The high nano-dispersity of metal sulfides in CSC anode underlies the cocktail-like mediation effect, enabling the circumvention of intrinsic drawbacks of different metal sulfides. By utilizing ether-based electrolyte, the reversibility of metal sulfides is greatly improved, sustaining a long-life effectivity of cocktail-like mediation. As such, CSC effectively overcomes low-rate flaw of SnS2 and high-plateau demerit of CoS2, simultaneously realizes a high rate and a low plateau. In half-cells, CSC delivers an ultrahigh-rate capability of 327.6 mAh g−1anode at 20 A g−1, far outperforming those of monometallic sulfides  and their mixtures. Compared with CoS2 phase and SnS2/CoS2 mixture, CSC shows remarkably lowered average charge voltage up to ca. 0.62 V. As-assembled CSC//Na1.5VPO4.8F0.7 full-cell shows a good rate capability  and a high average discharge voltage up to 2.57 V, comparable to full-cells with alloy-type anodes. Kinetics analysis verifies that the cocktail-like mediation effect largely boosts the charge transfer and ionic diffusion in CSC, compared with single phase and mixed phases. Further mechanism study reveals that alternative and complementary electrochemical processes between nano-dispersed SnS2 and CoS2 phases are responsible for the lowered charge voltage of CSC. This electrolyte/structure-dependent cocktail-like mediation effect effectively enhances the practicability of metal sulfide anodes, which will boost the development of high-rate/-voltage sodium-ion full batteries.As promising anodes for sodium-ion batteries, metal sulfides ubiquitously suffer from low-rate and high-plateau issues, greatly hindering their application in full-cells. Herein, exemplifying carbon nanotubes (CNTs)-stringed metal sulfides superstructure (CSC) assembled by nano-dispersed SnSThe online version contains supplementary material available at 10.1007/s40820-021-00686-4. Howe_ENREF_6 –8. At th_ENREF_6 –12. Thus_ENREF_6 . Therefo−1) in ether-based electrolyte, holding a great promise in SIBs (Scheme +), largely lowering the discharge plateau of full-cells  is engineered to combine the merits of FMS- and AMS-type anode materials, aiming at simultaneously solving the dual-problems of poor rate capability/output-voltage characteristics  and a high average discharge voltage up to 2.57 V, comparable to full-cells with alloy-type anodes. Kinetics and mechanism studies reveal that the cocktail mediation effect largely boosts the charge transfer and ionic diffusion in CSC; along the diffusion direction of Na+ carriers, alternative and complementary electrochemical processes between different nano-dispersed metal sulfides  and Na+ carriers are responsible for the lowered average charge plateau of CSC. This exhibited cocktail-like mediation effect evidently improves the practicability of metal sulfide anodes, which will boost the development of high-rate/-voltage sodium-ion full batteries.Herein, CNTs-stringed metal sulfides superstructure anode assembled by nano-dispersed SnSs Scheme b-c. The xSyn−) and cobalt-based zeolitic imidazolate framework (ZIF-67) followed by annealing treatment  exist in solution and these are referred to collectively as ‘SnxSyn−’  [2+ in ZIF-67 reacts rapidly with SnxSyn− species, forming a unique superstructure comprised of nano-dispersed CoS2 and SnS2 phases. The overall reaction follows Eq.  . Within 2 (PDF No. 00-41-1471), SnS2 (PDF No. 00-23-0677), and carbon, verifying their presence in the CSC. Compared with standard phase, the reflection for the (0 0 1) plane of SnS2 registers a slight shift toward lower angles, implying an expanded interlayer spacing [2 results in the expanded interlayer spacing. Also, the relatively low annealing temperature (450 °C) is beneficial to retain the expanded interlayer spacing of SnS2. The content of carbon nanotubes in CSC is obtained by thermogravimetric analysis (TGA), which is ca. 3.75 wt% . By inductively coupled plasma-mass spectrometry (ICP-MS), the elemental content of CSC is analyzed, revealing that the mole ratio of Co/Sn/S is ca. 1.00/1.73/5.46 (Table S1). The corresponding mass content of CoS2 and SnS2 in the CSC is 26.95 and 69.30wt%, respectively  can be evaluated roughly according to the equation: CT-CSC = xCT-CoS2 + yCT-SnS2, where x and y is the percentage content of CoS2 and SnS2 in the CSC. The CT-CoS2 and CT-SnS2 are the theoretical capacity of CoS2 and SnS2, which is 872 and 1136 mAh g−1, respectively. Thus, CT-CSC = 0.2695 × 872 + 0.695 × 1136 = 1024.5 mAh g−1.As shown in Fig.  spacing . The exp2 in the CSC can be tuned to some extent by varying the concentration of thiostannate solution . Transmission electron microscope (TEM) image shows the typical radial morphology of the CSC  and SnS2 (0 0 1) lattice planes, verifying such nanosheets assembled by nano-dispersed SnS2 (red) and CoS2 (blue-green). The TEM-EDS line-scan profiles show matched peaks with Co, Sn, S elements, further suggesting the superstructure of shell co-assembled by SnS2 and CoS2 phases . Corresponding to SEM image of CSC (inset), the core of CSC shows an abundant microstructure, in which the pore (green) can be observed . As shown in Fig. 2 (0 0 1) and CoS2 (2 1 0), which accord with the corresponding selected area electron diffraction (SAED) pattern . Such results verify that the core of CSC is also assembled by nano-dispersed SnS2 and CoS2 phases. The CSC was further analyzed by X-ray photoelectron spectroscopy (XPS). As shown in Fig. S9, compared with commercial CoS2 sample, the high-resolution of XPS of Co 2p of CSC shows a ca. 0.45 eV shift toward higher binding energy. Moreover, the high-resolution of XPS of Sn 3d of CSC also appears a 0.61 eV shift toward higher binding energy. Such results imply the presence of chemical effect between CoS2 and SnS2 in CSC anodes [Figure CSC Fig. f. EnergyCSC Fig. g. The deFig. 2 2  0 and SnC anodes .2 and CoS2 powders with well-matched XRD patterns to standard phases are also tested . Compared with the CSC, the N2 isotherms of commercial SnS2 and CoS2 samples typically exhibit no evident hysteresis loop, whereby the corresponding pore diameter distributions display nonporous properties . After initial three scans at 0.1 mV s−1, mono-component metal sulfides (CoS2 and SnS2) and anodes composed of both compounds show gradually stabilized CV curves . The initial CV curve of the CSC anode shows three oxidation peaks, which are associated with SnS2 phase at 0.70–1.55 V and CoS2 phase at 1.70–2.10 V, respectively. The reduction peak at 1.60–1.80 V is correlated with the CoS2 phase, while the peaks at 0.50–1.10 V are linked to SnS2 and formation of solid electrolyte interphase . In subsequent scans, the reduction peak related to CoS2 gradually disappears, which could result from electrochemical activation of nano-dispersed SnS2 and CoS2 phases [2 (0.80–1.45 V) but remarkably lower than that of CoS2 (1.30–2.18 V) and SnS2/CoS2 mixture (1.25–2.15 V). Correspondingly, CSC anode displays an average charge voltage of ca. 1.30 V, which is close to that of SnS2 but lower than that of CoS2 (ca. 1.92 V)  with excellent long-life cyclability, specifically, 410.8 mAh g−1anode at 10 A g−1 over 500 cycles without decay  was conducted at 0.05 A g−1 for 0.5 h, followed by relaxation for 2 h. The typical GITT discharge profiles of CSC and SnS2/CoS2 mixture are shown in Fig. L is Na+ diffusion length (cm), τ is the current impulse time (s), t is relaxation time (s), ΔES is steady-state potential