Due to their physical and chemical properties  they have been used for image guided therapy for cancer treatment monitoring. There are various limitations that make many theranostic agents unable to be used for the extended periods of time required for enhancing theranostic capabilities. Some of these are due to inherent characteristics  present upon continuous irradiation and others are due to environmental  conditions that can lead to physical instability  affecting the amount of particles that can accumulate at the target site and the overall contrast that can be achieved. In this study, perfluorohexane (PFH) nanoemulsions (NEs) were synthesized with silica coated gold nanoparticles (PFH-NEs-scAuNPs) in order to give both stable and enhanced signals for cancer imaging by increasing vaporization of the emulsions into bubbles through the process of optical droplet vaporization (ODV). The resulting perfluorohexane bubbles could be imaged using nonlinear ultrasound (NL US) which significantly increases the signal to noise ratio due to the nonlinear scattering properties of oscillating bubbles. The NL US signals from PFH bubbles were found to be more stable compared to conventional bubbles used for contrast imaging. In addition, the vaporization of PFH NEs into bubbles was shown to cause significant cancer cell death reflecting the theranostic capabilities of the formed PFH bubbles. Since cell death is initiated with laser excitation of PFH-NEs-scAuNPs, these nanoparticles can specifically target cancer cells once they have accumulated at the tumor region. Due to the type of theranostic agent and imaging modality used, the PFH-NEs-scAuNPs can be used to provide higher specificity compared to other agents for locating the tumor region by minimizing tissue specific signals while at the same time being used to treat cancer.Nanoparticles have extensively been used for cancer therapy and imaging ( PFH-NEs from PFH-NEs-scAuNPs can vaporize upon laser excitation leading to formation of PFH bubbles that can be used for contrast enhanced US imaging and therapy. Research has expanded substantially in the development of various nanoparticles that can be used both for therapy and imaging of various diseases.3–6 An important class of nanoparticles are those made of inorganic material  as their unique electric, magnetic and plasmonic properties enable them to destroy cancerous cells at the same time as being used to monitor tumor growth/regression through imaging.7,8 For example, iron based nanoparticles can induce hyperthermia in tissue through heat generation when under a magnetic field9 and/or be used as contrast agents for magnetic resonance imaging (MRI).10 Semiconductor nanoparticles such as quantum dots use their energy band gaps for fluorescence imaging with an alloy core encapsulated in an insulating inorganic shell to enhance their quantum yield.11 Due to their narrow fluorescence emission and broad absorption spectra, multiple quantum dots can be excited at a time for in vivo imaging of multiple components in biological systems.12 Such different types of nanoparticles show great potential for biomedical applications. Having said this, there continues to be a need for different methods/techniques and technologies for imaging and synthesizing nanoparticles that can greatly enhance specific target signals  while minimizing background signals . The NPs and imaging modality at the same time should allow for greater depths of imaging in tissue compared to conventional imaging.Nanotechnology is a very fast growing and interdisciplinary field bringing together knowledge from chemistry, biology, physics and engineering. It has found many applications in industries such as agriculturein vivo are gold nanoparticles, able to release non-radiative heat from localized surface plasmon resonance, a phenomenon in which electrons oscillate coherently in resonance with the incident electromagnetic wave at a specific frequency. In most cases for deeper penetration and imaging in vivo, the frequency of oscillation is shifted from the visible to the near-infrared (NIR) region by increasing the aspect ratio of the nanoparticles13 and/or aggregation through the interparticle coupling effect.14 Both methods have been shown to enhance NIR absorption, with photothermal properties from absorption used for destroying cancer cells.15–17 A popular imaging modality used to detect signals from nanoparticles is photoacoustic (PA) imaging.18,19 Taking advantage of the absorption properties of nanoparticles and the resulting pressure generated upon optical excitation, PA imaging is able to distinguish the regions of nanoparticle accumulation for monitoring of cancer tumor therapy. Compared to other imaging modalities ), PA imaging can provide greater spatial resolution and deeper tissue penetration due to detection of ultrasonic signals, which attenuate less compared to visible electromagnetic waves.20 In our previous work we synthesized perfluorohexane nanoemulsions (PFH-NEs) that were able to give photoacoustic signals through the intrinsic near-infrared absorption properties of the fluorosurfactant shell of particles.21 The photoacoustic signals detected were from the vaporization of PFH-NEs into PFH bubbles due to conversion of the volatile PFH liquid into gas through the process of optical droplet vaporization (ODV).22 Coupling PFH-NEs with silica coated gold nanoparticles (scAuNPs) was shown to further increase PA signals in tissue-mimicking phantoms23 due to the enhanced absorption from scAuNPs surrounding the NEs. However even with the ability to provide strong acoustic signals, when using photoacoustic imaging in vivo, the background signal from blood and other tissue chromophores can reduce the contrast between tissues in which PFH-NEs-scAuNPs have accumulated and the surrounding tissues, even after spectral unmixing is applied. An approach with a greater target-to-background ratio is needed.One kind of inorganic nanoparticle able to provide deeper imaging 24 CEUS imaging specifically detects signals from the contrast agent  targeting the region, suppressing unnecessary signals from background tissue. This is because signals from CEUS imaging are from nonlinear bubble subharmonic and superharmonic scattering,25 providing a highly specific signal confined to the location of the contrast agent . In this work we show that coupling scAuNPs to PFH-NEs can be used to give strong CEUS signals after laser induced vaporization of NEs into PFH bubbles through ODV . The bimodal imaging capability of PFH-NEs-scAuNPs for CEUS and photoacoustic imaging23 can complement each other and increase treatment success by efficiently locating the tumor (using photoacoustics), and then imaging treated areas where signals are greatest (using CEUS). The imaging feedback would facilitate altering treatment conditions and dose, if required. The approach is similar to other important nanoparticle systems where CEUS is combined with other imaging modalities where the conversion of perfluorocarbon nanoparticles into bubbles upon laser excitation can be used to induce cancer cell death.26,27 The agents' ability to be used for deep tumor treatment and imaging by absorbing penetrating NIR light and the ability to provide ultrasound signals specifically from PFH bubbles make them highly advantageous over other theranostic agents. Moreover, compared to other nanoparticles, the PFH-NEs-scAuNPs developed have very good biocompatibility, enabling higher concentrations for improving theranostics. The nanoparticles can be easily coupled without the requirement of covalent linkage, minimizing the synthesizing steps and can be applied to a broad range of nanoparticles where theranostic capability can be provided through simple electrostatic interactions.An imaging modality that provides high signal to noise for bio-imaging is contrast enhanced ultrasound (CEUS) imaging.ough ODV , with OD2.2.1.28 was used by adding chloroauric acid (HAuCl4) while vigorously stirring sodium citrate in Milli-Q water at boiling. A coating of silica was added to gold nanoparticles by adding (3-aminopropyl)trimethoxysilane (APTMS) with a sodium silicate solution.29 The silica thickness of these nanoparticles were found to be around 10 nm with a 5 nm gold core sphere, to give a total size of 25 nm and absorption in the visible electromagnetic region23  in methanol  were made by mixing for 24 hours a solution of 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES) (80 μL) , ammonium hydroxide (70 μL)  and scAuNPs.30,31 To remove excess silica and unreacted products after silica coating and fluorination, particles were repeatedly washed using centrifugation. To make PFH-NEs with scAuNPs, particles were either mixed together after first emulsification of PFH-NEs (referred to as unfluorinated nanoparticles or unfluor. NPs) or first fluorinating scAuNPs and then suspending in PFH (600 μL) and adding Zonyl FSP (150 μL) and MilliQ water (4250 μL) prior to vortexing and sonication (using above settings) (referred to as fluorinated nanoparticles or fluor. NPs). To achieve the required concentrations for nanoparticles, samples were diluted or concentrated  in terms of weight over volume of solution after determining the mass of solutions of PFH-NEs and scAuNPs.To synthesize nanoemulsions, microemulsions were first made by mixing for 1 minute (2700 rpm using vortexer) a solution containing 600 μL perfluorohexane (PFH) , 150 μL of Zonyl FSP fluorosurfactant (with anionic phosphate group)  and 4250 μL of Milli-Q water. A sonicator  was then used for 2 minutes using 10 seconds on/20 seconds off cycles at 4 °C in an ice water bath . To make gold nanoparticles, the sodium citrate reduction method2.2.