We discovered odorous 16-androstenes (Androstenone and Androstenol) in endangered mouse deer during a captive breeding program. This study examined the molecular characteristics, their synthesis pathway, and the possible functional role of these compounds in the reproduction of mouse deer. CYP17A1 and CYB5 genes were cloned and expressed in HEK-293, COS-7 cell lines, and gonads of mouse deer to investigate the CYP17A1 gene’s andien-β-synthase activity towards the synthesis of 16-androstenes in mouse deer. An enzyme immunoassay was further developed and standardized to measure fecal androstenone during the reproductive cycles of mouse deer. Results showed that the mouse deer CYP17A1 gene possesses andien-β-synthase activity and could transform pregnenolone into 5,16-androstadien-3β-ol. The expression of the CYP17A1 gene upregulated in the testis and ovary compared to other tissues in mouse deer. Significantly elevated androstenone and estrogens were recorded prior to delivery and postpartum estrus/mating in mouse deer. Further, there were weak correlations between fecal androstenone and estrogens/androgens in mouse deer during the breeding season. These findings suggest that androstenone probably plays a role in the reproductive activities of mouse deer. This knowledge can be used for captive breeding programs of mouse deer in India and elsewhere. Pheromones are chemical messengers secreted by an individual to modulate the behavior and physiology of conspecifics . In mammCalled “boar pheromones”, 16-androstenes are odorous steroids first discovered in pigs that play a major role in social, sexual interactions, and reproduction, ,22,23,24Four key candidate genes are involved in the production of 16-androstenes in pigs, including Cytochrome P450C17 (CYP17A1), Cytochrome B5A (CYB5A), 3β hydroxy dehydrogenase (3β-HSD), and 5α reductases (SRD5A) . The firHowever, despite these dual activities, CYP17A1 possesses a third specific andien-β-synthase activity that catalyzes the transformation of pregnenolone into 5,16-androstadien-3β-ol without any intermediate precursors in humans and pigs . CYP17A1Moschiola indica), a primitive deer, belong to a distinct family, Tragulidae, listed in Schedule I of the Wildlife Protection act 1972, India and face severe threats due to habitat loss and hunting. As part of the conservation breeding and species recovery program, the Laboratory for Conservation of Endangered Species, CSIR-CCMB, Hyderabad is involved with Nehru Zoological Park, Hyderabad to breed them in captivity. With a founder population of eight individuals, the breeding center has bred more than 400 individuals, of which, more than 100 individuals were released into the wild. We studied the reproductive characteristics of mouse deer as part of the captive breeding program [Mouse deer  housed at the mouse deer conservation breeding center of Nehru Zoological Park, Hyderabad (Bambusa vulgaris), acalypha , and royal palm (Roystonea regia). The animals were fed twice a day with apple (50 g), banana (100 g), carrot (50 g), sweet potato (50 g), soaked grams (50 g), lucerne (100 g), dry grass (500 g), and peepal (500 g) and had free access to clean water as per the Central Zoo Authority of India norms. All behavioral activities, including mating and estrus, were monitored 24 × 7 using CCTV cameras. Females in an advanced stage of pregnancy were housed with adult males to facilitate mating following parturition [The experimental group comprised ten adult females and four adult males of mouse deer  on ice and stored at −80 °C until further analysis. For GC-MS analysis, 16-androstenes (androstenone and androstenol) were extracted using the previously modified procedure [Gonad tissues (testes and ovary) and heart tissues were collected opportunistically from Nehru Zoological Park during the study period. The tissues were harvested during postmortem in RNArocedure . Approxig for 20 min, transferred to a fresh tube, and kept at −20 °C until further analysis. Fecal hormone metabolites were extracted using the previously described procedure . The pulThe 16-androstenes (androstenone and androstenol) were identified using the GC-MS system . Before injecting into GC-MS, approximately 10 mL of pooled fecal extracts were purified by solid-phase extraction using a Sep-Pak C18 matrix column . In brief, fecal extracts were passed through the C18 column at 1 mL/5 min flow rate (per manufacturer’s instructions) and eluted with 5 mL of 90% methanol in order to remove the contaminants. The purified supernatant samples were then evaporated under nitrogen gas and resuspended in 100 µL of absolute methanol and kept at −20 °C until further analysis . This meExtracted fecal samples were analyzed using an HP-5 MS capillary column with a 30 m × 250 µm × 0.25 µm film thickness . Helium was used as a carrier gas with a head pressure of 15.8 psi and injector temperature of 300 °C. The interface and ion source temperature were maintained at 300 °C and 200 °C, respectively. The injection was made, and after 3 min, purge valve was turned on, then 2 µL of each sample extract was injected in the split-less mode with an inlet temperature of 270 °C. The oven temperature was raised to 300 °C at 15 °C/min. The mass spectra were recorded in full scan mode and identified by computer MS library search (Wiley MSD Chem 7th Edition) and authentic standards  were used to confirm the desired compounds by comparison of spectra and retention time of samples.Total RNA was extracted from the tissues  using the trizol  method. RNA concentration and quality were evaluated by an ND-1000 Spectrophotometer . After DNase treatment , cDNA synthesis was carried out using the first-strand cDNA synthesis kit  from 2 µg of the total RNA and incubated at 42 °C for 1 h followed by 95 °C for 5 min. As no CYP17A1 and CYB5A gene sequences of mouse deer or any closely related species were available in Genbank, shotgun sequencing was performed of mouse deer genomic DNA to assemble the candidate gene exons for designing primers specific to mouse deer. Briefly, genomic DNA was isolated from a tissue sample using DNeasy Blood and Tissue Kit  and quantified using a dsDNA assay in Qubit 4 Fluorometer . About 1µg of genomic DNA was taken as input for library preparation using Truseq DNA PCR-free kit  following the manufacturer’s protocol. The purified library was quantified using the NGS library quantification kit  and pooled with other libraries to obtain about 50× coverage. The pooled libraries were sequenced on an S4 flow cell on the Illumina Novaseq 6000 platform. The first and last exons of CYP17A1 and CYB5A were assembled by taking the corresponding exons from pig reference gene sequences as a template using the selective recursive local assembly of homologous genomic regions (SR Assembler) . The priThe cDNA fragments possessing the entire coding region of CYP17A1 and CYB5A of mouse deer and pig testis were amplified using LA taq DNA polymerase (Invitrogen) with primers listed in To quantify levels of gene expression of CYP17A1 in different tissues of mouse deer and pig, we designed primers in the conserved exon-–exon junctions of mouse deer and pigs. CYP17A1 and β-actin were used as reference genes for control. The primers spanning the exon–exon junction would only amplify cDNA and would not amplify any contaminated genomic DNA. RT-PCR was performed to examine the expression levels of CYP17A1 in testis and ovary in mouse deer using the TB Green Premix Ex Taq II  following the manufacturer’s protocol. Heart tissue was used as internal control. The PCR reaction composed of 5 µL of SYBR Premix Ex Taq, 1 µL of cDNA template, 0.5 µL of reverse primer (10 µM conc.), 0.5 µL of forward primer (10 µM conc.), and 3 µL of milliQ water in a total volume of 10 µL using Roche Lightcycler 480 II . Quantitative PCR conditions were set at 95 °C hot start for 30 s, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 30 s, 95 °C for 5 s, 60 °C for 1 min, and at 50 °C for 30 s. The PCR amplification products were analyzed by cp value and melting curves. 3H pregnenolone was added in the fresh media to the culture plates and kept for incubation at 30 °C in 5% Co2 incubator for 16 h [g for 5 mins. Further, steroids in the medium were extracted twice with 4 mL of diethyl ether and organic phase was pooled and evaporated to dryness using a stream of nitrogen. The extracts were reconstituted with 100 µL of 85% acetonitrile:15% H2O [HEK-293 and COS-7 cells were grown in DMEM medium supplemented with 10% fetal bovine serum. Cells were transfected using lipofectamine  as a transfection agent with pcDNA3.1  and pcDNA3.1 encoding mouse deer CYPA17A1 and CYB5A gene constructs. After 48 h of incubation, fresh medium was added and 50,000 cells/well of 50 µg of for 16 h . After i:15% H2O . The sepAndrostenone polyclonal antibody was produced to develop the sensitive indirect competitive enzyme immunoassay (EIA). Two adult New Zealand white rabbits were immunized with 16, (5α)-androsten-3-one-carboxymethyloxime: BSA conjugate . The first injection was prepared at a concentration of 1 mg/mL of conjugate dissolved in saline and emulsified with an equal volume of Freund’s complete adjuvant . The emulsified solution was injected subcutaneously at four sites on the rabbit’s back. The booster shots (0.5 mg/mL) were administered with Freund’s incomplete adjuvant every two weeks after the initial dose on days 14, 28, 42, 56, and 70. Blood was collected every one week of booster dose on days 35, 49, 63, and 76. ,48. The 2O2  was added and incubated in the dark for 5–10 min (for color development). The reaction was stopped using 50 µL of stopping solution (1N HCL) and absorbance was read at 450 nm in the ELISA reader .Fecal androstenone was measured using polyclonal anti-androstenone antibody diluted to 1:25,600, androstenone standards (1000–3.9 ng/mL), 16(5α)-androsten-3-one-carboxymethyloxime: BSA conjugate (1 mg/mL), and horseradish peroxidase (HRP)–conjugated goat antirabbit IgG secondary antibody  diluted to 1:10,000. The procedure of 5α-androst-16-en-3-one indirect competitive ELISA was performed as previously described . The 96-Fecal progestogens, estrogens, and androgens were measured using the previously described methods ,50,51,52The cross-reactivity of 5α-androst-16-en-3-one antibody with other steroids was assessed using the half displacement method as described earlier ,53. The 2 = 0.99) (2) and slope (m) values for the exogenous androstenone, progesterone, testosterone, and estradiol were r2 = 0.99, m = 0.86; r2 = 0.99, m = 0.93; r2 = 0.99, m = 1.05; r2 = 0.98, m = 1.15, respectively. The intra- and interassay coefficients of variation (CV) were 6.09% and 10.26% (n = 10), 7.64% and 14.63% (n = 10), 8.87% and 14.93% (n = 10), and 8.385 and 15.89% (n = 10) for androstenone, progesterone, testosterone, and estradiol, respectively. The presence of androstenone, progesterone, testosterone, and estradiol in fecal samples was confirmed by HPLC profile and eluted fractions showed the immunoreactivity of fecal hormones with the corresponding antibody  test was performed to calculate the correlation between individual females androstenone and estrogens and male androstenone and androgens, respectively. Mann–Whitney U test (M–W test) was used for testing differences in fecal progestogens concentrations in pregnant and nonpregnant animals. Statistical analyses were carried out using SPSS 17.0 for hormone analysis. m/z 79, m/z 257 and m/z 79, m/z 148, respectively. Following