Both electrode and cell designs werefound to have a significant impact on the rate performance and cyclelife of Li-ion half- and full cells. A higher specific capacity, improvedrate performance, and a longer cycle life were obtained in both anodeand cathode half-cells by lowering the electrode porosity throughcalendaring. MNO/Li half-coin cells displayed excellent cyclability,reaching 80% state of health (SOH) after 600 cycles at C/2 chargeand 1C discharge. MNO/NMC622 full-coin cells displayed a high capacityof 179 mAh g–1 at 100 mA g–1 (0.5mA cm–2) and excellent cyclability at 25 °C,reaching 70% SOH after over 1000 cycles at 1 mA cm–2 after optimizing their N/P ratio. Excellent cyclability was obtainedat both 1C/1C and fast 2C/2C cycling, reaching 80% SOH after 700 and470 cycles, respectively. Full-coin and small pouch cells had outstandingrate performance as they could be charged from 0 to 84% capacity inless than 5 min at 10 mA cm–2 and to 70% SOC in120 s at 20 mA cm–2.Niobium oxides are an emerging class of anode materialsfor usein high-power lithium-ion batteries. Galvanostatic cycling and electrochemicalimpedance spectroscopy (EIS) were used in this study to investigatethe influence of electrode porosity, electrode mass ratio, and cyclingrate on the capacity, cycle life, and ionic conductivity of Li-ionbattery cells based on a modified micron-sized MoNb Indeed, the growth of a thicksolid-electrolyte interface (SEI) layer formed on the surface of graphiteanodes results in increased impedance and irreversible consumptionof a large amount of Li+ ions.7 Moreover,the low electrochemical potential for lithium intercalation in graphite(<0.1 V vs Li/Li+) leads to lithium dendrite formationduring fast charging, which limit the cycle life of graphite-basedLIBs and raises serious safety concerns.9Commercial LIBs currentlyused in EVs are based on cell chemistrieswith an intercalation-type graphite anode against various possiblelayered transition-metal oxide cathodes such as LCO, LMO, NMC, NCA,or LFP.10 Lithium metal for solid-statebatteries is highly promising due to its ultrahigh specific capacity(3860 mAh g–1), but significant improvements stillneed to be made to mitigate Li-dendrite growth.11 Similarly, pure silicon and Si oxides (SiOx) have been investigated extensively as the theoreticalspecific capacity of Si-based anodes is 1 order of magnitude higherthan that of graphite. However, the practical implementation of Si-basedanodes for commercial applications is limited by the dramatic volumeexpansion of 100–300% experienced by these materials upon cycling,which results in extensive particle cracking, low initial Coulombicefficiency, and poor cycle life.13 Therefore,the use of silicon/graphite composite anodes with 0–20 wt %of Si nanoparticles has been favored, although it tends to increaseboth manufacturing complexity and cost.16Severalalternative anode materials have been investigated andimplemented to improve the cycle life, safety, and rate performanceof LIBs.4Ti5O12 (LTO) has a high intercalationpotential (1.55 V vs Li/Li+) resulting from Ti4+/Ti3+ redox couple that makes it inherently safer thangraphite by eliminating dendrite formation but results in lower voltageand hence lower energy density in a full cell.17 LTO also has excellent cyclic stability and has been widelydemonstrated as a good anode material candidate for high-rate applications.18 However, the low theoretical capacity of LTO(175 mAh g–1) limits its suitability for high energyand high voltage applications. Moreover, it needs to be nanosizedand/or undergo surface modification19 ordoping20 for high charge/discharge capabilitydue to its low Li-ion diffusion coefficient, which results in increasedcost and severe limitations on achieving high electrode volumetriccapacities  and volumetricenergy densities . This workinvestigated a material that belongs to a nascent class of intercalation-typeanodes: Wadsley–Roth crystallographic shear structure niobiumoxides, which much like LTO have a dominant Li+ intercalationpotential above 1.0 V (vs Li+/Li). Furthermore, niobiumoxides have a higher theoretical specific capacity than LTO and atheoretical volumetric capacity up to three times as high as LTO,21 which could significantly increase the energydensity of high-power LIBs.Li22 Since TiNb2O7 was proposedas an alternative anode by Goodenough’s group in 2011,23 a large number of Wadsley–Roth niobiumoxide materials were developed and demonstrated in recent years, includingTi2Nbx2Ox4+5 ,24 Al0.5Nb24.5O62,25 PNb9O25,26 WNb12O33,27 and MoNb12O33,28 GeNb18O47,29 Cr0.5Nb24.5O62,30 AlNb11O29,31 andFeNb11O29.32Wadsley–Roth phases basedon niobium oxide were first reportedfor use in lithium-ion batteries by Cava et al. in 1983.3-like channels that enable high capacity and fastLi+ diffusivity, combined with crystallographic shear planesthat enable excellent stability and reversibility.33 The double Nb5+/Nb4+ and Nb4+/Nb3+ redox couple of niobates also contributes to a hightheoretical capacity, alongside the potential to include other redox-activecations.Wadsley–Roth crystallographic phases are characterized byopen ReO12O33 molybdenumniobium oxide (MNO) powder provided by Echion Technologies Ltd., whichhas developed and patented a range of mixed niobium oxide materialsfor high-power Li-ion cells.34In this work, we investigated the electrochemical performanceofa modified Wadsley–Roth MoNb12O33 was first proposed by Zhu et al.in 2019 in the form of porous microspheres synthesized through a one-stepsolvothermal method followed by low-temperature calcination.28 Their MNO/Li half-coin cell had a high specificcapacity (∼320 mAh g–1) with a minimum voltagecutoff of 0.8 V, excellent rate capability with a capacity retentionof 73% at 10C relative to 1C, and exceptional cyclability at both1C and 10C. These properties were retained in an MNO/LiMn2O4 full-coin cell. However, they used extremely thin electrodeswith a mass loading of only 1.0 mg cm–2, while theweight ratio of conductive carbon black (25 wt %) in both the anodeand cathode electrodes was significantly higher than typically usedin commercial battery electrodes, which can lead to misleading results.35 Furthermore, only coin cells assembled in aglovebox under argon were tested, and no information was given eitheron the volume of electrolyte used or on the porosity of their electrodes.Finally, compared to the literature on similar materials, a low minimumvoltage cutoff of 0.8 V was used, which increases the experimentalcapacity but is typically a disadvantage in full-cell applications.Indeed, the wider the voltage window for practical use, the more complex,costly, and heavy power electronics and pack-level modifications arerequired.MoNb–2 was targeted, which was significantly higherthan most previously published work on similar niobate anode materialsin the literature.37 A capacity of 1–2 mAh cm–2 is commerciallyrelevant for high-power applications, such as an A123 M1A high-powerbattery, which was found to have an anode areal capacity of ∼1.25mAh cm–2.38 We investigatedthe influence of electrode design (porosity) and cell design, includingthe negative-to-positive capacity balance (N/P ratio), on the rateperformance and cycle life of MNO/Li half-cells and MNO/NMC622 fullcells, with practical voltage limitations in use.Here, we proposed to evaluate the electrochemicalperformance ofMNO anodes with a 90:5:5 ratio by weight of active material, conductivecarbons, and PVDF, respectively, used in coin and pouch cells assembledin a dry room environment to evaluate and demonstrate the applicabilityof this anode toward commercialization. These cells were designedfor power applications, and therefore an areal capacity of ∼1mAh cm22.1A scanningelectron microscope  was usedto observe the size and morphology of raw anode and cathode activematerial powders. The microstructure of the anode and cathode electrodesand the distribution of the active material, carbon black powder,and carbon nanotubes were investigated by conducting the scanningelectron microscopy (SEM) analysis of cross-sectioned anode and cathodecoatings. The cross sections were performed using a Hitachi Ion MillingSystem (IM4000 plus) at an accelerating voltage of 5 kV for 3 h. SEMimaging was conducted on pristine (noncycled) electrode coatings ata working distance of 8 mm in an FEI Quanta scanning electron microscopeunder an acceleration voltage of 15 and 20 kV for the cathode andanode, respectively.2.212O33 (MNO) molybdenum niobium oxide powder . The cathode material was a commercialgrade NMC622 powder . The electrolyte used in allcells was a battery-grade solution of 1.0 M lithium hexafluorophosphate(LiPF6) in ethylene carbonate (EC) and diethyl carbonate(DEC) in a volume ratio of 50:50 .Theactive anode material was a modified MoNb40Both anode and cathode electrodes were made of 90 wt % active material,5 wt % poly(vinylidene fluoride) (PVDF) binder, and 5 wt % conductivecarbon additives—of which 4.5 wt % was carbon black (TIMCALSuper C45 in anode inks and TIMCAL Super C65 in cathode inks) and0.5 wt % were thin multiwalled carbon nanotubes . The carbon nanotubes were added to all anode and cathodeink formulations as it has been shown in the literature to improvethe electrochemical properties and increase the electrical conductivityof battery electrodes.–1 onto battery-grade aluminum foil(16 μm thickness). The blade gap was set so as to obtain anactive material mass loading of 5.5 ± 0.5 mg cm–2 for MNO anodes and 6.0 ± 0.5 mg cm–2 forNMC622 cathode coatings. After coating, electrodes were immediatelydried at 80 °C on a hot plate and then transferred in a vacuumoven at 120 °C overnight. Dry coatings were then calendared tothe desired electrode density using a hot rolling press .Anode and cathode activematerial powders were dried in a vacuumoven at 120 °C for 24 h before ink preparation. First, the carbonblack powder was dispersed in PVDF and 1-methyl-2-pyrrolidinone (NMP)using an ARE 250 Thinky centrifugal mixer . CNTs and more NMP were then added and mixed before the progressiveaddition of the active material powder (mixing at 2000 rpm for 3 minbetween each step). Electrode inks had a powder solid loading of ∼40wt %. Inks were coated using a draw-down coater with a micrometeradjustable doctor blade  at 1 cm s2.3Calendared coatingswere cut into individual electrode disks with a diameter of 14.8 and15 mm for cathodes and anodes, respectively.