−1) given the much greater concentration of PFH-NEs used relative to scAuNPs. The size distributions represented for each type of nanoparticle were from more than 500 nanoparticles to obtain a representative size distribution, with size reported as mean ± standard deviation from each distribution. To characterize the PFH bubbles, unfluorinated or fluorinated samples  were placed in 60 mm × 15 mm polystyrene dishes  before laser excitation using 680 nm wavelength using a preclinical Vevo LAZR commercial imaging system . Nanoemulsions from each sample were vaporized for 5 minute intervals at three different locations in the dish before being imaged using a ZOE Cell Imager (Bio-Rad). For all brightfield images a gain of 8, exposure time of 300 ms, and LED intensity of 40 was used. The average size (±standard deviation) for PFH bubbles from unfluorinated and fluorinated samples were determined from more than 100 bubbles (for each type of nanoparticle) and quantified using ImageJ. The morphology of scAuNPs and PFH-NEs-scAuNPs were determined using transmission electron microscopy  immediately after placing a few drops of sample on a grid with the absorption spectra of scAuNPs determined using a Perkin Elmer Lambda 20 UV/vis spectrometer. To image nanoparticles in MCF-7 cancer cells, an Olympus CKX41 microscope was used with 10× phase contrast objective after 1.5 hours incubation with unfluorinated (0.6 mg mL−1 PFH-NEs with 0.09 μg mL−1 scAuNPs) and fluorinated nanoparticles (0.6 mg mL−1 PFH-NEs with 0.036 μg mL−1 fluorinated scAuNPs). Cells were first grown at a concentration of 125 000 cells per mL for 24 hours before incubation with particles. Images were created using the given red, green, blue intensities for representing signals, with brighter intensities representing nanoparticles from the lighter background.To determine the absorption spectra of unfluor. and fluor. NPs, a NanoDrop spectrophotometer (ThermoFisher Scientific) was used. The size of PFH-NEs with scAuNPs were characterized using an Archimedes Particle Metrology System , which can measure size of particles less than ∼1 μm. The size distributions of negatively buoyant unfluorinated and fluorinated nanoparticles were determined by assuming the particles had the density of perfluorohexane  to determine NL US signal enhancement. Furthermore, ultrasound signals were also analyzed after vaporization from PFH bubbles compared to before vaporization from PFH-NEs-scAuNPs using the Vevo LAZR  with the time/spatial domain signals used to convert into amplitude spectra using an established method through fast Fourier transform of RF data.32 To determine the stability of PFH bubbles from particles, unfluorinated (5 mg mL−1 PFH-NEs with 0.75 μg mL−1 scAuNPs) and fluorinated nanoparticles (5 mg mL−1 PFH-NEs with 0.3 μg mL−1 fluorinated scAuNPs) were mixed with 10% Type A gelatin with 2% v/v formaldehyde before the NEs were vaporized directly after making inclusions at day 0, and after 24 (day 1) and 48 hours (day 2). Before acquiring the NL US signals from PFH bubbles, each inclusion with particles was excited for 10 seconds at 680 nm at each day using the Vevo LAZR commercial system. Between days inclusions were placed in a 37 °C water bath.For contrast enhanced ultrasound (CEUS) imaging of perfluorohexane bubbles formed after laser excitation of unfluorinated and fluorinated samples (PFH-NEs plus scAuNPs), a Vevo LAZR imaging system was used (with LZ250 transducer using 18 MHz frequency). The nonlinear contrast mode of the Vevo LAZR uses amplitude modulation sequences to generate the images with significant background signal suppression. The nanoparticles were injected in a tissue mimicking phantom made using 10% w/v Type A gelatin  and 2% v/v formaldehyde  by first inserting nanoparticles in ∼1 mm inclusions, followed by imaging PFH bubbles after 10 s vaporization at 680 nm  for fluorinated samples. After incubation of the nanoparticles with cells at the above time points, cells were washed three times with PBS to remove any external particles prior to trypsinization using 0.05% trypsin–EDTA. To make cell inclusions, the cells were mixed with Type A gelatin to create inclusions on the surface with the same concentration of gelatin  prior to measurements in a heated water bath at 37 °C  and imaged with the same experimental and NL imaging conditions previously mentioned for imaging in cell inclusions. To measure NL US signals from cell inclusions in tissue mimicking layers, hemoglobin (0.67 mg mL−1)  and 20% intralipid (7 mg mL−1)  were used to mimic the optical and acoustic properties of breast tissue.33–36 The same experimental conditions were used as above for incubation of nanoparticles with cells after 24 hours. A PFH-NEs concentration of 125 mg mL−1 and silica coated gold nanoparticle concentration of 18.8 μg mL−1 were used for unfluorinated samples and 7.5 μg mL−1 of fluorinated scAuNPs with perfluorohexane nanoemulsions (125 mg mL−1) for fluorinated samples, for incubation with MCF-7 cells. All imaging of cells with nanoparticles were carried out at non-cytotoxic concentrations as measured by trypan blue viability test (viability above 90% for both types of nanoparticles).To analyze CEUS images from MCF-7 cancer cells that incorporated the nanoparticles, a 125 000 cells per mL solution was first incubated for 24 hours followed by incubation for 4, 24 and 48 hours with nanoparticles. For experiments in cells, a PFH-NEs concentration of 10 mg mLin vitro and in vivo experiments in channels and with cells were determined to be statistically significant compared to controls  using two sample t-test .To compare NL US signals from bubbles (formed from the nanoparticles by ODV) to those conventionally used for ultrasound imaging, Definity microbubbles  at a concentration of 3.3% volume/volume were used and the same procedure followed for experiments and analysis. To activate Definity bubbles vials were shaken using the Vialmix activation system from the company using their defined instructions for 45 seconds. For all experiments involving Definity bubbles, only NL US imaging was applied with no prior laser exposure. The averages reported for nonlinear ultrasound signals are the mean ± standard deviation from three replicates from gray scale values from a 3 mm × 1 mm region within the inclusion . NL US signals from 2.4.−1 insulin) in 35 mm polystyrene dishes . Samples were irradiated using 680 nm wavelength for two intervals of 10 minutes each using the Vevo LAZR at a focus of 11 mm. The Vevo LAZR sends 5 ns pulses of light (20 mJ cm−2) at a repetition frequency of 20 Hz. Samples (cells plus nanoparticles) were mixed between the two intervals of excitation to ensure previously untreated cells could be treated. A final concentration of 10 mg mL−1 of PFH-NEs were used for both unfluorinated (with 1.5 μg mL−1 of scAuNPs) and fluorinated samples (with 0.6 μg mL−1 of fluorinated scAuNPs). As a control MCF-7 cells were treated with scAuNPs only (1.5 μg mL−1) with same laser excitation conditions as above. Propidium iodide  was used to determine nonviable cells after treatment by incubating 10 μL of a solution of 1 mg mL−1 of propidium iodide with cells for 5 minutes prior to washing cells with media. To image cells, a ZOE fluorescent imager was used with excitation of 556/20 nm and emission filter of 615/61 nm with viability analyzed using a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter). To measure viability, 600 μL samples containing treated cells were placed in sample cups before quantifying nonviable cells based on the brightness from trypan blue labelled cells (after removing any NPs and bubbles using repeated centrifugation at 100 × g for 5 minutes each multiple times). The percent viability is reported as mean ± standard deviation from three replicates.To determine the potential of nanoparticles to cause cancer cell death through stresses caused by vaporization, nanoparticles were mixed with 926 000 MCF-7 cells in suspension (in DMEM media with 10% FBS and 0.01 mg mL−1 with 3 μg mL−1 of silica coated gold nanoparticles (ScAuNPs) for unfluorinated samples or NEs concentration of 20 mg mL−1 with 1.2 μg mL−1 of scAuNPs for fluorinated samples were used. For each replicate, 50 μL of cell solution with NPs were placed on 35 mm × 10 mm polystyrene dishes  with NPs vaporized at 680 nm using the Vevo LAZR at different treatment times  for which cancer cells were continuously treated. The viability of cells was then quantified using Vi-Cell XR Cell Viability Analyzer (Beckman), using trypan blue as the dye for nonviable cells. Viability was quantified as percent of control PC-3 cells (without any NPs and treatment with laser), to determine the percent of viable cells after laser treatment with NPs (after removing any NPs and bubbles using centrifugation at 100 × g for 5 minutes each for three times). Viability of non-treated cells, with laser only and with NPs only (for both unfluor. and fluor. NPs) at the above treatment times were above 90%. The viability (% of control cells) is reported as mean ± standard deviation from three replicates. Additionally, the sizes of treated cells were recorded to determine the extent of cell damage using the Vi-Cell XR Cell Viability Analyzer.To determine the ability of PFH-NEs-scAuNPs to cause cancer cell death upon laser excitation and their effectiveness in treating other cell types, NPs were directly mixed with PC-3 prostate cancer cells (grown using RPMI-1640 media with 10% FBS). Using 125 000 cells either a NEs concentration of 20 mg mL2.5.in vivo experiments for NL US imaging, BALB/c mice (6 weeks old) were purchased from Charles River Laboratories for determining the imaging capability of PFH bubbles. Tumors were grown using 150 000 4T1 breast cancer cells injected subcutaneously in the hindlegs of 6.5 weeks old mice. Tumors were then allowed to grow for 10 days before injecting nanoparticles intravenously through tail vein. For imaging each tumor, a dose of 4 mg of NEs per gram weight of mouse was used using a solution of 480 mg mL−1 of PFH-NEs with 0.08 mg mL−1 scAuNPs for unfluorinated nanoparticles, and 480 mg mL−1 of PFH-NEs with 0.03 mg mL−1 scAuNPs for fluorinated nanoparticles. Doses used for nanoemulsions and nanoparticles for in vivo experiments were below those used in other in vivo experiments for imaging and therapy.37–40 Furthermore, in vitro tests with cells at these doses showed high viability (>90% using trypan blue viability assay) in order to minimize cytotoxic effects. For therapy, tumors were irradiated (after 1.5 hours incubation of NPs) with 680 nm laser excitation (20 mJ cm−2 at 680 nm for 5 min for each treatment and time point for each day) using the Vevo LAZR system before collecting NL US images using the scanner. Multiple 2D CEUS images were collected from each tumor using the scanner from the Vevo LAZR imaging system. For CEUS and linear US imaging and acquiring signals, a CEUS gain of 35 dB and linear US gain of 50 dB were used. Signals are from PFH bubbles from maximum averaged gray scale signals from different slices within tumors compared to signals before injection of nanoparticles or before vaporization of nanoemulsions for each day. The Vevo LAB software from Vevo LAZR  was used for analysis of in vivo signals from both types of nanoparticles. For determining the therapeutic effect from laser excitation of PFH-NEs-scAuNPs and PFH bubbles, volume measurements of tumors (mm3) were determined using the 2D slices of images (collected using the Vevo LAZR for tumors) using the Vevo LAB software. All mice were handled according to the protocol (SMH protocol 870) approved by St. Michael's Hospital Animal Care Committee and by the Canadian Council on Animal Care.For proof of concept 3.3.1.21 while scAuNPs were synthesized and fluorinated using APTMS, sodium silicate and PFDTES ,23 which absorb in the visible to near-infrared region  and fluorescence cross-correlation spectroscopy (FCCS). By fluorescently labelling both the NEs and scAuNPs and using the latter technique (FCCS), it was determined that the PFH-NEs and scAuNPs are co-diffusing. The ability of these nanoparticles to form clusters is advantageous as the PFH-NEs can be vaporized into PFH bubbles after passing through larger sized endothelial gaps in the tumor vasculature,41 where they can be used for theranostics after accumulation at the tumor