GS-MS mass spectra analysis, 16-androstenes (Androstenone and Androstenol) were identified in the feces of mouse deer of both sexes  as well p = 0.024, n = 5) and COS-7 cell line . Interestingly, no increase was found in 5,16-androstadien-3β-ol in the presence of CYB5A. Further, no significant difference was observed in the transformation of 5,16-androstadien-3β-ol in HEK-293 and COS-7 cells . Overall, results showed that mouse deer P450C17 has a catalytic activity to transform 5,16-androstadien-3β-ol in the absence of oxidoreductase enzymes POR and CYB5R. To evaluate the enzymatic activity of CYP17A1 in mouse deer for conversion of pregnenolone into 5,16-androstadien-3β-ol, two mammalian cell lines, HEK-293, and COS-7, were transfected with an expression construct of CYP17A1. In mouse deer, the CYP17A1 enzyme is able to transform pregnenolone into 5,16-androstadien-3β-ol in the presence of cofactors, glucose-6-phosphate salt, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADP) salt . HoweverMouse deer CYP17A1 mRNA transcript shows tissue-specific expression in the testis and ovary as compared to the heart (as a control) . The expA total of 1627 fecal pellets were collected from 10 adult females and four adult male mouse deer for 12 months . Of the p = 0.03, n = 8; 2 week vs. 0 week, M–W U = 43, p = 0.05, n = 8) and significantly decreased on the third week from 0 week . In addition, the presence of androstenone was confirmed in the fecal samples and examined androstenone antibody immunoreactivity against the fecal androstenone using high-performance liquid chromatography (HPLC).The mean fecal androstenone concentrations ranged between 47.66 ± 4.08 ng/g and 226.11 ± 14.55 ng/g. p = 0.05, n= 8; −3 vs. 0 week, M–W U = 30, p= 0.04, n = 8; 0 week vs. 3 week, M–W U = 25, p = 0.03, n = 8). However, all individual females show estrogens peak within 1.33 ± 0.18 (n = 12) days following delivery. The peak observed in fecal estrogens levels could be of follicular origin, as mating was observed within 4–6 h of parturition.Overall, fecal estradiol metabolite concentration ranged widely from 57.10 ± 2.65 ng/g to 189.91 ± 8.42 ng/g . There wp = 0.008; rs = 0.24, p = 0.489; rs = 0.87, p = 0.001; rs = 0.24, p = 0.489; rs = 0.18, p = 0.603; rs = 0.40, p = 0.244; rs = 0.09, p = 0.803; rs = 0.30, p = 0.385; rs = 0.15, p = 0.676; rs = 0.22, p = 0.533, n = 6 individuals, 10 deliveries, Overall, 10 out of 13 deliveries from six female individuals showed a weak positive correlation between fecal androstenone and estrogens concentrations one week prior to parturition, postpartum estrus, and mating  Individual mean fecal androstenone concentrations ranged from 5560.84 ± 278.44 ng/g to 16,027.87 ± 1109.119 ng/g and began increasing 5-6 weeks prior to mating. Moreover, individual mean fecal androgens concentrations ranged from 311.31 ± 29.85 ng/g to 1069.76 ± 26.32 ng/g . Overallp = 0.00, n = 8, Overall, fecal progesterone metabolite concentrations ranged from 4116 ± 204.97 to 20,175 ± 1083.92 ng/g . The meaPheromones play a major role in reproduction in mammals ,9,13; hoIt has been previously shown that CYP17A1 catalyzes the production of 17-α-hydroxyprogesterone (17αOHP) and DHEA from pregnenolone using the 17α-hydroxylase and C17,20 lyase reactions, including the conservation of 5,16-androstadien-3β-ol in a single step via the andien-β-synthase pathway ,37,54. IThe homology of the CYP17A1 amino acid sequences between mouse deer and pigs was 73.87%, with five extra amino acids and the deletion of two amino acids at the 21st and 22nd positions in the mouse deer as compared to the pig . The moun = 12) days following parturition. However, the peak in fecal estrogens levels could be of follicular origin and presumably associated with luteinizing hormones, as mating was observed within 4–6 h of parturition. A similar observation was reported in tammar wallabies (Macropus eugenii), whereby mating occurred within the first 1–6 h (1.3 ± 0.8 h) of birth [To understand the significance of androstenone in the reproductive cycles of mouse deer, an enzyme immunoassay was specifically developed and standardized by raising the antibody against androstenone to measure the fecal androstenone using a noninvasive method. The fecal androstenone concentration followed a cyclic pattern in which delivery and postpartum estrus could be clearly distinguished from consistently elevated androstenone levels compared to baseline prior to parturition, postpartum estrus, and mating . Dehnharof birth . In addiof birth . In tammof birth , and speof birth ,65,66. Aof birth .n = 6) between mean fecal androstenone and estrogens concentrations one week prior to parturition and postpartum estrus (n = 3) between fecal androstenone and androgens one week prior to mating in females. As fecal estrogens and androstenone peak precisely following estrus, the decline in progesterone levels is an effective stimulant for estrus behavior and provides necessary preconditions for estrus and mating (In addition, the present study observed a positive correlation in 10 deliveries out of 13 (m estrus . This pod mating . Our horIn conclusion, the current study discovered 16-androstenes in mouse deer and further sheds light on the molecular pathway of androstenone and a possible role of 16-androstenes in the reproductive cycles of mouse deer. Furthermore, mouse deer are solitary and nocturnal animals, and they can possibly use odorous 16-androstenes for sexual communication and the advertisement of their reproductive status to the opposite sex. However, further study is warranted to investigate the effects of 16-androstenes on the behavioral responses or physiological changes in free-range mouse deer. Nevertheless, our findings might help in various captive breeding programs undertaken by Indian zoos and elsewhere.


Haemophilus influenzae, and by constructing a high-quality genome-scale metabolic model, we rationally identified new metabolic drug targets in this organism. Contextualization of available gene essentiality data within in silico predictions identified most genes involved in lipid metabolism as promising targets. We focused on the β-ketoacyl-acyl carrier protein synthase III FabH, responsible for catalyzing the first step in the FASII fatty acid synthesis pathway and feedback inhibition. Docking studies provided a plausible three-dimensional model of FabH in complex with the synthetic inhibitor 1-(5-(2-fluoro-5-(hydroxymethyl)phenyl)pyridin-2-yl)piperidine-4-acetic acid (FabHi). Validating our in silico predictions, FabHi reduced H. influenzae viability in a dose- and strain-dependent manner, and this inhibitory effect was independent of fabH gene expression levels. fabH allelic variation was observed among H. influenzae clinical isolates. Many of these polymorphisms, relevant for stabilization of the dimeric active form of FabH and/or activity, may modulate the inhibitory effect as part of a complex multifactorial process with the overall metabolic context emerging as a key factor tuning FabHi activity. Synergies with antibiotics were not observed and bacteria were not prone to develop resistance. Inhibitor administration during H. influenzae infection on a zebrafish septicemia infection model cleared bacteria without signs of host toxicity. Overall, we highlight the potential of H. influenzae metabolism as a source of drug targets, metabolic models as target-screening tools, and FASII targeting suitability to counteract this bacterial infection.Expediting drug discovery to fight antibacterial resistance requires holistic approaches at system levels. In this study, we focused on the human-adapted pathogen IMPORTANCE Antimicrobial resistance drives the need of synergistically combined powerful computational tools and experimental work to accelerate target identification and drug development. Here, we present a high-quality metabolic model of H. influenzae and show its usefulness both as a computational framework for large experimental data set contextualization and as a tool to discover condition-independent drug targets. We focus on β-ketoacyl-acyl carrier protein synthase III FabH chemical inhibition by using a synthetic molecule with good synthetic and antimicrobial profiles that specifically binds to the active site. The mechanistic complexity of FabH inhibition may go beyond allelic variation, and the strain-dependent effect of the inhibitor tested supports the impact of metabolic context as a key factor driving bacterial cell behavior. Therefore, this study highlights the systematic metabolic evaluation of individual strains through computational frameworks to identify secondary metabolic hubs modulating drug response, which will facilitate establishing synergistic and/or more precise and robust antibacterial treatments. Haemophilus influenzae is a human-adapted Gram-negative bacterial pathogen. Asymptomatic colonization begins in the upper airways, but it can spread through the respiratory tract and lead to invasive infections. The polysaccharide capsule vaccine has driven the almost complete disappearance of capsulated H. influenzae type b (Hib) in countries with established child immunization programs (H. influenzae serotypes and nontypeable strains (NTHi), untargeted by the Hib vaccine, emerge as important causes of infections. NTHi causes otitis media, conjunctivitis, sinusitis, and lower respiratory infections in children; exacerbations of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) in adults; and invasive disease in neonates, immunocompromised adults, and the elderly .protocol  and the protocol . A detai cytosol . Compareabolites . Reactioabolites . iNL638 ote tool  to definiNL638, we evaluated its capability of predicting well-known metabolic traits of RdKW20, such as the production of acetate and formate  only played a limited role  as a main source was taken by the PDH, which completely replaced PFL at oxygen uptakes higher than 7 mmol/gDW·h−1. Finally, under optimal oxygen availability, we observed a stable growth rate with acetate as the sole end product. These predictions agree with the switch in pyruvate metabolism from PFL to PDH, observed in H. influenzae as oxygen becomes available  and in the complete formulation (cCDM). We found 260 and 169 essential genes using minimal and complete CDM, respectively  Quantitative comparison of available experimental datasets of essential genes in H. influenzae. Download FIG S1, TIF file, 2.7 MB.Gene essentiality analysis. (A) Copyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in silico and experimental data sets to identify subsystems overrepresented in all cases  and β-hydroxyacyl-ACP products, which are key components of the bacterial membrane. Acyl-ACP is used by the 1-acyl-sn-glycerol-3-phosphate (PlsB) and 1-acyl-sn-glycerol-3-phosphate (PlsC) acyltransferases to generate phosphatidic acid, the precursor of H. influenzae phospholipids phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) , 6 genes encoding enzymes required for phospholipid biosynthesis, and 12 genes encoding enzymes involved in lipid A biosynthesis . The H. rol (PG) ; β-hydrorol (PG) . Predict10.1128/msystems.01459-21.6FIG S2H. influenzae lipid metabolism. Genes commonly predicted to be essential by the iNL638 model in both complete or minimal CDM  determined for some of them (H. influenzae FabH activity (IC50 of 0.82 μM), high solubility, acceptable human plasma protein binding, easy chemical accessibility . Fa. Fa42). th Arg36  to D. Tholi FabH , as doesoli