We propose an etching route and compareit to conventional fluoride etching products. Additionally, the studyexplores the possible application and energy storage mechanism ofMBenes in supercapacitors, marking the first investigation of itskind. At room temperature, 1/24-MoAlx1–B with terminal groups −OH exhibits ∼25% Al removalin 1 wt % NaOH for 24 h, outperforming traditional etching technology.Increasing the Al removal exposed more open space, resulting in ahigher capacitance. Compared to LiF/HCl-MoAlx1–B (etched by LiF + HCl), 1/24-MoAlx1–B has a higher energy storage capability. The multilayered1/24-MoAlx1–B film electrode exhibitsultrahigh conductivity with a rapid relaxation time of 0.97 s andhigh areal capacitance (2006.60 mF cm–2) while maintaining80.2% capacitance after 5000 cycles. The MoAlx1–B all-solid-state supercapacitor (ASSS) deliversa high capacitance of 741.6 mF cm–2 at 1 mV s–1 for a single electrode and exhibits stable capacitanceeven at a 90° bending angle, highlighting its potential practicaluse. Our research represents an important step in the synthesis ofMBenes and highlights their potential applications in supercapacitors.The present study describes the synthesis of multilayeredMBenesMoAl They concluded that boron-based nanosheets perform better thangraphene for supercapacitors due to their low molecular weight andmetallic nature. Furthermore, boron-doped nanosheets have been ofgreat interest in energy storage applications, as Chi et al.8 have reported boron-doped graphene for high-voltageaqueous supercapacitors. The high specific capacitance of 336 F g–1 was found for boron-doped mesoporous graphene whereit reduced to 169 F g–1 at a current density of0.1 A g when boron was removed by calcination.9 This study revealed that boron plays a vital role in the chargestorage capability. In this aspect, most recently, 2D-layered boron-basedmaterials have been widely researched for energy storage applications.By applying jointdensity functional theory (JDFT) to the electrochemicalsystem, Zhan et al.10 A new groupof transition metal boride layered nanomaterials (MBenes) is exploredhighly due to their exceptional electronic conductivity, outstandingstability, and rich surface chemistry.12 2D multilayerMBenes are derived by chemically etching and exfoliating 3D MAB phases,which typically contain a transition metal (M), IIIA and IVA groupelements (A), and boron (B). The first report on MBenes dates backto 2017,13 where they were considered analogousto early transition metal carbides, nitrides, and carbonitrides (MXenes).On the other hand, MBenes have received less attention than MXenesbut have immense potential in many areas which benefit from the multiplevalence state of the boron atom and its electron-deficient nature.14 Theoretical calculations and preliminary experimentssuggest a plethora of applications for 2D MBenes in magnetic devices,15 electrocatalysis,16 and batteries,18 making the exploration of theseclasses of compounds promising. MBenes are also predicted to buildnewer, more efficient supercapacitors with long-cycle stability andhigh-power density.14 MAB, specificallyMoAlB, exhibit extremely low electronic resistance,19 but their use, particularly in the bulk form, is oftenhindered by poor exposure to active sites due to their low specificsurface area. Numerous attempts have been made to synthesize its deintercalationproducts (MBenes) by etching aluminum layers from the MoAlB phase.Several studies have demonstrated that the etching process can effectivelyremove approximately 25% of the aluminum content in MoAlB,22 leading to improved electrochemical properties. However, currentetching methods usually involve the use of fluoride etchants or highcorrosion solutions at elevated temperatures,23 which are either not cost-effective or environmentally unfriendlyand pose safety hazards. As a result, the development of a more cost-effectiveand environmentally friendly etching process remains an open challenge.One potential approach to improving the electrochemical propertiesof MoAlB is by deintercalation of aluminum from MoAlB with largerspacing. However, to the best of our knowledge, the promising applicationand energy storage mechanism of MBenes in supercapacitors are yetto be reported.The development of energy storage device is dependent on the designof novel electrode materials.x1–B with different Al deintercalationrates (up to ∼25%)were synthesized by removing Al from MoAlB using dilute alkali NaOHand traditional fluoride etchant LiF/HCl  displayed a lower rate of Al removal compared to 1/24-MoAlx1–B. Following on, the propertiesand energy storage mechanism of MBenes in supercapacitors were investigatedfor the first time. The capacitance of MoAlx1–B enhanced with the increase of the Al defective rate due tothe open space. The 1/24-MoAlx1–B film electrode exhibited ultrahigh conductivity, superior cycleperformance, and high areal capacitance (2006.60 mF cm–2). 1/24-MoAlx1–B had a higherand more stable energy storage capability than LiF/HCl-MoAlx1–B. Diffusion-controlled and surface-controlled mechanismscoexisted during charge–discharge. Additionally, an all-solid-statesupercapacitor device  into20 mL of 1 wt % NaOH at room temperature (RT) and stirred for 1, 12,and 24 h, 10 wt % NaOH for 24 h, and 30 wt % NaOH at 50 °C for48 h. The products were denoted as 1/1-MoAlx1–B, 1/12- MoAlx1–B, 1/24-MoAlx1–B, 10/24-MoAlx1–B, and 30/48/50-MoAlx1–B, respectively. LiF/HCl-MoAlx1–B was obtained by etching MoAlB in 6 M HCl + 1.2 g of LiF usuallyused for preparing MXenes at 35 °C for 24 h.24 The prepared materials were rinsed with deionized waterto pH ∼ 7, then separated from the solution via centrifugation,and dried at 60 °C overnight.Different Al removal rates in multilayer MoAlThe elemental composition and morphologicalanalysis were characterized for the prepared sample by a scanningelectron microscope  equipped with energy-dispersiveX-ray analysis (EDX). The X-ray diffraction  has beenperformed to confirm the formation of the prepared phase and determineits crystallinity. X-ray photoelectron spectroscopy has been employed to estimate the surface composition and electronicstate of the elements in the prepared sample. Atomic absorption spectroscopy was used to further confirm the Al etching rates,and the specific surface area of samples was measured by a surfacearea and porosity analyzer (Micromeritics TriStar 3000). Surface terminationgroups have been verified by a Fourier transform infrared spectrometer. The electrochemical characterizationand data analysis were performed using electrochemical workstations(Solartron SI 1280 and SI 1260) equipped with software Corrware andZplot (Scribner Associates Inc.).x1–B/MoAlB powder, conductive agentacetylene black (AC), and PTFE binder (60 wt % in H2O).The slurry contained 70 wt % MoAlx1–B, 10 wt % PTFE, and 20 wt % AC. The slurry was rolled into a filmwith a thickness of about 120 μm and then tailored into rectangularsheets (1 × 1.5 cm2). These electrodes were driedat 60 °C for 24 h. The mass density for the active materialsper unit area was approximately 30 mg cm–2.The thick film electrodes were prepared by a mechanicalprocess of a premixed slurry, containing MoAlx1–B film electrodes withsolid-state poly (PVA)–LiCl electrolyte andseparated by an NKK membrane . Specifically, 1 g of LiCland 0.5 g of PVA powder were added to 5 mL of deionized water. Then,the mixture was heated at 70 °C under stirring until it becamea transparent gel. The gel was cooled naturally to room temperatureand cast onto one side of the 1/24-MoAlx1–B film electrode. After solidification, two copper foils wereattached as current collectors and directly connected to the electrochemicalstation.The ASSS was fabricated by sandwiching two pieces of rectangular1/24-MoAl2 Pt mesh as the counter electrode, 1 M Na2SO4/1 M H2SO4/2 M NaOH as electrolyte,and Ag/AgCl (saturated KCl) as a reference electrode. A separatorwas sandwiched between a couple of film electrodes in a two-electrodesetup. All electrochemical measurements for ASSS were performed ina symmetric two-electrode configuration. The electrochemical studiesfor supercapacitor application were done using cyclic voltammetry(CV), galvanostatic charge–discharge (GCD), and electrochemicalimpedance spectroscopy (EIS). CV curves were obtained at differentscan rates: 1, 2, 5, 10, and 20 mV s–1 in a suitablevoltage window. EIS was demonstrated in the Nyquist plot in the frequencyrange from 0.1 to 106 Hz by applying a fixed potentialof 20 mV. GCD curves were obtained at different current densitiesranging from 2 to 24 mA cm–2. The capacitance, energydensity, and power density of the film electrodes and the deviceswere calculated by eqs S1–S10 of the Supporting Information. The flexible properties of ASSS have also beentested by comparing the CV curves after bending the flexible devicefrom 0° to 30°, 60°, and 90°.Electrochemical experimentsof film electrodes were performed in a three-electrode setup and atwo-electrode setup. As for the three-electrode setup, the synthesizedfilm electrode is considered as the working electrode, 1 × 1.5cmS1). SEM-EDX analysis revealed a0.992:1 Al:Mo ratio (k0) reflections weakened with increasing etching time. The(040) reflection also weakened and almost disappeared after 24 h ofetching, and higher temperature or longer etching time did not furtherimprove the etching efficiency, indicating that the Al removal ratereached equilibrium. Despite previous reports showing only ∼25%etching by fluoride treatment or concentrated alkaline,25 our study found that using 1 wt % NaOH at room temperature was themildest method that also achieved ∼25% Al removal. SEM imagesin x1–B slabs with thicknessesranging from 50 to 500 nm exhibited periodic gaps with 10–200nm spacings. We propose an etching pathway (20 The remaining MoAlx1–B slab canbe viewed as (MoB)2-(Al2(MoB)2)n-Al2(MoB)2 , which increased Al retentionin MoAlx1–B and stopped furtheretching due to surface coatings of AlF3 and Al oxide.25 The dark phase EDS spectrum showed a high Aland F atomic ratio , indicating Al removal relied on the reaction of HF and Al atoms,generating insoluble AlF3 after etching  computer results, the energy requiredto generate the first Al defect in the Al layer of MoAlB is 5.03 eV,which is lower than that of the Ti3AlC2 case(6.38 eV).28 Therefore, it is more feasibleto remove Al atoms from MoAlB. The use of 1 wt % NaOH to etch Al fromMoAlB is a crucial step toward achieving chemical delamination ofa MAB phase22 and even a MAX phase,31 and it will broaden their usage in various research fields suchas