–1 for NMC622 and 200 mAh g–1 for MNO at 20 mA g–1 (∼0.1 mA cm–2) in theirrespective voltage window of 2.7–4.25 and 1.1–2.4 V.In half-coin cells, lithium foil (PI-KEM) was rolled to 70 ±10 μm thickness and cut into 15 mm discs to be used as the counterelectrode.In full-coin cells,anode and cathode disks were individually matched to provide the desirednegative-to-positive areal capacity ratio (N/P ratio) in each cell,assuming a rated reversible specific capacity of 170 mAh g–2); two stainless steel spacers (1 and 0.5 mm); and a 1000 psi crimpingpressure. Cell components, including casing, spacers, spring, andseparator, were stored in a vacuum oven at 50 °C before use.CR2032 coin cells were prepared using the following:a single trilayerpolymer Celgard H1609 separator disk with a diameter of 16 mm; 70μL of electrolyte  used a double-layeredPP fiber/PE membrane separator , an ultrathinLi reference ring, and were filled with 100 μL of electrolyte.2 cathode coating and a 31 × 31 mm2 anode,with a Celgard H1609 separator and 380 μL of electrolyte (∼40μL cm–2).Single-layer pouch cells were manufactured using a 30 × 30mmTable S1).The main characteristic ofour cells, including the dimensionsof the positive electrode (PE) and a negative electrode (NE), is summarizedin the Supporting Information (The entire preparation from ink mixing and electrode coatingtocoin and pouch cell manufacturing was performed in a dry room (dewpoint between −45 and −60 °C). Three-electrodecells were assembled under an argon atmosphere in a glovebox.2.45 to 10–2 Hz.After cellassembly, a resting period of at least 6 h was allowed for the electrolyteto fully soak in the porous electrodes and separator. Electrochemicaltesting was conducted on BCS and VMP3 battery testers .Cells first underwent two CCCV formation cycles at 25 °C usinga constant current density of 10 mA/g; the current was allowed todecay to 4 mA/g during the constant voltage step. Potentiostatic electrochemicalimpedance spectroscopy (PEIS) was performed in half-cells and fullcells at ∼50% SOC after formation and at 80% SOH by using anamplitude of 5 mV in the applied frequency range of 5 × 10–1 .The evolution of internal resistances and rate performance throughoutthe life of full cells was investigated by performing EIS and ratetesting at various SOHs in coin and pouch cells.Asymmetric charge anddischarge rate tests were performed in half- and full cells usingcurrent densities ranging from 20 to 4000 mA g–1 (∼1.0 mA cm–2) on both charge and discharge with a short rest after each chargeand discharge to observe the evolution of the IR drop. In full cells,the current density was calculated relative to the mass of cathodeactive material. Cycling was also performed at a higher rate on anotherset of cells using a discharge current density of 1000 mA g–1 (∼5.0 mA cm–2).In full cells,long-term cycling was performed at 25 °C using200 mA g33.13.2–1. The capacity plot of formation is shown in Q/dV plot in –1 at 10 mA g–1, with a Coulombic efficiency of 90.0% in the first cycle and 98.5%in the second cycle. The initial Coulombic efficiency is higher thanwith graphite electrodes because the potential at the anode is abovethe reduction potential of the electrolyte, which keeps the SEI formationto a minimum.40 Therefore, the primary source of the first cycle loss is likelydue to irreversible intercalation and trapping of lithium ions inthe crystal structure (an activation process). Side reactions withthe electrolyte could still be a secondary source of first cycle loss,as is seen with the formation of SEI or SEI-like layers constitutedof both organic and inorganic compounds formed at the surface of LTO.43The effect of electrode porosity on the electrochemical propertieswas studied in MNO vs Li coin cells using a series of galvanostaticcycles at various charge and discharge rates. First, cells were formedat 25 °C by performing two CCCV cycles between 1.1 and 2.4 Vwith a constant current density of 10 mA g–1 and CC discharging (lithiation) at 200 mA g–1 between1.1 and 2.4 V. MNO electrodes with a 40 ± 2% porosity reachedthe key state-of-health levels of 90, 80, and 70% SOH after 130, 374,and 473 cycles, respectively, while reducing the porosity down to24 ± 1% through calendering led to significant improvements,as the same SOH levels were reached after 307, 600, and 689 cycles,respectively, representing an improvement of 200 ± 25 cyclesthroughout the life cycle of the cell.–1) up to 20C (∼4000mA g–1) while applying a constant discharge or chargerate of C/2 (∼100 mA g–1). –1 on a 5C delithiation compared with only58 mAh g–1 upon lithiation at the same rate. Besides,while calendaring initially had little effect on the specific capacityof MNO/Li half-cells at C/10 and C/5, it had a significant impacton capacity retention at higher rates. Indeed, as the porosity ofthe anode was reduced from 40 ± 2 to 30 ± 2%—i.e.,an increase of the anode density from 2.6 to 3.0 g cm–3, which corresponded to a reduction of the thickness from 23 ±2 to 19 ± 1 μm—the gravimetric delithiation capacitywas increased from 161 to 182 mAh g–1 at 5C andfrom 1 to 98 mAh g–1 at 20C. Moreover, the electrodecalendared to 24 ± 1%  displayed further improvementsin capacity retention with 191 mAh g–1 at 5C and129 mAh g–1 at 20C. Therefore, the 24% porosityelectrode displayed exceptional delithiation rate performance witha 93% capacity retention at 5C (relative to the capacity at C/2) andas much as 87 and 63% capacity retention at 10C and 20C, respectively.Lithiation rate performance was still excellent although lower thanin delithiation, with 67 and 44% capacity retention at 5C and 10C,respectively. However, the lithiation capacity was only 10 mAh g–1 at 20C (∼20 mA cm–2); thiswas likely due to current rate limitations in half-cells caused bythe use of lithium metal with a liquid organic electrolyte, as describedin the literature.45 Therefore, resistances and rateperformance were further investigated in full-cell setups.Asymmetric charge anddischarge rate tests, respectively, wereperformed between 1.1 and 2.4 V by incrementally increasing the ratefrom C/10 alongside PEIS results for both MNO/Li and NMC622/Li half-cells .A similar investigationof the electrochemical behavior, rate performance,and cycle life of NMC622/Li half-cells as a function of electrodeporosity was also conducted to provide a benchmark of the cathodeproperties for subsequent full cells. The results are provided inthe Supporting Information  the potential at the anodeshould remain above 1.0 V as it is thought to limit the growth ofthe SEI layer on niobate anode. As the full cell was discharged to1.1 V, the potential at the cathode and anode reached 3.4 and 2.4V, respectively, where both appeared to be ideal from the shape ofthe formation curves. It can be observed that there was a slight voltagecreep at both the anode and the cathode from one discharge to thenext, with the potential at the anode increasing from 2.33 to 2.39V and then 2.41 V during the first, second, and third cycles, respectively,while the potential at the cathode reached 3.43, 3.49, and 3.51 V.This “voltage walking” could be explained by lithiuminventory loss, where Li+ extracted from the cathode duringcharge remains permanently within the anode, thus incurring a lossof cyclable Li+, coupled with the loss of active anodeand cathode sites due to structural disordering.A three-electrode full cell was constructedin an argon glovebox to obtain a better understanding of the individualpotentials reached at the anode and cathode, respectively, when cyclingan MNO/NMC622 full cell (N/P ratio of 1.1) within the chosen voltagewindow of 1.1–3.1 V. The three formation cycles of the three-electrodefull cell are displayed in 3.4Full-coincells were constructed by individually matching MNO anodes and NMC622cathodes to obtain the desired N/P ratio for each cell. The N/P capacityratio was varied between 0.9 and 1.2 for two C/20 formation cyclesfollowed by 1C/1C cycling tests at 25 °C. Further cycling wasthen performed using selected N/P ratios at faster charge/dischargerates, including 1C/5C and 2C/2C.Figure S3 in the Supporting Information shows the galvanostatic charge–dischargeformation curves and their associated dQ/dV graph of multiple MNO/NMC622 full-coin cells with N/Pratios of 0.9, 1.0, 1.1, and 1.2 cycled at 10 mA g–1 between 1.1 and 3.1 V. The N/P ratio had no impact on the overallshape of the formation curves, including the first cycle loss, polarization,and location and amplitude of peaks on the dQ/dV plots. However, it appears clearly that a higher specificcapacity was obtained as the N/P ratio was increased. Indeed, a reversiblecapacity of 160, 165, 170, and 180 mAh g–1 was obtainedfor N/P ratios of 0.9, 1.0, 1.1, and 1.2, respectively. The FCL wasconstant across all N/P ratios at ∼12%.Q/dV plots in Figure S3 show that upon the first charge, allfull cells displayed a small peak at 1.65 V followed by a much largerpeak with an amplitude of 4 mAh V–1 at ∼2.1V. Specifically, the 1.65 V peak loses intensity after the first cycle,which indicates that the intercalation process at this voltage isirreversible. Also, the amplitude of the 2.1 V peak reduces to 3 mAhV–1, which indicates that intercalation is not fullysymmetrical and that irreversible loss can also occur at other stagesin the intercalation process.The d–1 current density on both charge and dischargeat 25 °C. –1 charge and 1000mA g–1 discharge. Full cells experienced materiallyfaster capacity fade at this higher rate of discharge, reaching 54%SOH on average after only 200 cycles for cells with an N/P ratio of1.1, while cells with an N/P ratio of 1.2 degraded slower, reaching72% SOH on average after 200 cycles. Nevertheless, After formation, MNO/NMC622 full-coincells were cycled using a200 mA g3.5–1 was obtained withboth cell formats; the FCL in the small pouch cell was ∼2%lower than in the coin cell. Furthermore, postformation PEIS performedat 2.2 V in both cell formats revealed the remarkably lower internalresistance in the pouch cell, although the Nyquist plots were almostidentical when expressing the resistances in Ω cm2 rather than in Ω.A full-coin cell and a small single-layer pouch cell with optimizedelectrode porosities (24 ± 1%) and N/P ratio (1.2) were constructedand tested to evaluate the influence of the cell format on the rateperformance and cycle life of MNO/NMC622 full cells 8. A reve–1 charge–discharge, reaching80% SOH after 720cycles and 70% SOH after 1080 cycles . The full-cell rate capabilityon charge is significantly better than in lithiation of the MNO/Lihalf-cells displayed in Full-coin cells displayed excellent cyclabilityat 200 mA g0 cycles 9a. Howev0 cycles 9b. This 0 cycles 9c,d. Mor3.612O33. Electrochemical tests were carried out in half- and full-coincells, MNO/Li and MNO/NMC622, respectively, as well as in three-electrodePAT cell and small single-layer pouch cell formats.In this work, we characterizedand tested lithium-ion half- and full cells based on a novel niobiumoxide anode material: a micron-sized, modified MoNb–1,thickness of 17 ± 1 μm) compared to electrodes with 30± 2%  and40 ± 2% porosity .Furthermore, anode porosity also affected the cyclability of MNO electrodesat 25 °C, and the most porous MNO electrode reached 80% SOH afteronly 374 cycles at 200 mA g–1, while the denserelectrode reached 600 cycles to 80% SOH. The difference between NMC622and MNO in sensitivity to porosity could be explained by their differencein electrode microstructure, as observed in the SEM images in First, resultsobtained from NMC622/Li half-coin cell testing were in line with theliterature and showed the excellent performance of this material interms of both high-rate performance and cyclability. The extent ofelectrode calendaring had no significant influence on neither therate performance nor the cycle life of NMC622 cathodes, although theinitial Coulombic efficiency and absolute gravimetric capacity couldbe increased significantly by reducing the electrode porosity. Onthe other hand, the rate performance of MNO/Li half-coin cells dependedstrongly