change (V), ΔEt is the instantaneous potential change (V) used to deduce IR drop [ca. 0.5 × 10–9 cm2 s−1, which is around twice that in half-cell with SnS2/CoS2 mixture . Compared with electrodes made from commercial samples of SnSfer Fig. a. Based  IR drop , 56. Cori) and scan rates (v) was assessed against the relationship i = avb, where a and b are adjustable constants [b-values are 0.98, 0.81, and 0.93, respectively, which implies the presence of a substantial pseudocapacitive contribution. The latter can be quantified through the equation i = k1v + k2v1/2, where k1v and k2v1/2 represent pseudocapacitive and ion-diffusion controlled contribution, respectively [−1, specifically, 64.0%, 67.0%, 71.6%, 78.3%, and 86.3%, respectively. Figure −1, in which the shaded region represents the pseudocapacitive contribution. This, together with the small charge transfer resistance and high Next, the pseudocapacitive contribution to charge storage in the Na//CSC half-cell was evaluated, on the basis that this component gives rise to faster charge transfer kinetics. CV curves at different rates are shown in Fig. onstants . As showectively –59. As s2 and CoS2), the samples after electrochemical activation exhibit dramatically different XRD patterns, indicating the occurrence of phase transition . For CoS2, the relevant electrochemical reactions are as follows: CoS2 + xNa+  + xe− → NaxCoS2, NaxCoS2 + (4 − x)Na+  + (4 − x)e− ↔ 2Na2S + Co [2, the corresponding electrochemical reactions are as follows: xNa+  + SnS2 + xe− → NaxSnS2, NaxSnS2 + (4-x)Na+  + (4-x)e− ↔ 2Na2S + Sn, Sn + yNa+  + ye− ↔ NaySn [2, while the peaks from the CoS2 diffraction pattern are difficult to discern. This could be associated with differences in crystallinity between products derived from SnS2 and CoS2. For investigating the mechanism of activated CSC, original CSC anodes were activated for at least 3 cycles to obtain phase-transformed materials. Corresponding to the discharge–charge-time curves in Fig. 2 and CoS2 [To investigate the mechanism that underpins the superior electrochemical behavior of CSC anodes, samples were at various states-of-(dis)charge characterized by ex situ XRD. The copper current collector in a Na//Cu half-cell discharged to 0.4 V shows only the intrinsic diffraction peaks for metallic copper, verifying no evident electrochemical reaction between Na and Cu collector in ether-based electrolyte . Compared with original samples  of Co. Selected area electron diffraction (SAED) patterns reveal the lattice plane (2 1 1) of Co, (5 1 3) and (1 3 1) of Na29.58Sn8 in the discharged product ’ of NaxSnS2 (with CoS2 and SnS2 standard phases as reference), respectively. The SAED pattern exhibits typical polycrystalline features, in which lattice plane (2 2 0) of NaxCoS2, (1 0 3) and (1 0 0) of NaxSnS2 can be identified , which act as active materials for subsequent discharge/charge cycles. Based on the analysis above, the exotic property mediation beyond rule-of-mixtures [2 and CoS2 phases in CSC is schematically illustrated in Fig. 2 and CoS2 phases in CSC will alternatively react with Na+ carriers, as schematically illustrated in Fig. 2 and CoS2 phases effectively shortens the ion diffusion path, which can kinetically boost electrochemical processes of both metal sulfide anodes. Due to intrinsic thermodynamics difference, the electrochemical competition is present between SnS2 and CoS2 phases. Also, it does not exclude one of the two phases could show local kinetic merit owing to the diameter difference between them. Thus, in the CSC anode, the alternative electrochemical reaction processes could coexist between the two phases. It enables complementary charge voltage plateau of different metal sulfide phases, resulting in lowered charge plateau of CSC anode.As shown in Fig. uct Fig. d. When cied Fig. f. Based mixtures , 36 (coc1.5VPO4.8F0.7 was employed to assemble CSC//Na1.5VPO4.8F0.7 full-cells. Synthesis of Na1.5VPO4.8F0.7 followed a modified literature method (Supporting Information), and yielded a micro-particle morphology with a well-matched XRD pattern with the standard phase  [1.5VPO4.8F0.7 cathode shows ca. 3.9 V discharge plateau with low electrochemical polarization, which is suitable for demonstrating the practicability of different anodes . The Na1.5VPO4.8F0.7 cathode delivers a good rate capability from 0.05 to 0.5 A g−1, showing a high reversible capacity of 124.1 mAh g−1electrode at 0.05 A/g . Over 350 cycles at 0.1 A g−1, the Na1.5VPO4.8F0.7 cathode shows a capacity of 106.4 mAh g−1electrode, corresponding to a low capacity decay of 0.02% per cycle . Figure 2//Na1.5VPO4.8F0.7, SnS2//Na1.5VPO4.8F0.7, and CSC//Na1.5VPO4.8F0.7 full-cells at 0.5 mV s−1. Evidently, the main redox peaks of CoS2//Na1.5VPO4.8F0.7 appear at 1.0–2.5 V, implying that its average discharge voltage is in the range. In contrast, the ranges of main redox peaks of SnS2//Na1.5VPO4.8F0.7 and CSC//Na1.5VPO4.8F0.7 full-cells are in 2.0–4.0 V, which imply a higher average discharge voltage than that of the former. Figure 1.5VPO4.8F0.7 cell, while the voltage is above 2 V, is ca. 61.7 mAh g−1electrode, which is 1.62 times that of CoS2//Na1.5VPO4.8F0.7. As displayed in Fig. 1.5VPO4.8F0.7 full-cells present an average discharge voltage of 2.57 V, which is close to that of SnS2//Na1.5VPO4.8F0.7 and ca. 0.62 V higher than that with CoS2 anode. The CSC anode confers a significantly higher average voltage during discharge of full-cells when compared with CoS2 cells. Compared with other full-cells reported previously, CSC//Na1.5VPO4.8F0.7 full-cells also show obvious merits in terms of discharge voltage and capacity  . Correspdensity.  Specific2 and CoS2 phases is engineered as a case study in ether-based electrolyte, simultaneously realizing high-rate and low-plateau properties. The high nano-dispersity of metal sulfides endows CSC anode with evident cocktail mediation effect similar to high-entropy materials, effectively circumventing intrinsic drawbacks of different metal sulfides. The utilized ether-based electrolyte greatly enhances the reversibility of metal sulfides, sustaining a long-life effectivity of cocktail-like mediation. In half-cells, CSC delivers an ultrahigh-rate capability of 327.6 mAh g−1anode at 20 A g−1 and remarkably lowered average charge voltage up to ca. 0.62 V, far outperforming CoS2 phase and SnS2/CoS2 mixture. The as-assembled CSC//Na1.5VPO4.8F0.7 full-cell shows a good rate capability  and a high average discharge voltage up to 2.57 V, comparable to full-cells with alloy-type anodes. Kinetics and mechanism studies further verify that the cocktail-like mediation effect largely boosts charge transfer and ionic diffusion in CSC, while alternative and complementary electrochemical processes between different nano-dispersed metal sulfides (SnS2 and CoS2) and Na+ carriers account for the lowered charge plateau of CSC. This work shows a unique electrolyte/structure-dependent cocktail-like mediation effect of metal sulfide anodes, which will boost the development of high-rate/-voltage sodium-ion full batteries.Despite with high-capacity and low-cost merits, the ubiquitous low-rate and high-plateau issues greatly lower the practicability of metal sulfide anodes in full-cells. Herein, enlightened by electrolyte/structure-dependent properties of metal sulfides, CSC anode assembled by nano-dispersed SnSSupplementary file1 (PDF 3447 kb)Below is the link to the electronic supplementary material.


