Paenibacillus jamilae DSM 13815T DSM (LA22), Bacillus subtilis ssp. spizizenii DSM 15029T DSM (MA3), and Pseudomonas aeruginosa A07_08_Pudu FLR (SN36). A test was conducted to evaluate the effect of (1) isolated heavy metal–resistant bacteria (soil application), (2) a foliar spray with silicon dioxide nanoparticles (Si-NPs), and (3) moringa leaf extract (MLE) on the production, antioxidant defense, and physio-biochemical characteristics of spinach grown on heavy metal–contaminated soil. Bacteria and MLE or Si-NPs have been applied in single or combined treatments. It was revealed that single or combined additions significantly increased plant height, shoot dry and fresh weight, leaf area, number of leaves in the plant, photosynthetic pigments content, total soluble sugars, free proline, membrane stability index, ascorbic acid, relative water content, α-tocopherol, glycine betaine, glutathione, and antioxidant enzyme activities  compared with the control treatment. However, applying bacteria or foliar spray with MLE or Si-NPs significantly decreased the content of contaminants in plant leaves , malondialdehyde, electrolyte leakage, superoxide radical 2O2). Integrative additions had a more significant effect than single applications. It was suggested in our study that the integrative addition of B. subtilis and MLE as a soil application and as a foliar spray, respectively, is a critical approach to increasing spinach plant performance and reducing its contaminant content under contaminated soil conditions.Microorganism-related technologies are alternative and traditional methods of metal recovery or removal. We identified and described heavy metal–resistant bacteria isolated from polluted industrial soils collected from various sites at a depth of 0–200 mm. A total of 135 isolates were screened from polluted industrial soil. The three most abundant isolate strains resistant to heavy metals were selected:  Spinacia oleracea L.) is a critical vegetable for the human diet, owing to its richness in carbohydrates, protein, and mineral nutrients (Spinach (utrients . Spinachutrients . Spinachutrients . Each 10utrients . Spinachutrients .−), superoxide radical 2O2), which are highly toxic to plants, are the main consequence of heavy metal stress , lead (Pb), copper (Cu), and arsenic (As). Therefore, the strain Paenibacillus sp. RM can be used as a heavy metal bioremediator  promotes plant growth because it is rich in plant hormones, including large amounts of zeatin, carotenoids, phenols, antioxidants, osmoprotectants, and nutrients  application enhances the ability of the plant to cope with stress . It was Bacillus, Paenibacillus, and Pseudomonas; (2) foliar spray with MLE or Si-NPs; and (3) both treatments combined in spinach grown on heavy metal–contaminated soils. Specifically, we evaluated the vegetative growth, physiological properties, antioxidant defense system, biochemical components, and heavy metal accumulation of spinach plants grown under such circumstances. We hypothesize that applying Bacillus, Paenibacillus, and Pseudomonas as supplements and MLE or Si-NPs as foliar spray would enhance spinach growth and production. This result would happen by adjusting ion imbalance and raising osmoprotectant contents and antioxidant activities, which play critical roles in alleviating heavy metal stress on spinach.To our knowledge, no study has verified the co-addition of microorganisms and plant extracts or Si-NPs in relieving heavy metal stress on spinach plants growing in soil polluted with heavy metals. Our study aimed to assess the influences of (1) the soil additions of Strains were routinely grown in a Luria Bertani (LB) medium, according to the method described by Soil samples were obtained from industrially contaminated soil from the 10th of Ramadan, Sharqia, Sharkia , Egypt. Then, the soil samples were instantly brought to the laboratory.−1 was preserved for each heavy metal. The segregated colonies were regrown to prepare pure cultures  was estimated using the microdilution broth method following the European Committee on Antimicrobial Susceptibility Testing. A total of 30 ml of each concentration of minerals was added to tubes containing 9 ml of Mueller Hinton broth, and 100 ml of bacteria (1.5 × 108 CFU/ml) was added to the tubes. The control treatment was composed of only Mueller Hinton broth tubes. The tubes were incubated for a day at 37°C. The MIC was the minimal mineral concentration that inhibited bacterial growth, and it was tested at 30°C for 6 days. The three bacterial strains with the highest MIC estimates for Cr, Cd, Pb, Zn, Ni, and Hg were then selected for further experimental steps . The agaThe bacteria were identified using morphological and biochemical characteristics for motility activities, following the manual of systemic bacteriology by The ideal growth conditions were assessed on the basis of temperature and pH. The selected isolates were developed in an LB agar medium at temperatures of 25°C, 30°C, 35°C, and 40°C. The pH values were 5.0, 6.0, 7.0, and 8.0. The log phase of the developed cultures under these conditions for 12–14 h was observed for the optical density at 600 nm using a spectrophotometer .A field experiment was conducted during 2021 growing season on a private farm in Abu-Zabal City, El-Qalyubia Governorate (N 30° 14′ 58″ and E 31° 21′ 16″), Egypt. The area is located within a semi-arid region, with rainfall ranging between 6 and 10 mm and daily average temperatures of 18°C ± 2°C and 12°C ± 3°C at both day and night, respectively, during the trial duration (from October to December). The relative humidity ranged between 35.4% and 38.2%, and the day length was 10–12 h. Before the settlement of the trial season, soil samples were collected randomly from the study site. The samples were preliminarily analyzed before the experiment, following the methodologies by Spinacia Oleracea L., c.v. Polar bear R Z, F1-Hybrid) seeds were brought from the Vegetative Research Section, Horticulture Research Institute, Agricultural Research Centre, Giza. Before sowing the seeds on 25 October 2021, ammonium sulfate at 280 kg ha−1 (20% N) and potassium sulfate at 200 kg ha−1 (48.5% K2O) were broadcast-applied. The experiment followed the split plot design with bacteria treatments as main plots and the foliar amendments as subplots. The treatments were triplicated. After washing with distilled water, the seeds were sterilized with NaClO for 2 min, cleaned out by distilled water, and then left to dry at room temperature (25°C). After air drying, the seeds were sown at a rate of 15 kg ha−1 in hills (spaced 15–20 cm) with spacing of 60 cm between rows in plots with an area of 10.5 m2 for each (3.0 m long × 3.50 m width). Four seeds per each hill were used, which were thinned later to two seedlings before the first irrigation using a drip irrigation system.Healthy spinach , at three equal doses at 15, 30, and 45 days after sowing. The bacterial strains were applied during the last 10 min of drip irrigation.The selected bacterial strains  were grown on T- medium Moringa oleifera leaves were mixed with 80% ethanol at a rate of 200 g/2 L and stirred using a homogenizer as recommended by Moringa oleifera leaf extract was filtered out using Whatman No. 2 filter paper, which was within 5 h from extraction process, or stored at −20°C until use. The used nano-silicon dioxide   was 180–600 m2 g−1 as the surface area. MLE and Si-NPs were applied as foliar spray at rates of 5 ml L−1 and 1.5 mM nano-SiO2, respectively, at three times during the experiment, 15, 30, and 45 days after sowing. MLE and Si-NPs were sprayed using a pressurized spray bottle (0.1% Tween 20 as a surface spreader). Foliar application of distilled water was set as a control.The The plant height for 10 spinach plants in each treatment was estimated after cutting the plants in addition to plant height, leaf area, number of leaves, and fresh and dry weight of shoot (grams per plant). The shoot was dried in an oven at 70°C until constant weight.−1 leaf fresh weight (FW). The membrane stability index (MSI) was determined using 200 mg of fresh leaf (two sets) in test tubes containing 10 cm3 of double-distilled water. One group of samples was heated at 40°C for 30 min. EC was recorded on a conductivity bridge (C1). The second group of samples was boiled at 100°C for 10 min in a boiling water bath, and Electrical conductivity (EC) was measured (C2). As in the study by Photosynthetic pigment contents  were determined using the acetone extract method proposed by The method by −1 FW) was measured in 0.1 g leaf homogenized in a Na-phosphate buffer. The homogenate was centrifuged under cooling at 20,000 × g for 25 min. The supernatant was read at 532 nm and corrected for non-specific turbidity at 600 nm, following the methods proposed by The malondialdehyde (MDA) content (μmol gElectrolyte leakage (EL) was measured in a solution of 20 leaf discs 0.5 cm subjected to room temperature, 45°C–55°C for 30 min, and 100°C for 10 min to measure ECa, ECb, and ECc , respectively. EL was calculated using the following formula, as proposed by −1 FW) was determined by immersing leaf fragments in 10 mM K-phosphate buffer (pH 7.8), 0.05% Nitro blue tetrazolium chloride (NBT), and 10 mM NaN3 for 1 h. After heating the immersed solution (at 85°C for 15 min) and rapidly cooling, the absorbance was read at 580 nm (The superoxide t 580 nm .2O2 content (µmol g−1 FW) was determined in the acetone extract. After adding titanium reagent and ammonium, the extract was dissolved in 1 M H2SO4, and the absorbance was measured at 415 nm (The Ht 415 nm . The dri3–COO–CH2–CH3). The standard solution and dilutions of 0.02–0.2 mg/ml were prepared using R-TOC (0.05 g/0.1 L of n-hexane). The ascorbic acid (AsA) level (μmol/g of leaf FW) was determined following the method by 2SO4 ], boiled for 10 min in a water bath, and cooled. Then, the contents (mg/g of leaf DW) were measured using a spectronic spectrophotometer (Bausch and Lomb-2000) at 625 nm (−1 of leaf DW) was determined using standard L-proline prepared according to The content of α-tocopherol (α-TOC) in the dried leaves in micromoles per gram was determined by a high-performance liquid chromatography system with a mobile phase (94 ml of methanol and 6 ml of water) and a 1.5 ml/min flow rate at 292 nm of a UV detector . Nearly t 625 nm . The prog for 15 min. The supernatant was then referred to as the enzyme extract. The activity of catalase (CAT) was assayed spectrophotochemically according to 2O2, and the total volume was made up of 1 ml of 250 mM phosphate buffer (pH 6.8). The decrease in the optical density at 240 nm against blank was recorded every minute. Ascorbate peroxidase (APX) was assayed spectrophotochemically according to 2O2, and the decrease in the optical density at 290 nm against blank (without extract) was continuously recorded every minute (for 2 min). 