Spondias mombin (SM) as feedstock for the synthesis of biosurfactants by Pseudomonas spp. The cultures were composed of basic mineral medium amended with SM, SM + glucose, glucose (GLC), and nutrient broth (NB) as carbon sources. Biosurfactant production was determined by surface-active properties such as hemolysis, emulsification index (E24), drop collapse, oil-spreading assays, and reduction of surface tension. The stability of the biosurfactants was monitored across different temperature and pH regimes while chemical components of the extracted biosurfactants were determined by thin-layer chromatography. Biosurfactants synthesized from SM as sole substrate showed the highest emulsification index (56.35%), oil-spreading capacity (4.4 ± 1.31 cm), hemolysis (3.10 ± 0.02 cm), the shortest time for drop collapse (30 s), and surface tension reduction (24 mN/m). Biosurfactant concentrations ranged from 0.07 ± 0.01 in the NB to 2.08 ± 0.01 g/L in the media amended with SM. Chemical characterization revealed significant concentrations of carbohydrates and lipids in the biosurfactant produced from SM  when compared to SM + glucose , glucose , and nutrient broth . The biosurfactants were stable over a wide range of temperature while E24 increased with pH. Our results show the viability of SM fruit pulp as low-cost feedstock for industrial-scale production of biosurfactants using Pseudomonas spp.In this study, we explored the possibility of utilizing the succulent pulp of  Spondias mombin is a wild tree that is common in southern parts of Nigeria. The fruit, which is rich in carbohydrates and lipids, are hardly harvested and often left to rot as waste [Spondias mombin fruit pulp as an important substrate for the industrial production of feedstock chemicals including biosurfactants.as waste . The goaMicrobially produced surfactants (biosurfactants) are surface-active amphiphilic molecules consisting of a structurally diverse group of biomolecules including glycolipids, lipopeptides, lipoproteins, lipopolysaccharides, and phospholipids . They arSpondias mombin fruits adds value to large-scale production of biosurfactants via microbial fermentation [Pseudomonas species, an established metabolic workhorse for microbial production of biosurfactants can utilize alternative low-cost substrates for fermentation [Spondias mombin fruit waste as viable feedstock to produce biosurfactants by Pseudomonas species. To the best of our knowledge, this is the first attempt at using Spondias mombin fruit as a carbon source for the microbial production of any feedstock chemical.Unfortunately, many surfactants used today are produced chemically and pose serious environmental and toxicological challenges due to their recalcitrance ,8. To oventation . Importaentation . In the 2.2.1Spondias mombin (SM) fruits was carefully separated from the seeds. An optimized quantity (147 g) of the fruit pulp was extracted with 250 mL of methanol. The methanol SM extract was dried in an oven set at low heat, 50°C for 24 h. The dry powder was stored in an air-tight container and subsequently used as a carbon source for the preparation of Pseudomonas spp growth media. To identify the active constituents of the fruit, qualitative phytochemical analysis of the fresh and dry SM samples was performed following a standard protocol [The pulp of freshly collected quantity 7 g of thprotocol  was used2.2Pseudomonas spp slant culture was re-activated using established protocol [Pseudomonas spp into 100 mL of nutrient broth  followed by incubation at a temperature of 25°C and 150 rpm for 6 h. The basal mineral medium (BMM) consisted of 12.5 g/L K2HPO4, 3.8 g/L KH2PO4, 1 g/L (NH4)2SO4, 0.1 g/L MgSO4.7H2O, and 5 ml of trace element solution containing 0.232 g/L H3BO3, 0.174 g/L ZnSO4.7H2O, 0.116 g/L FeSO4, 0.096 g/L CoSO4.7H2O, 0.022 g/L (NH4)6Mo7O24.4H2O, 8.0 mg/L CuSO4.5H2O, and 8.0 mg/L MnSO4.4H2O. Four basal mineral media (BMM) containing different carbon sources were prepared as follows: (1) 14.4 g/L SM only, (2) 7.2 g/L each of SM and glucose, (3) 14.4 g/L glucose only, and (4) 14.4 g/L nutrient broth. Media were autoclaved at 121°C for 15 min. Fermentation was carried out in loosely capped 250-mL Pyrex culture bottles using 6% (v/v) of Pseudomonas spp pre-culture in 50 ml of culture medium . All cultures were grown in triplicate at 32 ± 1°C and 150 rpm for 12 days. BMM without carbon source served as the negative control. Samples were collected every 24 h to determine the growth of the cells and to analyze biosurfactant production and activity.Laboratory stock of protocol . In a st2.3Two ml culture was centrifuged at 10,000 x g for 2 min to remove cell pellets. Biosurfactants were isolated from the cell-free supernatant using the acid precipitation method by adjusting the pH to 2.0 using 6 N HCl . Afterwa2.4Pseudomonas spp. These include hemolysis, oil drop collapse, emulsification capacity, oil-spreading assays, and surface tension measurement.Biosurfactants consisted of a structurally different groups of biomolecules including glycolipids, lipopeptides, lipoproteins, lipopolysaccharides, or phospholipids . Consequ2.4.1.The hemolysis assay was performed following the method of Mulligan et al. . Biosurf2.4.2.The assay principle involves using surfactants to destabilize oil droplets. Drop collapse assay was done following the method described by Khan et al. : Briefly2.4.3.This test was done as previously described . To make2.4.4.24) is defined as the percentage of the height of the emulsified layer (in cm) divided by the total height of the liquid column (in cm) [This assay was performed following the protocol described by Kumari et al. . Cell-fr (in cm) .E24=Hei2.4.5.24 value of biosurfactants in the culture supernatant was measured. Similarly, the pH stability was measured by adjustment of pH from 2 to 12 using 1 M HCl or NaOH, followed by determination of E24.To determine the thermal stability, culture supernatants were subjected to varying temperatures (30°C to 90°C) for 1 h and then cooled to 25°C. After subjecting to each temperature, the E2.4.6.A KRUSS KIOT Tensiometer  equipped with a 6 cm De Nuoy platinum ring was used to measure the surface tension of the culture broth. During the study, an average of triplicate samples was used in other to increase accuracy.2.4.7.The different biosurfactants produced were characterized following the thin-layer chromatography method ,23. Brie2.5All data were reported as mean ± standard deviation of triplicate determinations. The general linear model of Minitab version 17  was used for all statistical analyses to compare the differences between carbon sources as they pertain to biosurfactants, carbohydrates, and lipids concentrations, hemolysis, oil-spreading activity, surface tension measurement, and emulsification indices. Significant differences were established at p ≤ 0.05.3.3.1.Phytochemical and proximate analyses were conducted to determine the bioactive components of SM. The qualitative data indicate that flavonoids, proteins, and steroids were present in moderate quantities . In addi3.2.Pseudomonas in the different media was determined by measuring culture optical density for a 12-day period. As shown in Pseudomonas spp for up to 10 days after which a decline in growth was observed. The maximum cell growth for the SM-only culture was ~1.6-fold higher than in the nutrient broth (NB) culture. However, the maximum growth of Pseudomonas spp in SM-only medium mirrored that observed for the glucose-only medium. Nonetheless, the SM-only medium supported an extended growth of Pseudomonas spp as indicated in the prolonged stationary growth phase observed in the SM-only medium relative to the glucose-only medium (Pseudomonas spp in SM supplemented with glucose (SM+GLC) medium also had a prolonged stationary growth phase  of biosurfactants on oils ranged from 43% in NB to 56.35% in SM media. The highest E24 value was obtained with kerosene whereas the least was recorded with crude oil (24 of SM biosurfactant in kerosene (56.35%) was comparable to the E24 obtained with Tween-80 (60%). The E24 of SM biosurfactant in kerosene was ~1.1-, 1.2-, and 1.2-fold higher than the E24 of SM+GLC (53%), GLC (48%), and NB (46.35%) biosurfactants, respectively. Similar emulsification patterns were observed with crude and olive oils  at different temperatures are presented in 24 value and remained relatively unchanged at all the temperatures (30–90°C) tested. Temperature change appears to have no significant impact on the E24 values of biosurfactants synthesized from SM+GLC, GLC, and NB substrates.The E24 of biosurfactants in the pH regime test showed that biosurfactants produced from SM substrate had the highest values and ranged from 38% at pH 2 to 56% at pH 12 , cultures grown in SM had a longer stationary phase . The lono et al.  where thPseudomonas spp showed superior characteristics. For instance, the SM biosurfactant exhibited a significant (p < 0.05) hemolysis clearing zone  recorded with SM biosurfactant. The least emulsification index was observed with NB biosurfactant. Furthermore, it was noted that E24 among the different hydrocarbons followed the order: kerosene> olive oil > crude oil  involved in the production. Furthermore, the extracted biosurfactant was a glycolipid molecule. Therefore, we conclude that Spondias mombin pulp extract is a good raw material for biosurfactant (glycolipid) production by Pseudomonas spp used in the present study.Based on the results obtained in this study, the yield of biosurfactant produced by


