This nonlinear behavior, combined with ultrafast dynamicfor excited carriers, proved to be essential for third harmonic generationin the sub-THz and low (<2.5 THz) THz range, using moderate (60kV/cm) fields and at room temperature. Here, we show that, for monochromatichigh peak power (1.8 W) input THz signals, emitted by a quantum cascadelaser, the nonlinearity can be controlled using an ionic liquid gatethat tunes the graphene Fermi energy up to >1.2 eV. Pump and probeexperiments reveal an intense absorption nonlinearity at 3.2 THz,with a dominant 3rd-order contribution at EF > 0.7 eV, hence opening intriguing perspectives per engineeringnovel architectures for light generation at frequencies > 9 THz.Graphene is a nonlinearmaterial in the terahertz (THz)frequencyrange, with χ This usually requires alteringthe material absorption and refractive index through electronic effects,9 thermally induced effects,10 or through optical pumping with intense (hundreds kV/cm)electric fields.11Nonlinear optical devices, needed forultrashort pulse generation,(3) ∼ 10–9 m2/V2 in the far-infrared)12 and it is thus ideal for integration into nonlinear optical devices.15 SLG has zero band gap and large electrical tunability, which shiftsthe Fermi energy EF, and induces Pauliblocking of the optical transitions.18 It also exhibits saturableabsorption across a broad range of frequencies,21 large nonlinearoptical response22 with a power transmission modulationof ∼ 50% per monolayer,21 ultrafast photoexcitation dynamics and recovery time,23 high chemical and mechanical stability,24 large thermal,25 and optical thresholddamage,26 and it is also an extremely efficientTHz frequency multiplier, with a huge 3rd-order nonlinearity,28 allowing for the direct generation of multiple harmonics in the0.85–2.12 THz range.28Atomic thin activelayers offer the advantage to prevent dephasingand dispersion of the propagating wave, if compared with III–Vsemiconductors. Single-layer graphene (SLG) is a highly nonlinearmaterial ,31 exhibitinglow saturation intensity (<6.7 W/cm2)31 and transparency modulations up to 80%,31 tunable SA mirrors,31 opticalfrequency combs,32 for drivingsemiconductor heterostructure lasers in the mode-locking regime,33 for engineering SAs for compact pulsed lasersin the microwave region,34 and for manipulatingthe polarization state of THz waves.35The strong opticalnonlinearity38 This, in turn, depends on EF and canbe controlled through electrostatic gating,37 a property that discloses the potential of graphene for THz all-electronicreconfigurable modulators.30 Increasingthe density of free carriers in SLG, and hence EF, enhances the power absorption of the THz driving field.16 Once the SLG carrier density is increased upto extremely high values (n > 1014 cm–2),40 it induces a strongly metallic-likebehavior with a larger electronic heat capacity,25 which reduces the SLG thermodynamic nonlinearity, meaningthat, at THz frequencies, an optimal EF exists, which favors nonlinearity.At the low THz photon energies (4–12 meV), the SLG opticalconductivity can be assumed to match the electrostatic one.4 four-wave mixing,41 and self-phase modulation42 via saturablephotoexcited-carrier refraction have been reported in graphene, asan effect of single or multicycle driving fields with peak electricfields up to MV/cm and peak intensities up to TW/cm2 inthe near-infrared.4 At low (≤2.15THz) THz frequencies, the SLG nonlinearity, both for ultrashort single-cycleand quasi-monochromatic multicycle input signals, has been controlledusing electrical gating and peak electric fields up to 80 kV/cm.28A variety of opticallyinduced nonlinear phenomena such as HGG,EF. For low gate voltage (VG) (<1 V) and negligible leakage current (<100 nA/cm2), we reach EF ∼ 1.2 eV, modulatingthe SLG broad-band linear optical conductivity in the THz range. The EF dependence of the SLG absorption saturationis then assessed by means of open-aperture z-scanexperiments. Pumping with a high-power (1.8 W) 3.2 THz quantum cascadelaser (QCL), in a pump and probe configuration, reveals signaturesof intense 3rd-order nonlinear absorption at 3.2 THz. We hence demonstratethe effects of EF on SA and provide evidenceof the associated nonlinear carrier dynamics in gated graphene, elucidatingthe role of hot electrons in eliciting thermal and field-driven 3rd-ordernonlinearities in highly doped SLG. This may lead to the on-chip integrationof nonlinear electrically pumped laser sources in unexplored frequencydomains (6–10 THz).Here, we investigate the nonlinear responseof SLG at frequencies> 3 THz, exploiting an ionic liquid gate to tune 44 is fabricated on a0.5 mm-thick quartz substrate, showing a 75% transparency in the THzrange and no evidence of optically induced nonlinear effects.21 An electrolyte ionic liquid N,N-diethyl-N-methyl-N-(2-methoxyethyl ammonium-bis-trifluoromethanesulfonyl imide) of99.9% has a large electrochemical power owing to the large surfaceelectric field achievable12 (∼10–20MV/cm), yielding a tunable EF up to 1.2eV.45 A self-forming ultrathin electricaldouble layer (EDL) at the SLG- and metal–electrolyte liquidinterfaces generates electric fields of ∼109 V/mat nanometer scales without electrical breakdown.45 The electrical double layer ensures high charge accumulationcapability over a large area (∼mm2) with a negligible(∼pA) leakage current.45 An 8 × 8 mm2 SLG film is grown on Cu via chemicalvapor deposition (CVD) and then transferred on the quartz substrate,using poly(methyl methacrylate) (PMMA)-assisted wet-transfer, as discussedin 2 , VD = +0.24 V. The residual conductivity stems from backgroundcharge carriers36 and is not altered by VG. By applying increasing VG – VD, the carrierdensity varies, inducing a resistance modulation.T(VG) is normalizedwith that acquired at the CNP, revealing a broad-band modulation acrossthe entire spectral range from 0.1 to >4.5 THz, reflecting theconductivityvariation associated with EF tuning. Wethen extract the modulation depth (47T(ν) at a given VG is related tothe EF-dependent SLG optical conductivityσ(ν) as12Z0 = 377 Ω is the free-space impedance, and n1 = 1.98 and n2 =1.45 are the refractive indexes of the quartz substrate48 and of the ionic liquid gate,49 respectively. The EF-dependentσ(ν) comprises both interband, σinter, and intraband, σintra, terms. At THz frequencies,σinter is dominant, with a constant value of 2.3%per graphene layer, resulting in more than three orders of magnitudelower than σintra.36 Consequently,it is here neglected. The total conductivity can be therefore calculatedas40D0 = EFe2/ℏ2 is the linearDrude weight, e is the electroncharge, ℏ is the reduced Planck constant,Γ0 = evF2/EF μ is the scattering rate, sensitiveto EF and to the carrier mobility μ,and vF is the Fermi velocity. The broad-bandoptical response is slightly modulated (∼20%) by the frequency-dependentintraband conductivity, which, as expected, induces a weaker modulationat higher frequency,17 visible in EF ≤ 1 eV, the experimentaltransmission modulation closely agrees with that calculated from themodel. For EF > 1 eV, the trend ofthetwo curves remains in good agreement, but with the linear model exceedingthe experimental maximum MD by ∼8%.40 Such a discrepancy, related to a decrease in the transmitted signalat higher VG, suggests a dynamic morecomplex than that explained by the aforementioned model when EF > 1 eV, related to the dependence of μfrom EF.50The transmittanceis measured using a purged time domain spectrometer(TDS) over the 0.1–2.5 THz range (Menlo System TeraK15). Eachtransmission trace 2d T(VG) on depth 1d as:46,z-scan experiment  with a few μm-precision stage, and simultaneouslycollecting the transmitted light impinging on the 3 mm diameter opticalwindow of a pyroelectric detector, positioned behind the focal planeat a distance set on purpose to allow the entire scanning of the focalregion. To unveil the nonlinear behavior, we extract the transmittancemodulation along the z-axis, around the focal region,by normalizing the traces acquired from the SLG device, as a functionof VG, with that acquired on a referencedevice with an identical architecture but with no SLG variation of the peak transmittance retrieved at z = 0 for the traces obtained at different EF in VG in The light absorptionas a function of beam intensity is then measuredby moving the sample within and outside the focal region at the oppositecavity vertex, scanning the position along the optical beam axis  = I0/(1 + (z/zR)2) can be written as a function of the beam intensityat the focal point I0 and of the Rayleighlength, zR, which expresses the distance,along the propagation direction of a beam, from the waist to the placewhere the area of the cross section is doubled and is extracted froma knife edge measurement that provides a circular spot size of 0.4mm diameter. We then estimate IS at theCNP by assuming that the normalized transmission intensity, as a functionof the optical power intensity, T(I), follows the same dependence of the absorption coefficient on thepump intensity52 . Both T0 and TS are extracted bynormalizing the z-scan traces from the SLG and fromthe reference sample, hence T0 ≡1. From the fitting procedure ,despite the significantly larger pumping intensity. This is partlyan effect of the longer optical path and mainly the consequence ofthe fact that the detector signal is encoding both the pump on andoff terms, with the on-term contributing only for a small portionof the pulse .We calculate the pump-enhanced transmission 3d by norionband 3c. ThetVG – VD| = 1.0 V, EF =1 eV. We extract the transmission modulation, ΔT(VG)/T = [SSLG (VG) – αSref]/αSref, where SSLG(VG) is the transmission acquired with the SLG at VG, Sref is the signal fromthe reference sample, and α is a normalization parameter sothat ΔT/T = 0 at the onsetof the nonlinear behavior, accounting for the difference of signalmagnitude from the two samples, in the linear regime  optical beam leadsto a nonequilibrium excess distributions of carriers at the energyof the optical pump. At first, the ultrafast (∼20 fs) carrier–carrierscattering27 drives the initial redistributionof the energy absorbed by the photons, leaving the system in a nonequilibriumstate characterized by a hot electron distribution with electronsat temperature Te.53 Then, depending on the type of excitation and on the availablecooling channels, the system relaxes to an equilibrium state.53 Here, owing to pulse duration in the μsscale, the optical excitation varies on a time scale much longer thanthat associated with the heating–cooling dynamics of the SLGcarriers. This effectively realizes a steady excitation state at Te, where a nonlinear modulation of the SLG opticalresponse can emerge, stemming from the temperature-dependent opticalconductivity.Both the Te at the steady excitationstate canbe written as25Tsub isthe temperature at equilibrium (which approaches room temperature25), Pin is the excitationpower density, and τcool is the cooling time. Ce, the SLG electronic heat capacity, dependson EF and on the temperature itself.13 For moderate  to highly doped SLG (EF = 1 eV), this can be expressed as5512 cm–2, corresponding to EF ∼ 110 meV. Therefore, the assumption EF > kbTe for the validity of 25)is essentially always satisfied. Hot electron cooling typically occursvia electron–optical phonon through a biexponential decay,53 with a subpicosecond initial decay related todirect coupling to optical phonons, and a few-picosecond decay dueto the hot-phonon bottleneck.56 It is alsoinfluenced by the dielectric environment and by disorder.58 An accurate estimate of this parameter would require ultrafast pumpand probe experiments. Here, we use τcool to describethe hot electron dynamics in a steady-state excitation, in agreementwith previous reports on SLG on quartz,59 and by combining Te dependence from the power density, shown in EF = 50 meV) and high doping (EF = 1 eV). In order to maximize the temperate increase,it is convenient to operate at low EF <50 meV and, more generally, under low Ce ∼ 0.17 J/(m2·K).sure see 1b and insure see 1c, we reT = Te.61 The transmission variation is defined as:63T0 is theequilibrium transmittance calculated from Tth is the nonlinear transmittancedue to thermal effects, calculated by considering the hot electrontemperature Te in the optical conductivityof Our experimental observations can be described by the complex conductivity of hot electrons. Thetemperature dependence of the intraband conductivity is accountedin E0 is theelectric field amplitude, defined as Thefield-dependent conductivity is then:Jgra (ν) = σfield (ν) × E0 (ν), one can see that the nonlinearterm of the absorption coefficient  of interest for ultrahigh-speedcommunications.We show that the doped SLG pumped withQCLs at >3 THz has a nonlinearbehavior. The retrieved nonlinearity is dominated by hot carriers,with a 3rd-order field-driven nonlinear contribution that affectsthe optical conductivity at high 67 and wet-transferred ona z-cut quartz substrate. Both the as-grown and transferredSLGs are characterized by Raman spectroscopy . A statistical analysis on the as-grown SLGon Cu and on transferred SLG on z-cut quartz is performedto estimate EF and defect density. Fromthe Raman study, we extracted p doping with EF = 250 ± 70 meV,68 which corresponds ton = 4.8 ± 2.6 × 1012 cm–2 68 and a defect density nD = (3.0 ±1.8) × 109 cm–2.69SLGis grown by CVDSI) is realized as follows:CVD SLG is transferred on z-cut quartz, and source,drain, and coplanar gate metal electrodes (5 nm Cr/50 nm Au) are depositedusing a shadow mask. The electrically switchable optical window hasan area of 6 × 6 mm2. The modulator stack is sealed,positioning a 0.1 mm thick quartz substrate on top of the structure).The SLG-gated devicemodulator  and top  contacts.Laser bars are 2 mm long and 180 μm wide.The single-plasmon THz QCL, emittingat 2.68 THz (λ = 111 μm), employed for the experimentsin 18 cm–3) GaAs top contact layer liesbetween the active region and substrate.70The high-powerTHz QCL employed for the experiments described in


Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm’s depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium  Chemical gradients form inevitably during multicellular growth, leading to the establishment of distinct subzones–with different conditions of resource availability–in cellular aggregates and complex organisms . The conThe predominant multicellular structure of the microbial world is the biofilm, an aggregate of cells attached to each other and encased in a self-produced matrix . There hPseudomonas aeruginosa, which is a major cause of biofilm-based and chronic infections in immunocompromised individuals  in a 10 cm × 10 cm × 1.5 cm square Petri dish . Plates were incubated for three days at 25°C in the dark and imaged using a VHX-1000 digital microscope .™ Diamond Antifade Mountant P36965) and overlaid with a coverslip. Fluorescent images were captured using an LSM800 confocal microscope . Samples were illuminated with 488 and 561 nm lasers, respectively. eGFP acquisition parameters were: 400–548nm (emission wavelength range). mScarlet acquisition parameters were: 593–700nm (emission wavelength range). Each strain was prepared in this manner in at least biological triplicates.After three days of growth as described above, biofilms were overlaid with 1% agar and sandwiched biofilms were lifted from the bottom layer and fixed overnight in 4% paraformaldehyde in PBS at 25 °C for 24 h in the dark. Fixed biofilms were washed twice in PBS and dehydrated through a series of 60-min ethanol washes  and cleared via three 60-min incubations in Histoclear-II ; these steps were performed using an STP120 Tissue Processor (Thermo Fisher Scientific). Biofilms were then infiltrated with wax via two separate 2-h washes of 100% paraffin wax (Paraplast Xtra) at 55 °C, and allowed to polymerize overnight at 4 °C. Trimmed blocks were sectioned in 10-μm-thick sections perpendicular to the plane of the biofilm, floated onto water at 42 °C, and collected onto slides. Slides were air-dried overnight, heat-fixed on a hotplate for 30 minutes at 45 °C, and rehydrated in the reverse order of processing. Rehydrated colonies were immediately mounted in (Thermo Fisher Scientific ProLongwww.python.org). Each image was viewed in FIJI for quality control and then binarized using a custom Python script. For each sample, a fixed-threshold binarization was applied to the fluorescence image using a multi-level version of Otsu’s method, which separates pixels of an input image into several classes obtained according to intensity  in a 10 cm × 10 cm × 1.5 cm square Petri dish . Plates were incubated for one day at 25°C in the dark. Biofilms were overlaid with 1% agar, and sandwiched biofilms were lifted from the bottom layer, and fixed overnight in 4% paraformaldehyde in PBS at 25 °C for 12 hours in the dark. Fixed biofilms were washed twice in PBS then incubated for three hours in 70% (V/V) glycerol solution deionized water solution for optical clearing. The clarified biofilms were mounted on a standard microscopy slide, and were imaged with ClearScope  (MBF BioSheared pilin and cell lysate proteins were extracted as previously described  with somBiofilm samples (25 biofilms per strain) were gently scraped from agar, resuspended in 1 mL sterile PBS , and centrifuged at 4000 × g for five minutes at room temperature. Pellets were resuspended in 4.5 mL of sterile PBS, vortexed for 30 s to shear cell surface proteins, and centrifuged at 4000 × g for 15 min at room temperature. Supernatants containing the sheared surface proteins were subjected to multiple rounds of centrifugation at 11,600 × g for 15 min to remove residual cells from the samples. Next, 5 M NaCl and 30% (w/v) PEG  at a volume ratio of 0.1 each were added to the total supernatant, and incubated on ice for 90 min before pilin was collected by centrifugation at 11,600 × g for 30 min at 4 °C. The pelleted pilin was resuspended in 1X LDS sample buffer (Invitrogen) with 5% β-mercaptoethanol (Sigma) and boiled for 5 min before separation on a 4–12% NuPAGE Bis-Tris gel (Invitrogen) for immunoblotting.600 ~ 4.0, centrifuged, and pellets were resuspended in 1X LDS sample buffer with 5% β-mercaptoethanol. Samples were boiled for five min, centrifuged, and aliquots from the supernatants were resolved on a 4–12% NuPAGE Bis-Tris gel for visualization and immunoblotting.Cellular proteins were extracted from the cell pellets after surface pilin was sheared and removed (described above). Briefly, cell pellets were resuspended in 0.2 M Tris-Cl (pH 8.0), normalized to ODFor PilA immunoblotting, both sheared and whole-cell pilin preparations were resolved using 4–12% NuPAGE Bis-Tris gels at 125V for 2.5 h and transferred onto 0.22 μm nitrocellulose membranes. Protein bands were normalized to total protein visualized by BLOT-FastStain (GBiosciences). Membranes were blocked using TBS-T  containing 5% milk before probing with an anti-PilA rabbit polyclonal antibody  at 1:1000, followed by an IR conjugated goat anti-rabbit antibody at 1:10,000 (LI-COR Biosciences). Bands were visualized using the LI-COR Odyssey Clx imager and Image Studio.Overnight precultures were diluted 1:100 in LB and grown to mid-exponential phase (OD at 500 nm ~ 0.5). OD values at 500 nm values were read in a Spectronic 20D+ spectrophotometer  and cultures were adjusted to the same OD. Adjusted cultures were then mixed in a 2.5:97.5 ratio of constitutively fluorescent eGFP cells : rhamnose inducible mScarlet fluorescent cells. Five microliters of mixture were spotted onto bilayer plater. Plates were incubated for three days at 25 °C in the dark, then transferred to medium containing 0.5% L-rhamnose for five hours. Samples were subsequently processed using standard paraffin embedding and thin sectioning protocol.® Multifluorescent Microspheres 0.2 μm (PolySciences 24050–5) were spread onto a bilayer plate using sterile glass beads and dried for 30 minutes. Five microliters of bacterial culture mixture were spotted onto plate and incubated for three days at 25 °C in the dark and macrocolony biofilms were subsequently imaged live or prepared for cryosectioning. Samples were imaged on a ZEISS Axio Zoom.V16 microscope, equipped with a mercury lamp. eGFP was detected using a 450/490nm excitation filter, 500/550nm emission filter, and 495nm dichroic mirror. Fluoresbrite® Multifluorescent Microspheres were detected using a 538/562nm excitation filter, 570/640nm emission filter, and 570nm dichroic mirror.Overnight precultures were diluted 1:100 in LB and grown to mid-exponential phase (OD at 500 nm ~ 0.5). OD values at 500 nm values were read in a Spectronic 20D+ spectrophotometer  and cultures were adjusted to the same OD. Adjusted cultures were then mixed in a 2.5:97.5 ratio of fluorescent:non-fluorescent cells. Bilayer plates of 45 mL (bottom layer) and 15 mL (top layer) of 1% tryptone, 1% agar [Teknova  A7777] in a 10 cm × 10 cm × 1.5 cm square Petri dish  were made and solidified overnight. 300 μl (10^10 microspheres/mL) of FluoresbriteMacrocolony biofilms were overlaid with 1% agar, and sandwiched biofilms were cut and lifted from the bottom layer, and placed in disposable cryomold (Tissue-Tek). They were then covered with an embedding agent (Tissue-Tek OCT), and flash frozen in liquid nitrogen. Cryoembedded biofilms were subsequently sectioned with a Leica CM1950 cryostat set at −18 °C. Ten-micrometer-thick sections perpendicular to the plane of the macrocolony were then transferred onto slides and immediately imaged using a ZEISS Axio Zoom.V16.Overnight precultures were diluted 1:100 in LB and grown to mid-exponential phase (OD at 500 nm ~ 0.5). OD values at 500 nm were read in a Spectronic 20D+ spectrophotometer  and cultures were adjusted to the same OD. Adjusted cultures were then mixed in a 2.5:97.5 ratio of eGFP fluorescent:non-fluorescent cells. Five microliters of mixture were spotted onto bilayer plate. Plates were incubated for three days at 25 °C in the dark, then transferred to medium containing 50 μM PI with and without 100 μg/mL tobramycin for five hours. Samples were subsequently processed using standard paraffin embedding and thin sectioning protocol. Samples were imaged on a ZEISS Axio Zoom.V16 microscope, equipped with a mercury lamp. Propidium iodide staining was detected using a 538/562nm excitation filter, 570/640nm emission filter, and 570nm dichroic mirror.Supplement 1


Glioblastomas are highly aggressive brain tumors for which therapeutic options are very limited. In a quest for new anti-glioblastoma drugs, we focused on specific structural modifications to the benzoyl-phenoxy-acetamide (BPA) structure present in a common lipid-lowering drug, fenofibrate, and in our first prototype glioblastoma drug, PP1. Here, we propose extensive computational analyses to improve the selection of the most effective glioblastoma drug candidates. Initially, over 100 structural BPA variations were analyzed and their physicochemical properties, such as water solubility (− logS), calculated partition coefficient (ClogP), probability for BBB crossing (BBB_SCORE), probability for CNS penetration (CNS-MPO) and calculated cardiotoxicity (hERG), were evaluated. This integrated approach allowed us to select pyridine variants of BPA that show improved BBB penetration, water solubility, and low cardiotoxicity. Herein the top 24 compounds were synthesized and analyzed in cell culture. Six of them demonstrated glioblastoma toxicity with IC50 ranging from 0.59 to 3.24 µM. Importantly, one of the compounds, HR68, accumulated in the brain tumor tissue at 3.7 ± 0.5 µM, which exceeds its glioblastoma IC50 (1.17 µM) by over threefold. According to the World Health Organization (WHO), glial tumors are classified as grade I and grade II (low-grade gliomas), grade III (anaplastic), and grade IV (glioblastoma)2. Current standard-of-care therapies include maximal surgical resection, followed by radiotherapy plus concomitant and maintenance treatment with temozolomide (TMZ)3. In addition, a large variety of different genetic and epigenetic modifications have been found in glioblastomas, among which p53, EGFR, PTEN, and IDH mutations are the most common9. However, these validated molecular targets, as well as immunotherapies, including immune checkpoint inhibitors10, tumor vaccines11, and chimeric antigen receptor T cell (CAR T) therapies12, have all been extensively studied, but failed to significantly improve the therapeutic outcome in glioblastoma patients.Glioblastomas are the most aggressive brain neoplasms with a dismally low 5 year patient survival rate of below 5%13: (2) Glioblastomas are highly infiltrating and heterogenous tumors that are very difficult to remove by surgical resection without compromising the function of surrounding brain areas14; (3) It is difficult to diagnose glioblastomas in their early stages, therefore, large highly infiltrating and vascularized tumors are often present at diagnosis15; (4) Use of rodent syngeneic and patient-derived models are common to optimize clinical protocols. One major problem is that these experimental tumors are typically ~ 103–104 smaller than actual tumors in humans. Therefore, data from drug delivery, drug retention, and tissue penetration experiments obtained from these small animal models are difficult to extrapolate to glioblastoma patients16; and finally, (5) The blood brain barrier (BBB) prevents the majority of anticancer drugs from reaching tumor sites