Craugastor evanesco) in captivity has been challenging. Therefore, the objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this species in the near future. The Gonadotropin-Releasing Hormone agonist, at a concentration of 4 micrograms per gram of body weight, stimulated the production of high-quantity and quality spermatozoa. This study also described the morphology of C. evanesco spermatozoa for the first time. These results contributed new and valuable knowledge on the reproductive biology of C. evanesco and for the development of other assisted reproductive technologies that might be applied to this and other species’ conservation programs.Recurrent successful breeding of the critically endangered Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9—GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.The Vanishing Rainfrog ( Agalychnis lemur (CR), Tripion spinosus (NT-decreasing populations), Pristimantis museosus (VU), Atelopus limosus (CR), Craugastor evanesco (CR), Strabomantis bufoniformis (EN) and Cruziohyla calcarifer (LC-decreasing populations) were highlighted as conservation priorities for the country [Craugastor evanesco (Batrachochytrium dendrobatidis (Bd), and its common name, Vanishing Rainfrog, refers to the fact that it had mostly disappeared from its distribution range from the time it was discovered (2004) to its formal description (2010). It is estimated that 90% of its population has disappeared from the wild [In response to the continued decline of its amphibian species, Panama has developed a Conservation Action Plan to help mitigate the severity of the problem . An asse country ,3. Of thevanesco  was the the wild .C. evanesco and are important tools to help mitigate this crisis [In situ conservation programs for persisting amphibian species and populations in the wild are not enough to avoid ongoing declines and extinctions due to chytridiomycosis; thus, ex situ conservation programs have become the only hope for species like s crisis . Due to s crisis .C. evanesco are based on scarce information on the natural histories of related species. To date, accomplishing recurrent reproductive events for this species has been challenging [The primary goal of an ECPs is to maintain a self-sustainable captive population, that is reliably reproductive and which can retain at least 90% of the genetic variation present in the wild population for at least 100 years ,9. Howevllenging ,10.Assisted Reproductive Technologies (ARTs) are tools designed to help reproduce endangered species, and their development is usually species-specific ,11,12. HAtelopus [C. evanesco spermic urine, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this critically endangered species in the near future.Previous studies by our laboratory have produced valuable information on the effect of hormonal stimulation on the production of spermic urine in endangered species from the genus Atelopus ,12,22, rAtelopus . TherefoC. evanesco (29.00 ± 7.32 g) housed at the Smithsonian Tropical Research Institute’s (STRI) Panama Amphibian Rescue and Conservation Project, located in Gamboa, Panama, were used for this study. Animal use protocols were submitted for the STRI’s Institutional Animal Care and Use Committee (IACUC) approval. Tanks , housing up to two frogs, containing rocks and substrate, were kept at 23–25 °C and misted with carbon-filtered water every 8 h for 3 min and drained through a bulkhead. UV lighting was set on a 12 h cycle with Zoomed 10.0 T8 high-output UV-B bulbs to avoid D3 hypovitaminosis and help with proper bone growth. Frogs were fed ad libitum three times per week with live crickets (Acheta domestica) and roaches (Blaptica dubia) dusted with a calcium carbonate powder containing vitamin D3  or multivitamins with beta carotene powder  on a rotating basis.From December 2018 to December 2019, thirty-one adult male 10, D-Ala6, Pro-NHEt9—GnRH , Amphiplex—a cocktail of 0.4 μg GnRH-A plus10 μg/gbw metoclopramide hydrochloride, a dopamine antagonist (Sigma-Aldrich Corporation) or hCG   injections A with onoration) . Two difThroughout spermic urine collection periods, animals were kept individually in temporary ventilated plastic containers (10″ × 6″ × 4″) lined with a dampened layer of paper towel (~15 mL of water) and covered from light to reduce stress. Spermic urine samples were collected at 10 different time points—0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 24, 28 and 48 h—post-injection by cloacal catheterization  B. SampleQuality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality and pH. Sample volumes were measured with a 200-μL pipette. Sperm concentration was assessed by adding 10 μL of the spermic urine sample to a hemocytometer. Osmolality and pH were measured using an Osmocheck urine analysis unit  and pH indicator strips , respectively. Percentage sperm motility was determined by counting all the cells with any type of movement from a total of 100 cells under a Zeiss AXIO Scope A1 microscope at 400× magnification. Percentage FPM was obtained by counting all the cells with forward progression relative to the 100 cells that were assessed for motility.In preparation for morphological assessments, 10 μL of spermic urine was fixed in 80 μL 4% paraformaldehyde (PFA) in phosphate-buffered solution (PBS). Fixed sperm cells were then stained with Coomassie blue  for 90 s, air dried and mounted with Permount™ Mounting Medium  on Superfrost slides  . EvaluatSperm concentration–response curves describe the magnitude of the response in terms of sperm concentration as a function of exposure to different hormonal treatments and doses over time. This information denotes if and when a specific treatment’s effect begins, peaks and ends. Curves were calculated using descriptive statistics. For each collection time point, replicates were averaged and the standard deviation was calculated.Identification of sperm concentration peaks allows researchers to identify the specific window of time in which collections of highly concentrated sperm samples should occur. When applying ARTs such as sperm cryopreservation and artificial fertilization, the use of such samples is required. Therefore, in this study, sperm concentration peaks are defined as the time points where the number of spermatozoa per unit volume of urine is higher compared to other time points. To obtain sperm concentration peaks for each treatment, cutoff values were used and calculated as follows: total mean sperm concentration for each time point was averaged and added to one times the standard deviation (SD). Time points with sperm concentrations equal to or higher than the cutoff were considered part of the peak and removed for the following round of calculations. The process was repeated with the remaining values until there were no time points equal to or higher than the cutoff ,23. Sperp < 0.05.Statistical analysis was conducted using GraphPad Prism 9.0 software . Statistical analysis for concentration, percentage motility, percentage forward motility, osmolality and pH were based on and applied to metrics obtained during the time points where peak concentrations occurred. If more than one peak was identified for a single treatment, all the metrics obtained during these peaks were included in the analysis. The effects of hormonal treatments on sperm concentration and percentage motility were determined using one-way ANOVA with Tukey’s post hoc test. The effects of hormonal treatments on percentage motility and FPM, osmolality, spermic urine pH and morphology parameters were determined with the Welch ANOVA test followed by Dunnett’s post hoc test (when variances were not homogeneous). The effect of hormonal treatments on sperm head and tail size was determined with two-way ANOVA followed by Šidák’s multiple comparison test. Values for all data were expressed as mean ± SD. Means were considered statistically different if C. evanesco, with 100% of males responding to all concentrations of GnRH-A and Amphiplex, 44% to 10 IU/gbw hCG and no males responded after treatment with 5 IU/gbw hCG. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, followed by GnRH-A at 2 and 6 μg/gbw. However, the hCG concentrations used in this study were not reliable inducers of sperm production and consistently had higher percentages of sperm abnormalities. Furthermore, spermic urine induced with hCG was more acidic than spermic urine induced by GnRH-A and Amphiplex.The results of this study indicate that all tested hormones , but not all concentrations, elicited the release of sperm in C. evanesco  = 17.31, p < 0.0001)  .3 ± 5.33 × 103 (10 IU/gbw hCG) to 8.64 × 106 ± 2.00 × 107 (4 μg/gbw GnRH-A). Control samples were devoid of sperm at all time points. GnRH-A at 4 μg/gbw induced a significantly higher response than 2 and 6 μg/gbw GnRH-A, 5 and 10 IU/gbw hCG, and both controls but was not different from Amphiplex. There was no significant difference in sperm concentration between 6 μg/gbw GnRH-A and the treatments with 2 μg/gbw GnRH-A and Amphiplex and the controls. Similarly, no difference was found among 2, 6 μg/gbw GnRH-A and Amphiplex, while 2, 4 μg/gbw GnRH-A and Amphiplex were significantly different to 5 and 10 IU/gbw hCG and both controls  (4 ± 4.74 × 104)  A,C. No s5 × 105) D, while 4 × 104) E,F. Cont4 × 104) A–F.6 ± 1.53 × 106 to 3.04 × 106 ± 6.03 × 106) and 28 h (range: 4.55 × 105 ± 3.18 × 105 to 2.92 × 106 ± 3.88 × 106) post-injection, with Amphiplex stimulating the production of higher concentrations at this time point (2.92 × 106 ± 3.88 × 106). While still present, the response to stimulation with 10 IU/gbw hCG was lower compared to GnRH-A and Amphiplex treatments at both 24 h (7.78103 ± 2.20 × 104) and 28 h (1.67 × 103 ± 4.71 × 103) post-injection. At 48 h post-injection, 4 μg/gbw GnRH-A showed a good response (1.30 × 106 ± 2.04 × 106), followed by Amphiplex (7.14 × 105 ± 9.57 × 105), 2 μg/gbw GnRH-A (4.31 × 105 ± 4.58 × 105), 6 μg/gbw GnRH-A (2.55 × 105 ± 2.83 × 105) and 10 IU/gbw hCG (no response)  and Amphiplex remained high at 24 h (range: 1.28 × 10esponse) A–F.GnRH-A was the most efficient elicitor of sperm responses compared to hCG and Amphiplex. At 2 μg/gbw, GnRH-A induced the most prolonged response . GnRH-A at 4 μg/gbw had similar effects but with a slightly shorter response , while GnRH-A 6 μg/gbw had a slight delay in the initiation of a response . At the lower concentration of 5 IU/gbw, hCG was unsuccessful in stimulating any sperm production. However, 10 IU/gbw hCG showed similar but shorter chronological patterns of sperm induction  than that of 2 μg/gbw GnRH-A, while Amphiplex showed a