Numerous studies have been reported on single- and multicolored highly fluorescent carbon nanoparticles (FCNPs) originating from various sources and their potential applications in bioimaging. Herein, multicolored biocompatible carbon nanoparticles (CNPs) unsheathed from date palm fronds were studied. The extracted CNPs were characterized via several microscopic and spectroscopic techniques. The results revealed that the CNPs were crystalline graphitic and hydrophilic in nature with sizes ranging from 4 to 20 nm. The unsheathed CNPs showed exemplary photoluminescent (PL) properties. They also emitted bright blue colors when exposed to ultraviolet (UV) light. Furthermore, in vitro cellular uptake and cell viability in the presence of CNPs were also investigated. The cell viability of human colon cancer (HCT-116) and breast adenocarcinoma (MCF-7) cell lines with aqueous CNPs at different concentrations was assessed by a cell metabolic activity assay (MTT) for 24 and 48 h incubations. The results were combined to generate dose-response curves for the CNPs and evaluate the severity of their toxicity. The CNPs showed adequate fluorescence with high cell viability for in vitro cell imaging. Under the laser-scanning confocal microscope, the CNPs with HCT-116 and MCF-7 cell lines showed multicolor fluorescence emissions, including blue, green, and red colors when excited at 405, 458, and 561 nm, respectively. These results prove that unsheathed CNPs from date palm fronds can be used in diverse biomedical applications because of their low cytotoxicity, adequate fluorescence, eco-friendly nature, and cheap production. Fluorescent carbon nanoparticles (FCNPs) have unique optical and chemical properties compared to previously used organic fluorescence probes. Carbon nanoparticles (CNPs) are emerging green nanoparticles with superior consonance emission capabilities compared to fluorescence semiconductors . There aSynthesized CNPs based on polymers, metals, silica, and semiconductors are known to possess better multiplexity abilities, making them excellent tools for biomedical applications, such as intracellular monitoring. However, their morphology and concentration remain their major drawbacks, leading to higher cytotoxicity effects. In this regard, it is necessary to investigate the balancing of organic CNP morphology and their doped chemical concentration to reduce their cytotoxicity through selectivity and sensitivity . In addiThe synthesis of CNPs from natural sources using green biosynthesis methods could be better for biomedical applications. The synthesis of CNPs based on trees as a carbon source as an alternative to chemical carbon sources could be environmentally and economically beneficial. Thus, in this work, we synthesized CNPs from date palm fronds. Millions of date palm trees are available worldwide. Date palm fronds are organic and represent a renewable resource, but these trees are burnt as waste for various reasons; therefore, utilizing them as a propitious bio-source for various biomedical applications would be beneficial. Furthermore, the biomass of date palm fronds was found to be rich in lignin content compared with the biomass of other fruits, making them an ideal biomass for carbon extraction. The biomasses extracted from date palm fronds are biocompatible with potential applications in bioimaging and theragnostics ,13. The In this report, novel CNPs with sizes of less than 10 nm were extracted from date palm fronds. The cell viability of the cell lines HCT-116 and MCF-7 in the presence of CNPs was also studied for potential applications in bioimaging. To the best of our knowledge, no study has reported the cell viability and dose-response curve under the application of high concentrations of CNPs to assess their biocompatibility. We also reported the bioimaging potential of crystalline graphitic CNPs in HCT-116 and MCF-7 cell lines through their fluorescence properties. Further investigations should explore the potential applications of these CNPs for cancer diagnosis, such as in multiphoton endomicroscopy , and tre3 with filter caps were purchased from SPL Fife-Sciences, Pocheaon, South Korea. CytoOne 96-well plates (Microtiter plates) with covering lids for cell culture were purchased from Scientific Incorporation, Ocala, FL, USA.Date palm fronds (Phoenix dactylifera) were collected from date farms in Madinah, Saudi Arabia. Thiazolyl blue tetrazolium bromide (MTT) dye, trypsin-EDTA (0.25%), fetal bovine serum (FBS) growth media, phosphate-buffered saline (PBS) solution (pH 7.4), penicillin–streptomycin (Pen/Strep) antibiotic solution, dimethyl sulfoxide (DMSO) media solvent, and high-glucose Dulbecco’s Modified Eagle Medium (DMEM) containing phenol red and L-glutamine were obtained from Invitrogen . SPL tissue culture flasks of sizes 75 and 25 cmAs illustrated in The characterizations of CNPs were performed at the Center of Nanotechnology, Jeddah, Saudi Arabia. The topographies of CNPs were determined using a scanning electron microscope (SEM) model JSM-7600F  and a high-resolution transmission electron microscope (HR-TEM) model JEM 2100F . The XRD was carried out using an Ultima IV model  at room temperature in continuous mode. It emitted graphite-monochromatized Cu-Kα radiation with a wavelength of λ = 1.54 Å. The applied current was 40 mA, and the applied voltage was 40 kV. The scanning axis for XRD patterns at 2θ had an angular range of 10 to 80°, and the first step was 0.05°. FTIR was used to trace the functional group present in the CNPs by applying the attenuated total reflection (ATR) sampling technique using a Nicolet iS10 model . To record the photoluminescence emissions of the CNPs we used a spectral fluorophotometer model RF-5301 . Absorption spectra of the CNPs were recorded with an ultraviolet–visible spectroscope model Lamda 750 . The zeta potential of the CNPs was measured to determine the charges on the CNPs and their colloidal nature with cells using a Malvern particle size analyzer model v2.3 .We estimated the carbonization by investigating the chemical elements using energy dispersive X-ray (EDX) spectroscopy, as shown in 3 with lids were used for culturing cell lines. To maintain the cell growth, 10% (v/v) FBS in DMEM, as well as 1% Pen-Strep solution, was added. The culture flask was incubated under a humidified atmosphere at 37 °C, maintaining 5% CO2. The culture media were replaced when cells attained a high confluence of 90%. Finally, cell harvesting was performed using trypsin solution.The malignant cells, namely the HCT-116 and MCF-7 cell lines, were obtained from the Regenerative Medicine Unit, King Fahad Medical Research Center, Jeddah, Saudi Arabia, where the biological experiments of this study were conducted. Originally, the Regenerative Medicine Unit purchased the cell lines from the American Type Culture Collection . Cell culture flasks of 75 cmFollowing cell harvesting, the cells were seeded using microtiter plates containing ~5000 cells/well. These plates were supplemented with cell growth media and incubated for 24 h under the incubation conditions mentioned above, along with 95% air for adherence. Furthermore, CNPs were prepared at different concentrations , with a CNP concentration of 0 as the control. An MTT assay kit was used to estimate cell viability at time intervals of 24 and 48 h. The principal reaction of the MTT assay is based on the catalyzed conversion of soluble MTT tetrazolium into insoluble crystals of formazan, which are purple in color and produced by the succinate dehydrogenase within mitochondria. The MTT assay was conducted according to the manufacturer’s guidelines.2, maintaining a humid atmosphere. Later, 25 µL of media was removed from each well to dissolve formazan in 50 µL of DMSO [The MTT solution was prepared by dissolving 5 mg/mL of PBS in MTT powder; each well was supplemented with 10 µL of the prepared MTT solution, followed by a further 4 h of incubation at 37 °C with 5% CO of DMSO , mixed w of DMSO . The cel of DMSO :(1)CV%=AData obtained from the cell viability test were applied to plot the dose-response curve for cell lines HCT116 and MCF7 using the logarithmic function; the curve was plotted using cell viability % values versus the different concentrations of aqueous CNPs applied. The estimation of the CNP concentration doses applied to the cell lines was presented in the dose-response curve. The curves for EC20, EC50, EC80, and EC90 showed that these concentrations of CNPs caused the cell viability count to be 20, 50, 80, and 90%, respectively. A non-linear four-parameter logistic (4PL) sigmoid curve (Hill equation) was used to fit the dose-response curve. The curve was fit using the equation provided below .