-Gamasy et al., Gopal et al., and Alaro et al. [If kidney function is impaired, urea and creatinine cannot be removed from the body, and their blood levels increase. Frequently impaired renal function occurs as a result of acute kidney injury (AKI), which concerns 7–72% of asphyxiated infants and alters their urinary biomarker profile . It is wo et al. –37. Inteo et al. . Generalo et al. . Moreovep < 0.0001). Also, Gunes et al. found that TnT concentrations in the first hours of life were significantly higher in HIE grade 3 compared with that in grades 1 and 2 [Myocardial damage occurs in 28 to 73% of neonatal asphyxia cases and is the main cause of neonatal mortality associated with hypoxia–ischemia . Asphyxi 1 and 2 . Signifi 1 and 2 . Another 1 and 2 . Moreove 1 and 2 . Importa 1 and 2 .p = 0.003), which suggests a correlation between morbidity and severity of HIE. These findings were in agreement with data from Bala et al., who showed reduced platelet counts in HIE and noted that during the first 48 h of life it is related to severity of HIE [Thrombocytopenia, defined as a platelet count < 150,000/mL, is associated with increased risk of bleeding, and severe thrombocytopenia  is associated with significant morbidity . As a reAs the endpoint, we tried to assess whether severity of HIE and selected biomarkers can be helpful in the prognosis of infants’ outcomes. Although the results of our analysis showed significantly reduced survival for cases with severe HIE compared with that of patients with moderate HIE, there was no correlation between biomarkers and mortality. There are data showing that several biochemical markers, including serum S100b and neuron-specific enolase (NSE), are predictors of death, neurological abnormalities, or MRI-detected brain injury in hypothermia-treated HIE , but theIn conclusion, the severity of the hypoxic-ischemic injury can be evaluated based on selected markers; however, their levels do not correspond with future prognosis of newborns.


P < 0.01). More common postoperative complications included hypertensive phase (38.1%), corneal decompensation (23.8%), and tube exposure (14.3%).  The cumulative failure rates were 9.5%, 20.0%, 32.5%, and 46.0% at six months, one year, two years, and three years of follow-up. At final follow-up, complete success and qualified success rates were 23.8% and 33.3%, respectively; mean IOP and number of medications decreased by 5.6 mmHg (23.9%) and 1.7 mmHg (54.8%), respectively, from preoperative baseline ( An additional AGV implant had good short and modest long-term effectiveness in reducing IOP following a failed glaucoma tube shunt in Asian eyes, with the mentioned common postoperative complications to be actively monitored and managed. The tube versus trabeculectomy (TVT) study showed that the glaucoma tube shunt group experienced fewer early postoperative complications, were less likely to undergo additional glaucoma surgery, and had higher success rates when compared to using trabeculectomy with MMC [Glaucoma tube shunts are effective in lowering intraocular pressure (IOP) for eyes with refractory glaucoma –4. One owith MMC , 6. Thiswith MMC , 8.If the first glaucoma tube shunt implant fails to control IOP adequately, there are limited surgical options for subsequent IOP control which includes revision of the primary tube shunt, implantation of a second GDD, and cyclodestructive laser procedures. Shah et al. reported that additional tube shunt has better IOP control than tube shunt revision by excision of an encapsulated bleb . LevinsoCurrently, several studies have reported the surgical outcomes of a second AGV, with varying success, but there is a lack of studies in an Asian glaucoma population. The purpose of this study is thus to evaluate the short- and long-term outcomes of a second AGV implant and to define risk factors for failure of the second AGV implant in an Asian population.This is a retrospective study that is approved by the National Health Group Domain Specific Review Board. It was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and that are consistent with the Singapore Good Clinical Practice and the applicable regulatory requirements.We reviewed the medical records of 569 patients who consecutively underwent an AGV implantation between January 2008 and March 2019, at the Eye Surgery Centre, National University Hospital, Singapore. In total, 32 eyes of 31 patients received a second AGV implantation (FP7 model) after their first GDD has failed to control the IOP. Patients who underwent a second AGV implantation during this period were excluded from this study if their postoperative follow-up period was less than 3 months, or if their medical records were missing or insufficient for the purposes of this study.The demographic and ocular characteristic data collected included the age, gender, race, eye laterality, history of hypertension and diabetes mellitus, glaucoma diagnosis, history of trabeculectomy, date of first GDD implantation, lens status , visual field results, clinical vertical cup-to-disc ratio, best