FabH . Single of FabHi , support10.1128/msystems.01459-21.7FIG S3RdKW20 structure are highlighted by colors. N-terminal domain core, alpha-helices (Nα1-Nα3) in light cyan and βeta-strands (Nβ1-Nβ5) in orange. C-terminal domain core, alpha-helices (Cα1-Cα3) in dark cyan and beta strands (Cβ1-Nβ54) in magenta. Elements of N-terminal insertions are in yellow (Lβ1-Lβ2) and light blue (Lα1-Lα2), and C-terminal ones in light pink (Lβ3-Lβ4) and green (Lα3). Download FIG S3, TIF file, 2.8 MB.Multiple alignment of FabH variants. Variants were identified in a previously whole-genome-sequenced NTHi clinical isolate collection . PolymorCopyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msystems.01459-21.8FIG S4RdKW20 interaction mode. FabHi-FabHRdKW20 interaction mode compared with crystal structures of the E. coli enzyme in complex with close structural analogues of the inhibitor. (A) FabHi bound to FabHRdKW20 (green) superimposed to E. coli FabH (cyan) in complex with compounds 23 (violet) and 36 (cyan)  Comparison of ligand disposition in the FabHRdKW20 docking complex and the FabHE. coli complexes shown in panel A. Protein residues at hydrogen-bonding distance are also depicted (same color code). (C and D) Ligplot representation of hydrogen bonds (dashed lines) and hydrophobic contacts (brown arcs with radiating spokes) in the FabHi:FabHRdKW20 complex and the 5BNSE. coli complex, respectively. Polymorphisms of FabH may affect FabH stability and activity. (E) Structure of the FabH dimer with FabHi (sticks) bound to the active site of each monomer. Loops comprising residues 84 to 89 or 185 to 207 are colored in blue and orange, respectively. (F to H) Details of contacts at the dimer interface potentially sensitive to polymorphisms at positions 85 (F), 197 to 198 (G), 105, 118, and 186 (H). (I) Details of contacts relevant for the monomer stability potentially affected by polymorphisms of residues 169, 286, 287, and 296. Ser169 of RdKW20, together with E167, keep the orientation of strands Nβ1 and Nβ5  by hydrogen bonding to Asn2 and Arg4 residues. Network of hydrogen bonds participated by Ala286, Ile287 and Glu296 helping orientation of Cβ2, Cα3, Cβ3, Cβ4 and configuration of loop connecting Cα3 to Cβ3  is shown. Residues affected by polymorphisms are underlined and catalytic residues  are shadowed. Elements of secondary structure are indicated in brown lettering . Download FIG S4, TIF file, 2.8 MB.FabHi-FabH6 (cyan) , E. coliCopyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. influenzae viability.Before antimicrobial effect testing, we tried to improve FabHi solubility by preparing different salts . Assays RdKW20:FabHi complex structure relied on the H. influenzae RdKW20 genome content and FabHRdKW20 crystal structure  , with different frequency and CT distribution but intra-CT conservation  , 46. Beianalysis –50. FourH. influenzae strains to FabHi upon growth in CDM, being the medium used to interrogate the iNL638 model ; (ii) sharp slope without complete viability loss ; (iii) not-as-sharp slope but reaching complete viability loss ; and (iv) not-as-sharp slope, without complete viability loss .We first tested the susceptibility of the 14 selected cubation  to F, we10.1128/msystems.01459-21.9FIG S5H. influenzae selected strains carrying FabH variants A1 to A14 in two different media, CDM (A) or sBHI (B). NTHi strains grown on PVX agar for 16 h were inoculated (2 to 3 colonies) in 10 mL sBHI or CDM using 100-mL flasks and incubated for 11 h with shaking (100 rpm). Cultures were then diluted to OD600 of 0.07 in a final volume of 25 mL sBHI or CDM using 250-mL flasks, grown with shaking (180 rpm), and OD600 was recorded every hour for up to 8 h. (C to F) Antimicrobial effect of FabHi on H. influenzae. Determination of FabHi inhibitory effect on a panel of representative NTHi strains carrying different FabH allelic variant (A1 to A14), grown in CDM. Strains were shown to be susceptible to FabHi in a dose-dependent manner and were classified into four groups  based on increasing resistance. FabHi concentrations tested range from 10 to 600 μg/mL. Results are shown as log10 CFU/mL (means ± SD). For each strain, statistical comparisons of means were performed by one-way ANOVA and Dunnett’s multiple-comparisons test. A significant reduction on bacterial viability was observed: A1-NTHi375, at [FabHi] 200 μg/mL and higher, P < 0.0001; A2-P621, at [FabHi] 250 μg/mL and higher, P < 0.0005; A3-RdKW20, at [FabHi] 250 μg/mL and higher, P < 0.0001; A4-P652, at [FabHi] 75 μg/mL and higher, P < 0.0001; A5-P673, at [FabHi] 125 μg/mL and higher, P < 0.0001; A6-P642, at [FabHi] 100 μg/mL and higher, P < 0.0001; A7-P665, at [FabHi] 150 μg/mL and higher, P < 0.05; A8-P657, at [FabHi] 200 μg/mL and higher, P < 0.005; A9-P645, at [FabHi] 200 μg/mL and higher, P < 0.0001; A10-P851, at [FabHi] 150 μg/mL and higher, P < 0.0001; A11-P593, at [FabHi] 125 μg/mL and higher, P < 0.05; A12-P607, at [FabHi] 125 μg/mL and higher, P ≤ 0.0005; A13-P590, at [FabHi] 125 μg/mL and higher, P < 0.05; A14-P626, at [FabHi] 125 μg/mL and higher, P < 0.0001. (G) Expression of the fabH gene in a panel of representative NTHi strains carrying different fabH allelic variant, grown in CDM to half of each strain’s maximal OD600. This gene expression was shown to be variable among strains and not linked to the differences observed in terms of susceptibility. Statistical comparisons of means were performed for each previously established group by one-way ANOVA and Tukey’s multiple-comparison test . Antimicrobial effect of FabHi on strains A2-P621 (H), A7-P665 (I), A9-P645 (J), and A13-P590 (K), grown in sBHI (gray circles) or CDM (white circles). For each strain, statistical comparisons of means were performed by two-way ANOVA and Sidak’s multiple-comparisons test. A significant difference on bacterial viability was observed: A2-P621, at [FabHi] 275, 300 and 400 μg/mL, P < 0.005; A7-P665, at [FabHi] 600 μg/mL, P < 0.0001; A9-P645, at [FabHi] 125, 200, 275, 400 μg/mL and higher, P < 0.05; and A13-P590, at [FabHi] 400 μg/mL and higher, P < 0.005. Download FIG S5, TIF file, 2.8 MB.Effects of growth medium in bacterial phenotypes. Growth of Copyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. influenzae growth and antimicrobial susceptibility testing  to those forming poorly detectable biofilms (A4-P652 and A5-P673). FabHi 50 μg/mL (+), which allowed growth of all tested strains , loweredH. influenzae clinical strains representative of the observed FabH variation decreased when exposed to FabHi in a dose-dependent manner and with variable efficacy among strains. The susceptibility of H. influenzae to FabHi irrespective of the culture conditions argues in favor of FabH as a condition-independent drug target and highlights our gene essentiality analysis as a robust computational approach to identify drug targets.In summary, the viability of RdKW20 structure showed that amino acid substitutions were out of the substrate-binding cavity phenyl)pyridin-2-yl)piperidine-4-carboxylic acid showed increased susceptibility upon inactivation of the AcrAB-TolC efflux pump system . AcrAB-Tibiotics , this ism RdKW20  and the fabH gene allelic variation, we tested strains carrying the same variant. Variants A1 (NTHi375 as reference), A3 (RdKW20 as reference), A5 (P673 as reference), A6 (P642 as reference), and A8 (P657 as reference) were used as representative examples (To further assess if the observed heterogeneity would match only to examples . No signin vivo assays (see below), to become resistant to FabHi through serial independent passage in sBHI broth containing three different inhibitory concentrations of FabHi  medium aiming to mimic cultured epithelial cell infection conditions, showing a significant dose-dependent decrease . Based o10.1128/msystems.01459-21.10FIG S6P < 0.0001); 0 to 40%, high toxicity; 41 to 60%, intermediate toxicity; 81 to 100%, no toxicity. Download FIG S6, TIF file, 0.5 MB.FabHi toxicity analyses. (A) Cytotoxicity measured by the release of lactate dehydrogenase by A549 cells incubated for 16 h with increasing doses of FabHi in serum-starved medium. FabHi did not cause a cytotoxic effect on cells under the conditions tested (means ± SD). Statistical comparisons of the means were performed with one-way ANOVA and Dunnett’s multiple-comparison test. (B) Effect of FabHi on the mortality of zebrafish embryos exposed for 96 h postfertilization. Zebrafish embryos (20 per concentration) were distributed in 96-well microplates (one embryo/well), and exposed to FabHi at concentrations ranging from 0.5 to 12.5 μg/embryo . Mortality was assessed at the indicated time. C-, untreated negative control; C+, paracetamol-treated positive control; DMSO, vehicle solution by adding a volume equivalent to the one used with the highest drug concentration. Figure represents means ± SD from three biological triplicates  with 10 μL of bacterial suspensions containing 7 × 1010 CFU/mL or 0.9% saline solution as a control. NTHi P642 severely reduced zebrafish survival, and an infection dose consisting of 7 × 108 CFU/zebrafish caused progressive death of the animals after injection; however, strain P673 did not modify zebrafish survival (data not shown), precluding us from using this strain in this model system.Our final goal was to evaluate drug efficacy at the preclinical level, on a FabHi protective assay in adult zebrafish infected with NTHi, by following a previously established sepsis model , 58. We P < 0.0001)  test. Results showed that FabHi doses lower than 5.7  μg/embryo were nontoxic . Based o 0.0001) . This moH. influenzae as a basic computational framework to identify essential genes (among other possibilities) suitable for further exploitation as drug targets. The exponential increase of pathogen genomes and the emergence of high-throughput technologies are providing a myriad of large data sets. Among the numerous computational methods developed to generate meaningful outcomes of these data, metabolic modeling is emerging as a promising approach for the coherent organization of this information while providing mechanistic insights on biological systems  and second  substrates and the feedback inhibitor , all of which will compete with FabHi binding. Hence, the antimicrobial effect of FabHi depends on the available concentrations of all potential ligands and their respective affinities, which may vary among strains and growth conditions. Indeed, as shown above, several polymorphisms in FabH variants locate at regions relevant for activity and structural stability, and sequence changes at such positions may alter substrates and inhibitor binding as well as FabH catalytic efficiency. Additionally, strain variability associated with different CTs might modify, among others, the efflux pump system, with a subsequent impact on the amount of FabHi available to interact with FabH. Likewise, variations in expression level, conformation, or potency of other components of the FASII biosynthetic pathway, including peripheral enzymes involved in production of FabH substrates , may modNonetheless, variations observed among strains carrying the same FabH variant indicate that heterogeneity is multifactorial. Those strains are diverse in terms of genomic content, i.e., they do not cluster by CT , and sucRdKW20 complex structure indicates that FabHi affinity could be increased by introducing a hydroxyl group ortho to the N-atom of the piperidine ring, which could lead to an extra hydrogen bond with the side