on electrode porosity, with significantly better capacityretention at a high rate for electrodes that were calendared downto 24 ± 1% porosity . Lithiation rate performancewas also excellent although lower than in delithiation. However, thiswas resolved when the Li disk was replaced by an NMC622 cathode toform full cells. Indeed, in both coin and single-layer pouch MNO/NMC622full cells, asymmetric rate testing showed superior capacity retentionat high rates of both charge and discharge, with 84% retention ofcapacity at 2 A g–1 (wrt NMC) in full-coin cells.MNO/NMC622 cells could be charged from 0 to 92% capacity in as littleas 12 min and to 70% capacity in ∼120 s at 4 A g–1 (∼20 mA cm–2). However, while scaling upfrom a CR2032 coin cell to a 30 × 30 mm2 single-layerpouch cell resulted in materially lower impedance and internal resistances,it also led to accelerated capacity fade as 350 cycles correspondedto 90% SOH in coin cell compared to 70% SOH in pouch cell. This islikely due to the electrolyte quantity and degradation reactions,with less excess of electrolyte in the pouch cell due to excess packagingvolume and surface area of separator in pouch cells compared withtight coin cells (folded separator in pouch cell result in more than3 times more separator-to-electrode surface ratio than in coin cell),as well as a much more accurate electrolyte deposition method in coincells than in pouch cells. This, however, needs further investigation.Full-coin cells with various N/P ratios were also cycled at 25 °Cat ∼1C/1C and 1C/5C charge and discharge rates , showing excellentcyclability at 200 mA g–1 but accelerated capacityfade with the five times faster discharge rate, with a significantimprovement in cycle life with an N/P ratio of 1.2 compared with thatof 0.9–1.1.MNO/Li half-coin cells using a 24% porous electrode exhibited outstandingrate performance in delithiation with as much as 87% capacity retentionat 10C and 63% at 20C , on the rate performance and calendarlife of MNO should also be characterized. It would be ideal to furtherinvestigate full cells in single- and multilayer pouch cell configurationsto better understand the various implications of these scaled-up cellgeometries on the electrochemical behavior of niobate anodes. Finally,it would be extremely interesting to investigate how switching fromthe PVDF/NMP system to a more sustainable and cost-effective aqueousbinder system would impact the electrochemical performance of MNOelectrodes.Future works need to focus on the effectof the electrolyte uponcycling, particularly the electrolyte concentration, degradation reactions,and quantity. The increased electrolyte ratio in the coin cell likelyimproved cycled life when compared to the pouch cell, and optimizationof the pouch cell design needs further investigation. In addition,the improved understanding of the electrochemical diffusion kineticsand thermodynamics of this MNO material and electrodes is requiredfor further optimization. The impact of the cycling temperature, aswell as the influence of higher mass loadings (areal capacity of 2–4mAh cm46 in 50:50 EC/DEC electrolyte. Charge rates of 2 min to 70%capacity were observed and over 1000 cycles to 70% of initial capacityat 1C rate of charge and discharge.To conclude, a high-ratemolybdenum niobium oxide vs NMC622 infull-cell configuration is demonstrated for the first time at coincell and single-layer pouch scale. Through careful electrode and celldesign optimization and reduction of internal resistances of cellformats, improved cycle life and rate capabilities are observed. Porosity,in particular, had a significant impact on the rate performance ofMNO half-cells, and performance was much improved upon electrode calendaring,as the micron size of this MNO powder resulted in easier calendaringcompared to competing anodes such as nanosized LTO. Regarding thefull-cell design and N/P ratio, a higher specific capacity as wellas improved rate and cycling performance were obtained in this workwith an N/P ratio of 1.2 for MNO/NMC622 using a standard 1 M LiPF


Acinetobacter baumannii infection is becoming a thorny health problem in hospitals. However, there are no licensed vaccines against A. baumannii. Acinetobacter trimeric autotransporter (Ata) is an important known virulence factor located on the outer membrane of bacteria. Herein, we carried out a series of experiments to test the immunogenicity of a short C-terminal extracellular region of Ata  in a murine model. The short peptide Ataα was fused with the cholera toxin B subunit (CTB), which has been reported to have immunoadjuvant activity. The fusion protein showed no inflammation and organ damages, and have the ability to elicit both Th1 and Th2 immune responses in mice. The bactericidal activities against A. baumannii and prophylactic effects of the fusion protein were further evidenced by a significant reduction in the bacterial load in the organs and blood. In addition, the candidate vaccine could provide broad protection against lethal challenges with a variety of A. baumannii strains. Moreover, when CpG was added on the basis of aluminum adjuvant, the immune response, especially cellular immunity, could be further strengthened. Overall, these results revealed that the Ataα is a promising vaccine target against A. baumannii infection.With the emergence of multidrug-resistant strains,  Acinetobacter baumannii, a Gram-negative bacterium, is an opportunistic pathogen that usually causes nosocomial infections   , 21 impeAcinetobacter trimeric autotransporter (Ata) of A. baumannii ATCC 17978, belongs to the trimeric autotransporter adhesin superfamily and is secreted through a type V secretion system. Trimeric autotransporter adhesins act as key virulence factors in many Gram-negative bacteria; participate in adhesion, biofilm formation, immune evasion, angiogenesis, and cell death; and mediate adhesion to extracellular matrix proteins in a murine pneumonia model  are prone to interfere with the carrier resulting in protein misfolding and structural instability. Therefore, a shorter epitope is beneficial for the further design of the vaccine.ia model , 23. Ata strains , 24. Furaumannii . This evThe B subunit of cholera toxin (CTB), which forms a pentameric ring structure, is widely used as a delivery vehicle and adjuvant for various antigens –30. StudA. baumannii infection.In this study, we used only 39 amino acids of Ata as an antigen and evaluated its immunogenicity by coupling with CTB. After confirming its safety, a series of animal experiments were carried out, and they indicated that both Th1 and Th2 immune responses were induced and efficient protective effects were demonstrated through lethal and non-lethal infection models. In particular, we found that when CpG was added with aluminum adjuvant, both cellular immunity and humoral immunity could be further strengthened, among which the cellular immunity response was more enhanced. Overall, our results reveal that the candidate antigen, prepared based on 39 amino acids of Ata, is a promising protective target for vaccination against Escherichia coli DH5α was used to clone plasmids, and BL21(DE3) was used for the expression of the recombinant protein CTB-Ataα. A. baumannii ATCC 17978 was purchased from ATCC, and the clinical isolates XH733 and MDR-ZJ06 were kindly provided by professor Yunsong Yu . All of the E. coli and A. baumannii strains were grown in Luria–Bertani broth or on solid medium containing 1.5% agar. The Ataα peptide was fused to the C-terminus of CTB (GenBank: X76390.1) with a flexible linker (GGSG). The full-length CTB-Ataα coding sequence was codon-optimized, synthesized by Sangon Biotech (Shanghai) Co., Ltd., and inserted into pET30a between NdeI and XhoI. pGEX4T-ata was constructed to produce GST-Ataα, which was used in the ELISA assay.E. coli BL21(DE3)/pET30a-CTB-Ataα cells were cultured in a shake flask at 37°C to an OD600 of 0.6. CTB-Ataα expression was induced by 0.5 mM IPTG at 30°C for 12 h. Cells were collected by centrifugation, resuspended by 20 mM Tris-HCl (pH 8.5), and disrupted through a homogenizer with four passes at 700 bar. The CTB-Ataα inclusion bodies were solubilized by lysis buffer (8 M urea and 5 mM DTT), diluted with refolding buffer (20 mM Tris (pH 8.5), 1 mM GSSG, and 1 mM GSH), and purified by anion-exchange chromatography and size-exclusion chromatography. The recombinant CTB-Ataα was analyzed by RP-HPLC  with a gradient of 0 to 100% elution buffer (95% acetonitrile 0.1% TFA) in 40 min and SEC-HPLC  with PBS.2SO4 to stop the reaction, and the dual-wavelength detection was performed at 450 nm and 630 nm.A 96-well plate was coated with 100 μL GM1 solution (2 μg/mL) overnight at 4°C. After washing three times, 300 μL of 5% skim milk was added to each well. After incubation at 37°C for 1 h and washing three times again, samples with different dilutions from 3.3 nM were added and incubated at 37°C for 1 h. After another washing step, 100 μL of anti-CTB antibody  was added to the plate, followed by incubation at 37°C for another hour. Then, the plate was washed again three times, 100 μL of HRP-labeled goat anti-rabbit antibody  was added, and the plate was incubated at 37°C for 1 h. At last, after washing the plate three times, 100 μL of TMB solution  was added to the plate, followed by adding 50 μL of 2 M HSix-week-old female BALB/c mice (SPF) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All of the animal experiments and procedures were performed in accordance with the guidelines of the Academy of Military Medical Sciences Institutional Animal Care and Use Committee . Mice were randomly grouped and for the evaluation of immune effects, and mice were immunized three times at an interval of 2 weeks. Two weeks after the second and third immunizations, blood was collected and the serum was separated for further analysis.