(2)y=A12 cell flask containing media. The detaching process was performed by applying 0.25% trypsin EDTA when a high cell confluency was reached. Then, cells were suspended in DMEM-based media before collection onto the slide. Ten microliters of cells was dropped onto the slides and left for 2 h. Finally, the slides containing the cells were placed into a petri dish and incubated under the abovementioned conditions before imaging with and without aqueous CNPs. Images of cells were captured using a high-resolution scanning confocal laser microscope, model LSM 780, from Carl Zeiss, Aalen, Germany; the laser system was controlled digitally using ZEN 2010 software.The in vitro imaging of cell lines HCT-116 and MCF-7 was carried out using a laser-scanning confocal microscope. The cell lines were cultured in a 25 cmt-test, resulting in a value of p > 0.05. All statistical analyses were performed through Origin version 2022b .Standard deviations (SDs) were taken as the average value of the input data. The significance of the statistical analysis was evaluated using the Student’s −1, which could be assigned to the stretching vibrations of C-O, C-O-C, C=C, and C=O [−1 could be assigned to the O=C=O stretching vibrations; this band was reported as background carbon dioxide CO2 [2 in porous CNPs [−1 could be assigned to the stretching vibration of C-H, while the sharp band at 3400 cm−1 was attributed to vibrations of C-OH [The CNPs showed homogeneous distribution; they appeared to agglomerate as balls, as shown in the SEM image in  and C=O ,27,28,29xide CO2 ,31,32 orous CNPs . The ext of C-OH ,34. The  of C-OH , which wAs was found in the results of the physiochemical properties of the CNPs presented in The CNP dye at a 16 mg/mL concentration diluted in deionized water (DI water) to different concentrations  is presented in The cell viability of the aqueous CNPs was assessed for different concentrations  of CNPs. In this study, CNPs were applied to two famous carcinoma cell lines, namely HCT-116 and MCF-7. The CNPs were dispersed with MCF-7 and HCT-116 cell lines in a 96-well microtiter cell plate. Each cell line was cultured in three wells to obtain triplicate results, with each concentration applied. The average absorbance value of the three wells was used to determine the cell viability, which was calculated using Equation (1). Dose-response curves were generated using the experimentally obtained values of cell viability % at 24 and 48 h of incubation versus the logarithmic concentrations of CNPs for both cell lines. The curves were fitted using the fitting equation. EC50 represents 50% cell viability in response to the applied concentration, so the sigmoid curve at EC50 was denoted as the Hill slope. The dose-response curves for the HCT-116 and MCf-7 cell lines demonstrated a decreasing order of cell viability with corresponding concentrations, as observed from the EC90, EC80 EC50, and EC20 values. The values for EC90, EC80, EC50, EC20, and R2 are summarized in Unsheathed aqueous CNPs from date palm fronds were evaluated for biocompatibility via an MTT colorimetric assay. Both carcinoma cell lines (HCT-116 and MCF-7) were incubated for 24 and 48 h with high CNP doses. The cell viability of the two cell lines was very high, with a survival rate greater than 95% at a 0.5 mg/mL concentration for both incubation times, as depicted in The CNPs reported in this study contained oxygen as the only surface functional group. These CNPs were implemented for bioimaging using a laser-scanning microscope at wavelengths of 405, 458, and 561 nm and emitted mainly blue, green, and red colors, respectively, as depicted in The in vitro cellular uptake and the interaction of CNPs with individual HCT-116 and MCF-7 cells were imaged. A few drops of CNPs at a safer concentration dose of 0.5 mg/mL were applied to the cells. A laser-scanning microscope is used for cell imaging in DAPI mode. The samples were exposed at different excitation wavelengths, including 405, 458, and 561 nm. The images of both cell lines revealed that the CNPs have potential for bioimaging, since they had a zeta potential value of −24.10 (cationic) and a pH of 7.2, demonstrating their ability to penetrate into the cell membrane ,48,49,50The CNPs investigated in this study are environment friendly and originate from date palm trees, with the potential to replace expensive toxic, metallic, and chemical fluorescent dyes for bioimaging. In this study, crystalline graphitic CNPs extracted from date palm fronds demonstrated exemplary PL properties at a pH of 7.23 for bioimaging. Investigations of the CNPs’ fluorescence lifetime, quantum yield at different wavelengths, and fluorescence mechanism need to be conducted in future studies. The immense bioimaging potential and biocompatibility of the CNPs were assessed by an MTT assay with two cell lines, HCT-116 and MCF-7. The adopted concentrations were high compared with those used in most published works due to CNPs’ organic nature. A lower toxicity was recorded under the effect of the CNP concentration of 0.5 mg/mL. The cell viability recorded at 24 h of incubation was 115.86 and 96.47% for HCT-116 and MCF-7 cell lines, respectively, while that at 48 h of incubation was 101.38 and 95.97%, respectively. Both cell lines showed higher cell viability at 48 h of incubation. Both the cell lines were stained with CNPs and then imaged via a fluorescence laser-scanning confocal microscope, which proved that the cell organelles were labeled. However, further studies on efficient strategies for the surface functionalization of CNPs are required for biolabeling, in vivo bioimaging, and various other biomedical applications.


Triatoma and Rhodnius transmit Trypanosoma cruzi, the causative agent of Chagas disease in humans in tropical Latin America. We have developed new cell lines from two of the major vectors of T. cruzi, Triatoma infestans and Rhodnius prolixus, and have shown that they do not harbour any contaminating bacteria or known insect-borne viruses. We propose that the new cell lines may be suitable host cells for Triatoma virus, a natural pathogen of T. infestans that has been proposed as a biocontrol agent but cannot be grown in existing cell lines. The new cell lines may also be used to study interactions between trypanosomes and cells of their vectors. Arthropod vector cell lines play an important role in research on the biology and control of blood-feeding insects and the diseases they transmit. Kissing bugs of the genera Triatoma and Rhodnius are vectors of Chagas disease, a neglected tropical disease of humans in South America caused by Trypanosoma cruzi. Triatoma virus (TrV), a natural pathogen of Triatoma infestans, has been proposed as a possible tool for the bio-control of triatomine bugs, but research into this virus has been hampered by a lack of suitable host cells for in vitro propagation. Here we report establishment and partial characterisation of continuous cell lines from embryos of T. infestans (TIE/LULS54) and Rhodnius prolixus (RPE/LULS53 and RPE/LULS57). RNAseq screening by a sequence-independent, single primer amplification approach confirmed the absence of TrV and other RNA viruses known to infect R. prolixus, indicating that these new cell lines could be used for propagation of TrV. Triatomine bugs of the genera  Trypanosoma cruzi. T. cruzi is the causative agent of Chagas disease, a zoonosis widespread in Latin America [Triatomine bugs of the order Hemiptera, family Reduviidae, also known as kissing bugs, are vectors of the kinetoplastid protozoan parasite  America . Followi America , indicatTriatoma infestans [T. infestans increases their T. cruzi infection rate [T. infestans in Argentina [Rhodnius prolixus [in vitro system for laboratory propagation of the virus outside the insect host [Triatoma or Rhodnius spp. were available to test.Triatoma virus (TrV) , a membenfestans  and therion rate . TrV infrgentina  and in srgentina ,8. Interect host . To dateect host , possiblect host , and no Triatoma maculatum embryo-derived cell cultures surviving for up to 49 days were reported over 50 years ago [T. infestans [T. cruzi [Nosema disstria [Rhodnius.Primary ears ago , and a cnfestans . This ceT. cruzi  and contdisstria . HoweverT. infestans and R. prolixus obtained from laboratory colonies. Screening by a sequence-independent, single primer amplification (SISPA) approach [R. prolixus [Here we report the establishment and partial characterisation of three continuous cell lines derived from embryos of approach  revealedprolixus .T. infestans eggs were harvested from a laboratory colony maintained at LSHTM since 2009. The colony originated from T. infestans insects collected in Paraguay by Dr Nidia Acosta, Universidad Nacional de Asunción—UNA, Paraguay. The T. infestans insects were maintained in glass jars measuring 9.5 cm × 14 cm at 26 °C ± 2 °C, at 70% relative humidity with a 12 h light:12 h dark cycle, and fed every 6–8 weeks on defibrinated horse blood  using a Hemotek® membrane feeding system with 5 mL reservoirs . R. prolixus eggs were harvested from a laboratory colony maintained at Swansea University [R. prolixus insects were fed via a membrane feeding system on human blood. Whole blood was collected by phlebotomists at the School of Medicine, Swansea University, from anonymous healthy adult volunteers by venipuncture into vacuette® tubes containing CPDA . All human blood sampling procedures were reviewed and approved by the South West Wales Research Ethics Committee, REC ref 13/WA/0910. For both insect species, the eggs were used to generate primary cell cultures when the eyes of the majority of developing embryos were visible as reddish spots inside the eggshells.iversity . This coiversity . The R. Three complete culture media were used: L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth, 20% foetal bovine serum (FBS), 2 mM L-glutamine (L-glut), and 100 units/mL of penicillin and 100 µg/mL of streptomycin (antibiotics) (L-15); Hanks balanced salt solution (HBSS) supplemented with 0.5% lactalbumin hydrolysate, 20% FBS, L-glut and antibiotics (H-Lac); and a 1:1 mixture of L-15 and H-Lac (L-15/H-Lac). Complete media were prepared freshly each week. All ingredients were sourced from Invitrogen  or Sigma-Aldrich . T. infestans and R. prolixus eggs were surface-sterilised by immersion in 0.1% benzalkonium chloride for 1–3 min, 70% ethanol for 1 min, sterile ultrapure water for 10 s and HBSS for 10 s in a 30 mm plastic petri dish. The eggs were immersed in fresh HBSS and dissected aseptically by carefully removing the operculum of each egg and gently squeezing out the embryo using sterile watchmakers’ forceps under a binocular dissecting microscope in a horizontal laminar flow cabinet. Embryos with pale or red eye spots were selected for further processing; older embryos with black eye spots (both species) and/or developing reddish exoskeleton (R. prolixus) were rejected. The embryos were collected in a drop of HBSS in a second petri dish and macerated using the watchmakers’ forceps. The macerated tissues were diluted with 1 mL of HBSS and centrifuged at 200× g for 5 min. The tissue pellet was resuspended in 0.5 mL of a solution of 500 µg/mL trypsin  in PBS and incubated at 37 °C for 5 min (R. prolixus) or 10 min (T. infestans). Then 1 mL of complete culture medium was added to inactivate the trypsin and the tissue suspension was centrifuged as before. The tissue pellet was resuspended in 2.2 mL of complete culture medium and placed in a flat-sided culture tube . The sealed tubes were incubated in ambient air at 28 °C. Medium was changed weekly by removal and replacement of ¾ of the total volume. Cultures