chain of Asn246 without introducing steric clashes. While of interest for further analysis, exploring the effect of FabHi modifications exceeds the scope of this study.On the other hand, inspection of FabHi:FabHH. influenzae and show how iNL638 can be used (i) as a powerful tool to better understand the metabolism of this pathogen, (ii) as a computational framework for large experimental data set contextualization, and (iii) for the discovery of condition-independent drug targets. We shed light on the mechanism of inhibition of FabH mediated by FabHi, and, beyond allelic variation, we suggest the strain-specific metabolic context is a key factor modulating drug performance. Therefore, the systematic metabolic evaluation of individual strains through computational frameworks will be of critical importance in the near future to design more precise and accurate treatments.Overall, we present here a high-quality metabolic model of 600) of 0.07 in a final volume of 25 mL sBHI or CDM using 250-mL flasks, grown with shaking (180 rpm), and OD600 was recorded every hour for up to 8 h. When necessary, bacterial viability upon host cell infection conditions was tested. To do so, phosphate-buffered saline (PBS)-normalized bacterial suspensions (OD600 of 1) were prepared by using NTHi grown on PVX agar; 100-μL aliquots were incubated in 1 mL Earle’s balanced salts  in the absence/presence of FabH inhibitor (FabHi) for 2 h at 37°C, serially diluted, and plated on supplemented Haemophilus Test Medium (sHTM) agar for CFU/mL determination. When needed, media supplements refer to with 10 μg/mL hemin and 10 μg/mL nicotinamide adenine dinucleotide (NAD).RdKW20 is a genome-sequenced capsule-deficient laboratory strain . NTHi37547–2O (dH2O), filtered, and used at the indicated working concentrations.FabHi was used by freshly preparing a panel of stock solutions, customized to each type of assay (7 to 35 mg/mL) in dimethyl sulfoxide (DMSO), and diluted to the working concentrations specified in each assay in CDM, sBHI, or RPMI 1640 . Antibiotics and FabHi working concentrations are specified for each assay (described below). Ampicillin and azithromycin dihydrate  were used to assess FabHi synergic effects (described below). For this purpose, 10-mg/mL stock solutions of each antibiotic were prepared in distilled H600 of 0.07 in a final volume of 55, 40, or 25 mL CDM using 250-mL flasks, grown with shaking (180 rpm) for ∼3, 4, or 6 h, respectively. Next, 45, 30, and 15 mL was collected at OD600 of 0.3, 0.4 and 0.8, respectively. Bacterial suspensions were centrifuged at , and pellets were instantly frozen in liquid N2, directly transferred to a lyophilizer , lyophilized for 24 h at 0.020 × 105 Pa and −80°C, and weighed. Using this procedure, we established a lineal relation (R2 = 0.99) between OD and DW: DW (g/liter) = (0.8627 × OD600) + 0.0279 (see Two to 3 colonies of the RdKW20 strain grown on PVX agar for 16 h were inoculated in 10 mL CDM using 100-mL flasks and incubated for 11 h with shaking (100 rpm). Cultures were diluted to OD10.1128/msystems.01459-21.3TABLE S1Table S1, DOCX file, 0.03 MB.Bacterial cultures dry weight. Download Copyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. influenzae lacks the GPR relationship, it could not be used as the template for draft construction. Instead, we used the high-quality and well-curated GEM of E. coli K-12  and peptidoglycan biosynthesis, were manually mass and charge balanced and added to the reconstruction. Furthermore, the reactions involving strain-specific cofactors were updated, such as those reactions using menaquinones or ubiquinones from the E. coli model, which were replaced by reactions accounting the cofactor usage of H. influenzae, i.e., demetylmenaquinone and demethylmenaquinol, respectively. Finally, the network gaps across the metabolic pathways were filled out by manual gap filling by adding new reactions based on the information stored in biological databases, scientific literature, and the functional annotation of strain RdKW20. For instance, metabolic gaps in fatty acid and molybdopterin biosynthesis pathways were manually completed during this step. Additionally, transport reactions were manually curated considering the connectivity of the cognate metabolites and experimental evidence. For instance, transport reactions for certain amino acids, fatty acids, uracil, pantothenate, biotin, dihydrofolate, protoporphyrin, pyridoxine, riboflavin, nucleotides, or NAD transporters were added manually based on available biochemical and physiological evidence. On the contrary, transport reactions for several compounds initially included in the automatic draft were removed due the lack of physiological evidence. The evaluation of iNL638 was done with MEMOTE v0.13.0 (https://memote.io/) (Since the available GEM of (iJO1366 ) for thiote.io/) .H. influenzae RdKW20 was used as a base for the construction of a highly complete biomass reaction  were used for solving the linear programing problems. The constraint-based model consists of a 1,161 by 1,385 matrix containing all the stoichiometric coefficients in the model of 1,161 metabolites and 1,385 reactions (S). Flux balance analysis (FBA) was used to predict growth and flux distributions , whereas mCDM formulation only allowed uptake of inorganic salts and essential vitamins for growth . Source code to simulate the iNL638 with cCDM and mCDM media and the MEMOTE full report are available at https://github.com/SBGlab/Haemophilus_influenzae_GEM.Formulation of ll cases , 27, 89.singleReactionDeletion function implemented in the COBRA Toolbox. FBA  was performed on silica gel 60 . Compounds were purified by flash column chromatography with silica gel 60 (230 to 400 mesh) (Merck). The purity of the compounds was analyzed using a high-performance liquid chromatography mass spectrometer (HPLC-MS) and was performed on an HPLC Waters 2695 instrument connected to a Waters Micromass ZQ 2000 spectrometer and a photodiode array detector. The column used was a Sunfire C18 , and the flow rate was 1 mL min−1. All retention times are quoted in minutes. Nuclear magnetic resonance (NMR) spectra were recorded with Varian XL-400 spectrometer operating at 400 MHz for 1H-NMR and at 100 MHz for 13C with Me4Si as an internal standard. Mass spectra were measured on a quadrupole mass spectrometer equipped with an electrospray source . Microanalyses were obtained on a Heraeus CHN-O-RAPID instrument. The purity of novel compounds was also determined to be >95% by elemental analysis.CH4, filtered, and evaporated to dryness. The final residue was purified by flash column chromatography  to give 1  as a colorless oily solid. 1H-NMR  δ 8.19 , 7.76 , 6.95 , 4.00 , 2.92 , 2.35 , 1.84 , 1.76 , 1.52 − 1.41 , 1.23 . HPLC 9.02 min (98%) (H2O/CH3CN from 15/85 to 95/5 in 10-min flowrate of 1 mL/min). MS (ES+) m/z 327.0 (M)+, 329.0 (M + 2)+. Anal. for C14H19BrN2O2 : C, 51.39; H, 5.85; N, 8.56. Found: C, 51.22; H, 5.91; N, 8.63.A solution of ethyl piperidine-4-acetate , 5-bromo-2-chloropyridine , and triethyl amine  in dry acetonitrile was heated under microwave irradiation to 125°C for 2 h. The crude was diluted with ethyl acetate (EtAcO) (10 mL) and washed with 1N HCl. The combined organics were dried over MgSO2CO3  in anhydrous DMF (12 mL) was stirred at 75°C for 16 h. The reaction was cooled to room temperature, and the residue was filtered through Celite and evaporated to dryness. The final residue was purified by flash column chromatography  to give 2  as a white solid. 1H-NMR  δ 8.40 , 7.71 , 7.39 , 7.25 , 7.13 , 6.73 , 4.75 , 4.28 , 4.17 , 3.12 , 2.54 , 2.00 , 1.83 , 1.27 . HPLC 5.03 min (99%) (H2O/CH3CN from 15/85 to 95/5 in 10-min flowrate of 1 mL/min). MS (ES+) m/z 373.2 (M + 1)+. Anal. for C21H25FN2O3 : C, 67.72; H, 6.77; N, 7.52. Found: C, 67.51; H, 6.80; N, 7.65.A suspension of 1 , 2-fluoro-5-(hydroxymethyl)phenylboronic acid , palladium(II) dichloride , K2O . The aqueous phase was acidified to pH 5, and the organic phase was separated. The aqueous phase extracted with EtAcO, and the organic phase combined was dried over MgSO4, filtered, and evaporated to dryness to obtain 3 . 1H-NMR  δ 8.24 , 7.65 , 7.38 , 7.22 , 6.87 , 4.47 , 4.30 , 2.80 , 2.14 , 1.90 , 1.70 , 1.12 . 13C-NMR  δ 174.1, 158.6 , 158.5, 147.7 , 139.7 , 138. 1 , 128.3 , 127.2 , 125.6 , 119.8, 116.2 , 107.1, 62.7, 60.3, 45.2, 41.2, 33.3, 31.5. HPLC 1.35 min (96%) (H2O/CH3CN from 15/85 to 95/5 in 10-min flowrate of 1 mL/min). MS (ES+) m/z 345.4 (M + 1)+. Anal. for C19H21FN2O3 : C, 66.27; H, 6.15; N, 8.13. Found: C, 66.31; H, 6.05; N, 8.19.A solution of 2  in THF-methanol (MeOH)  and 1 M NaOH (1.5 mL) was refluxed for 4 h. The mixture was evaporated to dryness, and the residue was dissolved in a mixture of EtAcO-HRdKW20; Protein Data Bank entry 3IL3) as receptor and FabHi as ligand. In detail, the protein was considered rigid and the ligand flexible. FabHi structure was carefully built using the PyMOL visualizer  from the atomic coordinates of ligand cocrystallized with E. coli FabH in PDB entry 5BNR  and diluted 1:100. Next, 50-μL bacterial aliquots were transferred to each well. A vehicle solution control consisting of a volume of DMSO equivalent to that of the highest FabHi concentration tested was performed in parallel (indicated as 0 [FabHi] μg/mL). Blank controls (sBHI or CDM added) were used. Plates were incubated for 24 h at 37°C, 5% CO2, without shaking. Culture samples were serially diluted and plated on sHTM agar. Data are shown as log CFU/mL; experiments were performed in duplicate at least three times (n ≥ 6). Alternatively, FabHi antimicrobial effect was assessed on H. influenzae biofilm grown in sBHI. For this purpose, assays were set as indicated above, and bacterial growth was determined by measuring OD600 on a SpectraMax 340 microplate reader. The liquid portion in each well (containing planktonic bacteria) was then discarded, and plates were washed 3 times through gentle submersion in distilled water and allowed to air dry. Next, 150 μL/well 0.5% crystal violet dissolved in dH2O was added, and plates were incubated for 20 min with gentle shaking at room temperature, followed by plate washing as previously described. Finally, 150 μL/well 95% ethanol (Merck) was added, plates were incubated for 20 min with gentle shaking at room temperature, and the OD570 was determined as a measure of biofilm growth. The OD570/OD600 ratio for each strain and independent assay was calculated to normalize biofilm biomass to overall growth; experiments were performed in triplicate at least three times (n ≥ 9).A 25-mg/mL FabHi stock solution in DMSO was used to prepare 40-, 200-, 300-, 400-, 500-, 600-, 800-, 1,000-, 1,100-, 1,200-, 1,600-, 2,000-, and 2,400-μg/mL working solutions in CDM or in sBHI. Fifty-microliter aliquots of sBHI or CDM were transferred to individual wells. Next, 50 μL of each FabHi working solution was added and mixed and 50 μL was discarded. A suspension of PVX agar with freshly grown bacteria was generated in sBHI or CDM, adjusted to 0.5 MacFarland  index of each FabHi-Amp or FabHi-Azm combination was calculated to determine the existence of synergy (ΣFIC ≤ 0.5), additive (0.5 > ΣFIC ≤ 1), indifferent (1 > ΣFIC < 4), or antagonic (ΣFIC ≥ 4) effects. Three independent assays were performed (n = 3).A 7-mg/mL FabHi stock solution was used to prepare 300-μg/mL working solutions in sBHI; 10-mg/mL Amp or Azm stock solutions were used to prepare 4-μg/mL and 2-μg/mL working solutions in sBHI, respectively. After