Fusarium spp., is a major disease of field pea (Pisum sativum). The development of genetic resistance is the most promising approach to manage the disease, but no pea germplasm has been identified that is completely resistant to root rot. The aim of this study was to detect quantitative trait loci (QTL) conferring partial resistance to root rot and wilting, caused by five fungal isolates representing Fusarium solani, F. avenaceum, F. acuminatum, F. proliferatum, and F. graminearum. Evaluation of the root rot-tolerant cultivar “00-2067” and susceptible cultivar “Reward” was carried out with the five species. There was a significant difference (p < 0.001) between the mean root rot values of the two cultivars inoculated with the F. avenaceum (F4A) and F. graminearum (FG2) isolates. Therefore, in the QTL study, the F8 recombinant inbred line (RIL) population derived from “Reward” × “00-2067” was inoculated in the greenhouse (4 ×) with only F4A and FG2. The parents and F8 population were genotyped using 13.2K single nucleotide polymorphisms (SNPs) and 222 simple sequence repeat (SSR) markers. A significant genotypic effect (p < 0.05) and high heritability (79% to 92.1%) were observed for disease severity, vigor, and plant height following inoculation with F4A and FG2. Significant correlation coefficients were detected among and within all traits. This suggested that a high proportion of the genetic variance was transmitted from the parents to the progeny. However, no significant QTL (LOD > 3) were detected for the RILs inoculated with F4A. In the case of the RILs inoculated with FG2, 5 QTL for root rot severity and 3 QTL each for vigor and plant height were detected. The most stable QTL for plant height (Hgt-Ps3.1) was detected on Chrom5/LGIII. The two most stable QTL for partial resistance to FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in a 15.1-cM and 11.2-cM genomic region, respectively, on Chrom4/LGIV. The most stable QTL for vigor (Vig-Ps4.1) was found in the same region. Twenty-five major and moderate effect digenic epistatic interactions were detected. The identified region on chrom4/LGIV could be important for resistance breeding and marker development.Fusarium root rot, caused by a complex of  Fusarium spp., Aphanomyces euteiches, Pythium spp., Phytophthora spp., Rhizoctonia spp., Didymella spp. (formerly Mycosphaerella spp.), and Ascochyta spp.  in the major field pea cultivation regions in Canada and worldwide  includes the major pathogens causing Fusarium head blight (FHB) of wheat, barley, oats, and other small grain cereals , while four minor QTL were found on LGIII, IV, VI, and VII . These QTL explained 44.4–53.4% of the total variance for resistance  in a rough map generated with 14 SSRs. The QTL identified in most of these studies had very large confidence intervals due to the limited number of markers used. The low marker density makes it difficult to apply the identified markers in pea breeding.Genetic resistance offers the most promising way to control FRR and wilt in pea. However, there is no complete resistance to FRR in field pea, and only a few studies have reported QTL associated with partial resistance to this disease , 2019. Csistance . McPhee Fusarium spp. an important research objective.On the Canadian prairies, cereals are grown in tight rotations with canola, while the cultivation of field pea and other pulses is increasing . Boom-anFusarium spp. recovered from surveys for root rot in Alberta, Canada; (2) map the QTL associated with partial resistance to FRR using a segregating recombinant inbred line (RIL) pea population genotyped by Fusarium isolates; and (3) determine the stability of the QTL, accounting for disease severity, vigor, and plant height.Therefore, the objectives of this study were to: (1) evaluate the partially resistant pea cultivar “00-2067” for resistance to different 1 plants, which were then used to develop an F8 RIL population (One-hundred thirty-five RILs used by pulation  by the spulation .F. solani), F4A (F. avenaceum), F037 (F. acuminatum), F039 (F. proliferatum), and FG2 (F. graminearum), representing the Fusarium species most frequently recovered from symptomatic pea plants in root rot surveys in Alberta, were used to screen the parental cultivars “00-2067” and “Reward.” Briefly, to obtain the SSIs, surface-sterilized pieces of root tissue with disease lesions were placed on potato dextrose agar (PDA) and incubated at 25°C for 2–3 days and then transferred to the peptone-pentachloronitrobenzene (PCNB) medium for further selection. Mycelial tips of the fungal isolates were cut from selected colonies under a stereomicroscope , and the water agar (WA) procedure was used to obtain SSI (Five single-spore isolates (SSI), S4C , with the resulting suspension decanted into 250-ml Erlenmeyer flasks, containing a 100-ml autoclaved CMC medium . The flasks were covered in aluminum foil to block light and incubated on a rotary shaker at room temperature for 2 weeks. The suspension was centrifuged to collect conidia. The concentration of conidia was estimated with a hemocytometer and adjusted to a final concentration of 2 × 106 spores ml–1 with sterile-deionized water.Conidial suspensions of the five isolates were generated following Plastic cups (9-cm diameter and 10.5-cm depth) were filled with a sterilized potting mixture . In the greenhouse tests with each SSI , the roots of seven 5-day-old seedlings of the partially resistant parent “00-2067” and the susceptible parent “Reward” were immersed in the conidial suspension for 15 min and transplanted into the soilless mixture in a cup. An aliquot (1 ml) of conidial suspension was pipetted onto the roots before they were covered with the potting mix. The plants were kept in a greenhouse at 20–25°C/15–18°C day/night and a 16-h photoperiod with daily watering to maintain the potting mix at saturation conditions conducive for FRR development. Each experiment was repeated two times. After 3 weeks, disease severity was estimated for the parental cultivars on a scale of 0–4, where: 0 = completely healthy; 1 = brown or black spots on the main root; 2 = lesions covering the main root, but the rootlets still healthy; 3 = lesions spread to the entire root system; and 4 = root totally dead .Fusarium isolates was used as inoculum to screen the 135 RIL population and parents under greenhouse conditions. The inoculation and the maintenance of the plants were as described above. The pots were arranged in a randomized complete block design (RCBD) with four replicates. The greenhouse experiment was repeated four times. After 3 weeks, plant height (cm) was measured from the base of the stem to the top leaf. Plant vigor was evaluated as a measure of the wilting severity on a scale of 0–4  (The most virulent of the ly dead) . The plaP-value of the Shapiro–Wilk test was used to determine the normality for each variable using R  searches of the Pulse Crop Database1 to determine their possible functions.Linkage analysis was carried out using the filtered SNP and SSR markers, following F. graminearum (FG2). This was then repeated for the RILs inoculated with F. avenaceum (F4A). The analysis was conducted using means for the four single greenhouse experiments, LSM, and BLUPs of the total data by Composite Interval Mapping (CIM) using WinQTL Cartographer v2.5  from the RILs inoculated with her v2.5 . The prop < 0.05 . The LODFusarium isolate was indicated, followed by “Ps” = Pisum sativum, the first number = the pea linkage group (Fg-Ps4.1” represents the QTL for disease severity caused by F. graminearum located on linkage group IV of the pea genome. The chromosomes and pseudomolecules were named in accordance with Vig-Ps2.1) and plant height .The QTL names were defined according to the QTL detection studies by ge group , and theR2) was determined for each QTL. Furthermore, QTL with R2 > 10%, 5–10%, and <5% were arbitrarily classified as major-, moderate-, or minor-effect QTL, respectively. The origins of favorable alleles for individual traits were assigned to different parents, following R2 > 15%, 7.5–15%, and <7.5%, respectively. Epistatic-effect QTL were named with the prefix “E,” followed by the QTL name and a serial number .Quantitative trait loci identified in at least two of the four environments were classified as stable. The percentage of variation (p < 0.001) between the mean root rot values of the tolerant parent “00-2067” and the susceptible parent “Reward”, following inoculation with F. graminearum isolate FG2  (p < 0.001)  ,b. This < 0.001) ,b. The G< 0.001) ,b.t-test indicated a significant difference between the parents for disease severity in all four experiments. Frequency distribution  existed among the single experiments, pooled, and BLUPs for vigor  of “00-2067” were 234.5 ± 54.6 cm, 157.3 ± 50.6 cm, 155.5 ± 59.5 cm, 159.7 ± 6.7 cm, 176.7 ± 56.6 cm and 158.9 cm, respectively. For “Reward”, the plant heights were 177.5 ± 36.1 cm, 120.7 ± 31.5 cm, 129.5 ± 26. cm, 178.5 ± 34.6 cm, 151.5 ± 36.9 cm, and 100.8 cm, respectively. The frequency distribution of plant height of the RIL population for all six variables was not normal and slightly skewed  was found for plant height among the single experiments, pooled, and BLUPs data  on the parental cultivar “00-2067” inoculated with FG2 were 1.5 ± 0.7, 1.3 ± 0.6, 2 ± 1.2 and 1.5 ± 0.7 for the four greenhouse experiments, 1.6 ± 0.8 for LSM and 1.2 for the BLUPs. This was comparable with the estimated mean of 1.1 ± 0.4 obtained in the preliminary screening of the parents . On the ribution  indicateribution . High coribution . The difribution . The Sharibution . A signior vigor . The heiy skewed . A high UPs data .r < –0.90, p < 0.001), indicating the adverse effect of FG2 on root and aboveground growth. Plant height showed low to moderate correlation with disease severity  and vigor .Collectively, the correlation analysis among traits indicated that root rot caused by FG2 was negatively correlated with vigor and plant height. High correlation coefficients were detected between disease severity and vigor in all conditions   for “00-2067” were 4.0 ± 0.0, 3.7 ± 0.5, 3.5 ± 0.6, and 4 ± 0 for the four individual greenhouse experiments, 3.8 ± 0.5 for LSM for the pooled data and 4.0 for BULPs of the pooled greenhouse experiments (p < 0.001) (The estimated means (±SE) of disease severity for “00-2067” were 1.0 ± 0.0, 1.0 ± 0.8, 1.3 ± 0.5, 1.0 ± 0.0, 1.1 ± 0.4, and 1.0, while, for “Reward”, they were 3.3 ± 0.5, 3.3 ± 0.5, 3.5 ± 0.6, 3.0 ± 0.0, 3.3 ± 0.4, and 3.0 for DSGH1, DSGH2, DSGH3, DSGH4, LSM of pooled data, and BLUPs, respectively . These vribution , althougribution . The cor< 0.001) . Based oeriments . The esteriments . Additioeriments . The cor< 0.001) .t-test. The estimated plant height for “00-2067” for the individual experiments, LSM, and BLUP was 210.8 ± 128.2 cm, 174.5 ± 104.8 cm, 159.5 ± 13.5 cm, 210.0 ± 53.1 cm, 188.7 ± 82.8 cm, and 208.0 cm, respectively. The estimated plant height for “Reward” was 118.3 ± 100.2 cm, 193.5 ± 104.5 cm, 125. ± 98.9 cm, and 194.0 ± 38.4 cm for the individual experiments, 157.7 ± 88.5 cm for LSM and 133.1 cm for the BLUP. The frequency distribution for the RIL population was continuous and slightly skewed to the right. In addition, HGH2, HGH3, HGH Pooled, and HGH BLUPs followed a normal distribution   .r < –0.95, p < 0.001) and with plant height . Plant height was positively correlated with vigor .The correlation among the traits for plants inoculated with F4A was similar to that of plants inoculated with FG2. Disease severity was highly correlated with vigor  retained markers were as described by F. avenaceum isolate F4A. As such, no QTL likelihood profiles are shown. In the case of RILs inoculated with F. graminearum isolate FG2, a total of 11 QTL were detected for the three parameters and six variables  by the CIM using Win QTL Cartographer v2.5 (R2) values ranging from 4.05 to 36.35%  for disease severity, vigor, and plant height were detected for the RILs inoculated with her v2.5 ; Table 3F. graminearum isolate FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in the middle of Chrom4/LGIV at positions 59.3–74.4 cM and 74.0–85.2 cM, respectively  of Chrom5/LGIII and with flanking markers of AA5 and PsCam036163_21311_1095) and Fg-Ps3.2  explained 9.62–9.88% of the total variance (Fg-Ps5.1 [detected on the top part (0.9–9.2 cM) of Chrom3/LGV and flanked by the SNP markers PsCam059449_39630_321 and PsCam011153_7569_125] explained 14.2% of the total variance in greenhouse Experiment 1 (Fg-Ps5.1) had a negative additive effect, indicating that genomic regions for resistance in Fg-Ps4.1, Fg-Ps4.2, Fg-Ps3.1, and Fg-Ps3.2 originated from “00-2067,” while Fg-ps5.1 derived its resistance from “Reward”  > Vig-Ps3.2  > Vig-Ps3.1  both on Chrom5/LGIII  of the same chromosome or linkage group, was flanked by the SNP marker PsCam013763_9362_423 and the SSR marker AD270 . This QTL was located on the bottom segment of Chrom5/LGIII  of four greenhouse experiments (R2 = 7.04–20.04%). These QTL were located 142.3–168.0 cM on Chrom7/LGVII and were flanked by the SNP markers PsCam002756_2184_427 and PsCam045262_28962_162  (R2 = 13.63–14.06). The additive effect was negative for Hgt-Ps3.1, but positive for Hgt-Ps7.1 and Hgt-Ps7.2  , was detgt-Ps7.2 . This suTwo hundred eight putative digenic epistatic pairs were identified using all variables for disease severity, vigor, and plant height. These comprised 65 (12-24) for disease severity, 57 (10–21) for vigor, and 86 (15–28) for plant height. The 208 putative digenic interactions consisted of one major epistatic effect (PVE ≥ 15%), 13 moderate epistatic effects (7.5% ≤ PVE ≤ 15%), and 194 minor epistatic effects (PVE ≤ 7.5%). BLUPs for disease severity, vigor, and plant height detected 20, 18, and 21 putative digenic interactions, respectively. Within the 59 digenic interactions among BLUPs of all traits, epistatic analysis identified one major QTL pair, three moderate QTL pairs, and 55 minor QTL pairs . In contFg-Ps3.1, Fg-Ps3.2, and Vig-Ps3.1). The remaining 15 epistatic QTL were not related to any of the additive-effect QTL , followed by E.Hgt-Ps7 (R2 = 19.1%) and E.Hgt-Ps4 (R2 = 13.5%). The fourth was E.Hgt-Ps3 (R2 = 13.5%), which was linked to Fg-Ps3.2 and Vig-Ps3.1. Only E.Fg-Ps7 and E.Vig-Ps7 were linked to Fg-Ps3.1, showing moderate epistatic effect .Twenty-five digenic epistatic interactions with major and moderate effects were identified by 33 flanking markers, of which 10 epistatic-effect QTL with 14 flanking markers were linked to three additive-effect QTL  of all the Fusarium species isolated from 1,812 wheat, 71 durum, 596 barley, and 177 oat samples  only for the isolates representing F. avenaceum and F. graminearum. Therefore, the F8 RIL population derived from “Reward” × “00-2067” was screened with F4A (F. avenaceum) and FG2 (F. graminearum) for the detection of partial resistance to the two Fusarium species. The greenhouse experiments were repeated four times to determine the G × E interaction for all traits. In addition, the best linear unbiased predictors (BLUPs) and LSM were applied to minimize environmental effects  was comparable. R2 = 68–80%) identified by F. oxysporum were also located on Chrom4/LGIV, while three minor QTL (R2 = 2.8–5.4%) were located on Chrom5/LGIII. Despite identifying the same chromosomes, the similarity of the location of the QTL cannot be confirmed, given the different markers used in the two studies. However, the coincidence of the QTL is not surprising, since very few partially resistant pea cultivars are used in breeding programs across the world. F. avenaceum were located on Chrom7/LGVII. F. solani were located on Chrom6/LGII, while several minor QTL were located on Chrom5/LGIII, Chrom4/LGIV, Chrom6/II, and Chrom7/LGVII.In this study, 11 QTL accounting for disease severity, vigor, and plant height were identified. The major QTL for disease resistance were located on Chrom4/LGIV, while two minor QTL were detected on Chrom5/LGIII and one QTL on Chrom3/LGV. These QTL were coincident with the QTL detected for resistance to Aphanomyces root rot . The majSignificant QTL × QTL interactions were found between the minor QTL for disease