site.Perfluorohexane nanoemulsions were synthesized previously using sonication and found to have a uniform size distribution around 50 nm 21 while 42,43 The size of PFH bubbles formed from unfluorinated and fluorinated samples were 2.4 ± 0.5 μm and 7.3 ± 1.9 μm, respectively, determined from images captured 5 minutes after irradiation. The larger microbubbles formed are important for enhancing US scattering for locating tumor specific regions for imaging and therapy after nanoparticles have accumulated at the target site. To demonstrate that the NPs can efficiently target/internalize in cancer cells, NPs were imaged using phase contrast with MCF-7 cells. A significant number of highly scattering nanoparticles from both unfluorinated and fluorinated samples were localized at the membranes of cells   imaging. CEUS imaging (unlike ultrasound B-mode imaging) is a technique which is sensitive predominantly to the nonlinear scattering signals from bubbles (subharmonic and superharmonic generation from bubble scattering) and is able to provide high signal to noise compared to the linear scattering detected from the surrounding tissue. When ultrasound (US) contrast agents are used, CEUS is effective at locating the region the bubbles have accumulated, important in monitoring tumor size. In this application, the CEUS signals allow the spatial mapping of the locations where optical droplet vaporization (ODV) has occurred. To demonstrate this, the US signals were compared before   leads to a decrease in the vaporization threshold of nanoparticles51 resulting in a greater amount of bubbles formed. Since most conventional bubble contrast agents are unstable, with dissolution (disassembly) occurring within few hours,52 unfluorinated and fluorinated nanoparticles can be used for long-term NL US imaging. Since nanoemulsions from these nanoparticles convert into bubbles only upon laser excitation they can have much longer physical stability compared to other contrast agents, and hence the bubbles formed from vaporization of NEs can be utilized for imaging for longer time periods. The longer stability of PFH bubbles after vaporization of NEs might be due to differences in composition for the shell (Zonyl FSP fluorosurfactant) and core (perfluorohexane), as the PFH bubbles may not be affected to the same extent by chemical and physical properties 52,53 compared to other micro- and nanobubble contrast agents  commonly used in previous cell studies, found to be both optimal and non-cytotoxic for cell experiments. bubbles , NL US s bubbles  and had  bubbles . This su bubbles  in cancei.e., Definity microbubbles). Compared to Definity bubbles which have previously been used for comparison with extravascular US contrast agents,54 the PFH bubbles from nanoparticles are much more stable in terms of rate of NL US signal decay  after laser treatment with NPs. Viability for unfluorinated and fluorinated samples were 67 ± 2% and 58 ± 2% (21 Since the viability of MCF-7 cells after laser exposure using the same concentration of scAuNPs (1.5 μg mL−1) as that from unfluorinated samples showed viability of 93 ± 1% (after laser excitation), this suggests that cell death is primarily from the formation of the PFH bubbles. Results show that the addition of scAuNPs with PFH-NEs, can not only be used to increase CEUS signals for imaging of tumors but also can lead to more cell death (10–20% decrease in viability) compared to when using PFH-NEs alone. Compared to other therapeutic nanoparticles, PFH-NEs-scAuNPs can provide immediate treatment without waiting for NPs to internalize in cancer cells. Their ability to provide significant stress induced damage only after accumulating at the tumor site (due to the enhanced permeability and retention of tumor vasculature)41 and after laser excitation within the irradiated region make these NPs advantageous, minimizing cellular damage in healthy tissue.Since PFH NEs from unfluorinated and fluorinated samples can vaporize and have the potential to cause intracellular disruption due to the bubble expansion, the viability of MCF-7 cells was determined (directly after mixing cells with particles and treatment). Experiments from both types of particles showed a significant amount of cell death in breast cancer cells after exposing cells with nanoparticles and laser light. Significant changes in cell morphology and size were seen depending on the amount of NPs localized with cancer cells leading to different types of damage to their cell membranes compared to untreated cells . Membran 58 ± 2%  respectii.e., prostate cancer cells). After treatment there was significant number of smaller nonviable cancer cells/cell fragments seen only after laser irradiation of NPs  ultrasound signals from the presence of NEs . The signals from PFH bubbles were stable in vivo for at least four days, with future work involving experiments of biodistribution of nanoparticles for explaining the stability. The PFH-NEs-scAuNPs can simultaneously be used for imaging and therapy as theranostic agents as the PFH bubbles formed can create strong non-linear ultrasound signals and cause damage to cell membranes of cancer cells. The vaporization of NEs can be tuned  to cause significant cancer cell death without the addition of any therapeutic agents. As the mechanism for cell death is through the stress induced by the vaporization of NEs, treatment using the NPs provides an effective alternative in inhibiting tumor growth, through permanent physical damage to cells. The ability to add optically absorbing agents  to nanoemulsions has the potential to be applied to other nanoparticle systems for enhancing theranostic outcomes and due to the characteristics of these theranostic agents can serve as a better non-invasive alternative compared to other nanoparticle-based techniques.PFH-NEs-scAuNPs can be used to increase both nonlinear ultrasound signals and cell death due to the reduction of the vaporization threshold and energy required for converting PFH-NEs into bubbles . This is accomplished by the addition of scAuNPs which can efficiently transfer energy to PFH-NEs to convert their liquid PFH core into the gas phase, creating greater amount of PFH bubbles. The NL US signals from PFH bubbles generated from vaporization of PFH-NEs are greater and more stable than common lipid-based bubbles used for medical imaging (The authors declare they have no conflicts of interest.RA-011-D0RA08009H-s001


Bedaquiline is a crucial drug for control of rifampicin-resistant tuberculosis. Molecular drug resistance assays could facilitate effective use of bedaquiline and surveillance of drug resistance emergence. To facilitate molecular assay development, we aimed to identify genomic markers of bedaquiline resistance.Mycobacterium tuberculosis isolates. All studies reporting on the assessment of variants in the four genes of interest  and phenotypic bedaquiline data in both clinical and non-clinical samples were included. We collated individual isolate data from eligible studies to assess the association between genomic variants with phenotypic bedaquiline resistance, using a standardised method endorsed by WHO. Risk of bias of the extracted data was independently assessed by two authors using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies. The primary outcome was to identify mutations associated with resistance in four genes of interest ; for each genomic variant, the odds ratio (OR), 95% CI, and p value were calculated to identify resistance markers associated with bedaquiline resistance. This study is registered with PROSPERO, CRD42020221498.In this systematic review and individual isolate analysis, we searched Europe PubMed Central and Scopus for studies published from the inception of each database until Oct 19, 2020, that assessed genotypic and phenotypic bedaquiline resistance in clinical or non-clinical Rv0678, 14 in atpE, 28 in pepQ, and 11 in Rv1979c. Most clinical isolates with a single variant reported in Rv0678 (229 [79%] of 287 variants), atpE (14 [88%] of 16 variants), pepQ (32 [100%] of 32 variants), or Rv1979c (115 [98%] of 119 variants) were phenotypically susceptible to bedaquiline. Except for the atpE 187G→C  and Rv0678 138_139insG  variants, phenotypic–genotypic associations were not significant (p≥0·05) for any single variant in Rv0678, atpE, pepQ, and Rv1979c.Of 1367 studies identified, 41 published between 2007 and 2020 were eligible for inclusion. We extracted data on 1708 isolates: 1569 (91·9%) clinical isolates and 139 (8·1%) non-clinical isolates. We identified 237 unique variants in M tuberculosis isolates, especially in patients who have received unsuccessful bedaquiline-containing regimens. Treatment regimens should be designed to prevent emergence of bedaquiline resistance and phenotypic drug susceptibility tests should be used to guide and monitor treatment.Absence of clear genotypic–phenotypic associations for bedaquiline complicates the development of molecular drug susceptibility tests. A concerted global effort is urgently needed to assess the genotypic and phenotypic drug susceptibility of Research Foundation Flanders, South African Medical Research Council, Department of Science and Innovation - National Research Foundation, National Institute of Health Institute of Allergy and Infectious Diseases, and Doris Duke Charitable Foundation. To date, many countries still do not have phenotypic DST capacity for bedaquiline.7Half a million people were diagosed with rifampicin-resistant tuberculosis in 2019.Mycobacterium tuberculosis ATP synthase—encoded by the atpE gene—inhibiting energy production.8 Laboratory experiments have shown an association between atpE variants and bedaquiline resistance.9 Clinical studies found that resistance was also associated with variants in the Rv0678 (mmpR) gene, which encodes a repressor protein regulating efflux pump expression via the mmpS5-mmpL5 operon, thereby implicating a drug efflux mechanism in bedaquiline resistance.11 In 2016, pepQ (rv2535c) and Rv1979c were identified as genes that might be associated with bedaquiline resistance.13 Mutations in pepQ (encoding a putative Xaa-Pro amino-peptidase) and Rv1979c (encoding a putative permease) have been implicated in bedaquiline resistance, but the underlying mechanisms remain unknown.15Development of an accurate molecular assay requires a strong understanding of the genetic correlates of bedaquiline resistance. Bedaquiline targets subunit C of Rv0678, atpE, pepQ, and Rv1979c genes.To facilitate the development of a molecular bedaquiline resistance diagnostic tool, we did a systematic literature review and pooled individual isolate data analysis to assess the association between phenotypic resistance and variants in the Rv0678, atpE, pepQ, and Rv1979c) in both clinical and non-clinical M tuberculosis isolates. We excluded conference abstracts and book chapters or studies not reporting original data. We searched Europe PubMed Central and Scopus for articles published from inception of each database to Oct 19, 2020, using the terms (“bedaquiline” OR “sirturo” OR “TMC207” OR “R207910”) AND (“tuberculosis” OR “TB”) AND (“MIC” OR “MICs”) OR (“minimum” AND “inhibitory” AND concentration*) OR resist* OR susceptib*) AND (muta* OR “genetic” OR “genome”: OR sequenc*) without language or date restrictions. When information was missing authors were contacted for clarification . A full search of the grey literature was not done, but WHO publications were eligible for inclusion. Two authors independently screened articles for eligibility. After removal of duplicate entries, article titles and abstracts were reviewed to exclude studies not related to M tuberculosis or bedaquiline; basic science research articles not focusing on phenotype–genotype association and studies reporting on general clinical and epidemiological topics of tuberculosis were also excluded. The full text of selected articles was reviewed to confirm eligibility. The reference lists of meta-analyses, review articles, and included manuscripts were searched (By ER) for eligible publications missed by the search.In this systematic review and individual isolate analysis, we included studies reporting phenotypic DST or minimal inhibitory concentration (MIC) and assessment of variants in the four genes of interest ; nucleotide and amino acid changes; presence of co-occurring variants; bedaquiline MIC; phenotypic DST; and sequencing method. Data on isolate lineage was not extracted because these were available for only a minority of isolates. When either nucleotide or amino acid change was not provided, missing data were inferred using Expasy,16 or through contact with the author. Data were extracted manually by one investigator (ER) and was checked by a second (AVR). Authors were contacted when information was missing. For isolates reported by multiple studies, only the first study was chosen unless the later study reported additional phenotypic or genotypic data.Variables extracted at the individual isolate level were geographical origin of isolation; isolate type ; bedaquiline exposure status; variants reported in 18 Quality was not assessed for the in-vitro studies because no comprehensive and standardised guidelines were available.Two investigators (ER and AVR) independently assessed the risk of bias and concern of applicability using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies (QUADAS-2) and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies , resazurin microtiter assay (REMA) and Thermo Fisher Scientific microtiter plates.21 Phenotypic results were classified as indeterminate out of quality concern (silent mutations classified as resistant),22 or when the method was not specified.23 Classification agreement of isolates as phenotypically resistant or susceptible by different methods was investigated for isolates assessed by multiple methods. For isolates with information on at least the atpE and Rv0678 genes, we described the MIC distribution of (1) wild-type isolates; (2) isolates with one or more variants in the Rv0678 gene; (3) one or more variants in the atpE gene; and (4) variants in both atpE and Rv0678. MIC distributions of isolates with only pepQ or Rv1979c variants were not described due to scarce data.We classified isolates as phenotypically susceptible or resistant to bedaquiline according to the breakpoint concentrations of 1 μg/mL for mycobacteria growth indicator tube (MGIT), 0·25 μg/mL for 7H11,24 Because of the large number of variants and the paucity of data per variant, we included clinical and non-clinical isolates.25 Isolates were excluded if variants were not reported for both atpE and Rv0678 genes. Association with phenotypic resistance was investigated for variants that were observed at least once when occurring alone in phenotypically resistant isolates and for variants reported independently of the presence of co-occurring variants for phenotypically susceptible isolates. We also investigated the association of combinations of mutations observed more than once in phenotypically resistant isolates with bedaquiline resistance.We collated the genotypic and phenotypic data of individual isolates and applied the standardised method for interpreting the association between variants (exposure) and phenotypic resistance (outcome).atpE, Rv0678, pepQ, and Rv1979c. We also investigated the association with bedaquiline resistance for combinations of variants.The primary outcome was to identify mutations associated with resistance in four genes of interest: Odds ratios (OR) were used to evaluate the association of the genotypic and phenotypic data. 95% CIs and p values were calculated using the fisher test function in the stats package R (version 4.0.0). Except for non-sense mutations and frameshift mutations, p values were adjusted for false discovery rate using the Benjamini-Hochberg procedure. Associations with phenotypic resistance were considered significant when the p value was less than 0·05 and the OR CIs did not cross 1  pp 2–3. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.58 Additionally, a technical report published by WHO was identified and included  of 28 studies58 due to a possible absence of masked interpretation of mutations; risk of bias was high for 11 studies53 that used a phenotypic DST method not approved by WHO  were eligible for inclusion.included .19 QUADAd by WHO  pp 3–7.  of bias  pp 8–14.M tuberculosis isolates: 1569 (91·9%) clinical and 139 (8·1%) non-clinical isolates. Of the 1569 clinical isolates  reported on Rv0678 only, 11 (27%) on Rv0678 and atpE, nine (22%) on Rv0678, atpE and pepQ, and 11 (27%) studies reported on all four genes of interests  study on atpB.43 15 studies (933 samples) used MGIT, 13 (913 samples) used 7H11 media, eight (70 samples) used MABA, eight (158 samples) used REMA, three (423 samples) used Thermo Fisher Scientific microtiter plates, three (42 samples) used 7H10 media, and one (35 samples) used 7H9 media with tetrazolium chloride to determine the bedaquiline MIC; seven studies used two methods and two studies used three methods for all isolates  of the studies reported on variants in d Rv0678 . Of the d Rv0678 . MIC disd Rv0678  pp 17–18Rv0678 variants in 1653 isolates. 1214 (73·4%) of these isolates were Rv0678 wild type and 439 (26·6%) contained one or more Rv0678 variants  unique SNPs (including ten silent mutations) and 85 (36%) unique insertions or deletions. 142 (60%) of the 237 unique variants were reported only once. 395 (90%) of the 439 isolates with mutations had either a single (320 [73%]) or multiple (75 [17%]) Rv0678 variants with no variants reported in the other genes of interest. 23 (5%) of 439 isolates contained both Rv0678 and atpE variants, and 21 (5%) isolates had co-occurring Rv1979c variants. 79 (95%) of the 83 isolates with multiple variants in the Rv0678 gene were clinical isolates, of which 57 (72%) were retrieved from patients who had previously received bedaquiline. The MIC of isolates that had one or more Rv0678 variants but were atpE wild type ranged from less than 0·25 to more than 4 μg/mL, and 49 (34%) of these 145 isolates were phenotypically resistant on MGIT  were clinical isolates, 198 (51%) were retrieved from patients who were bedaquiline naive and 174 (45%) from patients who had previously received bedaquiline; bedaquiline exposure was unknown for the remaining 14 (4%) isolates. 229 (80%) of 287 clinical isolates with any type of Rv0678 variants (143 had SNPs and 144 had insertions or deletions) but no variants in the other genes of interest were phenotypically susceptible isolates: 120 (83%) of 144 with insertions or deletions and 109 (76%) of 143 with SNPs.37 (90%) studies reported on variants . 386  were atpE wild type and 88 (7·1%) contained one or more atpE variants  were only reported once. Possible hotspot regions were located at positions 82 and 83 and 183–198. In addition, three mutations were identified in the 200 base-pair upstream region of the atpE gene. 65 (74%) of 88 isolates with atpE variants had either a single (n=62) or multiple (n=3) variants in this gene but none in the other genes of interest; the remaining 23 (26%) isolates had variants in both atpE and Rv0678, whereas none had co-occurring variants in pepQ or Rv1979c. 12 (86%) of 14 isolates carrying only atpE variants and 14 (78%) of 18 isolates carrying atpE and Rv0678 variants were phenotypically resistant on MGIT  were retrieved from patients who were bedaquiline naive, 18 (69%) from patients who were bedaquiline exposed, and five (19%) had an unknown exposure status. Of the 16 clinical isolates with an isolated atpE variant and no other mutations, 14 (88%) were phenotypically susceptible to bedaquiline.35 (85%) studies reported on variants . Overallvariants  p 20. On on MGIT , D. MIC pepQ  of the seven murine isolates with a pepQ variant were phenotypically resistant to bedaquiline (20 (49%) studies reported on 1061 isolates with variants in pepQ , of whicepQ gene  pp 21–22aquiline .Rv1979c  studies reported on variants in Rv1979c . Overallisolates  pp 23–24isolates .mmpS5-mmpL5 genes.32 Two variants were reported in clinical isolates  studies reported on variants in the isolates . The isoatpE and Rv0678. Of the 1143 included isolates, 659 (57·7%) were wild type, 292 (25·5%) were phenotypically susceptible and contained one or more variants in the genes of interest, and 192 (16·8%) were phenotypically resistant. 1071 (93·7%) of 1143 isolates were clinical isolates, of which 724 (67·6%) were from patients who were bedaquiline naive and 277 (25·9%) from patients who were bedaquiline exposed. Only one insertion mutation in Rv0678 (138_139insG) was associated with phenotypic resistance . There was no evidence of association with resistance (p≥0·05) for the other 59 insertions or deletions and any of the 102 assessed SNPs in the Rv0678 gene , which was reported in ten in-vitro isolates and one clinical isolate  were excluded from the statistical analysis of the genotype–phenotype association due to quality concerns on the phenotypic DST and 529 (31·0%) were excluded because of an absence of data on both 678 gene . The onl isolate . There w p≥0·05; .Rv0678 variants, one dual atpE variant, three combinations of variants in atpE and Rv0678, three combinations of variants in Rv0678 and Rv1979c, and one combination of three Rv0678 variants. All four combinations containing a variant in atpE were associated with resistance  that were both reported in susceptible isolates when occurring alone. One combination contained the 187G→C atpE variant, which was associated with resistance when occurring alone, together with the 141_142insC variant in Rv0678, which was reported as a single variant in 13 phenotypically susceptible and four resistant isolates. One combination contained a deletion in Rv0678 with the 83A→C variant in atpE, which also occurred alone in a resistant isolate. One combination contained the 188C→T atpE and the 425T→G Rv0678 variants, both of which were only reported in phenotypically susceptible isolates when occurring alone. Of the eight dual Rv0678 variant combinations, two were associated