generating each FabHi-Amp or FabHi-Azm concentration matrix, each well of a 96-well microtiter plate contained 80 μL final volume. A suspension of PVX agar and freshly grown bacteria was generated in sBHI, adjusted to OD2, without shaking. Cultures were then passaged (50 μL in 50 μL fresh sBHI with FabHi or vehicle solution) every day for 12 days. At each time point throughout the cycling, each well’s absorbance (OD600 at 24 h minus OD600 at 0 h) was measured. Nine replicates per condition were made (n = 9).A 14-mg/mL FabHi stock solution was used to prepare 550-, 1,100-, and 2,200-μg/mL working solutions in sBHI. A vehicle solution control (indicated as V) consisting of a volume of DMSO equivalent to that of the highest FabHi concentration tested was performed in parallel. Blank controls (sBHI) were used; 50 μL sBHI was transferred to individual wells and mixed with 50 μL of each FabHi working or vehicle control solution, and 50 μL was discarded. A suspension of PVX agar and freshly grown bacteria was generated in sBHI, adjusted to 0.5 MacFarland, and diluted 1:100. Next, 50-μL bacterial aliquots were added to individual wells. Plates were incubated for 24 h at 37°C, 5% CO600 of 0.07, and grown to half of each strain’s maximal OD600 for ∼5 h. Bacterial total RNA was isolated using TRIzol reagent (Invitrogen) and total RNA quality evaluated using RNA 6000 Nano LabChips . Reverse transcription (RT) was performed using 1 μg RNA by PrimerScript RT reagent kit (TaKaRa). To amplify fabH, 1:10 diluted cDNA was used as the template (including gyrA endogenous control). In all cases, 20-μL reaction mixtures containing 1× SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) and the adequate primer mix were used. Fluorescence was analyzed with an AriaMx real-time PCR system (Agilent Technologies). The comparative threshold cycle (TC) method was used to obtain relative quantities of mRNA that were normalized using gyrA as an endogenous control. The fabH gene was amplified with primers fabH-F and fabH-R in all cases except for the P851 and P642 strains, where fabH-P851-P642-F and fabH-P851-P642-R primers were used (n = 3).Two to three colonies of bacterial strains grown on PVX agar were inoculated in 10 mL CDM or sBHI, grown for 11 h, diluted into 25 mL fresh CDM or sBHI to ODere used . Samples10.1128/msystems.01459-21.4TABLE S2Table S2, DOCX file, 0.06 MB.Primers used in this study. Download Copyright © 2022 López-López et al.2022López-López et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 cells/well for 32 h. To assess FabHi cytotoxicity, a 35-mg/mL FabHi stock solution was diluted in serum-starved RPMI 1640 (Sigma-Aldrich) to 125, 275, and 500 μg/mL (1 mL/well), cells were incubated for 16 h at 37°C, 5% CO2, and cytotoxicity was determined by measuring the release of lactate dehydrogenase  and microscopy. To assess the effect of FabHi during epithelial infection by NTHi, cells were maintained and seeded as indicated above and treated with FabHi in serum-starved RPMI 1640 for 16 h before infection. For infection, PBS-normalized bacterial suspensions (OD600 of 1) were prepared by using NTHi grown on PVX agar. Adhesion and invasion assays were performed as previously described (n = 9).A549 cells were maintained as described previously  and seedescribed , 48. Conhttps://www.ikanbiotech.com) animal housing facility were performed as previously described (n = 10/group). Infected groups were intraperitoneally injected with 10 μL of an exponentially grown (OD600 of 0.3) H. influenzae suspension containing 7 × 1010 CFU/mL (7 × 108 CFU/zebrafish) prepared in PBS. At 1 hpi, one infected and one uninfected group were intraperitoneally administered FabHi at a dose of 4 μg/zebrafish in 10 μL PBS by using a 35 mg/mL FabHi stock solution; the other two groups were administered PBS or vehicle solution. The survival rate for each group was monitored three times per day for 4 days after infection. Experiments were performed on three independent occasions.Animal experiments conducted at the Ikan Biotech , and are detailed in each figure legend.In all cases, a


Despite ceasing intravenous glucose infusion on the protein night, the BGLs peaked at 9.6 ± 1.6 mmol/L, with a hypoglycemia risk period mean of 7.8 ± 1.5 mmol/L compared to 5.9 ± 0.4 mmol/L (p = 0.028) on the water night. The mean plasma glucagon levels were 51.5 ± 14.1 and 27.2 ± 10.1 ng/L (p = 0.028) on the protein and water night, respectively. This suggests that an intake of protein is effective at reducing the post-exercise hypoglycemia risk, potentially via a glucagon-mediated stimulation of glucose production. However, 50 g of protein may be excessive for maintaining euglycemia.Dietary protein causes dose-dependent hyperglycemia in individuals with type 1 diabetes (T1D). This study investigated the effect of consuming 50 g of protein on overnight blood glucose levels (BGLs) following late-afternoon moderate-intensity exercise. Six participants (3M:3F) with T1D, HbA1c 7.5 ± 0.8% (58.0 ± 8.7 mmol/mol) and aged 20.2 ± 3.1 years exercised for 45 min at 1600 h and consumed a protein drink or water alone at 2000 h, on two separate days. A basal insulin euglycemic clamp was employed to measure the mean glucose infusion rates (m-GIR) required to maintain euglycemia on both nights. The m-GIR on the protein and water nights during the hypoglycemia risk period and overnight were 0.27 ± 043 vs. 1.60 ± 0.66 mg/kg/min ( The benefits of exercise in healthy individuals and individuals living with type 1 diabetes (T1D) are well known. However, the fear of hypoglycemia continues to be a barrier in the uptake of exercise by individuals living with T1D ,3,4. IndConversely, protein ingested alone or as part of a meal causes a late postprandial rise in blood glucose levels that require additional insulin to maintain euglycemia in individuals living with T1D ,8,9,10. There is also evidence that the ingestion of protein after exercise helps optimize muscle protein remodeling and repair  and thatThus, based on Patterson’s and McMahon’s work ,11, we cThe aim of this pilot study was to compare the difference in the mean intravenous glucose infusion rate to maintain overnight euglycemia following a bout of late afternoon MOD, with and without the consumption of 50 g of protein post-exercise, using a basal insulin glucose clamp protocol ,15. We hThis pilot study was approved by the Child and Adolescent Health Service Human Research Ethics Committee, Western Australia. Participants and/or their parents provided informed consent to participate in the study.Six participants with confirmed T1D duration > 1 year, who were hypoglycemia aware and otherwise healthy, participated in this study. Five participants were c-peptide negative (<0.05 mmol/L) pre-lunch, and one had a c-peptide level of 0.16 mmol/L. This in-laboratory pilot study assessed the effect of consuming 50 g of protein in water vs. water alone on overnight glycemia and especially during the expected LOPEH risk period, following late-afternoon MOD, using a randomized counter-balanced study design. On two separate testing days, following a familiarization visit, participants performed MOD in the late afternoon followed by the intake of water only or a 50 g protein drink 3.25 h after exercise. The participants thus acted as their own controls, with female participants performing both of their exercise sessions in the same phase of their menstrual cycle. An established glucose clamp protocol, described in more details in earlier studies from our laboratory ,15, was 2 peak test, lactate threshold assessment, and the collection of baseline demographic and clinical measurements. On the two subsequent exercise testing days, one month apart, participants attended the laboratory at 1000 h. Hypoglycemia, exercise, alcohol, and caffeine were avoided in the 24 h preceding these testing days ) during the LOPEH risk period were 0.27 ± 0.43 mg/kg/min  and 1.60 ± 0.66 mg/kg/min  on the Pr-night and Wa-night, respectively . The overnight m-GIR were 0.51 ± 0.16 mg/kg/min  on the Pr-night and 1.34 ± 0.71 mg/kg/min  on the Wa-night,  (The pre-drink m-GIR ( = 0.63) A,B. p = 0.028). The overnight mean BGLs were 7.2 ± 1.0 mmol/L and 6.0 ± 0.4 mmol/L (p = 0.028) on the Pr-night and Wa-night, respectively. The BGLs on the Pr-night peaked at 9.6 ± 1.6 mmol/L (range 7.7–11.9 mmol/L) despite ceasing the intravenous glucose on the Pr-night for 380 ± 112 min. The mean BGLs during the LOPEH risk period on the Pr-night and Wa-night were 7.8 ± 1.5 mmol/L and 5.9 ± 0.4 mmol/L (p = 0.028) on the Pr-night than the Wa-night, with plasma glucagon levels of 51.5 ± 14.1 ng/L vs. 27.3 ± 10.1 ng/L, GIP levels of 26.8 ± 7.2 pmol/L vs. 13.9 ± 5.1 pmol/L, and GLP-1 levels of 18.8 ± 2.2 pmol/L vs. 10.2 ± 4.7 pmol/L. On the Pr-night, the glucagon levels peaked at 82.6 ± 26.9 ng/L at 65 ± 31 min and returned to the pre-drink levels within six hours. This peak in the glucagon response preceded the peak in BGLs on the protein night (The post-drink hormone responses were higher (in night C–F. Despite the many published recommendations available to people living with T1D for the prevention of post-exercise hypoglycemia , there iThis pilot study, involving adolescents and youths with a mean duration of diabetes of 11.1 ± 4.9 years, shows that the ingestion of 50 g of whey protein markedly reduced the m-GIR needed to prevent LOPEH. We found that when protein was ingested approximately three hours after 45 min of MOD, the m-GIR on the protein night was six-fold lower during the 4 h LOPEH risk period [We also found that the ingestion of 50 g of protein resulted in transient hyperglycemia overnight. This hyperglycemia occurred despite the complete cessation of intravenous glucose whilst trying to maintain euglycemia on the protein-intake night. In contrast, a continuous infusion of intravenous glucose was invariably required to maintain euglycemia when water alone was ingested. This observed protein-mediated increase in the BGL is consistent with previous studies reporting that the ingestion of protein in non-exercise and basal insulin conditions resulted in a sustained and dose-dependent elevation of the blood glucose levels in individuals with T1D ,11. This increase in glycemia following a protein ingestion is likely to result from the prompt and major, albeit transient, rise in plasma glucagon , as incrA strength of this pilot study is the use of the glucose clamp protocol to quantify the m-GIR required to maintain euglycemia under individualized near-basal insulin levels, a condition that replicates real life scenarios and has been used effectively in previous studies investigating LOPEH ,15. AddiHowever, having demonstrated in this pilot basal insulin glucose clamp study that the ingestion of 50 g of whey protein in the evening is more than sufficient to maintain overnight euglycemia after a bout of late afternoon MOD, further studies can now investigate the effect of smaller doses of protein which could also be individualized to the weight of the individual in free-living conditions. There is also a substantial variation in the amino acid profile of different dietary protein sources . As suchConsuming protein in the evening, after late-afternoon moderate intensity exercise, may provide a novel and effective strategy to prevent late-onset post-exercise hypoglycemia. Real life studies are now required to define the overnight blood glucose responses to different doses and types of protein ingested following late-afternoon exercise.