α  overnight at 4°C, washed three times with PBST (PBS with 0.05% Tween 20), and blocked with 300 μL of 5% skim milk in PBST at 37°C for 2 h. Next, the wells were incubated with 100 μL 3-fold serially diluted serum from 1:50 at 37°C for 1 h. After another washing step, 100 μL of HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b  was added to each well, and plates were incubated at 37°C for 1 h, followed by washing five times with PBST, and then 100 μL of TMB solution  was added to each well. The reactions were stopped with 50 μL of 2 M H2SO4, and the dual-wavelength detection was performed at 450 nm and 630 nm.First, 96-well plates were coated with GST-Ata5 cells/mL) were differentiated in RPMI1640 containing 0.8% N N-dimethylformamide  for 4 days. HL60 cells (4 × 105 cells per well), A. baumannii ATCC 17978 (1 × 103 CFUs per well), and complement  were added into the wells along with the heat-treated mouse serum (56°C for 30 min for the inactivation of endogenous complement components). After 45 min incubation at 37°C in 5% CO2, the microtiter plates were placed on ice for 20 min to terminate the reaction. Finally, the mixtures were diluted and plated in duplicate on LB plates for bacterial counting. Serum killing rates were calculated by comparing the number of CFU with naïve serum samples.The opsonophagocytic assay was performed as previously described . BrieflyFor cell surface marker staining, draining lymph nodes (dLNs) of each mouse were individually collected and triturated into a single cell suspension. Then the cells were stained with different combinations of flow cytometry antibodies for 30 min at 4°C, which included APC-conjugated anti-mouse CD3 , eFluor450-conjugated anti-mouse CD4 , PE-conjugated anti-mouse CD8a , APC-conjugated anti-mouse B220 , Pacific Blue-conjugated anti-mouse GL-7 , PE-conjugated anti-mouse CD95 , FITC-conjugated anti-mouse CD4 , PE-conjugated anti-mouse PD-1 , and Brilliant Violet 421-conjugated anti-mouse CXCR5 . After washing with staining buffer , the cells were dispersed in 500 μL of staining buffer  and analyzed by flow cytometry .A. baumannii ATCC 17978 was cultured at 37°C to an OD600 of about 2.0 and then diluted with saline to approximately 2.0 × 107 CFU/200 µL. At 12 h after intraperitoneal inoculation, blood (10 µL) was collected and mixed with saline (990 µL), and organs (spleen and lungs) were removed, homogenized with saline (1 mL), and then collected in 2.0-mL microcentrifuge tubes as initial samples. After sample was placed at 4°C for 15 min, the supernatant of each initial sample was diluted and cultured on solid LB medium. The bacterial colonies were counted after culturing overnight at 37°C. Blood samples were collected, and the serum levels of IL-1β, TNF-α, and IL-6 were determined using ELISA kits (Dakewe). ATCC strains (ATCC 17978) and clinical isolates (MDR-ZJ06 and XH733) were used to evaluate the survival rates. Each mouse was intraperitoneally injected 14 days after the third immunization with 4.9 × 107 CFU/200 µL of A. baumannii ATCC 17978, 1.0 × 107 CFU/200 µL of MDR-ZJ06, and 4.5 × 107 CFU/200 µL of XH733. Survival rates were monitored continuously for 7 days.To evaluate the bacterial load, P < 0.05 .GraphPad Prism 8 was used for statistical analysis. The data are presented as mean ± SD. Data were analyzed by one-way ANOVA with Dunn’s multiple-comparison test for the multiple group comparison. Differences were considered statistically significant at A. baumannii strains, and its extracellular translocator domain forms an α-helical structure. The peptide consisting of 39 amino acids (Ataα), which used as a candidate antigen in this study, acts as a neck between the β-barrel membrane-inserted anchor and the surface-exposed passenger domains  of the sequences -inducible vector, pET30a-CTBAtaα, in which CTB was fused at the N-terminus of Ataα. The recombinant protein CTB-Ataα was highly expressed in the form of inclusion bodies  and a series of indicators were detected at different time points , alanine aminotransferase (ALT), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and alanine aminotransferase (ALP), were in the normal range  of A. baumannii strain ATCC 17978, the mice were dissected and the bacterial loads in the lung, spleen, and blood were detected. Significant reductions in bacterial load were observed in the lung, spleen, and blood in the two CTB-Ataα-immunized groups compared with the control, while there was no significant difference between the CTB-Ataα groups , Ataα+Al, or CTB-Ataα+Al, on days 0, 14, and 28. Blood was sampled on days 13, 27, and 41 to facilitate quantitation of antibodies against Ataα. ELISA-based measurement of the IgG titers showed that only CTB-Ataα-treated mice showed an Ataα-specific antibody response α, we next evaluated the protective effects of the vaccine against bacterial infection. All of the immunized mice were challenged intraperitoneally with a non-lethal dose of A. baumannii strain ATCC 17978 (2.0 × 107 CFU/mouse) 14 days after the third immunization. The blood of mice was collected 12 h after infection and the inflammatory factors  in serum were measured . To evaluate the immune response types after CpG addition, we analyzed the proportions of CD4+ and CD8+ T cells from draining lymph nodes (dLNs) 7 days after the secondary immunization according to the previous immunization protocol. We observed significant changes in the ratio of CD4+/CD8+ T cells between CTB-Ataα + Al and CTB-Ataα + Al + CpG mice  cells and B cells in the germinal center (GC) had increased when CpG was added 7 days after the last immunization  with CTB. After confirming the safety of CTB-Ataα, a series of animal experiments were performed, which proved that the vaccine can elicit an effective immune response and provide great protection against A. baumannii strains, including two clinical isolates, while Ataα alone has no effect. Therefore, our work not only provides a simple and promising A. baumannii antigenic peptide, but it also reveals two effective immune enhancement strategies (delivery carrier coupling and adjuvant mixing) for short peptide antigens.At present, sistance , which lA. baumannii clinical isolates, assists in the formation and maintenance of biofilm , and a conserved membrane anchoring domain, and are extremely diverse in amino acid length (from hundreds to thousands) , 35. Ata biofilm  and adhe biofilm , human c biofilm . Therefo biofilm , 25. In with CTB . Thus, bα with CTB, and a strong immune enhancement effect was observed. Thus, our next research direction is the development of more efficient delivery vectors to further improve the immune response to Ataα. In conclusion, the efficient vaccine delivery system is of great significance to improve the immune effects of antigens, especially weak immunogenic antigens.According to our results, the immune response is greatly improved when the peptide antigen is fused with CTB, suggesting that the carrier plays an important role in the enhancement of antigen immunogenicity. The same effect can be found in conjugate vaccines, which are produced by coupling the weakly immunogenic polysaccharide antigen with carrier protein –39. Polyα were enhanced when fused to CTB and mixed with CpG. Besides, MHC I-restricted antigen presentation plays a key role for CD8+ T cell activation. Previous studies have reported CTB has the capacity to enhance MHC I-restricted antigen presentation, due to GM1 binding-dependent cytoplasm transportation .Conceptualization and theoretical analysis, JW, HW, and LZ. Methodology, PS, XL, CP, JW, and ZL. Carried out experiments and data analysis, PS, XL, CP, and ZL. Writing—original draft preparation, PS and CP. Writing—review and editing, CP, JW, HW, and LZ. Funding acquisition, CP and LZ. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Natural Science Foundation of China (82171819 and U20A20361).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Leishmania. Clinically, the disease presents a broad spectrum of symptoms, the mechanisms underlying the development of lesions remaining to be fully elucidated. In the present work, we performed a correlation and multiparametric analysis to evaluate how parasite- and host-related aspects associate with each other, and with the different clinical manifestations of tegumentary leishmaniasis. This cross-sectional study involved 75 individuals from endemic areas of Brazil, grouped according to their symptoms. Leishmania species were determined by DNA sequencing, and parasite load, antibody production, and cytokine profile were evaluated by kDNA qPCR, ELISA, and flow cytometry. Data were analyzed using the Chi-square test, principal component analysis, canonical discriminant analysis, and correlation analysis. Among the recruited patients, 23 (31%) were asymptomatic, 34 (45%) had primary cutaneous leishmaniasis, 10 (13%) presented recurrent cutaneous leishmaniasis, and eight (11%) had mucocutaneous leishmaniasis. Leishmania species identified included L. amazonensis, L. braziliensis, and L. guyanensis. Surprisingly, no Leishmania RNA virus infection was detected in any sample. In summary, our work showed that parasite load, antibody production, and cytokine levels alone are not determinants for tegumentary leishmaniasis symptoms. However, the correlation analysis allowed us to observe how these factors are correlated to each other within the groups, which revealed a unique network for each clinical manifestation. Our work reinforces the complexity of tegumentary leishmaniasis outcomes - which are associated with multiple host and parasite-related elements and provides a holistic model of the disease.Tegumentary leishmaniasis is a tropical disease caused by protozoa of the genus  Leishmania. TL occurs in 92 countries, and it is the sixth most prevalent dermatological disease in the world (Tegumentary leishmaniasis (TL) is a vector-borne neglected tropical disease caused by species of the genus he world .TL shows a broad spectrum of clinical manifestations: cutaneous leishmaniasis (CL), disseminated leishmaniasis (DL), diffuse cutaneous leishmaniasis (DCL), and mucocutaneous leishmaniasis (MCL) . CL is tLeishmania strain, parasite load, and even the presence of the Leishmania RNA virus (LRV1) (The symptoms observed in TL are of complex etiology and the mechanisms involved in the development of lesions are not completely elucidated . At thiss (LRV1)  Besides,s (LRV1) .Several works have been carried out to investigate the role of specific clinical parameters over TL outcomes. Nevertheless, most studies fail to assess concurrent variables and don’t use multiparametric analysis. In recent years, the complex and multifactorial nature of TL pathophysiology has become increasingly clear, underscoring the need for experimental and statistical approaches that enable the investigation of how multiple aspects of the disease influence each other and how all variables taken together explain the broad spectrum of clinical manifestations observed in TL.Leishmania species, parasite load, and LRV1 presence), and host elements  correlate with each other and how this correlation is associated to the severity of the symptoms which is represented by different clinical manifestations.The present study aims to shed light into the TL pathophysiology, investigating how different parasite  primary CL, individuals whose first lesions were detected at the time of recruitment; (3) recurrent CL, defined as patients who developed TL after previous adequate treatment and lesion healing, and; (4) MCL, patients with lesions in mucosal areas. HIV+ individuals, patients younger than 18 years, and indigenous subjects were excluded.The study population consisted of 30 patients from Corte de Pedra , and 45 participants treated at the Dermatology Ambulatory of the University Hospital of Brasília (UHB) . All patients lived in TL endemic areas. Blood samples were collected from all patients and biopsies were collected from 51 individuals showing lesions during the medical appointment. Patients were classified as TL positive if serological or molecular tests were positive, and grouped into four categories, according to clinical manifestations: (1) Leishmania antigen prepared in the Laboratory of Immunology of the University Hospital of Salvador, Salvador, Bahia, Brazil, using L. amazonensis (strain MHOM/BR/86/BA125) promastigotes, at a concentration of 5x106 promastigotes per ml, equivalent to 250 µg ml−1 antigen. An induration of ≥5 mm was considered positive  for posterior RNA and DNA extraction, ELISA, and cytokine quantification.Blood samples were collected in tubes containing