2O2 (20 mM), 50 µl of enzyme extract, and 50 µl of guaiacol (20 mM). The reaction mixture was incubated in a cuvette for 10 min at room temperature. The optical density was measured at 436 nm, and the enzyme activity was expressed as the number of absorbance units g−1 FW of leaves. Superoxide dimutase (SOD) activity was measured on the basis of the absorbance peak of superoxide–nitro blue tetrazolium complex  containing 0.5 mM EDTA . Under c complex . The actp ≤ 0.05 using Duncan’s multiple range test. COSTAT computer software  was used to carry out the statistical analysis.The significance of differences between the means of variables was statistically compared at B. subtilis (SA6) and Paenibacillus (AT26) strains were gram-positive, rod-shaped motile bacteria. The Pseudomonas aeruginosa (MM40) strain was gram-negative and rod-shaped. These isolates had optimum growth at 35°C ± 2°C and pH 7.0.In this study, 135 bacterial colonies were examined using an LB agar medium with heavy metals to identify, isolate, and characterize resistant bacteria in polluted industrial soils. Among the analyzed colonies, 61 isolates were chosen for a secondary examination. Later, three strains were selected according to their resistance to heavy metals and were further utilized in this study. −1) prepared from stock solutions. The measurements from the cultures incubated for 48 h agreed with bacterial resistance for every heavy metal. All isolates were sensitive to Ag, Hg, and Ni. Consequently, microorganisms with simultaneous resistance to heavy metals accompanied by their biotransformation into non-toxic forms would be potentially helpful for the detoxification of soils contaminated by heavy metals.Growth studies of the strains SA6, AT26, and MM40 were conducted in an LB medium with Zn, Ni, Pb, Cd, Ag, Hg, and Cr . The bacterial isolates displayed a high ability of heavy metal resistance while we screened these isolates utilizing the second concentration . Primary identification for the three bacterial isolates was made on the basis of 36 tests, including gram reaction, motility, cell morphology, oxygen requirement, spore formation, and biochemical and physiological tests for Bacillus, Paenibacillus, and Pseudomonas identifications , P. jamilae (AT2), and P. aeruginosa (MM40) added as soil supplementation or MLE and Si-NPs applied as foliar spray)] or integrative treatments (bacteria soil application + MLE or Si-NPs) significantly increased plant height, leaf area, leaf number, shoot fresh, and dry weight of spinach plants grown on heavy metal–contaminated soil , phytohormone , cytokinin , and total soluble sugar . It was B. subtilis applied as soil application in integration with MLE used as foliar spray resulted in increments in chlorophyll a (98.6%), chlorophyll b (32.4%), carotenoids (103%), RWC (54.6%), and MSI (42.5%).Chlorophyll a and b and carotenoids, along with the RWC and the MSI of spinach plants grown on heavy metal–contaminated soils, had significant responses to both single and integrative amendments compared with the control (without bacteria application + without foliar spray) Table 4.Heavy metal toxicity in the planting medium causes excess toxic impacts on plants, such as the induction of leaf chlorosis . This revia connecting heavy metals to the cell walls and prohibiting their movement to plants. This reduces the pervasive effects of heavy metals on plants, counteracts premature leaf senescence, and increases leaf area and photosynthesis  of the cell walls in the root system. A high CEC indicates more metal adsorption by the cell walls, which causes more available metal ions for the membrane transfer. In addition, high CEC can amplify the metal content in the cell cytoplasm and thus impact metal tolerance in the plant . RemediaOn the other hand, MLE contains Fe, which activates enzymes involved in the chlorophyll biosynthesis pathway, and some antioxidant enzymes, such as APOX and GSH, which release ROS and protect chlorophyll from degradation . The maj2O2, and B. subtilis as a soil application integrated with MLE as the foliar spray was the best treatment. In that treatment, MDA decreased by 74.1%, EL by 57.8%, 2O2 by 73.5% compared with the control treatment plants.Compared with the control treatment, EL, MDA, H2O2) and raised in spinach plants under heavy metal stress , Pseudomonas aeruginosa (SN36), and Paenibacillus jamilae (LA22) in producing these antioxidants (enzymatic and non-enzymatic) under Cd and/or Pb stresses.Heavy metal stress (Cd and/or Pb) in plants results in the overproduction of ROS, which is regulated by antioxidant defense systems that include non-enzymatic antioxidants including GSH, enzymatic antioxidants , carotenoids, α-TOC, and proline . AccordiThe application of MLE increased the soluble sugar and proline of spinach plants under heavy metal stress, and this may be attributed to the fact that moringa is a good source of amino acids, soluble sugar, and antioxidants . In addiBacillus subtilis as soil amendment with MLE as foliar spray recorded the highest reductions in Fe by 83.7%, Zn by 71.1%, Cu by 44.4%, Mn by 51.4%, Pb by 92.8%, Cd by 87.2%, and Ni by 75.6%, as compared with control , iron (Fe), copper (Cu), manganese (Mn), cadmium (Cd), lead (Pb), and nickel (Ni) in spinach leaves grown on heavy metal–contaminated soil. Both single and integrative treatments significantly declined the contents of Fe, Zn, Cu, Mn, Pb, Cd, and Ni as compared with control, with superior effects of combined application than single ones. The combined application of  control Figure 2via transporters , Paenibacillus (AT26), and Pseudomonas aeruginosa (MM40), and/or foliar spray with MLE or Si-NPs, which play a key role in plant growth due to its beneficial influences on mineral nutrition and mechanical strength and, therefore, plant resistance to abiotic stresses. The leverage of heavy metal–resistant bacteria in combination with MLE or Si-NPs in alleviating the stress in plants reflecting better growth and yield is found to be due to the enhanced antioxidant defense systems, non-enzymatic and enzymatic antioxidants , to decline the ROS damages. The role of heavy metal–resistant bacteria and/or foliar spray with MLE or Si-NPs on supporting the antioxidative defense systems in plants under stress is also reported in our study as a “stay-green effect” due to their usage of an eco-friendly, rapid, and low-cost method.Stress tolerance in spinach plants grown on soil contaminated with heavy metals was effectively enhanced by the addition of heavy metal–resistant bacteria, i.e., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceived and designed the experiments: AE, E-SA, AI, A-RM, and E-SD. Performed the experiments: AE, A-RM, and E-SD. Contributed reagents/materials/analysis tools: AE, A-RM, and E-SD. Wrote the paper: AE and A-RM. Revised the paper: AE, E-SA, AI, A-RM, and E-SD. All authors contributed to the article and approved the submitted version.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To detect viral load in human cytomegalovirus (HCMV) infection children after hematopoietic stem cell transplant (HSCT) by chip digital PCR (cdPCR).  The plasmid pUC57-UL83 containing the HCMV-UL83 gene and HCMV AD169 strain were used to evaluate the sensitivity of cdPCR. Either HSV-1, HSV-2, VZV, EBV, HHV-6, or HHV-7 was used to evaluate the specificity of HCMV cdPCR. The cdPCR was compared with quantitative PCR (qPCR) by detecting HCMV infection in 125 children's whole blood samples following HSCT.  The limit of detection (LOD) of HCMV cdPCR was 103 copies/ml and the qPCR LOD was 297 copies/ml for plasmid pUC57-UL83. The result of HCMV cdPCR was 146 copies/ml for the HCMV AD169 strain, indicating that the sensitivity of cdPCR was higher than that of qPCR. There is no cross-reaction between HCMV cdPCR and other herpes viruses. The incidence of HCMV infection was 30.40% in 125 children following HSCT by cdPCR. The range of the HCMV viral load was from 107 copies/ml to 6600 copies/ml by cdPCR.  