corrected visual acuity (BCVA), IOP, and the number of IOP-lowering medications. Intraoperative complications and placement location of the second AGV were also collected. Postoperative data collected included BCVA, IOP, and IOP-lowering medications at each follow-up visit and any postoperative complications and surgical interventions.After informed consent, all the patients underwent AGV implantation at a selected quadrant which did not have significant conjunctiva scarring or preexisting scleral thinning. The AGV implants were all primed with balanced salt solution injected from a blunt cannula. After adequate conjunctival peritomy and dissection, the anterior portion of the AGV plate was sutured to the sclera (avoiding the recti muscles) between 8.5 and 9.0 mm posterior to the limbus. After anterior chamber paracentesis was performed, a 25 or 23G needle was used to create a scleral track into the anterior chamber. The tube was shortened and inserted into the anterior chamber just above the iris plane or into the mid-anterior chamber depth. The tube was sutured down onto the sclera and a piece of sterilised bovine pericardium  was fixated over the tube. The conjunctiva was approximated and sutured to the limbus.The primary outcome measure was the surgical success rate, classified as complete success (IOP ≥5 mmHg and ≤21 mmHg without the use of IOP-lowering medications) and qualified success (IOP ≥5 mmHg and ≤21 mmHg with the use of IOP-lowering medications). Failure was defined as any of the following: IOP >21 mmHg or <5 mmHg for 2 or more consecutive visits after three months of follow-up, surgical intervention for IOP-related indications, removal of the second AGV implant, or loss of light perception.We defined early postoperative complications as those occurring within the first 12 postoperative weeks, and late postoperative complications as those occurring after this time period. We defined the presence of a hypertensive phase as having an IOP >21 mmHg in the first three postoperative months , 15.Kaplan–Meier survival analysis was applied to evaluate the cumulative failure rates based on the criteria above. All BCVA measured by the Snellen chart were converted to logMAR (logarithm of the minimum angle of resolution) for analysis, with any visual acuity worse than or equal to 6/60 on the Snellen chart being allocated a logMAR value of 1.00. Wilcoxon signed rank test was used to compare the continuous variables of IOP, number of medications, and BCVA at each follow-up time point with baseline levels.P value of <0.2 in univariate analysis were included in the multivariate analysis. Risk factors with a P value of <0.05 in the multivariate analysis were considered statistically significant.Univariate and multivariate cox proportional-hazards regression analyses were used to evaluate risk factors for failure. The following preoperative characteristics were evaluated as possible risk factors: age, gender, race, history of hypertension and diabetes mellitus, glaucoma diagnosis (primary vs. secondary glaucoma), history of trabeculectomy surgery, duration between the first and second GDD implantation, lens status, visual field results, clinical vertical cup-to-disc ratio, best corrected visual acuity (BCVA), IOP, and number of IOP controlling medications; other risk factors considered were intraoperative location of second AGV implantation and the presence of postoperative corneal graft. Risk factors with a The records of 32 eyes of 31 patients were reviewed. 11 eyes were excluded . In total, 21 eyes of 20 patients with a second AGV implantation were included in the final analysis of this study. Demographic characteristics of the study sample are shown in The Kaplan–Meier survival analysis demonstrated cumulative failure rates of (standard errors) of 9.5% (6.4%), 20.0% (9.0%), 32.5% (11.2%), and 46.0% (12.4%) at six months, one year, two years, and three years of follow-up according to the criteria defined above . The medP=0.006) at three months, 18.1 ± 5.0 mmHg (P=0.003) at six months, 16.7 ± 3.9 mmHg (P=0.003) at one year, 16.8 ± 4.1 mmHg (P=0.003) at two years, and 16.2 ± 3.0 mmHg (P=0.011) at three years (P < 0.001). Out of 21 subjects, 17 (81.0%) have a lower IOP at final follow-up than their baseline preoperative IOP (The mean (±SD) IOP decreased from a baseline preoperative value of 23.4 ± 5.4 mmHg to 19.0 ± 5.0 mmHg (ee years . The meative IOP .P < 0.001) at three months, 0.7 ± 1.1 (P < 0.001) at six months, 0.8 ± 1.0 (P=0.001) at one year, 1.0 ± 1.4 (P=0.003) at two years, and 1.5 ± 1.5 (P=0.023) at three years (P < 0.001).The mean (±SD) number of medications decreased from a baseline preoperative value of 3.1 ± 0.8 to 0.6 ± 0.9 .The mean (±SD) BCVA increased from a baseline value of 0.47 ± 0.34 logMAR to 0.53 ± 0.29 logMAR at three months, 0.56 ± 0.35 logMAR at six months, 0.49 ± 0.37 logMAR at one year, 0.60 ± 0.37 logMAR at two years, and 0.62 ± 0.40 logMAR at three years of follow-up. The increase in BCVA at each time point compared to baseline was statistically insignificant  experienced a hypertensive