discontinuous, 4 h peak  .W = 61.56, p < 0.0001), with Amphiplex (81.86 ± 18.58%) and 4 μg/gbw GnRH-A (68.39 ± 11.90%) showing a higher percentage motility than the rest of the treatments  = 36.75, p < 0.0001), with Amphiplex (66.52 ± 30.64%) and GnRH-A at 4 μg/gbw (57.44 ± 17.14%) presenting a higher percentage FPM, followed by 6 and 2 μg/gbw GnRH-A , and 10 IU/gbw hCG (1.11 ± 3.33%) , stimulated the production of sperm with percentage motility >59% (eatments A. Forwar± 3.33%) B. These W = 15.41, p < 0.0001) (p = 0.0863) and between 10 IU/gbw hCG and the control with no injection (p = 0.0964).There was a significant effect of hormonal treatments on spermic urine osmolality ( 0.0001) . TreatmeW = 6.33, p = 0.0009)  . The pH Craugastor evanesco spermatozoa have a filiform shape  = 10.94, p = 0.0002) with Amphiplex showing a significantly lower percentage of intact acrosomes (72.63 ± 5.82) than 4 and 6 μg/gbw GnRH-A , while not differing from 2 μg/gbw GnRH-A and 10 IU/gbw hCG. Although treatment with 10 IU/gbw hCG showed no statistical difference due to its larger SD, a lower acrosome integrity than that of the other treatments was noticeable.To understand if there is an effect of hormonal treatments on morphological parameters, three different traits were examined: acrosome, head and tail integrity . All horW = 5.89, p = 0.0051)  C. Treatm = 20.13, p < 0.0001), suggesting that the size of a spermatozoon’s head and tail are hormonally dependent  = 12.61, p < 0.0001). Due to the limited number of spermatozoa obtained with the 10 IU/gbw hCG treatment, we were not able to include it in this analysis. Our results indicate that treatment with Amphiplex stimulated the production of spermatozoa with longer tails (114.21 ± 7.39) compared to all other treatments (p < 0.0001).An analysis of size (head and tail) indicated that there was a significant main interaction effect between size and hormonal treatments , regulated by the production of Gonadotropin-Releasing Hormone (GnRH). GnRH is a decapeptide produced by GnRH neurosecretory cells and it is released into the portal capillary vessels. GnRH acts on the anterior lobe of the pituitary, stimulating the production of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH). LH and FSH act on the gonads stimulating the production of sex steroids and gametogenesis ,26,27. IIn our study, a concentration-dependent response was observed in all GnRH-A treatments, where 4 µg/gbw GnRH-A stimulated the production of more than double the sperm concentration obtained with 2 and 6 µg/gbw GnRH-A. This could be explained by the fact that the physiological responsiveness to GnRH, in terms of the number of GnRH receptors in the cell membrane of GnRH neurons and gonadotrophs and the secretion of LH and FSH, is regulated mainly by GnRH itself . The actC. evanesco compared to that of 4 µg/gbw GnRH-A. On the other hand, 6 µg/gbw GnRH-A appears to be on the verge of the supraphysiological concentration for this species. Supraphysiological concentrations of GnRH have been shown to produce desensitization or downregulation of the GnRH receptors, pituitary inhibition and/or suppression of testosterone production due to LH and FSH receptor reduction in the testes [Litoria booroolongensis, treatment with 2 µg/gbw GnRH-A shows a nearly significantly higher response than that of 4 µg/gbw GnRH-A, while the lowest concentration of 0.5 µg/gbw GnRH-A produced the best results in this species [C. evanesco. Studies on hormonal stimulation in captive Atelopus zeteki [Agalychnis callidryas [GnRH-A 2 µg/gbw appears to be a sub-physiological concentration, not strong enough to elicit a physiological response in e testes , capable species , suggests zeteki  and wildllidryas  have shoC. evanesco; the lower concentration of 5 IU/gbw did not elicit a response at all, whereas an incipient response was observed after stimulation with a higher concentration (10 IU/gbw). Several studies in anurans have shown highly variable responses to hCG stimulation with fewer species responding successfully to this treatment compared to GnRH-A ; others show no response or respond poorly to hCG (Pseudophryne corroboree and Geocrinia rosea), while some respond positively to both hormones, with GnRH-A showing a better response  [Rhinella arenarum suggested a more efficient role of FSH than that of LH and hCG in stimulating spermiation, showing a differential sensitivity, dose-dependent response to these different gonadotropins and an FSH receptor-mediated response with higher affinity for FSH than for LH and hCG [Rana temporaria and Lithobates pipiens [β subunit of hCG appears to be less compatible with LH receptors than the LH β subunit. These explanations would support why GnRH-A is more efficient in stimulating sperm production than hCG since GnRH-A stimulates the production of endogenous LH as well as FSH and both gonadotropins are acting in the testes. Exploring the possibility of utilizing higher hCG concentration may be necessary to better understand if the lack of or incipient response to hCG in this species was due to a sub-physiological dose response or due to low affinity to an FSH receptor-mediated response.Contrastingly to GnRH-A, Human Chorionic Gonadotropin (hCG) circumvents the HPG axis and acts directly on the gonads, binding to the same receptors as LH on the Leydig cells . Our resgnifera) ,37,38,39 and hCG . One exp pipiens , and as Atelopus zeteki) with a GnRH-A concentration 10 times lower than that of the best-working GnRH-A treatment [Lithobates pipiens, Ceratophrys ornata, Ceratophrys cranwelli and Odontophrynus americanus have shown a positive spawning and breeding behavior response as well as a biphasic increase in the gene expression of lhb, fshb and gnrh1 over time after treatment with Amphiplex higher than that observed with GnRH-A alone [C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH, 5 and 10 IU/gbw hCG as non-optimal treatments.Although not significantly different from any of the GnRH-A treatments, Amphiplex showed the second-best response at stimulating the production of highly concentrated sperm samples in this species, despite the fact that the GnRH-A concentration in this treatment is 5, 10 and 15 times lower than that of 2, 4 and 6 µg/gbw GnRH-A treatments, respectively. Despite some contradictory evidence, previous studies in different taxonomic groups, including amphibians, suggest a possible inhibitory action of the central dopaminergic system on the biosynthesis of gonadotropins. Dopamine, through its DA-2 receptors in the pituitary, might exert a regulatory role on the GnRH and GnRH receptor gene expression, directly impacting LH and FSH release ,19,41,42reatment . Other a-A alone ,43, sugg6 ± 1.90 × 107 cells/mL) contained 6.2-, 19.6-, 298.9- and 2.4-fold higher sperm concentration than 2, 6 µg/gbw GnRH-A, 10 IU/gbw hCG and Amphiplex, respectively, at the same time point, gradually decreasing until reaching the lowest point of the first peak at 6.5 h pi, still producing sperm concentrations in the order of 106 cells/mL. The efficacy of the 4 µg/gbw GnRH-A was such that the highest sperm concentration obtained during peaks was produced by the first time point (0.5 h pi). A study in Rana catesbiana provided evidence of a rapid (10 min) GnRH dose-dependent LH and FSH concentration increase in circulating blood after GnRH-A stimulation, followed by a spermiation response 30 to 45 min post-stimulation [Anaxyrus americanus [All samples were collected over a period of 10 time points, starting at 0.5 h pi and ending at 48 h pi. At the 0.5 h pi time point, all treatments, except for 5 IU/gbw hCG, stimulated the production of spermatozoa, as observed in the Panamanian Golden Frog at the same time point . The sammulation . Obringeericanus . This raericanus , insuffi6 cells/mL) from most treatments at this time point, two more time points (28 and 48 hpi) were added to learn how long the hormone treatment effects lasted in this species. Out of the five treatments that were successful in stimulating sperm production, only one (10 IU/gbw hCG) failed to produce any spermic urine at 48 hpi but still produced spermic urine at low concentrations (104) at the 28 hpi time point. The optimal treatment of 4 µg/gbw GnRH-A was the only one to maintain the production of spermic urine with concentrations in the order of magnitude of 106 cells/mL at 48 hpi, followed by Amphiplex, 2 µg/gbw GnRH-A and 6 µg/gbw GnRH-A with concentrations of 105 cells/mL (listed in a descendent manner). To our knowledge, most studies but one [lhb, fshb and gnrh1 after treatment with GnRH-A in female Lithobates pipiens, finding a biphasic response to stimulation with lower doses of this hormone alone and with 0.4 µg/gbw GnRH-A+10 µg/gbw Metoclopramide at 12, 24 and 36 hpi [C. evanesco.Initially, collection time points for this species were set up to 24 hpi. Due to the high sperm concentrations collected  at 3, 7 and 12 hpi, with the peak sperm concentration produced at 7 hpi. Subsequently, they tried the same time points at a lower GnRH-A concentration of ~0.04 µg/gbw  and observed a sperm production peak at 12 hpi [All treatments differed in their sperm production peak pattern and duration. Two of the treatments showed two different sperm production peaks (4 µg/gbw GnRH-A and 10 IU/gbw hCG), two treatments showed three different sperm production peaks (2 µg/gbw GnRH-A and Amphiplex) and one treatment showed four sperm production peaks (6 µg/gbw GnRH-A) over the 10 collection time points. Other studies have reported different sperm production peaks using similar hormones, similar or different concentrations, time points and species. Obringer et al., first measured sperm concentration in t 12 hpi .Crinia georginia, Heleioporus albopunctatus and Neobatrachus pelobatoides) peaked at 7 hpi, two other species (Pseudophryne guentheri and Geocrinia rosea) peaked at 3 hpi, one species (Helioporus eyrei) peaked at 12 hpi and two species (Crinia pseudinsignifera and Crinia glauerti) showed two different separated peaks at 3 and 12 hpi and 3 and 7 hpi, respectively. As for the hCG treatment, three species , two of which coincided with the GnRH-A treatment, showed a sperm production peak at 7 hpi, four species , one of which coincided with the GnRH-A treatment, peaked at 12 hpi and one species  showed two separate peaks at 3 and 12 hpi. These studies utilized the same collection time points with a significant time gap between them that could have missed one or more peaks; nevertheless, at least one peak was identified and species-specific hormone concentration and time-dependent differences were evident.Another