cdPCR is more sensitive than qPCR for detecting HCMV viral load. Furthermore, the cdPCR could be used to detect the viral load of HCMV infection before or after HSCT in children. Most peountries –4. As anountries –7. The rountries –10.For high-risk children following HSCT, detection of HCMV infection should precede the appearance of clinical symptoms. For the reason that the higher or more rapidly changing viral load would correlate with both the development of HCMV disease and the higher risk of severe HCMV disease in children following HSCT –14. ThusOur study established a chip digital PCR (cdPCR) method to detect the viral load of HCMV infection before or after HSCT in children and validate its sensitivity, specificity, and repeatability by plasmids and herpes viruses.6 copies/ml plasmid was a 10-fold serial dilution. The plasmid was double diluted 1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32, 1 : 64 by 3.2 × 102 copies/ml (The copy number of plasmid was obtained by the following formula: (6.02 × 1023) × plasmid concentration (g/ml)/(DNA length × 660)). HCMV AD169 strain (5.67 × 106 TCID50/ml) was 10-fold serially diluted from 5.67 × 106 TCID50/ml to 5.67 TCID50/ml. Herpes simplex virus 1 , herpes simplex virus 2 , varicella zoster virus , Epstein–Barr virus , human herpesvirus 6  (HHV-6 foundation), and human herpesvirus 7  (HHV-6 foundation) stored at −80°C. Each experiment has negative and positive controls. The ddH2O was used as the negative control and HCMV AD169 DNA was used as the positive control. The cdPCR and qPCR experiments were repeated three times and these experiments were completed in the second-level biosafety laboratory.The UL83 gene of HCMV was cloned into the plasmid pUC57 to yield pUC57-UL83. The 3.2 × 10+ & R+), 31 cases of D+& recipient negative (R−), 18 cases of D− & R+, and 2 cases of D− & R−. There were 37 cases of acute myelocytic leukemia (AML), 17 cases of acute lymphocytic leukemia (ALL), 22 cases of aplastic anemia (AA), ten cases of myelodysplastic syndrome (MDS), three cases of lymphoma, five cases of neuroblastoma (NB), six cases of Wiskott–Aldrich syndrome (WAS), 25 cases of mucopolysaccharidosis (MPS). HSCT children were collected from May 2018 to May 2020 at the Beijing Capital Institute of Pediatrics Children's Hospital. HCMV viral load in the serum of all recipients was detected by qPCR and cdPCR before HSCT. All children were confirmed according to diagnostic criteria. All subject inclusion was approved by the Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC.One hundred twenty-five children following HSCT were enrolled in this study. Male/Female: 73/52, the median age is 7.5 years old. Among 125 children following HSCT, 122 children were allogeneic HSCT, and three were autologous HSCT. And in three autologous HSCTs, both the CMV status (IgG) of the donors (D) and recipient (R) patients prior to transplantation were negative. Among 122 allogeneic HSCT, there were 68 cases of both donor and recipient positive . The final elution volume was 60 μL and stored at −80°C for use. Eight μL DNA templates of each sample were added into a 25 μL qPCR reactions system [μL of Premix Ex Taq ™ , 200 nM of each primer and probe. The cycling procedure is as follows: 95°C for 30 s; 45 cycles of 95°C for 5 s and 60°C for 30 s .140 s system –21, inclμL DNA was added into the 25 μL cdPCR reaction system containing 5 μL ToughMix buffer , 2.5 μL fluorescein , 200 nM of each primer and probe. The cycling procedure is as follows: 95°C, 10 min; followed by 45 cycles of 94°C-5 s and 60°C-30 s. cdPCR was run in Naica™ Crystal Digital PCR system . Data were analyzed using CrystalMiner.Eight P < 0.05.The data was analyzed by SPSS 20.0 software. Quantitative data were assessed by mean ± standard deviation. Count variables were assessed by the chi-square test. Relations between the expected value of diluted plasmid and those values detected by cdPCR or qPCR were assessed by Spearman's correlation. The linear relation between qPCR and cdPCR was assessed by linear regression. And each result was determined to be significantly different when 6 to 3.2 × 10 copies/ml. Eight μL of plasmid in each dilution was detected by qPCR and cdPCR. The plasmid with dilution from 3.2 × 106 copies/ml to 3.2 × 102 copies/ml can be detected by qPCR and cdPCR simultaneously. The 32 copies/ml diluted plasmid could not be detected by qPCR and cdPCR. To accurately understand the sensitivity of qPCR or cdPCR, plasmids 3.2 × 102 copies/ml were diluted in a 2-fold series by 1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32, 1 : 64. The limit of detection (LOD) of cdPCR was 103 copies/ml (2.0 copies/reaction) and the LOD of qPCR was 297 copies/ml. The results indicate that the sensitivity of cdPCR was higher than that of qPCR.The linear dynamic range of the plasmid DNA containing the pUC57-UL83 gene was from 3.2 × 10R = 0.979, P < 0.05 for cdPCR and the expected value of diluted plasmid, R = 0.939, P < 0.05, for qPCR and the expected value of diluted plasmid).To understand the repeatability of cdPCR, a standard curve of HCMV DNA copy number was established using plasmids. Copies of serially diluted plasmids were detected by cdPCR and the coefficient of variations  of copy number detected by cdPCR was analyzed with a standard curve . The resTo determine the specificity of cdPCR for HCMV, 7 herpes viruses, including HSV-1, HSV-2, VZV, EBV, HCMV, HHV-6A, and HHV-7, were tested by cdPCR, respectively. The results showed that cdPCR only detected the HCMV AD169 strain but not the other 6 herpes viruses, suggesting that cdPCR for HCMV has no cross-reaction with other herpes viruses.μL HCMV DNA each dilution by 10-fold dilution from 5.67 × 106 TCID50/ml to 5.67 TCID50/ml was tested by qPCR and cdPCR, respectively. HCMV DNA from 5.67 × 106 TCID50/ml to 5.67 × 10 TCID50/ml virus could be detected by qPCR but not for 5.67 TCID50/ml. However, HCMV DNA of 5.67 TCID50/ml virus could be detected by cdPCR, which was 146 copies/ml (2.7 copies/reaction). The results showed that the sensitivity of cdPCR was better than that of qPCR.Eight To verify the sensitivity of the cdPCR method and whether it can be used for HCMV detection in the blood of HSCT patients, 125 children's whole blood samples following HSCT were tested by qPCR and cdPCR. Thirty-four samples were positive through qPCR and cdPCR, ninety-one samples were negative through qPCR. However, 4 out of 91 qPCR negative samples were positive by cdPCR. The results showed that the method of cdPCR was more sensitive than qPCR for HCMV detection .+ & R+, HCMV was detected in 28 cases (41.18%), and the viral load was 107–6600 copies/ml. HCMV was detected in 7 cases (22.58%) with a viral load of 437 to 5314 copies/ml in 31 D+ & R− cases. There were 18 cases of D− & R+, and three cases (16.67%) of HCMV were detected, and the viral load was 463–883 copies/ml. There were 5 cases of D− & R−, and HCMV was not detected. The HCMV infection rate was 40.54% (15/37) among AML cases, and the HCMV viral load was from 107 copies/ml to 5314 copies/ml. The HCMV infection rate was 41.18% (7/17) in ALL cases, and the HCMV viral load was from 137 copies/ml to 1779 copies/ml. The HCMV infection rate was 22.73% (5/22) among AA cases, and the HCMV viral load was from 154 copies/ml to 6600 copies/ml. The HCMV infection rate was 40% (4/10) among MDS cases, and the HCMV viral load was from 999 copies/ml to 5957 copies/ml. The HCMV infection rate was 33.33% (1/3) among lymphoma cases, and the HCMV viral load was 2494 copies/ml. There was no HCMV infection in NB cases. One case (16.67%) was infected with HCMV in WAS cases, and the HCMV viral load was 801 copies/ml. The HCMV infection rate was 20% (5/25) among MPS cases, and the HCMV viral load was from 351 copies/ml to 5100 copies/ml  in 125 children following HSCT, and the range of HCMV viral load was from 107 copies/ml to 6600 copies/ml. The detection rate in the male group was 30.14% (22/73) and in the female group was 30.77% (16/52). The detection rate of HCMV was 89.47% (34/38) in the HCMV-positive children following HSCT aged 0–12. In the aged 0–6 group, the detection rate in males was 25.64% (10/39) and in females was 22.58% (7/31). In the aged 7–12 group, the detection rate in males was 39.29% (11/28) and in females was 40% (6/15). For over 12 years old children following HSCT, the HCMV detection rate was 33.33% (4/12), the detection rate in males was16.67% (1/6), and the detection rate in females was 50% (3/6) , suggestP > 0.05). HCMV-positive children were slightly more likely to develop GVHD than HCMV-negative children. Among the 38 positive children, five children died following HSCT. The coinfection with EBV occurred in 2 of the 5 deaths.The prognosis of 125 children following HSCT was analyzed retrospectively. GVHD was found in 13 children  (34.21%) of 38 HCMV-positive children and 25 children (28.74%) of the 87 HCMV-negative children  and cdPCR are two types of commercial digital PCR technic. As our results, many studies show that the sensitivity of digital PCR is significantly higher than that of qPCR , 26, 27.It is well known that HCMV is the most common transmissible virus in children following HSCT and is considered to be the major risk factor for transplantation. Studies have shown that almost all HCMV viremia after bone marrow transplantation occurs in HCMV-positive recipients, and only a few patients can be transmitted from the donor . For patIn this study, up to 30.40% of samples were positive for HCMV by cdPCR. About 90% of HCMV infections occurred before the age of 12 in these children following HSCT. Among 125 children following HSCT, neoplastic diseases  have a higher HCMV infection rate than non-neoplastic diseases . It is reasonable that the immune status of neoplastic children is weaker than that of non-neoplastic children, which makes HCMV infection more likely to occur. Our results suggest that HCMV-positive children were more likely to develop GVHD than HCMV-negative children, consistent with previous studies. In other words, it is necessary to pay attention to children following HSCT with HCMV and HCMV-infected children after bone marrow transplant. Therefore, timely monitoring and accurate quantification of HCMV viral loads in children following HSCT can provide an important basis for clinical antiviral treatment.In conclusion, the cdPCR method of HCMV DNA was established and confirmed for detecting HCMV viral load in this study.