were monitored for growth by weekly inverted microscope examination. When significant cell multiplication became evident, subcultures were initiated. An equal volume of fresh complete medium (2.2–2.4 mL) was added to the culture, adherent cells and tissue clumps were resuspended by pipetting and half of the resultant suspension was transferred to a new daughter tube, leaving the remainder in the parent tube. When sufficient numbers of tubes were available, half of the contents of two tubes were pooled and transferred to a T25 flask with a total of 5.5 mL of medium. The subculture series were maintained thereafter in parallel in tubes and T25 flasks; cells in flask cultures were passaged 1:1 by resuspending the monolayer using a cell scraper instead of pipetting. Occasionally, cells were resuspended and used to prepare Giemsa-stained cytocentrifuge smears as described previously .g for 5 min. The cell pellet was resuspended in ice-cold complete medium, held on ice and an equal volume of ice-cold complete medium with 20% DMSO was added dropwise with swirling. The resultant suspension was immediately dispensed as 1 mL aliquots into ice-cold cryovials and frozen rapidly on dry ice, before being transferred to vapour-phase liquid nitrogen storage within 30 min. The contents of three culture tubes or one T25 flask yielded three cryovials. Cells were resuscitated from liquid nitrogen storage by rapid thawing in a 37 °C water bath, added immediately to 9 mL of complete medium at room temperature, centrifuged at 300× g for 5 min, resuspended in 2.2 mL of complete medium, placed in a flat-sided tube and incubated at 28 °C. The first medium change was carried out after 5–7 days.Cultured cells were cryopreserved using a modification of a previously-described technique , as follT. infestans and R. prolixus cell DNA using forward primer EukA: 5′-AACCTGGTTGATCCTGCCAGT-3′ and reverse primer EukB: 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ [cytB [Ehrlichia ruminantium-infected bovine endothelial cell culture [T. infestans and R. prolixus cultures were screened for contamination with Mycoplasma spp. using two commercial tests, the Mycoalert Mycoplasma Detection Kit  and the PCR Mycoplasma Test Kit , following the manufacturers’ instructions.Genomic DNA was extracted from harvested cells using a DNeasy Blood and Tissue Kit  following the manufacturer’s instructions for tissue cultures and stored at −20 °C. To confirm the species origin, a fragment of the eukaryotic 18S rRNA gene was amplified by a conventional PCR from the CCTAC-3′ . PCR reaCCTAC-3′  and cytB3′ [cytB  genes we3′ [cytB . All PCR culture . PCR pro culture . RandomlTotal RNA was extracted from cells harvested from two flat-sided tubes or one T25 flask using an RNeasy blood and tissue kit  following the manufacturer’s instructions. Following a TurboDNase treatment, the RNA was reverse transcribed using SuperScript IV Reverse Transcriptase  using primer Sol-A, according to the manufacturer’s instructions. Klenow Fragment  was used for second strand synthesis . The cDNhttps://github.com/spond/gb_taxonomy_tools (accessed on 29 January 2022) based on taxids searched by BLAST and visualised with Krona [https://bugseq.com/free (accessed on 29 January 2022) using the NCBI nt database and visualised with Recentrifuge [NanoFilt was used to filter out sequencing readings less than 100 nucleotides and to then trim 30 nucleotides off the end of the read using the --headcrop and --tailcrop parameters . The firSequences obtained from PCR products sequenced successfully in both directions were deposited in GenBank with accession numbers OP123038 (TIE/LULS54) and OP123039 (RPE/LULS53) for the eukaryotic 18S rRNA gene. The SISPA RNAseq datasets obtained for the three cell lines were deposited in the NCBI Sequence Read Archive (SRA) database under BioProject number PRJNA843092.T. infestans primary cultures, one in H-Lac and two in L-15 derived from a single batch of ~90 eggs, were set up in flat-sided tubes and survive at the time of writing (30 months post-initiation). The primary cultures comprised adherent tissue clumps, some clearly identifiable as sections of limbs, and individual cells with a range of phenotypes including round, epithelial-like, fibroblast-like and contracting muscle cells. As time passed, epithelial-like and muscle cells came to predominate in all cultures. Subculture series were initiated from two of the primary cultures, the one in H-Lac and one of the ones in L-15, after 12 and 9 weeks respectively; growth of the H-Lac culture was slow and this series only reached passage 3 after a total of two years in culture. Three The L-15 culture grew more rapidly, reaching passage 2 after a further 6 weeks and giving rise to a continuous cell line, designated TIE/LULS54. At passage 3, cells of this line were transferred to a T25 flask, where they grew almost as well as in the flat-sided tubes and were easier to dislodge for subculture using a cell scraper. At 10 months after initiation, cells at passage 3 were cryopreserved and successfully resuscitated, and DNA was extracted from a passage 1 culture at that time and from a passage 8 culture at 22 months, for species confirmation and microorganism screening. At 20 months after initiation, RNA was extracted from two passage 6 cultures for RNAseq analysis. At the time of writing, the TIE/LULS54 cell line has reached passage 14; subcultures are carried out at 6-week intervals.TIE/LULS54 cultures comprise a mixture of phenotypes a, includR. prolixus primary cultures were initiated from batches of 12–80 eggs; of these, two in L-15/H-Lac and two in L-15 survive at the time of writing (38–40 months after initiation). In the failed primary cultures, the cells and tissue clumps did not attach well to the surface of the culture tube and did not metabolise beyond the first few weeks, as indicated by rising pH of the culture medium. In the successful primary cultures, tissue clumps attached within the first few days and cells began to migrate out from them, forming sheets of epithelial-like cells and small patches of contracting muscle cells. Areas of large epithelial-like cells containing red pigment, visible to the naked eye, were present in some of the cultures; these persisted for more than 20 months in one of the surviving L-15 cultures. In contrast, the areas of muscle cells ceased to contract after 12–18 months. Attached, and occasionally floating, multicellular vesicles appeared in most of the primary cultures after several months, but did not persist to the time of writing. The subculture series were initiated from three of the surviving primary cultures after 10, 13 and 26 months; of these, the first two gave rise to the continuous cell lines described below while the third, in L-15, has reached passage 4.Eleven R. prolixus cell lines to become established, RPE/LULS53, was generated from 30 eggs in L-15. The first subculture was carried out at 10 months after initiation, with the second and third passages following at 5- and 3-month intervals respectively. Cells at passage 4 were transferred to a T25 flask to facilitate subculturing, 19 months after initiation. The first cryopreservation attempt, carried out with passage 4 cells 20 months after initiation, was successful. DNA was extracted from a passage 1 culture at that time, and from a passage 13 culture at 33 months, for species confirmation and microorganism screening; at 32 months after initiation, RNA was extracted from a passage 12 culture for RNAseq analysis. The RPE/LULS53 cells have reached passage 20 at the time of writing.The first of the two R. prolixus cell line, designated RPE/LULS57, was generated from 44 eggs in L-15/H-Lac. The first subculture was carried out after 19 months, passage 3 cells were successfully cryopreserved at 23 months and the cells have reached passage 14 at the time of writing. DNA was extracted from a passage 6 culture at 30 months for species confirmation and microorganism screening, and RNA for RNAseq was extracted at 29 months from pooled cultures at passages 5 and 7.The second R. prolixus cell lines comprise cells that are considerably smaller than those of the T. infestans line TIE/LULS54 and do not exhibit muscle contractions. Several cell types are present, forming a monolayer of spindle-shaped and fibroblast-like cells , the R. prolixus cell lines now grow faster than the T. infestans cell line. Further study would be required to investigate these contrasting characteristics. All three cell lines are grown in relatively simple culture media, for which all ingredients are commercially available, and are incubated in sealed containers in ambient air, making them suitable for use in laboratories with only basic cell culture facilities and therefore making them applicable in a range of settings in countries where T. cruzi and its vectors are endemic.In the present study, we generated continuous cell lines from two medically-important triatomine bug species. The approach used to generate the TIE/LULS43, RPE/LULS53 and RPE/LULS57 cell lines was relatively simple, and based on techniques used previously to generate a range of insect and tick cell lines ,21,37,38T. infestans and R. prolixus are among the four main vectors of T. cruzi in Latin America [T. cruzi with triatomine cells, trypanosomes cultured with T. infestans TI-32 cells underwent a pattern of differentiation similar to that seen in the vector but not seen in axenically-cultured trypanosomes, indicating that the TI-32 cells produced factor(s) influencing parasite development [Trypanosoma grayi, isolated from a Glossina pallipides tsetse fly, that could not be maintained in axenic culture [T. cruzi for mammalian and insect cells was compared using the non-vector Aedes albopictus mosquito cell line C6/36; in all cases the percentage of cells infected was lower in C6/36 cells than in mammalian cells, but the numbers of parasites associated with the infected cells was similar overall [T. cruzi and the new triatomine cell lines TIE/LULS54, RPE/LULS53 and RPE/LULS56. America  and, as elopment . TI-32 c culture ,42. The  overall . It willT. cruzi [Blastocrithidia triatomae, originally isolated from T. infestans