electrocatalysis, energy storage, and biochemistry.23 After more aluminum was removed, we observedan increase in the specific surface area of MoAlx1–B, as shown in 3 andaluminum oxide. However, the aluminum removal rate of LiF/HCl-MoAlx1–B was found to be lower comparedwith the products obtained from NaOH etching. Furthermore, the presenceof the terminal functional group −OH on the surface of MoAlBafter etching, as shown by the band at ∼3350 cm–1 of FTIR spectra 2 3. Bubblel2(MoB)2 1:1Higherer slabs2f,g, so er slabs2h showed etching 2.2621 wter slabsS1, whichTables S2–S5. After etching for 24 h,the Al:Mo ratio measured by XPS was down to 0.764:1 in 1/24-MoAlx1–B . The fitted peaks at a binding energy (BE) of 228.53eV (231.58 eV) are assigned to MoAlx1–B  element when selectively etching the A element in MAX phases,forming MXenes.33 As shown in x1–B are also shifted to higher values due to the introductionof the electron-withdrawing group −OH and the increasing valenceof the remaining Al. The fitted XPS spectra of the O 1s regions alsoshow the presence of the terminal group −OH after etching  analysis, presented in MoAl1–xB 4band ar etching 4e, whichx1–B for supercapacitor applications was initially evaluatedby investigating the effect of different Al etching rates on the propertiesof MoAlB and the fluoride etching product in a three-electrode system. x1–B at a scan rate of 10 mV s–1 increasesas the rate of Al removal increases. However, the capacitance of 1/24-MoAlx1–B and 30/48/50-MoAlx1–B remains almost the same due to a very similar Alremoval rate (∼25%). S2 demonstrate thatMoAlx1–B exhibits significantlyimproved performance compared to its precursor MoAlB due to a largersurface area, better exposure of active sites after etching, and morespace available for electrolyte infiltration. Furthermore, 1/24-MoAlx1–B outperforms LiF/HCl-MoAlx1–B due to higher Al removal and lower conductivityof AlF3 remaining in LiF/HCl-MoAlx1–B. Moreover, the presence of the terminating −OHin 1/24-MoAlx1–B enhances its supercapacitorproperties. Bulk MoAlB exhibits lower resistivity than MoB (0.36–0.49μΩ·m19 vs ∼6 μΩ·m34). As metal Al acts as “electron bridges”in MoAlB bulk materials, the reserved Al ensures high interlayer conductivityafter partial Al removal from MoAlB. The solution resistance of MoAlBand MoAlx1–B is extremely low,as shown in D) by fittingthe real part of the impedance (Zre) withthe square root of the radial frequency (ω–1/2) according to eqs S11 and S12. Since MBene-based and MoB-based materialsare good electrocatalysts for hydrogen (HER) and oxygen evolutionreactions (OER),36 HER and ORR easily occur in acidand alkaline electrolytes, limiting the voltage window largely. Theareal capacitance results are summarized in 2SO4 >1M Na2SO4 > 2 M NaOH. This can be attributedto the cation migration rate, with H+ having a faster migrationrate than Na+ due to its smaller size. However, long-durationelectrochemical tests in acidic and alkaline electrolytes caused severecapacitance attenuation at high scan rates, making them unsuitablefor energy storage.The electrochemical performances of the as-prepared1/24-MoAlx1–B in a 2-electrode configuration. Two 1/24-MoAlx1–B film electrodes sandwiched with an NKK separatorwere fabricated in a symmetric two-electrode configuration and testedin 1 M Na2SO4. The charge storage capacity ofthe electrode was primarily evaluated by CV measurement at differentscan rates in a potential window of 0–1V (x1–B andcompares it to other MXene/molybdenum/boron-based electrodes (Table S6), with an areal capacitance of 2006.60mF cm–2 (1 mV s–1) calculatedfrom CV curves. Despite its higher loading mass (30 mg cm–2) due to the much higher molecular weight of MoAlB, the film electrodehas a thickness of only 120 μm, which meets the thickness limitof thin film electrodes37 and achievesan outstanding volumetric capacitance of 166.67 F cm–3 for a single electrode at the scan rate of 1 mV s–1. The specific mass capacitance of 66.89 F g–1 hereis considered moderate when compared to some low molecular weightactive materials, such as active carbon39 and MXene/molybdenum/boron-basedelectrodes (Table S6), but the excellentareal capacitance suggests promising applications of MBenes in supercapacitors.Moreover, the specific mass capacitance could be significantly enhancedby further removing more Al and exposing more surface, demonstratingthe potential for future improvements.Further study has been conducted for thesupercapacitor performanceand energy storage mechanism of 1/24-MoAl of 0–1V 6a. The q–2 at a charge–discharge rate of 2 mAcm–2 and a high charge–discharge rate of24 mA cm–2, respectively (Table S7). The high performance could be attributed to its good iondiffusion and ultralow resistance as shown in RS) and the charge transfer resistance(Rct), respectively, which are calculatedto be equal to 0.0037 and 0.0023 Ω cm–2 basedon the equivalent circuit fitting shown in the inset of –2 for bimetallic-sulfide@layered Ti3C2Tx-MXene,40 1.109 and 0.0075 Ω cm–2 for Ti3C2Tx@Al,26 and 0.392 and 0.164 Ω cm–2 for PANI/small-sizedMXene41). The complex model of capacitancewas utilized to further confirm the impedance behavior (eqs S13 and S14).43 The relaxationtime constant τ0 can be calculated according to thepeak position for imaginary capacitance C″(ω)from the equation τ0 = 1/f0, where f0 is the frequency. A noticeablyshort time constant of τ0 = 0.97 s  versus log(υ). For an ideal surface capacitanceprocess, the b value is 1. The b value is 0.5 when only the pseudocapacitance process is observed.The b value is between 0.5 and 1 when both the capacitanceprocesses coexist. As shown in b values are in the range of 0.6–1,indicating that the surface-controlled capacitance and diffusion-controlledcapacitance coexist during charge–discharge. Furthermore, thesurface-controlled contribution to the overall current response wasfurther quantified by conducting Dunn’s method (eqs S17 and S18),44 where k1v represents the surface-controlled contribution and k2v1/2 represents the diffusion-controlledcontribution. The top current (inset is bottom current) responds tothe square root of the scan rate versus the square root of the scanrate shown in –1, the diffusion-controlledarea still accounts for 30.78%, and the surface-controlled capacitiveaccounts for 69.22%, illustrated in S3). Large interlayer structurecontributes to the surface capacitance, and the high diffusion-controlledpseudocapacitance benefits from its ultrahigh conductivity. The hybridcharge storage mechanism is beneficial for increasing energy and high-powerdensities with excellent stability.Generally, capacitance can be divided into surface-controlledcapacitanceand diffusion-controlled pseudocapacitance.x1–B film (1 × 1.5 cm2) electrodes were assembledinto an ASSS by sandwiching an NKK separator with LiCl–PVAas the solid-state electrolyte and Cu foils as current collectors.The ASSS retains stability at different bending states, as shown in –2 is 741.6 mF cm–2 or 61.8 F cm–3 (370.8 mF cm–2 for the ASSS device), which exceeds many MXenes orother active material previously reported in symmetric ASSS (Table S8). When the device is bent from 0°to 30°, 45°, and 90°, the CV curves show negligiblechange, and the capacitances almost remained at 81% when bending toa high angle of 90°  have significant potential for use in supercapacitorsand that their properties could be further improved by increasingthe Al deintercalation rate, thus advancing their practical applicationsin energy storage.This research presents a novel approachfor synthesizing multilayeredMBenes MoAl


Furthermore, survival of malaria transmitting Anopheles mosquitoes is strongly decreased after feeding on humans recently treated with ivermectin. Currently, mass drug administration of ivermectin is under investigation as a potential novel malaria vector control tool to reduce Plasmodium transmission by mosquitoes. A “post-ivermectin effect” has also been reported, in which the survival of mosquitoes remains reduced even after ivermectin is no longer detectable in blood meals. In the present study, existing material from human clinical trials was analysed to understand the pharmacokinetics of ivermectin metabolites and feeding experiments were performed in Anopheles stephensi mosquitoes to assess whether ivermectin metabolites contribute to the mosquitocidal action of ivermectin and whether they may be responsible for the post-ivermectin effect.Ivermectin  to produce ivermectin metabolites. In total, nine metabolites were purified by semi-preparative high-pressure liquid chromatography. The pharmacokinetics of the metabolites were assessed over three days in twelve healthy volunteers who received a single oral dose of 12 mg ivermectin. Blank whole blood was spiked with the isolated metabolites at levels matching the maximal blood concentration (Cmax) observed in pharmacokinetics study samples. These samples were fed to An. stephensi mosquitoes, and their survival and vitality was recorded daily over 3 days.Ivermectin was incubated in the presence of recombinant human cytochrome P2B1a) was mainly produced by CYP3A5. Both desmethyl-H2B1a (M1) and hydroxy-H2B1a (M2) killed all mosquitoes within three days post-feeding, while administration of desmethyl, hydroxy-H2B1a (M4) reduced survival to 35% over an observation period of 3 days. Ivermectin metabolites that underwent deglycosylation or hydroxylation at spiroketal moiety were not active against An. stephensi at Cmax levels. Interestingly, half-lives of M1 (54.2 ± 4.7 h) and M4 (57.5 ± 13.2 h) were considerably longer than that of the parent compound ivermectin (38.9 ± 20.8 h).Human CYP3A4 metabolised ivermectin more rapidly than CYP3A5. Ivermectin metabolites M1–M8 were predominantly formed by CYP3A4, whereas metabolite M9 (hydroxy-HAn. stephensi mosquitoes and could be responsible for the “post-ivermectin effect”.In conclusion, the ivermectin metabolites M1 and M2 contribute to the activity of ivermectin against The online version contains supplementary material available at 10.1186/s12936-023-04624-0. Plasmodium parasites are the causative agents of malaria, and are transmitted to humans through the bites of female Anopheles mosquitoes. Although the disease is both preventable and curable, malaria remains a significant global public health problem. Globally, there were an estimated 241 million malaria cases in 2020, 6% more than in 2019, and deaths increased by 12% to about 627,000 deaths, mainly due to service disruptions during the COVID-19 pandemic [pandemic .Loa loa co-endemic regions [Ivermectin is a broad-spectrum antiparasitic drug, used in a wide range of infestations such as helminths, scabies, and mites. It has been licensed for human use for more than thirty years. Its safety profile has been thoroughly assessed in over 70 trials and extensive post-marketing surveillance. Billions of doses have been distributed worldwide for the control of neglected tropical diseases with a good safety profile, with the important exception of its use in  regions –8.Anopheles gambiae, Anopheles albimanus, Anopheles arabiensis and Anopheles stephensi [50) within 7-days ranges from 3 to 55 ng/mL in Anopheles spp., and 178–187 ng/mL in Aedes aegypti [After a mosquito has ingested ivermectin with a blood meal from a treated subject, ivermectin binds to the glutamate-gated chloride ion channels, leading to hyperpolarisation of the neuronal membrane, paralysing and potentially even killing the insect . Numeroutephensi , 10–15.  