at clinically relevant concentrations, and current methods to enhance BBB penetration are not very effective for glioblastoma patients17.There are several reasons why it is difficult to develop more effective glioblastoma therapies: (1) Glioblastomas are characterized by many dysregulated pathways that cannot be blocked simultaneously via a single therapy18 produces a value of -0.7, which indicates a high capability of the compound to cross the BBB. In spite of these positive features, TMZ-treated glioblastoma patients develop TMZ-resistance and recurrent tumors are practically incurable19.One drug that readily crosses the BBB is temozolomide (TMZ). Upon oral administration, TMZ maximum plasma concentration can be reached in about one hour, and the elimination half-life is approximately 1.8 h. Importantly, penetration efficiency of TMZ into the central nervous system (CNS) is experimentally estimated to be about 20% of plasma levels. This is important because applying this estimate to calculate logBB (Brain-Blood Distribution)20 and fibrates, like for instance fenofibrate (FF), which has strong anti-glioblastoma activity in cell culture, and in glioblastoma intracranial mouse models21. However, we have also found, that the ability of FF to cross the BBB is low, and the compound is quickly processed by blood and tissue esterases to form the PPARα agonist, fenofibric acid (FFA), which is no longer effective in triggering tumor cell death22. We made numerous modifications to the FF chemical structure and selected our first drug candidate, PP123, which similar to FF, blocks mitochondrial respiration and triggers a severe ATP depletion. With the use of both substances, ATP depletion is followed by phosphorylation/activation of AMP-activated protein kinase (AMPK—intracellular energy sensor), blockade of p70S6K phosphorylation (marker of active protein synthesis), activation of autophagy (p62 degradation), and extensive glioblastoma cell death24. In spite of these promising results, and in spite of the fact that we detected PP1 in the brain at therapeutically relevant concentrations , PP1 anti-glioblastoma therapeutic effects were only marginal when the treatment was applied to large intracranial tumors in mice24. These data indicate that additional adjustments to the BPA scaffold, focused on improving compound cytotoxicity, BBB penetration and retention in the brain tumor tissue, are continuously needed.In addition, TMZ has been used in combination with other drugs, which enhanced its therapeutic effects. One example is a combination of TMZ with lipid lowering drugs, including statins25. For drugs associated with the CNS, their ability to cross the brain blood barrier (BBB) is the most important characteristic and is not directly addressed by the Lipinski’s rule of five. Therefore, two computational scoring systems have been introduced to evaluate the probability of new drug candidates penetrating the CNS. One is CNS Multiparameter Optimization (CNS-MPO) algorithm26, which uses 6 physicochemical properties  for estimating the probability of entering the CNS. The CNS-MPO score range is between 0 and 6, and values ≥ 4.0 have been used as cut-offs for compounds with increased ability to penetrate the CNS26. Another scoring system is the blood–brain barrier score (BBB_SCORE)27. BBB_SCORE is based on five physicochemical parameters including number of aromatic rings, heavy atoms, MWHBN , pKa and topological polar surface area. Similar to CNS-MPO, BBB_SCORE also considers the value of 4.0 as the cut-off for acceptable BBB penetration27. Consequently, CNS-MPO and BBB_SCORE both serve as complementary algorithms in which CNS-MPO provides information regarding probability of the compound to be found in the CNS, and BBB_SCORE is oriented towards specific physiochemical properties that increase probability of the compound crossing the BBB.In early stages of drug design it is important to evaluate relevant physicochemical properties of prospective drug candidates. For over two decades, Lipinski’s rule of five was the gold standard in drug design29. Based on the compound structure, the hERG algorithm gives a score which is indictive of the compound’s inhibitory effect towards hERG. In other words, for the drug to be considered safe, it’s hERG IC50 should be significantly higher than its therapeutic IC5028.In regard to toxicity, one of the most common hurdles in drug testing is cardiotoxicity triggered by inhibition of the cardiac potassium channel that is coded by the human ether-à-go-go-related gene (hERG). This toxicity test became a mandatory requirement for drug design and development, and can be calculated by the hERG algorithm30. Results in Fig. 32, and in our first glioblastoma prototype drug, PP124.In order to determine glioblastoma-specific and therapeutically relevant values for all proposed algorithms/scores, including hERG, we have selected fifteen drugs  that are either currently used as glioblastoma drugs, or are in clinical trials with glioblastoma patientsBPA) derivatives, we have decided to explore pyridine variants  one with a directly attached unsubstituted pyridine moiety (pyridine-BPA), (II) and one with a separation unsubstituted pyridine moiety with one carbon (methylenepyridine-BPA), (III) with two carbons (ethylenepyridine), (IV) a benzo fused pyridine  a hydroxy substituted pyridine (hydroxypyridine-BPA)  Fig. .Figure 236; brain penetration capability (CNS-MPO and BBB_SCORE), and water solubility (− logS). The values for the best 24 BPA-pyridines are presented in Fig. These structural variations of BPA-pyridines were designed and tested using calculated values from four algorithms: cardiotoxicity (hERG)37. There are two major problems in performing FFA coupling with aminopyridines: (a) FFA has exceptionally low reactivity due to steric constraints generated by the two methyl groups located alpha to the carbonyl group, and (b) amino groups of aminopyridines are exceptionally weak nucleophile due to amino group electron delocalization through the pyridine ring. The difficulty of performing this kind of coupling reaction was recently examined by others38. However, FFA can be converted into corresponding fenofibric chloride  and its derivatives have exceptionally low reactivity toward nucleophilic acyl substitution. The obvious synthetic pathway for preparation of these compounds is by coupling FFA with corresponding amines39. We have used density functional theory to compute HOMO energies. Results provided in Supplementary Materials (page 153) indicate that aminopyridines should be less reactive (low HOMO) in comparison to aniline while aminophenol is more nucleophilic. Based on our calculations, the coupling of sterically hindered FFA through the reactive fenofibric chloride (FFC) with 4-aminophenol should give a product with a highly isolated yield. Our previous studies40 demonstrated that FFC is a viable, reactive FFA intermediate for amine coupling. For these reasons, we have developed a new synthetic methodology, which is shown in Fig. In molecules of similar structural framework, it is possible to compare frontier molecular orbitals to determine order of nucleophilic and electrophilic reactivityHR66-HR70, HR82, HR83, HR88-HR90), and (b) one with separation of an aromatic heterocyclic ring by one or two aliphatic carbons . Preparation of the second group of compounds (with reactive amines) is straightforward by mixing FFC with corresponding amines in presence of a base. Because the corresponding amines are more nucleophilic than water and reactions are completed in several minutes at room temperature, these reactions can be performed with an environmentally friendly base such as sodium carbonate in water. Isolated yields are nearly quantitative and this method (Method A) is well suited for small (milligrams) and large (hundreds of grams) scale synthesis. Also, this synthetic procedure does not require any special precautions or preparations. Preparation of the first group, with the heterocyclic aromatic ring directly attached to the amino group, requires certain precautions. Because water is a better nucleophile than these amines’, reaction must be performed in water-free (dry) conditions. In addition, less nucleophilic amines are also prone to oxidation in basic conditions40. Because of this, such reactions should be carried out in an oxygen free atmosphere (Method B). Under dry conditions, a pyridine solution of a corresponding aminopyridine was mixed with anhydrous sodium carbonate and kept under nitrogen atmosphere overnight. A separate dichloromethane solution of FFC was also prepared in dry conditions under nitrogen atmosphere. The dichloromethane solution was slowly added to cold (~ 0–5 °C) pyridine suspension of a corresponding aminopyridine and sodium carbonate under a nitrogen atmosphere. Nearly quantitative yields were obtained with reactions performed at 0–5 °C for 3 h, then at room temperature overnight, and finally, at 60 °C for additional 3 h. The product was isolated after solvent evaporation and water addition by simple filtration and extensive water washing. This method gave products in high yield, more the 97% purity, and did not require additional purification by either extraction or chromatography. It is also applicable to milligram and multigram preparation scales for amines with wide ranges of reactivity  one with an aromatic heterocyclic ring directly attached to the nitrogen atom (LUMO) and energy of Highest Occupied Molecular Orbital (EHOMO) were determined. We have included MPA based on studies showing that if a compound does not interact with cell membranes and has a MPA lower than 60 Å2, it should be able to penetrate the CNS via passive diffusion43. Therefore, MPA is considered as a better parameter than molecular weight in discriminating the compound ability of entering the CNS44.The first group of pyridine-BPA variants are presented in Fig. 46.In the context of comparing compounds with high structural similarity, frontier orbital energies (HOMO and LUMO) can be used in determining which of the similar compounds may have a stronger protein binding ability, and therefore a higher chance of BBB penetrationHR67 and HR68 , HR74 should have the best binding ability. In addition, all HR compounds in Fig. The next group of compounds belong to methylenepyridine-BPA Fig. . All of HR83 is 7.29). As the size of the molecule increases, MPA values follow, suggesting that HR81, HR82, and HR83 may have a low probabilities of penetrating the CNS. Considering the energies of both frontier molecular orbitals from three similar compounds  HR80 should be the most active. Indeed, obtained cell viability (CV) data for these compounds correlated with computational data and suggest that best drug candidate from this group is HR80. Although both benzopyridine-BPAs (HR82 and HR83) have encouraging IC50 values, 1,4 and 2.75 µM, respectively, their computed physical properties such as ClogD and MPA suggest that there is very low probability for these compounds to penetrate the CNS, further supporting HR80 as the best glioblastoma drug candidate in this group. In addition, HR81, which has low glioblastoma-specific cytotoxicity . There are noticeable differences between the two stereoisomers: R isomer HR84 being three times more cytotoxic than S isomer HR85  and optically pure R isomer HR87. This finding is reasonable because of the pyridine nitrogen proximity to the chiral center. One could argue that pyridine nitrogen binding is sterically diminished in HR85 in comparison with HR84. Because of the position of the pyridine nitrogen regarding chiral center, steric difference is diminished in HR87, which is associated with improved cytotoxicity. The hydoxypyridine-BPAs can exist in both the hydroxypyridine and the amide form with the amide form being preferable by 8.97 kcal/mol according to the DFT ωB97X-D/6-31G* computational method. Computational studies as well as NMR spectroscopy indicate that HR88 is in the hydroxypyridine form while HR89 and HR90 are in their amide form.We have also explored the importance that chirality may have on the activity of these compounds Figure . For insHR89 and HR90 can be compared because they are in the amide form while HR88 is in the hydroxypyridine form. All computed parameters suggest that both HR89 and HR90 should easily penetrate the CNS. However, computed frontier orbital energies for HR90 are lower, indicating it should be more effective in penetrating the BBB. In addition, HR90-induced glioblastoma cytotoxicity is nearly 10-fold greater in comparison to HR89, making HR90 the better drug candidate. In conclusion, HR87 and HR90 are the best drug candidates in this group with corresponding, glioblastoma-specific IC50 values of 5.38 and 2.05 µM, respectively . Reaction types involved in Phase I metabolism are hydrolysis, oxidation, and reduction50. All predicted metabolites of HR67 and HR68 have better or comparable computed water solubility, as well as computed abilities to penetrate the CNS. Importantly, all these metabolites are predicted to be relatively safe  with the exception of amide hydrolysis, which may trigger some cardiac toxicity and should be further analyzed (hERG=3.91) .When pursuing new drug candidates, it is very important to envision the possible metabolites and estimate their physicochemical properties, including toxicity24, and a positive control (caffeine). We also compared the negative control (FF), for which we have previously demonstrated an inability to accumulate in the brain tumor tissue22. Although CNS-MPO scores for HR67 (3.71) and HR68 (3.71) are slightly lower, compared to the CNS-MPO of our prototype drug, PP1 (CNS-MPO = 3.9)24, these two compounds can cross the BBB model membrane  Institutional Biosafety Committee , and LSU Institutional Animal Care and Use Committee . In addition, our IACUC approved animal experiments follow 10 essential recommendations included in the ARRIVE guideline.https://www.ambeed.com), Millipore Sigma , and TCI America . 