Streptomyces ipomoeae (SiLA) and a fungal laccase from Myceliophthora thermophila (MtL), for kraft lignin polymerization. To study the influence of some variables in these processes, a central composite design (CCD) with two continuous variables (enzyme concentration and reaction time) and three levels for each variable was used. The prediction of the behavior of the output variables (phenolic content and molecular weight of lignins) were modelled by means of response surface methodology (RSM). Moreover, characterization of lignins was performed by Fourier-transform infrared (FTIR) spectroscopy and different nuclear magnetic resonance (NMR) spectroscopy techniques. In addition, antioxidant activity was also analyzed. Results showed that lignin polymerization (referring to polymerization as lower phenolic content and higher molecular weight) occurred by the action of both laccases. The enzyme concentration was the most influential variable in the lignin polymerization reaction within the range studied for SiLA laccase, while the most influential variable for MtL laccase was the reaction time. FTIR and NMR characterization analysis corroborated lignin polymerization results obtained from the RSM.Kraft lignin, a side-stream from the pulp and paper industry, can be modified by laccases for the synthesis of high added-value products. This work aims to study different laccase sources, including a bacterial laccase from  Then, for each of the output variables, the obtained data from the CCD were used to fit a quadratic regression equation representing the influence of the two input variables. Each equation was plotted by means of response surface methodology (RSM). The design of the experiments of the CCD, the quadratic regression equations, and the plots obtained from modelling the equations by RSM were obtained using Minitab 19.1 software .2CO3. The reaction mixture was incubated at 50 °C for 10 min, and absorbance at 760 nm were measured after cooling, using a UV–Vis spectrophotometer . The total phenolic content of samples was quantified using a calibration curve prepared from a standard solution of gallic acid (1–200 mg/L) and expressed as mg gallic acid equivalent (GAE)/g of lignin (on a dry basis).The total phenolic content of kraft lignins was determined following the Folin–Ciocalteu method with some modifications, according to Jiménez-López et al. . Firstly−1, was employed as mobile phase at 25 °C for 30 min. Samples were dissolved at a final concentration of 0.5 g/L in NaOH (0.05 M). Polystyrene sulfonated standard  were used for the calibration curve [SEC analysis (weight-average (Mw), number-average (Mn) molecular weights, and polydispersity (Mw/Mn)) of kraft lignins was carried out in an Agilent Technologies 1260 HPLC. The samples were analyzed at 254 nm  using two columns (Phenomenex) coupled in series  and a safeguard column (35 × 7.8 mm). NaOH (0.05 M), pumped at a rate of 1 mL min−1, 400 scans, and a spectral range of 600–2000 cm−1 [FTIR spectra of kraft lignins were acquired by a JASCO FT/IR 460 Plus spectrometer , equipped with an accessory single-reflection diamond, working with a resolution of 1 cm000 cm−1 .−1, was carried out using a UV–Vis spectrophotometer , and the absorbances were measured between λ 200 and 800 nm.UV–Vis analysis of kraft lignins, dissolved in 0.1 M NaOH to a final concentration of 50 µg mL13C nuclear magnetic resonance (13C NMR) spectroscopy of kraft lignins was performed at 25 °C in a Bruker Avance III 400 MHz  at 100.64 MHz with the cross polarization/magic angle spinning (CP/MAS) technique. Lignin samples were prepared in 4 mm rotors and the spinning rate was 10 KHz. The contact time was 2 ms and the delay between scans was 5 s. The number of scans was 10,240 [Solid-state s 10,240 .13C–1H two-dimensional nuclear magnetic resonance (2D NMR) spectra of kraft lignins were recorded at 25 °C in a Bruker AVANCE 500 MHz  equipped with a z-gradient double resonance probe. Around 40 mg of each lignin was dissolved in 0.75 mL of deuterated dimethylsulfoxide (DMSO-d6) and an HSQC (heteronuclear single quantum correlation) experiment was recorded. The spectral widths for the HSQC were 5000 Hz and 13,200 Hz for the 1H and 13C dimensions. The number of collected complex points was 2048 for 1H-dimension with a recycle delay of 5 s. The number of transients for the HSQC spectra was 64, and 256 time increments were always recorded in 13C-dimension. The J-coupling evolution delay was set to 3.2 ms. Squared cosine-bell apodization function was applied in both dimensions. Prior to Fourier transform the data matrices were zero filled up to 1024 points in the 13C-dimension. Residual DMSO (from DMSO-d6) was used as an internal reference (δC/δH 39.6/2.5 ppm) [2.5 ppm) .+• methods according to Re et al. [+• was produced by the reaction between 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) stock solution (7 mM) and potassium persulfate (2.45 mM). Prior to use, the ABTS+• stock solution was diluted with ethanol to obtain an absorbance of 0.7 ± 0.02 at 734 nm. Then, 1 mL of the ABTS+• stock solution was mixed with 10 μL sample (32 mg mL−1) or control and allowed to react for 6 min. The change in absorbance of the reaction mixture was measured at 734 nm and the antioxidant activity was expressed as trolox equivalent antioxidant capacity (TEAC). To calculate TEAC capacity, the gradient of the plot of the percentage inhibition of absorbance vs. concentration for lignin samples was divided by the gradient of the plot for trolox.The antioxidant activity of kraft lignin samples was estimated following the ABTSe et al.  and Ratae et al. . ABTS+• M. thermophila (MtL) laccase, with a high pH range and noticeable thermal stability, is widely employed for lignin polymerization [S. ipomoeae (SiLA) laccase, with similar properties to MtL laccase, has been also assessed for this purpose [There is an increasing interest to evaluate the potential of novel laccases, including those from bacterial origin, for residual lignin polymerization. Presently, the fungal rization ,20,21,35 purpose , althougAs it can be observed in The quadratic regression equation obtained from the phenolic content values a,b when mg GAE/g lignin = 434.8 − 0.306 · Time (min) − 0.786 · Dosage (IU/g) − 0.000339 · Time (min) · Time (min) + 0.00089 · Dosage (IU/g) · Dosage (IU/g) + 0.002381 · Time (min) · Dosage (IU/g)mg GAE/g lignin = 222.5 + 0.1458 · Time (min) − 0.588 · Dosage (IU/g) − 0.000343 · Time (min) · Time (min) + 0.00157 · Dosage (UA/g) · Dosage (IU/g) − 0.000776 · Time (min) · Dosage (IU/g)®) was used to polymerize alkali lignins from birch and spruce materials.As it is widely known, laccases can oxidize free phenolic lignin units, yielding resonance-stabilized phenoxyl radicals via a single electron transfer process . Thus, tTrametes (syn. Coriolus polyporus). A similar trend was reported by Huber et al. [Contrary to this study, the effects of laccase dosage and reaction time on the phenolic content of lignin are usually evaluated separately. Gillgren et al.  showed tr et al. , using ar et al.  also repThe molecular weight distributions of laccase-treated lignins are displayed in The quadratic regression equation obtained from the Mw values a,b when Mw (Da) = 3371 + 14.40 · Time (min) + 30.7· Dosage (IU/g) − 0.0050 · Time (min) · Time (min) − 0.0959 · Dosage (IU/g) · Dosage (UA/g) − 0.0223 · Time (min) · Dosage (IU/g)Mw (Da) = 3936 + 7.42 · Time (min) + 25.46 · Dosage (IU/g) + 0.00040 · Time (min) · Time (min) + 0.1201· Dosage (IA/g) · Dosage (IU/g) − 0.0471 · Time (min) · Dosage (IU/g)® laccase was used to polymerize alkali spruce lignin. Mayr et al. [As previously commented, the stabilized phenoxyl radicals, generated from lignin by laccase oxidation, undergo radical–radical coupling through phenyl ether–carbon and carbon–carbon linkages, yielding the observed increase in Mw values of kraft lignin by both laccases. Moreover, the polydispersity increase is also expected due to the non-selective radical–radical coupling reactions, which link lignin end groups to each other spontaneously with low or no control and, consequently leading to higher polydispersity values . MtL lacr et al.  achievedSimilarly to phenolic content, the effects of laccase dosage and reaction time on the molecular weight of lignin are generally studied separately, reporting different results in function of both laccase and lignin sources. Gouveia et al.  observedWhile the significant phenolic content of kraft lignin can translate into good reactivity for producing phenol-formaldehyde resins, epoxy resins, polyester systems, and polyurethanes, among others, the molecular weight increase of kraft lignin by laccase enzymes enables new applications as lignin-based dispersants providing better adsorption properties, stabilizer for emulsions, and in thermoplastic blends or copolymers enhancing thermal and mechanical performance .The antioxidant ability of lignins  promotes their use as natural additives in food, cosmetics, pharmaceuticals, and polymeric formulations as an alternative to synthetic compounds such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), among others . It is w−1 associated with aromatic ring vibrations, and at 1455 cm−1 attributed to C−H asymmetric vibrations and deformations (−1 (S and G units), a shoulder at 1270 cm−1 (G units), 1220 cm−1 (G units), 1115 cm−1 (S units), 1025 cm−1 (G units) and 820 cm−1 (S units).FTIR spectra of untreated and laccase-treated kraft lignins (MtL-KL and SiLA-KL resulting from experiments with maximum decrease in phenolic content and increase in molecular weight achieved) are displayed in rmations a. Bands −1) and unconjugated (1715 cm−1) linkages  in C/δH 45.0–95.0/2.5–6.0 ppm) and the aromatic (δC/δH 90.0–150.0/5.0–9.0 ppm) regions in 13C–1H lignin correlation signals identified in HSQC spectra are displayed in The HSQC spectra of untreated and laccase-treated kraft lignins (MtL-KL and SiLA-KL resulting from experiments with maximum decrease in phenolic content and increase in molecular weight achieved) are shown. They included the whole spectra , overlapping with aryl-glycerol, could also be found.The oxygenated aliphatic region of the kraft lignin spectrum exhibited information about the different interunit linkages present a, includ1-1′ , G1-1′ (3-dimethoxy-para-diphenol) and S1-G1′/G5′ were tentatively identified as a result of Cα-C1 breakdown in a retro-aldol reaction, followed by a radical coupling reaction, during kraft pulping [The aromatic region of kraft lignin spectra displayed the typical correlation signals of S, G, and H lignin units a, the us pulping ,45,46.Trametes villosa and Trametes hirsuta laccases. Wang et al. [®) bacterial laccase. In addition to the cleavage of interunit linkages, a new signal could also be observed in the aliphatic oxygenated region of both MtL-KL and SiLA-KL spectra, which was tentatively attributed to α-5′ condensed structures. The appearance of this structure is probably due to lignin condensation/polymerization reactions by laccases action. In this sense, Wang et al. [Significant changes in the oxygenated aliphatic region of MtL-KL and SiLA-KL spectra a, were fg et al.  also desg et al.  already 13C–1H correlation signals , especially in the SiLA lignin sample as previously described by FTIR analysis  in new 4-O-5′ structures formed during laccase (T. villosa) treatment of lignosulfonates, whereas Magina et al. [5 in 5-5′ structure during MtL laccase treatment of lignosulfonates. lignins  also shoanalysis , resultia et al.  endorsedThen, these observations arising from NMR analysis suggest lignin condensation/polymerization reactions by laccases action, supporting the molecular weight increment observed by SEC . The pheS. ipomoeae (SiLA) and a commercial fungal laccase from M. thermophila (MtL) to polymerize kraft lignin (referring to polymerization as lower phenolic content and higher molecular weight). Specifically, the enzyme dosage was the most influential variable in the kraft lignin polymerization reaction, within the range studied, when the bacterial SiLA laccase was used, while for MtL fungal laccase the most influential variable was the reaction time. FTIR and NMR characterization spectra verified lignin polymerization, observing new condensed structures such as α-5′, 5-5′, and 4-O-5′.In order to produce the most appropriate lignin for each possible application, structural modification of this molecule is needed. A possible way to achieve this goal is by using laccase enzymes, which is considered a sustainable and environmentally friendly approach. Concerning this, this study confirmed the ability of a bacterial laccase from