aegypti –19.50 for a considerable time [Approximately 4 h after a single oral dose of 150 µg/kg ivermectin, the peak concentration is around 40 ng/mL , 21. Aftble time , 22–24. ble time . Ivermecble time , 26–29. ble time , 31. A cble time .Anopheles stephensi is expanding its geographic range worldwide and has been implicated in outbreaks of urban malaria [An. stephensi was detected in Kenya for the first time [ malaria . The Worrst time .50 values, for example, even at 28 days post-dosing in the IVERMAL trial [Interestingly, the blood of treated individuals reduces the survival of the mosquitoes beyond what is expected from plasma pharmacokinetics of the parent compound, even up to 28 days post-dosing , which iAL trial . This isAL trial .2B1a (M1), 4-hydroxy-H2B1a (M2), 26-hydroxy-H2B1a (M3), 3″-O-desmethyl, 4-hydroxy-H2B1a (M4), 24-hydroxy-H2B1a monosaccharide (M5), 3″-O-desmethyl, 26-hydroxy-H2B1a (M6), 26-hydroxy-H2B1a monosaccharide (M7), 4, 26-dihydroxy- H2B1a (M8), and 24-hydroxy-H2B1a (M9) [450 3A4 (CYP3A4) is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes. In brief, ivermectin can be O-demethylated at the disaccharide moiety, undergo deglycosylation, and can be hydroxylated at the aglycone portion. The hydroxylation takes place at the hexahydrobenzofuran, the spiroketal portion of the molecule or both. Tipthara et al. confirmed these results and identified four additional metabolites including ketone and carboxy formation [Zeng et al. identified the structure of nine ivermectin metabolites in the presence of human liver microsomes, namely 3″-O-desmethyl-HB1a (M9) . Cytochrormation .An. stephensi. Since no reference standards for ivermectin metabolites were available, a method was set up to produce and purify nine different metabolites. Furthermore, a screening assay was established to estimate whether the metabolites are active against An. stephensi at levels observed in humans.This study investigated whether metabolites may contribute to the activity of ivermectin against An. stephensi mosquitoes to assess the mosquitocidal activity over 72 h.First, an in vitro system was developed using recombinant CYP3A4 and 3A5 isoforms to produce ivermectin metabolites, which were then purified and enriched by semi-preparative high-pressure liquid chromatography (HPLC). Secondly, we studied the pharmacokinetic properties of those metabolites in healthy volunteers who received a single oral dose of 12 mg ivermectin . Finally2 were products of Toronto Research Chemicals . Gradient grade water and methanol as well as formic acid (98–100%) were purchased from Merck . Dimethyl sulfoxide (DMSO), ketoconazole, ammonium formate , bovine serum albumin (BSA), potassium phosphate monobasic (1 M KH2PO4), and potassium phosphate dibasic (1 M K2HPO4) were obtained from Sigma-Aldrich . Recombinant human (rh) CYP3A4 supersomes, rh CYP3A5 supersomes, nicotinamide adenine dinucleotide phosphate (NADPH) regenerating solution A  and solution B  were purchased from Corning Life Sciences B.V. . Drug-free human blood, stabilised by citrate–phosphate-derivative with adenine was acquired from the local blood donation centre .Ivermectin and ivermectin-d2B1a (M1), hydroxy-H2B1a (M2), hydroxy-H2B1a (M3), desmethyl, hydroxy-H2B1a (M4), hydroxy-H2B1a monosaccharide (M5), desmethyl, hydroxy-H2B1a (M6), hydroxy-H2B1a monosaccharide (M7), dihydroxy- H2B1a (M8), and hydroxy-H2B1a (M9) was evaluated by high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) based on mass transitions published by Zeng et al. [2, which was used as internal standard (ISTD solution). After centrifugation , sample supernatants (10 µL) were analysed by LC–MS/MS. Metabolite production was quantified by dividing the metabolite peak area with the ISTD peak area. Each metabolism assay was performed in triplicate.Ivermectin (10 μM) was incubated in the presence of rh CYP3A4 and rh CYP3A5 supersomes. Production of nine ivermectin metabolites namely desmethyl-Hg et al. . The reag and 15 °C. Supernatants of all reactions were combined and evaporated at 40 °C for about 4 h using a TurboVap LV evaporator . The sample was concentrated by resuspending the residuals in 1 mL methanol. Overall, 10 µL of the sample was injected repetitively (about 50 times) to purify the ivermectin metabolites by semi-preparative high-performance liquid chromatography (HPLC). Nine metabolite fractions were collected, which were evaporated to dryness and resuspended in 1 mL of methanol  was incubated in the presence of human recombinant CYP3A4 and CYP3A5 supersomes. Multiple reactions were prepared for each CYP isoform as described above. The 500 µL reactions were stopped after 2 h of incubation by the addition of 1.5 mL methanol. Samples were vigorously mixed for 30 min and centrifuged  for 30 min at 3220×0 μM was g and 15 °C (5810 R centrifuge). Twenty microlitres of supernatant were injected into the LC–MS/MS system to determine the signal intensity of the metabolites.The signal intensity (peak height) time course of nine ivermectin metabolites (M1–M9) were recorded in blood samples originating from a clinical trial . In brieIvermectin and its metabolites were analysed by LC–MS/MS based on a previously validated bioanalytical method . The metZeng et al. reported on the structure of nine ivermectin metabolites and published the corresponding mass transitions . These mA Kinetex C8 analytical column  was used for pharmacokinetic assays. A Luna C8(2) column  was employed for the metabolism assays and fractioning of the ivermectin metabolites. Mobile phase A was an aqueous solution of 20 mM ammonium formate with 0.1% formic acid. Methanol plus 0.1% formic acid was used as mobile phase B. Chromatography was performed at 55 °C. The dual binary flow programmes used for the different assays are summarised in Additional file Metabolite fractions were isolated with a FRC-12A fraction collector that was coupled to the HPLC system. LabSolutions software 5.97 (Shimadzu) was employed to operate the fractioning. The following nine fractions (F1-F9) were collected: F1; 4.50–4.85 min, F2; 4.90–5.15 min, F3; 5.15–5.50 min, F4; 5.50–5.70 min, F5; 5.75–6.00 min, F6; 6.10–6.40 min, F7; 6.50–6.75 min, F8; 6.95–7.35 min, and F9; 7.40–7.80 min. Metabolite M7 was predominantly present in F1, M8 in F2, M6 in F3, M3 in F4, M5 in F5, M9 in F6, M4 in F7, M2 in F8, and M1 in F9. Metabolite composition of the fractions was assessed by LC–MS/MS so as to quantify carry-over effects and the purity of each fraction. Additional file http://www.lixoft.com, Antony, France), for the most abundant metabolites. The integral method “linear up log down” was used on area/internal standard area ratio. Metabolites were included when peak height at Cmax was above 500 counts per second (cps), hence M5, M7, M8 and M9 were excluded from the NCA analysis.The pharmacokinetic parameters of ivermectin metabolites in twelve healthy volunteers were calculated in the non-compartmental analysis (NCA) framework of PKanalix  from the blood level-time curves. Afterwards, the mean metabolite peak area of the spiked blank blood was compared with the mean peak area of the Cmax samples  that was spiked with a known amount of the metabolite fraction. In brief, an aliquot of 10 µL of each of the nine ivermectin metabolite fractions was evaporated and resuspended in 50 µL of blank blood. In total, three T0 blank blood samples originating from different PK study participants were used to resuspend the metabolites  for 20 min at 39 °C. A Petri dish  was filled with 3 mL of warm blood, sealed with a piece of sealing film . The Petri dish was placed upside down on top of the net sealing the paper cup. Female mosquitoes were allowed to feed for 20 min on the blood through the net and the sealing film. The feeding was conducted in a climate chamber  at 27 °C, 70% humidity, and 30% light intensity. After feeding, the cups were put on ice to immobilise the mosquitoes and only the fully engorged mosquitoes were selected for the assay. The mosquitoes were returned to the climate chamber to assess their activity and survival for 3 days.max levels observed in PK samples. Afterwards, the LC50 was estimated for the most active metabolite fractions in three independent assays. The mortality effect of the nine metabolite fractions, ivermectin (positive control), and blank blood (negative control) on mosquito survival and activity was assessed at 24 h, 48 h and 72 h post-feeding. In screening assays, eleven treatments were evaluated with three replicates using three batches of 50–70 mosquitoes. At each time point, dead and alive mosquitoes were counted and their activity was rated with the following scores: + 2; the mosquito is flying and resides mainly on the top of the paper cup, + 1; the mosquito is moving but stays on the bottom of the paper cup, 0; the mosquito is classified as dead and does not move upon physical contact.In the framework of screening assays, mosquitoes were treated with blood containing each metabolite corresponding to C50) was evaluated for ivermectin and the most active metabolites M1 and M2. Therefore, three replicates using three batches of 50–70 mosquitoes were treated with different dilutions of ivermectin , M1  and M2 . A pilot test was performed to evaluate optimal dilutions  matching the expected LC50 values (data not shown). A Cmax of 50 ng/mL was used for ivermectin [50 was evaluated using ivermectin concentrations ranging between 1 and 12.5 ng/mL.The concentration of compound required to kill 50% of adult mosquitoes in 3 days . Survival analyses, summary and visualisation of survival analysis were computed using the GNU R packages survival (version 3.1-8) and survminer (version 0.4.9). Replicates were