study compared the same time points of 3, 7 and 12 hpi in eight different species using the concentrations of 2 µg/gbw GnRH-A and 13 IU/gbw of hCG . After tC. evanesco and Atelopus zeteki [C. evanesco with those observed in A. zeteki, similar hormones and concentrations were utilized, and differences in the type of response were noted, with the most apparent being the number and duration of sperm production peaks. The longest continuous peak observed in C. evanesco lasted 6 h (2 µg/gbw GnRH-A) and all treatments showed at least two peaks, while in A. zeteki the longest peak lasted 2.5 h (10 IU/gbw hCG) and only one treatment showed more than one peak (2 µg/gbw GnRH-A), although it is important to note that collection time points did not exceed 24 hpi in this species. Taken together, these results suggest a species, hormone, concentration and time-dependent pattern in sperm production peaks. Spermatogenic cycles inside testicular cysts (intra-cystic) are mostly synchronous in anurans; nevertheless, some studies have shown the existence of spermatogenic intracystic asynchronicity in some species [As observed in s zeteki , hormona species ,49. A po species ,50 at a  species  after hoC. evanesco, critical to the future development of other assisted reproductive technologies such as sperm cryopreservation or the synchronization of breeding events.The occurrence of one or more sperm concentration peaks and the duration-dependence of these after hormonal stimulation could be the result of several factors acting simultaneously: (1) the species; (2) the hormone type and concentration, which will stimulate receptors in a sub-optimal, optimal or supra-optimal manner ; (3) timRana temporaria showed that a dose of 11.7 IU/gbw hCG produced lower motility parameters than GnRH-A treatments. Although not significant, a higher concentration of the hormone increased motility parameters over a period of 24 hpi [Anaxyrus baxteri, reaching motility percentages of ~80% [Rhaebo guttatus [Litoria booroolongensis [Anaxyrus fowleri [Atelopus zeteki [C. evanesco, drastically reducing the sperm quality in this species, also evidenced by our sperm concentration assessment. In addition to hormonal effects, it is possible that the lower FPM percentages observed in C. evanesco, compared to those of other aquatic breeders, are adequate for the species due to its terrestrial reproductive mode, where the cells need not swim to reach the eggs and FPM is only needed to penetrate the thick jelly layers of the eggs. Low testicular sperm motility parameters have also been reported for Eleutherodactylus coqui , which is a terrestrial and direct-developing species [During the sperm production peaks, all treatments, with the exception of 10 IU/gbw hCG, produced spermic urine with percentage motility in a range between 59 and 82%. Although not significantly different, Amphiplex showed the highest percentage of motile sperm, followed by 4 µg/gbw GnRH-A. Across treatments, the percentage FPM (<66%) was lower than the percentage motility as has been observed in other amphibian species ,36,46,54f 24 hpi . Other songensis , Anaxyru fowleri  and Atels zeteki . Some sts zeteki ,58,59. T species . Another species .C. evanesco, favoring sperm quality.Osmolality, pH and ionic composition are some of the major factors related to motility activation and quality in amphibians and fishes ,62,63,64Atelopus zeteki, an aquatic breeder, found that neither hormonal treatments nor injection with ARS affected spermic urine osmolality in this species, while treatment with 4 µg/gbw GnRH-A decreased the spermic urine pH, although this decrease remained higher than pH 7.0 [Craugastor evanesco is a riparian amphibian of the tropical rainforest that lays eggs in a terrestrial environment. The osmoregulatory apparatus of terrestrial amphibians works differently than that of biphasic species, helping them to prevent dehydration by rapid water absorption from the environment as well as water reabsorption from the bladder [Lithobathes sylvatica, an aquatic breeder, showed a beneficial osmolality reduction window favoring sperm motility (~185–55 mOsm/kg), with higher or lower osmolalities drastically reducing this parameter [C. evanesco sperm motility did not appear to be as strong as in the species that breed in water, evidenced by the low spermic urine osmolality and high motility percentage of the 2 µg/gbw GnRH-A treatment and the average osmolality and low percentage motility of the 10 IU/gbw hCG treatment. Interestingly, spermic urine pH seemed to have a stronger influence on C. evanesco sperm motility.A previous study on n pH 7.0 . Craugas bladder ,66. The arameter . The effC. evanesco sperm motility and viability. Taken together, these results support species specificity in terms of sperm quality with respect to hormonal treatments, highly influenced by the reproductive mode and the physiological adaptations to the external environment.In addition to osmotic pressure, spermic urine pH is an important factor that influences sperm motility in amphibians and fishes ,62,63. ICraugastor evanesco has a terrestrial reproductive mode with females laying their eggs in humid sand burrows while the amplexed male excretes the spermic urine on top of the egg mass. The eggs, with a diameter of ~1.5 cm each . Taken together, our results suggest that treatment with 4 µg/gbw GnRH-A was the best in terms of quantity and quality parameters for the stimulation and collection of C. evanesco spermatozoa.Treatment with 10 IU/gbw hCG, although not significant due to its elevated SD, showed lower percentages of morphological integrity on the three parameters compared to all other treatments, confirming once more that this treatment is not recommended for the collection of C. evanesco, as well as a successful sperm stimulation and collection protocol. Stimulation via the intraperitoneal injection with 4 µg/gbw GnRH-A using an injection volume of 10 µL/gbw, followed by sperm collection via cloacal catheterization between 0.5 and 5.5 h pi, will allow for the collection of spermic urine with motility parameters >60% and morphological integrity >80%. This new knowledge can be used to increase the reproductive success of the species, forming the base for the development of other, more advanced ARTs such as Artificial Fertilization for the production of viable offspring.The amphibian extinction crisis is causing the loss of valuable species and populations. Ex situ conservation programs and the development and application of Assisted Reproductive Technologies are efficient tools to mitigate these declines. This study produced new information on the reproductive biology of the species


Large bone defect treatments have always represented an important challenge in clinical practice and created a large demand for more efficacious regenerative approaches. The bone tissue engineering approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Among the most used biomaterials for bone tissue engineering, polylactic acid scaffolds have been considered the most promising ones due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells, in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed polylactic acid scaffolds, obtained by fused deposition modelling. The obtained data suggested that 3D-printed polylactic acid scaffolds represent promising biomaterials for medical implantable devices in the orthopaedic field and have the potential to increase patients’ quality of life.Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field. Annually, numerous bone grafts are required for the treatment of severe bone fracture, secondary to bone loss from trauma, congenital anomalies, illnesses including cancer, and aging . To dateAliphatic polyesters, such as polylactic acid (PLA), represent the most common and most promising polymers, due to their biocompatibility, non-toxic biodegradability and bioresorbability in the production of sutures, manufactured orthopaedic devices and support for tissue regeneration ,25. AlthIn addition, with the aim of improving this desired outcome, scaffolds can be suitably plasma treated to enhance surface properties and further promote a positive biological response. In this respect, cold atmospheric plasma, carried out in the presence of helium, was previously assessed as a modification tool for PLA scaffolds to evaluate osteoblast and mesenchymal stem cell attachment . The effIn this work, the potential of plasma treatment was implemented, exposing 3D-printed PLA scaffolds, obtained by FDM, to oxygen, with the aim of clearly assessing the role of the induced modifications on a simple and repetitive pattern, not resembling the physiological architecture of bone tissue. The physiological response of human foetal osteoblast cells (hFOB) was then evaluated in terms of cell proliferation and osteogenic differentiation. PLA scaffolds were also analysed by means of mechanical simulations to evaluate their behaviour to traction, flexural or torque solicitations, in order to assess the potential capability for future implantable applications.3). The geometric model was imported in ideaMaker  to prepare the GCode file to drive the N2 FFF 3D printer  and process a polylactic acid (PLA) filament  for the fabrication stage. The nozzle temperature (0.4 mm diameter) was set at 205 °C while the build platform was set at 40 °C. The morphological assessment of the 3D-printed PLA scaffolds was carried out by means of optical microscopy (Nikon Eclipse 80i) to measure and verify the geometrical consistency of the collected layers with the CAD design.Testing scaffolds were designed by stacking polymeric layers in the −60°/0°/60° directions  models and are reported in Material properties were imposing by using data from Final loads were calculated by performing benchmarks simulations while continuously increasing the load in a tensile test, since the critical value of stress was reached. The normal load was imposed by fixing 500 nodes at the base of the model and imposing a distributed vertical load of 700 N, as depicted in 3/min of O2 gas were introduced to expose the scaffolds to a 100 W power, 10 Pa plasma for 10 min. After completing the process, the chamber was ventilated, and the scaffolds were removed and used for the cell adhesion step.The oxygen plasma treatment of scaffolds was performed using a RIE plasma etcher SI 591 . 