We found that α- and β-santalol synergize to reduce wild-type (WT) and Val30Met (V30M) mutant TTR aggregates in novel C. elegans strains expressing TTR fragments fused with a green fluorescent protein in body wall muscle cells. α- and β-Santalol extend the lifespan and healthspan of C. elegans strains carrying TTRWT::EGFP and TTRV30M::EGFP transgene by activating the SKN-1/Nrf2, autophagy, and proteasome. Moreover, α- and β-santalol directly interacted with TTR and reduced the flexibility of the thyroxine-binding cavity and homotetramer interface, which in turn increases stability and prevents the dissociation of the TTR tetramer. These data indicate that α- and β-santalol are the strong natural therapeutic intervention against TTR-associated amyloid diseases.Transthyretin (TTR) is a homotetrameric protein found in human serum and is implicated in fatal inherited amyloidoses. Destabilization of native TTR confirmation resulting from mutation, environmental changes, and aging causes polymerization and amyloid fibril formation. Although several small molecules have been reported to stabilize the native state and inhibit TTR aggregation, prolonged use can cause serious side effects. Therefore, pharmacologically enhancing the degradation of TTR aggregates and kinetically stabilizing the native tetrameric structure with bioactive molecule(s) could be a viable therapeutic strategy to hinder the advancement of TTR amyloidoses. In this context, here we demonstrated α- and β-santalol, natural sesquiterpenes from sandalwood, as a potent TTR aggregation inhibitor and native state stabilizer using combined  TTR) (4). TTR transports T4 and the retinol-RBP complex to the brain and various body parts  amyloidosis with polyneuropathy, commonly known as transthyretin-type familial amyloid polyneuropathy (FAP), is a sparse condition mostly found in Japan, France, Portugal, Sweden, Spain, and descendants of these regions . FAP is TTR) . FAP is TTR) . Over 14TTR) . TTR is dy parts . Two funogenesis . Therefo4-binding sites within TTR and kinetically stabilize the native quaternary structure, which limits the tetramer destabilization and aggregate formation (V30M) amyloidoses and diseases associated with other TTR variants  oil as described (3), and dimethyl sulfoxide (DMSO) were purchased from HiMedia Laboratories Pvt. Ltd. . Tolcapone, chloroquine, Epigallocatechin gallate (EGCG), isopropyl β-D-1-thiogalactopyranoside (IPTG), 5-fluoro-2′-deoxyuridine (FUdR), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), prealbumin from human plasma, and MG-132 were obtained from Sigma Aldrich .α- and β-Santalol were purified from sandalwood (escribed . NematodC. elegans were cultured on standard nematode growth media (NGM) agar plates carrying E. coli OP50 and maintained at 20°C using the standard techniques . The novel strains expressing several type of TTR fragments fused with GFP in body wall muscle cells viz., zyeEx1069[pCKX3307(unc-54p::nsTTRWT::EGFP)], zyeEx1070[pCKX3311(unc-54p::nsTTR1-80::EGFP)], zyeEx1073[pCKX3303(unc-54p::nsTTR81-127::EGFP)], zyeEX1076[pCKX3316(unc-54p::nsEGFP)], zyeEx1078[pCKX3309(unc-54p::nsTTRV30M::EGFP)], and zyeEx1082[pCKX3313(unc-54p::nsTTR49-127::EGFP)] were gifted by Professor Kunitoshi Yamanaka   . Synchro, Japan) .Lifespan was measured at 20°C as previously described . In brieTo measure the body bends, control and santalol isomers treated worms were gently washed off from the plates and released on the agar plates to crawl for 5 min at 20°C. Individual worms (40 worms per experiment) were transferred to 24-well plates containing 1 ml M9 buffer. After 1 min recovery time, the number of body bends, the reciprocating motion of bends at the mid-body, were scored for 30 s. Mechanosensory neuronal aberrations were measured in the soft-touch sensitivity assay . On day 3 at a final concentration of 25 mM. Fluorescence images were obtained, and the visible TTR::EGFP aggregates were counted. The TTR::EGFP aggregates are spherical-shaped discrete structures, and their boundaries are clearly differentiated from nearby fluorescent signals  were grown on NGM plates with and without santalol isomers. On day 2 and day 6 of adulthood, the worms were collected, washed with M9 buffer, and mounted on glass slides for fluorescent microscopy . Worms were anesthetized using NaN signals . At leasC. elegans were measured using the molecular probe H2DCF-DA. The worms were maintained and treated as described in the lifespan assay. After treatment, the worms were washed off from the plates with PBST buffer (PBS + 1% Tween 20) and pelleted by centrifugation. The worms were quickly frozen in liquid nitrogen and immediately sonicated for 5 min to disrupt the outer cuticle. The homogenized samples were then transferred to 96-well plates and incubated with 50 μM H2DCF-DA solution for 30 min in the dark at 20°C. Fluorescence intensity was measured using a microplate reader at an excitation/emission wavelength of 490/530 nm.Intracellular ROS levels in E. coli strain HT115(DE3) transformed with either the RNAi-encoding plasmid (test) or the empty pL4440 vector (control) were grown in LB media containing 100 μg ml−1 ampicillin for 12 h at 37°C. Bacteria were concentrated fivefold and seeded onto NGM plates containing 100 μg ml−1 ampicillin and 1 mM IPTG, hood-dried and cultured for 12 h to induce the expression of dsRNA. Synchronized L1 stage worms were transferred to RNAi plates and raised until the young adult stage for efficient knockdowns.The regular feeding RNAi method was performed to inactivate specified genes, as previously described . Individt (ΔΔCt) method. The housekeeping gene act-1 was chosen as the reference gene.The worms were frozen in liquid nitrogen, thawed on ice, and homogenized at 4°C. Total RNA was extracted using TRIzol reagents (Invitrogen)  and was WT (PDB ID: 4D7B) and TTRV30M (PDB ID: 4TL4)  to identify the binding affinity of ligands α- and β-santalol towards the TTRD: 4TL4) . The molp < 0.05 was considered to be statistically significant between the means.The lifespan of worms was graphed as Kaplan-Meier survival curves with MedCalc statistical tool  and analyzed using the log-rank (Mantel-Cox) test. The differences between control and santalol isomers-treated groups were statistically compared by one-way analysis of variance  followed by Bonferroni’s post hoc test  and presented as mean ± standard deviation (SD)/standard error of the mean (SEM) of at least three separate experiments, unless otherwise noted. The probability level of in vitro conditions at low pH (4.2). This acidic pH induces TTR fibril formation, thus increasing sample turbidity and light scattering. The results showed that α- and β-santalol were effective at inhibiting both TTRWT  and TTRV30M aggregation  under acidic denaturation conditions   and 75.9% (TTRV30M) in the absence of santalol isomers or tolcapone. In the positive control , however, more than 90% of TTR remains in the native state  . Under sectively , as measociation . Howeverociation . Unfortuociation . With threscence . The resve state .in vitro results, we, therefore, performed molecular dynamics simulations to identify the possible interaction of santalol isomers with T4 binding sites. The crystal structures of tolcapone bound to TTR variants (TTRWT and V122I) were solved previously , SER117 (2.49 Å), and THR118 (3.31 Å) were involved in hydrogen bonding, whereas residues ALA108 (3.80 Å) and LEU110 (3.84 Å) were hydrophobic contacts. β-Santalol showed a binding affinity of −5.5 kcal/mol towards the TTRWT and formed five hydrogen bonds with residues ALA108 (2.36 Å), LEU110 (2.85 Å), SER117 (2.48 Å), THR118 (3.29 Å), whereas the residues THR119 (3.34 Å), and LYS15 (3.53 Å), LEU17 (3.71 Å), ALA108 (3.60 Å), and LEU110 (3.42 Å) were hydrophobic contacts. The binding affinity of α- and β-santalol towards the TTRV30M binding site were −5.4 and −6.1 kcal/mol, respectively. α-Santalol interacts with the residue THR119 (2.71 Å) through hydrogen bonding and has strong hydrophobic interactions with residues VAL93 (3.67 Å), PHE95 (3.58 Å), PHE95 (3.70 Å), THR118 (3.74 Å), ALA120 (3.62 Å), and VAL122 (3.48 Å). β-Santalol forms two hydrogen bonds with residue THR96 , and residues PHE95 (3.40 Å), THR105 (3.59 Å), THR118 (3.65 Å), and ALA120 (3.31 Å) were hydrophobic contacts. Hydrogen bonding and hydrophobic contacts play a crucial role in forming and stabilizing energetically-favored protein-ligand complexes  of the complexes made the most contribution to the total binding free energy; a similar result was previously observed  in body wall muscle cells viz., TTRWT::EGFP (the full-length wild-type TTR), TTRV30M::EGFP (the full-length TTR but containing V30M mutation), TTR1–80::EGFP (the 1–80 residue fragment), TTR81–127::EGFP (the 81–127 resides fragment), TTR49–127::EGFP (the 49–127 residue fragment), and EGFP alone (as control) were used , which is approved in Europe and the United States for the treatment of Parkinson’s disease. It has been shown to prevent the onset of TTR misfolding and TTR-induced toxicity by specifically binding and stabilizing the native tetrameric structure of TTR more effectively than clinically approved drugs for early-stage FAP, diflunisal and tafamidis (WT::EGFP and TTRV30M::EGFP across various adulthood stages (day 2 and day 6). However, α- and β-santalol treatment exhibited a negligible effect on the aggregation of TTR1–80::EGFP, TTR81–127::EGFP, and TTR49–127::EGFP when compared to that of untreated control. It was interesting to note that the TTR inhibitory potential of santalol isomers was found to be gradually increase as the worms aged. In earlier developmental stage (day 2 adulthood), α- and β-santalol treatment marginally decreased TTRWT [21.6% (p = 0.44) and 19.8% (p = 0.59)] and TTRV30M [16.8% (p = 0.84) and 29.1% (p = 0.0.06)] aggregates (WT [33.3% (p < 0.01) and 40.2% (p < 0.01)] and TTRV30M  aggregation in day 6 adulthood worms. The effect of santalol isomers in preventing TTR aggregation was on par with the positive control tolcapone. Tolcapone treatment inhibits the aggregate formation in TTRWT::EGFP , TTRV30M::EGFP , and TTR81–127::EGFP  worms; however, it did not significantly affect TTR1–80::EGFP, TTR49–127::EGFP aggregates, but tolcapone treatment slightly reduce TTR formation (p > 0.05). We also explored the efficacy of combining α- and β-santalol on the aggregation of TTR in C. elegans. We