pooled and analysed by the logrank test and the hazard ratio with 95% confidence intervals. LC50 in An. stephensi mosquito were approximated with regression analysis, using GNU R package drc [50 estimation .Student's t test was adopted to test the significance of all mean survival differences between groups, using the R package kage drc  , GraphPad Prism , and Adobe Illustrator .Data analysis and visualisation were performed with GNU R  than recorded for ivermectin (Tmax: 4.4 h). An average terminal elimination half-life of 38.4 h (T½ range: 27.1 h–57.5 h) was calculated for the metabolites. This is close to the half-life of ivermectin (T½: 38.9 h) determined in the same subjects [last) was 24.2 h (MRTlast range: 13.9 h–33.8 h). The measurement of the maximal metabolite peak intensities in PK samples permitted the preparation of the blood samples with equal amounts of metabolite. Consequently, blood samples were produced with a pharmacologically relevant amount of metabolite, as observed after administration of a regular ivermectin dose. An absolute quantitative determination was not possible as no ivermectin metabolite reference standards were commercially available, and none could be synthesised in-house due to the structural complexity and by consequence very involved chemical synthetic process.The pharmacokinetics of nine ivermectin metabolites, M1–M9, were assessed in blood samples of twelve healthy volunteers who received a single oral dose of 12 mg ivermectin . The measubjects . SimilarBoth CYP isoforms were required to produce sufficient amount of all nine metabolites, considering that M9 was mainly produced by CYP3A5. The metabolites were purified by semi-preparative HPLC collected in nine fractions , i.e. the concentration at which 50% of mosquitoes die over a defined observation period. Multiple doses/concentrations of compound were orally fed to mosquitoes to determine their LC50 in An. stephensi from 24 to 72 h after treatment. The experiment was performed in triplicate, with an average of 24 mosquitoes per compound dilution. A total number of 485, 516 and 424 mosquitoes were fed for ivermectin, M1 and M2. The 1–2–3-day LC50 of ivermectin and of the most promising candidates, M1 and M2, were investigated in An. stephensi —that is mainly formed by CYP3A5. All metabolites apart from M9 are measurable in human blood after a single oral dose of 12 mg ivermectin. Pharmacokinetic analysis reveals that the mean residence time of the metabolites is shorter (MRTlast range: 13.9–33.8 h) as reported for ivermectin after a single dose of 150 µg/kg (89.5 h) [This study set out to develop a protocol to produce and isolate nine different ivermectin metabolites. It also shows that CYP3A4 and CYP3A5 metabolise ivermectin, while CYP3A4 readily produces all metabolites except M9 (Hydroxy-H(89.5 h) . Finally2B1a) and forms the metabolites M3 and M6—yet to a lesser extent than CYP3A4. The involvement of CYP3A5 in metabolising ivermectin might be of clinical relevance considering that its expression greatly varies between individuals according to their ethnicity and geographical location because of genetic polymorphisms in CYP3A4 and/or CYP3A5 genes [Zeng et al. have previously reported on the metabolic fate of ivermectin in human in vitro systems, and recently Tipthara et al. published in vitro as well as in vivo data , 42. TheA5 genes . PossiblA5 genes . Since n2B1a) and M2 (hydroxy-H2B1a). However, this does not necessarily indicate that those two metabolites are the most abundant. Yet, assays with radiolabeled ivermectin imply that M1 and M2 are the major in vitro metabolites of ivermectin [Aedes aegypti mosquitoes [The results of this study show that the most intense signals in the in vitro metabolism assays were recorded for M1 , hydroxy-H2B1a (M2), and desmethyl, hydroxy-H2B1a (M4) in human blood samples collected 24 h post-treatment [The LC–MS/MS method presented here was able to assess the kinetic disposition of eight ivermectin metabolites over a period of up to 72 h post-treatment in human whole blood. A previous study also detected the most abundant metabolites namely desmethyl-Hreatment . Only trreatment . Neverthmax (5–7 h) are shifted in time and occur later than ivermectin (4.4 h). Moreover, some metabolites elimination half-lives are longer (15.9–57.5 h) than ivermectin (38.9 h), allowing for an overall longer timeframe when mosquitoes can potentially be killed by certain ivermectin metabolites  than what would be expected from ivermectin alone. As another measure for the length of exposure to a substance, the mean residence times (MRT) of ivermectin metabolites (M1–M6) were calculated by non-compartmental analysis (NCA) of pharmacokinetic profiles (Table last) is shorter for some of the metabolites (MRTlast range: 13.9–33.8 h) compared to ivermectin. A reported MRT of 89.5 h was measured for ivermectin after a single dose of 150 µg/kg ivermectin [The drug concentration in the blood and the time the drug remains in circulation are important factors affecting the pharmacological effect. The results of the clinical pharmacokinetic study in humans indicates that ivermectin metabolites Termectin . BecauseAn. stephensi mosquitoes. In addition, M4 and M6 exhibited minor mosquitocidal properties either because the systemic exposure was lower or the metabolites are less mosquitocidal. M4 underwent demethylation on the disaccharide and hydroxylation on the hexahydrobenzofuran moiety like M1 and M2, respectively. All metabolites, which were hydroxylated on the spiroketal portion deglycosylated or both, were not or only slightly active (M6) at concentrations observed in clinical pharmacokinetic samples. However, the results presented here do not exclude that those metabolites are active at increased concentrations. In this study, a 3-day-LC50 of about 3 ng/mL was measured for ivermectin, which is slightly lower compared to observations made by Dreyer et al., with a 4-day-LC50 of 7 ng/mL against An. stephensi [50 value varies greatly between Anopheles species, insectary conditions, and with the feeding method or observation period. In An. gambiae, it was reported that the 7-day-LC50 of ivermectin is 3.4 ng/mL with blood fed from treated humans, and 15.9 ng/mL from spiked ivermectin experiments (in vitro mixture) [An. stephensi were more susceptible to ivermectin in comparison to assays done with other Anopheles species [Anopheles species.Kobylinski et al. demonstrated first evidence that metabolites might contribute to the activity of ivermectin as they observed that ivermectin spiked blood was less mosquitocidal than blood from treated humans with matching ivermectin levels . In supptephensi . The LC550 value, and thus contribute significantly to the overall mosquito-lethal effect of ivermectin. This time above the lethality target can be considered as a “mosquitocidal window” of ivermectin, M1 and M2. The mosquitocidal window of ivermectin after a single oral 150 µg/kg dose for a vector with a LC50 of 6 ng/mL was predicted to be of 55 h and 7 h for a vector with a LC50 of 25 ng/mL [50 and thus ivermectin’s efficacy in killing mosquitoes such as increasing the dose of ivermectin, employing repeated dosing regimens, and using a long-lasting drug formulation) [The current study estimated that M1 and M2 stay on average 69 h and 34 h above their LCulation) . Since Mulation) . StudiesThe present study has three evident limitations. First, the isolated fractions were analysed by multiple reaction monitoring, which was needed to reach the sensitivity to measure low-abundant metabolites. However, this detection mode is very selective and may not have recorded co-eluting constituents of the different fractions. Consequently, the overall purity of the isolated fractions could not be determined and it cannot be excluded that undetected metabolites or byproducts of the bioassay may have caused the observed mosquitocidal effects. However, the preparation of nine different blank fractions would have been very laborious, complicated considerably by the execution of the activity assays and would have substantially increased the required amount of consumables. Moreover, most of the fractions used for the treatments were not entirely pure and contained residues of the previous fraction  and hydroxy-H2B1a (M2) are active against An. stephensi at concentrations attained in the blood circulation of humans treated with a regular oral dose of 12 mg ivermectin. Pharmacologically relevant amounts of metabolites were used to allow the prediction of their in vivo activity, circumventing laborious and costly synthesis of reference substances. The methodology developed here can be applied to assess the activity of ivermectin metabolites against other parasites, which may further the development of novel chemotherapies. Overall, this study provides a better understanding of the pharmacokinetics of ivermectin metabolites and might facilitate further insight into efficacy for neglected tropical diseases and malaria, as ivermectin has been suggested as a possible vector control tool to assist malaria elimination. This approach can be applied to other drugs to study the activity of their metabolites. It should be emphasised that actual concentrations would still need to be assessed in PK trials to understand if relevant exposures can be achieved. For complex small molecular drugs such as ivermectin, the synthesis of references can generate considerable costs and operational complexity. The described approach adds value as a directed screening tool to focus resources on the most promising candidates.In conclusion, this study established a protocol to produce, isolate and test the activity of nine ivermectin metabolites. These results demonstrate that ivermectin metabolites desmethyl-HAdditional file 1: Table S1. Specific tandem mass spectrometry settings applied for the detection and quantification of ivermectin and its metabolites. Table S2. Dual binary gradient flow program used for metabolism and fractioning assays. Table S3. Dual binary gradient flow program used for metabolism assays. Figure S1. Chromatogram before/after fractioning of the ivermectin metabolites. Table S4. Ivermectin metabolite compositionof each fraction. Table S5. Comparison of ivermectin metabolite Cmax pharmacokinetic levels with spiked blank blood samples. Figure S2. Screening for ivermectin metabolites effect on mosquito activity. Figure S3. Fit of the nonlinear model to calculate ivermectin, M1 and M2 LC50. Figure S4. Compound level above LC50 in whole blood for ivermectin, desmethyl-H2B1a (M1) and hydroxy-H2B1a (M2).