Bacillus velezensis-amended and Bacillus subtilis-amended diets. In the first trial, immune gene expression indicated a significant down in B. velezensis AP193-fed fish for il1β, tnf-α, and tlr9 expression within splenic tissue, compared to that of the basal and B. subtilis diets. In the second trial, no substantial up-or down-regulation of immune-related genes was observed in B. subtilis-amended feed at different inclusion levels.Two trials were performed, utilizing channel catfish fed probiotic-supplemented diets in flow-through systems under natural rearing conditions. There were no significant improvement in growth performance, survival, hematocrit, and blood chemistry parameters in channel catfish fed Ictalurus punctatus) farming industry is the largest and one of the oldest aquaculture industries in the United States. Despite being an established industry, production issues stemming from disease outbreaks remain problematic for producers. Supplementing fish diets with probiotics to enhance the immune system and growth potential is one approach to mitigating disease. Although considerable laboratory data demonstrate efficacy, these results do not always translate to natural modes of disease transmission. Hence, the present work was conducted in the laboratory but incorporated flow-through water from large catfish pond production systems, allowing for natural exposure to pathogens. Two feeding trials were conducted in an 18-tank aquaria system housing two different sizes, 34.8 ± 12.5 g and 0.36 ± 0.03 g, of channel catfish. Channel catfish in the first trial were fed three experimental diets over six weeks. Commercial diets were top-coated with two selected spore-forming Bacillus spp. probiotics, Bacillus velezensis AP193 (1 × 106 CFU g−1) and BiOWiSH (3.6 × 104 CFU g−1), or a basal diet that contained no dietary additive. In the second eight-week trial, diets were top-coated with BiOWiSH at three concentrations , along with one basal diet (no probiotic). At the completion of these studies, growth performance, survival, hematocrit, blood chemistry, and immune expression of interleukin 1β (il1β), tumor necrosis factor-alpha (tnf-α), interleukin-8 (il8), transforming-growth factor β1 (tgf-β1), and toll-like receptor 9 (tlr9) were evaluated using qPCR. Trial results revealed no differences (p > 0.05) among treatments concerning growth, survival, or hematological parameters. For immune gene expression, interesting trends were discerned, with substantial downregulation observed in B. velezensis AP193-fed fish for il1β, tnf-α, and tlr9 expression within splenic tissue, compared to that of the basal and BiOWiSH diets (p < 0.05). However, the results were not statistically significant for anterior kidney tissue in the first trial. In the second trial, varied levels of probiotic inclusion revealed no significant impact of BiOWiSH’s products on the expression of il1β, tnf-α, il8, and tgf-β1 in both spleen and kidney tissue at any rate of probiotic inclusion (p > 0.05). Based on these findings, more research on utilizing probiotics in flow-through systems with natural infection conditions is crucial to ensure consistency from a controlled laboratory scale to real-world practices. The channel catfish ( Ictalurus punctatus) industry is well-established in the domestic aquaculture sector. This industry has a long developmental history and socioeconomics, coupled with vital research and extension programs. Modernized practices, such as applying intensive aeration in production, adopting split-pond systems, and breeding programs, has propelled channel catfish farming to become one of the largest and oldest aquaculture sectors in the US [Aeromonas septicemia (MAS), enteric septicemia of catfish (ESC), and columnaris disease [Concentrated in the southern part of the US, the channel catfish  was formulated to 32% protein and 6.5% lipid . The BD Afterward, the moist pellets were put in a forced air oven (<45 °C) overnight to achieve less than 10% moisture content. Dry pellets were crumbled, packed in bags, and kept in a freezer (−20 °C) until needed. The proximate composition and amino acid (AA) profile of all diets were determined at the University of Missouri Agriculture Experiment Station Chemical Laboratories  per g was suspended in 10 mL of distilled, deionized water and sprayed onto feed for a final concentration of 1 × 106 CFU g−1 , which was suspended in distilled, deionized water, according to manufacturer’s specifications, and sprayed onto feed for a final concentration in the first experiment of 3.6 × 104 CFU g−1 , 1.8, 3.6, and 7.2 × 104 CFU g−1 , temperature, and salinity were monitored twice daily using a YSI 55 multi-parameter instrument . Total ammonia N (TAN) and nitrite-N were measured twice per week using YSI 9300 photometer . The pH of the water was measured twice weekly during the experimental period using the EcoSense pH10A .B. velezensis AP193 at 1 × 106 CFU g−1 (B-AP), and for BiOWiSH, the final concentration was 7.2 × 104 CFU g−1 (B-BW). The control or basal diet without probiotic amendment was coated with distilled water. The diets were then air-dried for at least 12 h, stored at 4 °C, and used within 3 days of mixing. Each experimental diet was administered to 6 replicate tanks for the study duration.The first 6-week experiment took place in a biosecure wet lab at E. W. Shell Fisheries Center of Auburn University, Auburn, Alabama, from March to May 2021. Ten juvenile channel catfish (34.8 ± 12.5 g) were randomly stocked into twelve aquaria (75 L) in a flow-through system utilizing natural water sourced from channel catfish production ponds. Catfish were hand-fed twice daily at ~4% body weight, and the ration was adjusted every 2 weeks. The probiotic spores were top coated on fish feed with a final concentration of −1. During the trial, the water quality was within range for normal growth , 0.03 ± 0.02 mg L−1 nitrite, 0.14 ± 0.03 g L−1 salinity, and pH 8.36 ± 0.71), except for temperature (20.01 ± 0.33 °C) [The study tanks received water from channel catfish production ponds with a mean water flow rate of 1 L min0.33 °C) .At the end of the feeding trial, fish were bulk weighed, and three fish were randomly collected from each tank, anesthetized with buffered tricaine methanesulfonate (MS-222), and bled from the caudal vein with a 1 mL syringe, and then fish were euthanized, and the spleen and anterior kidney tissues were collected.Blood samples were collected in a 1.5 mL microcentrifuge tube without anticoagulant for biochemistry analysis. For hematocrit analysis, blood was collected in heparinized soda-lime glass micro-hematocrit capillary tubes  that were wax-sealed . Spleen and kidney tissues were collected and preserved in DNA/RNA Shield  within 1.5 mL microcentrifuge tubes for gene expression analysis. All growth metrics were calculated as follows:−1 using a natural water source from channel catfish production ponds. Fish were hand-fed twice daily at ~4% body weight, and the ration was adjusted every 2 weeks. There were four experimental diets, basal diet, and three inclusion levels of BiOWiSH FeedBuilder Syn3, with a final dosage on feed of 1.8 × 104, 3.6 × 104, and 7.2 × 104 CFU g−1 top-coated on feed. The inclusions represented 50, 100, and 200% of the recommended dose . The diets were left air-dried for at least 12 h, stored at 4 °C, and used within 3 days. The experimental diet was administered to 4 replicate tanks for 0.25 g kg−1 and 0.5 g kg−1 inclusion levels, while 0 g kg−1 and 1 g kg−1 had five replicate tanks for the study duration.The second 8-week growth trial was conducted from August to October 2021, using fingerling channel catfish (0.36 ± 0.03 g) randomly stocked into 18, 105 L fiberglass tanks in a flow-through system with the stocking density at 40 fish tank−1. Similar to the first trial, the water quality was within range for the normal growth for channel catfish  [The study tanks received water from channel catfish production ponds, with the mean water flow rate at 1 L min0.19 °C) .At the end of the feeding trial, fish were bulk-weighed, and three fish were collected, as previously described, for blood, spleen, and kidney samples. All growth parameters were calculated as similar to the first trial, with the addition of:Wax-sealed capillary tubes were spun down using a hematocrit IEC Clinical Centrifuge  in 5 min using the instrument setting. The hematocrit percentage results were then read using a micro-capillary reader .g for 5 min to collect serum. Three serum samples from each tank were then pooled into one 100 μL composite sample. The serum biochemical parameters  were determined by using Abaxis VetScan Mammalian Liver Profile on the Abaxis VetScan VS2 analyzer .Blood samples were allowed to clot at 4 °C overnight, followed by centrifugation at 15,000× c microvolume spectrophotometer . Extracted RNA samples were then diluted and standardized to 50 ng μL−1. All samples were then converted into cDNA using the High-Capacity cDNA Reverse Transcription Kit , according to the manufacturer’s instructions. A total of 20 μL was used in the reaction, which included 2 μL of 10× R.T. buffer, 0.8 μL of 25× dNTP Mix, 2 μL of 10× R.T random primers, 1 μL of multiscribe reverse transcriptase, and 4.2 μL of nuclease-free water. The cDNA was synthesized using a MiniAmp Plus thermal cycler . The thermal program was set at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. The RNA with a 25 ng μL−1 concentration was diluted to reach the concentration of 2.5 ng μL−1. Experiment A utilized four genes: il1β (interleukin 1 beta), tnf-α , tlr9 (toll-like receptor 9), and tgf-β1 (transforming growth factor beta 1) with a housekeeping gene (18s rRNA), while Experiment B used four genes il1β, tnf-α, il8, and tgf-β1 with two housekeeping genes: ef1α  and actb (beta-actin) (−ΔΔCt) [RNA of spleen and kidney samples were extracted and purified using Quick-RNA Miniprep Kit . Sample concentration was measured using a NanoDrop Onea-actin) . The eff (−ΔΔCt) .