systematically evaluated 12 possible combinations that can be formed out of four doses of α-santalol  and three doses of β-santalol . It was observed that α-santalol combined with β-santalol, each at its half-optimal dose , lead to a significant reduction in the TTRWT::EGFP  and TTRV30M::EGFP  aggregates compared to the groups treated with single molecule and the control group. A few of the tested combinations also produced a noticeable impact on TTR aggregation, while their effect was not found to be synergistic than the half-optimal dose of santalol isomers, and some combinations showed no benefits  and TTR1–80  did not obviously alter compared to their wild-type counterparts (16.1 ± 0.5 days). However, worms expressing TTRWT , TTRV30M , TTR81–127 , and TTR49–127  fragments had a significantly shorter lifespan  and 20.8% (p < 0.0001), respectively, compared to the control groups (WT (V30M (p < 0.0001) and 27.8% (p < 0.0001), respectively. However, α- and β-santalol treatment would not further extend the lifespan of worms expressing TTR1–80    . Firstly, we observed that 16 µM α-santalol and 8 µM β-santalol together were the minimal dose to synergistically extend the lifespan of wild-type C. elegans by 28.0% under standard laboratory conditions (p < 0.01) than the largest effect produced by each compound under similar conditions , TTR81–127 (p = 0.2477), and TTR49–127 (p = 0.2497) worms  , TTR81–1 0.5074) , and TTR 0.6365) . We nextnditions . The comnditions . Interesnditions . Secondl7) worms . In contp < 0.0001) (p = 0.5247) and 5.5% (p = 0.0310), respectively. Furthermore, worms cultured on 8 and 10 µM tolcapone displayed a significantly different right-shifted survival curve when compared with unexposed control, and the mean lifespan was increased by 13.8 and 7.8%, respectively. Therefore, 6 µM tolcapone was selected as the optimal concentration for most subsequent studies. On the other hand, tolcapone feeding increased the mean lifespan of TTRWT (V30M (81–127 (p < 0.0001), 23.0% (p < 0.0001), and 41.7% (p < 0.0001) respectively, and it would not further extend the lifespan of TTR1–80  and TTR49–127  worms. Together these data imply that tolcapone can extend the lifespan of wild-type worms and worms expressing TTR at a relatively low dose; this may reduce or minimize the potential side effects  with a maximum lifespan of 30 days and increased by 21.2% in mean lifespan ( 0.0001) . Worms tof TTRWT , TTRV30MWT   and TTRV30M (p < 0.01) worms (WT::EGFP and TTRV30M::EGFP worms depleted of lgg-1 (lgg-1 RNAi) and bec-1(ok700) (mutant). lgg-1 and bec-1 are mammalian homologs of MAP-LC3 and Beclin1, respectively, and are required for the breakdown of cellular components by autophagy. It was observed that animals silenced with lgg-1 RNAi were unresponsive to α-+β-santalol treatment, since the number of TTR aggregates in TTRWT (p = 0.82) and TTRV30M (p = 0.27) worms were statistically similar to unexposed groups that received control RNAi or solvent  (p < 0.0001) (bec-1 gene (p = 0.3404), identifying the underlying role of bec-1 in santalol isomers-mediated TTR inhibition  (We next investigated the mechanism(s) by which the combination of α-+β-santalol extends lifespan and inhibits TTR aggregation in e (H2O2) , neurotoe (H2O2) , and proe (H2O2)  stressesinocytes . Autophainocytes . Recent inocytes . In C. er levels . Extracer levels . The pro1) worms , suggest solvent . lgg-1 R 0.0001)  and V30M 0.0001)  TTR. Furhibition . To furthibition . The resctively) . These rC. elegans. In addition, HLH-30 regulates the expression of several genes involved in activating autophagy and lysosomal functions (hlh-30(tm 1978) loss-of-function allele by 11.4% (p < 0.0001) , and hsp-4 (ER chaperon)  in TTRWT and TTRV30M worms treated with santalol isomers. After treatment with α-+β-santalol, the transcript levels of pas-3 and rpn-5 were significantly upregulated (p < 0.05) (wormbase.org). RPN-5  is a conserved proteasome subunit required for proper proteasome localization and assembly , was shown to regulate longevity, stress resilience, and protein aggregation in unctions . α-+β-Sa 0.0001) , but thenificant . These rhaperon) . We also < 0.05) . PAS-3   compared to age-matched control worms, indicates the healthy status of sensory neurons. Consistent with the previous observation, however, the expression of TTRWT and TTRV30M significantly reduced memory and learning ability in C. elegans (p < 0.01) than non-TTR control and wild-type animals, suggesting the neurotoxic property of TTR aggregates  . Next, w elegans . In FAP  elegans . Similar elegans . TTRWT aemotaxis  and foodemotaxis  during tgregates . To testwith age  and TTR with age . We obse in life . To furtC. elegans was established , and the overproduction of MDA indicates oxidative damages  in the tissues of patients with TTR deposits and ablished . Santaloablished . Therefol values . We alsol values . Day 5 a damages . These rin vitro. We found that the combination of santalol isomers  acts synergistically to prevent TTR-induced toxicity, lifespan reduction, and other functional deficits in C. elegans. Moreover, santalol isomers interact with TTR variants (TTRWT and TTRV30M) and reduce the flexibility of the T4 binding cavity and the homotetramer interface, which in turn confers the conformational stability of TTR and prevents the dissociation of the tetramer and subsequent self-assembly into toxic aggregates. Santalol isomers inhibit TTR aggregation and help maintain proteostasis in C. elegans by activating the SKN-1/Nrf2 protective pathway, autophagy, and proteasome. SKN-1/Nrf2, autophagy, and proteasome are highly conserved between C. elegans and mammals, and hence it is not unfair to hypothesize that santalol isomers may also be protective against TTR-induced toxicity in patients with FAP, FAC, and/or SSA. Therefore, preclinical studies on mammalian models of TTR amyloidosis using a combination of santalol isomers are justified.In summary, we have shown that santalol isomers are potent inhibitors of TTR amyloidogenesis. Santalol isomers effectively inhibit fibril formation and stabilize the native tetrameric form of TTR under denaturation conditions


Lysinibacillus sp. BP1 and Lysinibacillus sp. BP2, respectively, within 72 h, under conditions of optimal inoculum size and/or Cu concentration. Apart from a higher estrogen removal rate compared to free laccased, the biocatalysts were more resistant to temperature, pH and other environmental perturbations, and had enhanced storage ability and reusability. In comparison to clay, beads had a higher potential for recyclability and were more stable under certain experimental factors such as pH, reuse, and temperature, as well as storage conditions. Immobilized enzymes were able to remove 100% of E2, as well as over 90% of E1 and EE2, in 24 h, indicating that they could be scaled up to benchtop bioreactor levels.Against the backdrop of towering ecological health implications of estrogen pollution and the inefficacies associated with cost-intensive treatment techniques, this study recorded the earliest attempt of developing an inexpensive bacterial laccase-based biocatalysts for biodegradation of EDCs (Endocrine disrupting compounds), particularly estrogens. First, a central composite design was used to investigate the interactive effects of pH (6.0–8.0), inoculum size (100–500 U/mL), and copper (Cu) (25–75 mg/L) on laccase activity and estrogen degradation respectively. Thereafter, biocatalysts was synthesized comprising laccase and glass beads or silver impregnated clay granules (SICG), which was further used to treat estrogen infused aquatic matrices under different reaction conditions. Maximum laccase activities and estrogen removal for the two tested laccases were 620 U/mL (85.8–92.9%) and 689.8 U/mL (86.8–94.6%) for  For the elimination of EDCs, conventional methods such as adsorption, electrochemical oxidation, improved chemical oxidation, membrane filtration, and photocatalysis have been developed. However, subsequent research have revealed that these approaches have numerous downsides, including a significant environmental effect, operational difficulties, and costs . Although biological treatment methods are hypothetically considered the best option due to their cost and environmental friendliness, they are significantly overridden by expensive physico-chemical methods for various real-life environmental applications, including the removal of Endocrine disrupting compounds (EDCs) from contaminated sites . Neverthvia simultaneous adsorption and biocatalytic conversion , followed by moulding into microscopic granules. The mixture was fired at 887 °C for 8–9 h, then incubated at 90 °C for 48 h to allow the granules to dry out. Finally, the dried clay granules were rinsed with synthetic water containing 0.9 percent w/v salt water and re-dried at room temperature.All reagents, cartridges, and filter papers were purchased from Sigma-Aldrich , as were the compound standards (>98%) for E1, E2, and EE2. Bisphenol A-d16 was used as an internal standard, as well as HPLC grade methanol, ethanol, and acetone, formic acid, and hydrochloric acid (99% purity). Sigma-Aldrich also provided ABTS , Tri-chloro acetic acid (TCA), and glass beads. SICG granules were synthesized at CSIR SA  as described by Lysinibacillus sp. BP1 and Lysinibacillus sp. BP2, respectively. Measurement of laccase activity was according to an previous study . The choice isolates, based on the aforementioned processes, were identified as us study . BrieflyBased on optimal parameters observed from a one-variable-at-a-time (OVAT) approach in a previous study , estrogen degradation was further optimized by computing the interactive effects of estrogen concentration (100–500 ng/L), pH (6.0–8.0), inoculum (100–500 µL) and copper (25–75 mg/L) in a central composite design (CCD), where responses measured were laccase activity (R1), E1 degradation (R2), E2 degradation (R3) and EE2 degradation (R4). Design expert software v13 trial version  was used to evaluate the design in terms of single, interaction, and quadratic effects. Each factor was varied at three levels: 1, 0, and +1, corresponding to low, medium, and high values. The design consisted of 30 duplicated runs to optimize the levels of the selected variables. The experiment was run for 72 h; thereafter, aliquots of pure estrogens and their degraded residual concentrations were