1H-NMR spectra were recorded on Varian Mercury 300 and Varian Mercury 400 Plus instruments in CDCl3 and DMSO-d6, using the solvent chemical shifts as an internal standard. NMR solvents were purchased from Cambridge Isotope (https://www.isotope.com). In DMSO-d6 solvent our final compounds such as HR68 show two sets of signals due to restricted rotation of C-N amide bond53. This restriction is not present in CDCl3 as NMR solvent . All studied compounds have 96% or higher purity as determined with H-NMR and HPLC. All computed molecular descriptors were generated by ChemAxon MarvinSketch version 22.21 (https://chemaxon.com/products/marvin). All calculated values for each and every compound were performed with MarvinSketch and are included in the Supplementary materials. Frontier orbital energies, conformational studies, energy differences between various isomers and their electrostatic potential maps were calculated with ωB97X-D/6-31G* Density Functional Theory (DFT) method as implemented in Spartan ’18 v 1.1.0 (https://www.wavefun.com) and are included in the Supplementary Materials (pages 150–152). 1H-NMR and 13C-NMR spectra for all HR compounds generated in this study are included in Supplementary Materials. Phase I metabolites prediction was performed with BioTransformer 3.0 (https://biotransformer.ca/new) -2-methyl-N-(pyridin-3-yl)propenamide (HR67). Dry pyridine suspension of 3-aminopyridine  and anhydrous sodium carbonate  was sonicated for thirty minutes and left at room temperature in a closed system overnight under nitrogen atmosphere to ensure that the pyridine suspension remained dry. Separately, fenofibric chloride (FFC) was prepared as follows: Dichloromethane (500 ml) suspension of fenofibric acid , oxalyl chloride , and DMF (few drops) were stirred at room temperature overnight. After approximately 1.5 h, the reaction mixture became light brown. The majority of solvent was removed by distillation at atmospheric pressure and the remaining solvent was removed under Argon flow at room temperature. The resulting solid material was dissolved in dichloromethane (150 ml), under nitrogen atmosphere and with slow stirring, was added to the previously prepared pyridine suspension of 3-aminopyridine and sodium carbonate cooled with ice-water. The resulting suspension was stirred at 0–5 °C for 3 h, then at room temperature overnight, and was followed by stirring at 60 °C for additional 3 h. The resulting solvent was removed under reduced pressure to separate the solid residue. This residue was mixed with water (1 L) and stirred via sonication for 4 h. The insoluble white crystalline product was separated by filtration, extensively washed with water (20 × 100 ml), and dried at 60 °C, under vacuum. The isolated yield was 90% (53.3 g). 1H-NMR  δ 10.31 , 8.83 , 8.26 , 8.06 , 7.72 , 7.67 , 7.54 , 7.31 , 7.04 , and 1.63  ppm. 13C-NMR  δ 193.7, 172.9, 159.4, 145.2, 142.6, 137.6, 136.6, 135.6, 132.3, 131.6, 130.6, 129.0, 128.0, 123.9, 118.8, 81.5, and 25.2 ppm.phenoxy)-2-methyl-N-(2-(pyridin-4-yl)ethyl)propenamide (HR80). Fenofibric chloride (FFC) (0.3 mol) was prepared by the following procedure described in Method A from fenofibric acid  and oxalyl chloride  in dichloromethane (1 L). Prepared FFC (0.3 mmol) was slowly added dropwise over a period of 45 min at ice-water bath temperature into a magnetically stirred mixture of sodium carbonate  in water (500 ml) and 1-(pyridine-4-yl) ethanamine  in tetrahydrofuran (500 ml). After addition was complete, the resulting reaction mixture was stirred at room temperature overnight. The reaction mixture volume was reduced by 75% via solvent evaporation under air flow (produced by air pump). The resulting white suspension was mixed with water (500 ml) and the insoluble product was separated by filtration, washed with water (10 × 50 ml), and dried at 50 °C overnight. The isolated yield was 97% (82 g) of pure product. The filtrate was acidified with hydrochloric acid to pH = 2. The resulting white solid precipitate was separated by filtration, washed with water (10 × 20 ml) and dried at 50 °C overnight to give 34.2 g (95% recovery) of fenofibric acid. 1H-NMR  δ 8.36 , 8.21 , 7.66 , 7.13 , 6.86 , 3.85 , 2.73 , and 1.45  ppm. 13C-NMR  δ 193.7, 173.1, 159.6, 149.7, 148.7, 137.5, 136.7, 132.1, 131.6, 130.2, 129.1, 124.6, 118.7, 81.1, 39.4, 34.3, and 25.5 ppm.General preparation of HR66-HR90. Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-2-methyl-N-propanamide (HR89). Freshly prepared dichloromethane (5 ml) solution of fenofibric acid chloride was made from fenofibric acid  and oxalyl chloride (1 mmol) as described above. These were added, under a nitrogen atmosphere while stirring, to a pyridine (10 ml)—tetrahydrofuran (10 ml)-sodium carbonate  of 3-aminopyridin-2(1H)-one  solution. The resulting mixture was stirred at room temperature in the nitrogen atmosphere for 3 h, followed by stirring under the nitrogen atmosphere at 60 °C for an additional 3 h. After cooling to room temperature, the solvent was evaporated under air flow (produced by air pump) at room temperature yielding a solid residue. This solid residue was mixed with dichloromethane (30 ml) and? water (100 ml). The water layer was discarded, and the organic layer was washed with water (3 × 100 ml), 5% sodium carbonate (3 × 100 ml), and dried over anhydrous sodium carbonate. The drying material was separated by filtration. The volume of the filtrate was reduced to ~ 2 ml, then hexanes (~ 10 ml) were added. The resulting solution was left uncovered at room temperature, allowing the solvent to slowly evaporate. The resulting white crystalline product was separated by filtration, washed with hexane (3 × 3 ml) and air-dried overnight. Isolated yield = 93% (95 mg). 1H-NMR  δ 12.08 , 9.29 , 8.24 , 7.73 , 7.71 , 7.59 , 7.12 , 7.10 , 6.26 , and 1.58  ppm. 13C-NMR  δ 193.8, 172.4, 158.5, 157.6, 137.7, 136.4, 132.3, 131.7, 131.6, 129.1, 128.7, 128.6, 123.1, 120.3, 105.9, 82.3, and 25.2 ppm.Small scale preparation applicable to all 2 atmosphere. Prior to treatment with HR compounds, cells were plated in 96-well plates  at an initial density of 2 × 104 cells/cm2. Twenty-four hours after plating, stock solutions of HR compounds were prepared in DMSO, diluted in cell culture medium and added to previously plated cells in triplicate for every experimental condition . DMSO (0.5%) was used as vehicle control. After 72h incubation, an MTT assay was performed to measure cell metabolic activity (surrogate for cell viability). Following a 1.5 h incubation with 0.5 mg/ml MTT in serum free low glucose DMEM, the resulting formazan crystals were dissolved in 5mM HCl in isopropanol and the absorbance read at 540 nm. Data represent mean values expressed as the percentage of vehicle control ± SD. Phase contrast images of treated cells were taken 72 h after treatment with HR compounds using a BZ-X800 fluorescence microscope (Keyence) equipped with a 20 × objective. The drug dose resulting in 50% inhibition of cell metabolic activity (surrogate for cell viability) was measured using MTT assay, at 72-h time point, and half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 8.Human glioblastoma LN-229 cells (ATCC CRL-2611) were maintained as a semi-confluent monolayer culture in DMEM with 1 g/L glucose, sodium pyruvate and L-glutamine (Corning), supplemented with 10% heat-inactivated FBS (Gibco) and P/S (50 units/mL of penicillin and 50 µg/mL of streptomycin) at 37 °C in a 5% CO.55. Briefly, 24-well transwell inserts  were coated with 10 μg/cm2 of Collagen Type IV (Sigma) for 24 h at 4 °C. Inserts were washed with sterile water and air-dried for 2 h. Next, the inserts were coated with 2 μg/cm2 poly-l-lysine (ScienCell) for 1 h at 37 °C, then washed twice with sterile H2O and air-dried for 2 h. Primary human astrocytes (1.5 × 105) and 3 × 104 primary human pericytes (both ScienCell) were resuspended in 25 μl of astrocyte medium and pericyte medium (ScienCell), respectively, then combined in a 1:1 ratio for 50 μl total volume. Dried, coated inserts were turned upside down such that the basolateral surface was exposed at the top, and 50 μl of the cell mixture was added to the membrane, covered with the plate lid, and incubated for 2 h at 37 °C to allow cell adhesion. Any medium remaining on top of the membrane was carefully removed before returning inserts to their upright position with the apical surface facing upward, as they were placed in a 24-well plate containing 500 μl per well of astrocyte/pericyte medium (1:1). An additional 300 μL of medium was added to the apical compartment. Four days after plating, the apical compartment medium was removed, and 3.75 × 104 of telomerase-immortalized vain endothelial cells  in 50 μl of TIVE medium56 were added and incubated for 5 h at 37 °C to allow cell attachment, followed by the addition of an extra 250 μl of TIVE medium. Half the volume of the corresponding media in the lower and upper compartment was replaced with fresh media every third day. Ten days after initial plating, trans-endothelial electric resistance (TEER) was measured using a EVOM2 meter with a STX3 electrode (World Precision Instruments). The ability of selected HR compounds to pass through the in vitro BBB was tested using inserts with effectively reconstructed BBB as confirmed by TEER values57.The BBB was re-created in vitro using a modified protocol provided by Stone et al22, was used as a negative control, and 50 µM caffeine was used as a positive control58. Plates containing the inserts were returned to the incubator , and after 3 h of incubation, conditioned media from the well and insert  of the in vitro BBB model Fig. A was repert Fig. A were co2O) and solvent B (acetonitrile) mixed at predetermined ratios for each compound  equipped with an analytical YMCbasic, 3µm, 150 × 4.6 mm column . Isocratic elution of the compounds was performed using a mobile phase composed of solvent A  and were cultured and propagated according to the recommended protocols59. The cells were injected into the striatum region using 5 μl of PBS containing 1 × 105 of the tumor cells guided by the stereotactic approach  as reported in our previous study24. The treatment started when the intracranial tumors were well-established . HR68 injection solution was prepared from the 50 mM DMSO stock solution diluted in 20% cyclodextrin (2-Hydroxypropyl-β-cyclodextrin) in sterile PBS and delivered intraperitoneally (ip) at 15 mg/kg. Blood, liver, kidneys, spleen, heart, brain and brain tumor were subsequently collected, solid tissues were washed from blood in PBS, and placed on ice for an immediate sample preparation for HPLC analysis (see above). Tissues were prepared for HPLC by mixing 150 μl of sample tissue (~ 120 mg) that had been mixed with 3 volumes of Methanol : H2O mix (4:1), well-blended using TissueRuptor II (Qiagen), and centrifuged at 15,000g for 10 min at 4 °C. Supernatants were collected in 1.5 ml Eppendorf tubes and incubated at 95 °C for 3 min. Following flash cool on ice, samples were centrifuged again at 15,000g for 10 min at 4 °C and supernatants were used for HPLC-based measurement.Foxn1nu female immunodeficient mice at 6 to 8 weeks of age were used in this study . The mice were inoculated with patient-derived glioblastoma, GBM12-TMZ-resistant, which stably express luciferase reporterP values of ≤ 0.05.The data were analyzed with a homoscedastic Student t test. Differences between control and experimental groups were considered significant at Supplementary Information.