α = 0.05 was used for all statistical analyses. The pooled standard error (PSE) was calculated as follows:All data were analyzed using R Version 4.2.1 . The natural log transformation was performed on two parameters for blood biochemistry (ALP and ALT) and all relative gene expressions to meet the normality requirement ,41. OutlB. velezensis B-AP and B-BW B. subtilis probiotics of Experiment A revealed no statistically significant changes (p > 0.05) in the final weight, percent weight gain, survival rate, or thermal-unit growth coefficient among treatments (p = 0.122), percent weight gain (p = 0.090), survival rate (p = 0.715), feed conversion ratio (p = 0.228), and thermal-unit growth coefficient (p = 0.123)  .B. velezensis-supplemented diet (B-AP). However, there was no significant difference discovered   .B. velezensis AP193-amended diet, with a significant down-regulation observed for the il1β, tnf-α, and tlr9 transcripts from spleen tissue, compared to that of the basal diet (p < 0.05). No significant changes were observed for tgf-β1 in spleen tissue in the control, compared to the B. velezensis AP193-amended diet. At the same time, there was no significant difference, in comparison to the B-BW diet (p > 0.05) (p > 0.05)   . Althoug > 0.05) . In Expeeatments . Further > 0.05) .B. velezensis AP193 was observed to reduce eutrophication in channel catfish ponds, showing the possibility for employing this probiotic to improve culture systems water quality [B. velezensis AP193’s protective potential against pathogenic bacteria [Probiotics have been promoted and employed as a solution to accelerate growth and improve immune responses, given that antimicrobial resistance has resulted in a limited number of drugs that can be used to battle pathogen infections, as well as the cost and scale concerns of immunization ,43,44,45 quality . On the bacteria .B. velezensis AP193 or BiOWiSH probiotics and the basal diet, in terms of final weight (g), weight gain (%), survival rate (%), feed conversion ratio, and thermal-unit growth coefficient. Furthermore, despite amending feed with 2× the recommended level , the second study showed the same outcome, with no significant difference between probiotic-supplemented and basal diets in the flow-through system. This is consistent with prior research by Peterson et al. (2010), in which channel catfish provided Lactobacillus spp. and Bacillus spp. displayed no discernible change in growth and survival rates [Oncorhynchus mykiss) fed Bacillus licheniformis and Bacillus subtilis [Penaeus latisulcatus) [The current study found no significant difference in the growth performance of fish offered diets with al rates . Furthersubtilis . Concernulcatus) . Concernulcatus) .Lates calcarifer) fed a probiotic diet exhibited considerably greater levels of all hematological indices and hepatic enzyme activity, including hematocrit, red blood cell, white blood cell, hemoglobin, alanine aminotransferase, and alkaline phosphatase [Hematological and blood serum parameters have been used to assess the physiological conditions of the animals; evaluating the related parameters may bring a better understanding of the impacts of probiotic-amended diets on the health of the vital organs and, ultimately, the cultured animals ,50. Regasphatase . Neverthsphatase ,55. Bothil1β (p = 0.029), tnf-α (p = 0.024), and tlr9 (p = 0.040) genes were observed for the B. velezensis AP193-amended diet group, while kidney tissue expression remained the same for all four genes (p > 0.05). Given that the investigated genes are proinflammatory cytokines and cell receptor genes, particularly il1β, tnf-α, tlr9, tgf-β1, and il8, the up- or down-regulation of any of these genes suggests the modulation of an immune response [il1β. Hence, a decrease in il1β was found alongside a decrease in tgf-β1 [tgf-β1 for head kidney tissue in shabout (Tor grypus) fed a probiotic diet [tnf-α in the kidney tissue of the B. velezensis AP193 group in this study, a contrast finding in olive flounder  fed Lactobacillus signified a noticeable upregulation of tnf-α in the anterior kidney, compared to the control group [Lactococcus lactis subsp. showed a noticeable upregulation in spleen tissue, while no significant impact was found for kidney tissue [il8 in the head kidney, the study of Mohammadian et al. (2021) and Rodríguez et al. (2009) contrasted with the outcome of this trial and that of Lu et al. (2020), for which no up- or down-regulation could be observed even with different level of BiOWiSH and MOS, respectively [B. velezensis AP193 contributed to a decrease in tlr9 expression in channel catfish in the spleen, but not in anterior kidney tissue, but this was different with the findings of Liu et al. (2020) in the kidney of golden pompano (Trachinotus ovatus), which expressed upregulation in the probiotics added diet group [tlr9 in yellow catfish (Pelteobagrus fulvidraco) after a challenge with Flavobacterium columnare [B. velezensis AP193 had no substantial deleterious influence on the integrity of essential organs, such as the spleen.Although the regulation remained similar for varying degrees of BiOWiSH inclusion, significant downregulations in spleen tissue for the response ,56. IL-1response . The curresponse . Furthern tgf-β1 ,58,59. Itic diet . Tumor ntic diet . Concernol group . Furthery tissue . IL8, a y tissue . Despiteectively ,65,66. Cectively . The finet group . Furtherolumnare . In the B. velezensis AP193. Various diets with diverse component matrices may boost or hinder the development of bacteria; studies on inoculating beneficial microorganisms with regularly used aquaculture ingredients should be conducted, and both ingredients used in the current study can be further examined for optimized dosing and life-stage specific administration for channel catfish production.Probiotic administration has been shown to boost growth rate and the immunological response in cultured aquatic animals. Not all strains that demonstrate good benefits in vitro would function similarly on a larger production scale. Additionally, different species may react differently to probiotic treatments, especially when the required rearing conditions are quite different. Our current study findings did not discern growth and survival differences with probiotic treatment additions, but did reveal changes to immune gene expression in the splenic tissue of fish fed the diet containing


At 100 µM and 10 µM , cana increased the ADP/ATP ratio, whereas dapa and ertu  showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa  had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs’ expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1. The selective sodium-glucose co-transporter-2 inhibitors (SGLT2) canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) are approved as therapeutic glucose-lowering agents for the treatment of type 2 diabetes patients ,5,6,7,8.Synthetic selective SGLT2 inhibitors, such as cana, empa, and others, have been developed and their clinical development has progressed. Cana, as a highly potent and selective SGLT2 inhibitor, has shown to lower the renal threshold for glucose, increase urinary glucose excretion, improve glycemic control and beta-cell function in rodent models of type 2 diabetes mellitus, and to reduce body-weight gain in rodent models of obesity . Cana, tFurthermore, sotagliflozin, a non-selective SGLT inhibitor acting on both SGLT1 and 2 , with stSGLT1 is responsible for a major part of the dietary glucose uptake in the intestine , and theSGLT2 is responsible for the majority of glucose reuptake in the tubular system of the kidneys , and selTo address potential differences in the clinical profiles between the SGLT2 inhibitors, several in vitro studies have investigated the cellular effects of SGLT2 inhibitors on energy metabolism ,32,33,34Endothelial cells make up the lining of the vascular system, and their dysfunction may lead to complications that are observed in diabetes and other metabolic disorders ,39,40,41The inhibition of mitochondrial function by cana has also been observed in previous metabolomics studies on several cell lines, which showed alterations in the fatty-acid and purine/pyrimidine metabolism ,36,37. DHowever, currently, no data are available for the effects of different SGLT2 inhibitors on mitochondrial activity, cellular glucose uptake, and metabolic processes in endothelial cells; effects that could potentially influence the clinical profiles of these SGLT2 inhibitors.Therefore, in this study, we aimed to investigate the effects of cana, dapa, ertu, and empa on mitochondrial oxidative phosphorylation and GU in human-umbilical-vein endothelial cells (HUVECs). To assess the putative impact of SGLT2 inhibitors on the function of the endothelium, we also examined the impact of the SGLT2 inhibitors cana, dapa, and ertu on the metabolomes of these cells.The HUVEC cells treated with a supra-pharmacological concentration (100 µM) of the SGLT2 inhibitor cana showed inhibition of OCR of nearly 60% A, simila50) of 27 µM. The IC50 values for dapa, ertu, and empa were greater than 100 µM (data not shown). The OCR inhibition became apparent at concentrations above 10 µM of cana , cana, dapa, ertu, and empa lowered the ECAR by 45%, 13%, 32%, and 17%, respectively A,B. The max elicited by its pharmacological dose (10 µM), and an even larger statistically significant response at the supra-pharmacological concentration (100 μM). The Dapa treatment, both at the pharmacologically relevant concentration of 0.3 µM and at the supra-pharmacological concentration of 3 μM, failed to cause any significant changes to the ADP/ATP ratio. However, the treatment with ertu at 1 µM  and 10 μM displayed a weak, concentration-dependent increase in the ADP/ATP ratio , with IC50 values > 100 µM (14C-DOG uptake and cana (100 µM) inhibited 97% of the 14C-DOG uptake (Both cana and ertu inhibited ectively A. At 100> 100 µM A. The GLG uptake B.The treatment of the HUVECs with cana (10 and 100 μM) had no significant effects on the expression of the glucose transporters GLUT 1, 3, 6, 8, and 10, nor detectable cytotoxic effects (data not shown). The rotenone did not cause detectable cytotoxicity.p-value < 0.01) in the cana (100 µM)-treated HUVECs . The same metabolites and metabolite classes were also changed in the HUVECs treated with a lower concentration of cana (10 µM), although fewer metabolites were affected . All the metabolite specifications are listed in detail in the The four main classes of ontology distribution for the MVA_UVA metabolites measured were amino acids, peptides and analogues (16 metabolites), nucleotides and analogues (21 metabolites), carbohydrates and conjugates (ten metabolites), and metabolites related to energy metabolism (seven metabolites). One cana (10 µM) sample was excluded due to a technical problem during the HPLC-MS measurement. Seventeen metabolites were altered significantly  and glutamate   . Cana atp-value < 0.01; The metabolites used for glycolysis were