Meloidogyne species occurring at the highest frequency. Nematodes were sampled from roots and the rhizosphere of one stunted Kiwi tree, extracted using the tray method and then fixed. The morphological characters fit well with those of M. hapla. The molecular approach using ITS and 28S rDNA, along with the related phylogenetic analysis, placed the examined population in a group with other populations of M. hapla. Kiwi is being reported as a new host for M. hapla in South Africa.Kiwi is becoming one of the most important fruit in subtropical regions of South Africa with altitudes that confer sufficient chilling requirements. During a survey on biodiversity of plant-parasitic nematodes of kiwi in Magoebaskloof in Limpopo Province, several plant-parasitic nematodes were discovered, with  Actinidia spp.) fruit trees are increasingly being cultivated in temperate regions. The genus Actinidia comprises more than 70 species  . Roots w species .6 generations for ITS and 28S rDNA. The trees were visualized with the TreeView program. The original partial ITS rDNA and 28S (D2-D3 expansion) sequence of M. hapla were deposited in GenBank under the accession numbers MT256109 and MT258534, respectively. The morphological and molecular analyses confirmed that the species was M. hapla.The molecular characterization followed the methods described in M. hapla J2 were (n = 10): L = 337 ± 15.5 (322-353) μm; a = 30.8 ± 2.4 (28-32); b = 3.7 (n = 1); c = 8.5 ± 1.0 (7.4-9.5); stylet length = 12.6 ± 0.6 (11.9-13.0) μm; center of the median bulb to anterior end = 48.8 ± 2.6 (46-51) μm; excretory pore to anterior end = 67 ± 1.0 (66-68) μm; length body to the middle of genital primordium = 208.3 ± 8.6 (199-216) µm, hyaline part of tail length = 11.2 ± 1.3 (10.0-12.6) μm and tail length = 39.6 ± 3.1 (37-43) μm. The J2 had the smooth and spherical head, with a tail tapering to a blunt or rounded terminus. This species already has been studied from tropical areas of Africa , excretory pore to anterior end (66-68 vs 60.7-82.4 µm), hyaline part of the tail (10-12.6 vs 12-18.5 µm), and tail length (37-43 vs 50.2-54.8 µm). Although, our population of M. hapla showed no significant differences with those second-stage juveniles of M. hapla studied by Morphometric mean, standard deviation and range values of f Africa , India , Japan (LC030357), and Taiwan (KJ572385), all with 99% similarity. Despite high similarity (99%) with M. hapla populations, our sequence of M. hapla showed the lowest similarity, 85% with M. incognita (KJ739707) and M. javanica (KJ739709), and 79% with M. enterolobii (KM046989) using ITS rDNA marker. The nBlast of 28S rDNA showed four bp differences with 98% similarity with the Chinese  and Ethiopian population (KP410845). Despite high similarity (98%) with M. hapla populations, our sequence of M. hapla showed the lowest similarity, 89% with M. incognita (JX100425), M. javanica (JX100426), and M. enterolobii (KJ146862) using 28rDNA marker. Therefore, molecular result confirmed our populations as M. hapla.The sequence lengths flanked by the forward primer TW81 [5′-GTTTCCGTAGGT GAACCTGC-3′] and AB28 [5′-ATATGCTTAAGTTCA GCGGGT-3′] ; D2A 5″ of the IM. hapla population in a clade together with other M. hapla populations (M. hapla suggested that they formed a monophyletic group. Findings in the current study were in agreement with the phylogenies of Meloidogyne species studied using 18S rDNA, ITS, 28S rDNA and COII of mtDNA (M. hapla were deposited in the Nematology collection of the University of Limpopo, South Africa. According to literature, this is the first record of M. hapla from kiwifruits in South Africa. Besides, ITS and 28S rDNA information of this species are being reported for the first time. M. hapla was associated with kiwifruits in Limpopo Province and therefore, host-status studies are necessary to find out the severity of this root-knot nematode.The phylogenetic analysis using ITS and 28S rDNA, placed the South African ulations . The molof mtDNA . Two per


Clinical use of the combination therapy of the neprilysin inhibitor sacubitril and angiotensin II type 1 receptor blocker valsartan is known to be associated with albuminuria. Albuminuria is both a risk factor for and an indicator of kidney injury. Earlier work from our laboratory reported that the agonist of angiotensin II type 2 receptor Compound 21 (C21) prevents proteinuria, albuminuria, and is reno-protective in obese Zucker rats fed high salt diet (HSD). Thus, we hypothesized that sacubitril/C21 combination provides superior reno-protection compared to sacubitril/valsartan. Male obese Zucker rats 10–11 weeks old were treated daily with vehicle, sacubitril + C21, or sacubitril + valsartan while fed HSD for 16 days. HSD-feeding caused kidney dysfunction, evident by significant increases in urinary protein, osteopontin, and cystatin C. HSD-feeding lowered plasma cystatin C and creatinine concentrations suggestive of hyperfiltration, which was not affected by either treatment. Unlike sacubitril/valsartan, sacubitril/C21 treatment significantly decreases proteinuria, albuminuria, the expression of nephrin, and kidney weight, independent of hyperfiltration, compared with HSD alone. Moreover, sacubitril/valsartan therapy increased plasma renin and did not prevent HSD-induced increases in renal angiotensin II, while sacubitril/C21 completely prevented these changes. Together, this study suggests that sacubitril/C21 afforded superior reno-protection compared to sacubitril/valsartan therapy in high salt-fed obese Zucker rats. While oactivity . Togetheactivity . For theactivity . Despiteactivity  and someactivity .2R) and receptor Mas are similarly classified as components of the alternative RAS axis. The actions of the alternative RAS axis oppose those of the classical RAS axis, which includes Ang II and AT1R. This knowledge led to the identification of novel RAS targets, such as AT2R. Developed by Vicore Pharma, the agonist of AT2R Compound 21 (C21), is a non-peptide agonist that binds selectively to the receptor, activating the nitric oxide-cyclic guanosine monophosphate pathway  and it’s generating enzymes ACE2 and neprilysin, angiotensin II type 2 receptor , the urinary albumin to creatinine ratio was significantly increased . Therefore, the goal of this study was to investigate and compare the kidney-specific outcomes of the current SAC/VAL therapy to our novel approach of SAC/C21 in chronically treated OZR fed a high salt diet (HSD). Outcomes from this study suggest that C21 in combination with neprilysin inhibition is superior in preventing decline in kidney function, albuminuria, and proteinuria, as well as in reversing the changes in Ang II and renin, while similarly preserving ANP.Natriuretic peptides including atrial natriuretic peptide (ANP) similarly drive diuresis and natriuresis and are known to lower blood pressure . The benncreased . The knoncreased , taken t2R agonist C21, 1 mg/kg/day (a gift from Vicore Pharma) & sacubitril, 10 mg/kg/day  or valsartan, 10 mg/kg/day  & sacubitril, 10 mg/kg/day. Thus, there were four groups of OZRs which each consisted of an n = 8; NSD + vehicle (NSD), HSD + vehicle (HSD), HSD + sacubitril/C21 (SAC/C21), and HSD + sacubitril/valsartan (SAC/VAL). Dosages for SAC and VAL in combination, and for C21 were chosen based on prior publications , 10–11 weeks of age, were purchased from Envigo in Indianapolis. Following arrival, the rats were housed in the animal care facility at University of Houston. Experimental protocols were approved by the IACUC at the University of Houston (protocol number 15-035) and were conducted in accordance with the NIH Care and Use of Laboratory Animal Guidelines. All rats were acclimatized for 7–10 days, prior to beginning any treatment. Simultaneously on the day that treatment began, all rats were also placed onto a 0.4% normal salt diet (NSD)  or 4% high salt diet (HSD) . Based on prior publications from our laboratory, 2 weeks of HSD-feeding in OZR resulted in kidney functional injury, including proteinuria . Therefoications . Urine wp < 0.05, **p < 0.01, ****p < 0.0001; n = 6–8 per group.GraphPad Prism Version 8.4.3 was used for data analysis via one-way ANOVA with Fisher’s LSD test. Data is presented as mean ± SEM, *In comparison to OZR fed NSD, those on HSD drank significantly more water over the 16 days study period (NSD 366 ± 15 ml vs HSD 742 ± 15 ml), which resulted in an increased daily urine volume (NSD 8.8 ± 1.4 ml/24 h vs HSD 28.4 ± 2.6 ml/24 h). Though water intake was not impacted by either treatment, SAC/VAL treated OZR produced modestly less urine volume over a 24 h period. HSD-fed OZR took in less food over the duration of the study compared to the NSD-fed OZR (HSD 394 ± 11 g vs NSD 517 ± 38 g), neither treatment affected total food intake. While initial body weight did not differ between the groups, one interesting outcome was that SAC/C21 treatment significantly prevented weight gain (HSD 57.6 ± 6.8 g vs SAC/C21 37.3 ± 3.9 g) .Kidney weight at the time of euthanasia in SAC/C21 treatment group was lower compared to that of HSD-fed rats (HSD 2.7 ± 0.1 g vs SAC/C21 2.3 ± 0.05 g) . Total uUrinary concentrations of osteopontin, a marker used to predict incident chronic kidney disease (CKD) were normalized to 24 h urine volume and determined to be increased by HSD-feeding and modestly decreased by treatment with SAC/C21 (NSD 163 ± 19.8 vs HSD 333.3 ± 62.3 vs SAC/C21 216.1 ± 17 ng/24 h urine) . UrinaryCirculating ANP was decreased by HSD-feeding, but the treatments preserved plasma levels of ANP . Renal ap = 0.0538)  . Only SA 0.0538) . Plasma  0.0538) . There wted rats .2R, with the neprilysin inhibitor SAC and AT2R agonist C21 (SAC/C21), compared to SAC and AT1R blocker VAL (SAC/VAL) therapy in OZR. SAC/VAL therapy is commonly associated with albuminuria  RAS axis have been increasingly studied in multiple animal models. ACE2 and neprilysin are peptidases that metabolize Ang II and Ang I, respectively into the reno-protective Ang-(1–7). However, neprilysin is also involved in one of the mechanisms of ANP elimination via enzymatic degradation; natriuretic peptide receptor type C (NPR-C) is involved in the receptor-mediated clearance of ANP renin receptor expressed in the kidney, heart, adipose, and other tissues of the body (1R regardless of its origin, studies have found that use of ARBs is associated with significant increases in Ang II, and that when given in combination with ACE inhibitors, ACE inhibition dose-dependently prevents the increases in Ang II induced by AT1R blockade (1Rs (1R expression in SAC/VAL treated OZR suggests that it is likely that the dose of VAL used in this study might not have been sufficient to offset and block the AT1R from the 3-fold increase in intrarenal Ang II levels. Though this should be taken into consideration for the development of reno-protective approaches, our study presents an innovative approach that eliminates the kidney specific problems associated with ARB monotherapy and SAC/VAL therapy via a novel combination of SAC with C21.Use of ARBs via a feedback loop drives a compensatory increase in plasma renin levels , in addiwith VAL . Renal Athe body . Also, athe body . Moreovethe body , influenthe body , and incthe body . Despiteblockade . Other rade (1Rs . This ev