severity and plant height but not for vigor. An interaction of the major QTL for disease severity, vigor, and height was not observed. F. graminearum. The use of high-density SNP markers and SSR anchor markers contributed to the construction of a fine linkage map and the identification of two stable QTL located on Chrom4/LGIV associated with partial resistance to F. graminearum. The identified QTL showed broad resistance to F. graminearum, F. solani, F. avenaceum, F. acuminatum, and F. proliferatum, as well as to A. euteiches. This study, together with our previous report (To the best of our knowledge, no genetic studies have been carried out to determine the genomic regions associated with the partial resistance of field pea to s report , suggestHeritability of each trait, marker information, linkage information, and QTL profiles are available in the main manuscript or as Fusarium spp., greenhouse screening of RIL population and parents for resistance to Fusarium root rot, disease rating, measurement of vigor and plant height, phenotypic data analysis, DNA extraction, PCR, genotyping with SSR markers, and writing of the manuscript. RF-A: supervision of molecular marker work, molecular data analysis, linkage map construction, QTL mapping, and writing of the manuscript. SS: principal investigator, grant application, supervision and provision of technical support to graduate student, and revision of the manuscript. K-FC: grant application, supervision, and provision of technical support for graduate students for RIL population screening in the greenhouse. S-FH: principal investigator, grant application, supervision and provision of technical support for graduate students for RIL population screening in the greenhouse, and revision of the manuscript. All authors contributed to the article and approved the submitted version.LW: inoculum preparation of 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.


RC48 contains the novel humanized anti-HER2 antibody hertuzumab conjugated to MMAE via a cleavable linker. A phase I study was initiated to evaluate the toxicity, MTD, PK, and antitumor activity of RC48 in patients with HER2-overexpressing locally advanced or metastatic solid carcinomas, particularly gastric cancer.This was a 2-part phase I study. Successive cohorts of patients received escalating doses of RC48 . Dose expansion proceeded at the dose of 2.0 mg/kg Q2W. The efficacy and safety set included all patients who received at least one dose of RC48.Fifty-seven patients were enrolled, the MTD was unavailable due to termination of 3.0 mg/kg cohort; 2.5 mg/kg Q2W was declared the RP2D. RC48 was well tolerated, the most frequent grade 3 or worse TRAEs included neutropenia (19.3%), leukopenia (17.5%), hypoesthesia (14.0%), and increased conjugated blood bilirubin (8.8%). Four deaths occurred during the whole study, three of which were believed to be related to RC48. Overall, ORR and DCR were 21.0% (12/57) and 49.1% (28/57). Notably, patients who were HER2 IHC2+/FISH- responded similarly to those who were IHC2+/FISH+ and IHC3+, with ORRs of 35.7% (5/14), 20% (2/10), and 13.6% (3/22), respectively. In patients who were pretreated with HER2-targeted drugs, RC48 also showed promising efficacy, with ORR of 15.0% (3/20) and DCR of 45.0% (9/20).RC48 was well tolerated and showed promising antitumor activity in HER2-positive solid tumors, including gastric cancer with HER2 IHC 2+/FISH- status.NCT02881190.The online version contains supplementary material available at 10.1007/s10120-021-01168-7. Approximately 20% of metastatic gastric cancer patients have HER2 overexpression or amplification. Although trastuzumab in combination with chemotherapy has become the standard of care in first-line treatment of HER2-positive metastatic gastric cancer , until rCurrently, novel agents and combinations are being actively investigated in HER2-positive gastric cancers. Antibody–drug conjugates (ADC), comprised of an antibody against the antigen of interest, a linker, and a payload cytotoxic agent, are designed for specific delivery of cytotoxic agents to malignant cells –12. RC48We initiated a dose escalation and expansion phase I study of single-agent of RC48. First, we aimed to assess the safety, tolerability, and PK of this drug in patients with HER2-positive solid tumors, particularly in HER2-positive gastric cancer. Second, based on the greater antitumor activity and bystander effect observed in preclinical models, RC48 is considered useful for treating tumors with heterogeneous antigen expression. Therefore, we aimed to investigate the clinical efficacy of RC48 in gastric cancer with low HER2 expression, including IHC 2+/FISH−.This was a phase I, open-label, multicenter, interventional two-part study, which included dose escalation and dose expansion parts. Patients were enrolled from six study sites in China.According to the “Guiding Principles for Clinical Trial Techniques of Anti-Tumor Drugs” and “Technical Guidelines for Non-Clinical Studies of Cytotoxic Anti-Cancer Drugs” issued by China, the initial dose of Phase I clinical trials was estimated as follows: the MTD obtained from rat multiple-dose toxicological study was 12 mg/kg, with the equivalent dose for human was 1.92 mg/kg and the safety factor was 10. The calculation of 1/10 gave the initial dose of 0.192 mg/kg. As a related animal, crab-eating macaque was given multiple doses (Q2W × 7) during toxicological study, providing an MTD of 10 mg/kg. The human equivalent dose was 3.2 mg/kg and the calculation of 1/6 gave the initial dose of 0.53 mg/kg. According to the FDA's “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, the starting dose was calculated to be 0.096 mg/kg, which was approximately equal to 0.1 mg/kg.t1/2) and PK results of ADC and free MMAE of the two subjects enrolled in the 0.1 mg/kg and 0.5 mg/kg single-dose group, the Q2W was selected as dosing frequency for further evaluation. Considering combining RC48 with other drugs in future trials, particularly immune checkpoint inhibitors, most of which were given Q3W, the investigation of Q3W dosing of RC48 was designed to add convenience for patients, which was a preliminary investigation with only three patients administered with 2.0 mg/kg Q3W in this trial.The following dose escalations were proposed: 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, and 4.0 mg/kg. To reduce the patient's exposure to an ineffective dosage, one subject was enrolled in the first-dose group (0.1 mg/kg) and the second-dose group (0.5 mg/kg), respectively. Starting with the third-dose group (1.0 mg/kg), the subjects were enrolled using the traditional 3 + 3 model. Based on the mean half-life (3 accompanied by a fever higher than 38.3 °C or a fever higher than 38 °C that persists for more than 1 h); grade 3 neutropenia with confirmed infections; grade 3 thrombocytopenia with a bleeding tendency; grade 4 thrombocytopenia; a non-hematologic toxicity of grade 3 or higher except for nausea, vomiting, and hair loss after supportive care; renal toxicity ≥ grade 3; neurotoxicity ≥ grade 2, with no response before the next medication; cardiac toxicity ≥ grade 2; and for patients with hepatic transaminases reaching grade 2 at baseline, hepatic transaminases increasing to a level ≥ 10 × ULN was considered a DLT.Dose-limiting toxicity (DLT) was defined as the following toxic reactions observed in the DLT evaluation window (observation period of days 1–21 after the first dose) that were believed to be related to RC48 treatment by the investigators, wherein the grading was based on the 5-level system of the CTCAE v4.0: grade 4 neutropenia lasting more than 3 days after G-CSF treatment; grade 4 neutropenia recurring after recovery to a normal level through G-CSF treatment; neutropenic fever . The MTD was defined as dosage below the dose with which DLT occurred in two or more out of six patients during the DLT evaluation window.The study was performed in accordance with the Declaration of Helsinki and the International Conference on Harmonisation Guidelines for Good Clinical Practice. Approval by an independent