In each case, the sensitivity was similar, whereas the signal stability was better for the surfactant. This study indicates that careful control experiments need to be designed to ensure reliable results and that the reliability can be improved by using Triton X-100 and a long equilibration time.Using DNA staining dyes such as SYBR Green I (SGI) and thioflavin T (ThT) to perform label-free detection of aptamer binding has been performed for a long time for both binding assays and biosensor development. Since these dyes are cationic, they can also adsorb to the wall of reaction vessels leading to unstable signals and even false interpretations of the results. In this work, the stability of the signal was first evaluated using ThT and the classic adenosine aptamer. In a polystyrene microplate, a drop in fluorescence was observed even when non-binding targets or water were added, whereas a more stable signal was achieved in a quartz cuvette. Equilibrating the system can also improve signal stability. In addition, a few polymers and surfactants were also screened, and 0.01% Triton X-100 was found to have the best protection effect against fluorescence signal decrease due to dye adsorption. Three aptamers for Hg Developing assays to study aptamer binding is of great importance to validate newly selected aptamers, characterize aptamer binding, and design biosensors ,2,3,4,5.However, the dye-staining assay also has complications. First, it may not work for all aptamers and the applicability of the assay is a trial-and-error process. Sometimes, the change in fluorescence is very small, which can compromise the reliability of this method and make quantitative measurement difficult. For many such assays, the fluorescence intensity drops when target molecules are added and the change in fluorescence intensity is within one-fold, such as in the adenosine aptamer with ThT ,19,20,21In this study, we studied the signal stability problem and explored two methods to establish stable background fluorescence. The first method is simply to wait for the signal to stabilize before titrating target molecules, where we observed time-dependent signal change even without adding target molecules. The second method is to add surfactants to cover the surface of reaction vessels. For this, we screened various surfactants and demonstrated the stabilization effect of Triton X-100 for a few representative aptamers.2. Buffer 2 (for T30 Hg2+ binding DNA): 20 mM pH 7.5 MOPS. Buffer 3 (for CSS.1-42 aptamer): 20 mM pH 7.5 HEPES, 100 mM NaCl, 10 mM MgCl2. Buffer 4 (for Ade and Ade-M2 aptamer): 10 mM HEPES buffer, pH 7.6, 50 mM NaCl, and 4 mM MgCl2. Buffer 5 (for Ade aptamer): 10 mM HEPES buffer, pH 7.6, 50 mM NaCl, 4 mM MgCl2, and 5% human AB serum.All of the DNA samples were ordered from Integrated DNA Technologies . The DNA sequences and modifications used are in Time-dependent stabilization was evaluated using a 96-well black plate. The sample solutions contained 100 nM adenosine aptamer or Ade-M2 aptamer and 2 μM ThT in Buffer 4 or Buffer 5 (when testing the effect of 5% serum on the titration of the adenosine aptamer by adenosine). Fluorescence intensity was monitored at 420 nm excitation and 490 nm emission using a Horiba Fluoromax 4 fluorometer  maintained at 20°C by a water bath or a Tecan Spark plate reader . Adenosine, guanosine, and MilliQ water were gradually titrated up to a final target concentration of 500 μM. Titration was either performed immediately or equilibrated by waiting for 10 min to reach a stable baseline before titration.The protective effects of different surfactants were evaluated using a 96-well black plate (Corning). The sample solution (100 µL) contained 200 nM DNA1 aptamer, 0.02× SGI in Buffer 1 with different concentrations of Triton X-100 . The fluorescence intensity was monitored under excitation at 488 nm and absorption at 532 nm for 30 min using a Tecan Spark plate reader. The effects of different assay materials were tested using 96-well black plates (Corning and Greiner) and a quartz cuvette. To test the effect of different surfactants, a 96-well black plate (Corning) and a quartz cuvette were used. For the plate method, 100 µL of the sample solution contained 200 nM DNA1 aptamer, 0.02×SGI in Buffer 1, and 0.01% surfactant . For the cuvette method, 500 µL sample solution contained 1 µM DNA1 aptamer, 0.1× SGI in Buffer 1, and 0.01% of the various surfactants. To test the influence of Triton X-100 on the adsorption of dyes on sample containers, 0.5×SGI in Buffer 1 was incubated for 15 min in a 96-well black plate, and then the solution was fully removed. Next (0.01% Triton X-100 or no Triton X-100) in Buffer 1 was added along with a 70-mer double-stranded DNA into the same wells used initially. In clean wells, 70-mer double-stranded DNA, in the presence of 0.01% Triton X-100, was titrated up to a final concentration of 0.4 µM SGI. Fluorescence intensity was monitored using a Tecan Spark plate reader at 488 nm excitation and 532 nm emission.2+ by T30 DNA was evaluated using a 96-well black plate (Corning) in Buffer 2 containing 200 nM T30 aptamer, 0.1× SGI, and 0% or 0.01% Triton X-100. The sample solution (100 µL) was titrated with mercury acetate up to a final concentration of 3 µM. Fluorescence intensity was monitored using a Tecan Spark plate reader at 488 nm excitation and 532 nm emission. For signal stability measurement, 100 µL of sample solution contained 200 nM T30 aptamer, 0.1× SGI, 2 µM mercury acetate, and 0% or 0.01% Triton X-100 in Buffer 2. Fluorescence intensity was monitored using the microplate reader with 488 nm excitation and 532 nm emission for 30 min.The effect of surfactants on the binding of HgThe effect of surfactants on the binding of adenosine by its aptamer was evaluated using a 96-well black (Corning) plate in Buffer 1 containing 1 µM aptamer, 1 µM ThT, and 0% or 0.01% Triton X-100. The sample solution (100 µL) was titrated with adenosine up to a final concentration of 150 µM. Fluorescence intensity was monitored using the microplate reader at 488 nm excitation and 532 nm emission. For signal stability measurement, 100 µL of sample solution contained, 1 µM aptamer, 100 µM adenosine, 1 µM ThT, and 0% or 0.01% Triton X-100 in Buffer 1. Fluorescence intensity was monitored using the microplate reader with 488 nm excitation and 532 nm emission for 30 min.The effect of surfactants on the binding of cortisol to CSS.1-42 DNA was evaluated using a 96-well black plate (Corning) in Buffer 3 containing 1 µM CSS.1-42 aptamer, 0.1× SGI, and 0% or 0.01% Triton X-100. The sample solution (100 µL) was titrated with cortisol up to a final concentration of 6 µM. Fluorescence intensity was monitored using the microplate reader at 488 nm excitation and 532 nm emission. For signal stability measurement, 100 µL sample solution contained 1 µM CSS.1-42 aptamer, 5 µM cortisol, 0.1× SGI, and 0% or 0.01% Triton X-100 in Buffer 3. Fluorescence intensity was monitored using the microplate reader at 488 nm excitation and 532 nm emission for 30 min.The classic adenosine aptamer stained by ThT was used to study the effect of time-dependent signal stability on the dye staining method ,25. A scFirst, we conducted the titration in a 96-well microplate without waiting (adenosine was titrated immediately after adding the aptamer and ThT mixture). Adding adenosine produced a 65% decrease in this case , compared to a 33% drop without stabilization (fitted Kd = 1.3 ± 0.3). This is concerning since in theory water does not impact the aptamer binding to ThT and thus no fluorescence drop should have occurred. This suggests that the system had a time-dependent signal change even without adding target molecules and we reason that two processes occurred simultaneously to contribute to the fluorescence drop: dye adsorption and aptamer binding.As a further control, water was titrated into the aptamer/ThT samples C. The wiKd compared to adenosine.With stabilization, the effect of dye adsorption was minimized. However, without stabilization, the interpretation of the results can be misleading. Despite this, it was still possible to differentiate between binding and non-binding targets based on the results in Kd of adenosine binding to the aptamer was 16.6 ± 8.4 μM with stabilization and 17.5 ± 8.7 μM without stabilization. In serum, the Kd was 20.9 ± 2.7 μM with stabilization and 9.5 ± 2.1 μM without stabilization Using ITC, we obtained a Kd of 16.4 μM under similar buffer conditions and DNA samples ,3433,34]Recently, many new aptamers have been reported, ,35,36,37Kd and kinetic information, the dye adsorption problem can be mitigated using either a long waiting time with agitation or by adding a surfactant such as Triton X-100. Using this surfactant, we tested three aptamers for Hg2+, adenosine, and cortisol. In each case, a more stable signal was observed with the surfactant, although a target concentration-dependent fluorescence change was achieved even without the surfactant. In addition, using a quartz cuvette is recommended over a plastic microplate to achieve more accurate Kd measurement.Second, for quantitative measurements to obtain values such as Overall, we articulated the signal stability problem of DNA staining dye-based aptamer binding assays and provided a few solutions to solve this problem to generate more reliable measurements and biosensors.