We tested the added value of 3D-vision on procedure time and surgical performance during robotic pancreatoduodenectomy anastomoses in biotissue. Robotic surgery has the advantage of articulating instruments and 3D-vision. Consensus is lacking on the added value of 3D-vision during laparoscopic surgery. Given the improved dexterity with robotic surgery, the added value of 3D-vision may be even less with robotic surgery.In this experimental randomized controlled cross-over trial, 20 surgeons and surgical residents from 5 countries performed robotic pancreaticojejunostomy and hepaticojejunostomy anastomoses in a biotissue organ model using the da Vinci® system and were randomized to start with either 3D- or 2D-vision. Primary endpoint was the time required to complete both anastomoses. Secondary endpoint was the objective structured assessment of technical skill  rating; scored by two observers blinded to 3D/2D.p < 0.001; 20.8 min reduction, 95% confidence intervals 12.8–28.8 min). This reduction was consistent for both anastomoses and between surgeons and residents, p < 0.001. Robotic 3D-vision improved OSATS performance by 6.1 points  compared to 2D .Robotic 3D-vision reduced the combined operative time from 78.1 to 57.3 min  contains supplementary material, which is available to authorized users. The morbidity rate of pancreatoduodenectomy is to a large extent caused by postoperative pancreatic fistula . In receAs stated by the 2020 international Miami evidence based guidelines on minimally invasive pancreatic resection, it is currently unclear whether the outcomes of general laparoscopic PD could benefit from 3D-vision . More reWe aimed to determine the added value of 3D-vision, as compared to 2D-vision, when performing robotic pancreatoduodenectomy anastomoses in biotissue.We conducted an experimental randomized controlled cross-over trial. The Consolidated Standards of Reporting Trials (CONSORT) guidelines where followed . All par., 3D-vision capabilities, were assessed using a Randot Test . Reported side effects and preferences, and baseline demographics were collected using questionnaires . Participants (n = 0) were excluded if they had no 3D-vision abilities, > 200 s of arc . Th. Th13]. .  validated by Birkmeyer et al. and Tam et alRandomization was done with SPSS  by the study coordinator. Participant data were anonymized by using a 4-digit code, and the principal investigator and study coordinators were the only ones with access to the decoding document.The primary outcome was the difference in total operative time expressed in minutes and percentages. Secondary outcome was surgical performance according to the OSATS score (attainable range 12–60). Other outcomes were the difference in operative time for the PJ and HJ; participant’s preference for 3D or 2D, and side effects of 3D-vision.The sample size estimation for this trial was based on a previous randomized study assessing 3D laparoscopy (LAELAPS-3D2D) . Assuminp value of less than 0.05 was considered statistically significant. Missing data were corrected by excluding the corresponding missing part of the video of both the intervention and control procedure into the analysis . Differences in anastomosis times were analyzed paired-wise according to the performing participant.Data were analyzed using IBM SPSS statistics for Windows version 24 . Normally distributed continuous data were presented as means and standard deviations (±). Non-normally distributed continuous data were presented as medians and interquartile ranges (IQRs) or 95% confidence intervals (95% CI). Categorical  data were presented as frequencies and percentages. Likert-Scale ordinal data were also presented in means and standard deviations, as this allows more insight into the effect size . A two-tU test for non-normally distributed data. Primary outcomes were analyzed using the Wilcoxon signed-rank test, since the comparison was paired-wise. Secondary outcomes were analyzed using Wilcoxon signed-rank test and analysis was performed with a Student’s t-test after normality was assessed.Baseline demographics were compared with Student’s t-test for normally distributed data, Chi-squared test for frequencies in one or more categories, and Mann–Whitney In the period December 2017 to January 2019, over 30 surgeons and surgical residents were invited, resulting in 20 participants of which 16 participants completed the experiment in a single session. For detailed information on the numbers of participants randomly assigned, their performed procedures, and data analyzed, see Fig. n = 14) and residents (n = 6) from 5 countries  participated. The two groups, i.e., start with 3D or 2D, were comparable for baseline characteristics, robotic experience , hand dominance, vision correction and 3D-vision abilities. Mean age was 35 years (± 8) and 16/20 participants were male (see Table Surgeons (p < 0.001). The relative reduction in operative time with 3D was 24.6% (± 17.3), 95% CI 15.6–33.5 (p < 0.001), see Fig. The mean operative time to complete both anastomoses was 57.3 (± 14.3) min  with 3D-vision and 78.1 (± 20.5) min  with 2D-vision. The median reduction in total operative time with 3D was 20.8 min, 95%CI 12.8–28.8, (p < 0.001), or 25.2%. The median operative time for the HJ was 19.5 min (IQR 16.4–27.2) in 3D and 27.5 min (IQR 23.5–40.1) in 2D. The median reduction in HJ operative time in 3D was 7.2 min, 95% CI 4.4–13.2 (p < 0.001), or 29.6%. The benefit of 3D did not differ between both anastomoses (p = 0.163) , for PJ, and 29.6%, 95% CI 14.5–40.4 (p < 0.001), for HJ.The median operative time for the PJ was 37.2 min (IQR 30.5–43.5) in 3D and 49.2 min (IQR 44.3–58.0) in 2D. The median reduction in PJ operative time with 3D was 11.2 min, 95% CI 6.1–15.5 (p = 0.003. The relative improvement was 20.8%.The mean OSATS for both anastomoses was 45.1 (± 6.2) points using 3D-vision and 39.4 (± 9.2) points using 2D-vision, with attainable scores between 12 and 60. The mean overall improvement in the OSATS for both anastomoses with 3D-vision was 6.1 points, ± 5.5, Of all participants, 20/20 participants stated to prefer 3D-vision, whereas 4/20 reported side effects. Minor side effects, e.g., minor eyestrain, were reported by 3/20 participants and 1/20 participants reported serious side effects, e.g., serious headache in relation to 3D-vision. Surprisingly, 2 participant reported side effects in relation to 2D-vision (see eTable 2 in Supplements).p = 0.027.The validation grading mean OSATS for both anastomoses was 39.0 (± 4.3) points using 3D-vision and 31.7 (± 1.9) points using 2D-vision with a mean overall improvement in the OSATS for both anastomoses with 3D-vision of 7.3 points, ± 3.2, p = 0.002), or 29.8%, for surgeons and 21.0 min, 95% CI 8.2–35.0 (p = 0.138), or 19.4%, for residents. The reduction in operative time with 3D did not differ significantly between surgeons and residents (p = 0.279).The median reduction in operative time with 3D-vision was 20.0 min, 95% CI 14.6–33.1 , as compared to 2D-vision. The benefit of 3D-vision on operative time, and its effect size, was similar between surgeons and residents during robotic surgery in an experimental setting, similar between surgeons with more and less experience, and was present for both the pancreatic and bile duct anastomoses.It seems that 3D-vision had a similar, or even somewhat higher, advantage for surgical time and surgical performance with robotic surgery as compared to laparoscopic surgery. The present study followed the LAELAPS-3D2D randomized trial that studied the same outcomes for 3D laparoscopy . In that. established that the complication rates nearly halves for every point increase in the numerical rating [Birkmeyer et all rating . Lower Ol rating , 22. In l rating . ClinicaWhat are disadvantages of 3D-vision? As confirmed in this study, some surgeons report discomfort when using 3D-vision. These side effects are mostly minor and appear less than previously reported for 3D laparoscopy potentially due to the more stable camera position with robotic surgery. Then again, in this study all participants had at least average 3D-vision capabilities, explaining the limited amount of complaints. People who are unable to see stereoptically experience adverse effects of nausea and eye strain . This stThis study has some limitations. First, this was a randomized trial in an experimental and not in a clinical setting. A clinical study comparing the benefits of 3D-vision would, however, seem unlikely especially in robotic surgery where 3D-vision is standard of care. This study is rather theoretical since 3D-vision is standard for current robotic surgery. The reported observations could also be translated to conventional laparoscopy which does not include 3D-vision routinely and to new robotic systems that aim to improve viewing conditions in minimally invasive surgery. A bias in familiarity with robotic surgery combined with 3D-vision seems unlikely, as there was no significant difference between surgeons and residents, and sensitivity analysis revealed that the benefit of 3D-vision was similar when stratifying the groups for experience. Second, participants were recruited on an invitational basis. This could have caused a selection bias toward participants with better skills or better stereoptic capabilities (mean seconds of arc of stereopsis of 50 (IQR 20–130)) . Third, Strengths of the current study include the randomized design, the use of international participants with varying expertise, observers blinded for 3D or 2D regarding OSATS outcomes, and standard description of stereoptic capabilities. With the use of artificial organs, confounders of patient variables are eliminated, e.g., variations in pancreas morphology.In conclusion, 3D-vision has a substantial, probably larger than expected, contribution to the value of robotic surgery, as compared to 2D-vision, with 25% reduction in operative time of completing both robotic biotissue PJ and HJ and 21% improved surgical performance. New robotic platforms and articulating laparoscopic instruments are advised to include 3D-vision.Supplementary file 1 (DOCX 356 kb)Below is the link to the electronic supplementary material.