S100a14 mRNA level. The proteomics study suggested that ScF diminish keratinocyte proliferation via the mTOR pathway by downregulating mTOR protein, corresponding to the modulation of TNF-α, Wnt, and IL-1β levels, leading to the enhancement of anti-inflammatory environment by IL-17 downregulation. Hematology data demonstrated the safety of using these biomaterials, which provide a potential therapeutic-option for psoriasis treatment due to desirable effects, especially anti-proliferation and anti-inflammation, functioning via the mTOR pathway and control of IL-17 signaling.Therapeutic treatment forms can play significant roles in resolving psoriatic plaques or promoting wound repair in psoriatic skin. Considering the biocompatibility, mechanical strength, flexibility, and adhesive properties of silk fibroin sheets/films, it is useful to combine them with anti-psoriatic agents and healing stimulants, notably silk sericin. Here, we evaluate the curative properties of sericin-coated thin polymeric films (ScF) fabricated from silk fibroin, using an imiquimod-induced psoriasis rat model. The film biocompatibility and psoriatic wound improvement capacity was assessed. A proteomics study was performed to understand the disease resolving mechanisms. Skin-implantation study exhibited the non-irritation property of ScF films, which alleviate eczema histopathology. Immunohistochemical and gene expression revealed the depletion of β-defensin, caspase-3 and -9, TNF-α, CCL-20, IL-1β, IL-17, TGF-β, and Wnt expressions and  Psoriasis is a chronic, non-contagious, and T cell-mediated autoinflammatory skin disease, which manifests in the form of macroscopic red and scaly plaque and microscopic epidermal lesions, particularly hyperkeratosis, acanthosis, dermatitis, and folliculitis. A single treatment cannot provide the best effective therapeutic outcome. In addition, most treatments cannot be used for prolonged periods of time, and need to be used in rotation to minimize adverse effects. Research to devise new therapeutic regimens has been ongoing for several decades, and the current treatment armamentarium includes topical preparations, ultraviolet therapy, and systemic therapies. The choice of suitable treatment methods depends on disease pattern and severity, comorbidities, patient preference, and other related factors such as the presence of complicated wounds in patients with psoriatic skin conditions. This includes for example, individuals who have undergone trauma, infection, or surgery.1. The combination treatment of hydrocolloid dressing, a hydrated-gel like matrix, with anti-psoriatic cream-based preparations such as corticosteroids or calcitriol presents some benefits such as ease of application, patient acceptability, relief from itching and traumatic plaque, limited use of steroids, and cost effectiveness3. However, bacterial contamination with long-term use of the occlusive materials is an important contraindication1. Presently, dual purpose dressings for simultaneous alleviation of psoriatic skin conditions and facilitating wound healing are being developed.Different types of psoriatic lesions, with or without associated complications, require appropriate treatment and care. Many adjunctive therapeutic dressings, each associated with their own advantages and disadvantages, have been developed to enhance treatment outcomes. Occlusion therapy with colloidal dressings can be used for some patterns of psoriasis, such as palmoplantar, localized pustular, and pustulosis psoriasis, to enhance the epidermal absorption of active ingredientsBombyx mori cocoons, are natural biopolymers with several medicinal properties, and are especially known to enhance wound healing4. Recently, we have demonstrated the efficacy of cream-based as well as polyvinyl alcohol-based preparations of sericin for treating psoriasis in preclinical studies6. The main mechanisms of anti-psoriatic action of sericin occur via the inhibition of epidermal cell overgrowth, epidermal inflammation, and imbalance of epidermal cell homeostasis, through immunomodulative and antioxidative effects.Sericin and fibroin, silk proteins derived from 10. Silk films are flexible and mechanically robust, and can also be formed in micropatterned configurations via photolithography. Owing to desirable properties such as mechanical flexibility, adhesion, biocompatibility, and non-toxic nature7, 2D fibroin sheets can potentially be combined with bioactive agents such as sericin, for treating psoriasis. Being biodegradable under proteolytic conditions, these films can also form a sustainable, zero-waste system.Two-dimensional (2D) silk protein  polymeric films and membranes have been explored for various biomedical applications including drug delivery, biofiltration, biosensors, biomimetic cellular construction, wearable devices, smart skin, and tissue regenerationTherefore, based on an in vivo approach—“imiquimod-induced psoriasis rat model,” this study aims to examine the therapeutic properties, toxic effects, and disease-resolving mechanisms of sericin-coated thin fibroin films for psoriasis treatment. Histopathology, immunohistochemistry, RT-qPCR (quantitative reverse transcription polymerase chain reaction), and proteomics were performed in the study. This study provides an alternative therapeutic form ScF—a combination of sericin extraction and flexible, photocrosslinked 2D silk fibroin films, for application in psoriasis treatment. Study outcomes may be useful for further development of a new prototype treatment which can be evaluated in clinical trials.7.The amino acid composition of sericin was evaluated to be: Serine (29.66%), aspartic acid 18.06%), glycine (10.01%), threonine (8.56%), glutamic acid (6.65%), tyrosine (6.35%), arginine (6.35%), lysine (4.02%), valine (3.94%), alanine (3.81%), and leucine (2.54%). The sericin was coated on photocrosslinked fibroin films to form sericin-coated films (ScF) , apoptotic marker, proinflammatory cytokine, chemokines, anti-inflammatory cytokine, antioxidation protein, and antiproliferation marker were examined using immunohistochemical staining Fig. . ImmunolFor the toxicological assessment of ScF application 7 days post-treatment, hematological and blood clinical chemistry was examined Table . All groS100a14 gene expression, compared to other groups  inflammation, (2) epidermal cell apoptosis, (3) cellular hyper-proliferation, and (4) cytoskeletal alteration. Below are some observations regarding these 17 up- and 38 down-expressed proteins based on their biological functions and properties:7 upregulation and 10 downregulation proteins were directly involved in the four disease mechanisms mentioned above Table . Some ofAccording to the proteomic analysis and immunohistochemical study, there is a definite connection between (1) mTOR (mechanistic target of rapamycin) protein Table , (2) theApart from the main influence of ScF on psoriasis treatment via the mTOR signaling and Th17 differentiation pathways, ScF also demonstrated therapeutic effects via another mechanism. An increment in decorin Table  resultedChemico-protein interaction analysis demonstrates that some amino acids in sericin, such as serine, leucine, alanine, arginine, and glycine have interactions with proteins in the mTOR pathway Fig. A. ArginiIn this study, we investigated the therapeutic and non-toxic properties of sericin-coated thin polymeric films (ScF) fabricated using photocrosslinked fibroin. This includes understanding its effects on disease progression and mechanisms in a psoriasis rat model based on several approaches. The use of the 2D fibroin substrates is important in terms of providing a mechanically robust, flexible, and compatible platform for the application of sericin. The films themselves are conformable to soft tissue and may be degraded under proteolytic conditions. We can safely postulate that the ScF relieves the severity of psoriasis by the reduction in epidermal cell overgrowth with anti-proliferative effect through the mTOR pathway through: 1) upregulation of anti-proliferative protein (decorin), (2) downregulation of antimicrobial peptide (β-defensin), apoptotic proteins (caspase-3 and -9), chemokine (CCL-20), proinflammatory cytokines , inflammatory modulator , proliferative related proteins , and keratinized proteins (KRT14 and KRT75). According to these underlying mechanisms, the reduction in hyperkeratosis, epidermal edema and swelling, squamous cyst, and folliculitis were distinctly observed in ScF-treated rats compared with the non-treatment group , induce aberrant mTOR activity, leading to epidermal cell overgrowth and abnormal epidermal organization. In agreement with our study, psoriatic skin-treated with ScF had a significantly low levels of mTOR protein alongside the downregulation of TNF-α, IL-1β, and IL-17  pathway for corneal resolution27. This pathway is well known as the main signaling pathway for proliferation, similar to PI3-K/Akt and the mTOR signaling pathway.Studies on the proliferative and other related effects of sericin based on PI3-K/Akt and mTOR signaling have not been much established. It was earlier reported that sericin inhibits breast cancer cell  proliferation via the PI3-K/Akt pathway, by inducing cell cycle arrest and promoting cellular apoptosis28. IL-17 promotes the S100A8 and S100A9 gene expression as an alarm signal during the inflammatory response of keratinocytes in psoriasis29. Our previous study demonstrated that a cream-based sericin formulation reduces the severity of psoriasis through Th17 cells by interfering with the Janus kinase (JAK)-signal transducer and the activator of transcription (STAT) pathway5. Sericin cream also modulates epidermal immunity via galectin-3 (LGALS3) and sphingosine-1-phosphate lysate1 (SGPl1), and epidermal proliferation via nucleoside diphosphate kinase B (Nme2). In the present study, the connection between an upregulation of mTOR protein (from proteomics approach) and the reduction in IL-1β, IL-17, and TGF-β (from immunohistochemistry) was observed, which is most relevant to the Th17 cell differentiation pathway  have a critical role in the pathogenesis of several autoimmune diseases such as psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease32. Apart from the common lesions in the psoriasis rat model, epidermal edema or swelling, and squamous cysts were also observed. Epidermal edema or spongiosis, with widening of the intercellular space, is a common feature of skin inflammation, in association with the rupture of the desmosome and formation of intraepidermal vesicles33. Squamous cysts most likely arise from injured pilosebaceous units, in which squamous epithelial cells producing keratin are trapped in the dermis33. Our results highlight the ability of ScF in the reduction of acanthosis, hyperkeratosis, and squamous cysts when compared with the calcitriol- and non-treatment groups -mediated inhibition of Na+-Dependent Alanine Cotransporter (ASCT1)42. In agreement with our study, the enrichment of arginine, leucine, and alanine, which are the components of sericin, could inhibit mTOR signaling, as demonstrated by a downregulation of mTOR protein in ScF-treated rats.Chemico-protein interactions indicated the connections between the amino acid composition in sericin extraction to proteins in the mTOR pathway Fig. . In the In summary, we propose that ScF—a combination of sericin extraction and 2D flexible, photocrosslinked fibroin films, inhibits the mTOR pathway and Th17 differentiation signaling to promote psoriasis resolution. The underlying mechanisms contributed to several factors: proliferative related proteins , antimicrobial peptide (β-defensin), apoptotic proteins (caspase-3 and -9), chemokine (CCL-20), proinflammatory cytokines , inflammatory modulator , and keratinized proteins (KRT14 and KRT75). Therefore, ScF provides a potentially promising modality for psoriasis treatment due to the favorable properties of sericin in terms of anti-psoriatic effects and the stimulation of healing.Preparation methods for each component used to formulate the dressing material in this study are mentioned below.7. In brief, pure silk fibroin obtained from Bombyx mori cocoons after a standard extraction protocol43 was dissolved in 1 M LiCl/DMSO (lithium chloride/dimethyl sulfoxide). It was then treated with 2-isocyanatoethyl methacrylate (IEM) for 5 h at 60 °C, maintaining a constant flow of nitrogen. The solution was then added to cold ethanol, to precipitate out the methacrylated protein44. The product was washed with a 1:1 ratio of cold ethanol and acetone, centrifuged, and lyophilized for 24 h.The fibroin films used in this study are based on photocrosslinkable fibroin (photofibroin) which provides unique tunability in terms of mechanical properties as well as surface morphology. The photofibroin was prepared according to the procedure described by Xu et al.