3D-printed PLA scaffolds were placed inside the high vacuum reaction chamber where 60 cm2 at 37 °C. The medium was replaced twice a week and cells were split at about 80% of confluence.Human foetal osteoblast cell line hFOB 1.19 were obtained from the American Type Culture Collection . hFOB 1.19 were cultured in 1:1 mixture of Ham’s F12 Medium—Dulbecco’s Modified Eagle’s Medium , supplemented with 2.5 mM L-glutamine , 0.3 mg/mL G418 ; 10% Foetal Bovine Serum  and 1% penicillin/streptomycin/amphotericin  and incubated in a humidified atmosphere containing 5% COBefore performing the in vitro studies, the 3D-printed PLA scaffolds underwent UV sterilization treatment for 2 h to eliminate any possibility of contamination,6 cells were cultured on 3D-printed PLA scaffolds, in a 24-well plate, with a specific medium and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for 4 h. Successively, the growth medium was added to completely cover the scaffold and the plates were re-incubated, as above, for 1, 3 and 7 days. After 1, 3 and 7 days of incubation, the medium from each well was removed and replaced with 200 μL of MTT solution (1 mg/mL in FBS-free medium). Following, 2 h incubation at 37 °C and 5% CO2, MTT solution was removed, each well was washed two times using cold PBS 0.01 M, and the formed crystals were melted using 200 μL of DMSO . Next, the absorbance at 540 nm was read using a synergy HT plate reader .Cell viability assay of hFOB cells seeded on 3D-printed PLA scaffolds was performed by MTT  assay . Specifically, 1 × 10hFOB cell proliferation on 3D-printed PLA scaffolds was determined using DAPI staining as previously reported in . BrieflyFor cell viability and proliferation assays, the cells cultured on the well with the same seeding density as the scaffolds were used as control .6 hFOB cells were plated on 3D-printed PLA scaffolds in culture medium and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. The culture medium was completely replaced twice a week and scaffolds were analysed on day 1, 3 and 7.For osteogenic induction, 1 × 102O, 2% Alizarin Red S stain solution was added and the plate with scaffolds was incubated for 30 min at room temperature. Finally, scaffolds were washed five times with H2O to remove the staining solution surplus and mounted. The stained scaffolds were acquired by using a Leica DMI 4000B microscope  and optical density was quantified using Fiji image J software (three sections/scaffold).The extracellular matrix (ECM) mineralisation of hFOB cells on 3D-printed PLA scaffolds was analysed by Alizarin Red S staining. Alizarin Red S solution  was prepared according to the manufacturer’s protocol and performed as reported in ,33. Brie−ΔΔCt method. RNA from hFOB cells without PLA scaffolds (only cells-control) was used as reference for relative quantification. The results were expressed as fold change with respect to control cells. The following primers for real-time PCR were used: Osteocalcin (BGLAP), Alkaline Phosphatase (ALPL), Transforming Growth Factor Beta 1 (TGFb1), Collagen Type 1 (COL1A1) and Collagen Type 2 (COL2A1). Oligonucleotide sequences are reported in For gene expression analysis, total RNA from hFOB cells growth on 3D-printed PLA scaffolds was isolated by using Trizol Reagent , according to the manufacturer’s instructions. The concentration and quality of RNA were determined by Nanodrop 1000 Spectrophotometer . cDNA was synthesised from an equal amount of total RNA using ImProm-II Reverse Transcription System . Quantitative real-time PCR (qRT-PCR) was performed using a 7500 Fast Real-Time PCR System with Sso Advanced universal SYBR1 Green supermix (2X) . Specific primers for each of the investigated gene were designed using primer blast and selecting exon-exon junctions on mRNA as the target region for annealing, as already reported ,34. EachRaman spectra were acquired by using a high-resolution micro-Raman spectrometer (LabRAM HR800 from Horiba Jobin-Yvon) equipped with a Peltier cooled CCD detector (Synapse—Horiba). Laser radiation from a He-Ne (632.8 nm) was focused with a power of 0.9 mW onto the samples by a long working distance 50X objective mounted on an Olympus microscope BX41. The spectroscopic signals were collected in the backscattering configuration with integration times of 30 s.p < 0.05 was considered to be significant.Data were analysed either as raw data or as mean ± standard error (SE), as appropriate. Differences between several timepoints of hFOB cultured on 3D-printed PLA scaffolds were evaluated by using One and two-way ANOVA with post hoc Holm test, where appropriate. The 3D-printed PLA scaffolds and the related pattern are shown in Results of the Finite Element (FE) analysis for tensile load are reported in 2 plasma treatment on 3D-printed PLA scaffolds. Cell viability and proliferation of hFOB cells cultured for 1, 3, and 7 days  on PLA not treated (PLA_NT) and PLA plasma treated (PLA_PT) scaffold surfaces were assessed. The results are shown in To increase the rate of hFOB cells’ adhesion and distribution along scaffold cavities, we performed OCell viability performed by MTT assay showed that the viable cells in PLA_PT and PLA_NT increase over time from D1 to D7, even if the respiratory activity in PLA_NT was lower for both the PLA_PT and control . Specifically, the percentage of viable cells was 88.78 ± 4.84% at D1, 93.93 ± 5.24% at D3, and 117.85 ± 2.75% at D7 for FDM4_PT; while 11.47 ± 1.66% at D1, 9.11 ± 5.21% at D3, and 32.95 ± 4.35% at D7 for PLA_NT compared to 100% of the CTRL group. Further, these data show that the respiratory activity of hFOB cells cultured on PLA_PT scaffolds was 7.7-, 10.3- and 3.57-fold higher than PLA_NT, respectively, from D1 to D7.These results highlight that the plasma treatment creates available hydroxyl groups on the PLA surface, increasing its wettability and so favouring greater cell adhesion, attachment, and growth, which is consistent with the literature ,38. staining. AR S staining was performed after 1, 3, and 7 days of hFOB growth on scaffolds. AR S staining representative images are shown in To further evaluate the osteogenic differentiation of hFOB cells cultured on PLA_PT scaffolds, we performed gene expression profiling by quantitative Realtime-PCR (qRT-PCR) at early (D1) and late (D7) time points. Specifically, we evaluated osteogenic differentiation markers functionally related to early osteogenic differentiation (TGFβ1 and ALPL), mineralisation and extracellular matrix maturation  ,41. The These data showed that hFOB cells cultured on PLA_PT scaffolds exhibited a TGFβ1 expression level higher than the control  only at D1 (1.36 ± 0.03 vs. 1.07 ± 0.07), while at D7, no difference was detectable (5.13 ± 0.1 vs. 5.06 ± 0.02). Instead, at D7, both samples (CTRL and PLA_PT) showed an increase in TGFβ1 expression of about 5-fold compared to D1.The early osteogenic marker ALPL increased its expression from D1 to D7 on PLA_PT scaffolds by 1.67 ± 0.07 and 18.7 ± 0.22, respectively, compared to the control . Finally, expression levels of mineralisation and extracellular matrix maturation markers, COL1A1 and COL2A1, showed a significant increase, only at D7, of about 1.84 ± 0.34 for COL1A1 and 34.5 ± 0.16 for COL2A1. In contrast, BGLAP was overexpressed, starting as early as day 1, by about 2.62 ± 0.21 and 29.6 ± 0.11 at D7, compared to the CTRL (about 1.1 ± 0.10).+2 transport. Further TGFβ1 plays a critical role in bone remodelling, stimulating matrix protein synthesis [The improved expression of TGFβ1 at the early stage of cell growth (D1) and the reduction at D7 is coherent with previous reports, suggesting that this gene is important during the early differentiation of osteoblasts, but it inhibits their late differentiation into osteocytes ,43. TGFβynthesis ,45. Furtynthesis ,47. ThesTo detect the simultaneous presence of the mineral and organic composition in bone cultures, cell cultured scaffolds were analysed by Raman spectroscopy. In −1 due to the stretching vibration of the C–COO group. We also observed the bands centred at 1454 cm−1 and 1771 cm−1 arising from the CH3 asymmetric bending and the C=O stretching modes, respectively. After depositing the cells on the PLA scaffold and leaving them in culture for 7 days, we noticed the appearance of a band at 935 cm−1. It is attributed to the heterogeneous stretching vibration of P–O–C in the amorphous calcium phosphate, as proof of the incipient mineralisation process. Other deformation modes of phosphate are associated with the peaks at 404 and 568 cm−1 [−1 , we accounted for the substitution of carbonate through culture time [−1 ; specifically, they are due to the collagen β-sheet and α-helix, respectively. In the figure, the band at 1320 cm−1 due to CH deformation is highlighted, as well as one at 1445 cm−1, which is due to CH2 wagging. Furthermore, the range between 1580 and 1730 cm−1 is typical of the amide I vibrations of the protein.The peak at 1252 cm2 plasma treated 3D-printed PLA scaffolds. The 3D-printed PLA scaffolds were obtained by fused deposition modelling (FDM) and mechanical simulation to investigate their behaviour when traction, flexural or torque solicitations were performed. Our findings show that hFOB seeded on plasma treated PLA scaffolds are not only able to adhere and grow on their surfaces, but over time, are able to penetrate along strands, differentiate and form a tissue-like fibrous matrix between the gaps. Furthermore, the results of FE simulations showed that the 3D-printed PLA scaffold demonstrates good mechanical behaviour, in each analysed configuration, that can be used in future applications for medical implantable devices. The above reported findings clearly demonstrate that the osteoconductive and osteoinductive properties of the engineered 3D-printed PLA scaffold could represent a promising biomaterial for bone tissue engineering applications.In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, cultured on O


Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.Cyclohexanone monooxygenase (CHMO) from  NCIMB 9871 cyclohexanone monooxygenase (Ac CHMO), has been extensively characterized in particular due to its versatile substrate scope and superior activity. However, one of the fundamental limitations to large-scale use of this enzyme is its well documented temperature instability [Baeyer–Villiger monooxygenases (BVMO) are valuable biocatalysts known for catalyzing the oxidation of (cyclic) ketones into esters and lactones through the utilization of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H). These enzymes have been studied for their industrial potential in the production of nylon monomers , (Z)-11-oic acid , and prooic acid . An NADPtability ,5.Ac CHMO less labile by introducing computationally predicted disulfide bonds [Ac CHMO and its more thermostable homologs [To address the poor thermostability, two main strategies have been approached. First, there have been extensive efforts to expand the substrate scope of BVMOs which have good stability but relatively narrow substrate ranges ,6–8. Secde bonds ,10 and chomologs .Further engineering with these methods is constrained by the available sites for potential disulfide bond introduction and the limited substrate scope of the homologs . While tE. coli strain MX203 by re-routing glucose metabolism through the pentose phosphate pathway; in addition, NADPH recycling is blocked by disrupting NADPH:quinone oxidoreductase and transhydrogenase. As a result, the cells cannot grow unless a NADPH-consuming enzyme is introduced to restore the NADPH/NADP+ balance. This selection platform employs growth as an easily measurable readout and features a >106 sample throughput per round. However, it has not been used at an elevated temperature to engineer enzyme thermostability.Recently, we described the construction and application of an aerobic growth-based, high-throughput selection platform for engineering NADPH-dependent oxygenases . This seHere, we sought to leverage the high throughput of the growth-based selection and mine beneficial mutations from a larger protein sequence space generated by error-prone PCR. Through a single round of selection at 42 °C , we disc2.2.1.CHMO wild type (WT) can restore growth of the NADPH-dependent selection strain (MX203) at 37 °C in minimal medium on glucose, but failed to restore the growth at 42 °C , suggest7 variants was subjected to selection in strain MX203 on agar plates with 2 g/L D-glucose in M9 minimal medium and 2 g/L cyclohexanone at 42 °C. The selection resulted in a single colony with superior growth, while ~18 additional colonies were observed with significantly slower growth. Due to the drastic growth difference between colonies, only the single fastest growing colony was extensively characterized. Sequencing of this variant revealed two mutations, A245G and A288V, previously unidentified as relevant to protein stability. The double mutant Ac CHMO A245G-A288V (CHMO GV) demonstrated reliable growth restoration of strain MX203 at 42 °C  bound in the Ac CHMO structure using Rosetta Comparative Modeling, based on Rhodococcus sp. CHMO [We examined the impact of the GV mutations on CHMO conformational dynamics to understand how they contribute to enhanced thermostability. Since there are no crystal structures of sp. CHMO , which sIn CHMO WT, A288 leaves excess space for water molecules to enter and causes undesired side-chain flexibility . In contAc CHMO homologs indicates that alanine and glycine are sampled in 97% of homologous sequences, with glycine in 47% of sequences and alanine in 50%  media consisted of 20 g/L Tryptone, 5 g/L Yeast Extract, 0.5 g/L NaCl, 4.94 g/L MgSO4· 7H2O, and 0.2 g/L KCl. Super Optimal Broth with Catabolite Repression (SOC) media shared the same composition of SOB media with the inclusion of 20 mM D-glucose. M9 Wash Buffer consisted of 1 mM MgSO4, 0.1 mM CaCl2, trace metal mix A5 with Co 2·6H2O 49 μg/L, and BD Difco M9 salts . M9 Selection Media shared the same composition as M9 Wash Buffer with the inclusion of 2 g/L D-glucose, 0.01 g/L thiamine, 0.04 g/L FeSO4 · 7H2O. For solid media M9 Selection Plates, 15 g/L agar was added in addition to M9 Selection Media composition. In overnight cultures, the substrate cyclohexanone was added at a concentration of 1 g/L and in growth restoration experiments 2 g/L was added from a concentrated stock when appropriate. Concentrations for antibiotic selection were 100 mg/L for ampicillin, 50 mg/L for kanamycin, 50 mg/L for spectinomycin, and 10 mg/L for tetracycline. All strains were cultured at 37 °C with 250 r.p.m agitation unless otherwise noted. Induction in overnights was initiated with final concentrations of 0.01% arabinose for strains with arabinose-inducible promoter.Cloning was carried out with Acinetobacter sp. NCIMB 9871 chnB gene was amplified from an E. coli codon optimized gBlock (IDT). After PCR and gel extraction, the Ac chnB gene fragment was inserted into the pRSF vector backbone which contained a 6×His tag at the C-terminus, using the Gibson isothermal DNA assembly method, resulting in plasmid pLS301. Plasmid pLS302 was generated using pLS301 as a template and applying site directed mutagenesis to introduce mutation T415C. Plasmid pLS305 was generated by the addition of mutation T415C using pLS304 as a template. Construction of strain MX203 was described previously [All PCR fragments were generated using PrimeSTAR Max DNA Polymerase (TaKaRa) unless otherwise noted. Splicing-by-overlap extension (SOE) PCR and degenerate codon PCR was conducted using KOD Xtreme Hot Start DNA Polymerase (Novagen). The eviously .Ac CHMO Error Prone PCR Library was cloned using the GeneMorph II Random Mutagenesis Kit (Agilent) to generate 1 to 2 mutations on gene Ac chnB. Using 180 ng of template DNA . Purified library inserts and backbone fragments were ligated overnight and then transformed into ElectroMAX DH10β competent cells (Invitrogen) by electroporation. Transformed cells were rescued in 600 μL SOC medium by shaking for 1 h at 37 °C. After rescue, cells were added to 20 mL 2xYT medium in a 250 mL baffled shake flask and 2 μL, 20 μL, and 200 μL was plated on 2xYT agar plates with appropriate antibiotics. Colonies formed on these plates were counted to estimate library size, 2.43 × 107. The liquid culture was incubated at 37 °C for 10 h before plasmid extraction to generate the plasmid library, pLS303. Ten colonies from the library estimation plates were cultured in liquid medium for plasmid extraction and sequencing to confirm low mutation rate.The 5 cells/mL, 104 cells/mL, and 103 cells/mL were prepared in M9 Wash and 5 μL aliquots were dispensed in series on an agar plate of M9 Selection Media, with appropriate antibiotics and inducers. Plates were grown at designated temperatures and photos were taken to document growth progress. Plates were sealed with parafilm to minimize evaporation of substrate and media. This procedure was applied after selection to verify that variant expression conferred growth restoration.The growth rescue condition used is as follows: Briefly, the strains tested were first cultured in 2xYT under aerobic conditions at 30 °C overnight with appropriate antibiotics and inducers. Next, overnight cultures were washed 3 times and re-suspended in M9 Wash Buffer. For solid growth, targeted serial dilutions of 10E. coli MX203 cells, a selection strain cultured in 200 mL SOB medium with appropriate antibiotics at 30 °C with shaking at 250 r.p.m. until OD600nm reached 0.4–0.6. The culture was chilled on ice for 15 min and the cells were pelleted at 4 °C, 4000× g. The cells were washed at 4 °C three times with 40 mL 10% glycerol solution (ice cold). After, cells were finally resuspended with 500 μL 10% glycerol solution (ice cold), and aliquoted for transformation.To generate electro-competent 6. Remaining cultures were grown at 30 °C for ~5 h or until OD600nm = 0.6 was reached. Subsequently, controls and the library were induced by addition of arabinose. Cultures were grown for an additional 4 h (until OD600nm = ~2.2).After electro-competent cells were prepared, 20 μL of library DNA (pLS303) was added to 200 μL competent cells. Cell-DNA mixture was added to four ice chilled 1 mm gap electroporation cuvettes (55 μL per electroporation cuvette). Cells were electroporated at 2 kV, 129 Ω, 50 μF, resistance 2.5 kV; 200 μL of SOC medium was immediately added and transferred to a microcentrifuge tube at room temperature. This step was repeated twice more. Cells were rescued at 37 °C with shaking for 1 h. After that, cultures were combined in 20 mL 2xYT with appropriate antibiotics in a 250 mL baffled shake flask while controls were added to 5 mL 2xYT in a 50 mL conical tube . Serial dilution of the library culture was performed and then plated on 2xYT agar plates with appropriate antibiotics. After incubation at 37 °C overnight, colonies formed were counted to estimate transformation efficiency, ~1 × 107 cells/mL. Twenty 100 μL aliquots of this cell suspension were plated on separate M9 Selection Plates supplemented with 2 g/L cyclohexanone and incubated at 42 °C for 48 h. Expression of CHMO WT from plasmid pLS301 was observed in parallel at 37 °C to serve as a positive control and at 42 °C to serve as a negative control. The colony growth was monitored periodically. A single colony was observed with rapid growth comparable to the positive control, while ~18 additional colonies were observed with significantly slower growth. Selected colonies were picked and re-streaked onto the fresh selection media and again incubated at 42 °C to obtain single colonies. Single colonies were cultured in liquid media to extract plasmids using the QIAprep Spin Miniprep kit (Qiagen) to yield pLS304. Due to the drastic growth difference between colonies, only the single fast-growing colony was extensively characterized.To prepare cells for the selection condition, 1 mL of each culture was pelleted in 2 mL microcentrifuge tubes and washed three times with M9 Wash Buffer. After wash, cells were finally re-suspended in 1 mL M9 Wash Buffer. Cells were diluted with M9 Wash Buffer to a final cell concentration of ~10After electroporation and rescue, all transformants containing library plasmid were added to a flask containing 20 mL 2xYT. From this culture, 2 μL was plated and incubated overnight. The number of isolated MX203 colonies formed on this plate was counted as 50 colony forming units (CFU). The library size contained in the entire culture was calculated using E. coli strain BW25113 [E. coli strain MX203 [600nm of 0.6–0.8 with 0.2% arabinose. The 50 mL main culture was then incubated at 25 °C or 30 °C and 250 r.p.m for 24 h. The expression was stopped by harvesting the cells . Cell pellet was resuspended in HisPur™ Ni-NTA equilibration buffer  and mixed with 1 mL of 0.1 mm glass beads  and homogenized using a benchtop homogenizer . Cell debris was separated from the crude extract by centrifugation at 4 °C, 20,000 g for 15 min. Protein purification was performed using the HisPur™ Ni-NTA Protein Miniprep (Thermo Fisher Scientific) according to the manufacturer’s instructions. The histidine-tagged protein was eluted in HisPur™ elution buffer . The concentrations of purified protein were quantified by Bradford assay using bovine serum albumin (BSA) as standard. For stock protein, 20% (v/v) glycerol was added; and the protein was stored at −80 °C for future use or used immediately for temperature gradient incubation