ethics committee or institutional review board was obtained before study initiation.Patients with incurable, locally advanced or metastatic solid cancers were eligible for inclusion if their tumors showed HER2 protein overexpression by IHC (3+ or 2+), regardless of whether FISH was positive or negative. However, FISH results were necessary in the dose expansion section of the study for efficacy analysis. Eligible patients were refractory to standard treatment, had no standard treatment, or for whom standard treatment was intolerable or unavailable. Trastuzumab combined with chemotherapy has been considered the standard regimen in first-line treatment. However, trastuzumab is not covered by medical insurance in some areas of China, and some patients could not afford it. On the other hand, there is no standard of care established for HER2-positive gastric cancer in later lines of treatment. Therefore, these patients were allowed to participate in this clinical trial to determine potential benefits.9/L; platelet ≥ 100*109/L; serum total bilirubin ≤ 1.5 times the upper limit of normal value[ULN]; for those without liver metastasis, ALT AST or ALP ≤ 2.5 ULN, for those with liver metastasis, ALT AST or ALP ≤ 5 ULN; serum creatinine was normal; INR ≤ 1.5 ULN, APTT ≤ 1.5 ULN).Eligible patients were with an Eastern Cooperation Oncology Group (ECOG) performance status of 0 or 1, had a left ventricular ejection fraction (LVEF) of at least 50%. There were measurable or evaluable lesions according to RECIST 1.1, and adequate bone marrow, liver, and kidney function  ≥ Grade 2. Patients who had uncontrolled primary or metastatic brain tumors were also excluded. Patients with the following treatments were permitted to be enrolled with adequate washout period: major surgery (≥ 4 weeks), radiotherapy (≥ 2 weeks), chemotherapy (including antibody drug therapy [≥ 3 weeks]).Full inclusion and exclusion criteria are listed in the study protocol. Written informed consent was obtained from all patients before study enrollment.Serial blood samples for the PK characterization of total antibody (TA), binding antibody (BA), and free MMAE (FM) were collected during cycles 1 and 4 at the following timepoints: predose, and 0.25, 1 (end of infusion), 1.5, 12, 24, 48, 72, 120, 168, 240, and 360 h after the start of infusion. The TA and BA concentrations in serum were measured using a validated mesoscale discovery (MSD) method and liquid chromatography-tandem mass spectrometry (LC–MS/MS) for detection of FM. Several additional blood samples were chosen to detect the conjugated MMAE (CM) using the same LC–MS/MS method from three patients who received multiple doses of RC48 at 2.5 mg/kg.Cmax) and time to Cmax (Tmax) were directly obtained from the measured plasma concentration. Other main PK parameters were calculated from the plasma concentration–time data using a noncompartmental analysis (NCA) method with WinNonlin 6.3  and included elimination half-life (t1/2), the area under the plasma concentration–time curve from zero to the last time (AUCt0−) and from zero to infinity (AUC0−Inf), apparent total body clearance (CL/F), and apparent volume of distribution (Vd/F).PK parameters such as maximum observed serum concentration  of RC48 in HER2-positive advanced solid tumors and to explore the recommended phase 2 dose (RP2D) in future studies. Secondary outcomes included assessment of pharmacokinetic (PK) characteristics and preliminary clinical efficacy of RC48 in patients with advanced HER2-positive solid tumors.All subjects were observed for any adverse events that occurred during the clinical study according to NCI-CTC AE (4.0), including clinical symptoms, signs of life, and abnormal in laboratory tests. Radiologic assessment was evaluated at baseline and every 2 cycles  or 3 cycles  of treatment. Tumors were assessed by CT or MRI of the chest, abdomen, and pelvis. RECIST v1.1 criteria were used to evaluate the objective response rate (ORR), disease control rate (DCR). Initial response evaluation as complete response (CR) or partial response(PR) need to be confirmed after 4 weeks.Descriptive statistics were mainly used because of the small sample size. Descriptive statistics for pharmacokinetic parameters  were provided for the list of drugs tested. The incidence of all adverse events (AE) was listed by treatment group. The adverse events were recorded using the terms of the investigator and a statistical description of MedDRA. All statistical analyses were performed using SAS®9.3.From December 22, 2015, to June 27, 2019, 68 patients were screened. Fifty-seven patients met the inclusion criteria and were sequentially enrolled into six dose groups. The most common tumor type among enrolled patients was gastric cancer. All 57 patients had measurable lesions at baseline. Baseline characteristics are summarized in Table Each of the 0.1 mg/kg and 0.5 mg/kg cohorts enrolled one patient, with no observed drug-related adverse events of Grade > 2. From 1.0 mg/kg dose cohort 3 + 3 dose escalation was used. No DLT occurred in 1.0 mg/kg and 2.0 mg/kg cohort. Three patients were enrolled in 2.5 mg/kg group and 1 DLT occurred due to Grade 4 neutropenia. After discussion with investigators and with approval by ethics committee, G-CSF was allowed from the 2.5 mg/kg group in order to observe safety of long-term therapy. After three patients were treated at the dose of 3.0 mg/kg, one DLT occurred. This patient died 13 days after the first dose of RC48. The cause of death was considered to be septic shock by investigators, possibly related to marrow toxicity of RC48. Considering there was no trend of improving efficacy after adding the dose to 3.0 mg/kg dose compared with 2.0 mg/kg and 2.5 mg/kg cohorts, and continuing enrolling patients in 3.0 mg/kg group may significantly increase the risk of subjects, after discussion the investigators decided to terminate the enrollment of the 3.0 mg/kg cohort and reported to institutional review board. The MTD was unavailable due to the termination of the 3.0 mg/kg cohort and 2.5 mg/kg was declared the RP2D. After discussion about PK, adverse events and efficacy of RC48 in the dose escalation part, 2.0 mg/kg Q2W was chosen as the dose used in the expansion part. Finally, the expansion cohort was expanded to a total of 38 patients. The process of dose escalation and expansion is summarized in Fig. N = 57).In the adverse event (AE) analysis, patients who received at least one dose of RC48 were analyzed altogether (N = 2) and thrombocytopenia (N = 1). DLT was observed in only the high-dose cohorts (2.5 mg/kg and 3.0 mg/kg) (Table S1), indicating DLT of RC48 was dose-dependent.A total of 94.7% (54/57) of patients had AEs in the first 21 days after treatment. Three (5.3%) patients had DLT-related AEs, including neutropenia (Types and incidence of related AEs in each cohort were listed in Table Treatment-related serious adverse events (SAEs) occurred in 17 patients (29.8%): all cases occurred in 2.0 mg/kg and 2.5 mg/kg cohort. The most common treatment-related SAE was fever (in 5.3% patients), incomplete ileus (3.5%), elevated ALT (3.5%), elevated AST (3.5%), and death (5.3%).Four deaths occurred during the whole study. One death was due to biliary tract infection, and two deaths were due to febrile neutropenia. Another patient developed vomiting of Grade 3 during the third cycle of treatment and died one day after hospitalization in another hospital. This death was considered related to tumor progression instead of the study drug by the investigator. Three of the four deaths were in the 2.0 mg/kg cohort, and one was in the 3.0 mg/kg cohort (febrile neutropenia).TRAEs resulting in dose reduction or interruption occurred in the 2.0 mg/kg (7.9% or 47.4%), 2.5 mg/kg (36.4% or 45.5%), and 3.0 mg/kg (33.3% or 66.7%) cohorts. Common AEs included neutropenia, leukopenia, hypoesthesia, fatigue, and elevated ALT/AST. Permanent treatment discontinuation due to TRAEs occurred in 31.6% and 9.1% of patients in the 2.0 mg/kg and 2.5 mg/kg cohorts, respectively. The most commonly reported AEs leading to permanent treatment discontinuation were hypoesthesia, fatigue, and death.N = 57). In all the 57 patients, including 47 gastric cancer patients, 4 urothelial cancer patients, and 6 other cancer patients. 