in Argentina; interaction with the trypanosomes appeared to kill the TI-32 cells [Trypanosoma rangeli and R. prolixus haemocytes in vivo and ex vivo [R. prolixus cell lines could be exploited to further examine the role of reactive oxygen species in the pathogenicity of T. rangeli for its host. In addition to trypanosomes, triatomine bugs could play a role in the transmission of bacterial pathogens such as Bartonella spp. and Mycobacterium leprae, the causative agent of leprosy [T. infestans and R. prolixus cell lines might prove to be suitable host cells for the in vitro propagation of M. leprae, as reported previously for a tick cell line [T. infestans may also harbour symbiotic intracellular bacteria of the genus Arsenophonus; Candidatus Arsenophonus triatominarum was isolated from adult female T. infestans haemolymph and propagated in a mosquito cell line [Ca. A. triatominarum, these cells could be used as a more biologically-relevant substrate than mosquito cells for in vitro propagation and the study of this symbiont.Triatomine bugs also harbour trypanosome species that are not known to infect humans but may be pathogenic for their insect hosts and potentially modulate infection with T. cruzi ,44. TI-3 ex vivo  suggest  leprosy ,46. Indeell line . T. infeell line . As the Metarhizium anisopliae, which took longer to kill bugs infected with T. cruzi than uninfected bugs [T. cruzi infection in TrV-infected T. infestans [The environmental costs of insecticides and the increase in reports of insecticide resistance among triatomine bug populations across South America ,49,50 unted bugs  indicatenfestans  deservesT. infestans and R. prolixus cell lines will support productive infection with TrV, we have carried out the first step in this journey by screening them for an existing TrV infection.The reported failure to propagate TrV in cell lines derived from non-triatomine insects , and theT. infestans and R. prolixus for TrV [R. prolixus [R. prolixus insects (0.1–0.6%). These authors used a different approach, generation of paired-end Illumina RNAseq libraries and Illumina HiSeq sequencing, which could have influenced the proportions of viral reads detected in their insect samples. Of likely greater significance, however, are the different origins of the insects used in our study and that of Brito and co-workers [R. prolixus came from a laboratory colony which originated in Brazil, presumably from insects collected from the field in Brazil, sometime prior to 1980. Our R. prolixus came from a laboratory colony originated with insects from Venezuela nearly 100 years ago [The Nanopore sequencing platform was applied in preference to the RT-PCR assays used by others to screen laboratory and field populations of  for TrV ,8, as weprolixus . Interesprolixus  in RNA e-workers . Their Rears ago ; it is tT. infestans cell line TIE/LULS54 harboured the least diverse virome of the three cell lines, as determined by RNAseq, with between 0.0001 and 0.0005% of reads assigned to putative viral sequences, indicating the absence of any complete, replication-competent RNA viruses. This finding suggests that TIE/LULS54 cells would be particularly useful not only for the propagation of TrV, but also for more general investigation of insect host cell responses to RNA virus infection, as any response detected following infection of the cells could be confidently attributed to the presence of the test virus.The T. cruzi and other trypanosomes with their vectors at the cellular level. All three cell lines are deposited in, and available from, the Tick Cell Biobank at the University of Liverpool.In conclusion, the three new triatomine cell lines reported here represent research tools with a high potential for application in studies both on bio-control using TrV and on interaction of


EGFR, PIK3CA, and KRAS, did not predict sensitivity . The machine-learning predictions of combinations that were compared with experimentally generated data showed a bias to the highest quartile of Bliss synergy scores (P = 0.0243). We confirmed feasibility of running such assays on 16 patient samples of freshly isolated NSCLC cells from pleural effusions. We have provided proof of concept for novel methods of using acute ex vivo exposure of cancer cells to targeted anticancer drugs to predict response as single agents or combinations. These approaches could complement current approaches using gene mutations/amplifications/rearrangements as biomarkers and demonstrate the utility of proteomics data to inform treatment selection in the clinic.We hypothesize that the study of acute protein perturbation in signal transduction by targeted anticancer drugs can predict drug sensitivity of these agents used as single agents and rational combination therapy. We assayed dynamic changes in 52 phosphoproteins caused by an acute exposure (1 hour) to clinically relevant concentrations of seven targeted anticancer drugs in 35 non–small cell lung cancer (NSCLC) cell lines and 16 samples of NSCLC cells isolated from pleural effusions. We studied drug sensitivities across 35 cell lines and synergy of combinations of all drugs in six cell lines (252 combinations). We developed orthogonal machine-learning approaches to predict drug response and rational combination therapy. Our methods predicted the most and least sensitive quartiles of drug sensitivity with an AUC of 0.79 and 0.78, respectively, whereas predictions based on mutations in three genes commonly known to predict response to the drug studied, for example,  EGFR  is the leading cause of cancer-related mortality  and is aEGFR , ALK 4)EGFR EGFR , cell transfections (siRNA/CRISPR) or large quantities of protein and extended analysis (mass spectroscopy). These limitations preclude use rapid testing of tumor samples from an individual patient against multiple drugs to enable decision making at any point in their treatment.ex vivo to patient cells. The clinically relevant concentrations and the short exposure of drugs used in these experiments are key to clinical translation of these assays. We establish proof of concept that such an approach is feasible and, in the future, may result in the establishment of platforms that will inform clinical decision making and personalized treatment within 24–48 hours of a biopsy of individual tumors.Here, we quantify dynamic signaling responses within cancer cells to predict drug sensitivity and rational combinations in NSCLC. The approach is applicable both to cancer cell lines and Thirty-five NSCLC cell lines were obtained from the ATCC or from collaborators and STR typed (details in Supplementary Table S1).l-glutamine  and 1x MEM non-essential amino acid solution . Cells were incubated at 37oC with 5% CO2. All cell lines used in experiments were between 4 and 28 passages. Cell lines were tested for Mycoplasma using MycoAlert  within 2 weeks before use.All cell lines were grown in RPMI-1640  except for SK-LU-1 that was grown in DMEM . In addition, all media were supplemented with 10% FBS , 1 mmol/L Cmax) normalized to the protein binding effect in 20% FBS media: Details are provided in the Supplementary Methods.Were obtained from Selleck chemicals. Drug concentrations used for our Luminex assays were based off the clinical maximum plasma concentration (2 tissue culture flasks (Corning Inc.) at 20% FBS until approximately 80% confluent then dosed with one of seven drugs (plus 3 DMSO controls) for 1 hour. Lysate was stored at −80°C until required.Cells were grown in 25 cmMILLIPLEX MAP Akt/mTOR phosphoprotein kit, MILLIPLEX MAPK/SAPK signaling kit, MILLIPLEX MAP RTK phosphoprotein kit  were combined with the following single-plex magnetic bead sets to produce three multiplex Luminex assays: phospho-NFkB, phospho-SRC, phospho-STAT3, phospho-STAT5 A/B, total HSP27 and GAPDH . Bio-Plex Pro phospho-PDGFRa, phospho-PDGFRb and Akt  were combined into a triplex assay. Manufacturer's protocols were followed throughout.Growth inhibition was assessed using 72-hour Sulforhodamine B (SRB) assay (details in Supplementary Methods).Up to 1,000 mL of ascites or pleural fluid was collected by the patient and immunomagentically separated using previously published methods .All patients who had pleural effusions drained for palliative purposes. Pleural fluid was used in the study after investigators has obtained written informed consent. The tissue collection protocols were approved by the Institutional Review Boards and conducted in accordance with the Decleration of Helsinki.To standardize the phosphoproteomic measurements, the control GAPDH measurements were normalized and median-centered, all other data normalized accordingly (see Supplementary Methods).For predictions and feature selection, we created and assessed the performance of a suite of AI-based predictors. First, we used Random Forest recursive feature selection to define the phosphoprotein changes that most contributed to prediction, then trained and validated Random Forest classifiers and regressor functions (details of implementation in Supplementary Methods). Moreover, we in addition used elastic net predictors to predict responses to drugs. Similar models were constructed using notable clinical genomic features of NSCLC to allow comparisons of model performance using the different feature types in predicting drug response.The environmental perturbation score (EPS) is an integrative function across the protein–protein interaction network neighbors. The protein networks were constructed using the highly curated interactome from canSAR . The absU test. Details in Supplementary Methods.Combinations of drugs were assessed using Bliss independence analysis to study synergy. Details in Supplementary Data. The different distribution of the EPS rankings in the highest and lowest quartiles of the combinations ranked by the Bliss independent analysis was tested by a Mann–Whitney Data generated in this study are available upon request from the corresponding author.Cmax adjusted for protein binding in culture medium) of 7 anticancer drugs: Gefitinib (EGFRi), trametinib (MEKi), pictilisib (PI3Ki), capivasertib (AKTi), everolimus (m-TORi), vemurafenib (BRAFi), and luminespib (HSP90i) for 1 hour