phase after the second AGV implantation. There were three eyes (14.3%) with tube exposure and were repaired with a corneal graft patch surgery. In total, two eyes (9.5%) developed corneal decompensation and bullous keratopathy, both of which subsequently underwent corneal endothelial transplant surgery. In total, nine eyes (42.9%) required further IOP-lowering interventions . There were no cases of diplopia or ocular movement limitation and no AGVs were explanted.P < 0.2) were a history of diabetes mellitus (P=0.19), reduced cup-to-disc ratio (P=0.01) and visual field index (P=0.17), higher preoperative IOP (P=0.14), and higher number of preoperative medications (P=0.19). However, there were no statistically significant risk factors for failure identified using the multivariate Cox proportional hazards model (P < 0.05).The risk factors identified with univariate analysis , while being on fewer IOP-lowering medications. Patients had a statistically significant reduction in both their IOP and number of medications at six months, one year, two years, and three years of follow-up.The cumulative failure rates of our study determined by Kaplan–Meier analysis was 9.5%, 20.0%, 32.5%, and 46.0% at six months, one year, two years, and three years of follow-up, respectively. Whilst the short-term success rates were good, the longer-term success rates of a second AGV implantation were relatively modest. We reviewed studies which investigated the surgical outcomes of second GDDs, specifically AGV . These sThe success rates of the second AGV at the last follow-up in these studies ranged from 65.2%  to 84.2% , while our study had a lower success rate of 57.1% at the last follow-up –18. ThisThe mean IOP reduction for our study (23.9%) was lower than that reported by the other studies, which ranged from 42.0% to 52.9%. This could be due to differences in study methodology, population demographics, and types of glaucoma drainage implants. Preoperative IOP in our study was measured while patients were on medication; the other studies could be measuring preoperative unmedicated IOP, thus leading to a higher preoperative IOP value and a greater degree of IOP reduction postoperatively. In their studies, Ko et al. and Smith et al. included second AGV implants which were of FP8 and S2 models; however, this may not fully account for the difference in IOP reduction. Koh et al. have shown that there is no difference in IOP reduction between FP7 and FP8 models . MackenzPrevious studies demonstrating the success rates of initial AGVs yielded similar results. Their cumulative success rates ranged from 75.0% to 87.0% at one year, 60.6% to 75.0% at two years, and 54.0% to 66.0% at three years , 23–27. n = 8) of the eyes. This was similarly reported by Jiménez-Román et al. but at a lower rate of 10.3% [In our study, there were no serious intraoperative or postoperative complications which led to a loss of light perception. However, the most frequent early postoperative event was the development of a hypertensive phase, which occurred in 38.1%  [We found tube exposure of the second AGV to be the most frequent late postoperative complication, occurring in three eyes (14.3%). A widely proposed mechanism of tube erosion and exposure is a combination of mechanical forces of the eyelid and tension of the tube on the conjunctiva . The higP=0.006) . LevinsoP=0.006) . When coP=0.006) –20. The P=0.006) .Another significant late postoperative complication which we observed was corneal decompensation and bullous keratopathy 9.5%). Jiménez-Román et al., Ko et al., and Smith et al., whose studies included S2 AGV models as well, reported similar findings, albeit at varying frequencies of 17.2%, 13.0%, and 5.3%, respectively , 17, 20..5%. JiméP=0.027) [P < 0.001) and a younger age at surgery  were risk factors for failure, respectively [Ko et al. reported prior trabeculectomy to be a significant risk factor for failure of the second AGV  . Fatehi ectively , 20. MeaThere were some limitations of our study. The small sample size could have affected the accuracy of the Cox proportional hazard model analysis, which could explain the lack of risk factors for failure identified. Our study sample consisted mostly of male eyes without prior trabeculectomy, which would limit its generalizability. The retrospective nature of the study design was also a limitation in evaluating outcomes, success rates, and determining causality of risk factors. A larger, prospective randomised study over a long follow-up period is needed to confirm the results of this study.In conclusion, the implantation of a second AGV following a previous failed AGV implantation has good short-term but modest long-term success rates in reducing IOP. Most of these patients will still require IOP-lowering medications, but with a fewer number than preoperative baseline, for optimal IOP control after the second AGV surgery. Hypertensive phase, tube exposure, and corneal decompensation are frequent complications which should be actively monitored and managed, especially among the Asian population.