21.1% (12/57) patients had an PR, 10 of which were confirmed. No complete responses were observed. DCR of the evaluable patients was 49.1% (28/57). Detailed efficacy was listed in Table The efficacy of RC48 was evaluated in FAS . Therefore, patients who received at least one dose of RC48 were analyzed altogether . 17.1% PR occurred in 2.0 mg/kg Q2W cohort and 27.3% in 2.5 mg/kg Q2W cohort.N = 48) are shown in Fig. Median PFS of 57 patients was 3.5 m (95% CI: 1.9–5.3). The efficacy and response duration of all patients who were available for the assessment of response , confirmed ORR was 17.0% (8/47), and the DCR was 46.8% (22/47). In the 2.0 mg/kg and 2.5 mg/kg cohorts, the ORR was 20.7% (6/29) and 27.3% (3/11), respectively. HER2 IHC 2+/FISH− gastric cancer patients were considered as a group that cannot benefit from trastuzumab in previous studies [In addition, there were two PR and two stable disease (SD) patients among the four urothelial patients, with a confirmed ORR of 50% and a DCR of 100%. There was no PR in the six patients of other cancer types.Cmax) almost at the end of the RC48 infusion. Cmax and AUC0−inf of TA were approximately 1- to 1.5-fold and 1- to 1.8-fold higher than those of BA within the dose ranging from 0.1 to 3.0 mg/kg . After reaching their Cmax, concentrations of the two analytes declined in a multi-exponential manner. TA and BA analytes showed similar decline profile in concentration . As a result, they exhibited approximately same half-lives while the generally shorter clearance were observed for TA  and decreased half-life (38.3 h) of FM at 3.0 mg/kg dose level were observed, compared with those  at 2.5 mg/kg dose levels. Additionally, the systemic exposures of FM was much lower than those of CM .Serum concentrations of FM were much lower than those of TA and BA following single dose at all does levels. Mean Cmax decreased by 30% and AUC0-t remained stable after multiple dose compared to those after single dose. For FM analyte, there were no remarkably changes for both Cmax and AUC0−inf between single and multiple doses (Table After multiple administration (Q2W) of RC48 at 2.5 mg/kg dose level, significantly elevated half-lives of TA(44.12 h), BA(103.05 h), and FM (81.26 h) were obtained, compared with those  of three analytes after single dose of RC48 at the same dose level. For both TA and BA, Cmax, AUCt0−, and AUC0−∞ of free MMAE in ADA-negative and positive patients were 5.81 ± 4.21 vs. 2.86 ± 0.83 (ng/mL), 820.83 ± 548.25 vs. 415.11 ± 125.33 (h*ng/mL), and 947.99 ± 701.28 vs. 430.94 ± 121.89 (h*ng/mL), respectively. Details about ADA and PK are shown in supplementary resources.Anti-drug antibody (ADA) was assessed in 56 patients(one patient had no post-treatment serum sample available). Of these 56 patients, 11(19.6%) had ADA response, 8 of the 11 were from 2.0 mg/kg dose cohort. Comparison of PK parameters between ADA positive and negative patients revealed that impact on PK only occurred in the single-dose cohort. Due to complex heterogeneity of HER2 expression in gastric cancer, there is no standard of care established for HER2 positive gastric cancer that has progressed after first-line treatment of trastuzumab. Antibody–drug conjugate (ADC) is one of the emerging treatment strategies in HER2-positive tumors in recent years. In May 2020, DS-8201, a HER2-directed antibody and topoisomerase inhibitor conjugate, received Breakthrough Therapy Designation (BTD) from the U.S. FDA for the treatment of patients with HER2 positive unresectable or metastatic gastric or GEJ adenocarcinoma who have received two or more prior regimens including trastuzumab based on the results of DESTINY-Gastric01 clinical trial . In our One of the purposes of the study was to conduct integrated analysis to characterize the PK and explore the exposure–response relationship of RC48. The PK characteristic of TA and BA showed that dose-proportional increases both in exposures and half-lives at doses of 0.1–3.0 mg/kg. The pharmacokinetic profiles of two analytes were similar, which may indicate that the ADC drug would be stable in serum. Furthermore, no evidences of accumulation in serum for TA and BA were observed after repeated dosing of ADC drug at 2.5 mg/kg Q2W.Cmax and AUC0−inf of FM compared to those of CM demonstrated that the majority of MMAE was conjugated with antibody. It was supposed that FA was released slowly from the ADC drug after the process of cellular uptake, lysosomal internalization and degradation. The apparent delay of FA in reaching its Cmax compared with CM also confirms the suppose as time is needed for the whole process. In addition, accumulation of FA in serum was not observed after multiple doses of ADC drug at 2.5 mg/kg Q2W.The PK characteristic of FM was also studied. The significant lower N = 11); thus, the efficacy of RC48 in the 2.5 mg/kg Q2W dose needs further validation in phase II trials.The safety profile of RC48 was consistent with what would be expected of a HER2-targeted antibody–drug conjugate that uses monomethyl auristatin E as its payload . The mosBased on previous studies, interstitial lung disease, also referred to as pneumonitis, is a rare but shared adverse event in several anti-HER2 drugs, including trastuzumab(9.9%), lapatinib (0.2%), T-DM1 (0.5%), DS-8201 (9.0%), and trastuzumab duocarmazine (7.7%) , 18. HowOne of the significant findings of this study is that RC48 might be effective in HER2 low expressed gastric cancers. In current clinical practice, these patients are classified and treated according to guidelines for HER2 negative gastric cancer. Researchers have found that over 40–60% percent of all gastric cancers express low levels of HER2 as IHC 2+/FISH− , but no This promising efficacy may be due to the unique characteristics of RC48 that differentiates it from other HER2-targeted drugs. These characteristics include Hertuzumab which had a higher affinity to HER2 than trastuzumab, and the VC linker which was designed for optimal stability in human plasma and efficient cleavage by human cathepsin B . TherefoThe researchers also saw signs of efficacy in the patients with other cancer types: PR occurred in 50% of patients with urothelial cancer, and DCR reached 100%. Phase II clinical trial is currently ongoing, and preliminary result has demonstrated a clinically meaningful ORR of 60.5% in pretreated HER-2 positive metastatic urothelial cancer patients including those who underwent failure to the immunotherapy .There are some limitations to this study. On the one hand, pretreatment biopsy was not mandatory when patients were enrolled. However, differences in HER2 expression before and after trastuzumab were reported in HER2-positive gastric cancer and it is related with efficacy of trastuzumab in cross-line therapy .ThereforIn summary, in this phase I study, RC48 showed preliminary anti-tumor activity and a manageable safety profile in patients with advanced HER2-positive gastric cancer, including those with HER2 2+/FISH−, and those pre-treated with HER2-targeted drugs. Findings from this study remain to be confirmed in further investigation. Phase II studies have been initiated to confirm these promising findings in HER2-positive gastric cancer (NCT03556345) and urothelial cancer patients (NCT03809013).RC48 is associated with mild toxicity and substantial clinical activity in HER2 positive solid tumors, particularly offering potential efficacy in HER2 low expression gastric cancers. Phase II trials in patients with advanced HER2-positive gastric cancer and urothelial cancer have been initiated to confirm these promising findings.Supplementary file1 (PDF 157 KB)Supplementary file2 (PDF 143 KB)Supplementary file3 (PDF 1193 KB)Supplementary file4 (PDF 374 KB)Supplementary file5 (PDF 601 KB)Supplementary file6 (PDF 105 KB)Supplementary file7 (PDF 132 KB)Supplementary file8 (DOCX 287 KB)Below is the link to the electronic supplementary material.