to recapitulate a clinical setting and eventual translational relevance of our experiments. We chose a limited panel of drugs with well-understood mechanisms of action that had been either licensed or evaluated in clinical trials. We used a panel of 52 relevant phosphoproteins based on the known action of our drug panel and previously validated signal transduction pathways. Using highly curated protein–protein interaction data  and 16 samples of immunomagnetically separated cancer cells from patients with NSCLC with pleural effusions. Cells were exposed to a single concentration  to define the key phosphoprotein changes that predict drug sensitivity in individual cell lines. For comparison, we applied the same algorithms to test the power of known genomic features to predict drug sensitivity. We divided the response data into four quartiles where the first quartile and fourth quartile contain the least and most drug sensitive outcomes, respectively . FeatureEGFR  (Supplementary Table S2). Equally, PIK3CA-mutated cell lines did not show an enrichment for sensitivities to the PI3K inhibitor pictisilib in Quartiles 1 and 2 . Although this may be due to the relatively small sample sizes of numbers of cell lines, these results highlight the limitations of using genotype alone to predict sensitivity to targeted drugs, even those that target a protein that can drive a cancer cell. These experiments were performed before KRAS G12C inhibitors becoming available; however, proteomic analysis outperformed KRAS mutations to predict sensitivity/resistance to all drugs studied. Thus, the predictive power of phosphoproteomic changes in the models studied shows that they could be used to augment current predictive biomarker paradigms based on genotype.In addition, we calculated the predictive performance of each of the three mutated genes when targeted with drugs against their specific protein. Despite EGFR  and PIK3 . The list of EPS values for each node per cell line per drug is presented in the Supplementary Table S3 and an example of proteomic changes caused by capivasertib and trametinib in the HCC827 cell line and the associated EPS score are shown in –D, respein vitro of the 7 drugs in 6 cell lines , resulting in 252 experimentally derived Bliss independence scores. The Bliss independence scores of all the combinations in the 6 cell lines are represented in To test and validate the power of the EPS in predicting synergistic combinations, we conducted blind unbiased pairwise combination screening R2 = 0.0132; Supplementary Fig. S4). However, we observed enrichment of correct predictions in the highest and lowest Bliss data quartiles: Predictions for these quartiles showed significantly skewed distributions . To test the statistical significance of this enrichment, we compared the concordance of our EPS ranking with synergy based on the experimental input data versus 10,000 equivalent rankings based on randomly simulated data (see Supplementary Methods). We found a clear difference between EPS concordance with the experimental data of P values of <0.1 with that of the random rankings  for 1 hour, we exposed immunomagnetically separated cancer cells isolate from fresh pleural effusion aspirates to the 7 drugs under identical conditions. The phosphoprotein analysis was conducted and PCA of phosphoprotein changes due to 7 drugs in established NSCLC cell lines (n = 35) and samples from patients (n = 16) were broadly similar  can be used to predict drug sensitivity: These data were able to outperformed known genetic biomarkers as predictors of sensitivity in the cell line panels and drugs studied. The tailoring of experiments to use clinically relevant concentrations adjusted to protein binding and an acute 1 hour exposure to be used clinically on biopsy specimens in the future, make our proteomic dataset and analysis different from other important recently published work on effects of drugs on proteomic perturbation . HoweverKRAS G12C-driven NSCLC but not colorectal cancer and this is related to feedback loops involving EGFR signaling , xenograft experiments need be done at a scale that is out the scope for academic groups. Showing the results of 1–2 xenograft models to show proof of concept, although conventional, we felt would be against the spirit of unbiased testing and thus we have not conducted these experiments for this article. Such experiments will have to be considered before using the assay in the clinical setting.There are biological complexities such as the role of stroma or the immune system that cannot be captured in the model system described previously in this article. However, we do believe that the current approach is a functional assay that can be delivered in the clinic, which intellectually lies in between genomics (finding mutations/amplifications/deletions or siRNA/CRISPR experiments) and truly phenotypic assays (cell culture/organoid and patient derived xenografts), with the added advantage of being able to near contemporaneously predict sensitivity and synergistic combination therapy. The EPS algorithm, based on acute phosphoproteomic changes, has been validated in To conclude, we have demonstrated for the first time that the use of a focused phosphoproteomic assay and machine learning approaches that has used dynamic phosphorylation in signal transduction to predict sensitivity to drugs and prioritize rational combinations tested on cancer cell lines and patient samples in NSCLC. This is a powerful approach that is orthogonal to genomic markers, is adaptive and individualized, with a clinically meaningful turnaround time. This feasibility study provides proof of concept; however, considerable technical validation is needed before use in patients. If developed further, that this methodology can potentially improve the outcomes of patients with cancer treated with targeted anticancer drugs as a single agent or as combination therapy.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.


Humanised mouse models of GVHD can mimic the clinical setting for GVHD development, with disease progression and tissues impacted like that observed in humans. This review will highlight key findings from preclinical humanised mouse models regarding the role of donor human immune cells, the function of cytokines and cell signalling molecules and their impact on specific target tissues and GVHD development. Further, specific therapeutic strategies tested in these preclinical models reveal key molecular pathways important in reducing the burden of GVHD following allogeneic HSCT.Graft-versus-host disease (GVHD) is a major complication that occurs following allogeneic haematopoietic stem cell transplantation (HSCT) for the treatment of haematological cancers and other blood-related disorders. GVHD is an inflammatory disorder, where the transplanted donor immune cells can mediate an immune response against the recipient and attack host tissues. Despite over 60 years of research, broad-range immune suppression is still used to prevent or treat GVHD, leading to an increased risk of cancer relapse and infection. Therefore, further insights into the disease mechanisms and development of predictive and prognostic biomarkers are key to improving outcomes and reducing GVHD development following allogeneic HSCT. An important preclinical tool to examine the pathophysiology of GVHD and to understand the key mechanisms that lead to GVHD development are preclinical humanised mouse models. Such models of GVHD are now well-established and can provide valuable insights into disease development. This review will focus on models where human peripheral blood mononuclear cells are injected into immune-deficient non-obese diabetic (NOD)- Graft-versus-host disease (GVHD) is an inflammatory disorder that can occur following allogeneic haematopoietic stem cell transplantation (HSCT) ,2. AllogAllogeneic HSCT is a curative therapy for haematological malignancies and other blood-related disorders. This therapy aims to regenerate the immune system following pre-conditioning . In addiDuring GVHD donor immune cells recognise the recipient tissues as foreign and mount an immune mediated attack on host tissues, leading to tissue damage and organ failure . GVHD isWhile several factors may contribute to the severity of GVHD in the recipient, including the level of human leukocyte antigen (HLA) mismatch, pre-conditioning regimes, and the source of donor cells , furtherMechanisms for GVHD progression and the development of treatments for the prevention of this disease have come mainly from allogeneic mouse models, which do not always translate to the clinical setting . HumanisThis article will briefly review current approaches in allogeneic HSCT, before discussing GVHD and humanised mouse models. Finally, this article will detail cellular and molecular mechanisms in GVHD as observed through studies in humanised mouse models using functionally related strains.Allogeneic HSCT is largely used to treat haematological malignancies . This trDespite the life-saving benefits of allogeneic HSCT in people with haematologic malignancies, GVHD occurs in 30–70% of recipients . To mana+ cell depletion), anti-proliferative reagents including mycophenolate mofetil or sirolimus (rapamycin), targeting activation with basiliximab [interleukin (IL)-2 receptor (IL-2R) blockade] or daclizumab (IL-2R blockade), using protease inhibitors like α1-antitrypsin or Janus-associated kinase (JAK) inhibitors, targeting tissue homing molecules including vedolizumab [alpha 4 β 7 (α4β7) integrin blockade] or using extra-corporal photopheresis or faecal microbiota transplantation  and tocilizumab (IL-6R blockade), use of the kinase inhibitor ruxolitinib (JAK 1/2 inhibition) or the anti-proliferative reagent azacitidine (DNA-methyltransferase inhibition), glycoprotein targeting with sitagliptin (CD26/dipeptidyl peptidase-4 inhibition), and regulatory therapies including low dose IL-2 and transfer of Tregs .Use of each new GVHD therapy or combination therapy needs to balance the risk of GVHD prevention with the risk of engraftment failure, infection and in the case of haematological malignancies, disease relapse. To this end humanised mice have provided valuable models in understanding the mechanisms of GVHD, as well as in the testing the efficacy of new GVHD therapies and their impact on GVT immunity.GVHD is still a major issue in allogeneic HSCT recipients, despite the numerous treatment options outlined above, with 30–50% of recipients developing acute GVHD  and 30–7Acute GVHD is mediated by host antigen-presenting cells (APCs) and donor T cells in a three-stage process, defined as the afferent, efferent and effector phases . The aff+ and CD8+ T cells, respectively, recognise host major histocompatibility complex (MHC) class II and I molecules on APCs, with mismatches between host and donor MHC molecules driving the activation of T cells [+ T cells can differentiate into Th1 and Th17 subtypes following T-cell receptor (TCR) stimulation and co-stimulation with cytokines like IL-12 [+ T cells into various subsets with differing cytokine profiles [The efferent phase begins when activated host APCs stimulate donor T cells. Donor CD4 T cells . Moreove T cells . Followi T cells . For exake IL-12  or transke IL-12 , respectprofiles . Regulatprofiles ,46. Brieprofiles .