either EDTA or clot activator and separator gel to obtain serum. Skin biopsies were taken from the borders of active lesions, using a 4-mm diameter punch, after the application of local anesthetic. Blood, serum and biopsy samples were immediately stored in RNABiopur Mini Spin Plus Kit . Samples were quantified in a NanoVue Plus  and DNA quality was evaluated by PCR targeting the β-actin gene using the following primers: 5’- ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3’ and 5’-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3’.DNA was extracted from blood and skin biopsies using the Biopur Mini Spin Virus RNA Kit . The resultant viral RNA samples were quantified in a NanoDrop . cDNA synthesis was performed using the SuperScript ™ IV First-Strand cDNA Synthesis Reaction Kit , and quantitative reverse transcription PCR (RT-qPCR) was performed using the following primers for the ORF1 region of the LRV1 genome: 5’- ATG CCT AAG AGT TTG GAT TCG -3’ and 5’ ACA ACC AGA CGA TTG CTG TG - 3’. The reactions were performed in a final volume of 20 µL, consisting of 1x Universal SYBR Green PCR master mix , 10 µM of each primer and 2 μL of cDNA. Reaction conditions were as follows: 95°C for 10 minutes and 40 cycles at 94°C for 15 s, 53°C for 40 s and 72°C for 10 s. The melting curve was standardized with denaturation of 95°C for 15 s, followed by 60°C for 60 s and 95°C for 15 s. The reactions were carried out in an Applied Biosystems™ QuantStudio 3 . L. guyanensis LRV1+ (MHOM/BR/1989/IM35) cDNA was used as positive control, and L. infantum (MCER/BR/79/M6445) cDNA was used as negative control sample in all assays. The PCR reaction mixture containing water in place of template (blank) was also used as contamination control in each assay.Viral RNA was extracted from blood and skin biopsies using the Leishmania kDNA minicircle to determine parasite load. The primer sequences were as follows: 5’-GGC CCA CTA TAT TAC ACC AAC CCC-3’ and 5’-GGG GTA GGG GCG TTC TGC GAA-3’. The reactions were performed in a final volume of 20 µL, consisting of 1x Universal SYBR Green PCR master mix , 0.2 µM of each primer and 50 ng of genomic DNA. The reaction conditions were 94°C for 12 minutes, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. The melting curve was processed with 95°C for 5 s, followed by 50°C for 15 s and 95°C for 5 s. All qPCR assays included positive (L. braziliensis DNA) (MHOM/BR/2000/LTCP13396) and negative controls (blank and non-infected HEK cell DNA). To determine the number of parasites, a standard curve was built using serial dilution of L. braziliensis DNA (5 x 102 to 5 x 10-3 ng/µL). The reactions were carried out in an Applied Biosystems™ QuantStudio 3 . The parasite load was calculated as equivalent parasites per 50 ng of DNA.Quantitative PCR (qPCR) was performed using primers to Leishmania IgG was detected in serum samples by ELISA. The assays were run in microplate wells sensitized with specific parasite antigens (0.1 μg/well of L. braziliensis soluble antigen) and incubated overnight in a humidified atmosphere at 37°C. Then, 150 μL of 1X Milk-PBS  was added. Serum samples were diluted 1:100 using 1X Milk-PBS  and added to the wells. After 2 h incubation at 37 °C in a humid chamber, the plates were washed and 50 μL/well of the secondary antibody was added. A second incubation was then performed with 50 μL of 1:500 dilution of peroxidase-conjugated anti-human IgG . Each well then received 50 μl/well of revealing solution (pnPP-p-nitrophenol phosphate - diluted in Diethanolamine buffer pH 9.8) and, after a 12-minute incubation in the dark, absorbance reading was performed at 490 nm using a BioTeK®-Synergy HT spectrophotometer. The following controls were used in all assays: two sera known to be positive for anti-Leishmania IgG, and ten sera from healthy individuals as negative controls. The cut-off values were determined using the mean value of the negative controls plus three standard deviations  of serum samples obtained from all 75 patients was determined using the CBA Human Th1/Th2/Th17 kit . Briefly, the reconstituted cytokine standards and the thawed serum samples were processed using the LSRFortessa™ BD cytometer, according to the manufacturer’s instructions. Three hundred events or more were acquired for each cytokine bead. Data were analyzed using the FCAP software 3.0 .2, 0.25 μM of each primer, 0.2 mM dNTPs and 2.5 units of Taq DNA Polymerase  in a final volume of 25 µL. All amplifications were carried out on a T-100 Thermal Cycler thermocycler  with an initial denaturation step at 95° C for 5 minutes, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s and a final extension step of 72°C for 5 minutes. Leishmania kDNA minicircle primers were not recommended for the identification of species, although it is the most sensitive target, the amplified sequence does not allow the differentiation of Leishmania species  and sequenced at the Genomic Molecular Engineering Institute . Sequences were edited using Geneious software (www.geneious.com) and analyzed using BLASTn algorithm (www.ncbi.nlm.nih.gov/BLAST).Conventional PCR (cPCR) was performed with LITS.R and L5.8S primers: 5’- TGA TAC CAC TTA TCG CAC TT-3’ and 5’- AAG TGC GAT AAG TGG TA-3’. The conditions were: 20 ng of DNA sample, 1x reaction buffer (20 mM Tris-HCl pH 8.4 and 50 mM KCl), 1 mM MgCl species . The amp® program . Statistical significance was assigned to p ≤ 0.05. Effect size achieved 0.39 and was calculated using GPower® software  and the following input parameters: 0.05 for α, 0.8 for power, 4 groups and sample size of 75 individuals. Hedges’g value was calculated using effect size calculator for T-Test on Social Science Statistics website (https://www.socscistatistics.com/effectsize/default3.aspx) and and obtained a mean Hedges’g value of 0.36.Qualitative dependent variables were submitted to the Chi-square test. Quantitative data were submitted to Shapiro-Wilk analysis and then, an one way ANOVA using Kruskal-Wallis test (PROC NPAR1WAY) was performed in order to verify differences in cytokines concentration between clinical manifestations. Pearson correlation analysis using PROC CORR, Principal Component Analysis using PROC PRINCOMP and canonical discriminant analysis using PROC CANDISC were used to evaluate quantitative data. All statistical analyzes were performed using the SASFrom the 75 patients investigated, 23 (31%) were classified as asymptomatic, 34 (45%) as primary CL, 10 (13%) as recurrent CL and 8 (11%) as MCL. There was no patient with MCL in Corte de Pedra and no asymptomatic individual was recruited at the UHB Table 1.Leishmania kDNA qPCR standard curve parameters were R2 = 0.970, slope= -3.228 and efficiency=104%,  and with MCL (1.96 ± 0.46 U/µL), when compared to asymptomatic (0.99 ± 0.30 U/µL) and primary CL patients (1.09 ± 0.20 U/µL).The correlation of clinical manifestations with parasitemia showed a different profile in the different patient cohorts. In Corte de Pedra only one blood sample, which was obtained from one asymptomatic individual, tested positive for qPCR. On the other hand, all patients with active lesions had positive results for skin biopsy qPCR for parasite detection. Surprisingly, though, the same patients did not show positive results for qPCR parasite detection in the blood. Considering the single blood sample that tested positive for qPCR, parasite load was estimated to be of 0.008 parasite equivalents per 50 ng of DNA (par. eq./50 ng of DNA). In skin biopsies, the mean number of par. eq./50 ng of DNA was of 196.4 for patients with primary CL, and 20.0 for individuals with recurrent CL   . Moreover, IFN-y was also directly correlated with IL-6 and IL-17A (p<0.001). The antibody titers had a greater effect on cytokine levels in patients from this group, presenting a positive correlation with IL-2, IL-4, and TNF (p<0.05) , while IL-6 was inversely related to IL-4 (p<0.05). It was also verified a positive correlation between TNF and IL-10 (p<0.05). As seen in all other clinical manifestations, once again IFN-y was directly related to IL17-A (p<0.01). The parasite load was correlated with the increase of IL-4 p<0.05). Of interest, the release of TNF and IL-10 were inversely associated with the IgG production (p<0.05),while a direct relation was observed for IL-2 (p<0.01), IL-6, and IFN-y (p<0.001) . The first two eigenvectors of PCA explained 45.8% of variation observed and showed a collaborative association between IL-17A and IFN-y as well as between IL-6, IL-10 and parasite load and, in another pictorial cluster, IL-4, IL-2 and TNF , as well as host-related factors  might contribute to the clinical manifestations of TL embodied here in four categories . These categories were used for all analysis and does not properly mean “disease severity” as for example, a patient could show a lot of skin lesions in any different severity ranges and does not show MCL.The largest number of recruited patients were men aged between 30 and 59 years. Such observation was expected since middle-aged men are usually involved in agricultural activities, close to areas with the presence of the vector. Besides, physiological factors such as hormonal differences may also contribute to this finding, as previously published .L. braziliensis. Nevertheless, in our hands, parasite burden could not be associated with clinical manifestations of TL, as reported by Pereira et al.  can be found in the article/The studies involving human participants were reviewed and approved by Ethical Committee of Research at Faculty of Medicine/UnB (CAAE: 53157115.5.0000.5558/2016 and CAAE: 56709316.3.0000.5558/2015). The patients/participants provided their written informed consent to participate in this study.BCC and NN conceived and designed the experiments. CMG, EN, JO, and NN collected clinical samples. ACR, BC, and TV carried out the experiments. BD performed statistical analysis. BC, JLC, LH, MH, NN and BD analyzed the data. BC, JC and NN wrote the manuscript. All authors read and approved the final manuscript.This study was financed by Foundation for Advancement of Science (FAPDF - N° 193.000.965/2015), The National Research Council, Ministry of Science and Technology (CNPq/MCT) and The Agency for Training Human Resources, Ministry of Education (CAPES/ME), Brazil.We thank Dr. Tatiana Karla Borges for her expertise and assistance with flow cytometry experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state  were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints , and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes  Neuroblastoma is the commonest extracranial malignancy of childhood and responsible for a disproportionate number of deaths from childhood cancer. Nearly 60% of neuroblastomas relapse in distant sites , most coMore recently, single cell capture techniques have allowed CTCs to be enriched from peripheral blood. However, as most CTC capture platforms employ affinity-binding methods, they are limited by low throughput, cell viability and an inherent selection bias –15. ThusIn this study, we enriched CTCs and DTCs that expressed neuroblastoma markers on immunohistochemistry and quantitative reverse transcription PCR (RT-qPCR). We identified distinct CTC and DTC expression signatures that distinguish neuroblastoma patients with bone marrow metastases at initial diagnosis, and that persist in patients with subsequent relapse.Following informed consent under an institutional review board-approved protocol (SHS/2016/2022), neuroblastoma patients treated at KK Women’s and Children’s Hospital were recruited at initial diagnosis. Tumor, venous blood and BMA samples were obtained at diagnosis, after induction chemotherapy following ANBL0032 protocol, and at relapse .