N,N’-methylenebisacrylamide (BIS) is a very popular cross-linker for the radical polymerisation in water. It is highly reactive but prone to alkaline hydrolysis and suffers from a low solubility. This study shows that with slow polymerising systems such as N,N-diallyldimethylammonium chloride, only inhomogeneous networks are formed. As a consequence, gels with very low cross-linking densities, i.e., high swelling capacities, disintegrate during the swelling test and firm, coherent gels are not accessible due to the solubility limit. A promising alternative are multivalent tetraallyl-based compounds, of which tetraallylammonium bromide (TAAB), N,N,N’,N’-tetraallylpiperazinium dibromide (TAPB) and N,N,N’,N’-tetraallyltrimethylene dipiperidine dibromide (TAMPB) are the subject of this study. With these, the cross-linking polymerisation appears to be statistical, as gels formed at low monomer conversion have essentially the same swelling properties as those formed at high conversions. This is not observed with BIS. However, gelation with the tetraallyl cross-linkers is much slower than with BIS and follows the order TAPB < TAMPB < TAAB, but the differences become significantly smaller with increasing content. At low contents, all three allow the preparation of gels with high swelling capacities of up to 360 g/g. N,N’-methylenebisacrylamide (BIS) as cross-linker. The gel itself forms a stationary cationic polyelectrolyte backbone with mobile hydroxide counterions. This mobility allows the hydroxide ions in the gel to be exchanged with the carbonate in old concrete, which is a common cause for corrosion of the steel reinforcement. Overall, the cementitious matrix is “realkalised”. This protects the reinforcement from further corrosion. A similar hydrogel was recently used as coupling material for electrochemical chloride extraction  structure . It is tG’ and loss moduli G’’ of all four polymerisations , while G” increases only marginally before the gel point  swollen in D2O were not decisive, as these vinyl signals partially overlap with the broad signals of the DADMA+ backbone, and the cross-linker concentrations are generally rather small  was used to follow the polymerisation over the course of 14 d. The use of EIS is possible, as the gel formation depletes the solution of positively charged monomers and immobilises them in the polymer network. Consequently, the impedance Z increases as the polymerisation progresses. From that, the real part Zreal can be calculated , the build-up of G’ is still accelerating after approx. 1 d . Potassium carbonate (99%), allyl bromide (99%), methanol (99%) and 1,3-bis(4-piperidyl)propane (97+%) were obtained from Alfa Aesar , diallyldimethylammonium chloride (65 wt% in H2O) and triallylamine (99%) were obtained from Sigma Aldrich. Piperazine, diallylamine (97%) and D2O (99.9%) were purchased from Merck KGaA . The polyester filter bags with a maximum mesh size of 90 µm were bought from Rosin Tech Products .The anion exchange-resin Lewatit Monoplus MP 800 was provided by Lanxess . Double-distilled water, potassium hydroxide, chloroform (≥99%), acetone (≥99%), dichloromethane (≥99%), potassium persulfate, sodium metabisulphite, sodium hydroxide (97%) and A solution of 7.41 mL  triallylamine in 50 mL acetone was placed in a 250 mL round-bottom flask and cooled in an ice-water bath. To this, 4.53 mL  allyl bromide were added over the period of one hour. Afterwards, the solution was heated to 88 °C for 48 h. The solution was filtered and the resulting solid was washed several times with acetone. Tetraallyl ammonium bromide was obtained as a white crystalline solid. Both thermogravimetry and differential scanning calorimetry show unanimously that 1a decomposes at 185 °C. Previous reports listed this as melting point .Yield: 6.90 g (61%); Decomposition at 185 °C.1H-NMR  δ 6.09 , 5.81–5.69 , 3.93 ..13C-NMR  δ 128.52, 123.98, 60.32..−1 ν(C-H), 1447 cm−1 γ (CH2), 1020 cm−1 ν(C-N).ATR-IR: 2937 cm.10 g (116.1 mmol) of piperazine was dissolved in 31 mL saturated potassium carbonate solution in a 250 mL round-bottom flask. The solution was cooled in an ice-water bath and 22.0 mL  allyl bromide was slowly added over the course of 1 h. When approx. half of the allyl bromide had been added, the solution separated into a lower white suspension and an upper oily yellow phase formed. After complete addition, the reaction mixture was stirred at room temperature for 72 h before adding 50 mL Dichloromethane. The phases were separated and the aqueous phase was extracted three times with Dichloromethane. The combined organic phases were dried with magnesium sulphate, filtered, and the solvent was removed under reduced pressure. 11.43 g (54%) of the crude diallylpiperazine was obtained as pale, yellow, oily liquid.1H-NMR  δ 5.85 , 5.13 , 2.98 , 2.47 ..11.43 g  diallylpiperazine was dissolved in 95 mL of acetone. To this, 17.82 mL  allyl bromide was slowly added. The reaction mixture was refluxed for 72 h at 88 °C. During this, the crude product precipitated as a beige solid, which was filtered, washed with acetone, and recrystallized from methanol to yield 28.06 g (65%) tetraallylpiperazinedibromide as white crystalline solid. Similar to 1a, the dibromide 1b was found to decompose at 210 °C, previously reported as m.p. 207 °C [1H-NMR  δ 6.10 , 5.90 , 4.26 , 3.99 ..13C-NMR  δ 130.78, 122.22, 50.66..−1 ν(C-H), 1473 cm−1 γ (CH2), 1150 cm−1 ν(C-N).ATR-IR: 2988 cm.The preparation follows the two-step procedure outlined for TAPB. 16 g (81.49 mmol) trimethylenedipiperidine afforded 8.51 g (38%) crude diallyltrimethylendipiperidine (DAMP) as yellow, oily liquid.1H-NMR  δ 5.82 , 5.07 , 2.91 , 2.85 , 1.81 , 1.60 , 1.19 ..In the second step, 8.51g DAMP yielded after recrystallization in methanol 11.53 g (72%) TAMPB as beige crystalline solid. Similar to 1a, the dibromide 1c was found to decompose at 220 °C.1H-NMR  δ 6.07 , 5.73 , 4.00 , 3.89 , 3.52 , 3.28 , 1.93 , 1.68 , 1.40 ..13C-NMR  δ 128.61, 128.22, 123.77, 65.22, 56.14, 34.31, 32.15, 25.25, 22.51..−1 ν(C-H), 1497 cm−1 γ (CH2), 1037 cm−1 ν(C-N).ATR-IR: 3013 cm.N,N’-methylenebisacrylamide  was stirred until the crosslinker had completely dissolved. Meanwhile, 60 mg KPS were dissolved separately in 1.5 mL H2O and then added to the monomer solution. The mixtures containing 1a–c were stirred for 10 min and those with BIS for shorter times, as gelation with BIS begins earlier. An example image of a swollen polyDADMAC gel is given in the The method is described using 2 mol% crosslinker as an example. Details on the compositions and further experimental details can be found in −1 with 12 scans at a spectral resolution of 4 cm−1. Before each measurement, the diamond ATR crystal was cleaned with isopropanol.Infrared spectra were recorded using a Perkin Elmer Spectrum Two UATR FTIR spectrometer equipped with a diamond ATR  window. All spectra were recorded in the spectral range of 4000–400 cmThe rheological data were recorded on an Anton Paar Modular Compact Rheometer 102. A plate-plate geometry with a diameter of 25 mm made of stainless steel was used. All samples were measured with a plate gap of 1 mm at 20 °C. The isothermal oscillating time-dependent measurements were performed at 1% amplitude and an angular frequency of 1 Hz over a period of 16 h. The set-up was covered with a solvent trap over this period.G’ and G’’ curves  [Sample preparation follows the above procedure for the preparation of DADMAC hydrogels. After adding the KPS initiator, the mixture was stirred intensively for 5–10 min , depending on the rate of polymerisation. 560 μL of this mixture was then transferred to the rheometer using an Eppendorf pipette. The gel point was calculated from the intersection of the ed data) ,30, by a2 (blocking electrodes). The solution was covered with a layer of silicon oil to exclude oxygen and prevent evaporation. Over a course of 16 days, the impedance Z was recorded every hour using a voltage of 141 mV. As shown in Zimag ≈ 0 and Z = Zreal.Impedance spectra were recorded on a Gamry Potentiostat 400 . Sample preparation followed the above procedure for the preparation of crosslinked DADMAC hydrogels using 1 mol% of the desired crosslinker BIS, TAAB, TAPB, or TAMPB). After adding the KPS initiator and stirring intensively for 10 min, the mixtures were then transferred to standard electroporation cuvettes (Gap width = 4 mm), equipped with two opposing aluminium electrodes of 9 × 18 mmApprox. 100 mg of lyophilized hydrogel were weighed into polyester filter-bags and submerged in 0.5 L of double-distilled water at 22 °C. The mass increase was initially recorded every 15 min, later every 30 min to one hour, depending on the previous mass changes. After removing the teabags from the solutions, they were carefully stripped and hung up for 5 min to drain the nonabsorbed liquid. The reported maximum swelling ratios were determined by averaging the last three recorded values. The swelling experiments were each repeated once. It was found that the determined values varied by ±5% at low degrees of swelling. For high degrees of swelling (>200 g/g), deviations of up to ±14 % were found.1H-NMR and 13C-NMR spectra were recorded on a Mercury 400 spectrometer . Chemical shifts were calculated using the HDO signal at 4.64 ppm or the CDCL3 signal at 7.26 ppm as a reference.


Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines. S. peucetius DM07 by the OSMAC strategy.Activation of peucemycin in  Streptomyces are Gram-positive filamentous bacteria with the ability to produce a wide range of secondary metabolites, many of them are antimicrobial or anticancer drugs.1–3 The analysis of the genome sequences of Streptomyces using bioinformatics tools such as antiSMASH4 indicates that they possess more than 20 biosynthesis gene clusters (BGC).5 However, most of these BGCs are either expressed or not expressed under laboratory conditions. Therefore, several approaches have been utilized to activate these silent BGCs and/or increase the titer of production to characterize the encoded compounds; these approaches include either culture conditioning or genetic manipulation.6–89,10 Many successful OSMAC approaches have been reported to produce new compounds using various media,11,12 salinity,13 and metal ions.14 In addition, we applied the OSMAC approach by varying the culture conditions as temperature is a very important factor in the activation of specific compounds.15,16One strain many compound (OSMAC) is a simple and powerful approach used to activate numerous compounds such as penicibrocazines A–I, and brocaryrrozins A-B.Streptomyces peucetius ATCC 27952 is known for producing important anthracyclines: daunorubicin (DNR) and doxorubicin (DXR).17 Several secondary metabolites have been explored through mass and NMR analyses; for example, 1,3,6,8-tetrahydroxynaphthalene, peucechelin, hopene, and geosmin.18–21 The in silico analysis of the S. peucetius genome exhibited that 68 BGGs exist in this strain including diverse metabolites such as PKS, NRPS, terpenes, and siderophores.2223 For example, candelalide A, B, C from Sesquicillium candelabrum play a role in a blocker of the voltage-gated potassium channel Kv1.3;24 aureothin from Streptomyces thioluteus has numerous biological assays and exhibits antifungal and antibacterial action;25 actinopyrone A, B, C from Streptomyces pactum has microbial activity;26 and prenylflavones, prenylflavonoids and xanthone psorospermin exhibit cytotoxic activity.27,28 Macrolides are a 14- to 16-membered lactone ring, such as erythromycin, azithromycin, and tylosin, and are used as an antibiotic in veterinary medicine.29 Macrolides are important in human therapeutics as they are safe to use in β-lactam-allergic patients, period pregnancy, pediatric and elderly patients.30Structure motif γ-pyrones have biologically active compounds.S. peucetius; the isolation and structure of which were elicited via LC-MS/MS and NMR analyses. Furthermore, we examined the biological activity of the novel compound, which shows antibacterial and anticancer activity.Herein, we applied the OSMAC approach by changing the culture conditions as temperature leads to the activation of a novel compound in S. peucetius DM07 was used for this experiment. S. peucetius DM07 was a disrupted doxorubicin biosynthetic gene cluster from S. peucetius ATCC 27952 (accession number CP022438.1) by Singh et al.21 The seed culture was cultivated in a 50 mL R2YE medium, shaking at 200 rpm in an Erlenmeyer flask at 28 °C for 48 h.31 To produce secondary metabolite, S. peucetitus DM07 was cultured in a 50 mL NDYE medium  and incubated in a shaking incubator at 200 rpm at various temperatures. For evaluating the effect of temperature we first selected 37 °C  and 28 °C (most suitable for S. peucetius for the growth and production of secondary metabolites such as doxorubicin and doxorubicin). Then, we created a gradient of temperature with 5 °C difference at a lower scale such as 23 °C, 18 °C and 13 °C, and 5 °C difference at a higher scale as 43 °C and 48 °C .S. peucetius DM07 strain was cultivated in the 50 mL NDYE medium via continuous shaking at 200 rpm at various temperatures for 72 h. After fermentation, 100 mL of ethyl acetate was added to extract compounds from the culture broth. Next, 1 mL of methanol was dissolved in the evaporating ethyl acetate of the extract. The resultant methanol extract was analyzed by a Thermo HPLC series 1100 with a Thermo-C18 column . The column was equilibrated with 100% solvent A 0.1% trifluoroacetic acid (TFA) in the water and 0% solvent B (acetonitrile (ACN)), and was then adjusted following a linear gradient  at a flow rate of 1 mL min−1 and with UV detection at 268 nm.S. peucetius DM07 strain was fermented at 18 °C for 72 h in a fermenter. The fermentation broth (5 L) was extracted with 10 L of ethyl acetate and evaporated under a reduced pressure. After that, the extract was dissolved in 50 mL methanol. The crude extract was used for mass analysis and in the purification process.The via reversed-phase chromatography ultra-high-performance liquid chromatography electrospray ionization quadrupole time of flight high-resolution mass spectrometry (UPLC-ESI-Q-TOF-HRMS). Samples were eluted with a gradient solvent mobile phase of 0.1% TFA in water and acetonitrile (0 to 12 min) at 35 °C. The 10 μL of the sample was injected. An LC-MS analysis was performed on a high-resolution mass spectrometer equipped with an electrospray ionization source with 3 kV, 300 °C, and 600 L h−1 as the capillary voltage, desolvation gas temperature, and flow rate, respectively.The mass analysis was performed 18 column  connected to a UV detector . The parameters used were binary gradient: 100% water (solvent A); 100% acetonitrile (solvent B); 0% B (0–3 min), 0% B to 10% B , 10% B to 45% B , 45% B to 75% B , 75% B to 100% B  100%, 100% B to 0% B (32–35 min). The flow rate to purify the pure compound was 10 mL min−1.The extract of the culture broth was examined using an ultimate 3000 UPLC (Thermo Fisher Scientific) with a Cd6), and accessed in a 700 MHz spectrometer using a Bruker BioSpin nuclear magnetic resonance (NMR) spectrometer  for analyses, including one-dimensional (1D) 1H-nuclear magnetic resonance (NMR),13C-NMR, and two-dimensional (2D) correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), rotational frame NOE spectroscopy (ROESY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) analyses . IR (Infrared Spectroscopy) spectra were recorded using a Bruker VERTEX 70 FTIR spectrometer using the analysis function group.The purified compound was dried, lyophilized, dissolved in dimethyl sulfoxide , and Gram-negative bacteria as Klebsiella pneumoniae subsp. pneumoniae ATCC 10031, Proteus hauseri NBRC 3851, and Salmonella enterica ATCC 14028. Peucemycin was added to paper discs in various amounts . Positive control erythromycin was added (73.3 μg per disc). The test compounds were dissolved in DMSO, and 2 μL of DMSO was added to a paper disc as a negative control. The plates were incubated at 37 °C for 16–24 h.33The antibacterial activity of peucemycin was investigated on a petri dish by the disc-diffusion method2 incubator. For the cell viability assay, various cancer cells were plated at a 2 × 103 cells per well density in 96-well culture plates. Compound peucemycin was added to each well in various concentrations (0–400 μM) and maintained at 37 °C for 72 h. A 3--2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was performed to check cell viability. Briefly, 50 μL of MTT (2 mg mL−1 stock solution) was added to the plates and maintained at 37 °C for 4 h. Each well was removed from the medium and then 100 μL of dimethyl sulfoxide was added. A microplate spectrophotometer was used to measure the absorbance wavelength at 540 nm (Thermo Scientific Multiskan® Spectrum). The cytotoxicity assays were executed on triplets, and the results are shown as mean values standard error (SE).Peucemycin was also evaluated for its anticancer activity against six types of cancer cell lines. Human cancer cell lines HeLa, A549, Hep3B, A375SM, U87MG, and AGS, and a human lung fibroblast cell line MRC-5 were purchased from the Korean Cell Line Bank . AGS gastric cancer cells were grown in an RPMI 1640 medium by adding 10% fetal bovine serum . HeLa cervical cancer cells, Hep3B liver cancer cells, A375SM skin cancer cells, A549 lung cancer cells and MRC-5 lung normal cells were maintained in a Dulbecco's modified Eagle's medium  containing 10% FBS. U87MG brains cancer cells were cultured in a minimum essential medium (MEM) supplemented with 10% FBS. All cells were incubated at 37 °C in a humidified 5% COS. peucetius DM07 was cultured for 72 h in the NDYE medium at various temperatures: 18 °C, 23 °C, 28 °C and 37 °C. The secondary metabolite of S. peucetius DM07 was extracted by ethyl acetate and analysed by HPLC and LC-MS. When comparing secondary metabolites at different temperatures, a new peak appeared at 17.5 min with the 18 °C culture conditions, while this peak was absent at other temperatures + 177.12 m/z, (M − A + 2H)+ 239.09 m/z, (B)+ 221.08 m/z  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O of the aliphatic esters that appear at 1738 cm−1, a C–O stretch appears at 1287 cm−1, a stretching vibration of the CC bond (olefins) at 1653 cm−1, a CH3-asymmetric stretching-vibration absorption at 2972 cm−1, a CH2-stretching-vibration absorption at 2936 cm−1, a CH-asymmetric stretching-vibration absorption at 2862 cm−1, and the carboxylic acid with –OH stretching absorption from 3522 to 3192 cm−1 , δH 0.97 , δH 1.02 ; two singlets δH 4.91 , and 6.08 ; five doublets at δH 3.73 , 4.97 , 5.02 , 6.21 , 6.29 ; three double of doublets δH 3.66 , 6.01 , 5.70 ; two triple of doublets δH 5.18 , and 2.15 ; one double of triplets δH 6.83 ; one quartet δH 4.32 ; and multiplets 2.12–2.06  , an ester carbonyl group at δC 167.6 (C-3), oxy-quaternary sp2 at δC 160.46 (C-1), 163.22 (C-11), oxy-methine at δC 74.86 (C-9), 72.16 (C-10), quaternary sp2δC 132.92 (C-6), 122.7 (C-12), 137.30 (C-17), methine sp2δC 128.71 (C-7), 130.71 (C-8), 114.78 (C-14), 136.88 (C-16), 132.63 (C-18), 119.22 (C-23), 138.74 (C-24), methylene δC 39.71 (C-2), 60.99 (C-5), 21.24 (C-19), 23.71 (C-21), 27.08 (C-25) and three methyl δC 14.78 (C-20), 13.71 (C-22), 14.01 (C-26) carbon -12-((E)-but-1-en-1-yl)-6-((E)-2-ethylpent-2-en-1-ylidene)-9,10-dihydroxy-4,15-dioxabicyclo[9.3.1]pentadeca-1(14),7,11-triene-3,13-dione (peucemycin). The chemical structure of peucemycin shows a novel chemical architecture, thus it was allocated as a novel compound.Based on the mass spectrometry and NMR spectroscopy, the compound was named as  but it exhibits antibacterial activity against Gram-positive bacteria (M. luteus and S. aureus) and Gram-negative bacteria (P. hauseri and S. enterica). It was observed that the compound exhibits antibacterial potency at different concentrations: 5.175, 10.35, 20.7, 41.4, and 82.8 μg per disc, whereas the standard erythromycin (7.33 μg per disc) was used as the positive control and DMSO was used as the negative control . Moreover, the IC50 value of peucemycin on MRC-5 normal lung fibroblast cells 316 μM was much higher than that on A549 lung cancer cells (135.5 μM) (50 0.1 μM), human lung cancer cells A549 (IC50 4.91 μM).35 The result indicated that peucemycin is less toxic than doxorubicin against the tested cell lines, which predicts the potential selectivity of peucemycin against the various cancer cells.In addition, 35.5 μM) . Thus peStreptomyces such as chloramphenicol, erythromycin, tetracycline, and streptomycin.36–39 The emergence and spread of antimicrobial resistance are serious problems for public health around the world;40 for example, Streptococcus pneumoniae and Streptococcus pyogenes are resistant to erythromycin;41Staphylococcus aureus and Neisseria gonorrhoeae are resistant to penicillin.42,43 Therefore, finding new bioactive compounds is an important strategy.Natural products from microorganisms have been proven to be clinically important in human therapy as antifungal, antibacterial, antitumor, and anticancer agents. Two-thirds of medical antibiotics were isolated and identified from 3,44 Secondary metabolites including activity compounds were influenced by the culture temperature in Pseudogymnoascus sp., Penicillium flavigenum, and Atradidymella sp.44 Sadeghy and Hatami reported that Streptomyces sp. isolates C-26 and C-11 and there was a maximum metabolite production such as antifungal compounds when the culture condition was changed from 40 °C to 70 °C.15Streptomyces sp. RUPA-08PR increased the antibiotic production at 39 °C.16 Previously, S. peucetius showed the highest doxorubicin and daunorubicin production at 28 °C.45 In general, bioactive compounds are produced higher at room temperature; however, in this study, we demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius , decreased accumulation by enhanced efflux mediated, and hydrolysis degradation to the inactivation of macrolide were found.athogens  and cytoathogens . For furvia LC-MS and NMR. In addition, we examined the biological activity of peucemycin, which can be considered as a broader range antibacterial molecule and cytotoxicity against various cancer cell lines.In conclusion, we successfully activated and identified a new compound (peucemycin) by varying the culture temperature. The structure of peucemycin was elucidated There are no conflicts to declare.RA-011-D0RA06099B-s001