with resistance . Two of the three combinations of a variant in Rv0678 and Rv1979c were associated with resistance  of 45 patients with wild-type Rv0678 and atpE at baseline acquired a variant during bedaquiline treatment. Treatment was classified as successful in 26 (46%) of 56 patients after culture conversion, but 17 (30%) patients did not have culture conversion; of these 17 patients, eight (47%) died. Three (5%) of 56 patients relapsed, of whom one (33%) died, and treatment was ongoing in eight (14%) patients. Two (4%) of 56 patients were lost to follow-up. Nine (35%) of 26 patients whose treatment was classified as successful had wild-type atpE and Rv0678 at the end of treatment. The other 47 (84%) of 56 patients for whom treatment outcome data were available had at least one variant in one of the genes of interest at the end of treatment.Treatment outcome data were available for 56 patients , of whom2 the association of genotypic variants with phenotypic resistance or clinical outcomes remains unclear due to scarce data and study heterogeneity. By summarising 13 years of data, we generated the most exhaustive catalogue to date: 14 unique variants in atpE, 237 in Rv0678, 28 in pepQ, and 11 in Rv1979c. Results of our systematic literature review confirmed that variants in the atpE gene result in high level resistance,31 but the evidence originates predominantly from in vitro and animal experiments, with few documented clinical cases. Our results show that variants in the Rv0678 gene are numerous and scattered throughout the gene, with most of the SNPs, insertions, and deletions occurring in phenotypically susceptible clinical isolates. Although pepQ and Rv1979c have been hypothesised to play a role in the development of bedaquiline resistance,13 the data we collated on these genes showed that it is unlikely that they play an important role in resistance.Our results show that 8 years after US Food and Drug Administration approval of bedaquiline for multidrug-resistant tuberculosis treatment,atpE, Rv0678, pepQ, and Rv1979c with phenotypic resistance. Using a standard methodology,24 two single variants (atpE 187G→C and Rv0678 138_139insG) were associated with resistance. However, this knowledge will not contribute substantially to clinical care because the atpE 187G→C variant was only reported once and the Rv0678 138_139insG variant three times in clinical isolates over the past 13 years. None of the other single variants assessed in atpE, Rv0678, pepQ, or Rv1979c were associated with phenotypic resistance (p≥0·05), likely due to scarce data. Eight combinations of variants were associated with resistance. One contained the atpE 187G→C variant and one the Rv0678 138_139insG variant, confirming their association with resistance. The only dual atpE combination contained two variants (82G→A and 183G→T) only reported in susceptible isolates when occurring in isolation. The Rv1979c 1226G→A variant, which occurred in 37 susceptible isolates and two resistant isolates and was associated with resistance when occurring in combination with two different Rv0678 variants that were not associated with resistance when occurring in isolation. These findings highlight the difficulty of translating the phenotype of a variant when it occurs in isolation to a co-occurring variant. An updated literature search done during the review process of this Article on March 27, 2021, yielded one additional study that would have been eligible for inclusion in our systematic review and analysis.59 This study reported one new variant (Rv0678 110A→V), with all other variants already included in our dataset. Data from this study were not extracted because they would not affect the results and interpretation of our study.Our analysis is the first to statistically evaluate the association between variants in 60 to 41, by stratifying information by bedaquiline exposure status, and by summarising the effect of genetic variants on treatment outcome. Despite these strengths, several limitations should be noted. To collect as much data as possible, we included both clinical and non-clinical studies. Although it is broadly accepted that in-vivo antibiotic resistance can be replicated in vitro, this assumption has yet to be proven for bedaquiline specifically.25 Likewise, we included data from multiple clinical studies of different designs to increase the amount of data. Regarding phenotypic DST, multiple methodologies were used, but only MGIT, 7H11, and Thermo Fisher microdilution plates have been validated.21 Although no provisional breakpoints have been endorsed for MABA and REMA, these methods were used for a minority of samples and inter-phenotypic DST agreement with other methods was high. Because not all studies investigated or reported all genes of interest, the sample size of the genotype–phenotype analysis was reduced by only including samples with data on both atpE and Rv0678. This restriction was not applied to the pepQ and Rv1979c genes due to scare data. Most variants were only reported in a small number of isolates, many co-occurred with other variants, and minor variants were not always assessed, complicating the assessment of the genotype–phenotype association. Variants in genes that might compensate for loss of function by another variant, as has been shown for mmpS5-mmpL5 variants co-occurring with Rv0678 variants,14 were rarely reported. Clofazimine exposure status was only reported in 19 (46%) of 41 studies. This impeded full description of previous selection pressure on Rv0678 because clofazimine can result in cross resistance with bedaquiline.34 Our aim to assess the effect of resistance conferred by specific variants on treatment outcome was limited by scarce data. Finally, multiple studies were at high risk of bias due to incomplete description of the phenotypic DST and genotyping methods used. Future studies should be designed and reported according to international guidelines for diagnostic accuracy studies.61Our review extends the knowledge on bedaquiline resistance by increasing the number of articles reviewed from 18–22In summary, our findings show that our current knowledge on the genomic basis of bedaquiline resistance is insufficient to develop a rapid molecular assay. To advance our knowledge on the phenotypic–genotypic association for bedaquiline, a concerted effort is needed to report comprehensive genotypic (preferably whole-genome sequencing) and phenotypic (using standardised methodologies) data together with treatment outcome information, especially in people who experience treatment failure. Alternative approaches to determine the genotypic–phenotypic association should be explored, and multidrug-resistant tuberculosis treatment regimens should be designed to protect bedaquiline; phenotypic DST should be used to guide and monitor treatment of patients suffering from multidrug-resistant tuberculosis.1234


The peroxisome proliferator-activated receptor (PPAR) family of transcription factors has been demonstrated to play critical roles in regulating fuel selection, energy expenditure and inflammation in skeletal muscle and other tissues. Activation of PPARs, through endogenous fatty acids and fatty acid metabolites or synthetic compounds, has been demonstrated to have lipid-lowering and anti-diabetic actions. This review will aim to provide a comprehensive overview of the functions of PPARs in energy homeostasis, with a focus on the impacts of PPAR agonism on muscle metabolism and function. The dysregulation of energy homeostasis in skeletal muscle is a frequent underlying characteristic of inflammation-related conditions such as sepsis. However, the potential benefits of PPAR agonism on skeletal muscle protein and fuel metabolism under these conditions remains under-investigated and is an area of research opportunity. Thus, the effects of PPARγ agonism on muscle inflammation and protein and carbohydrate metabolism will be highlighted, particularly with its potential relevance in sepsis-related metabolic dysfunction. The impact of PPARδ agonism on muscle mitochondrial function, substrate metabolism and contractile function will also be described. Peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors implicated in wide-ranging cellular functions, including lipid metabolism, inflammatory responses and cell proliferation and differentiation . Three PPeroxisome proliferator-activated receptors (PPARs) are a group of proteins that belong to the nuclear hormone receptor superfamily of ligand-activated transcription factors. PPAR transcriptional activity is mediated by heterodimers of PPARs with retinoid X receptor (RXR), which subsequently bind to DNA sequence elements (PPREs) in regulatory regions of target genes . Three PEach PPAR subtype has been attributed to different tissue-specific expression levels and functions. For example, PPARα is highly expressed in tissue types that undergo significant fatty acid catabolism, such as brown adipose tissue, heart and liver . ActivatThe role of PPARδ has remained relatively unclear until recently, where it has been associated with a wide range of metabolic functions in vivo ,18. It hThrough their importance in metabolic regulation, the role of all three PPAR subtypes in skeletal muscle metabolism has been established. For example, one link between PPARs and metabolic regulation in skeletal muscle appears to be through the upregulation of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the pyruvate dehydrogenase complex (PDC). The PDC activation status is regulated by various competing PDKs and pyruvate dehydrogenase phosphatase (PDP) proteins . These cIn addition to increased availability of endogenous fatty acids and their metabolites being associated with PPAR activation, inflammation has been proven to be a major site of PPAR regulation, which can occur through both direct and indirect mechanisms . As mentIn summary, all three PPAR subtypes have distinct yet overlapping roles in regulating metabolic function and inflammation see , and synSeveral in vivo animal studies have been performed with the aim of determining the impact of PPARδ agonism on skeletal muscle metabolism and function. We previously demonstrated in our laboratory that 6 days of administration of the PPARδ agonist, GW610742 , resulteIn line with the above findings relating to a PPARδ agonism induced switch in muscle fuel selection away from carbohydrate to increased fat oxidation, in another study, mice treated with the PPARδ agonist GW501516 exhibited increased PGC-1α levels, and improved prolonged low-intensity wheel-running performance. They also saw hypertrophy of oxidative slow-twitch myofibres, which are rich in mitochondria, perhaps suggesting increased reliance on the catabolism of FA through mitochondrial beta-oxidation . HoweverWe have also sought to determine whether PPAR transcription factors may be necessary for the high-fat feeding induced inhibition of PDC activation and carbohydrate oxidation during submaximal exercise in humans ,42. HealFollowing these findings, further work from