−2, and was then dipped in deionized (DI) water to obtain a crosslinked fibroin film. The films were kept in a desiccator until use. Films were typically 25–40 µm in thickness. Films were cut into identical 1.0 cm × 1.0 cm pieces and soaked in 5% sericin solution5 for 24 h at room temperature  and 2.5% w/v of photoinitiator . The solution was poured for casting on plain glass slides, evaporated by air drying for 20 min, crosslinked by exposure to a 365 nm UV lamp (Lumen Dynamics OmniCure 1000 system) for 3 s at 20 mW cm45. Briefly, Bombyx mori cocoon shells, received from Chul Thai Silk Co. Ltd., Phetchabun Province, Thailand, were autoclaved in distilled water for 1 h at 120 °C. Dissolved sericin was filtered and stored in a desiccator until use. Amino acid composition was examined by the Central Laboratory (Thailand) Co., Ltd., Bangkok, Thailand.Sericin extraction was performed by an autoclaving methodAnimal studies were approved by The Faculty of Tropical Medicine ACUC, Mahidol University . Animal experimental protocols were in accordance with the ARRIVE guidelines 2.0, the National Research Council of Thailand's Guidelines for the Use of Animals and the Thai Animals for Scientific Purposes Act, formulated in B.E. 2558. Forty 8-week-old female Wistar rats were obtained from the Nomura-Siam International company, Thailand. All rats were housed in an environment controlled-room with temperature maintained at 25 ± 2 °C, 65 ± 10% humidity, 12-h/12-h light/darkness cycles; and they were provided access to a standard diet and filtrated water at all times.46. Absorbable Chromic catgut No. 2-0 was used as a control material due to its appropriate biocompatibility and common use in surgery. Twenty-five rats were used in this study. They were randomly divided into five equal groups with differently timed endpoints . Following anesthesia with 50 mg/kg of thiopentone intraperitoneally, they were shaved along the dorsal midline of the thoracolumbar area. The surgical site was scrubbed with betadine and a 2 cm incision was made. 1 cm × 1 cm of ScF and 1 cm of Chromic catgut were inserted into the blunt socket of both sides. The epidermal incision was closed with Vicryl No. 4-0 sutures. All rats received 15 mg/kg of tramadol, intramuscularly as an analgesic for 3 days post-surgery. The wound was carefully observed daily until suture removal on day seven post-surgery.To investigate sericin-coated photofibroin film (ScF) interactions in terms of host inflammatory response, a bioimplantation study was conducted according to the ISO 10993-6:2016 guideline as mentioned in our previous studyAt the endpoint for each group, all rats were humanely euthanized by an overdose of carbon dioxide inhalation. All implanted materials were excised with some adjacent tissues. They were preserved in 10% neutral-buffered formalin (NBF) for histopathological study and 2.5% glutaraldehyde in sucrose phosphate buffer (SPB) for electron microscopy.To assess inflammatory or irritation levels induced by ScF and its control, a histopathological study was carried out. Fixed specimens were processed as per standard protocol for tissue processing, and were subjected to dehydration with ethanol, infiltration with xylene, embedding with paraffin, cutting into 5 µm sections, and staining with hematoxylin and eosin (H&E). The sections were examined under a light microscope  with a focus on cellular infiltrations  and histopathological changes  in the tissue interfacing area. All these criteria were observed and graded as follows: 0: absent, 1: rare, 2: mild, 3: moderate or advanced stage, and 4: severe or end stage. The final score was calculated by the subtraction between the total score of the control material and ScF.To characterize the ultrastructure of ScF both before and after implantation, scanning electron microscopy (SEM) was performed. Specimens were fixed with 1% osmium tetroxide, dehydrated with ethanol, dried in a critical point drying device , coated by a coat-sputter , and examined under an SEM  with emphasis on the material alteration at the surface.47, 62.5 mg of imiquimod was applied daily on the experimental site measuring 1.5 cm × 1.5 cm for seven days, which was considered as an induction period. Macroscopic skin lesions such as epidermal scales, redness, and thickening were monitored. All rats were randomly divided into three equal groups with different treatments  and were treated with the appropriate treatment for 7 days, which was the treatment period. The experimental site was wrapped with a Fixomull stretch to protect the test site. To maintain psoriatic skin conditions during the treatment period, imiquimod was continuously administered to the experimental site after the induction period.Imiquimod-induced psoriasis rat model was used for the evaluation of potential curative and toxic effects of ScF in connection with disease progression and resolution. Fifteen rats were included in this study. They were shaved along the dorsal midline in the thoracic area. Based on our recent studieslater stabilization solution for molecular analysis with RT-qPCR, and (3) in − 80 °C freezer for proteomics study.Seven days post-treatment, all rats were humanely euthanized by carbon dioxide inhalation. Exsanguinated blood collection was performed by a cardiac puncture. The blood samples were sent for hematological and blood clinical chemistry evaluation, performed by the National Laboratory Animal Center, Mahidol University. Skin experimental sites were removed, separated into three parts, and preserved (1) in 10% NBF for histopathological and immunohistochemical studies, (2) in RNAFixed skins were subjected to standard tissue processing. The skin sections were examined under a light microscope focusing on psoriasis-related epidermal lesions, particularly hyperkeratosis, acanthosis, pustules, epidermal edema and cysts, folliculitis, and dermatitis. These lesions were scored using H-score . Moreover, epidermal thickening was measured by image analysis using ImageJ software, version 1.36 . Colored images were acquired using a digital camera  at 400× magnification. Epidermal length was examined from stratum basale to stratum corneum by drawing a straight line and measuring the distance in µm unit (5 measurements/section).To characterize the expression of some markers involved with psoriasis pathogenesis, immunohistochemical staining was performed. Rabbit isotype polyclonal antibodies  were used, which measured the levels of: (1) Psoriasis-related protein: β-defensin , (2) Apoptosis: Caspase-3 and -9, (3) Proinflammatory cytokines: Tumor necrotic factor (TNF)-α, interleukins (IL)-1β, -6, -8, -17, -21, and -22, (4) Inflammatory chemokine: C–C motif chemokine ligand 20 (CCL20), (5) Anti-inflammatory cytokine: Transforming growth factor (TGF)-β, (6) Antioxidation: Nuclear factor erythroid 2-related factor 2 (Nrf-2), (7) Proliferation protein: Wingless-related integration site (Wnt). Sections were further subjected to microwave-induced antigen retrieval in citrate buffer (pH 6) following deparaffinization with xylene and hydration with ethanol. For blocking the endogenous peroxidase activity and non-specific binding, sections were treated with 1% v/v of hydrogen peroxide in methanol and 2% v/v of bovine serum albumin  respectively. Sections were incubated with each primary antibody, polymer HRP anti-mouse/rabbit labeling , diaminobenzidine visualization , and then counter-stained with hematoxylin. Finally, immunolocalization was examined under a light microscope.The level of expression of each protein was measured in terms of the H-score (percentage area of expression × intensity score). ImageJ software was used to quantify the immuno-distribution area in terms of percentage. Color images (10 images/group) were captured at 400× magnification. The immunolabeled area was measured by a threshold mode to obtain the percentage of positive pixels after the conversion of images to grayscale. Intensity was scored from 0 to 3 and was classified into four grading scales: 0–negative staining, 1–low-intensity staining, 2–moderate-intensity staining, and 3–high-intensity staining.FLG, caspase-14, S100a7a, and S100a14 genes were measured by RT-qPCR.To measure the expression of genes involved in psoriasis pathogenesis, mRNA expression levels of later solution. Briefly, samples were homogenized in a lysis buffer and loaded in a spin column. After RNA bound to the column, it was washed with the buffer. RNA was eluted in RNase-free water, RNA concentrations were measured on NanoDrop™ 2000/2000c spectrophotometer .An RNA purification kit  was used to extract mRNA from the rat skin samples preserved in the RNA−∆∆Ct method, the individual gene expression levels were calculated. The expression levels of β-Actin were used as a reference for accurate normalization of gene expression data.RT-qPCR was performed by using the iTaq Universal SYBR Green Supermix . In the CFX96 Touch™ Real-time PCR detector, the primer pairs were used as indicated; FLG: F-5′AGATGTGGACCACGATGACAA3′, R-5′TAGTGCTGGATCCTCGTCTTTT3′, β-Actin: F-5′CACTATCGGCAATGAGCGGTTCC3′, R-5′AGCACTGTGTTGGCATAGAGGTC3′, Caspase-14: F-5′CAGACCCTGACGGATGTGTTC3′, R-5′GCGAGGGTGCTTTGGATTTCGG3′, S100a7a: 5′TAGTGTGCCTCGCTTCATGGAC3′, R-5′CACAACTGCCGGTGAAACTGA3′, and S100a14: F-5′AACAATGGGACAGTGTCGGTC3′, R-5′ACTGCTGGGTAACCAGGTCTC3′ for amplification and denaturation . Using 2Pool samples of psoriatic skins from rats treated with or without ScF were subjected to proteomics study. The samples (30 mg/group) were ice cool ground in 300 μl of lysis buffer  in an ultrasonicator, and centrifuged at 12,000 rpm, 10 min at 4 °C. The supernatant solution was collected for the next step of proteomics analysis.Prior to gel electrophoresis, the total proteins in the supernatant solution were measured by Bradford protein assay  using a spectrophotometer . 