specific activity assay. SDS-PAGE was performed with purified protein samples as described in the Enzymes purified for residual specific activity assay  were exp BW25113  incubatein MX203  for exprTo determine thermostability, residual specific activity values were determined for CHMO variants according to previously described methods . Newly pcat/KM) for NADPH  were incubated in sodium phosphate buffer  with 5 mM NADPH and 5 mM cyclohexanone (1 M stock dissolved in ethanol) at incubation temperature (37 °C) in sealed glass vials. At 2, 4, and 12 h, reaction mixture was sampled. Caprolactone and cyclohexanone were extracted with an equal volume of ethyl acetate. To confirm productive NADPH oxidation, cyclohexanone conversion to caprolactone was determined for CHMO variants via gas chromatography–flame ionization detection (GC–FID) with octanol as an internal standard. Samples were analyzed using previously described GC method “KIP 5 Short” .Ac CHMO with cofactors FAD and NADPH bound was generated with Rosetta Comparative Modeling (Rosetta CM) [Ac CHMO as the query [Rhodococcus sp. , which share 57.8% sequence identity to Ac CHMO, were selected as input models [Ac CHMO sequence was threaded onto the template structure based on Multiple Alignment using Fast Fourier Transform (MAFFT) sequence alignment, segments of the protein structure were constructed through insertion of fragments drawn from the library provided by the templates through Monte Carlo evaluation, followed by minimization to relax the final output [Ac CHMO variants were produced through 1000 further Rosetta docking simulations on the homology model with backbone flexibility, side chain repacking, and ligand minimizations; final models were selected by lowest total Rosetta energies.The model of WT etta CM) ,38. Threhe query ,40. Cryst models ,41,42.Thl output . A totalAc CHMO variants were completed with PMEMD from the AMBER 18 package utilizing the ff14sb force field and 8 Å Particle Mesh Ewald real space cutoff [+/Cl− counter-ions. The Ac CHMO systems were minimized in two stages, first with 2500 steps of steepest decent and 2500 steps of conjugate gradient where all non-hydrogen solute atoms were restrained with a 20 kcal mol−1 Å−2 force to relieve solvent clash. The second stage minimization to remove solute steric clashes was run with the same cycle settings and restraints removed. Heating from 0 K to 300 K was performed over 0.5 ns with 10 kcal mol−1 Å−2 restraints on all non-hydrogen solute atoms under isothermal-isobaric constant number, pressure, and temperature (NPT) conditions at 1 atm pressure with Langevin thermostat and 1 fs timestep. Solvent density equilibration was carried out over 5 ns with 5 kcal mol−1 Å−2 restraints on all solute atoms and an unrestrained 10 ns equilibration using 2 fs timestep to clear remaining structural artifacts followed the heating stage. Production MD trajectories were each carried out for 400 ns with 2 fs timesteps, SHAKE restraints on hydrogens, constant number, volume, and temperature (NVT) ensemble, Langevin thermostat with collision frequency 1.0 ps−1, and periodic boundary conditions.MD simulations of the e cutoff –47. Cofae cutoff –50. The Ac CHMO systems and representative frames for the discovered states were identified through PCA dimensionality reduction and K-Means clustering [Ac CHMO, we compared MD trajectories of GV and WT Ac CHMO featurized on minimum heavy atom distance between the residues at 245 or 288 to neighboring residues having any atom within 5 Å contact. The residue positions around 245 were selected as: 62, 239, 242, 243, 244, 245, 246, 247, 250, 433, 434, 437, and 510. The residue positions around 288 were defined as: 280, 281, 286, 287, 288, 289, 290, 294, 482, 483, and 484. The distance array was standardized to zero mean and unit variance and transformed to lower dimensional space with components maintaining maximal variance through PCA. K-means clustering was performed to discretize the sampled conformations projected onto the free energy landscape of the first two PCA components into metastable states. The optimal number of clusters was selected by the elbow heuristic where clustering over a range of K values, from one to nine here, is completed and the sum of squared distances from the sample points to their assigned cluster center is computed. The value of K where the sum of squared distances decrease becomes linear is selected as optimal and indicates that increasing K further will result in over-fitting. Thermostability was evaluated through the metric of Rosetta residue energies, a score quantifying the favorability of van der Waals, electrostatic, and geometric interactions that functions as a surrogate for predicted stability, summed over all residues within 5 Å of the mutated positions [Metastable conformations sampled by the ustering ,52. To uositions . 200 fraMDTraj and cpptraj were utilized for trajectory analysis, data processing was completed with the NumPy and scikit-learn packages –57. PyMoAc CHMO were identified through BLASTP search over the non-redundant protein sequence database [Ac CHMO was completed with MAFFT [Homologous sequences to WT database . Sequencth MAFFT . Residue5.E. coli selection strain MX203 and applies additional selection criteria for the identification of enzyme variants with enhanced thermostability. Through the selection of a random library of CHMO variants at a 42 °C incubation temperature, we identified a pair of subtle mutations, A245G and A288V, with a pronounced impact on protein stability. These mutations greatly improved residual specific activity and substrate conversion at elevated reactions temperatures. Additionally, the pair of mutations exhibited a synergistic stabilizing effect when combined with a previously identified single mutant T415C. Currently, the platform is limited to the temperature ranges tolerated by the E. coli selection strain. Future work may extend this approach for selection at higher temperatures to identify biocatalysts with even greater thermostability.In summary, we established a high-throughput screening method to select for thermostable variants of NADPH-dependent oxidoreductases. This growth-based approach utilizes a recently established Supplemental Info


N4-substituted piperazinylaminoacid derivatives of norfloxacin (24 new compounds) were designed andsynthesized to attain structural surrogates with additional bindingsites and enhanced antibacterial activity. Synthesized derivativesshowed increased antibacterial and antimycobacterial activity comparedto their lead structure, norfloxacin. Molecular modeling studies supportedthe notion that the derivatives can establish additional bonds withthe target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds weresignificant inhibitors of these enzymes. Inhibition of gyrase andtopoisomerase IV was then confirmed in living bacterial cells usingbacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomeraseinhibition phenotype characterized by severe nucleoid packing defects.Several derivatives exhibited additional effects on the Gram-positivecell wall synthesis machinery and/or the cytoplasmic membrane, whichlikely contributed to their increased antibacterial activity. Whilewe could not identify specific cell wall or membrane targets, membranedepolarization was not observed. Our experiments further suggest thatcell wall synthesis inhibition most likely occurs outside the membrane-boundlipid II cycle.Two series of  Moreover,extensively and totally drug-resistant Mycobacteriumtuberculosis strains plague low-income countries.4Increasing antibiotic resistance has createdan urgent need fornovel antibacterial agents and strategies. Both Gram-positive andGram-negative bacteria, as well as mycobacteria, contribute to thisproblem.https://list.essentialmeds.org/). The success of fluoroquinolones is partly based on their dualmechanism of action on two essential bacterial enzymes,5 gyrase, and topoisomerase IV. Both proteins areinvolved in DNA packing: DNA gyrase, mainly in relieving DNA strandtension during replication and transcription, and topoisomerase IV,mainly in segregating replicated chromosomes. Inhibition of theseenzymes leads to DNA aggregates, inhibits cell division, and ultimatelyleads to cell death.7 In Gram-negative and mycobacteria,the primary target of fluoroquinolones is topoisomerase IV, whilegyrase is the secondary target. In Gram-positive bacteria, the oppositeis the case.9Fluoroquinolones are an important class of broad-spectrumantibioticsthat are effective against Gram-positive, Gram-negative, and mycobacteriaand are orally available. Three fluoroquinolones, namely ciprofloxacin,moxifloxacin, and levofloxacin, are on the WHO’s list of essentialmedicines, the latter two for the treatment of tuberculosis . This is similar to DNA gyrase andtopoisomerase IV, which both contain catalytic Mg2+ thatinteracts with norfloxacin. NagA is an N-acetylglucosamine-6-phosphatedeacetylase, which catalyzes the deacetylation of N-acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate(GlcN6P). It is involved in cell wall peptidoglycan turnover and hasbeen proposed as a new drug target in M. tuberculosis since, in contrast to other bacteria, this protein is essentialin this organism.12 LpxC is a UDP-3-O-(R3̅-hydroxymyristoyl)-N-acetylglucosaminedeacetylase that catalyzes the second step of lipid A synthesis. LipidA is the lipid anchor of lipopolysaccharides, which decorate the outermembrane of Gram-negative bacteria and confer its high impermeability.13 Its expression levels are tightly regulated,and both the deletion and overexpression of the lpxC gene are lethal.14In this work,we aimed at synthesizing novel amino acid derivativesof norfloxacin, one of the oldest fluoroquinolones and the most importantlead structure, with the aim of introducing new binding sites capableof interacting with additional targets in the bacterial cell. To thisend, we were inspired by the enzymes NagA and LpxC, both of whichcontain catalytic metal cofactors in their active centers  and amino acid and hydroxamic acid derivativesof different N-acylpiperazinyl derivatives of norfloxacin. These new derivatives werethen characterized with respect to their antibacterial activity, cytotoxicity,and mechanism of action by using in silico, in vitro, and in vivo assays.Thus, wekept the quinolone ring of the norfloxacin lead structureand introduced modifications at two sites: metal-chelating groupsat the carboxylic acid group and lipophilic tails at the o groups2: amino N-acyl, sulfonyl, and alkyl derivatives of norfloxacin weresynthesized, as outlined