our laboratory studied the role of PPARδ (and FOXO1) in palmitate-induced PDC inhibition and carbohydrate use using a skeletal muscle cell model . MyotubeAs discussed earlier, all three PPAR subtypes have the potential for treating inflammatory states, with their anti-inflammatory effects well characterised ,32. BaseIt is unclear what causes the decline in muscle protein synthesis early in critical illness and could feasibly be related to impaired mitochondrial function or tissue content . DecreasIn terms of muscle-specific effects of PPARδ agonism on metabolism and inflammation, there have been few studies to date. In a model using cultured muscle cells, one group tested the hypothesis that PPARδ upregulates FOXO1 activity in muscle, thereby upregulating MAFbx and MuRF1 expression during sepsis and glucocorticoid treatment . ActivatCardiac failure and decreased uptake and oxidation of fatty acids in the heart are common features of severe sepsis . In a moTo summarise, the effects of PPARδ agonism on skeletal muscle appear to be predominantly related to the switching of fuel utilisation towards increased oxidation of fatty acids, as well as declined carbohydrate oxidation. This can result in impaired function during prolonged muscle contraction where carbohydrate is an obligate fuel. Activation of PPARδ may also induce atrophy-related programmes in skeletal muscle. During sepsis, however, declines in PPAR activity may underlie some of the declines in FFA oxidation in various tissues, indicating that there may be some benefit to PPARδ agonism during these conditions. As described earlier, PPARγ plays a central role in adipogenesis, and TZDs have been shown to increase insulin-stimulated glucose disposal in T2D patients effectively. The effects of PPARγ agonism on skeletal muscle metabolic regulation remains poorly understood. One study assessed the impact of the PPARγ agonist, Rosiglitazone, on fatty acid transport and oxidation in rat muscle . Seven dThe impact of TZDs on skeletal muscle lipid and carbohydrate metabolism has been predominantly studied in relation to animal models of T2D ,59,60, aIn a study with T2D patients, the effects of three months of rosiglitazone treatment on insulin sensitivity and lipid metabolism were examined during a hyperinsulinaemic-euglycaemic clamp . InsulinAs described in a previous section, more work is required to determine whether different types of PPAR drug targets may have any potential benefit in alleviating inflammation in sepsis, thereby potentially preventing and/or improving certain deleterious consequences associated with the condition. In relation to specifically PPARγ agonists impacting on targeting inflammation, we previously assessed the impact of Rosiglitazone on muscle carbohydrate and protein metabolism in a rat model of LPS-induced endotoxaemia . Initial−1·day−1) for 2 weeks before and during 24 h continuous intravenous infusion of LPS (15 μg·kg−1·h−1) or saline [To assess the effects of PPARγ agonism on muscle protein and carbohydrate metabolism during endotoxaemia, rats were fed standard chow containing Rosiglitazone  investigated whether reduced fatty acid oxidation is the underlying cause for cardiac dysfunction in sepsis . LPS admIn relation to myocardial dysfunction, a separate study in rats assessed the mechanism of PPARγ-mediated cardiac protective effects in sepsis . Using rTo summarise, agonism of PPARγ in vivo appears to improve insulin sensitivity and may also increase fatty acid oxidation in skeletal muscle. However, improved insulin sensitivity may not be related to reductions in fatty acid accumulation in muscle tissue under certain conditions. In relation to inflammatory conditions such as sepsis, PPARγ agonists effectively suppress pro-inflammatory cytokine production and appear to be beneficial in alleviating organ injury and dysfunction, which has promising potential for therapeutic development. To conclude, the PPAR family of transcription factors have wide-ranging critical regulatory roles in skeletal muscle and other tissues, from inflammation to fuel selection and contractile function. In terms of PPARδ, evidence suggests that the agonism of PPARδ appears to be primarily related to the switching of substrate utilisation towards increasing the use of fatty acids. In contrast, PPARδ agonism can impair muscle contractile function by inhibiting carbohydrate oxidation during muscle contraction, where carbohydrate is an obligate fuel. Conversely, there may be benefits to PPARδ agonism during certain inflammatory conditions, such as sepsis, since declines in PPAR activity may underlie some of the reductions in FFA oxidation. Similarly, agonism of PPARγ in vivo appears to be an effective anti-inflammatory strategy during sepsis and has proven beneficial in improving organ/tissue function in pre-clinical models. Blunting muscle cytokine accumulation during endotoxaemia in rodents has also been demonstrated to result in metabolic benefits via reduced muscle wasting and lactate accumulation. Moving forwards, more studies will be required to better define the mechanistic roles of all the PPARs in different physiological and pathophysiological conditions. The potential benefits of PPAR agonism on skeletal muscle protein and carbohydrate/lipid metabolism during sepsis and other inflammatory conditions remains under-investigated and is, therefore, a promising area of research opportunity. Studies using dual or pan-PPAR agonists could also widen the therapeutic potential of these compounds, while cross-tissue studies will be important in evaluating potential off-target effects. Nevertheless, with ongoing drug developments and a greater understanding of the wide-ranging functions of PPARs, these transcription factors will undoubtedly remain critical therapeutic targets for a multitude of metabolic and inflammatory conditions.


Thyroid cancer is one of the most common endocrine malignancies, with increasing incidence all over the world. Papillary thyroid cancer (PTC) accounts for 85% of all thyroid cancers and is generally an indolent tumor, treated effectively with surgery, radioactive iodine (RAI) and thyroid-stimulating hormone suppressive therapy.This retrospective Swedish study comprised of 165 patients with PTC operated on at Karolinska University Hospital in 2009–2011 with total thyroidectomy, elective central lymph nodes dissection (CLND) and selective lateral neck dissection (LND) [Since the ATA's guidelines for the management of these disorders were revised in 2015, many aspects of thyroid cancer management remain not uniformly accepted . CurrentAccording to the recent ATA 2015 guidelines, prophylactic CLND clearance of clinically negative central LNs is recommended in patients with advanced primary tumors T3 or T4) and/or positive lateral nodes (N1b). Prophylactic CLND can be abandoned in patients with T1 and T2 tumors and clinically negative central LNs. In this study, authors have shown that LN metastases were associated with T stage and occurred mainly in T3 and T4 tumors. This observation supports the notion that prophylactic CLND can be refrained in patients with T1 and T2 tumors. Hence, the message presented in this Swedish study indicating that LNR might be useful in assessment of patients with T3 and T4 tumors is consistent with recent ATA 2015 recommendations. Some other earlier studies in the field provided matching evidence that central LN macro- but not micro-metastases together with positive to negative LNR ≥ 0.3  represent the strongest independent prognostic factors for the PTC recurrence in the lateral neck [ and/or pUnfortunately, biologically aggressive variants of PTC like hobnail, tall cell, columnar and solid were not taken into consideration in the analysis of this study cohort . As show


Corona virus disease of the year 2019 (COVID-19) is still devastating the world for more than 19 months since its declaration as a pandemic by world health organization. Its manifestations does not stand at respiratory system but involves other body systems including central nervous system and its vasculature. In the following case report, cerebral venous and arterial thrombosis is detected in a case just in convalescence from COVID-19 with still detected positive IgM.A 68-year-old female presenting with disturbed conscious level, bilateral convergent squint, single attack of generalized seizures, left sided dense weakness within a short time from catching COVID-19 and while still in quarantine hospital in recovery phase from infection. Magnetic resonance studies revealed bilateral cortical border zone infarcts as well as left lateral dural sinus and deep venous thrombosis.Along the forth wave, COVID-19 is still hitting hardly the central nervous system vasculature. Novel severe acute respiratory syndrome Corona virus 2 (SARS-CoV-2) has been associated with liability for coagulopathy and immunothrombosis through different means . Plenty To our knowledge dual pathology being venous and arterial thrombosis with a single common etiology that is COVID-19 in a single case is not commonly reported.A 68-year-old female, compliant and controlled on her medications for hypertension, diabetes, and with paroxysmal atrial fibrillation (AF) on new oral anticoagulants with a controlled rate diagnosed positive for COVID-19 on clinical basis as well as through polymerase chain reaction (PCR) in September 2021. Patient was admitted to quarantine hospital with an oxygen saturation of 90% and discharged home after 20 days. Few days later the patient began to complain of severe throbbing headache radiating to the back of head and increasing on lying flat that was followed by a single unprovoked generalized tonic clonic seizure followed by disturbed conscious level with a Glasgow coma scale (GCS) of 10/15. Patient was readmitted to an intensive care unit and on retesting PCR it was still positive.Ten days later the patient was discharged yet with a bilateral convergent squint more on her right eye and left sided weakness with national institute of health stroke scale (NIHSS) of 12. Patient sought medical advice at emergency room (ER) and was admitted to stroke center, where magnetic resonance imaging (MRI)  stroke protocol was done as well as magnetic resonance venography (MRV) that revealed bilateral cortical border zone infarcts with restricted diffusion along DWI Fig. .Fig. 1BiBasic laboratory samples were ordered and results were going with mild elevation of Prothrombin time / activated Partial thromboplastin time (PT / aPPT) 12.3/23.2 , D-dimer 0.95, ferritin 726.2, CRP 9.03, low normal platelet count 160.000, relative lymphopenia 14.9% .Laboratory blood samples for collagen disorders and thrombophilia were obtained including anti-thrombin III, Protein C and S, factor V Leiden, ANA, anti DNA, anti-cardiolipin antibody and lupus anticoagulant and all were within normal limits, beside absence of clinical history suggestive of collagen disorders or previous history of thrombosis.Neurosonology with color coded duplex study of both extracranial carotid tree