30 μg of sample in each group was loaded into the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) device, using 4% stacking and 12% separating gels. Gel electrophoresis was performed with a constant voltage of120 V and current of 400 mA for 80 min . The gel was then stained with Coomassie Blue-R (CBB-R) for 10 min, and then was subjected to destaining by soaking in 30% methanol and 10% acetic acid for 2 h. The gel was washed in distilled water for 15 min and photographed by gel documentary . Each lane of the gel was cut into 13 pieces, and each piece of gel was placed in a new 1.5 ml tube.The gel pieces were dehydrated with 50% acetonitrile . Protein reduction was performed by adding 7 mM DTT (Dithiothreitol) in 50 mM ammonium bicarbonate for 15 min at 60 °C. Iodoacetamide 250 mM was added to the samples for alkylation and incubated for 30 min in the dark at room temperature. The alkylation reaction was quenched by using 4 mM DTT in 50 mM ammonium bicarbonate and the solution was discarded. The gel was dehydrated by adding 100% acetonitrile and dried for 1 h at room temperature. For the tryptic digestion of protein, solution of trypsin in 50 mM ammonium bicarbonate was added to gel samples and incubated for 24 h at 37 °C. The peptides were added to acetonitrile and kept for 20 min. The peptide solutions were transferred in 1.5 ml tubes. All peptide samples were dried at 40 °C by CentriVap Vacuum Concentrators .Rattus norvegicus (search date 02 September 2022). For the search parameter settings, the methionine oxidation was a fixed modification and carbamidomethylation of cysteine was a variable modification. The exponentially Modified Protein Abundance Index (emPAI) value was used for semiquantitative determination of protein expression levels48. The fold changes in protein expression were reported by comparison of the emPAI between sericin film and non-treatment groups. The proteins that had a fold change in protein expression ≥ 2 were classified on the basis of the biological functions using the UniProt database (www.uniprot.org). The associated proteins in the pathogenesis and physiology of psoriasis were explored with respect to the biological pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG)51. The amino acids in sericin and associated protein interaction and protein–protein interactions were investigated via STITCH version 5.0  (www.stitch.embl.de/).The dried peptides were added to a solution of 0.1% formic acid for dissolution of the peptides, before being tested on a mass spectrometer. The samples were subjected to liquid chromatography in UltiMate® 3000 Nano-LC systems . The peptides were separated using an Acclaim PepMap RSLC C18 column, measuring 75 mm × 15 cm, at a flow rate of 300 nl/min. The mobile phase was composed of solutions A and B a mixture of 0.1% formic acid and 80% acetonitrile in 0.1% formic acid, respectively. The peptide solution was subjected to gradient elution using solution B from 4 to 50% for 30 min. The eluted peptides were identified using the peptide spectra using positive electrospray ionization system coupled with microTOF-Q II . The MS and MS/MS spectra covered the mass range of m/z 400–2000 and 50–1500, respectively. Afterwards, the proteins were identified and quantified using Mascot Daemon version 2.3.02 . The protein database was acquired from SwissProt specific to p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). The data were calculated and expressed by mean ± standard error (SD) with the appropriate parametric and non-parametric independent t-tests or analysis of variance (ANOVA).Statistical analysis was performed by GraphPad Prism, version 6.05. Significant levels were considered as Supplementary Figure S1.Supplementary Figure S2.Supplementary Table S3.Supplementary Table S4.


Its immense application poses harmful effects on various non-target organisms as well. Therefore, searching the effective methods for the degradation of fipronil is imperative and logical. In this study, fipronil-degrading bacterial species are isolated and characterized from diverse environments using a culture-dependent method followed by 16S rRNA gene sequencing. Phylogenetic analysis showed the homology of organisms with Acinetobacter sp., Streptomyces sp., Pseudomonas sp., Agrobacterium sp., Rhodococcus sp., Kocuria sp., Priestia sp., Bacillus sp., Pantoea sp. The bacterial degradation potential for fipronil was analyzed through High-Performance Liquid Chromatography. Incubation-based degradation studies revealed that Pseudomonas sp. and Rhodococcus sp. were found to be the most potent isolates that degraded fipronil at 100 mg L−1 concentration, with removal efficiencies of 85.97 % and 83.64 %, respectively. Kinetic parameter studies, following the Michaelis-Menten model, also revealed the high degradation efficiency of these isolates. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed fipronil sulfide, benzaldehyde, (phenyl methylene) hydrazone, isomenthone, etc., as major metabolites of fipronil degradation. Overall investigation suggests that native bacterial species isolated from the contaminated environments could be efficiently utilized for the biodegradation of fipronil. The outcome derived from this study has immense significance in formulating an approach for bioremediation of fipronil-contaminated surroundings.Fipronil (C Fipronil -4-[(trifluoromethyl)sulfinyl]- 1H-pyrazole-3-carbonitrile] is a systemic insecticide grouped under the phenylpyrazole family . It is aStreptomyces rochei AJAG7 showed degradation efficiency of fipronil at 500 mg L−1 in MSM and soil in 6 and 7 days respectively. Fungal species such as Trametes versicolor and Aspergillus glaucus have been reported for their degradation potential of fipronil  utilizing it as a solitary nitrogen and main carbon source. In a recent study, Bacillus amyloliquefaciens, which achieved 93% degradation of 10 mg L−1 of fipronil during 5 days of incubation period. Bacillus firmus, Burkholderia thailendensis, Acinetobacter calcoaceticus, Acinetobacter oleivorans, Paracoccus sp., Gamma Proteobacteria are some of the known bacterial isolates, involved in biodegradation of fipronil  was prepared in acetonitrile, filtered, and kept at 4 °C for further use  [NH4] citrate, 20 mg CaCl2, 0.5 g NaHCO3 and 1 mL trace element solution (in 100 mL stock solution) of 50 mg FeSO4.7H2O, 1 mg ZnSO4.7H2O, 0.3 mg MnCl2.4H2O), 3 mg H3BO3, 2 mg CaCl2.6H2O, 0.1 mg CuCl2.2H2O, 0.2 mg, NiCl2.6H2O, 0.3 mg Na2MoO4.H2O per liter in double distilled water and pH was maintained between 6.8-7 , with a purity of 95% was bought from Sigma-Aldrich, USA. HPLC grade acetonitrile, dichloromethane, and, ultra-pure HPLC water were purchased from E. Merck Limited, Mumbai, India. Acetonitrile and water were filtered through a 0.5 μm and 0.45 μm nitrocellulose syringe filter (Merck Millipore Ltd.) respectively before use. A stock solution of fipronil , samples of sludge from drainage  and activated sludge sample from Sewage Treatment Plant (STP) Bhagwanpur, Varanasi, India  83m above mean sea level (MSL). The topsoil layer samples (20 cm depth) were collected with the help of a sterile corer. Sludge samples were collected with the help of a sterile spatula. All the samples were placed in sterile poly bags, and transported to the laboratory. The collected samples were dried, passed through a 2 mm mesh sieve to remove debris, and stored at 4 °C for further investigation.−1 of fipronil in three different 250 ml Erlenmeyer flasks containing 45 mL of MSM media. All three flasks were incubated on a shaker (120 rpm) at 28±2 °C for 2 weeks. After the incubation period, 1 mL of suspension from each flask was put into their respective flasks containing 50 mL fresh MSM supplemented with 50 mg L−1 fipronil and incubated on a rotary shaker at 120 rpm at room temperature for overnight. Serial dilution of overnight samples with microbial growth was made from 10−1 to 10−9 and incubated in vials containing MSM and fipronil (50 mg L−4). Lower dilutions were again transferred into vials having 10 mL MSM and fipronil (50 mg L−4) and were incubated on a shaker at 120 rpm (28±2°C). The control included MSM, and fipronil but absence of bacterial inoculum. Vials showed turbidity as an indicator of bacterial growth. Thus, 100 μl of enriched samples from vials showing the best bacterial growth were spread on MSM agar plates supplemented with 50 mg L−1 fipronil . After the extraction of DNA, quantification was done using a spectrophotometer . Amplification of 16S rRNA gene (1.5 kb sized) was done in a Thermal cycler  using universal primers 27 F’ 5’ - AGAGTTTGATCMTGGCTCAG-3’ (forward) and 1492R’ 5’ - TACGGYTACCTTGTACGACTT-3’ (reverse) in a reaction mixture including 10X Buffer, 10 mM dNTPs, 500 U Taq DNA polymerase, 100 ng of template DNA and volume was maintained up to 50 μL with miliQ. The Polymerase Chain Reaction (PCR) cycling parameters were initial denaturation at 94 °C for 5 min that follows by 30 cycles of denaturation at 94 °C (1 min), further annealing at 55 °C (1min), extension step at 72 °C (1.30 min), and the last extension step at 72 °C for 5 min, uphold at 4 °C. DNA bands were observed on 1% agarose gel for PCR-amplified 16S rRNA amplicon size. Purification of amplified PCR products was done using MinElute® PCR Purification Kit (Qiagen) and sequencing was done by an automated sequencer at Centyle Biotech Pvt. Ltd., New Delhi using Sangers dideoxy nucleotide chain termination method. Processing of obtained sequence was done by BioEdit Sequence Alignment Editor (Version 7.2.5). Further identification of sequences was done based on reference species present in National Center of Biotechnology Information (NCBI) using BLAST . Finally, the sequences were submitted with NCBI database to get the GenBank Accession number. The phylogenetic tree was constructed for bacterial strains by the neighbor-joining method using MEGA X (Version 11.0.13) software . All these experiments were performed in replicates to minimize the chances of error. In order to determine the degradation potential of bacterial strains for fipronil, HPLC analysis was performed.To check the tolerance capacity of bacterial strains, a gradually increasing concentration range of fipronil was selected in which bacteria showed enormous growth in a short period. The inoculum for batch culture studies was prepared through LB overnight culture of bacterial strains. Cells were then centrifuged , pellets were washed with sterile saline (0.85%) and resuspended in sterile saline. A series of 100 ml flasks comprising 50 ml MSM were inoculated with 2 mL inoculum of individual isolates and supplemented with gradually increasing fipronil concentrations of 10, 25, 50, 100, 200, 300, & 400 mg L717plus Autosampler) HPLC  equipped with reverse phase C18 column (SunFire® 4.6 mm × 250 mm × 5 μm) and Photodiode Array (PDA) detector (Waters 2998). Acetonitrile and water  including 0.1% phosphoric acid were used as a mobile phase, and the flow rate was maintained at 1 mL min1−. 