+ and CD8+ T cells directly attack target host cells through the Fas/FasL pathway and perforin/granzyme pathways, respectively [The effector phase occurs when activated alloreactive donor T cells migrate to host tissues, including the gastrointestinal tract, skin, liver and lung, and initiate a severe inflammatory response . T-cell ectively . Furtherectively . For exaectively  and miR1ectively  increaseectively  and miR1ectively  decreaseectively . AltogetChronic GVHD has also been described to occur in three phases . The firDespite current prophylaxis and treatment options, GVHD is a prevalent complication following allogeneic HSCT and remains a major cause of morbidity and mortality. A better understanding of the mechanisms of GVHD pathogenesis is needed to overcome this limitation and improve the success of allogeneic HSCT as a treatment for haematological malignancies. To this end, humanised mice have provided valuable models in understanding the mechanisms of GVHD, as well as the testing of the efficacy of new GVHD therapies and their impact on GVT immunity in relation to haematological malignancies. However, such models are limited mainly to acute GVHD, which will be the focus of the remaining article, with acute GVHD denoted simply as GVHD hereafter.Humanised mouse models can be broadly classified as those that involve the expression of specific human gene products within mice, including instances in which a specific mouse gene is replaced by the human orthologue or those that involve the injection of human cells into immunodeficient mice . The latIl2rg gene [tm1WjlIl2rg) or a truncated IL-2R γ-chain lacking the intracytoplasmic domain (tm1SugIl2rg), both of which prevent murine NK cell development [scid mutation in the gene encoding the DNA-dependent protein kinase catalytic subunit (scidPrkdc) or the targeted tm1Fwa mutation in the recombination-activating gene 2 (tm1FwaRag2), both of which impair murine T- and B-cell development [Most commonly, GVHD is studied by the transplantation of hPBMCs into immunodeficient mice encoding targeted mutations in the 2rg gene , which relopment . The bacelopment . Mice arelopment . Mice enelopment ,71.scidIl2rgtm1WjlPrkdc (NSG) mice the most published. The first detailed report using this strain revealed that intravenous (i.v.) injection with as few as 5 × 106 hPBMCs into irradiated (2 Gy) NSG mice results in lethal GVHD [6 hPBMCs into non-irradiated NSG mice could induce lethal but delayed GVHD. A preliminary observation from the same group indicated that intraperitoneal (i.p.) injection of 20 × 106 hPBMCs into non-irradiated NSG results in human leukocyte engraftment, but details regarding GVHD development in this study were limited [scidIl2rgtm1SugPrkdc (NOG) mice. The i.v. or i.p. injection of at least 1 × 104 or 1 × 107 hPBMCs, respectively, into irradiated (2.5 Gy) NOG mice results in lethal GVHD  mice, with GVHD also able to occur in the absence of irradiation [The most commonly used hPBMC mouse model to study GVHD involves three related strains  with NODhal GVHD . This sa limited . Since thal GVHD , with thNOG mice . The thiadiation  (Table 1W-JTyr+PrkdcscidIl2rgtm1WjlKit (NBSGW) mice [The studies above indicate that the pre-conditioning of mice with irradiation and the subsequent tissue damage is not necessary for the induction of GVHD following hPBMC injection. These findings parallel reports in other models, where it was revealed that i.v. injection of human peripheral blood T cells into NOG mice  or the rGW) mice  could inGW) mice . LikewisGW) mice . Human TGW) mice . Thus, tGW) mice  and NBSGGW) mice  mice, bu6 hPBMCs into irradiated C.Cg-tm1FwaIL2rγtm1SugRag2 (BRG) mice (more commonly referred to as BALB/c-−/−IL2rγ−/−Rag2 mice) and which also leads to lethal GVHD [scid-IL2Rγnull mice. Use of these humanised mouse models has promoted insights into the mechanisms of GVHD  , which can impair autoimmune disease [In NSG mice injected with hPBMCs, Tregs are known to be reduced over time . Therefo of IL-2 , indicat of IL-2 , indirecuce GVHD . Finally8+ Tregs . This ef disease , can alsMDSCs are a heterogeneous population of myeloid precursors with emerging inhibitory roles in preventing GVHD . As for Finally, tolerogenic DCs may also have a role in preventing GVHD. Co-injection of human tolerogenic DCs (induced by active vitamin D) with hPBMCs reduced GVHD and corresponded to an increase in human Tregs . CollectMSCs are multipotent stem cells that can not only differentiate into various cell types but can also regulate the immune response in many diseases including GVHD . As suchex vivo manipulated MSCs prior to injection, reporting decreased GVHD [Numerous clinical trials investigating MSCs as a therapy for GVHD in allogeneic HSCT recipients have been conducted, with the vast majority electing to use allogeneic bone marrow-derived MSCs . These tsed GVHD .Inhibitors and ligands have been examined in a number of humanised mouse studies and have helped elucidate cell signalling mechanisms in GVHD. The majority of such studies have shown the prevention or reduction of GVHD following administration of these compounds, highlighting important roles for DAMPs (and their receptors), co-stimulatory molecules, cytokines (and their receptors) and intracellular pathways in GVHD development  reduced GVHD, which corresponded to a reduction in a number of human pro-inflammatory cytokines including IFN-γ, TNF-α, IL-2, IL-4 and IL-17A in humanised mice . InhibitIn contrast with the co-stimulatory molecules above, mAb blockade of the immune checkpoint molecule programmed cell death (PD) molecule-1  worsened GVHD in humanised mice, which corresponded to an increase in human IFN-γ . This suCytokines, including chemokines, and their respective receptors play a pleiotropic role in cell signalling. These have been indirectly studied in the context of GVHD development and progression with a limited number of studies directly investigating cytokines and their receptors in humanised mice. The latter will be the focus below.+ T cells, corresponding to a reduction in murine neutrophils and macrophages, and reduced C-X-C chemokine ligand (CXCL) 1 and CXCL5 in skin lesions [+ T cells alone could not engraft NOG mice, revealed that the transgenic expression of human IL-2 could support CD8+ T cell engraftment and GVHD development, with the authors suggesting that IL-2 from CD4+ T cells facilitates CD8+ T cell engraftment in this model following injection of hPBMCs. Consistent with this, transgene expression of human IL-2 promotes the expansion of both human CD4+ and CD8+ T cells and GVHD development in NSG mice [+ T cells and limits GVHD development compared with transgene expression of IL-2 alone [Blockade of cytokines or their receptors has revealed the role of signalling axes in the development of GVHD in humanised mice. Fusion protein blockade of the soluble TNF-α receptor delayed progression of GVHD, which corresponded to a reduction in human leukocyte engraftment and was interpreted to be an indicator of GVHD development . Blockad lesions . MoreoveNSG mice . HoweverNSG mice , an effe-2 alone . Paradox-2 alone . Collect+ T cells and decreased Tregs [Two studies have examined the impact of exogenous cytokines on GVHD development in humanised mice. Transgene delivery of human IL-4 was shown to reduce GVHD, which corresponded with a reduction in human T-cell engraftment , suggested Tregs , possiblFinally, blockade of C-C chemokine receptor (CCR) 7 (CD197), which facilitates leukocyte trafficking into secondary lymphoid tissues, reduced GVHD in humanised mice, which corresponded to a reduction in leukocyte engraftment in spleens of these mice . This st+ and CD8+ T cell engraftment and reduced human Treg engraftment [+ cells and increased human Tregs [By extension, the above studies of DAMPs, co-stimulatory molecules and cytokines indirectly implicate a number of intracellular signalling pathways in the development and progression of GVHD. As such, numerous studies have used small molecule inhibitors to investigate the role of various intracellular signalling molecules in GVHD . Blockade of JAK 1, which is downstream of IL-2, IL-4 and IL-6 receptor family members, demonstrates a role for this kinase in GVHD development. Blockade of JAK 1 corresponded to increased human CD4raftment . Dual blraftment . Likewisan Tregs . CollectOther studies of humanised mice indicate that kinases unrelated to the above are also involved in GVHD progression. Blockade of mTOR has demonstrated a role for this kinase in the development of GVHD through production of human IFN-γ and TNF-α and impairment in human Treg numbers . Finally+ and CD8+ T cells in the pathogenesis of GVHD are required, as evidence from current studies is still conflicting. Furthermore, the role of Th17 cells and the mechanisms behind their pathogenicity in a GVHD setting, as well as other Th subsets need to be further explored. Whilst there are numerous studies that investigate the role of CD4+ Tregs in alleviating GVHD, future studies should seek to determine the efficacy of other regulatory cells such as KIR+CD8+ T cells and MDSCs. Further investigation of key points within cell signalling pathways may also elucidate additional mechanisms through which GVHD arises in humanised mice. Outcomes of these studies may provide valuable insights into the best approach to prevent and reduce GVHD and lead to better biomarkers for predicting disease development in a clinical setting. Finally, studies that investigate such mechanisms need to assess the role of these mechanisms in the development of GVT immunity to prevent increased rates of disease relapse.It is evident that humanised mouse models are a useful preclinical tool to examine the mechanisms of GVHD development. Further studies investigating the specific role of CD4


Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a helical loop on the cell surface. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell and revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction. Several electron microscopic analyses revealed the presence of a possible multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. These results provide insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along tracks that may form a multi-rail system underneath the outer membrane. The insights may give clues as to how the SprB filaments get their driving force.Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, called gliding motility. In our previous study with the Bacteroidetes gliding bacterium  Electron microscopic visualization and movement analysis of the gliding machinery in Bacteroidetes provide insights into the mechanism of gliding motility, or the ability of these microbes to move on solid surfaces. Gliding motility requires cell contact with a solid surface, since cells suspended in liquid do not actively move. Genetic analyses have suggested that the Bacteroidetes gliding motility is unrelated to other well-studied bacterial motility mechanisms such as flagellar motility, type IV pilus-mediated twitching motility, myxobacterial gliding motility, and Mycoplasma gliding motility, but instead relies on novel machinery consisting of Gld and Spr proteins that are confined to members of the large and diverse phylum Bacteroidetes such as Flavobacterium johnsoniae6. Some Gld and Spr proteins are not only involved in gliding motility but are also components of the type IX secretion system (T9SS)7. T9SSs were first identified and studied in the nonmotile oral pathogen Porphyromonas gingivalis and in F. johnsoniae11. Recent studies have revealed that T9SS is a unique system that is clearly different from other secretion systems in terms of its supramolecular structure and secretion mechanism14. F. johnsoniae cells use the T9SS to secrete dozens of proteins, including soluble extracellular enzymes and motility adhesins that reside on the cell surface17.Cells of many bacterial species belonging to the phylum Bacteroidetes move over surfaces at ~1–5 µm per second in a process called gliding motilityF. johnsoniae has been studied as a model organism to understand the molecular mechanism of Bacteroidetes gliding motility. Genetic and molecular analyses demonstrated that SprB, a huge filamentous 6497 amino acid protein, is a primary cell-surface adhesin of F. johnsoniae18. Immunofluorescence microscopic analysis using antiserum against SprB revealed that the SprB filaments are propelled at ~2 µm per second with a ~19° tilt with respect to the long axis of the cell. Mathematic analyses suggested a helical-loop track model in which gliding motors act on SprB filaments that have attached to a surface-generating rotation and translocation of the cell body19. A recent cryo-EM study has revealed that GldL and GldM, a transmembrane core complex for gliding motility has a structural organization similar to that of the bacterial flagellar stator complex consisting of MotA and MotB, which acts as a proton channel for torque generation22. GldLM complex shows relatively static localization distributed in multiple foci along the cell body and fueled by the proton gradient to drive the helical motion of SprB adhesin24. However, the structural basis for why SprB moves along a helical track is still unknown.F. johnsoniae cell surface by immunofluorescence microscopy and morphological analysis of the gliding machinery by electron microscopy suggests the possibility of a multirail structure as a component of the helical-loop track involved in SprB movement. Similar multirail structures were seen in the distantly related gliding Bacteroidetes Saprospira grandis, suggesting that this may be a general feature of the Bacteroidetes gliding machinery. The results provide insights into the mechanism of this common form of bacterial gliding motility.In this paper, a detailed analysis of SprB movement on the F. johnsoniae where SprB adhering to a substratum was propelled along a helical-loop track on the cell surface, resulting in rotation and translocation of the cell19. Here, we investigated whether gliding cells actually rotate as proposed. To visualize cell rotation, we attempted to find a cell-surface protein that did not change position with respect to the long axis during gliding, and observe its behavior by total internal reflection fluorescence (TIRF) microscopy using fluorescently labeled antiserum. Cell-surface proteins were isolated and collected by centrifugation. The isolated cell-surface proteins were separated by SDS-PAGE  to reduce signals and track SprB signals easily, and movement of signals on the surface of gliding cells was observed by fluorescence microscopy. Under these conditions, about 58% of cells had 1–10 SprB signals and the rest had none. Consistent with the previous report19, the SprB signals were propelled between cell poles along an apparently helical-loop track on gliding F. johnsoniae cells  when the Fjoh_0697 signal made one revolution in the direction of the short axis of a cell  . Note that the helical pitch was measured by the SprB signals moving from the lagging pole to the leading pole. These signals followed CCW trajectories on the view from the rear end. Considering the left-handed helical-loop track, it is expected that the SprB signals moving from the leading pole to the lagging pole follow CW trajectories. However, we could not follow the CW trajectories because these signals moved slowly and the cells switched back their moving direction frequently. During cell migration, the rotational rate of Fjoh_0697 signals was directly proportional to the cell velocity  Fig.  left, d,97) Fig.  right, dity Fig. . To showity Fig. . Plots sity Fig. , VSprB+,ity Fig. , VSprB–.Some SprB signals exhibited unexpected movements such as mid-cell U-turns. Figure N = 96), which was similar to SprB movement on a normal-length cell   cells and on non-elongated control cells that had not been exposed to the antibiotic  Fig. . We then28) Fig. , this re28) Fig. , g. ThesN = 58). The multirail structure was easily peeled from the cell after osmotic shock. Thin filaments appeared to be attached to the multirail structure  extended from the outer membrane . Gold particles accumulated on the multirail structure only when anti-GldJ antiserum was used Fig. , suggestgldA, gldB, gldD, gldF, gldG, gldH, gldI, gldJ, gldK, gldL, gldM, and gldNO mutants showed no multirail structures . However, orthologs of the periplasmic lipoprotein, GldI and the cell-surface lectin, RemA38  were not found in S. grandis. TEM analysis revealed the presence of abundant bundle fibers in osmotically shocked cells as previously reported35 . Cryo-electron tomography also revealed the bundle fibers in intact S. grandis cells  is consistent with the cell rotation generated by the left-handed helical movement of SprB. Shrivastava et al.39 reported right-handed movements of SprB and cell movements. We do not have an explanation for this difference. Regardless of the handedness of the movements, it seems clear that SprB movement along helical tracks results in rotation and forward movement of gliding cells.In a previous study, we proposed a model for gliding motility of F. johnsoniae cells that were burst by osmotic shock to reduce cell thickness and were then negatively stained with 0.5% uranyl acetate. It must be noted that we did not observe such multirail structures by cryo-electron tomography. Instead, parallel filamentous structures were observed in the periplasm of F. johnsoniae cells. The relationship between the multirail structures and the filamentous structures is unknown. The location of the multirail structure on the periplasmic side of the outer membrane of F. johnsoniae was revealed by quick-freeze deep-etch electron microscopy. Considering this location, lipoproteins could be components of the structure. Immunoelectron microscopic analysis revealed that GldJ lipoprotein was associated with the multirail structure. It was previously reported that GldJ is organized in discrete bands that appear to form a helical structure33, which is consistent with the present results. GldJ lipoprotein shares 30% identity with GldK lipoprotein over 382 amino acids33. P. gingivalis PorK (GldK homolog) with PorN makes a ring structure of ~8 nm in thickness and 52 nm in diameter40. The thickness of fibers in the multirail structure involving GldJ is almost the same as that of the PorK/PorN ring. Various gld mutants in addition to the gldJ mutant lacked the multirail structures. GldJ was reported to be unstable in gldA, gldB, gldD, gldF, gldG, gldH, and gldI mutants33. The lack of GldJ in these mutants may explain the absence of the multirail structures. GldJ appeared to be part of a large complex in wild-type cells, whereas it was present in lower molecular weight form in cells of gldK, gldL, gldM, or gldNO mutants. GldK, GldL, GldM, and GldN may be required for the assembly or stability of a GldJ complex associated with the multirail structures.A detailed analysis of the SprB movement in this study shows that SprB can turn around in the middle, SprB can catch up with and overtake another SprB, and SprB can change its speed, suggesting that SprB does not always move at a constant speed on a single track. We observed the multirail structure