The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein’s tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of βγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with C To transmit and focus light onto the retina, the human eye lens needs to be transparent with a high refractive index of approximately 1.33 ,3.Ubiquitous crystallins include two large gene families, the α and βγ-crystallins. α-crystallins are members of the small heat shock protein family and act as chaperones , while β−1 to the protein when they are perpendicular to each other [−1 stability to the protein by parallel displacement stacking [Five aromatic pairs are present in β-hairpin sequences in human γS-crystallin. Of these, four pairs exist in Greek key motifs and are termed Greek key pairs  and one falls outside the Greek key motifs, termed a non-Greek key pair (Y21-Y33). Three of the homologous aromatic pairs  and two of the non-homologous aromatic pairs (Tyr/Phe) are present in the β-hairpins of the human γS-crystallin protein. Energetically, the optimal distance between these aromatic rings is 4.5–7 Å, either perpendicular or parallel to each other . They coch other  or aboutAny mutation that substitutes a non-aromatic residue for the crucial phenylalanine or tyrosine in an aromatic pair is predicted to destabilize βγ-crystallin folding severely. A dinucleotide substitution NM_017541.4:c.30_31delinsAA resulting in a NP_060011.1:p.(F10_Y11delinsLN) substitution in human γS-crystallin has been reported to be associated with autosomal dominant cortical lamellar cataracts with juvenile onset .The backbone conformation of γS-crystallin samples was monitored by following the circular dichroism (CD) in the far-UV region (185–260 nm). The far-UV CD spectrum of γS-crystallin displays a strong negative band at 218 nm and a small arm around 206 nm, both of these peaks being characteristic indicatives of antiparallel β-sheet and α-helices A. Wild-tDetails of the tertiary structure were assessed by measuring the circular dichroism of each protein in the near-UV range (250–350 nm) and recording the tryptophan fluorescence in the 300–400 nm range by selectively exciting the protein at 295 nm. Wild-type and mutant near-UV CD spectra showed similar peaks with differing intensities of molar ellipticities B. This dTo further investigate the exposure of hydrophobic amino acids to the protein surface, two extrinsic probes, bis-ANS   and Nilem values  shows similar results  at various temperatures using the equation ΔG° = −RTlnK, where R is the gas constant 1.987 kcal KN mutant . OverallThe thermal stability was also estimated by measuring the secondary structure of the wild type and F10_Y11delinsLN mutant at the transition midpoint of the mutant (61 °C) with different incubation times from 200 to 900 s. The spectra of the wild type were similar at all time points , but the−1 and 8.4 kcal mol−1, respectively, while for the wild type transition it is 11.7 kcal mol−1. The refolding curves and their derived ΔG plots show similar trends  on size exclusion chromatography . Its farAssociation of αA-crystallin with wild-type and mutant F10_Y11delinsLN γS-crystallin under benign and thermal stress conditions were tested by heating the protein samples at 60 °C and checking for light scattering by particles at 600 nm. Wild-type γS- and αA-crystallins show little light scattering over the entire 1200 s exposure, while the mutant alone shows increasing levels of light scattering particles beginning at 400 s . A 1:1 mDynamic light scattering was also used to check the particle size of wild-type and mutant γ-crystallin proteins with and without αA-crystallin under benign conditions and thermal stress. Wild-type γS-crystallin maintains small radius equivalents, below 20 nm through 50 °C, and shows particles with radius equivalents of 90 and 200 nm, at 60 °C. αA-Crystallin maintains a radius equivalent below 20 nm, as does a 1:1 mixture of wild-type γS- and αA-crystallin A,B,E,F. Association of αA-crystallin with wild-type and mutant γS-crystallin in their native and stressed states was also checked by analytical size exclusion chromatography. Mixed wild-type γS-crystallin and αA-crystallin displayed two elution peaks around 9–13 mL (500 kDa) and 16–18 mL (20 kDa) A, as didThe effects of reducing agents DTT and TCEP at various concentrations in suppressing thermal aggregation were examined using light scattering measurements. Both DTT and TCEP suppress aggregation to some extent compared to that seen in their absence, including the early linear phase of the curve and the later plateau region . While tThe γ-crystallins have extremely rigid three-dimensional structures with tight packing . Five ar2) (F10 to L10) and 229 to 158 (Å2) (Y11 to N11) [The total accessible surface area that is occupied by the changed amino acids is decreased from 218 to 180 (Å58 (Å2) Y to N11 [ to N11) , and sho to N11) . However to N11)  and the While the increased exposure of hydrophobic residues to the protein surface indicates an opening of the protein fold, the circular dichroism and tryptophan fluorescence studies indicate that the mutations have only a minor effect on the overall protein structure under benign conditions approximating physiological exposures. However, the decreased stability is still present and simply becomes more obvious under stress, which accelerates the structural changes. Thus, while denaturation and aggregation would be much slower under physiological conditions, proteins in the lens fiber cells do not turn over, and even minor instability will manifest itself in structural changes over the months of prenatal development and especially the months and years of childhood and adult exposure to environmental stress. While the lens has large amounts of α-crystallins that act as chaperones to bind unstable and partially denatured proteins as well as high concentrations of reducing agents such as glutathione, these are eventually overcome, resulting in clinical cataract, either by scattering light directly or through damaging the lens cells and disrupting the lens microarchitecture . In gene−1 and lower the transition midpoint to 12.7 °C in thermal unfolding experiments monitored by CD. The energy change observed from fluorescence-based thermal unfolding data was similar, 4.3 kcal mol−1. There is a slight difference in ΔH values and slopes in the unfolding procedures, although this is probably due to differences in the methods with regard to holding times and monitoring [The mutations do destabilize γS-crystallin significantly under both thermal and chemical stress. In combination, the mutations decrease the overall stability to 5.0 kcal molnitoring . The mutnitoring ,13.m [−1 to overall protein stability [m by 9.5 °C, similar to the results seen here. An even greater destabilization of the second Greek key motif was seen in the Y46D γC-crystallin mutant, which showed a ΔTm of −23 and 24.4 °C with CD and fluorescence, respectively, for the native to intermediate form and −9.9 and 9.3 °C for the intermediate to unfolded species corresponding to Δ(ΔG) values of −11 and −4.7 kcal mol−1 for the CD curves and −9.1 and −3.5 kcal mol−1 for the fluorescence curves. Similarly, GuHCl unfolding curves yielded ΔG values of 3.2 and 6.2 kcal mol−1 for the intermediate and unfolded Y46D mutant species, respectively vs. 10.5 for the wild type [The role of aromatic pair interactions on the stability of the human γD-crystallin protein has been well studied. N-terminal domain substitutions affect the stability of the protein by shifting transitions of that domain to lower GuHCl concentration while the C-terminal domain transitions remained relatively unaffected. In addition, Greek key pairs tend to contribute more to the thermal and thermodynamic stability of the domain than non-Greek key pairs . The Y11m , similarm  and a sitability ,9, suppotability , in whicWhile both intrinsic fluorescence and ANS and Nile Red binding studies demonstrate that the surface hydrophobicity of the F10_Y11delinsLN mutant γS-crystallin is increased, size exclusion chromatography of αA-crystallin alone and mixed with wild-type and F10_Y11delinsLN mutant γS-crystallins shows that αA-crystallin does not bind either the wild-type or mutant protein to any appreciable extent under physiological conditions. However, as the F10_Y11delinsLN mutant γS-crystallin is heated at 60° both light scattering at 600 nm and dynamic light scattering show that it forms HMW aggregates, and that this process is prevented by a 1:1 molar ratio of αA-crystallin. These studies show that the F10_Y11delinsLN mutant γS-crystallin does not escape binding by αA-crystallin as some rapidly denaturing proteins do ,25. In cNdeI/XhoI sites of pET-20b (+)  as described earlier [DpnI, and the remaining plasmid was transformed into E. coli DH5α. Human αA-crystallin cDNA was cloned into the NdeI/XhoI sites of pET-21a (+) (Novagen) using a reverse primer harboring a CACCACCACCACCACCAC (6X his tag) sequence just before the stop codon. Plasmids were isolated from the transformed E. coli colonies and bidirectionally sequenced to confirm the appropriate base pair changes and the absence of nonspecific mutations.Human γS-crystallin cDNA was cloned into the  earlier . To gene600 of 0.6–0.8. The cultures with the F10_Y11delinsLN bacterial clone were adjusted to 0.25 mM IPTG and grown at 25 °C for an additional 6 h, whereas the cultures with wild-type γS and αA clones were induced with a final concentration of 1 mM IPTG and grown at 37 °C for an additional 4 h. Induced cells were pelleted via centrifugation at 6000× g for 10 min at 4 °C and frozen at −80 °C until use.Proteins were overexpressed and purified as described earlier ,30. BL21g for 20 min at 4 °C, and the supernatant was loaded into an 8 mL 10,000 MWCO dialysis cassette  and dialyzed against 2 L of buffer A  for 6 h.Pellets containing overexpressed proteins were resuspended in 50 mM Tris-Cl pH 7.3, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.25 mM TCEP, and a protease inhibitor mix . A final lysozyme concentration of 0.3 mg/mL and a final DNase concentration of 7.5 µg/mL were maintained in the suspensions. Cell suspensions were freeze–thawed for 3 cycles, each cycle consisting of 5 min incubation on dry ice and 5 min incubation in a 37 °C water bath. The freeze–thawed cell suspensions were further sonicated at 4 °C and 20% amplitude with an Omni international Sonic Ruptor-400  using 9 cycles for the mutant, with each cycle comprising a 5 s pulse on and a 55 s pulse off, and using 6 cycles for the wild type, with each cycle comprising a 10 s pulse on 50 s pulse off. The cell lysate was centrifuged at 30,000× g for 20 min at 4 °C, and samples were loaded onto a 5 mL HiTrap SP FF  column equilibrated with buffer A at a flow rate of 1.0 mL/min. The column was washed with five column volumes of buffer A. Then, a linear gradient of buffer B  was applied and fractions were collected.Soluble extracts were purified at room temperature with ion-exchange and size-exclusion chromatography using an NGC™ Chromatography System . The dialyzed supernatant was centrifuged at 30,000× 2PO4/K2HPO4 pH 8.0, 300 mM KCl, 1 mM EDTA (buffer C) with a protease inhibitor mix . Sonication was carried out as described above for the wild-type protein , and the centrifuged cell lysate was loaded onto a HisTrap HP  column equilibrated with buffer C at a flow rate of 1.0 mL/min. Five column volumes of buffer C were passed through the column to wash away the unbound proteins completely, then, a linear gradient of buffer D  was applied and fractions were collected.