20 μL samples were injected and peaks were detected at a wavelength of 278 nm. All the samples were filtered through a 0.22 μm nylon syringe filter (Merck Millipore Ltd.) prior to their injection. For the quantification of fipronil concentration, a standard curve of peak area vs fipronil concentration was prepared. In order to know whether average degradation rate varied depending on concentration, the following formula  was chosen for the study of kinetic constants using the Michaelis-Menten model. MSM media containing individual isolates and amended with optimum fipronil concentration were incubated at 28±2 °C at 100 rpm for 13 days. For analyzing residue fipronil concentration using HPLC, samples were collected regularly at time intervals of 24 hr.For the residue analysis of fipronil in samples, HPLC was performed through a method described by −1 concentration of fipronil. These were incubated for 7 days at 28±2 °C at 100 rpm. After centrifugation (5000 rpm 15 min) of bacterial culture, the supernatant was taken for further extraction of fipronil and its metabolites through liquid-liquid partitioning by adding an equal volume of dichloromethane and then evaporated on a rotary evaporator to concentrate the sample. Further, it was resuspended in 4 mL MS-grade acetonitrile and analyzed on a triple quadrupole Gas Chromatography Tandem Mass Spectrometry  with autosampler /injector AOC-20i+s. Rxi®- 5 Sil MS Capillary column   was used for separation. 1 μL sample was injected in a linear velocity, split mode with a ratio of 10, and the injector temperature was 260°C. Carrier gas, Helium was used with a flow rate of 1.21 mL min−1. The oven temperature program was as follows: 100 °C (2 min hold) and increased up to 300 °C at a rate of 10 °C min−1 (18 min hold). Ion source temperature was 220 °C and the interface temperature was 270 °C with a solvent cut time of 4.50 min and operated in Flame Thermionic Detector/ Barrier discharge Ionization Detector (FTD/ BID) mode. MS has performed in Multiple Reaction Monitoring (MRM) mode. Data were processed and integrated through Shimadzu Real-time analysis software. On the basis of retention time (RT) and molecular weight , fipronil and its metabolites were identified using National Institute of Standards and Technology (NIST) library database.To determine the intermediates produced during the biodegradation of fipronil, FTIR and GC-MS analyses were performed. Two of the most efficient bacteria from the above study were selected for this experiment. 100 mL flasks containing 40 mL sterilized MSM were inoculated with 2 ml of both of the active bacterial strains individually and spiked with the 50 mg L−1 with a scan speed of 64 and resolution of 4 cm−1. For sample preparation, 20 mL sterilized MSM were enriched with fipronil (50 mg L−1) and 1 mL bacterial inoculum of both strains individually. A control sample was prepared without bacterial inoculum. After 7 days of the incubation period , the supernatant was collected by centrifugation (5000 rpm 15 min), and extraction of compounds was done by adding an equal volume of dichloromethane. The organic layer was evaporated on a rotary evaporator and residues were reconstituted in acetonitrile for analysis of the functional groups and any bond stretching due to fipronil biodegradation.For identification of functional group present in parent chemical and its intermediate metabolites FTIR analysis was performed through KBr pellet mode on Nicolet iS5 (THERMO Electron Scientific Instruments LLC). Spectra were recorded in the infrared region of 400-4000 cmTable S1. 16S rRNA nucleotide sequencing and phylogenetic tree analysis validated that isolate belonged to ten different bacterial genera Acinetobacter sp. (FIP_A1), Streptomyces sp. (FIP_A3), Pseudomonas sp. (FIP_A4), Agrobacterium sp. (FIP_A8), Rhodococcus sp. (FIP_B3), Kocuria sp. (FIP_B4), Priestia sp. (FIP_B10), Bacillus sp. , Aeromonas sp. (Fip_C8), Pantoea sp. (FIP_ C9)  and six  were gram negative and gram-positive, respectively. All the isolates showed positive responses for catalase test while in case of urease test, only three isolates  gave positive results. All the isolates except FIP_B3 and FIP_B4 showed positive results for citrate utilization test. For the oxidase test, FIP_A3, FIP_A4, FIP_A8, FIP_B4, and FIP_C8 were found positive except these all showed negative results. For the nitrate reduction test, only isolates FIP_A1, FIP_A3, FIP_A8, and FIP_C9 showed negative results. Biochemical test results of all isolates have been enlisted in FIP_ C9) .Streptomyces sp. competent for degrading fipronil in aqueous media and soil. Bacillus megaterium. Acinetobacter calcoaceticus and Acinetobacter oleivorans also showed their efficacy in degrading fipronil in soils , and FIP_B3 (0.114 d−1) at 100 mg L−1 while the lowest in case of FIP_C9 (0.075 d−1) at 50 mg L−1.The results of average degradation rate of fipronil degradation studies by bacterial isolates are shown in al cells . The var−1) and FIP_A8, FIP_B4, FIP_B10, FIP_C5, FIP_C6, and FIP_C9 (50 mg L−1)  . It was Stenotrophomonas acidaminiphila was reported by −1 fipronil in 14 days as a carbon source. A study reported by Staphylococcus arlettae and Bacillus thuringiensis were able to degrade 76.39% and 65.95% of 10 mg L−1 of fipronil after 7 days. These results were obtained at relatively lower concentrations in contrast to the present study. While A bacterial strain The kinetics of fipronil biodegradation at the optimum concentration was investigated by selecting the Michaelis-Menten paradigm of the microbial kinetics model because substrate concentration and oxygen limitation did not serve as inhibitory factors in the biodegradation process. For the biodegradation process, kinetic parameters were resolved as described by Gautam and Dubey (2022). The non-linear kinetic model for the Michaelis-Menten equation is as follows :(2)dSmax is maximum degradation rate of substrate (mg L−1 d−1), S0 and St are substrate (fipronil) concentrations (mg L−1) at time 0 and t respectively, Ks is Michaelis-Menten constant (mg L−1)  because biodegradation activity is accessed by virtue of intact bacterial cells instead of purified enzymes. Ks is half-saturation concentration of substrate or the concentration of substrate at which reaction achieves half of its Vmax (maximum degradation rate). Values of Ks and Vmax/KS were deduced through slope and intercept of the Lineweaver Burk equation respectively. 1/t (ln S0/St) was plotted against (S0-St)/t to obtain these kinetic parameters, Ks was calculated through the inverse of slope and Vmax was obtained through intercept of the best fit straight line of experimental data. Vmax/KS was calculated after getting the values of Ks and Vmax  can be considered as a better parameter instead of Vmax and Ks individually for measuring nutrient (fipronil) uptake efficiency and assimilation ability of bacterial isolates  as depicted in max, Ks, and, Vmax/Ks for all the bacterial isolates have been represented in Kinetic parameters were calculated with the help of the Lineweaver Burk equation, also known as the double reciprocal plot by linear regression plot .(3)1tand Vmax . The bioand Vmax  because,isolates . Higher s (mg L−1) was found between 135.13 to 166.67 mg L−1 for isolates FIP_A1, FIP_A3, FIP_A4, FIP_B3, and between 61.73 to 76.33 mg L−1 for isolates FIP_A8, FIP_B4, FIP_B10, FIP_C5, FIP_C6, FIP_C9. However, values of Vmax (mg L−1 d−1) were found highest for FIP_A4 (19.84) and FIP_B3 (19.13) followed by isolates FIP_A1 > A3 > A8 > C6 > B4 > C5 > C9 > B10 values ranged between 2.83 to 15.01 mg L−1 d−1. The values of Vmax/Ks (d−1) were found highest for isolates FIP_A4 and FIP_B3 i.e., 0.125 and 0.115 respectively whereas it was between 0.045 to 0.096 for the remaining strains with trend FIP_A1 > FIP_A3 > FIP_A8 = FIP_C6 > FIP_C5 > FIP_B4 > FIP_B10 = FIP_C9. The result obtained through the above kinetic study showed that the highest Vmax/Ks was observed in the case of FIP_A4 and FIP_B3, followed by other eight bacterial isolates. On that basis, it was concluded that Pseudomonas sp. FIP_A4 and Rhodococcus sp. FIP_B3 was the most efficient degrader of fipronil while the isolate Pantoea sp. FIP_C9 was found to be the least efficient among all the ten bacterial isolates.Results obtained through our present study indicated that the value of KBacillus sp. FA3 followed first-order reaction kinetics for degradation of 50 mg L−1 of fipronil concentration with a degradation constant (k) of 0.0891 day−1, half-life (t1/2) of 7.7 days, determination coefficient (R2) of 0.921 day−1 and Ks of 65.096 mg L-1. −1) and reported that Bacillus sp. FA4 followed the kinetics of first-order reaction for fipronil biodegradation with k, t1/2, R2, and Ks of 0.0861 day−1, 8.04 days, 0.970 days−1, and 12.08 mg L−1. However, sufficient data for comparison was unavailable related to the kinetic parameters of the Lineweaver Burk plot due to the lack of Michaelis-Menten kinetic study on fipronil biodegradation. Therefore, comparison of all the kinetic parameters was not discussed here. Gautam and Dubey (2022) reported kinetic parameters for the biodegradation of imidacloprid, a neonicotinoid insecticide i.e., Ks values ranged between 70.9 to 144.9 mg L−1, Vmax values ranged between 3.7 to 15.5 mg L−1 d−1 and Vmax/Ks(d−1) values ranged from 0.051 to 0.107. s, Vmax, and Vmax/Ks values found 92.07, 13.07, and 0.14 respectively. Based on the above result it was concluded that the result of present study was in line and showed conformity with other previous results.max/Ks) was found highest, metabolites formed by them were analyzed. Metabolome analysis using GC-MS provided information regarding the compounds formed after the degradation of fipronil by FIP_A4 and FIP_B3 . For FIP_A4 compound A1 was identified as fipronil sulfide at retention time (RT) 28.645 min with m/z of 417.51, compound A2 as N-[2--5-iminomethyl-4-trifluoromethanesulfonyl-2Hpyrazol-3-yl]-acetamide at RT 32.945 min with m/z of 495.45. The compound A3 was identified at RT 24.950 min with m/z of 467.40 as hydroxylated fipronil sulfone and the compound A4 at RT 13.8 min with m/z of 154.10 was identified as isomenthone . A peak at 1735 cm−1 was observed in the samples treated with bacterial strains which correspond to ester groups indicating the formation of esters upon degradation of fipronil. Another band in treated samples at 1221cm−1 also indicates formation of ester group in the samples. Peak formed around 1350 cm−1 showed the formation of sulfone derivatives of fipronil further proving the degradation of fipronil to its sulfone derivatives.The biodegradation of fipronil is confirmed by correlating the numerous changes in FTIR spectra of bacterial strain-treated samples  with control (MSM + Fipronil) Aspergillus glaucus strain AJAG1 observed similar bands in FTIR spectra around 1737 and 1271 cm−1 indicating formation of esters after degradation of fipronil. Control and bacterial-treated samples both include some similar peaks which could be due to the undegraded fipronil. −1 concentration) with degradation efficiency of ~ 61 to 86 % in 13 days. Among all the isolated bacterial species Pseudomonas sp. FIPA4 and Rhodococcus sp. FIPB3 were found to be the most promising for the bioremediation of fipronil. Their higher values for kinetic parameters such as Vmax and Vmax/Ks also ratified the efficiency of these two isolates for the removal of higher concentrations of fipronil. Identified intermediate metabolites provide evidence of bacterial degradation of fipronil. Thus, these isolates can aid in the degradation of fipronil and contribute to the reduction of its harmful effects on the environment and human health.Bacterial strains isolated from fipronil-contaminated environments showed high potential for removal of fipronil (10-400 mg L