associated with the SprB filaments in the S. grandis had a multirail structure similar to that of F. johnsoniae  as also observed here for F. johnsoniae. In M. xanthus, cytoplasmic and cytoplasmic membrane proteins migrate along a helical track in the cytoplasm, resulting in cell movement47. The cytoplasmic membrane motor proteins form a complex with periplasmic and outer membrane proteins that exert force on cell-surface adhesins that are attached to the substratum50, which is similar to the F. johnsoniae motility model.Gliding motility of the δ proteobacterium F. johnsoniae and M. xanthus cells51. There are numerous differences between the two systems. Many proteins have been identified that are involved in the gliding motility of both organisms but there is little if any similarity between them52. For F. johnsoniae, the cell-surface adhesin SprB appears to be propelled along a helical track associated with the periplasmic face of the outer membrane that may be comprised of the lipoprotein GldJ. There is no evidence for helical movement of components in the cytoplasm or cytoplasmic membrane and the current model involves motors, stationary on the cell, that propel SprB along the cell surface24. In contrast, for M. xanthus movement of cytoplasmic membrane proteins along a helical cytoplasmic track is proposed44, resulting in movement of surface proteins. Gaps in our understanding of the F. johnsoniae and M. xanthus motility machines remain. For M. xanthus the cytoplasmic membrane motor proteins  are responsible for force generation. In contrast, for F. johnsoniae the cytoplasmic membrane proteins GldL and GldM forms a nanoscale electrochemical motor55, which drives both gliding motility and protein secretion. Control of the motors in response to the environment is also distinct for each organism. The M. xanthus motors are controlled by the frz chemotaxis system56, whereas F. johnsoniae and other members of the Bacteroidetes lack critical chemotaxis proteins2 and must use another mechanism to control cell movement.Although the helical movement of motility proteins drives the helical movement of both 57 suggested that the gliding motor in F. johnsoniae is rotary because F. johnsoniae cells rotated when tethered to glass by anti-SprB. We do not yet know how a rotary motor results in the helical movement of SprB. One possibility is that the rotating motor pushes on a tread carrying SprB filaments and propels it along the helical GldJ track24. Alternatively, the SprB filaments could be anchored on the tracks, and the tracks could be propelled by the motor58.Shrivastava et al.19. However, it is not known how the motor(s) transmit force to the SprB filaments on the cell surface. The apparent linkage of SprB filaments with the outer membrane-associated periplasmic rails described here may explain part of this transduction. Further studies including the clarification of the force-transducing mechanism are needed for a more complete understanding of this common form of Bacteroidetes motility.Various unanswered questions regarding the Bacteroidetes gliding machinery remain to be solved. The proton gradient across the cytoplasmic membrane is required for SprB movement and cell glidingF. johnsoniae cells were grown in Casitone-yeast extract (CYE) medium at 25 °C with shaking59. To observe gliding motility, F. johnsoniae cells were grown in a motility medium  to an optical density of around 1.0 at 600 nm. S. grandis cells were grown in Marine medium  with 0.5% tryptone (BD)) at 25 °C with shaking to an optical density of around 1.5 at 600 nm.Bacterial stains are listed in Supplementary Table g for 1 min, and the pellet was suspended in fresh MM with 1:500 to 1:1000 dilution of antiserum against SprB, against pooled cell-surface proteins or against Fjoh_0697 and incubated for 5 min. Cells were collected by centrifugation at 5000 × g for 1 min and washed with fresh MM. The cells were suspended in fresh MM with 1:1000 dilution of Alexa Fluor 555-conjugated antibody against rabbit IgG . After incubation for 5 min, cells were washed and suspended in fresh MM.For immunofluorescence microscopy of live cells, cells were harvested by centrifugation at 5000 × F. johnsoniae cells were poured into a tunnel slide assembled by using double-sided tape to attach a coverslip onto a glass slide60, and were observed using an inverted microscope . TIRF images were acquired with UApoN 100×OTIRF objective lens (Olympus) using a 561 nm laser. Images were recorded with an iXon3 897 EMCCD camera  using MetaVue software . Time-lapse montage images and kymographs were processed with ImageJ 1.48r software (http://imagej.nih.gov/ij/).F. johnsoniae was grown in MM at 25 °C for 6 h. The cells were suspended in PBS  and passed several times through a 26 G-1/2 inch needle to shear cell-surface structures. After the removal of the cells by centrifugation at 9000 × g for 10 min, a fraction of surface proteins was collected by ultracentrifugation at 66,000 × g for 60 min. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant blue R250. Protein bands were excised and digested by trypsin. Proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry .F. johnsoniae was grown in 5 ml of MM at 25 °C for 3 h. Cells were suspended in 100 µl of ice-cold sucrose solution . The cells were placed on ice for 15 min and then mixed rapidly with 1.4 ml of ice-cold ultrapure water. Intact cells were removed by low-speed centrifugation at 9000 × g for 3 min. Osmotically shocked cells were collected from the supernatant by high-speed centrifugation at 20,000 × g for 5 min. For S. grandis, cells were first fixed with 1.5% paraformaldehyde for 30 min at room temperature and then washed twice with ice-cold ultrapure water or 50 mM MgCl2 solution. Intact S. grandis cells were removed by centrifugation at 2000 × g for 3 min and osmotically shocked cells were collected by centrifugation at 20,000 × g for 5 min. Centrifugation was done at 4 °C.33.Samples were negatively stained with 2% PTA (pH 7.0) or 0.5% uranyl acetate on a Butvar B-98 (Sigma-Aldrich) coated copper grid, and observed by transmission electron microscopy . Micrographs were taken at an accelerating voltage of 80 kV. For immunogold electron microscopy, osmotically shocked cells were treated with 1000-fold diluted antiserum against SprB protein in PBS containing 2% BSA and incubated on ice for 20 min. The cells were washed three times with PBS and treated on ice with 20-fold diluted goat anti-rabbit IgG conjugated to 5 nm diameter gold particles  in PBS containing 2% BSA for 20 min, washed three times, and then stained with 0.5% uranyl acetate. For anti-GldJ immunostaining, osmotically shocked cells were fixed with 1.5% paraformaldehyde before treatment with anti-GldJ rabbit polyclonal antiserum62. Samples were knife-fractured, deep-etched at −104 °C for 10 min, rotary-shadowed with Pt/C at an angle of 20°, and then backed with pure carbon by a freeze-fracture device (JFD-V: JOEL). Replicas were floated off the cover glass onto the surface of full-strength hydrofluoric acid. Household bleach was occasionally used to remove the remaining debris from the replica. Replicas were rinsed with three changes of water and picked up onto copper grids for electron microscopic examination.Bacterial cells, which were partly disrupted by osmotic shock, were mounted onto a cover glass and quickly frozen by metal contact at liquid-nitrogen temperature. Frozen samples were subjected to freeze-fracture deep-etch replication−/Å2 or less was used for each tilt series. Images were generally binned two-fold and 3D reconstructions were calculated using the IMOD software package63. Surface-rendering images were obtained using the three-dimensional modeling software Amira 5.2.2 .Quantifoil molybdenum 200 mesh R0.6/1.0 grids  were glow discharged and pretreated with a solution of 10 nm colloidal gold particles concentrated 1.5 times before use  for tomogram alignment. A 3 μl sample was applied to the grid, blotted with filter paper, and plunged into liquid ethane using Vitrobot . Images were collected at the liquid-nitrogen temperature using a Titan Krios FEG transmission electron microscope (FEI) operated at 300 kV on FEI Falcon 4 k × 4 k direct electron detector (FEI). The magnification was calibrated by measuring the layer-line spacing of 23.0 Å in the Fourier transform of images of tobacco mosaic virus mixed in the sample solution. The pixel size on the specimen was 0.57 nm. Single-axis tilt series were collected covering an angular range from −70 to 70 with a nonlinear Saxton tilt scheme at 4–10 μm underfocus using the Xplore 3D software package (FEI). A cumulative dose of 200 en-dodecyl-β-D-maltoside (DDM), 0.5 M sucrose, 10 mM Tris-HCl (pH 7.5), 1 mg per ml DNase, and protease inhibitor cocktail (Sigma-Aldrich). Soluble fractions were mixed with 10× Blue native sample buffer , and loaded on a native gel . Proteins on the gel were blotted on PVDF membrane and subjected to immuno-detection with anti-GldJ rabbit polyclonal antiserum.Cells were sonicated in lysis buffer containing 1% (weight per volume) sprD was amplified using primers 1296  and 1297 . The fragment was digested with SalI and SphI and ligated into pRR5164, which had been digested with the same enzymes, to generate pYT55. A 2.1-kb fragment spanning sprC and the first 57 bp of sprD was amplified using primers 1294  and 1295 . The fragment was digested with BamHI and SalI and fused to the downstream region of sprD by ligation with pYT55, which had been digested with the same enzymes, to generate the deletion construct pYT58. pYT58 was introduced into F. johnsoniae CJ1827 by triparental conjugation to construct the ∆sprD mutant CJ2246. To delete sprF, pYT31365 containing 1.8-kb regions upstream and downstream of sprF was introduced into F. johnsoniae CJ1827 by triparental conjugation to construct the ∆sprF mutant CJ2518.A 1.9-kb fragment downstream of and spanning the final 33 bp of Micrographs and electron micrographs in Figs. Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12Supplementary Movie 13Supplementary Movie 14Supplementary Movie 15Supplementary Movie 16Supplementary Movie 17Supplementary Movie 18Reporting Summary