® tubes  or Cell-Free DNA BCT tubes  and processed on the same working day using the ClearCell® FX system , as described  and human gastric cancer cell line AGS (RRID: CVCL_0139) were maintained in RPMI-1640 (Hyclone) containing 10% fetal bovine serum . Kelly was maintained in RPMI-1640 (Hyclone) with HEPES containing 10% FBS. IMR32 was maintained in MEM/EBSS (Hyclone) containing 1% NEAA, 1% sodium pyruvate and 10% FBS. BE2C was maintained in DMEM/F12 (Hyclone) containing 10% FBS. All cells were obtained from America Type Culture Collection (ATCC) and cultured in a 37°C, 5% CO+/CD45- cell  and Hoechst+/CD45+ cell  in the capture chamber were ejected into the recovery and recycling port, respectively.Normal blood samples spiked with NLF cells were subjected to 1% paraformaldehyde (PFA) fixation and staining with Anti-Human CD45-PE  and Hoechst 33342  prior to loading into the microfluidic device. Having 10 single-cell capture chambers, the device was mounted on a microscope  for isolation of single CTCs based on immunofluorescence and morphology. Two syringe pumps  were used to maintain constant flow rates . HoechstEach single CTC was transferred to 0.2 ml PCR tube and subjected to lysis and RNA extraction according to the manufacturer’s specifications . 2.5 μM oligo (dT) primers and 0.5 mM dNTP Mix  were added into the lysed CTC sample, which was subsequently incubated at 65°C for 5 min and cooled on ice for at least 1 min. 1x first-strand buffer, 5 mM DTT, 10 U RNaseOUT Recombinant RNase Inhibitor, and 50 U SuperScript III RT  were used, made up to a final volume of 20 μl in nuclease-free water. The final product was incubated at 25°C for 5 min, 55°C for 60 min, and 85°C for 5 min for reverse transcription on a C1000TM Thermal Cycler .Prior to preamplification, 1 μM primer mix comprising PHOX2B, TH, GD2 synthase, β2 microglobulin, GAPDH and UBB gene primers were prepared by adding 1 μl of 100 μM forward gene primer and 1 μl of 100 μM reverse gene primer up to a final volume of 100 μl in nuclease-free water. 1x PCRBIO Ultra Mix , 100 nM of each primer, and 10 μl of the reverse-transcribed products were added to a final volume of 20 μl in nuclease-free water. The final product was incubated at 95°C for 10 min, followed by 25 cycles of 95°C for 20 sec, 60°C for 1 min and 72°C for 20 sec with an addition of 1 cycle of 72°C for 7 min on a C1000™ Thermal Cycler . The amplified products were purified prior to quantitation using Agencourt AMPure XP beads  according to the manufacturer’s recommendations.1x FastStart SYBR Green Master mix (Roche), 300 nM of forward and reverse gene primer (Integrated DNA Technologies), and 1 μl of eluted DNA product were added to a final volume of 10 μl in nuclease-free water. The final product was incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 20 sec, 55°C for 30 sec and 72°C for 20 sec with an addition of 1 cycle of 72°C for 7 min on a CFX96 Real-Time PCR Detection System . Two housekeeping genes  were used for normalization of expression data. Each experiment was performed in duplicate.GAPDH forward: CAAGCTCATTTCCTGGTATGACGAPDH reverse: CAGTGAGGGTCTCTCTCTTCCTUBB forward: GCTTTGTTGGGTGAGCTTGTUBB reverse: CGAAGATCTGCATTTTGACCT® 480 System . PHOX2B , tyrosine hydroxylase (TH) , GD2 synthase , and β2 microglobulin , primers were designed as previously described and purchased from Sigma Aldrich  . Two µl of cDNA in triplicate was used for real-time quantitative PCR (RT-qPCR) in 384-well plate  and performed on LightCyclerGermany) –31. PrimGermany) . The samCTC and DTC cytospots were fixed in 10% buffered formalin , and stained with PHOX2B (1:100)  and TH (1:3200)  with the BOND-III Automated IHC stainer , using manufacturers’ default automated staining protocol, as follows: pre-treatment unmasking with BOND epitope retrieval solution 2 (for PHOX2B) or 1 (for TH) , wash with absolute alcohol and Bond Wash Solution, staining with primary antibodies and detection using Bond™ Polymer Refine Detection . After staining, slides were dehydrated in absolute alcohol, cleared with xylene and mounted in DEPEX medium.Samples (1ng) were amplified using nCounter Low RNA Input Amplification Kit followed by multiplexed target enrichment according to manufacturer’s instruction and underwent 17-hour hybridization and post-hybridization high-sensitivity cleanup with the nanoString nCounter Prep Station , and automated counting using the nCounter Digital Analyzer. Additional probes were added to the PanCancer Pathway panel encoding for KIF1Bβ, PHOX2B, TH, GD2 synthase, CHD5, LIN28B, CASZ1, BARD1, LMO1, and TP73; and to the Progression panel for KIF1Bβ, PHOX2B, TH, GD2 synthase, CHD5, CHRNA3, PTPN14, GAP43, DCX and DDC. Samples were hybridized for 17 hours and underwent nanoString nCounter gene expression assay, according to manufacturer’s instructions . Genes with fewer than the recommended minimum background threshold of 20 probe counts were filtered out. Custom gene signatures comprising genes of the ADR and MES neuroblastoma super enhancer state found within the PanCancer panels were defined and used to calculate custom ADR and MES signature scores across both panels. Raw data was analyzed using nSolver™ software  under standard settings and normalized against manufacturer’s respective default housekeeping genes.Pathway scores were generated using default annotations of the NanoString nSolver Advanced Analysis v2.0.115. Pathway scores are calculated as the first principal component of the pathway genes’ normalized expression, with a higher score indicating increased expression in the majority of pathway genes.Continuous variables were compared between clinical subgroups using one-way ANOVA. Univariate statistical significance was defined as a P-value of <0.05.Differential expression analyses were performed using NanoString nSolver Advanced Analysis v2.0.115 and DESeq2 (RRID : SCR_000154) v.1.28.1 on R v.4.0.1 , and pat2 fold-change values against a reference human genome. FDR was calculated using Benjamini-Hochberg procedure and an adjusted p-value of 0.05 was considered significant.Statistical over-representation testing was performed using Protein ANalysis THrough Evolutionary Relationships annotations (PANTHER (RRID : SCR_004869), v.14) . Fisher NOL7, COG7, NUBP1, DDX50 and USP39) were selected based on their respective average gene stability measure ~M  and 12 genes from the PanCancer Progression panel (2.28–5.31 log2 fold change) that were significantly upregulated in the CTCs of patients with bone marrow metastases compared to those without  (2 fold change >6.6) and GPCR downstream signaling (log2 fold change 4.4), and PANTHER pathways related to PI3K signaling (log2 fold change 6.5) . Given their known function in tumor metastases  . Applyintastases , 38, the® FX device, and immunohistochemical and gene expression of DTCs and CTCs from the same patients were compared. In 4 patients with clinically-proven metastatic disease in their bone marrow aspirates , DTCs stained positive for neuroblastoma immunohistochemical markers PHOX2B and TH, and DTC-enriched fractions from BMAs also expressed elevated levels of PHOX2B and TH on RT-qPCR  were significantly upregulated in CTCs compared to DTCs, and 6 genes , EVI2A, IL1B, SERPINA1, TNFSF10) were significantly downregulated   clustered separately from the third relapse patient who had a more protracted disease course (case NB13) and the non-relapse patients, with significant upregulation in a unique set of genes 3. Thus,Five genes  remained persistently upregulated despite treatment , VIVA Foundation for Children with Cancer (VIVA-KKH Paediatric Brain and Solid Tumour Programme), and Children’s Cancer Foundation (Singapore Childhood Cancer Registry).The authors thank Candy Choo, Esther Hee, Zhang’e Choo, Khin Mar Cho, Josh Jie Hua Loh, Lorraine Yun Lin Yeo, Joyce Ching Mei Lam, Soh Hong Ang and Wen Di Lee.CL is a co-founder of Biolidics, Singapore.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RBP > ATPase > 40S_S3. Their expression pattern correlation analysis showed that the coefficients among each pair of RBP, ATPase, and 40S_S3 were larger than 0.96, revealing consistent and stable expression patterns under various treatments. Then, the expression of three pathogenesis-related (PR) genes and seven MYB genes in rhizomes during postharvest storage and subjected to pathogen infection was normalized by RBP, ATPase, 40S_S3, RBP and ATPase, ATPase and 40S-S3, and RBP and 40S-S3. The results showed that PR and MYB genes were induced by postharvest deterioration and pathogen infection. The correlation coefficients of RBP/ATPase, RBP/40S_S3, ATPase/40S_S3, RBP and ATPase/ATPase and 40S-S3, RBP and ATPase/RBP and 40S-S3, and ATPase and 40S-S3/RBP and 40S-S3 were 0.99, 0.96, 0.99, 0.99, 1.00, and 1.00, respectively, which confirmed the stability of these three reference genes in postharvest biology studies of ginger. In summary, this study identified appropriate reference genes for RT-qPCR in ginger and facilitated gene expression studies under biotic and abiotic stress conditions.Gene expression analysis largely improves our understanding of the molecular basis underpinning various plant biological processes. Stable reference genes play a foundational role during the normalization of gene expression levels. However, until now, there have been few reference genes suitable for ginger reverse transcription-quantitative PCR (RT-qPCR) research. In this study, 29 candidate reference genes with stable expression patterns across multiple ginger tissues and 13 commonly used reference genes were selected to design RT-qPCR primers. After amplification specificity validation, 32 candidates were selected and further evaluated by RT-qPCR using samples from various organs subjected to NaCl, drought, heat, waterlogging, and chilling stress. Four strategies, including delta-CT, BestKeeper, geNorm, and NormFinder, were used to rank the stability of reference genes, and the ranks produced by these four strategies were comprehensively evaluated by RefFinder to determine the final rank. Overall, the top three stability reference genes indicated by RefFinder were  Gene expression analysis is an important approach for improving our understanding of the molecular basis underpinning various plant biological processes . RT-qPCRZingiber officinale Roscoe) contains many bioactive constituents and has been widely used in food, as spices, as flavoring agents, and as medicine  genes and seven MYB genes, were analyzed as a case study to investigate the application of these newly developed reference genes in postharvest biology studies. This study lays the foundation for further gene expression analyses of ginger in future research.In this study, we used comprehensive expression profiling