and vertebral system and Doppler spectral wave analysis as well as transcranial duplex  revealed diffuse atherosclerosis with no evidence of hemodynamic changes or stenosis.Electrocardiogram (ECG)  revealed normal sinus rhythm and long term monitoring revealed controlled rate paroxysmal AF. Transthoracic echocardiography  was within accepted limits with ejection fraction 76% and left atrium diameter of 32 with no evidence of intracardiac thrombus or wall abnormality.Patient was tested for COVID-19 by PCR that was negative on two successive tests within a 48 h interval as for IgG it was positive donating previous infection and IgM was weak positive donating convalescence phase.Patient was placed on full dose anticoagulation beside antiepileptic and dehydrating measures with initial improvement regarding sensorium yet recurrent deterioration took place with GCS 6. Follow-up MRI revealed cortical venous infarction donating unresponsive venous system thrombosis to treatment Fig. . PatientA final diagnosis was that the patient had dual arterial as well as venous thrombosis, venous thrombosis had no other possible explanation rather than COVID-19 induced hyper coagulopathy (CIC), while arterial thrombosis was either COVID-19 associated or secondary to the patient medical past history.Severe acute respiratory syndrome Corona virus 2 is no longer considered a novel virus after bombarding the globe for more than 22 months since December 2019 when it was first noticed in Wuhan province in China. Meanwhile, its novel manifestations are still going on.Along the current case report a dual pathology of venous and arterial thrombosis is diagnosed in a 68-year-old female patient that was just recovering from COVID-19.Accounting for 0.5% of all strokes; dural vein thrombosis is considered a rare etiological cause of stroke . Rough eSevere acute respiratory syndrome Corona virus 2 enters the body through receptors on endothelial cells causing damage in the endothelial cell wall with no longer properly functioning endothelial cells either as a smooth lining for blood vessels or as a source of pro or anticoagulant factors that either stabilize clot or reduce excessive coagulation .Virchow’s triad that was described in the nineteenth century which is composed of hyper coagulopathy, endothelial damage, and blood stasis is resurrected again with COVID-19 and its associated thrombosis .As for arterial border zone infarcts that were detected within the presented case, although having history of vascular risk factors being hypertensive and diabetic yet the patient’s vitals and laboratory results were within accepted ranges all through the hospitalization period and she was complaint and controlled on her medications.Also vascular investigations that can reveal stenosis that may have a role in border zone infarcts development as MRA and Neurosonology did not reveal any significant abnormality other than diffuse atherosclerosis.As for AF which is a possible explanation for bilateral cortical watershed infarcts that accounts for 5–10% of all strokes . Atrial Corona virus disease of the year 2019 is still presenting with novel manifestations along the forth wave. Two forms of strokes being dural and deep venous thrombosis as well as cortical border zone arterial infarcts are detected in a single case just in the convalescence phase of COVID-19.


Sindbis virus (Alphaviridae) is a plus-strand RNA virus that is dependent on the host cell for replication. Cannabinoid (CB) receptors are found on most human cells, including virally infected cells. Activation of cannabinoid receptors has been shown to alter normal cellular physiology. This study aimed to assess how agonist (ACEA) or antagonists/inverse agonist (AM251) of the cannabinoid receptors would alter the cellular environment and impact Sindbis virus replication.Human hepatoma (Huh7) cells were used as our model for viral replication. Cells were infected with Sindbis virus (SINV) and then treated with CB agonist (ACEA) (10 μM) or antagonist/inverse agonist (AM-251) (10 μM) and virus replication was monitored. A double subgenomic Sindbis virus containing a green fluorescent protein (GFP) reporter gene inserted into a 3′ subgenomic promoter was utilized for these assays to quickly measure viral replication. GFP fluorescent cells were analyzed using flow cytometry to measure the percentage of cells expressing the viral reporter and also quantify the levels of GFP fluorescence.Treatment of SINV-infected Huh7 cells with CB1 receptor antagonist/inverse agonist  resulted in a significant decrease in viral replication, while infected cells treated with a CB1 receptor agonist  resulted in a significant increase of viral infection. The data indicates that activation of CB1 receptor by cannabinoids significantly influences the ability of Sindbis virus to replicate in the host cell.Blocking CB1 receptor activity with 10 μM AM251 reduced viral replication, but activating the CB1 receptor with 10 μM ACEA resulted in an increase in viral infection. These results indicate cannabinoids may significantly impact a virus replicating in human liver cells. Future confirmation with other viruses and cell lines will be performed to better understand the impact of cannabinoids on viral infections. Alphaviruses, such as Sindbis virus (SINV), are plus-strand RNA viruses with an enveloped virion of about 70 nm diameter. Alphavirus is a broad genus of viruses that include Sindbis virus, chikungunya virus, Eastern, Western, and Venezuelan equine encephalitis virus, Ross River virus, and Semliki Forest virus. Alphaviruses infect millions of humans and animals each year  receptors Pertwee . There aActivation of CB1 receptors stimulates fatty acid synthesis and gluconeogenesis in human liver cells  cells and human embryonic kidney (HEK) cells were maintained in similar conditions. Expression of CB1R was verified in cultured cells by immunofluorescence assay with a primary antibody (1AB) rabbit anti-cannabinoid receptor 1 antibody (Abcam. Ab237303) and a secondary, goat polyclonal secondary antibody to rabbit IgG Alexa Fluor 488 (Abcam. Ab150077)Human hepatoma (Huh7) cells were cultured using DMEM media containing streptomycin/penicillin and FBS (10%). Huh7 cells are adherent and were cultured in T75 flasks or 24-well culture plates in an incubator at 37 °C and 5% COTogaviridae family and alphavirus genus of viruses. SINV is a typical prototype alphavirus, which was used as the model virus for all experiments. The GFP reporter is inserted into the viral genome and GFP fluorescence serves as an indicator for viral replication in infected cells. Double sub-genomic alphavirus reporters have been used for several years in alphavirus research and are great reporter viruses used in gathering data on virus replication. To infect cultured cells, the cells were removed from the plate using trypsin, suspended, and counted with trypan blue staining and a hemocytometer. The resulting cell concentration (cells/well) was used to calculate how much virus to add to each well. Cells were infected with a multiplicity of infection (MOI), which is the ratio of infectious virus to cell, of 1 or otherwise stated. Virus was taken from a stock vial and added to culture media and then placed on the cells and incubated for 1 h at 37 °C, which is sufficient time for the virus to enter the host cells. After 1 h of infection, the virus media was replaced with fresh media. This fresh media could contain the agonist/antagonist treatment. The infected cells were incubated for the specified time, usually 24 h, to allow the virus to infect the cells.A double sub-genomic promoter Sindbis virus (dsSINV) expressing a green fluorescent protein (GFP) at the 3′ end of the viral genome was used in the experiments. Sindbis virus (SINV) belongs to the Arachidonyl-2-chloroethanolamide (ACEA) and AM-251 were purchased from Tocris Bioscience. The compounds were suspended in DMSO at a 100 mM stock concentration. The 100 mM stock was diluted with solvent to generate stock concentrations at 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, and 1.56 mM. To treat the cells, the mM stocks were diluted at 1:1000, resulting in μM concentrations in cell culture media that could be added directly to the infected or mock-infected cells. A mock/control treatment was prepared by diluting only solvent (DMSO) 1:1000 in cell culture media. The dilutions of ACEA or AM-251 in cell culture media were always prepared fresh prior to setting up an experiment.Cytotoxicity and cell viability were measured with the Alamar blue Cell Viability Assay® from ThermoFisher according to manufacturer’s protocols. Briefly, cells were stained with a 100-μM concentration of Resazurin/Alamar blue and incubated for 1 h at 37 °C. After 1 h, the resazurin would be broken down to the fluorescent resorufin in living cells and the plates were analyzed on an EnSpire Multimode Plate Reader® from PerkinElmer for fluorescence at an excitation of 540-570 nm and an emission of 580-610 nm to measure the living cells. The mock-/control-treated cells were set to 100% and then the viability measurements from treated wells were calculated as a percentage of the control. All experiments were performed and analyzed multiple times in triplicate.Virus replication/infection was measured by looking at GFP fluorescence from the reporter inserted into the viral genome. Cells were analyzed using a Guava easyCyte® flow cytometer from Millipore. Briefly, cells were trypsinized, resuspended, and run over the flow cytometer to quantify the number and percent of cells infected (expressing GFP) and the relative levels of GFP fluorescence per cell. In total, 10,000 cells were analyzed for each sample and each condition was run in triplicate on the flow cytometer. The same settings  were used for all analysis to accurately quantify and measure dsSINV-GFP positive cells. Data from the flow cytometer allowed specific analysis of GFP fluorescence per cell, number of cells infected, percent infected, etc.Statistical significance was determined using a one-way ANOVA followed by a Tukey’s post hoc analysis.To investigate our hypothesis and determine the impact of cannabinoids on alphavirus replication, we utilized a human liver cell line (Huh7). Confirmation of CB1 receptor expression on Huh7 cells was verified through an immunofluorescent assay Fig. . Viral iArachidonyl-2-chloroethanolamide (ACEA) has been shown to be an effective agonist for CB1 receptors. The cannabinoid binds to the CB1 and transducing a signaling pathway through a family of G proteins and into the cytoplasm of the effected cell. AM-251 is an antagonist/inverse agonist to CB1 receptors and successfully blocks the receptor from being activated. Adding a ligand or compound to cell media can have off-target or cytotoxic effects to cultured cells. Cell viability was monitored by treating cells with increasing concentrations of either ACEA or AM251 and measuring their viability using the Alamar blue assay. For ACEA treated cells, there was no cytotoxicity observed until