collected and analysed using LCMS-MS.inf is the initial concentration and Ceff is the final concentration .The degradation efficiencies of the laccases were calculated using the equation presented below.3 at 60 C for 4 h before being thoroughly washed with water and dried at 60°C. A 10 ml solution of glutaraldehyde (10%) was added to the prepared clay and glass beads and incubated at room temperature for about 5 h. The particles were washed with deionized water to remove unbound laccases and excess glutaraldehyde and thereafter airdried. Immobilization yield was calculated by dividing the laccase activity measured in the biocatalysts by the total activity present in the liquid solution at the start of the immobilization process. This procedure resulted in yields of over 76 and 84% for glass beads and SICG, respectively.SICG was crushed with a crusher and pestle before to immobilization to obtain smaller particles. Glass beads ranging between 0.4–0.6 mm in diameter  were pre-treated with 1.2 M HNOThe storage stability of free and immobilized laccases was investigated by measuring enzyme activity during storage at 4 or 25°C for 30 cycles. The residual enzyme activities of free and immobilized laccases were measured after only 60 min of incubation with 0.1 M phosphate buffer (pH 6) at temperatures ranging from 10 to 100°C. Laccase activity was measured at room temperature after incubation, as previously described. The effect of pH on enzyme activity was studied at 25°C in the pH range of 2–10.Degradation ability of the designed biocatalysts was evaluated using a laboratory scale continuous fed-batch reactor (250 ml filtration collection flask). About 200 mg of both biocatalysts corresponding to 500 U/mL were added to the reactors separately. Thereafter, reactors were separately fed with a 100 ml solution containing mixture of estrogens (500 ng/L) in deionized water under optimised conditions. The aliquots were withdrawn using a 1,000 ml micropipette at 24 h intervals to evaluate enzymatic activities and estrogen degradation. An immobilized enzyme and a number of beads corresponding to the appropriate enzymatic activity were used to breakdown the EDCs combination. Repeated measurements (30 cycles) of the activities of designed biocatalysts at room temperature were used to assess the recyclability of the resulting biocatalysts. The reactors were emptied using a filtration vacuum pump after each cycle and washed three times  with deionized water before the next feed. The reactors were placed in a shaking incubated at an agitation speed of 100 rpm to create perturbation. The removal efficiencies of developed biocatalysts were calculated using the aforementioned equation presented below.Statistical analyses of data synthesized in the current study were performed using Design expert software v13 trial version  and microsoft Excel 2010.Lysinibacillus. BP1 elicited maximum enzymatic activity (689.8 U/mL) and degradation (86.8–94.6%) at pH 6.0, 100 ng/L estrogen concentration, 500 µL and Cu concentration of 75 mg/L.  and degradation (85.7–92.9%) under the central values of all the factors evaluated  for the quadratic model showed F-value of 131.88 in the case of enzymatic activity response. The significance of the model was validated by the p values less than of 0.0500 which were noted to be less than 0.05 in all the cases as shown in 2, B2, D2, BCD, A2B, A2D, AB2 are significant model terms. Based on the statistical significance, the quadratic equations can be written as:In this case A, B, C, D, BC, BD, CD, A. BP1 laccase activity (U/mL) = 370.896 + -0.177778 * A + -87.1 *B + 177.85 *C + 32.85 *D + -11.0062 * BC + 11.3688 * BD + -8.88125 * CD + -4.80789 * A2 + -32.6579 *B2 + 18.8921 *C2 + 12.1921 *D2 + 7.75625 * BCD + 30.7563 * A2B + -5.16875 * A2C + -20.1938 * A2DLysinibacillusLysinibacillus. BP2 laccase activity (U/mL) = 377.418 + -62.25 * A + -65.7 *B + 153.55 *C + 198.9 *D + -58.2625 * BC + 33.7125 * BD + 37.9375 * CD + -25.2045 * A2 + -27.2545 *B2 + 51.5455 *D2 + -11.025 * BCD + 76.4 * A2B + -28.6 * A2C + -75.575 * A2D + 64.0625 * AB22 + 0.59375 * BCD + -3.39375 * A2B + -1.58125 * A2C + -0.75625 * AB2BP1 E1 degradation (%) = 72.9833 + 0.05 * A + -1.3 *B + 9.8 *C + 1.27778 *D + 0.50625 * AD + 0.58125 * BC + 0.51875 * BD + -1.21875 * CD + -0.955556 *CBP2 E1 Degradation (%) = 75.9946 + -0.7 * A + -1.35 *B + 6.3 *C + 7.5 *D + 0.18125 * AD + -1.83125 * BC + 0.30625 * BD + 0.40625 * CD + -1.26774 *C2 + 0.782258 *D2 + -0.19375 * ACD + 2.29375 * A2B + -1.76875 * A2D + 0.79375 * AB22 + 0.969355 *D2 + -0.4875 * ABD + 0.4375 * BCD + -2.4375 * A2B + -0.6625 * A2C + -0.6875 * A2D + -0.5625 * AB2BP1 E2 degradation (%) = 76.4968 + 5.23364e-16 * A + -2.6 *B + 9.85 *C + 1.85 *D + 0.025 * AB + 4.55267e-16 * AC + 0.15 * AD + 0.225 * BC + 0.675 * BD + -1.525 * CD + -0.880645 * A2 + 0.4625 * BCDBP1 EE2 degradation (%) = 71.1083 + -0.188889 * A + -3.97778 *B + 9.32778 *C + 0.8 *D + -0.075 * AB + 2.41703e-15 * AC + 0.0375 * AD + 0.775 * BC + 1.0875 * BD + -1.7125 * CD + -1.01389 *C2 + -0.368421 *B2 + -0.718421 *C2 + -0.0684211 *D2 + 0.0125 * ABC + -0.1 * ABD + 0.0125 * ACD + -0.725 * BCD + 2.3125 * A2B + 0.15 * A2C + -1.3125 * A2DBP2 EE2 Degradation (%) = 74.4175 + -0.0111111 * A + -2.2 *B + 6.25 *C + 7.4 *D + 0.025 * AB + 0.0125 * AC + 0.075 * AD + -1.4 * BC + 0.6625 * BD + -0.125 * CD + -0.368421 * AAs depicted in The stability of both free and immobilised were studied and compared through incubation with buffer under varying thermal conditions (10–100°C) for 1 h. As shown in Enzyme activities of both free and immobilised were evaluated over a broad pH range (2–10) as shown in As shown in The efficiency of the immobilized laccases in the removal of estrogen in a medium containing mixture of estrogen. Both immobilized enzymes demonstrated a 100% removal for E2 and over 90% of E1 and EE2, within 24 h. EE2 was the least removed estrogen under both circumstances, followed by E1 as shown in The role of a putative bacterial laccase enzyme in EDC biodegradation is investigated in this study. This is the first known bacterial laccase enzyme that degrades estrogen, to our knowledge. Laccase is best known for catalyzing the oxidation of aromatic compounds and other classes of EDCs in lignin-biodegrading fungi . The seqLysinibacillus genus are widely distributed in the environment and have been implicated in the degradation of micro pollutants such as aromatics, PFCs, and other chemicals , activity greater than 600 U/mL, and copper concentration (75 mg/L). The current study also discovered that high estrogen concentrations have a negative impact on laccase enzymatic activity as well as estrogen degradation efficiency. In contrast, increased copper concentrations resulted in laccase induction as shown in In the current study, S. lavendulae and T. versicolor laccases, on the other hand, were at their peak around pH 6 and declined slightly at pH 8 (Inoculum dosage (B) and Cu concentration variation (C) had the maximum impact on laccase activity and degradation  thereby signifying their predominant role in estrogen degradation process. In contrast, pH (factor A) has the least significant contribution, which could be attributed to the study’s narrow pH range (pH 6–8). According to research, a large number of microbes with the ability to degrade micro pollutants are mesophilic, with maximum metabolic activities at pH levels between 6 and 8.  at pH 8 . Thus, i. To evaluate the storage stability of laccase, we stored free and immobilized peroxidase at 4 and 25°C for 35 cycles and measured their activities. These findings indicated that immobilized laccase is far more stable in storage than free form enzyme. As shown in Enzymatic activities of the two isolates revealed unequivocal involvement of bacterial laccases during estrogen degradation, implying that they could serve as alternative biological sources for estrogen removal in wastewater. Studies on their antagonistic effects on wastewater microbial communities are relevant to biotechnology applications. Enzyme storage stability is pivotal in industrial applications We also investigated the thermal stability of the biocatalysts in comparison to the free enzyme because it is important for industrial laccase applications. As shown in Immobilized enzyme should be stable and reusable for multiple cycles to make immobilization cost effective . After 2et al>, 2016). Throughout the study, the removal efficiency of beads immobilised laccase showed slightly higher removal percentages than SICG as depicted in Both biocatalysts were able to completely remove E2 as shown in Despite the numerous benefits of biocatalysis, commercial enzyme catalysis is only used if it improves the process’s operational economics . AccordiEnzymatic activities of the two selected isolates revealed unequivocal involvement of bacterial laccases during estrogen degradation, suggesting that they could serve as alternative biological sources for estrogen removal in wastewater. However, the individual or interactive effects of enzymatic inoculum size (B) and Cu concentration (C) were particularly crucial for enhanced secretion. More than anything, the synthesized biocatalysts using beads and SICG indeed improve enzyme stability and efficiency as they were found to be more tolerant to temperature, pH, and other environmental perturbations. Correspondingly, they possessed improved storage ability and reusability, in addition to enhanced estrogen removal. Both biocatalysts were able to remove 100% of E2, as well as over 90% of E1 and EE2, within 24 h. Notwithstanding, they shared similar estrogen degradation patterns with free laccases, with E2 being the most removed, followed by E1 and lastly EE2. In comparison to clay based biocatalysts, beads biocatalysts possessed a higher potential for recyclability and were more resistant to environmental factors such as pH, storage, reuse, and temperature. The differences in terms of net estrogen removal was insignificant across all estrogens ranging between 95–100% and 94–100% for beads biocatalyst and clay biocatalyst, respectively. In view of the above credentials of the composite, the adoption of SIGC biocatalysts would be most feasible for real-life large-scale applications, due to its inexpensiveness and its additional function as an antibacterial composite. Therefore, future studies should be tailored to reduce the cutbacks it currently encounters, Kibamb.