results from large-scale RNA-seq data to identify the most stably expressed genes from a variety of ginger tissues subjected to different treatments. Meanwhile, the expression levels of the selected genes under various treatments were further determined using RT-qPCR. Then, the expression stability of selected genes was first ranked by the four algorithms  and finally ranked by RefFinder by comprehensively considering the ranks produced by the four algorithms. Moreover, the expression patterns of genes related to the postharvest storage of ginger, such as three Zingiber officinale cultivars “Shandongdajiang,” “Yunnanluopingxiaohuangjiang,” and “Fengtoujiang,” which were collected from the North, Southwest, and Central China regions, were sown in 40 silica sand-filled pots . For greenhouse cultivation, the temperature was set to 28/25°C with a photoperiod of 14 h light/10 h dark. After 90 days of planting, healthy seedlings of uniform size (approximately 50 cm in height) were divided into five groups and subsequently subjected to different treatments: (a) water (control), (b) NaCl , (c) drought , (d) waterlogging (the pots were submerged 1∼2 cm under the water surface), (e) chilling (10°C), and (f) heat stress (ginger seedlings were treated with 40°C/32°C day/night temperature in a growth chamber) . Three chamber) . For eacTotal RNA was isolated from samples using the TRIzol reagent . The concentration and purity of RNA were evaluated using the NanoDrop Spectrophotometer , and their integrity and quality were also analyzed by performing 1.5% (w/v) agarose gel electrophoresis. The cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit with oligo-dT and 1 μg of total RNA  in a PTC-200 Thermal Cycler  .PRJNA592215) of four samples collected from the red stem inner portion, green stem, young yellow rhizome, and red stem surface section with three biological replications were produced and used to profile the expression patterns of ginger genes. The Fragments per Kilobase per Million Reads (FPKM), mean values (MV), and standard deviation (SD) of the individual genes were calculated according to 12 data sets. In order to select the considerably high and stable expression candidates, genes with MV values greater than 50 were selected. Then, according to the SD, these selected genes were ranked from the least to the highest SD. Furthermore, based on the functional annotations, 29 candidates with known functions were selected from the top 500 genes. The gene sequences of 13 commonly used reference genes, including PP2A2 (AT1G10430), TUBA4 (AT1G50010), LOS1 (AT1G56070), UBC35 (AT1G78870), SAM-Mtase (AT2G32170), ACT1 (AT2G37620), ACT2 (AT3G18780), TPL40B (AT3G52590), UBC10 (AT4G05320), UBC9 (AT4G27960), TUBB8 (AT5G23860), Clathrin (AT5G46630), and EF1α (AT5G60390), were collected and used as a query to perform BLASTn against ginger transcripts with E-value < 1e-10 (2(FPKM),’ xlab = ‘Gene’)]” ]” . To furt–1), 1 μL each of forward and reverse primers , and 5 μL ddH2O. The reaction program was set as follows: (1) denaturation at 94°C for 5 min; (2) then denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extensions at 72°C for 30 s, repeated for 35 cycles; (3) following a final extension at 72°C for 10 min  was used to design the gene-specific primers, and exon–exon fragments were specifically targeted while designing the RT-qPCR primers to avoid any possible mismatches and amplification of genomic DNA . To valir 10 min . Then, 81 was used to evaluate the stability of reference genes under various stresses  in ginger. Then, samples from cultivars “Shandongdajiang” and “Yunnanluopingxiaohuangjiang” were used to evaluate the stability of the top four stable reference genes. The pairs.panels function in the R package “psych” was used to perform the correlation analysis of the RT-qPCR results of the top four stable reference genes using three ginger cultivars. The standard curve was generated using a set of 10-fold dilutions of cDNA in a RT-qPCR assay according to the equation E = [10–(1/slope) − 1] × 100%. Then, based on the slope of the standard curve, the regression coefficient (R2) and the RT-qPCR efficiency (E) of primers were calculated  with the cDNA as a template on a CFX 96 Real-Time PCR system . The volume of the reaction mixture was 20 μL, which contained 0.4 μL of each of the forward and reverse primers (10 μM), 2.0 μL of diluted cDNA, 10 μL of SYBR Green I Master, and the addition of PCR-grade water up to 20 μL. The RT-qPCR reaction program was set as follows: (1) denaturation at 95°C for 30 s; (2) then denaturation at 95°C for 10 s, annealing at 60°C for 30 s, repeated for 40 cycles; and (3) a melting curve protocol (65–95°C with fluorescence measured every 0.5°C). Three biological replications were adopted for each sample. For RT-qPCR results, RefFinderlculated . While plculated .In order to verify the stability of the top three stable reference genes, two sets of samples were prepared. Uniform sized-young ginger rhizomes (cv. “Shandongdajiang” and “Yunnanluopingxiaohuangjiang”) with no visible injuries or vascular discolorations were selected for further analysis. For postharvest dehydration stress treatment, about 50 ginger rhizomes (cv. “Shandongdajiang”) were held in a growth chamber with 22 ± 2°C and 65% relative humidity conditions. Ginger rhizome samples were collected after 0, 7, 14, 21, and 28 days of postharvest dehydration stress.Fusarium solani strain was isolated from the surface of naturally decaying ginger rhizomes (cv. “Yunnanluopingxiaohuangjiang”) and was cultured [potato dextrose agar (PDA)] at 28°C. A spore suspension (1 × 108 spores mL–1) was prepared by washing 5-day-old sporulating cultures with sterile distilled water. For F. solani infection treatment, the different samples were treated as follows: ginger rhizomes were wounded with a sterilized borer (1.0 cm deep × 0.2 cm wide) at three points around the equator of each rhizome. Each wound was treated with 50 μL of spore suspension. The control rhizomes were wounded and treated with 50 μl sterile water. All treated rhizomes were incubated separately in a plastic box covered with preservative film at 28°C and 90 ± 5% relative humidity for 7 days. Rhizome tissues with a depth of 1.5 cm were collected at the cross area of healthy tissues and disease lesions using a sterilized cork borer. A sample annulus of 1.5–2.0 cm around the wound was collected after 7 days of treatment. All samples were frozen in liquid nitrogen immediately and stored at −80°C. The relative expression of investigated genes was calculated with the 2–ΔΔCq method  and standard deviation of the FPKM (SD) values. Genes were then ranked from the smallest to the largest according to SD, and 29 genes with MV greater than 50 were selected . To estice genes .40S_S15, acetyl-CoA, SERINC, D6PK, Dynamin, SF3B, ATPase, 40S_S3, MTN, NAD5, 26S1, RBP, CKIIα, RPB7, UCH2, β-APB, 26S2, CYP21, ILR1, CCR, RHC1A, 60S, Pex, CLTC, NAD-ME, TUBA4, LOS1, UBC35, ACT1, ACT2, TPL40B, and Clathrin) were retained for further stability validation. RNA-seq profiling data were further analyzed to illustrate the gene’s expression stability. As shown in the boxplot  ranged from 0.961 (40S_S3) to 0.974 (Dynamin). To further validate the stability of RBP, ATPase, 40S_S3, and Dynamin, their expression levels in the roots, rhizomes, and leaf tissues of another two ginger cultivars, “Shandongdajiang” and “Yunnanluopingxiaohuangjiang,” under different stress conditions were determined by RT-qPCR  genes, NbRbohA, WRKY7, and WRKY8, were determined by RBP, ATPase, and 40S-S3, respectively , while the order of stable genes was slightly different. RefFinder is a comprehensive tool that can be used to produce a final overall ranking of candidate reference genes according to the geometric mean of the weights of every gene generated by the NormFinder, BestKeeper, geNorm, and delta-CT programs  participate in many developmental and physiological processes, including modulating lignin biosynthesis and plant resistance against pathogens  genes  and seven MYBs . Similarly, seven MYB genes that were potentially functionally associated with lignin synthesis were upregulated in F. solani-inoculated rhizomes, regardless of which reference genes  were used  were used. For further validation, correlation analysis was performed using the expression data of three PR and seven MYB genes determined by RBP, ATPase, and 40S_S3  can be found in the article/GL, JY, and YZ designed the experiments. GL, JM, KX, and PY conducted the experiments and analyzed the data with supervision by FG, XC, QJ, and LL. JY wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gilliamella, while Lactobacillus Firm4 and Firm5 mainly altered amino acid metabolism pathways. We then analyzed the brain transcriptomes of bees mono-colonized with these three bacteria. These showed distinctive gene expression profiles, and genes related to olfactory functions and labor division were upregulated by Lactobacillus. Interestingly, differentially spliced genes in the brains of gnotobiotic bees largely overlapped with those of bees unresponsive to social stimuli. The differentially spliced genes were enriched in pathways involved in neural development and synaptic transmission. We showed that gut bacteria altered neurotransmitter levels in the brain. In particular, dopamine and serotonin, which show inhibitory effects on the sensory sensitivity of bees, were downregulated in bacteria-colonized bees. The proboscis extension response showed that a normal gut microbiota is essential for the taste-related behavior of honeybees, suggesting the contribution of potential interactions among different gut species to the host’s physiology. Our findings provide fundamental insights into the diverse functions of gut bacteria which likely contribute to honeybee neurological processes.The honeybee possesses a limited number of bacterial phylotypes that play essential roles in host metabolism, hormonal signaling, and feeding behavior. However, the contribution of individual gut members in shaping honeybee brain profiles remains unclear. By generating gnotobiotic bees which were mono-colonized by a single gut bacterium, we revealed that different species regulated specific modules of metabolites in the hemolymph. Circulating metabolites involved in carbohydrate and glycerophospholipid metabolism pathways were mostly regulated by IMPORTANCE The honeybee possesses a simple and host-restricted gut community that contributes to the metabolic health of its host, while the effects of bacterial symbionts on host neural functions remain elusive. We found that the colonization of specific bee gut bacteria regulates distinct circulating metabolites enriched in carbohydrate, amino acid, and glycerophospholipid metabolic pathways. The brains of bees colonized with different gut members display distinct transcriptomic profiles of genes crucial for bee behaviors and division of labor. Alternative splicing of genes related to disordered bee behaviors is also mediated. The presence of gut bacteria promotes sucrose sensitivity with major neurotransmitters being regulated in the brain. Our findings demonstrate how individual bee gut species affect host behaviors, highlighting the gut-brain