significantly depleted in the cana (100 µM)-treated HUVECs  in the cana (100 µM)-treated HUVECs (The cana (100 µM)-treated HUVECs showed a trend towards reduced levels of free fatty acids (FFA). Cana at 10 µM showed the same trend, but less pronounced. The acetyl-CoA levels were significantly increased  had a significantly increased ADP/ATP ratio and cana (10 µM) induced the same effect as the trend, but no significance was observed. Furthermore, no significant effect on redox equivalents was induced by the SGLT2 inhibitors ertu and dapa.At 100 µM, the cana significantly increased the NADp < 0.05). Cana (10 µM) showed a trend of increased glutamic acid and n-acetyl-glutamic acid, but no significance was perceived. No effects on apoptosis-associated metabolites were observed in the dapa- and ertu-treated HUVECs, irrespective of the applied SGLT2 inhibitor concentrations.The levels of the apoptosis-associated metabolites, glutamine, glutamic acid, and n-acetyl-glutamic acid, were significantly increased in the cana (100 µM)-treated cells (The cells treated with cana (both 10 and 100 µM) showed a trend towards increased levels of metabolites in the purine/pyrimidine pathways, whereas a trend towards reduced metabolite levels in the purine/pyrimidine pathways was seen for the cells treated with dapa (0.3 and 3 µM) and ertu (1 and 10 µM).In the present study, we investigated the differential cell autonomous effects of the selective SGLT2 inhibitors cana, dapa, ertu, and empa on parameters related to mitochondrial activity  and on GU in HUVECs. Further, a metabolomics analysis was performed on the cana-, dapa- and ertu-treated HUVECs to assess their effects on the metabolomes of these cells. Our results confirmed the effects of cana on the mitochondrial activity and GU in the endothelial cells, which have been described previously in other cell models, using supra-pharmacological and pharmacologically relevant concentrations. By contrast, dapa, ertu, and empa did not show any of these effects, even when applied at supra-pharmacological concentrations.In more detail, we reproduced the effects of cana on the OCR in the HUVECs. At 100 µM, the cana inhibited the OCR by 60% in our study, and weak OCR inhibition was also observed after the treatment with cana at 10 µM, which was consistent with the reported results from several other cell lines ,32,33. IThe reduction in the OCR by cana was likely the result of the electron transport chain (ETC) complex 1 inhibition in our study. Nonetheless, when interpreting the results, one needs to take into account that endothelial cells, such as HUVECs, do not predominantly rely on their mitochondrial energy supply ,45,46. IETC complex 1 inhibition by cana has also been reported in human renal proximal tubule epithelial cells . Shetty p < 0.01) used in the glycolysis, which was in agreement with the lower GU observed after the cana treatments.The metabolomics analyses of cana (10 and 100 µM), ertu (1 and 10 µM), and dapa (0.3 and 3 µM)-treated HUVECs showed that cana, in contrast to dapa and ertu, caused a significant decrease in the metabolites .The levels of the amino acids used for the production of glucose showed a trend towards elevated levels in the cana (10 µM)-treated HUVECs, and were significantly higher in the cana (100 µM)-treated cells, indicating reduced TCA-cycle activity due to its low usage in endothelial cells, as described previously , and/or A potential increase in the TCA-cycle activity could also have been caused by the increased activity of the electron transport chain complex II (ETC2), since ETC2 directly links the TCA cycle and the respiratory chain by catalyzing the oxidation of succinate to fumarate . A potenSecker et al. observed the inhibitory effects of cana (50 µM) on glutamate dehydrogenase (GDH) in human renal proximal tubule epithelial cells after treatment . This efThe inhibition of glutamic acid decarboxylase reduces cellular ATP levels and increases pyruvate and fatty-acid oxidation. The blockage of the glutamine metabolism has also been shown to perturb proliferation dynamics and the adoption of a senescence-like phenotypes .In agreement with the results from Hawley et al., who have studied human embryonic kidney cells , cana evTrends in a cellular-depleted redox-state caused by lower levels of metabolites, such as NADPH and NADH, in the cana-treated HUVECs were observed. In particular, the NADPH, a crucial cofactor for endothelial nitric oxide synthase , was lowThe HUVECs treated with dapa (3 µM) and ertu (10 µM) showed no significant changes regarding glycolysis, the metabolites used in cell respiration and beta-oxidation. However, these cells showed reduced metabolite levels in the purine and pyrimidine pathways, and these effects were more pronounced with increasing SGLT2 inhibitor concentrations. The levels of metabolites from the purine and pyrimidine pathways were significantly lower after the ertu (10 µM)- and dapa (3 µM) treatments, which were similar to the results described in ,60, thanmax) of the respective SGLT2 inhibitor elicited by its pharmacological doses. The maximal plasma concentration (Cmax) of cana at daily therapeutic dose of 300 mg at steady state is approximately 10 µM (4445 ng/mL) [max of dapa at daily therapeutic dose of 10 mg at steady state is about 0.3 µM (122 ng/mL) [max of ertu at daily therapeutic dose of 15 mg at steady state [max of empa at steady state is 0.63 µM (284 ng/mL) at a daily dose of 25 mg [The synthetic selective SGLT2 inhibitors, cana, empa, and ertu , were pu5 ng/mL) ,62, Cmax2 ng/mL) , and 1 µdy state . Cmax ofof 25 mg .In contrast to sodium-dependent glucose transporters (SGLT), glucose transporters (GLUT) are facilitative glucose transporters  that areHUVECs  were plated and expanded in culture medium according to the manufacturer’s instructions .Data were analyzed as change in OCR and ECAR as a function of SGLT2 inhibitor concentration. Effects on cellular respiration and medium acidification was measured with Agilent/Seahorse Extracellular Flux analyzer XF96 at 37 °C. Basal OCR and ECAR were recorded prior to injection of the respective SGLT2 inhibitor to yield final concentrations of 0.2, 1, 5, 25, and 100 µM (n = 4), followed by five repeated measurements of OCR and ECAR (during approximately 35 min). Rotenone (2 µM) was used as reference for the inhibition of the mitochondrial ETC complex I. DMSO was utilized as blank sample. In total, 15,000 and 25,000 HUVEC cells were seeded in 150-microliter cell culture medium per XF96 plate well 18–24 h prior to experiment. XF96 cartridge was equilibrated with calibration buffer overnight according to manufacturer´s instructions. Cell culture medium was changed to XF Base Medium Minimal DMEM  complemented with 5 mM glucose, 1 mM pyruvate, 1 mM glutamine, and 15 mM HEPES 30–40 min prior to first reading. After the last read-out, cells were fixed in 4% formaldehyde, and subsequently washed with 4% formaldehyde prior to adding Hoechst nuclear stain for cell counting with Image Xpress system from Molecular Devices. OCR and ECAR were normalized based on the cell numbers per well.3–104 cells) was added. After five minutes of incubation at room temperature while gently shaking, a total of 100 µL of the reaction mix was added and background luminescence was assessed. Fifty µL of cells (103–104) treated with Nucleotide Releasing Buffer were pipetted into a luminometer plate and samples were read after approximately two minutes in a luminometer. A 10 × ADP converting enzyme was diluted 10-fold with Nucleotide Releasing Buffer. ADP levels in cells were read by adding 10 µL of 1 × ADP converting enzyme and reading the output after two minutes. All samples were run as duplicates in four independent experiments. ADP/ATP ratios were calculated based on the manufacturer´s recommendation.The cellular energy status was assessed by measuring the ADP/ATP ratio, following SGLT2 inhibitor treatment for three hours. ADP and ATP levels in the samples were detected using the ADP/ATP Ratio Assay Kit . Cells were grown as described above. Prior to the measurement, the culture medium was removed from the plate, and Nucleotide Releasing Buffer (50 µL per 1014C-deoxyglucose (14C-DOG) as tracer. In total, 40,000 HUVECs per well were seeded in a Cytostar 96w SPA plate and allowed to adhere overnight prior to the experiment. Cytostar plates were pre-coated with collagen I (0.03 µg/uL). The cells were pre-treated for 15 min with 0.2, 1, 5, 25, and 100 µM of the respective SGLT2 inhibitor. Next, they were pre-diluted in assay medium consisting of XF Base MedSium Minimal DMEM (Agilent 102353) and complemented with 5 mM glucose, 1 mM Pyruvate, 1 mM Glutamine, and 15 mM HEPES prior to the addition of 18 µM 14C-DOG tracer (specific activity: 1 µCi/mL). Uptake of 14C-DOG was monitored in real-time using a micro beta scintillation counter (PerkinElmer) with four readings between 10 and 190 min. Cell monolayers were visually inspected for potential loss of adhesion after the last reading. A total of 25 µM BAY-876 (n = 16) was used as reference for effects on GLUT1-mediated uptake against cana  [14C-DOG uptake rate was calculated as fmol DOG per minute, and after subtracting the value for 14C-background. Data were fitted to a four-parameter logistic model to calculate IC50.Effects on the GU were assessed using , n = 8) , and DMS® QuantStudio™ at 50 °C for two minutes and at 95 °C for ten minutes. Then, denaturation cycles (40 cycles of 95 °C for 15 s) were performed. Subsequently, the annealing and extension were performed, during which fluorescence was measured (40 cycles of 60 °C for 60 s). Data expression levels were recorded as quantification cycles, CT. Data were acquired using real-time PCR software . The mean CT values of the triplicate reactions were used in the subsequent analysis. Protein Atlas was used for data comparison.Total RNA was extracted from HUVECs treated with cana (10 and 100 µM) after 4 and 24 h, and purified using the Qiagen RNeasy mini kit . Cell lysis was performed in an RNases inactivating buffer provided by the manufacturer. Until RNA extraction, the samples were stored at −70 °C. RNA concentrations and 260/280 absorbance ratios were measured spectrophotometrically using Nanodrop ND-1000. Reverse transcription of the purified RNA was performed immediately after extraction. The cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit . The reaction was set with 800 ng RNA in 20 µL of solution containing RT-random primers, 100 mM deoxyribonucleotide triphosphates (dNTPs) mix, 2.5 U/µL MultiScribe Reverse Transcriptase, and RT buffer. Real-time qPCRs were performed on Applied Biosystems5) were treated with cana (10 µM and 100 µM), ertu (1 µM and 10 µM), and dapa (0.3 µM and 3 µM) and with vehicle solution  as controls with six replicates per treatment (n = 6).Cultivated HUVECs . The cell samples were divided into two smaller batches of 24 samples each, and these two batches were measured in one run. Maximum measurement time per batch was 24 h.Two hours after treatment, the cell culture samples were extracted, dipped into isotonic NaCl solution with 4.5 g/mL glucose and freeze-dried for 9.5 h, as described previously ,70. The 2O), QCs, and