αA-Crystallin was also purified using a modification of the above-described method. The lysis buffer used in purifying αA crystallin was 50 mM KH® S-100 HR column  previously equilibrated with SEC buffer (50 mM NaH2PO4/Na2HPO4 (pH 7.3), 0.15 M NaCl, 1 mM EDTA, 1 mM DTT, and 50 μM TCEP) at a flow rate of 0.5 mL/min. Pooled wild-type αA-crystallin was injected on to a HiPrep™ 16/60 Sephacryl® S-300 HR column  equilibrated with SEC buffer at a flow rate of 0.5 mL/min. The columns were pre-calibrated with five standards: thyroglobulin, γ-globulin, ovalbumin, myoglobin, and vitamin B12 . The locations of recombinant proteins in column fractions were monitored using absorbance at 280 nm and by SDS-PAGE on 12% polyacrylamide gels. The purity of the protein was assessed and confirmed by the presence of a single band in SDS PAGE and then estimating the mass by using electrospray ionization mass spectrometry. Two independent preparations for each batch of αA, γS wild-types, and mutant proteins gave an average mass of 20,730.32 ± 0.5, 20,873.18 ± 0.5, and 20,789.76 ± 0.5 Da, respectively, in close agreement with the predicted monomer masses.The fractions containing the proteins of interest were monitored with absorbance at 280 nm and SDS-PAGE on 12% polyacrylamide gels, and were pooled and concentrated to 10–15 mg/mL. Wild-type and mutant γS-crystallin protein pools were separately loaded onto a HiPrep™ 16/60 SephacrylMass spectrometry data of 0.05 mg/mL proteins samples (in 50 mM Tris-Cl (pH 7.3), 150 mM NaCl, and 50 µM TCEP buffer) were acquired on an Agilent 6130 Quadrupole LC/MS System,  equipped with electrospray source, operated in positive-ion mode. Separation was performed on a 300SB-C3 Poroshell column . The analytes were eluted at a flow rate of 1 mL/min with a 5–100% organic gradient over 5 min and holding organic for 1 min. Mobile phase A contained 5% acetic acid in water, and mobile phase B was acetonitrile. Data acquisition and data analysis and deconvolution of mass spectra were performed using OpenLab ChemStation Edition software (version C.01.05).2PO4/Na2HPO4 (pH 7.3) were run between 260 and 185 nm at 25 °C in a 1 mm pathlength cuvette using a J-1500  circular dichroism instrument. For near-UV CD spectra, 1.0 mg/mL samples in 50 mM NaH2PO4/Na2HPO4 (pH 7.3), 150 mM NaCl, and 50 μM TCEP were analyzed between 350 and 250 nm at 25 °C in a 1 cm pathlength cuvette. A bandwidth of 1 nm, scanning speed of 20 nm/min for far-UV CD and 50 nm/min for near-UV CD and a digital integration time of 4 s at each point were maintained. At least five independent runs were measured and averaged, and spectra for the corresponding buffer for each protein were subtracted. For determining the secondary structure of wild-type and mutant proteins under GuHCl-induced stress, 0.15 mg/mL protein samples in 10 mM NaH2PO4/Na2HPO4 (pH 7.3) were incubated with varying concentrations of GuHCl (0–5 M) for 12–14 h at room temperature, and circular dichroism was measured between 260 and 185 nm at 25 °C in a 1 mm pathlength cuvette as described above. Three independent runs were measured and averaged, and blank spectra of the corresponding buffer were subtracted for each protein. Analysis of the CD spectra was performed as previously described by Fasman and coworkers [For determining secondary structure, 0.2 mg/mL protein samples in 10 mM NaHoworkers ,32.2PO4/Na2HPO4 (pH 7.3), 150 mM NaCl, and 50 μM TCEP. An equilibration time of 10 s at each temperature was maintained. Thermal denaturation curves were analyzed using GraphPad Prism 7 software according to the method of C.N. Pace [Thermal denaturation experiments were performed by heating the test proteins from 25 to 95 °C with a ramping rate of 1 °C/min and collecting the 218 nm molar ellipticity using the same CD instrument as above. The protein concentration was 0.5 mg/mL in 50 mM NaH.N. Pace . The ent2PO4/Na2HPO4, 150 mM NaCl, 1 mM EDTA, 5 mM DT, and 50 μM TCEP were excited with 295 nm wavelength light and emissions were collected between 300 and 400 nm using a FluouroMax-4C Spectrofluorometer .For measuring tertiary structure using tryptophan fluorescence emission, 0.05 mg/mL protein samples in 52.5 mM NaH2PO4/Na2HPO4 (pH 7.3), 150 mM NaCl, 1 mM EDTA, 5 mM DTT, and 50 μM TCEP were incubated with varying concentrations of GuHCl (0–5 M) for 12–14 h at room temperature and tryptophan emission was collected between 300 and 400 nm at 25 °C by exciting the samples at 295 nm. Three independent runs were measured and averaged, and the values for the corresponding buffer were subtracted as blanks for each protein.For determining the tertiary structure of wild-type and mutant proteins under GuHCl-induced stress, 0.1 mg/mL protein samples in 50 mM NaH2PO4/Na2HPO4 (pH 7.3), 150 mM NaCl, 1 mM EDTA, 5 mM DTT, and 50 μM TCEP were incubated with a final concentration of dye ranging from 0.58 to 74.31 μM. For Nile Red experiments 0.1 mg/mL protein samples in 55 mM NaH2PO4/Na2HPO4 (pH 7.3), 150 mM NaCl, 1 mM EDTA, 5 mM DTT, and 50 μM TCEP were incubated with between 0.61 and 19.63 µM of the dye in the dark at room temperature for 30 min. Spectra for bis-ANS were recorded between 400 and 600 nm by exciting the samples at 390 nm and for Nile Red samples were recorded between 570 and 700 nm after excitation at 540 nm. Fluorescence intensities at 490 nm (bis-ANS) and 650 nm (Nile Red) were plotted against dye concentration.Surface hydrophobicity and aggregation propensities were assessed using bis-ANS  and NileFor GuHCl unfolding experiments , samplesGuHCl unfolding and refolding curves were analyzed by plotting the concentration of GuHCl for each sample versus the ratio of fluorescence intensities at 355 nm (maximum for the unfolded protein) and 330 nm (maximum for the native protein). The ratio of fluorescence intensities at these wavelengths was chosen for the analysis to allow simultaneous monitoring of changes in the native and unfolded maxima. Equilibria unfolding/refolding data were analyzed using GraphPad Prism 7 software, and ΔG° and m values were calculated using the method of C.N. Pace [For determining thermal aggregation, a final concentration of 2.5 µM protein in 50 mM Tris-Cl (pH 7.3) and 150 mM NaCl was heated at 60 °C for 1200 s, and static (Rayleigh) light scattering at 600 nm was monitored using a spectrofluorometer for 1200 s with a 1 cm path length quartz cuvette. An equilibration time of 2 min at 60 °C prior recording the scattering was maintained. To check the effect of DTT and TCEP on thermal aggregation 2.5 µM protein in 50 mM Tris-Cl (pH 7.3), 150 mM NaCl with varying concentrations of TCEP and DTT was heated at 60 °C for 1200 s and light scattering at 600 nm was monitored.All fluorescence experiments were carried out at 25 °C with 2.5 nm excitation and emission slits while maintaining 1 s integration time at each point. The excitation and emission slits used in the GuHCl unfolding experiments were 5 nm, and 10 nm excitation and emission slits were maintained in the refolding experiments.Thermal denaturation was also probed with tryptophan emission using a differential scanning fluorimeter . The same protein concentrations and buffer strengths were maintained as for the CD thermal unfolding experiments. Samples were heated from 25 to 95 °C while being excited at 285 nm, and the 350/330 nm emission ratio was collected as a function of temperature.Wild-type and mutant protein particle sizes and distributions were assessed with dynamic light scattering using a DynaPro NanoStar dynamic light scattering instrument . Protein solutions of 25 µM in 50 mM Tris-Cl (pH 7.3) and 150 mM NaCl buffer were illuminated using a 658 nm laser, and light scattered at a 90° angle was measured from 30 to 60 °C in 10 °C increments. A minimum of 20 acquisitions at each temperature step were recorded. Each protein solution was analyzed twice, and the average of all acquisitions is presented. A 1 °C/min ramping rate and an equilibration time of 2 min at each temperature was maintained before collecting acquisitions. To obtain the hydrodynamic radii the intensity autocorrelation functions were fitted by a regularization algorithm using Dynamics software version 7.10 .Protein at a final concentration of 37.5 µM in 50 mM Tris-Cl (pH 7.3) and 150 mM NaCl was injected into a 24 mL ENrich SEC650 (Bio-Rad #780-1650) column equilibrated with 2 column volumes of 50 mM Tris-Cl (pH 7.3) and 100 mM NaCl. A flow rate of 0.5 mL/min was maintained, and the sample volume injected was not more than 1% of the column volume (240 μL). αA-crystallin was assessed by mixing αA-crystallin and F10_Y11delinsLN mutant γS-crystallin at an 8:1 molar ratio at room temperature and stress conditions before chromatography.https://www.rcsb.org, accessed on 30 May 2023) [The crystal structure of wild-type human γS-crystallin was taken from 7N36 in the RCSB PDB (ay 2023) . The modThe F10_Y11delinsLN change relaxes the protein fold of the N-terminal Greek key motif under benign conditions, decreasing its stability under thermal or chemical stress, and also that of the N-terminal domain overall, leaving the C-terminal domain relatively unaltered initially. The partially denatured F10_Y11delinsLN γS-crystallin exists in a molten globule state and is bound by α-crystallin, eventually forming light-scattering HMW aggregates or possibly actually overwhelming the supply of α-crystallin in the lens and precipitating and damaging lens cells themselves. A combination of these processes then leads to the cortical lamellar cataract seen in the family with this mutation.