Prolyl oligopeptidase (POP) is a large cytosolic serine peptidase that is altered in patients with Alzheimer’s disease, Parkinsonian syndrome, muscular dystrophies, and other denervating diseases. Thus, POP may represent a relevant therapeutic target for treatment of neuropsychiatric disorders and neurodegenerative diseases. Here, we report the characterization of five novel cyanopyrrolidine-based compounds  and show that they are potent inhibitors of POP and are predicted to penetrate the blood-brain barrier (BBB). Indeed, we show that CbzMetPrdN penetrates the rat BBB and effectively inhibits POP in the brain when administered intraperitoneally. Furthermore, molecular modeling confirmed these compounds likely inhibit POP via interaction with the POP catalytic site. We evaluated protective effects of the cyanopyrrolidine-based POP inhibitors using scopolamine- and maximal electroshock-induced models of amnesia in rats and showed that BocTrpPrdN, BocGlyPrdN, CbzMetPrdN, and CbzGlnPrdN significantly prolonged conditioned passive avoidance reflex (CPAR) retention time when administered intraperitoneally (1 and 2 mg/kg) before evaluation in both models of amnesia, although CbzAlaPrdN was not effective in scopolamine-induced amnesia. Our data support previous reports on the antiamnesic effects of prolinal-based POP inhibitors and indicate an important role of POP in the regulation of learning and memory processes in the CNS. Proteolytic enzymes play an important role in regulating the function of endogenous peptides. Proteases and peptidases control the concentration of neuropeptides by modulating their formation, modification, and inactivation. Numerous peptidases are present in brain tissue and contribute to the specific functions of neurons or glial cells. Even minor disturbances in the function of these enzymes can contribute to the development of central nervous system (CNS) pathological states, including dementia, Parkinsonism, epilepsy, cerebral ischemia, and Alzheimer’s disease .Prolyl oligopeptidase  is a large cytosolic serine peptidase that is widely distributed in the CNS, including in the hypothalamus, striatum, hippocampus, cortex, and amygdala. POP specifically cleaves peptide bonds at the carboxylic end of proline residues in proline–containing endogenous peptides [reviewed in ]. For exPOP activity correlates with several CNS functions, particularly those that involve memory, learning, and mood related responses . On the Data on variations in the activity of proline-specific enzymes in the clinical picture of major depression have served as a basis for the development of a laboratory model of the anxiety–depressive state in rats . For exaAlzheimer’s disease is the most prevalent disease of old age leading to dementia. Alzheimer’s disease pathogenesis is complex and involves multiple factors, such as hyperphosphorylated tau, a microtubule assembly protein that is deposited intracellularly in neurofibrillary tangles; β-amyloid plaque formation; and inflammation; ensuing degeneracy; and neuronal cell death . In earlN-acylated glycyl-(2-cyano)pyrrolidines displayed inhibitory activity toward fibroblast activation protein (FAP)  , a serinin (FAP) . CbzMetPin (FAP) .The catalytic triad of POP consists of Ser554, His680, and Asp641 , and it In the present study, we synthesized and evaluated the POP-inhibitory activity of five novel KYP-2047 analogs, including BocTrpPrdN, BocGlyPrdN, CbzMetPrdN, CbzGlnPrdN, and CbzAlaPrdN . Molecultert-butoxycarbonyl)-L-prolinamide (BocProNH2) (2) (2S)-pyrrolidine-2-carbonitrile (CbzAlaPrdN) (tert-butoxycarbonyl)glycyl-(2S)-pyrrolidine-2-carbonitrile (BocGlyPrdN) (tert-butoxycarbonyl)-L-tryptophanyl-(2S)-pyrrolidine-2-carbonitrile (BocTrpPrdN) (Compounds N-benzyloxycarbonyl-L-glutamine (CbzGlnOH) , N-(tertcProNH2) , N-benzyNH2) (2) , N-benzoAlaPrdN) , N-(tertGlyPrdN) , and N-(TrpPrdN)  were syn2S)-pyrrolidine-2-carbonitrile (CbzMetPrdN)N-Benzyloxycarbonyl-L-methionyl- of benzenesulfonyl chloride were added. The reaction mixture was stirred at 15–17°C for 16 h, the solvent was evaporated in vacuo, and the residue was dissolved in ethyl acetate. The resulting solution was washed sequentially with H2O, 5% H2SO4, H2O, and brine and then dried over MgSO4. The solvent was evaporated, and the residue was crystallized from ether to obtain CbzMetPrdN as a white crystalline powder. Yield 78%; m.p. 73°C; specific rotation ([α]D) –94.8° (methanol). Found, %: C 60.1; H 6.6; N 11.3. C18H23N3O3S. Calculated, %: C 59.83; H 6.42; N 11.62. IR bands, cm−1: 3,450 (NH2), 1725 (C=O), 1,660 (NH), 2,233 (CN). 1H NMR (CDCl3) δ ppm: 7.35 , 5.84 , 5.08 , 4.72 , 4.66 , 3.77 , 2.56 , 2.20 , 2.12 , 2.06–1.82 . ESI-MS, m/z: 362.1496 exp. ([C18H23N3O3S + H]+ = 362.1538 theor.).p-nitrophenyl esterN-Benzyloxycarbonyl-L-glutamine . To a solution of 1 mol of CbzGlnOH in dimethylformamide (DMF), 1.1 mol of p-nitrophenol and 1.1 mol of dicyclohexylcarbodiimide (DCC) were added at 0°C, stirred for 1 h at 0°C and left for 20 h at room temperature. The precipitated dicyclohexylurea was filtered, washed with ethyl acetate, and the solvent was removed under reduced pressure. The residue was crystallized from ethanol. M.p. 155–156°C [α]D –24° (DMF).N-Boc-L-proline nitrile (BocPrdN). To a solution of 20 mmol BocProNH2 in 20 ml of pyridine at 10°C, 4 ml (31.5 mmol) of benzenesulfonyl chloride were added and stirred for 12 h. The mixture was diluted with H2O, extracted with ethyl acetate (100 ml), and the extract was washed sequentially with 0.5 M H2SO4, H2O, and 1 M Na2CO3 and then dried. The solvent was evaporated, and the residue was crystallized from hexane. Yield 50%, m.p. 47–48°C [α]D –64.0° (methanol).L-proline nitrile trifluoroacetate. CF3COOH (2 ml) was added to a solution of 2 g BocPrdN in 2 ml of dichloromethane. After 30 min, the solvent was evaporated, and the residue was crystallized from ether. M.p. 98–99°C. [α]D –19.6° (ethanol), −25.4° (methanol).2S)-pyrrolidine-2-carbonitrile (CbzGlnPrdN)N-Benzyloxycarbonyl-L-glutaminyl-(. A solution of proline nitrile trifluoroacetate (0.6 g), 1.2 g of N-Cbz-glutamine p-nitrophenyl ester, and 0.5 ml of triethylamine in 5 ml of DMF was stirred for 16 h, diluted with ethyl acetate, washed sequentially with H2O, 0.5 M H2SO4, 1 M Na2CO3, H2O, and brine, and dried with MgSO4. The solvent was evaporated, and the residue was recrystallized from chloroform. Yield 30%, m.p. 119–120° [α]D –37.4°. Found, %: C 60.5, H 6.4, N 15.3; C18H22N4O4. Calculated, %: C 60.34; H 6.19; N 15.63. IR bands, cm−1: 3,450 (NH2), 1725 (C=O), 1,660 (NH), 2,233 (CN). 1H NMR (CDCl3) δ pm: 7.38, 7.30 d (5H); 6.16–6.0 m (1H); 5.58 d (1H); 5.12–5.08 m (2H); 4,72 d, 4.64 d, 4.50 m (2H); 3.86–3.50 m (2H); 2.40–2.0 m (5H); 1.92–1.82 s (2H). ESI-MS, m/z: 359.1714 exp. ([C18H22N4O4 + H]+ = 359.1719 theor.).To prepare emulsions of the inhibitors and scopolamine for injection in to animals, the amount of compound powder corresponding to a proper dose for the animal was mixed with 20 μl of Tween 80. Two ml of physiologic NaCl solution was added to create a suspension.POP was isolated from rat brain, as described previously  with modcat (sec−1) = 161.7 ± 13.5 and Km (μM) = 54 ± 4. The reaction was initiated by addition of the substrate (20 μM) in a final reaction volume of 2 ml. The incubation mixture consisted of 50 μl of the enzyme preparation in 20 mM Tris-HCl buffer (pH 7.5) with 1 mM EDTA, 1 mM dithiothreitol, and 0.1% NP–40; 50 μl of 1 mg/ml substrate solution in DMSO; and 1.9 ml of Tris-HCl buffer. Final concentration of DMSO in the reaction solution was 2.5%, which had no effect on POP enzymatic activity (data not shown). After a 30-min incubation at 37°C, the reaction was stopped by addition of 1 ml of acetate buffer (pH 4.0). The AMC concentration was determined on an LS–5B spectrofluorometer  with excitation and emission wavelengths of 380 and 460 nm, respectively. For all compounds tested, the concentration of inhibitor that caused 50% inhibition of the enzymatic reaction (IC50) was calculated by plotting % inhibition versus logarithm of inhibitor concentration (at least 5-6 points).Enzymatic activity was determined by measuring the release of 7-amino-4-methylcoumarin (AMC) from the synthetic fluorogenic POP substrate Cbz-Ala-Pro-AMC  which is an excellent substrate for POP from rat brain: kAll manipulations with experimental animals were performed in accordance with international and Russian regulatory documents: Order of the Ministry of Health of the Russian Federation No. 199n of April 1, 2016 and Directive 2010/63/EU of the European Parliament and the Council of the European Union for the Protection of Animals Used in Scientific purposes. Experiments were performed on 3 month old male Wistar rats. Animals were housed in accordance with the sanitary and epidemiological rules of SR 2.2.1.3218–14 “Sanitary and epidemiological requirements for the design, equipment and maintenance of experimental biological clinics (vivariums).” The experiments were approved by the Commission on Biomedical Ethics of Zakusov Institute of Pharmacology . Rats were housed in groups of five animals per cage (57 × 36 × 20 cm) in standard laboratory conditions  in cages with sawdust bedding, standard rodent feed, and ad libitum water access.n = 3 for each time-point) after a single 20 mg/kg intraperitoneal (i.p.) administration of CbzMetPrdN. The test animals were sacrificed, and their brains were harvested and immediately homogenized in phosphate buffer saline.The total drug concentrations for CbzMetPrdN and POP activity in the brain were determined as a function of time at 0, 20, 40, 60, 80, and 120 min . The cartridge was washed with 1 ml of 5% ammonia solution and then with 1 ml of methanol. The sample was eluted with 1 ml of 2% trifluoroacetic acid in methanol and analyzed using a Gilson 41 HPLC system  with a 125 × 4 mm Nucleosil C18 column (Macherey-Nagel). The column was washed for 3 min with 4 mM hexanesulfonate in water (pH 3.5), followed by a 0–100% gradient of 4 mM hexanesulfonate in acetonitrile:water (50:50) % (v/v) over 10 min. The concentration of the POP inhibitor is expressed in ng of CbzMetPrdN per mg of brain