concentrations reached into the mM range. For AM-251, cells were healthy until concentrations reached about 100 μM, and then concentrations above 100 μM started to inhibit cell viability  reduced viral replication significantly compared to the untreated control, while ACEA treatment (10 μM) increased viral replication Fig. . This loThe AM251-induced reduction in SINV replication was further confirmed in other cell lines. Human embryonic kidney (HEK) cells and baby hamster kidney (BHK) cells are traditional model cell lines used in virology research and are susceptible and permissive to a wide variety of viruses. Both cell lines express the CB1 receptor and have similar cytotoxicity thresholds with ACEA and AM251 (data not shown). Although the cell lines infected at different efficiencies, both cell lines displayed a similar trend in viral replication when being treated with ACEA or AM-251 Fig. . ACEA siViruses are the most abundant pathogen on the earth and are responsible for significant human infection and disease. As obligate intracellular parasites, viruses are completely dependent on the host cell for survival. Drugs or stimuli that affect cells will directly affect viruses that are attempting to infect and use the cell as a host. As medicinal cannabinoids become more and more utilized, it is important to understand how activation of cannabinoid receptors affects viral infections. THC and CBD are commonly used medicinally and recreationally and it is critical to know the impact of cannabinoid receptor activation and any implications to increasing or decreasing the risk of viral infections. While this study did not use THC and CBD directly, the compound ACEA serves as an agonist and activator for cannabinoid receptors and serves as a model for what may occur with other cannabinoids.Our results confirm that cultured human liver cells (Huh7) express CB1 and are capable of being infected by Sindbis virus Fig. . Cells hCB1 activation in liver cells has been previously reported to induce fatty acid synthesis and gluconeogenesis, which are important cellular anabolic pathways involved in building glucose and synthesizing biomolecules. Alphaviruses, in a similar fashion, have been described as utilizing bioenergetic pathways, to support macromolecule synthesis. Although both CB1 stimulation and virus infection have been reported to alter and increase normal cell metabolism, this is the first-time reporting what happens when both cell stimulants are occurring at the same time with alphaviruses. Huh7 cells that were infected with SINV and then treated with AM251 displayed a significant reduction in virus replication when compared to mock-treated or ACEA-treated cells Fig. . This isThese results were validated in two other cell lines, HEK and BHK cells, where AM251 treatment reduced viral replication similar to the Huh7 data. These findings are very promising, but further analysis and confirmation are definitely needed to verify and explore cannabinoid’s anti-viral effects. Viruses are very different from each other, especially in different genus’s or families, but even at the species to species level, so careful analysis will need to be performed to investigate how cannabinoids impact virus replication for a wide variety of viruses. Nonetheless, these initial results from this pilot study are very intriguing; AM251 treatment significantly reduced viral replication for Sindbis virus in human liver cells, while ACEA treatment increased viral infection. The strengths of this preliminary study are that despite only using 2 synthetic cannabinoids in a cell model system, the results are very robust and have been validated through repeat experiments and in different types of cells. Although more work needs to be done with actual living organisms and with other viruses and cannabinoids, this pilot study presents some rigorous data that highlights a very intriguing and critical need to investigate how cannabinoids affect infectious pathogens and other cellular functions.Stimulation of the CB1 receptor by treatment with 10 μM ACEA, resulted in an increase in Sindbis virus replication. Treatment with 10 μM AM251, a reverse agonist of CB1 receptor reduced viral infection.


Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model.Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured   The authors investigate the transcriptome of their established human pulmonary cell culture model (SALI) that recapitulates alveolar cell markers. RNA-seq analysis reveals striking parallels between gene expression profiles of SALI culture and primary ATII cells. The findings underline the suitability of SALI culture as a clinically relevant proxy for alveolar epithelium. A promising approach was the establishment of organoid-like spheres from isolated primary human ATII cells or stem cells,Further understanding the mechanistic outcomes of the disorders, as well as screening potential drug and treatment approaches, requires robust models of ILDs, based on the alveolar epithelium comprising alveolar type II (ATII) and I (ATI) cells. As primary human alveolar cells lose their functional characteristics when cultured in vitro cell culture model that replicated key characteristics of human ATII cells and was suitable for study of ILDs, such as surfactant protein B (SPB) deficiency.in vitro to successfully replace the deficient SPB in the SFTPB knockout (KO) SALI model.Recently, we described a new SFTPB KO on SALI cultures and how they correlate to the phenotypic changes we previously observed.Here, we explore the transcriptomic profiles of the wild-type (WT) and KO SALI culture models to assess how this platform relates to the primary human alveolar epithelium and how the gene expression profiles of H441 cells respond to the different culturing conditions. Through RNA sequencing (RNA-seq) analyses, we show that the SALI culture condition significantly alters the transcriptional profile of H441 cells, especially of the genes relating to the surfactant metabolism. We further compare the transcriptome of the SALI cultures to that of lung alveolar epithelium determined via single-cell RNA-seq (scRNA-seq).SFTPB KO H441 cells were grown in submerged or SALI conditions, comprising four sample types. Total RNA was extracted and enriched for poly(A)-containing mRNAs, after which a cDNA library was prepared, multiplexed, and sequenced using the Illumina NextSeq platform.In our previous study,SFTPB KO culture samples clustered closely with their respective WT counterparts implying a targeted, as opposed to, systemic dysregulation of gene expression pathways. Differential gene expression analysis of SALI cultures versus submerged H441 cells revealed that a total of 2,242 genes were significantly (p < 0.01) altered  between the two culture conditions  enrichment of ATII cell genes in SALI cultures  that are responsible for the correct processing of both SPB and SPC in multivesicular bodies were detected  was also upregulated  and 48  genes were significantly altered (p < 0.01) in SFTPB KO submerged H441 cells and SALI cultures, respectively, compared with their WT counterparts , and XBP1 at the RNA level indicating the induction of ER stress via the PERK and IRE1 pathways.,We then set out to investigate mechanisms by which loss of SPB could influence cellular mechanisms. SPB expression is known to be required for the correct processing of SPC as well as the genesis of lamellar bodies.cultures B and 5C.CASP3 and CASP9) as well as BAX (TJP1 (encodes zonula occludens 1), OCLN (encodes occludin), and CLDN1, 3, 4, and 7   .SFTPB KO of SALI cultures also led to a statistically significant increase in the mRNA expression of several key ATI cell markers, namely CAV1 (caveolin-1),,AGER (advanced glycosylation end-product-specific receptor),,HOPX (homeodomain-only protein homeobox)SFTPB KO potentially drives SALI cultures to modestly transdifferentiate from an ATII-like phenotype to one which is more akin to that of ATI cells.Surprisingly, In vitro cell cultures have the potential to be tractable models to study the human lung alveolar epithelium and related monogenic disorders. Their ability to form reproducible and sustainable long-term cultures makes them prime candidates for the establishment of disease models as well as rapid high-throughput screening assessment of therapeutic interventions. However, they must be physiologically relevant, reflect cellular composition, and parallel gene expression profiles of the primary cells.in vitro cell culture platform, SALI, that, unlike similar organoid-based models,,We have recently reported a new FOXA1 expression between two culture conditions  genes, the majority of which are related to the surfactant metabolism pathway  cells were obtained from the ATCC. 5 cells/well were seeded into 12-well Transwell inserts (Corning) and allowed to attach and proliferate for 48 h. On day 3, the medium on the apical side of the Transwell chamber was removed to “air-lift” the cells and the medium on the basolateral side of the chamber was replaced with polarization medium comprising RPMI 1640 supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 1% insulin-transferrin-selenium (Gibco), 4% FCS, and 1 μM dexamethasone (Sigma). Cells were grown under SALI conditions for 14 days after air lift before experimentation.SALI cultures were established as described previously.H441 cells and SALI cultures were grown in parallel. Total RNA was extracted from harvested cell pellets using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. cDNA was prepared using the NEBNext poly(A) mRNA magnetic isolation module (New England Biolabs). A multiplexed cDNA library was prepared using the NEBNext Ultra II DNA Prep kit for Illumina (New England Biolabs) according to manufacturer's instructions. The prepared library was analyzed on the Illumina NextSeq 500 with 150 base pair paired end read lengths.http://broadinstitute.github.io/picard). Reads mapping uniquely to Ensembl-annotated genes were summarized using featureCounts.,,Reads were aligned to the human reference genome (GRCh37) using HISAT2−ΔΔCT method was utilized to determine relative expression levels of target genes. β-Actin was used as the reference gene. The TaqMan gene expression assays used for each target gene can be found in RNA was extracted from ALI cultures and submerged H441 cells using a QIAGEN RNeasy Mini Kit (QIAGEN). On-column DNase I digestion was performed to prevent genomic DNA carry-over according to the manufacturer's instructions. qRT-PCR was carried out directly on extracted RNA samples using TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems) in a QuantStudio7 Flex Real-Time PCR System (Thermo Scientific). The 2Immunoblotting was used to confirm the expression of several target proteins in SALI cultures and submerged H441 cells as described previously.Immunofluorescence microscopy was performed on SALI cultures embedded in optimal cutting temperature compound as described previously.GSE171624).All sequencing data (Fastq files) are available on Gene Expression Omnibus (