Efficient small ruminant production depends upon the knowledge of re productive physiology. This study provides knowledge about the correlation among physical and behavioral signs of estrus with ovarian dynamics and hormonal profiles such as estradiol-17β and progesterone after induced luteolysis during the preovulatory period in Barbari goats. The findings of this study will be helpful in developing better estrus synchronization strategies for improving the reproductive efficiency of Barbari goats. There is an enormous goat population in Asia, especially in China, India, and Pakistan. Different estrus synchronization protocols such as Ovsynch and PGF2α injection, administered indiscriminately during the breeding season of Barbari goats. Ovarian dynamics were observed at every 12 h interval by using B mode ultrasonography, blood samples for hormonal analysis such as estradiol 17β and progesterone were collected at every 12 h interval, and bucks with aprons were used to identify standing estrus at every 6 h interval. Relative to PGF2α, the start of standing estrus and ovulation differ (p < 0.05) between early- (n = 7), intermediate- (n = 6), and late-responding (n = 6) goats. The highest plasma level of estradiol 17β was detected 12 h prior to ovulation. The average diameter of the ovulatory follicle and length of standing estrus were comparable (p > 0.05) between the goats. The corpus luteum degenerated more quickly (p < 0.05) in early- than intermediate- and late-responding goats. Dominant follicle diameter and estradiol 17β concentration also differ (p < 0.05) among groups. Although the plasma level of progesterone did not vary (p = 0.065), the variation in progesterone concentration with time differed (p < 0.05) amongst the goats. As a result, this research indirectly reveals that the beginning of standing estrus, end of estrus, and ovulation after PGF2α might fluctuate in Barbari goats because of follicular and hormonal dynamics during the luteal phase.The purpose of the present research was to define ovarian follicular dynamics and plasma endocrine profiles in response to a single PGF There is continued demand for increased meat production in the Asian subcontinent due to the enormous human population. In this circumstance, an efficient animal production system is required to ensure food security. Approximately 46.9% of the total world goat population belongs to China, India, and Pakistan FAO, 2017) [7 [1]. Ba2α (PGF2α) and its analogs are extensively used for estrus synchronization during the breeding season  2017-0044. Overall, nineteen healthy, multiparous, cycling goats with body condition score within the range 2.5 to 4 and body weight within the range 30 to 40 kg were raised in an unrestricted stall and were given green fodder , concentrate with corn gluten, corn grain, wheat bran, soybean meal, canola meal , and water ad libitum. This experiment was conducted during the breeding season (September–December 2021).2α  randomly upon detection of the corpus luteum. Ovaries were examined transrectally every 12 h interval after PGF2α (0 h) up to ovulation using a 7.5 MHz transducer  as defined earlier [These nineteen goats were selected from a flock of thirty goats based on detection of the corpus luteum via ultrasonography. All goats (n = 19) were administered a single dose of PGF earlier . Ovulati earlier . Standup earlier .2α was administered upon detection of the mature corpus luteum between (4–16) days, and the 1st blood sample was collected for hormonal analysis. The second blood sample was collected after a 24 h interval, and subsequent sampling was conducted every 12 h interval until ovulation. Ultrasound was performed every 12 h interval to define ovarian follicular and luteal dynamics throughout preovulatory time until ovulation. Bucks with aprons were used to distinguish goats in standing estrus every 6 h interval. These goats (n = 19) were further divided into early- (n = 7), intermediate- (n = 6), and late-ovulating (n = 6) goats due to the significant variation in onset of estrus and ovulation because of different ovarian and hormonal status at the time of synchronization protocol.This experiment was conducted during breeding season from September to December 2021. Overall, fifty (n = 50) Barbari goats at research and training station Pattoki were scanned to select appropriate goats for further experimentation. From (n = 50) goats, twenty goats (n = 20) were already pregnant and inappropriate for the experiment. Eleven goats (n = 11) were in the very early (≤4 days) or late (≥17 days) luteal phase, and some were already in estrus and were excluded from consideration. Nineteen goats (n = 19) used in experiments were physically healthy, free from any history of reproductive disorder, and multiparous with an age of approximately 3.5 ± 0.3 years. A single shot of PGFIn general, research related to reproductive physiology and biotechnology is conducted with a large herd size. We selected n = 19 goats, which could be considered a reasonable sample size if not for the fact that goats respond significantly differently from each other due to variable ovarian status. In addition, the small sample size of this research was due to funding limitations and management and seasonality issues in Barbari goats. The scope of the research could definitely be increased if it is conducted with a larger herd size with short blood sampling intervals for hormonal analysis and ultrasonography for more precise information about hormonal profile association with ovarian dynamics, estrus, and ovulation.2α administration from the jugular vein in 5 mL vacutainer tubes containing EDTA. Immediately after collection, all samples were centrifuged at 3000× g for 15 min to isolate plasma, which was kept at −20 °C until further use. A double antibody radioimmunoassay method was used to estimate the plasma concentration of estradiol 17β and progesterone  as defined earlier [Blood samples for the endocrine profile test were collected at 0, 24, 36, 48, 60, 72, 84, and 96 h following PGF earlier . These kp ≤ 0.05. Data were normalized before comparing the means. Data of the corpus luteum and progesterone were normalized with 1/x transformation, while estradiol 17β data were normalized by Log10 transformation. The Pearson coefficient of correlation was used to analyze relationships between estradiol 17β and the diameter of the preovulatory follicle as well as among progesterone and the corpus luteum. Variation in the interval to onset of standing estrus between early-, intermediate-, and late-responding goats was analyzed using one-way ANOVA. Analyses of pooled variances among early-, intermediate-, and late-responding goats were carried out for regression of the corpus luteum, progesterone concentration, and diameter of preovulatory follicles relative to PGF2α through the generalized linear model using SPSS .The data are presented as means ± SEM, and statistical significance was measured at 2α treatment (100% response). The overall length (n = 19) of standup estrus was 22.3 ± 2.8 h. The overall time from the start of estrus to ovulation was 32.5 ± 4.4. The overall time to ovulation from the end of standing estrus was 9.4 ± 1.5 h  in the beginning of standing estrus and time of ovulation were detected between the goats , at 51 ± 3.0 h , and at 60 ± 0.0 h . The mean duration of standing estrus and the interval to ovulation from the end of standing estrus did not differ (p > 0.05) between the early-, intermediate-, and late-responding goats (p > 0.05) between the early-, intermediate-, and late-responding goats (All goats (n = 19) showed standing estrus after 50.6 ± 4.8 h of PGF ± 1.5 h . Relativhe goats . The onsng goats . Similarng goats .2α. The diameter of the largest dominant follicle was greater (p = 0.00) than second-largest dominant follicle, i.e., 7.3 ± 0.3 and 6.6 ± 0.3, respectively (p = 0.003) with the mean diameter of preovulatory follicles. The highest plasma quantity of estradiol 17β was detected 12 h. previous to ovulation and standing estrus ended three hours later (p = 0.005) following PGF2ά (Follicular dynamics exposed that the majority of goats had two dominant follicles (13/19 = 68%) throughout the periovulatory period after PGFectively A. Howeveectively B. The plrs later . The meang PGF2ά . A similng PGF2ά .2α administration. The diameters of the first and second corpora lutea 12 h after PGF2α were 9.6 ± 0.2 and 9.2 ± 0.6, respectively. Both corpora lutea degenerated with time  after PGF2α. The diameters of the corpora lutea did not vary (p = 0.42) from each other until ovulation (p = 0.00) with a decline in the combined diameter of the first and second corpora lutea after PGF2α  (p = 0.065) between early-, intermediate-, and late-responding goats  . On the ng goats .2α injected indiscriminately through the luteal stage in Barbari goats. Overall, one hundred percent estrus response to single PGF2α was achieved in this study, which is similar to a prior report in Beetal goats synchronized with two injections of PGF2α [2α were also congruent with the findings in Shiba goats treated with a single dose of PGF2α [2α treatment in Beetal goats [2α during the luteal phase in Barbari goats. On the other hand, the overall estrus response of 100%, start of standing estrus of 50.6 ± 4.8 vs. 49.2 ± 3.7 h, and interval to ovulation of 82.3 ± 4.0 vs. 77.2 ± 3.4 h in our study are comparable with a previous study in Beetal goats using single PGF2α, injected upon confirmation of the mature corpus luteum using ultrasonography [The current study highlights the ovarian dynamics and plasma concentration of the hormonal profile in response to single PGFof PGF2α . The staof PGF2α . Howeveral goats , both thnography .2α was correlated with deviation in follicle expansion during the luteal stage [2α [2α administration as compared to the late-responding goats  or late (day 16) luteal phase than the mid-luteal phase (day 11) [2α in early compared to intermediate- and late-responding goats  ,36. In a(day 11) . Correspng goats . Evidencin goats . Moreoveresponse . Hence, 2α treatment in the breeding season.In our study, estradiol 17β concentration in relation to the start of standing estrus followed a similar trend to that of Shiba goats . However2α administration, significantly affect the outcome of reproductive physiology, estrus, and ovulation in Barbari goats. That distinction is considerably controlled by the size of the developing preovulatory follicle and corpus luteum on the day of PGF2α injection. In this study, the majority of goats (68%) had two dominant follicles at the time of injection, which grow significantly (p = 0.00) with the passage of time and became mature preovulatory follicles, but their growth rates did not diverge (p = 0.124) from each other until ovulation. Similarly, most of the goats 68% had two corpus lutea at the time of synchronization, which regressed significantly (p = 0.00) with the passage of time, but their regression rates did not fluctuate (p = 0.93) from each other until ovulation. Furthermore, there was a higher correlation (r = 0.98) between the combined diameter of preovulatory dominant follicles with estradiol 17β concentration after every ultrasonographic scan. Regression of the corpus luteum (CL) and a decline in progesterone level were also highly associated (r = 0.93) until ovulation. The diameter of preovulatory dominant follicles attained maximum ovulatory size faster in early- than late-responding groups (7.5 ± 0.4 mm vs. 6.5 ± 0.3 mm). Similarly, the preliminary concentration of estradiol 17β was dissimilar among the goats, which resulted in a variable surge of estradiol 17β of 48 vs. 60 vs. 72 h in goats. Contrary to that, diameter of the corpus luteum (CL) 12 h post-PGF2α shot was smaller in early than intermediate- and late-responding goats (8.4 ± 0.4 mm vs. 8.9 ± 0.5 mm vs. 10 ± 0.5 mm). Similarly, there was a lower plasma concentration of progesterone at the time of treatment in some goats, which resulted in a rapid decline in progesterone level within 24 h in early groups compared to other groups (11.2 ± 0.3 vs. 19.1 ± 0.1 vs. 21.5 ± 0.5 ng/mL). Due to this, the start of estrus, peak estradiol 17β concentration, decline in progesterone concentration, and ovulation timing were considerably diverse among different goats. Moreover, this is the first ever major study profiling follicular dynamics and ovarian steroids after induced luteolysis during the preovulatory period in Barbari goats. In future, the prospects of the research might be improved if PGF2α treatment is conducted on identified days of the estrus cycle with a larger sample size, frequent blood sampling, and ultrasonography to determine a more precise timing of estrus and ovulation in Barbari goats.In conclusion, the discrepancy of ovarian follicular dynamics and variability in hormonal profile, like estradiol 17β and progesterone, at the initiation of synchronization protocol, specifically PGF