in 2a–f were synthesized according to published procedures.22 Subsequently, amino acid ester derivatives 4a–f . The final products were purified by crystallizationusing ethanol and washed with diethyl ether.A series of amino acid esters of 4a–f showed absorption bands at 3450, 1748, 1655,and 1629 cm–1 attributed to NH, ester C=O,carbamide C=O, and quinoloneC=O, respectively. The 1HNMR spectra of all estercompounds were characterized by the disappearance of the broad singletsignal of the COOH groups of the intermediatecompounds 2a–e and the presence ofa triplet or doublet (in the case of serine amino acid ester) signalat δ 10.5–10.24 ppm assigned to amidic CONH. In addition, glycine ethyl ester compounds 4a, 4c–e showed triplet signals at δ 1.41–1.26 ppm assignedto CH3CH2OOC and quartet signals at δ 4.15–4.12 ppm assignedto CH3CH2OOC, which overlapped with the doublet signal assigned to NHCH2CO. For the serinemethyl ester compounds 4b and 4f, we observedsinglet signals at δ 3.78–3.10 ppm assigned to COOCH3 and a triplet peakat δ 5.24 assigned to CH2OH. Other signals, including aromatic protons, appeared at their expectedchemical shift in agreement with the reported data.22 In the case of the hydrazide compounds 5a–e, IR spectra showed absorption bands at 3315, 1693, 1669,and 1610 cm–1 assigned to NH, hydrazide C=O,carbamidic C=O, and quinolone C=O groups, respectively.In addition to the expected aromatic and characteristic protons oftheir parent compounds 4a–e, the 1HNMR spectra of the hydrazides 5a–e were also characterized by the disappearance of ester groupsignals, the presence of triplet or doublet (in the case of serineamino acid) signals at δ 10.32–10.17 ppm assigned toamidic CONH, broad singlet signals at δ9.19–9.13 ppm assigned to NH2NH of hydrazide, and a singlet signal at δ 4.23 ppm assignedto NHNH2 ofhydrazide.IR spectra of ester compounds 7a–i were prepared by the reaction of N-acyl norfloxacin derivatives 2a, c, d, and f with ethyl chloroformate indichloromethane in the presence of triethylamine to give mixed anhydrides.Then, two equivalents of amino acids  were added . Acid compounds were purified by column chromatographyusing 9.7:0.3 dichloromethane/methanol as the mobile phase. To preparehydroxamic acid derivatives 8a–d, N-acyl norfloxacin amino acid derivatives 7a, 7b, 7e, and 7g were treatedwith ethyl chloroformate in dichloromethane in the presence of triethylamineto produce mixed anhydrides. Then, hydroxylamine hydrochloride wasadded to obtain the amino acid hydroxamic derivatives of 8a–d. Compounds were purified by column chromatographyusing 9.5:0.5 dichloromethane/methanol as the mobile phase . Chemical structures of the newly prepared compoundswere assessed by 1H NMR, 13C NMR, mass spectrometry,and elemental analysis .The second series of amino acid and hydroxamic acidderivativesof norfloxacin were prepared, as depicted in 7a–i showed absorption bands at 3435–3420,3400–2500,1710–1725, 1655–1650, and 1640–1625 cm–1 attributed to NH, OH, acidic C=O, amidic C=O, andquinolone C=O, respectively. In addition to anticipated aromaticprotons, 1HNMR spectra of 7a–i also showed triplet (in the case of glycine amino acid)or doublet  signals at δ 10.44–10.16 ppm assigned to amidicCONH and broad singlet signals at δ 12.55–10.35ppm assigned to the COOH group. Compounds 7a–d showed doublet signals at δ4.50–4.04 ppm assigned to the NHCH2CO group, which appeared as δ4.46 ppm multiplet for compounds 7e–f, which also showed a doublet peak at δ 1.36–1.26 ppmassigned to the COCHCH3 group. Compounds 7g–h gavemultiplet signals at δ 4.10–4.70 ppm assigned to the CHCH2SH group, triplet peaks at δ 3.75–3.33ppm assigned to the CHCH2SH group, and singlet signals at δ 3.75–3.44 ppmattributed to the CHCH2SH group.The 1HNMR spectrum of compound 7i showed asinglet signal at δ 10.36 ppm assigned to the CH2CONH2 group,a multiplet signal at δ 3.86 ppm assigned to the CHCH2CO group, and a doublet peak at δ1.41 ppm attributed to the CHCH2CO group.IR spectra of the free amino acid derivatives 8a–d showed absorptionbands at 3420, 3230, 1670, 1650, and 1625cm–1 attributed to NH, OH, hydroxamic CO, amidicCO, and quinolone C=O, respectively. In addition to the expectedaromatic and characteristic protons of the corresponding amino acidderivatives 7a, 7b, 7e, and 7g, the 1HNMRspectra of their hydroxamic derivatives were characterized by thedisappearance of the broad singlet peak of COOH and the presence ofbroad singlet signals at δ 9.12–7.58 ppm assigned tothe NH of hydroxamic acid.IR spectra of the hydroxamic acidderivatives 2–8 was evaluated against Escherichia coli W3110, P. aeruginosa PAO1, K. pneumoniae ATCC 10031 (Gram-negativewild type strains), S. aureus CCUG1800T,and Enterococcus faecalis ATCC 19433(Gram-positive wild type strains), and M. tuberculosis MC26020 (live attenuated strain for use in BSL-II laboratories23). Additionally, all compounds were tested againsta norfloxacin-resistant clinical isolate of E. coli and an MRSA strain resistant to both norfloxacin and ciprofloxacin.24 Several of the new derivatives showed equalor enhanced activity when compared to norfloxacin and ciprofloxacin(Gram-negative and Gram-positive strains) or isoniazid (M. tuberculosis) , it should be noted that derivatives 2e, 4d, and 8a were 2.8–3.5 times more active thannorfloxacin against the tested norfloxacin-resistant clinical isolateof E. coli, suggesting that they aregood lead structures for further improvement.Antibacterial activity of norfloxacinderivatives 8a and 8c were more potent than norfloxacin againsta number of strains, yet 8b and 8d showedpoor or no activity. The latter two both carry sulfur , while 8a and 8c carry carboxy and methyl/hydrogen in these positions,suggesting that hydrophobicity could play a role in the differencein activity. Interestingly, 8a is more active againstGram-negative bacteria, while 8c shows better activityagainst Gram-positive bacteria and mycobacteria. The structural differencebetween these compounds is minute, defined only by a methyl groupor hydrogen as R′. It could be speculated that the increasedhydrophobicity conferred by the methyl group may facilitate uptakeinto Gram-negative cells.Given the structural similarity of some derivatives,it was surprisingthat their activities differed considerably. For example, compounds in silico.Lipinski’s rule of five states that a compound with a molecularweight under 500 Da, a coefficient of partition between octanol andwater lower than 5, no more than five hydrogen bond donors, and nomore than 10 hydrogen bond acceptors is likely to be a good drug candidate.Veber’s rule states that a compound that has 10 or fewer rotatablebonds and a polar surface area no greater than 140 Å2 is likely to exhibit good oral bioavailability.25 Except for compounds 4b, 5b–d, 7d, 7i, and 8b,all tested derivatives complied with both Lipinski’s and Veber’srules (Table S2).The compliance of the newly synthesized compounds to Lipinski’sand Veber’s rules was estimated Tables S3–S4 and discussed in Text S1–S3. Inessence, most newly synthesized derivatives showed properties similarto or better than norfloxacin. Following favorable toxicity predictions,we experimentally evaluated cytotoxic effects on the human neuroblastomaSH-SY5Y and human fetal lung fibroblast (WI-38) cell lines for exemplarycompounds 4f and 5b . Compounds 4f and 5b exhibited similar IC50 valuesas norfloxacin and were clearly less toxic than the apoptosis-inducingkinase inhibitor staurosporine . While norfloxacin has a muchgreater therapeutic window for fluoroquinolone-sensitive E. coli, 5f and 5b stillhave considerable windows against the same strain.Further pharmacokineticand toxicity predictions are shown in osporine 3A. This S. aureus DNA gyrase  and A. baumannii topoisomerase IV . Docking studies wereperformed on the crystal structure of S. aureus DNA gyrase, the primary target of fluoroquinolones in Gram-positivebacteria, in complex with moxifloxacin and DNA (PDB 5cdq).26 The docking protocol was validated by redocking of thecocrystallized moxifloxacin ligand , and the validated docking setup was thenused to investigate the interactions of norfloxacin . The main interactions of norfloxacin were two coordination bondswith Mg2+ (2.44 and 2.34 Å) and H-bonding betweenthe carbonyl of the carboxylic acid group and Ser B84 (2.26 Å),as well as a π–hydrogen bond with deoxyadenosine (DA)E2013 and π–π stacking between the quinolone ringand deoxyguanosine (DG) D2009.To assess the interaction of thenewly synthesized derivatives with their target enzymes, moleculardocking studies were performed. Test compounds were selected basedon their antibacterial activity and docked to 4f and 7f wereselected fordocking studies on gyrase . H-bonding with Ser B84 was mediated by the hydroxylgroup of 4f and the oxygen of the carboxylic carbonylgroup of 7f (1.83 and 2.52 Å). Interactions withthe nitrogenous bases DG D2009 and DA E2013 were mediated by π–πstacking and π–H bonding with the quinolone and piperazinerings, respectively. Additionally, H-bonds were formed between ArgA122 and the amidic carbonyl group of 4f and the oxygenof the carboxylate of 7f (1.64 and 2.34 Å).Compounds n gyrase 4, showinN4 of the piperazine ring and the carboxylicacid group of norfloxacin. This included H-bonding and π-cationbonds, such as H-bonds between Lys C417 and the amidic carbonyl groupof 4f and the sulfonyl group of the phenylsulfonyl moietyof 7f (2.27 Å), a π–H bond betweenDC C2010 and the phenyl ring of the phenylsulfonyl moiety of 7f, a H-bond with Arg C458 with the carboxylate group of 7f, and a H-bond with DG C2009 through the hydroxyl groupof the serine moiety of 4f. These additional interactionsmight stabilize the binding of compounds 4f and 7f to gyrase, possibly explaining their higher antibacterialactivity against Gram-positive bacteria.Further interactions were mediated by the structural moieties thatwere added to A. baumannii topoisomerase IV in complex with moxifloxacinand DNA (PDB 2xkk).27 Redocking of moxifloxacin gave aredocking rmsd of 0.3718 Å and a binding score of −10.62kcal mol–1 . Docking of norfloxacin gave a binding score of −9.32 kcalmol–1 and revealed the main interactions to be acoordination bond with Mg2+ (2.41 Å), H-bonding betweenthe carbonyl of the carboxylic acid group and Arg A1123 (3.45 Å),a π–hydrogen bond with DA C20, and π–πstacking of A D16, both with the quinolone ring .Similar results wereobtained for DNA topoisomerase IV, which isthe primary target of norfloxacin in Gram-negative bacteria. Dockingstudies were performed on the crystal structure of 4d, 4f, 5b, 7i, and 8c were