connections important for honeybee neurobiological and physiological states. The honeybee has been widely used as a model animal for perception, cognition, and social behavior studies . As eusoThe bidirectional microbiota-gut-brain axis influences various complex aspects of behavior across the animal kingdom. The gut microbiota can modulate homeostasis and behavior in its animal host through chemical communication with the nervous system. Previous studies have suggested that the honeybee gut microbiota contributes to host brain physiology and behavior phenotypes. Specifically, the gut microbiota can alter endocrine signaling and thus sucrose sensitivity in honeybees , as wellGilliamella, Snodgrassella, Bifidobacterium, Lactobacillus Firm4 and Firm5, and Bartonella are the major bacterial phylotypes, and can be cultivated in the laboratory. Recently, the causal roles of honeybee gut bacteria in host nutrition, weight gain, and endocrine signaling have been extensively studied , Bifidobacterium asteroides (Bi), Snodgrassella alvi (Sn), Lactobacillus Firm4 (F4) and Firm5 (F5), and Bartonella apis (Ba)  . To assoGilliamella group. The metabolites that comprised the red and pink M were mainly involved in carbohydrate, glycerophospholipid, and amino acid metabolism , and different bee groups exhibited distinctive brain gene expression profiles . Specifically, the heat shock protein gene (loc410620) was upregulated in the Gi group  encoded in the A. mellifera genome were significantly upregulated in the F4 and F5 groups, while bees colonized with Gilliamella exhibited decreased expression of mrjp genes  with upregulated genes in the brain, while metabolites involved in glycerophospholipid metabolic pathways showed significantly negative correlations with those genes . In the F4 and F5 groups, fexofenadine, cholesteryl sulfate, and 2′-hydroxy-5′-methylacetophenone showed significantly positive correlations with upregulated genes in the brain, while homovanillic acid and gamma-caprolactone showed significantly negative correlations with those genes . These results suggest that upregulated genes in the brain were correlated with specific metabolites altered by different gut bacteria, indicating the potential roles of circulating metabolisms in host-bacteria interactions.We then performed Pearson correlation analysis to analyze the relationship between gene expression and the metabolites which are significantly regulated by Gi, F4, and F5 separately . In the Gi group, carbohydrate metabolites showed significantly positive correlations  and voltage-gated ion channel (Scn1a) showed different inclusion rates, and these are mostly regulated in the Gi groups compared to in the MF bees  is a taste-related behavior that is fundamental for olfactory discrimination and colony performance in honeybees . We thenGilliamella regulate host carbohydrate metabolism, which consistent with its capability to digest mono- and polysaccharides in the bee gut , Snodgrassella alvi (W6238G3), Bifidobacterium asteroides (W8113), Bartonella apis (B10834G6), Lactobacillus Firm4 (W8089), and Lactobacillus Firm5 (W8172) in 25% glycerol stock at −80°C were resuspended in 1 mL 1×PBS  at a final optical density at 600 nm (OD600) of 1, and then mixed with 1 mL sterilized sucrose solution  and 0.3 g sterilized pollen. For the CV group, 5-μL homogenates of freshly dissected hindguts of nurse bees from their hives of origin were mixed with 1 mL 1×PBS, 1 mL sterilized sucrose solution , and 0.3 g sterilized pollen. Then, MF, MC, and CV bees were provided sterilized sucrose (0.5 M) with sterile pollen and kept in an incubator  until day 7. Brains, guts, and hemolymph of bees were collected on day 7 for further analysis. All bees used for behavior experiments and tissue collection came from the same colony. Bees coming from the same cup cage for each group were considered a single replicate. Here, we used n = 3 replicates per group, 20 to 30 bees per replicate. Colonization levels of MF and MC bees were determined by CFU from dissected guts, as described by Kwong et al.  glycerol on ice for bacterial load quantification or collected into an empty 1.5-mL centrifuge tube for metagenomic sequencing and metabolomics analysis. All gut samples were frozen at −80°C until analysis. Honeybee brains were collected using a dissecting microscope (Canon). Individual bees were fixed on beeswax using two insect needles through the thorax. After removal of the head cuticle, the whole brain was placed on a glass slide and soaked in RNAlater  or proteinase inhibitor  for gene expression profiling, proteome analysis, and neurotransmitter concentration quantification. Then, the hypopharyngeal glands, salivary glands, three simple eyes, and two compound eyes were carefully removed. Dissected brains were kept frozen at −80°C. Hemolymph was collected using a 10-μL pipettor  from an incision above the median ocellus. A minimum of 50 μL hemolymph was collected from 10 bees into a 1.5-mL centrifuge tube. During the collection process, tubes were temporarily preserved on dry ice and subsequently stored at −80°C until analysis. Brain and hemolymph samples were collected from different individual bees following the same colonization step.g at 4°C for 10 min. The supernatant was diluted to a final concentration containing 53% methanol by liquid chromatography-mass spectrometry (LC-MS) grade water. The samples were then transferred to a fresh vial and centrifuged at 15,000 × g at 4°C for 20 min. Finally, the supernatant was injected into the LC-MS-MS system, and the analyses were performed using an ExionLC AD system (SCIEX) coupled with a QTRAP 6500+ mass spectrometer (SCIEX). Samples were injected onto a BEH C8 column (100 mm × 2.1 mm × 1.9 μm) using a 30-min linear gradient at a flow rate of 0.35 mL/min for the positive-polarity mode. Eluent A was 0.1% formic acid-water, and eluent B was 0.1% formic acid-acetonitrile. The solvent gradient was set as follows: 5% B, 1 min; 5 to 100% B, 24.0 min; 100% B, 28.0 min; 100 to 5% B, 28.1 min; 5% B, 30 min. The QTRAP 6500+ mass spectrometer was operated in positive-polarity mode with curtain gas at 35 lb/in2, collision gas at medium, ion-spray voltage at 5,500 V, temperature at 500°C, ion source gas at 1:55, and ion source gas at 2:55. For negative-ion mode, samples were injected onto aHSS T3 Column (100 mm × 2.1 mm) using a 25-min linear gradient at a flow rate of 0.35 mL/min. The solvent gradient was set as follows: 2% B, 1 min; 2 to 100% B, 18.0 min; 100% B, 22.0 min; 100 to 5% B, 22.1 min; 5% B, 25 min. The QTRAP 6500+ mass spectrometer was operated in negative polarity mode with curtain gas at 35 lb/in2, collision gas at medium, ion-spray voltage at −4,500 V, temperature at 500°C, ion source gas at 1:55, and ion source gas at 2:55.Hemolymph metabolites were determined by quasi-targeted metabolomics using high-pressure liquid chromatography (HPLC)-tandem mass spectrometry (MS-MS). First, 50 μL of hemolymph samples were mixed with 400 μL prechilled methanol by vortexing. All samples were incubated on ice for 5 min and then centrifuged at 15,000 × Detection of the experimental samples using MRM was based on the Novogene in-house database. Q3 (daughter) was used for the quantification. Q1 (parent ion), Q3, retention time, declustering potential, and collision energy were used for metabolite identification. Data files generated by HPLC-MS/MS were processed with SCIEX OS (version 1.4) to integrate and correct the peaks. A total of 326 compounds were identified in the hemolymph samples. Metabolomics data analysis was then performed using MetaboAnalyst 4.0 .P < 0.01. Metabolites in each module were annotated on the KEGG database and classified into major categories using MetaboAnalyst 4.0 . RNA degradation and contamination were monitored on 1% agarose gels, and the purity was checked with the NanoPhotometer spectrophotometer . RNA integrity was assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 System . RNA sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina , and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on the cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS , and the library preparations were then sequenced on an Illumina NovaSeq 6000 platform  and 150-bp paired-end reads were generated.The sequencing quality of individual samples was assessed using FastQC version 0.11.5 with default parameters. An index of the bee reference genome (Amel_HA version 3.1) was built using HISAT2 version 2.0.5 , and the2-fold change between the MC and MF groups. For significance testing, differential gene expression was determined by the Wald test. The resulting P values were corrected for multiple comparisons using the Benjamini-Hochberg FDR method  were aligned against protein sequences of the honeybee genome using BLASTP (P < 0.05).To find overlaps between the differentially expressed or spliced genes of bee brain and those from human autism spectrum disorders, a total of 3,531 high-quality reference protein sequences corresponding to 948 known autism risk genes  for targeted metabolomics analysis of GABA, dopamine, and serotonin. Six brain tissues from one treatment group were put into one tube and centrifuged . A 100-μL volume of acetonitrile containing 0.1% formic acid and 20 μL ultrapure water was added and the tubes were vortexed thoroughly. Metabolites were sonicated in an ice-water bath for 30 min, followed by subsiding at −20°C for 2 h. Supernatants were collected after centrifugation . Next, 20 μL of supernatant was transferred to a new vial followed by incubation for 30 min after the addition of 10 μL sodium carbonate solution (100 mM) and 10 μL 2% benzoyl chloride acetonitrile. Then, 1.6 μL internal standard and 20 μL 0.1% formic acid was added and the samples were centrifuged . A total of 40 μL of the supernatants was transferred to an auto-sampler vial for downstream UHPLC-MS/MS analysis. Serotonin hydrochloride, γ-aminobutyric acid, and dopamine hydrochloride  derivatized with benzoyl chloride  were used for the construction of the calibration standard curve. The internal standards mixtures  derivatized with benzoyl chloride-d5   of the cThe UHPLC separation was carried out using an ExionLC System , and samples were analyzed on the QTRAP 6500 LC-MS/MS system (AB SCIEX). Two μL of samples was directly injected onto an ACQUITY UPLC HSS T3 column . The column temperature was set at 40°C, and the auto-sampler temperature was set at 4°C. Chromatographic separation was achieved using a 0.30 mL/min flow rate and a linear gradient of 0% to 2% B in 2 min; 2 to 98% B in 9 min, 98% B for 2 min, and equilibration for 2 min. Solvent A was 0.1% formic acid and solvent B was acetonitrile. For all multiple-reaction monitoring (MRM) experiments, 6500 QTrap acquisition parameters were as follows: ion-spray voltage at 5,000 V, curtain gas setting of 35, nebulizer gas setting of 60, and temperature at 400°C. Raw data were analyzed using Skyline .Seven-day-old MF, MC, and CV bees were used to measure responses to different concentrations of sucrose solution as previously described, with some modifications . Before PRJNA783076.Raw sequence reads have been deposited in the NCBI SRA database under the BioProject accession no.