protein.POP activity in the brain tissue homogenate was measured using Cbz-Ala-Pro-AMC, as described above, and is presented as nmole AMC released/min/mg of brain protein. Protein concentrations of the supernatants were determined with a Bio-Rad protein assay kit.The development of CPAR was performed on a standard passive avoidance device from Lafayette Instrument Co. (United States). The device consists of a small platform located at a distance of 1 m from the floor, illuminated by a special lamp, and a dark chamber with an electrode floor. During learning, a rat was placed on the small platform in front of the entrance to the dark chamber. Immediately after entering the dark chamber, the rat received a painful stimulation from an electric current of 0.6 mA through the electrode floor lasting 10 s or until the rat ran out of the chamber (learning). A rat was considered trained if it did not enter the dark chamber for the next 30 s. If the rat entered the dark chamber, the training was repeated. A test for the refiex reproduction was performed after 24 h. In this case, the rat was placed on a lighted area, and step-through latency time (STL) of its entry into the dark chamber was recorded. The observation time was 3 min. The criterion for refiex reproduction was considered to be the STL of entry into the dark box.n = 8) received 2 ml of vehicle  i.p. 40 min before the CPAR learning. Rats in the non-treated group  received scopolamine  20 min before the CPAR learning. Rats of groups 3–17 received a single injection of POP inhibitor at doses of 0.5, 1, and 2 mg/kg, i.p. , including CbzAlaPrdN , BocTrpPrdN , BocGlyPrdN , CbzMetPrdN , and CbzGlnPrdN  followed (after 20 min) by administration of scopolamine  20 min before the CPAR learning. Rats in group 18 (n = 8) received piracetam (300 mg/kg) followed (after 20 min) by administration of a single injection of scopolamine (1 mg/kg in 2 ml of vehicle) 20 min before the CPAR learning.Experiments were performed using two rat models of amnesia. In the first model, effects of POP inhibitors on scopolamine-induced amnesia were studied. The series included eighteen groups of animals (137 rats). Rats in the negative control group  received 2 ml of vehicle  i.p. 30 min before the CPAR learning. Rats in the positive control group  received MES just after CPAR learning. MES was induced using an electric shock generator from Harvard Apparatus  consisting of a current  by way of corneal electrodes. Rats of groups 3–14 received a single injection of POP inhibitor at doses of 0.5, 1, and 2 mg/kg, including BocTrpPrdN , BocGlyPrdN , CbzMetPrdN , and CbzGlnPrdN  40 min prior to the CPAR learning  with following MES just after CPAR learning. Rats in group 15 (n = 8) received piracetam  50 min before the CPAR learning and MES just after CPAR learning.In the second model, effects of POP inhibitors on MES-induced amnesia were studied. The series included fifteen groups of animals (110 rats). Rats in the negative control group -1-chloro-3-[(4-fluorobenzyl)oxy]-6-(pyrrolidin-1-ylcarbonyl)pyrrolopyrazin-4(6H)-one . The structure of POP complexed with ((6H)-one  was downhttp://www.swissadme.ch)  .rot was determined from the structural formulae. The predicted ability of the compounds to penetrate the blood-brain barrier (BBB) was determined in accordance with the model proposed by Molecular modeling for the POP inhibitors was used for calculation of the following parameters: aLogP was calculated using HyperChem 7 software based on additive scheme , tPSA t, the num2. This amide was converted into the target nitrile by the action of benzenesulfonyl chloride in pyridine. This final stage is a modification of the methodology by 2Cl to obtain N-protected proline nitriles.We synthesized two novel compounds: CbzMetPrdN and CbzGlnPrdN. The other proline nitrile derivatives  were synthesized as previously reported ; however2 were used as starting materials for the synthesis of CbzGlnPrdN according to p-nitrophelyl ether of Cbz-protected glutamine.CbzGlnOH and BocProNH50 ∼ 2 nM). Inhibitory activity of the reference POP inhibitor KYP-2047  was in same concentration range as reported previously by 50 = 6 ± 4 nM) and was similar to the activity of most of our new cyanopyrrolidine-based POP inhibitors.Five cyanopyrrolidine-based derivatives were evaluated for POP inhibitory activity in comparison with the known POP inhibitor KYP-2047, and the results are reported in 6S)-1-chloro-3-[(4-fluorobenzyl)oxy]-6-(pyrrolidin-1-ylcarbonyl)pyrrolopyrazin-4(6H)-one complexed in the POP binding site. A pyrrolidine ring of the co-crystallized inhibitor is located in the vicinity of Ser554, and the molecule itself occupies key cavities of the site denoted as S1, S2, and S3  , and the number of rotatable bonds (Nrot) .In accordance with the classification model , these vi.p. administration (20 mg/kg)  .Rat brain POP activity as a function of time after CbzMetPrdN administration is shown in The antiamnesic properties of prolinal-based POP inhibitors and KYP-2047 have been demonstrated previously . Here weThe four POP inhibitors that showed activity in scopolamine-induced amnesia  were evaluated for antiamnesic activity in MES-induced amnesia. MES resulted in a decrease in STL time (34.3 ± 14.0 s) in comparison with rats from the control group (119.6 ± 14.7 s). At a dose of 1 mg/kg, CbzMetPrdN, and CbzGlnPrdN had potent antiamnesic activity, which was comparable with the antiamnesic activity of piracetam. Although BocTrpPrdN and BocGlyPrdN were less potent in comparison with piracetam (300 mg/kg), these POP inhibitors exhibited antiamnesic activity at doses of 1 and 2 mg/kg .POP is a serine protease that has been studied particularly in the context of neurodegenerative diseases, including Alzheimer’s disease . Over thWe found that administration of POP inhibitors, including BocTrpPrdN, BocGlyPrdN, CbzMetPrdN, and CbzGlnPrdN, significantly improved scopolamine- and MES-induced memory impairments in the CPAR model, with the most potent being CbzMetPrdN. It should be noted that CbzMetPrdN was previously studied in a preclinical model of Parkinsonian syndrome, where it exhibited neuroprotective activity and abolished depressive symptoms caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) . Thus, Cin vitro and in vivo and that administration of these compounds can be beneficial in rat models of amnesia. However, the exact mechanisms involved are still not fully elucidated. For example, in vivo cleavage of substance P or neurotensin and that positive cognitive effects associated with POP inhibitors are not mediated through elevated extracellular levels of these peptides. In addition, POP can participate in protein-protein interactions with various neuronal proteins  independently of peptidase activity (Our results show that novel cyanopyrrolidine-based derivatives can inhibit POP enzymatic activity activity . Thus, P


Porin alterations were analysed with Provean v1.1.3. Specific alterations of interest in PBP3 included a YRIN or YRIK insertion after P333.A total of 179 ENSE (MIC ≥ 1 mg/L) isolates underwent susceptibility testing using reference CLSI broth microdilution. WGS was performed using the Illumina NextSeq platform. Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0. Alterations in 50/MIC90, 0.5/2 mg/L; 177/179 isolates inhibited at ≤ 8 mg/L) against ENSE with various antimicrobial resistance phenotypes. Thirteen (7.3%) of the 179 ENSE isolates demonstrated cefepime/taniborbactam MIC values ≥ 4 mg/L and possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Of the two Escherichia coli isolates that demonstrated a cefepime/taniborbactam MIC of 32 mg/L, one possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration and P333_Y334insYRIN in PBP3, while the second contained CTX-M-71, a truncated OmpF and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE).Cefepime/taniborbactam was highly active (MICCefepime/taniborbactam was highly active against ENSE with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Cefepime/taniborbactam is in Phase 3 clinical development for the treatment of complicated urinary tract infection.8–10Cefepime is a parenteral extended-spectrum cephalosporin that has been used clinically for decades.in vitro activities of cefepime/taniborbactam and comparator antimicrobial agents against ertapenem-non-susceptible Enterobacterales (ENSE) clinical isolates collected from the CANWARD study 2007–19, as well as associations between MIC and various mechanisms of β-lactam resistance identified using WGS.The current study assessed the in vitro activities of antimicrobial agents against bacterial pathogens isolated by clinical laboratories from patients attending tertiary care hospitals across Canada.3 Each isolate submitted was considered clinically significant by protocols in place at the submitting laboratory. The CANWARD surveillance study sets annual quotas for respiratory, wound, urine and bloodstream isolates and requires isolates be collected consecutively, one per patient, per site of infection, from both in- and outpatients attending emergency rooms, hospital clinics, medical/surgical wards and ICUs each year.3 All isolates are shipped to the CANWARD coordinating laboratory  where their identities are confirmed by colonial appearance, spot testing and/or MALDI-TOF MS .3 Tertiary care hospitals in 8 of the 10 Canadian provinces participate in the CANWARD surveillance study. The number of tertiary care hospitals participating in the CANWARD surveillance study by year was: 12 in 2007, 10 in 2008, 15 in 2009, 14 in 2010, 15 in 2011, 12 in 2012, 15 in 2013, 13 in 2014, 13 in 2015, 13 in 2016, 14 in 2017, 12 in 2018 and 10 in 2019. The CANWARD surveillance study receives annual approval by the University of Manitoba Research Ethics Board (H2009:059).CANWARD is a national, ongoing, Public Health Agency of Canada-National Microbiology Laboratory (PHAC-NML)/Canadian Antimicrobial Resistance Alliance (CARA) partnered surveillance study evaluating 3 The 179 ENSE included in the current study were Enterobacter cloacae (n = 96), Escherichia coli (n = 26) and 57 other species . Specimen sources were 43% respiratory, 39% blood, 10% urine and 8% wound.The CANWARD 2007–19 isolate collection contained 18 027 isolates of Enterobacterales of which 179 (0.99%) were ertapenem-non-susceptible (MIC ≥ 1 mg/L).E. cloacae (n = 26), E. coli (n = 7) and 18 other species } were randomly selected from the CANWARD 2007–19 isolate collection and tested as controls.Fifty-one ertapenem-susceptible (MIC ≤0.5 mg/L) Enterobacterales {in vitro activities of cefepime/taniborbactam (cefepime doubling dilution range 0.03–128 mg/L with taniborbactam fixed at 4 mg/L) and comparator agents were determined by reference CLSI broth microdilution  using 96-well custom designed microtitre plates.11 Antimicrobial agents were obtained as laboratory grade powders from their respective manufacturers or commercial sources. Stock solutions were prepared and dilutions made as described by CLSI.11 MICs were interpreted using CLSI M100  or FDA breakpoints.13 Isolates with cefepime/taniborbactam MIC ≤ 8 mg/L were deemed susceptible.Following two subcultures from frozen stock, the E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, E. coli NCTC 13353 (CTX-M-15)  and K. pneumoniae ATCC BAA-1705 .Colony counts were performed to confirm inocula. Quality control was assured using https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and contigs were assembled using SPAdes software.14 Sequencing yielded an average of 2 637 395 reads per genome and an average genome coverage of 82×. De novo assembly yielded an average contig length and N50 length of 128 583 and 318 077 bp, respectively.ENSE isolates, plus 51 ertapenem-susceptible Enterobacterales controls, were sequenced using the Illumina NextSeq platform. Quality control was assessed using the FastQC tool (https://github.com/tseemann/mlst). Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0 at an identity threshold of 90%.15 Alterations in genes ompC/F (encoding major porins), ompK37 (in K. pneumoniae only) and ftsI (encoding PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. PBP3 alterations of interest included four amino acid insertions after P333, as previously described.16 Porin alterations were analysed with Provean v1.1.3 (default settings) to predict those that may have a negative impact on biological protein function.17 Only alterations predicted by Provean as having a negative impact on Omp function are discussed further in this study.MLST alleles and STs were identified by scanning assembled contigs against available PubMLST databases  with 100% susceptibility (MIC ≤ 8 mg/L) for the 51 ertapenem-susceptible Enterobacterales used as control isolates  for the 179 ENSE . For these 179 ENSE, not surprisingly, the susceptibilities to other β-lactam or β-lactam-like comparator agents ranged from lows of 15.1% susceptibility to piperacillin/tazobactam and 20.1% to ceftolozane/tazobactam to highs of 92.2% susceptibility to imipenem/relebactam, 96.1% to meropenem/vaborbactam and 97.8% to ceftazidime/avibactam (Table The cefepime/taniborbactam MICn = 171), aztreonam (n = 157) and ceftolozane/tazobactam (n = 125), respectively, attesting to the challenging nature of this collection of isolates. Cefepime/taniborbactam was highly active against ENSE isolates with various antimicrobial resistance phenotypes including: amoxicillin/clavulanate-resistant , aztreonam-resistant , cefepime-resistant , ceftolozane/tazobactam-resistant , colistin-resistant , imipenem-resistant , levofloxacin-resistant , meropenem-resistant , piperacillin/tazobactam-resistant  and trimethoprim/sulfamethoxazole-resistant  with susceptibilities ranging from 92%–100% , 87.7% (157/179) and 69.8% (125/179) of isolates were also resistant to amoxicillin/clavulanate  possessed a carbapenemase, 21.8% (n = 39) possessed an ESBL and 97.2% (n = 174) possessed other β-lactamase gene(s) (Table JAC-AMR Online). Six isolates possessed a carbapenemase, an ESBL and other β-lactamase genes. All isolates with carbapenemases and 97.4% of isolates with an ESBL (n = 38) also possessed truncated or altered Omp proteins. Overall, 88.3% (n = 158) of ENSE had at least one altered/truncated porin. Only two isolates (1.1%) possessed previously described alterations in PBP3, one each of Y333_R334insYRIK and Y333_R334insYRIN.Of the 179 ENSE, 8.9% (n = 43) had at least one altered/truncated porin gene and 74.5% (n = 38) possessed a non-carbapenemase, non-ESBL β-lactamase gene  of ENSE isolates demonstrated a cefepime/taniborbactam MIC ≥ 4 mg/L, including seven K. pneumoniae, three E. coli, two E. cloacae and one S. marcescens (Table K. pneumoniae isolate)  breakpoint for cefepime/taniborbactam for Enterobacterales is ≤ 8 mg/L, this analysis also examined the characteristics of isolates within the cefepime SDD range (cefepime/taniborbactam MICs of 4–8 mg/L) as well as above 8 mg/L. Only 7.2% n = 1 of ENSE E. coli isolates had a cefepime/taniborbactam MIC of 32 mg/L (cefepime MICs > 64 mg/L). One possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration (D192G) and P333_Y334insYRIN in PBP3. The second contained CTX-M-71, a truncated OmpF due to a four amino acid duplication and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE) , ESBL genes (21.8%) and other β-lactamases (e.g. AmpC) (97.2%) as well as porin alterations (88.3%) and insertions in PBP3 (1.1%)  and was active against subsets of isolates with various β-lactam and non-β-lactam antimicrobial resistance phenotypes  potentiated cefepime activity 8- to > 1024-fold.Of the 18 027 Enterobacterales isolates collected from CANWARD from 2007 to 2019, we obtained and tested the 0.99% (179/18 027) of isolates that were ENSE (ertapenem MIC ≥1 mg/L).%) Table . The low8–10 Our data show that cefepime/taniborbactam , is significantly more active than cefepime (MIC50 and MIC90 ≥ 64 mg/L) against ESBL-producing Enterobacterales (92.3% of isolates containing CTX-M- and SHV-type ESBLs) with MICs of 0.06–4 mg/L. These data are consistent with previous reports including Hamrick et al.10 who reported cefepime/taniborbactam MIC50/MIC90 of 0.06/0.5 mg/L for Enterobacterales ESBL/AmpC producers (cefepime MIC50/MIC90 of 8/128 mg/L). Wang et al.18 reported cefepime/taniborbactam MIC50/MIC90s of 0.03/0.12, 0.06/ 0.25 and 0.12/1 mg/L for ESBL-, plasmid-mediated AmpC- and ESBL with AmpC-producing Enterobacterales isolates, respectively, with corresponding cefepime MIC50/MIC90 of 8/32, 0.12/2 and 32/256 mg/L. Kloezen et al.19 reported a cefepime/taniborbactam MIC90 ≤ 0.5 mg/L for ESBL producers with median cefepime MIC reductions of 5–9 doubling dilutions in the presence of taniborbactam, depending on the Enterobacterales ESBL genotype.As previously stated, taniborbactam—a boronic acid-containing β-lactamase inhibitor—inhibits class A, C and D (serine) β-lactamases and class B  β-lactamases, including VIM, NDM, SPM-1 and GIM-1 (but not IMP).et al.10 who reported cefepime/taniborbactam MIC50/MIC90 of 0.5/2 mg/L for carbapenemase-producing Enterobacterales containing KPC, NDM, VIM and OXA-48/48-like genes (corresponding cefepime MIC50/90 64/> 256 mg/L). Wang et al.18 reported cefepime/taniborbactam MIC50/MIC90s of 2/8, 16/64 and 4/32 mg/L against KPC- and NDM-producing Enterobacterales and non-carbapenemase-producing CRE with corresponding cefepime MIC50/MIC90 for all organisms of > 256/> 256 mg/L. Consistent with our data, Mushtaq et al.9 reported that cefepime/taniborbactam was active against carbapenemase-producing Enterobacterales containing KPC, NDM, VIM, OXA-48 (OXA-48 like) but not IMP genes. Kloezen et al.19 reported a cefepime/taniborbactam of MIC90 ≤ 1 mg/L for KPC and VIM producers with median cefepime MIC reductions of 7–10 doubling dilutions depending on the Enterobacterales carbapenemase genotype. Piccirilli et al.20 reported cefepime/taniborbactam MIC50/MIC90 of 1/4 mg/L against Enterobacterales harbouring a variety of NDM and VIM carbapenemases.17 However, cefepime/taniborbactam was not active against strains harbouring IMP, demonstrating MICs of 128 mg/L.20Our data show that cefepime/taniborbactam is significantly more active than cefepime against carbapenemase-producing Enterobacterales, demonstrating MICs of 0.12–4 mg/L for 93.8% isolates containing KPC, NDM, OXA-48 (OXA-48 like) carbapenemase genes. Our data are consistent with prior data including those of Hamrick et al.21 demonstrated that the in vitro activity observed with cefepime/taniborbactam against resistant Enterobacterales was translated to the in vivo setting. Using a cefepime/taniborbactam human-simulated regimen equivalent to 2 g/0.5 g q8 h administered as a 2 h infusion  in mice, against 26 clinical Enterobacterales expressing ESBLs, plasmid-mediated AmpC and/or class A (KPC) or D carbapenemases (OXA-48), the combination exerted potent in vivo activity  against cefepime-resistant isolates, including serine-carbapenemase producers.Abdelraouf 22Although the number of isolates tested in the present study was small, we report that cefepime/taniborbactam demonstrated MICs of 0.5–4 mg/L for 75% (6 of 8), 50% (2 of 4) and 50% (2 of 4) of isolates resistant to imipenem/relebactam, ceftazidime/avibactam and meropenem/vaborbactam, respectively. In our study, one isolate containing NDM-1 was resistant to imipenem/relebactam, ceftazidime/avibactam and meropenem/vaborbactam, but was provisionally susceptible to cefepime/taniborbactam (MIC 1 mg/L). This is not surprising as, unlike ceftazidime/avibactam and meropenem/vaborbactam, cefepime/taniborbactam inhibits Enterobacterales with MBL NDM and VIM as well as serine-β-lactamases KPC and OXA-48.K. pneumoniae, 3 E. coli, 2 E. cloacae and 1 S. marcescens) and clonal diversity (STs). All isolates possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF (reduced uptake into the periplasmic space) and/or PBP3 (reduced binding to target site)  and documented the presence of PBP3 mutations in 28/29 isolates. A variety of different mutations in PBP3 were documented.15 Unfortunately, other (non-PBP3 and non-β-lactamase-mediated) resistance mechanisms, such as porin changes or efflux pump expression, were not characterized in that study. Mushtaq et al.9 analysed Enterobacterales with cefepime/taniborbactam MICs > 8 mg/L (taniborbactam fixed at 4 mg/L) . These researchers noted both genetic diversity (a variety of STs) as well as no universal resistance mechanism in all isolates but rather combinations of carbapenemases  along with PBP3 insertions (e.g. after amino acid 333), and/or porin changes (e.g. OmpF).9 Kloezen et al.19 analysed three isolates of Enterobacterales with cefepime/taniborbactam MICs > 4 mg/L (taniborbactam fixed at 4 mg/L). One isolate harboured a VIM gene while the other two carried VIM-1, CMY-13 and qnrA1 genes. The authors concluded that the presence of VIM and AmpC may reduce cefepime/taniborbactam activity against Enterobacterales. The presence of other underlying resistance mechanisms such as porin alterations, which may reduce periplasmic uptake, or target site binding to PBP3 or efflux were not assessed.Only 13 (7.2%) ENSE isolates demonstrated a cefepime/taniborbactam MIC ≥ 4 mg/L. The 13 isolates represented both microbiological species diversity  and putative binding to the target site (PBP3), we did not assess efflux pump expression, which is known to confer increased cefepime MICs in Enterobacterales and may affect cefepime/taniborbactam activity.In summary, the current study demonstrated that cefepime/taniborbactam was highly active against whole genome sequenced ENSE isolates with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.dlab197_Supplementary_DataClick here for additional data file.