selectedfor docking on topoisomeraseIV (S62–S64), of which 4f, 5b, and 7i showed the lowest binding scores .Coordination bonds with Mg2+ were formed with the quinolonecarbonyl group of 4f (2.43 Å), the amidic carbonylgroup of 5b (2.24 Å), and both the quinolone andamidic carbonyl groups of 7i (2.54 and 2.36 Å).H-bonds with Arg A1123 were formed with the amidic carbonyl groupsof 5b and 7i (average length of 1.9 Å)and the ester carbonyl group of 4f (2.46 Å). Allinvestigated compounds formed a π–hydrogen bond withDA C20 and π–π stacking with DA D16 with theirquinolone and piperazine rings.Compounds 4f and the amidic carbonyl group of 5b and 7i (average length 2.15 Å) and π-cation bonds betweenArg B418 with the quinolone ring of 5b and the phenylring of the benzoyl moiety of 7i. Instead, compound 4f formed a hydrogen bond with Arg B418 through the sulfonylgroup of its phenylsulfonyl moiety. Additionally, 5b and 7i formed H-bonds with Asp B1083 through the NH2 of the hydrazide and carboxylate groups, respectively (2.27 and1.98 Å). These additional interactions might stabilize the bindingof compounds 4f, 5b, and 7i to topoisomerase IV, possibly explaining their higher antibacterialactivity against Gram-negative bacteria.In addition to these interactions,the added moieties of the newlysynthesized compounds formed H- and π-cation bonds, such asH-bonds with Ser B1084 mediated by the ester carbonyl group of Mycobacteriumsmegmatis NagA and P. aeruginosa LpxC . To furtherassess the ability of the new derivatives to interact with LpxC, ligand-basedpharmacophore modeling was performed . Details on these experimentsare described in Text S4–S6.To assess possibleinteractions with additional target enzymes,docking experiments were also performed with in vitro inhibition studies were performed on the purified E. coli enzymes .28 Compounds 4d, 5b, and 7i were tested against gyrase and 4f and 7f against topoisomerase IV  showed aspectral shift indicative of metal complexation at a 1:1 ratio . Thereby, affinityfor zinc was higher than that for magnesium and cadmium, and the derivativesshowed higher binding of metal ions than norfloxacin. Further, hydroxamicacid and hydrazide derivatives showed higher metal chelation thancarboxylic compounds, aligning well with the molecular docking results.Since coordination bonds withMgin vivo, we used bacterial cytological profiling (BCP).BCP is a phenotypic analysis method based on phase contrast microscopycombined with fluorescent staining of the cell membrane and DNA thatcan be employed to elucidate antibiotic targets.29 Gyrase/topoisomerase IV inhibition by fluoroquinolonesresults in characteristic and unique nucleoid packing defects thatare not observed with other antibiotics.30 Compounds 4d, 5b, 7d, 7g, 7i, 8a, and 8c weresubjected to BCP in Gram-negative E. coli (Table S8). Gyrase inhibition could be confirmedfor 5b, 7d, 7i, and 8c, but not for 4d, 7g, and 8a. Since 4d was active against gyrase in vitro, it may interact with a different target in vivo.To confirm that the new compounds inhibit DNA gyrase 31), and indeed no effects were observed.No membrane aberrations were observed in the FM4-64membrane stain,yet this dye does not discriminate between inner and outer membraneeffects. To confirm that there are no additional effects on the cytoplasmicmembrane, a green-fluorescent protein (GFP) fusion to the ubiquitousmembrane protein GlpT was used as a proxy for the inner membrane , suggesting that they do not permeabilize the outermembrane.Since moleculardocking revealed a possible interaction of the new compounds withLpxC, outer membrane integrity was studied by assessing synergy withmupirocin, a translation inhibitor that is able to inhibit in vivo screening assay for LpxC inhibition. To this end, MICs were determinedagainst a strain overexpressing the lpxC gene fromthe arabinose-inducible PBAD promoter.35 If LpxC is a targetof the tested compound and its inhibition contributes to its antibacterialactivity, the MIC should increase with rising arabinose concentrationsdue to the presence of more target molecules. This was indeed thecase for the positive control ACHN-975, but not for other controlantibiotics (polymyxin B and nitrofurantoin), demonstrating that theassay works and is specific . However, no arabinose-dependent MIC increase was observed forany of the test compounds, confirming that they do not inhibit LpxC.To confirm these results and rule out an inhibitionof LpxC that would not manifest in increased outer membrane permeability,we additionally employed a newly developed Bacillus subtilis (see Table S10 for MICs). Gyrase inhibition couldbe confirmed for compounds 5b, 7a, 7b, 7c, 7d, 7e, 7f, 7g, 7h, and 7i,but not 8a. Compound 4a showed slight DNApacking defects, indicating a possible gyrase inhibition, yet no clearphenotype could be observed even at higher concentrations . Compounds 5b, 7b, 7c, 7d, 7e, 7h, and 8a showed aberrations in themembrane stain, suggesting additional membrane effects. However, similarphenotypes were observed with norfloxacin and ciprofloxacin as well,indicating that membrane activity may be a shared feature of fluoroquinolones.Membrane aberrations are most commonly caused by depolarization.32 To assess whether the observed membrane aberrationsare indicative of dissipation of the membrane potential, the membranepotentiometric probe DiSC(3)5 was employed. Yet, no effects were observed, suggesting that membranedefects may rather be caused by phase separation or inhibition ofcell wall synthesis.33To validate topoisomerase inhibition as a mechanism ofthe compounds in Gram-positive bacteria, BCP was performed in themodel organism B. subtilis BCP could point to possible cell walldefects, we tested the effects of the new compounds on B. subtilis cell wall synthesis using different phenotypicassays. Since moleculardocking revealed a possible effect on NagA, which is involved in cellwall turnover, and membrane aberrations observed in the 37 If the cellwall is compromised, the protoplast can protrude through cell wallbreaches, which is promoted by the fixation and can be observed as“bubbles” on the cell surface in phase contrast microscopy.36 No strong effects comparable to those of thepositive control vancomycin were observed, yet compounds 7c, 7d, 7f, and 8a showed slightlyelevated numbers of bubbles .40 To visualize MreB mobility, twoimages of the same field of view were taken 30 s apart, false-coloredin green and red, and overlaid, resulting in a yellow signal wherethe images overlap. Distinct red and green foci indicate mobile MreB(see untreated control), while entirely yellow cells indicate lossof MreB mobility (see positive control vancomycin). Loss of MreB mobilitywas observed for compounds 5b, 7a, 7b, 7c, and 7i, which was accompaniedby clear clustering of MreB into distinct, immobile clusters at thecell membrane , themost likely causes of these effects are membrane phase separationor invaginations. Compounds 7d, 7f, and 8a, which tested slightly positive in the acetic acid/methanolfixation assay, retained MreB mobility, suggesting that their peptidoglycansynthesis may be mildly compromised by indirect effects, e.g., on the cell membrane.Acetic acid/methanol fixation only tests positivewhen cell wallautolysins are active, is strongly concentration-dependent, and doesnot react to all types of cell wall synthesis inhibition.e latter S96.40 Tomembrane 2, sugges43 We first examined the localization of MurG, which is a peripheralmembrane protein and the most sensitive out of the four. In fast-growingcells, MurG localizes in small spots at the membrane , while in slow-growing cells, itslocalization becomes smooth .If peptidoglycan synthesis is inhibited, it forms large clusters inthe membrane, accompanied by loss of membrane binding . Compound 7d showedthe strongest effect with clear clusters and a partially dispersedGFP signal, as well as signs of phase separation and invaginations, suggesting that they do notaffect peptidoglycan synthesis at the level of lipid II or its synthesisby the MurG enzyme.We then tested the effects on further cell wallsynthesis proteins,including the lipid I synthase MraY, the lipid II synthase MurG, andthe penicillin-binding proteins PbpB and PonA, which incorporate theprecursor into the peptidoglycan cell wall all around the cell orspecifically at the cell division septum, respectively.inations2. 5b, 7b5b, 7d, and 7h .While compounds 5b, 7a, 7b, 7c, and 7i stopped MreB motion and are thus likelyto impair cell wall synthesis, they did not elicit characteristicphenotypes on MurG, MraY, PbpB, and PonA, and no compound showed strongeffects in the acetic acid/methanol assay, which typically indicatesinhibition of the lipid II cycle.37 Thus, we concludethat they may rather inhibit a so-far unknown, likely intracellulartarget affecting cell wall synthesis.Most cell wall synthesis inhibitors inhibit thesynthesis of lipidII by binding to either lipid-linked cell wall precursors  or penicillin-binding proteins,and less often to membrane-bound (MraY and MurG) or intracellularlipid II synthesis enzymes  and/or the cytoplasmic membrane , which likely contributeto the increased antibacterial activity. Such polypharmacologicalproperties can be beneficial for antibiotic drug candidates, as resistancedevelops much faster against single-target molecules.5 Inhibition of cell wall synthesis appeared to occur outsideof the lipid II cycle, suggesting that the compounds have a novelsecondary target. This is an interesting finding, as new antibiotictargets are highly desirable to avoid cross-resistance with existingdrugs. Moreover, several of our compounds showed a narrow activityspectrum, being active against either Gram-negative bacteria  or Gram-positiveand mycobacteria . Modern fluoroquinolones are typically broad-spectrum antibiotics.While these are certainly very important drugs, they can put considerablestrain on the microbiome, making compounds with a narrower activityspectrum attractive for cases where the pathogen is known. Taken together,our novel compounds provide an interesting starting point for furtherderivatization approaches aimed at creating new fluoroquinolones withpolypharmacological properties.Here, we synthesized two series of aminoacid derivatives of norfloxacin.Several of the new compounds showed increased antibacterial and antimycobacterialactivity compared to their parent compounds. This could be attributedto the more efficient inhibition of gyrase and topoisomerase IV, likelydue to their ability to form additional bonds with these enzymes.Phenotypic analysis validated topoisomerase inhibition