UltimateMix (UM) were measured in a stratified randomized sequence and analyzed with a Dionex Ultimate 3000 HPLC  equipped with a reversed-phase Atlantis T3 C18 pre- and analytical column  with an injection volume of 10 µL, as described previously [Samples, blanks  was performed, centered and scaled to unit variance (R function prcomp). Missing values were imputed by regularized expectation-maximization . For ANOVA analysis, the treatment was taken as the fixed factor . For specific group comparisons, pairwise post hoc tests (R function lsmeans) were conducted. Technical variability was 15.9% median relative standard deviation (RSD) in the QC samples for 60 multivariate and univariate analysis (MVA_UVA) metabolites and yielded a further 36 univariate-analysis (UVA) metabolites. The MVA_UVA metabolites were classified according to the Human Metabolome Database .Results for OCR, ECAR, and GU were presented as mean ± SD. Results for ADP/ATP ratios were presented as mean ± SEM.p-value < 0.05 between groups was considered as statistically significant.Data were analyzed for statistical significance using one-way analysis of variance (ANOVA) corrected for multiple comparisons by Tukey statistical hypothesis testing . A Statistical analysis for metabolomics was performed with R  and TibcoSpotfire . All metabolites were controlled for their analytical quality  and gradTo correct for cell number differences and reduce technical variability, median normalization was performed. Each metabolite was normalized to the sample median using the following procedure. First, each metabolite´s peak area was scaled between 0 and 1 within one sample. Next, the median of all 0-to-1-scaled metabolites’ peak areas within one sample was calculated, and each peak area was divided by the sample median value.p-values corrected for multi-parameter testing via Benjamini–Hochberg are given in the Finally, normalized peak areas were log-transformed. Kolmogorov–Smirnov and Brown–Forsythe Levene tests were used to discern normality and homoscedasticity of data distribution. The log10-transformed, normalized data were normally distributed according to the Kolmogorov–Smirnov test  and homoscedastic according to the Brown–Forsythe-Levene-type test . The max evoked by their pharmacological doses. The supra-pharmacological concentrations of cana, but not dapa, ertu, or empa, affected multiple cellular pathways  and blocked the GLUT1.Our data confirmed the reported inhibition of mitochondrial activity by cana at supra-pharmacological and, to some extent, at pharmacological concentrations. By contrast, dapa, ertu, and empa did not show any measurable effect on the mitochondrial activity, glucose uptake, and ECAR in the endothelial cells, even at concentrations that were much higher than the CAt 100 µM, cana showed a combined reduction in glycolysis and mitochondria respiration. This diminished cellular-energy state could lead to insufficient angiogenesis  and coul


Biologic hybrid meshes can be used safely in case of possible contamination. BHM seems to reduce the risk of hernia recurrence compared to completely resorbable biologic meshes, but this has to be investigated further.In case of potential contamination, implantation of synthetic meshes in hernia and abdominal wall surgery is problematic due to a higher risk of mesh infection. As an alternative, a variety of different biologic meshes have been used. However, relevant data comparing outcome after implantation of these meshes are lacking. Between January 2012 and October 2021, biologic meshes were used for reconstruction of the abdominal wall in 71 patients with preoperative or intraoperative abdominal contamination. In this retrospective study, semiresorbable biologic hybrid meshes (BHM) and completely resorbable meshes (CRM) were compared and analyzed using a Castor EDC database. In 28 patients, semiresorbable biologic hybrid meshes were used; in 43 patients, completely resorbable meshes were used. Both groups showed no difference in age, gender, BMI, operation duration, hernia size and Charlson comorbidity index. The risk degree of surgical-site occurrences was graded according to the Ventral Hernia Working Group (VHWG) classification, and the median value was 3 (range 2–4) in the BHM group and 3 (range 2–4) in the CRM group. Hernia recurrence within 24 months after hernia repair was significantly lower in the BHM group (3.6% vs. 28.9%;  The outcome in this group compared to all other patients with biologic meshgraft hernia repair was analyzed in this study.In the surgical treatment of abdominal wall hernias, the use of meshes is recommended for larger hernias, and is associated with a significantly lower rate of hernia recurrence –3. Howevt test in case of normally distributed data, and otherwise the Mann–Whitney U test. The Chi-square test was used, if appropriate, to compare nominal variables. The recurrence-free survival rates were calculated with a Kaplan–Meier analysis and the log-rank method was used to compare these rates between groups. A p value of 0.05 or less was considered statistically significant. The study was approved by the ethics committee of the University of Regensburg, Germany (Nr. 19-1547-101).Between January 2012 and October 2021, 71 cases with biologic mesh implantation in the setting of preoperative or intraoperative possible wound contamination were identified. Only patients with reconstruction of the abdominal wall, which included ventral abdominal hernias and abdominal cavity closing after open abdomen situations, were enrolled in this study. Other indications for mesh implantation were excluded. To compare the outcome of the different meshes, the patients were retrospectively separated into a semiresorbable (BHM) and a completely resorbable meshes (CRM) group. The data were stored in a Castor EDC database and analyzed using IBM SPSS statistics 26. Statistical analysis was done using Student’s o™ RTM: porcine-derived acellular dermal matrixStratticeo™: cross-linked, acellular porcine-derived dermal collagen matrixPermacoloR integra: acellular collagen matrix derived from fetal and neonatal bovine dermisSurgiMendResorbableo™ 2s: sterile bioscaffold composed of ovine-derived extracellular matrix and monofilament polypropylene.OviTexSemiresorbableIn total, four different types of biologic meshes were used. Three of them were completely resorbable and 1 semiresorbable:Grade 1 (low risk): low risk of complications, no history of wound infectionGrade 2 (co-morbid): COPD, diabetes, immunosuppression, active smoker, obeseGrade 3 : previous wound infection, stoma present, violation of the gastrointestinal tractGrade 4 (infected): infected mesh, septic dehiscence.VHWG classification of ventral hernias , 14, 15:p = 0.01) (Table 2 (range 15–41.8 kg/m2) for patients treated with a biologic hybrid mesh compared to 27.5 kg/m2 (range 13.5–72.6 kg/m2) in the CRM group. Furthermore, a median of 3.5 points (range 0–8 points) were generated using the Charlson Comorbidity Index in the BHM group and 3 points (range 0–10 points) in the CRM group. While all patients had undergone abdominal surgical treatment in the past, the median number of previous operations was 4.5 (range 1–6) in the BHM group and 5 (range 1–6) in the CRM group. The risk degree of surgical-site occurrences according to the VHWG classification indicated a median value of 3 (range 2–4) in the BHM group and 3 (range 2–4) in the CRM group. The median hernia size was 220.7 cm2 (range 20.7–586.6 cm2) in the BHM group and 107.8 cm2 (range 0.2–505.5 cm2) in the CRM group. Preoperative wound conditioning, either through negative wound pressure therapy or regular lavage (Table p = 0.64).Both groups showed no statistical difference in age, gender, BMI, Charlson Comorbidity Index, or hernia size, and only the presence of stomata was significantly higher in the CRM group p = 0.0 Table 1p = 0.01 p = 0.00; 53.6% vs. 14%, p = 0.04), while both groups did not differ in intraabdominal position rate (Table The median operation time was 162 min (range 51–493 min) in the BHM group and 157 min (range 37–511 min) in the CRM group. The mesh grafts were placed as intraperitoneal onlay mesh (IPOM), sublay, bridging, and onlay in 11 (39.3%), 9 (32.1%), 8 (28.6%), and 0 (0%) cases in the BHM group and in 26 (60.5%), 5 (11.6%), 11 (25.6%), and 1 (2.3%) cases in the CRM group, respectively (Table p = 0.35). With 46.4% vs. 20.9% a higher number of patients developed a seroma, located either subcutaneously or around the mesh in the BHM group. However, statistical significance was just missed (p = 0.08). Despite this, no difference in the necessity of interventional treatment (BHM 61.4% vs. CRM 55.6%) or the rate of positive microbiological findings in the seromas (BHM 15.4% vs. CRM 15.1%) was seen when comparing both groups. Operative revisions had to be performed in 8 (28.6%) cases in the BHM group and 7 (16.3%) cases in the CRM group (p = 0.22). The median follow-up was 16 months (range 9–28 months) in the BHM group and 31.5 months (range 5–125 months) in the CRM group with a total hernia recurrence rate of 7.1% in the BHM group and 32.6% in the CRM group, respectively. Due to the different length of the follow-up, hernia recurrence within 24 months after hernia repair was chosen for statistical analysis for better comparability. During this period, hernia recurrence was significantly lower in the BHM group, with specifically only 1 patient (3.6%) in the BHM group and 12 patients (28.9%) in the CRM group (Table p = 0.03). A detailed stratification of hernia recurrence considering the respective operation technique is shown in Table p = 1.0).The median hospital stay was 28 days (range 6–271 days) for all patients with 29.5 days range 6–136 days) in the BHM group and 27 days (range 7–271 days) in the CRM group  in a mid-term follow-up period when compared to completely resorbable biologic meshgrafts in potentially contaminated wounds. Comparable results regarding hernia recurrence rates for meshes with non-resorbable components are found in the current literature [2 vs. 107.8 cm2) and is consistent with the current literature analyzing biologic meshgrafts [The results of this study suggest that semiresorbable biologic hybrid meshes show a significantly lower hernia recurrence rate  as well as the follow-up period (16 months vs. 31.5 months) differs in both groups.Nevertheless, a clear benefit of this study is the comparison of a new semiresorbable biologic hybrid mesh with other kinds of completely resorbable biologic meshes, which is a valuable comparison that has not yet been done before. However, since only ovine biologic hybrid meshes were used in this study, all conclusions must be limited to this type of meshgraft.In summary, we suggest that semiresorbable biologic hybrid meshes are safe to use in complex patients with possible wound contamination and potentially reduce the risk of hernia recurrence compared to CRM in a mid-term follow-up period. Despite the heterogeneity of the presented retrospective study, our data show actual clinical courses of complex patients. However, for definitive clinical and economic conclusions, further prospective randomized trials and long-term follow-up data are required.