The KB cell competitive binding experiments indicated that [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA had specificity to folate receptor. Biodistribution studies in KB tumor-bearing mice illustrated that [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA had specific tumor uptake. Compared with [99mTc]Tc-CN5FA, the tumor/muscle ratios of [99mTc]Tc-CNPFA were higher, resulting in a better SPECT/CT imaging background. According to the results, the two 99mTc complexes have potential as tumor imaging agents to target folate receptors.In order to seek novel technetium-99m folate receptor-targeting agents, two folate derivatives (CN5FA and CNPFA) were synthesized and radiolabeled to obtain [ Tumors are pathological changes caused by the uncontrolled and continuous proliferation of cells under the influence of various factors . Folate In recent years, nuclear medicine imaging has shown the advantages of being sensitive, specific, noninvasive, and able to detect changes in molecular biological behavior and pathophysiology in vivo. It has become one of the most important diagnostic methods for cancer. With the widespread use of single-photon emission computed tomography (SPECT) and positron emission tomography (PET), radionuclide tumor imaging has become one of the advantages of nuclear medicine ,6,7.99mTc is the most widely used SPECT radionuclide with good nuclide properties, including a half-life of 6.02 h, emission of 140 keV γ-rays, and the advantages of coordinated chemical diversity. Therefore, 99mTc complexes are often used in the clinical diagnosis of some diseases, accounting for more than 85% of nuclear medicine SPECT images Tc-CN5FA and [99mTc]Tc-CNPFA. Their potential for tumor imaging was evaluated for the first time.Considering the evidence above, we aimed to develop novel The reaction route is shown in 99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA (the proposed structures of the radiolabeled tracers are shown in 99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA were as follows: in anticoagulant acid citrate dextrose (ACD) solution, [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA stayed at the origin (Rf = 0–0.1), while 99mTcO4− (Rf = 0.9–1.0) and 99mTcO2·nH2O (Rf = 0.8–1.0) were moved to the front. The HPLC results of [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA are shown in 99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA were about 2.24–22.46 GBq/µmol and 2.74–27.42 GBq/µmol, respectively.[99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA was found, indicating their good stability in vitro.As shown in 99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA were −1.92 ± 0.15 and −2.28 ± 0.02, respectively. This result suggested that both complexes were hydrophilic and that [99mTc]Tc-CNPFA had a better hydrophilicity than [99mTc]Tc-CN5FA due to the ethylene glycol chain linker  of Tc-CN5FA and [99mTc]Tc-CNPFA are shown in 50 values of [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA were 37.92 ± 0.41 nM and 3.80 ± 0.07 nM with KB cells, respectively. This result suggests that both complexes specifically bound to the folate receptor.To confirm the FR binding property of acquired 99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA were 2.42 ± 0.44 %ID/g and 2.53 ± 0.12 %ID/g at 2 h post injection (p.i.), respectively, which were significantly reduced to 1.53 ± 0.10 %ID/g and 1.62 ± 0.08 %ID/g at 2 h p.i. by pre-administration of free folate acid (FA) (about 4 × 104 µg/kg). This indicates that they were specific to the folate receptor. As the results demonstrate, the tumor/muscle ratios of [99mTc]Tc-CNPFA were 2.82 at 0.5 h p.i. and 3.01 at 2 h p.i., which were better than 1.76 at 0.5 h p.i. and 1.91 at 2 h p.i. for [99mTc]Tc-CN5FA . Interestingly, the imaging effect of [99mTc]Tc-CNPFA was superior to that of [99mTc]Tc-CN5FA because the former had a higher T/NT ratio than the latter.SPECT/CT imaging studies of Tc-CN5FA and [99mTc]Tc-CNPFA) were successfully prepared, and the RCP of [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA was over 95% without purification. In this work, we used folate as a target molecule, isonitrile as a BFC, methylene chain and PEG chain as linkers, and In the biodistribution experiment stage, because of the poor solubility of ligand CN5FA, the biodistribution results showed obvious concentrations in the liver (52.35 ± 1.19 %ID/g at 2 h p.i.) and other abdominal organs when the labeled condition was under weak acid. Therefore, to reduce the concentration of radioactivity in the liver and other abdominal organs, the pH of the labeling solution was changed to weakly alkaline in the follow-up experiments so that the ligand CN5FA could be better dissolved.99mTc]Tc-CN5FA was 3.03 ± 0.10 %ID/g and that of [99mTc]Tc-CNPFA was 4.18 ± 0.36 %ID/g at 0.5 h p.i., suggesting that [99mTc]Tc-CNPFA had a higher tumor uptake. However, there was no significant difference in tumor uptake between the two complexes at 2 h p.i. The muscle uptake of [99mTc]Tc-CNPFA (0.84 ± 0.04 %ID/g) was lower than that of [99mTc]Tc-CN5FA (1.27 ± 0.03 %ID/g), thus leading to a higher tumor/muscle ratio of the former than that of the latter. In the inhibition experiments, with early injection of excessive FA, the tumor and kidney uptake of both complexes was shown to have an inhibitory effect of more than one-third at 2 h p.i.As shown in 99mTc]Tc-10 using isonitrile (–NC) as the BFC [99mTc]Tc-10 had higher tumor uptake (4.02 ± 0.80 %ID/g) and lower liver concentration (5.14 ± 1.79 %ID/g) at 2 h post injection. However, the preparation of [99mTc]Tc-10 was a two-step process in which the [99mTc(H2O)3(CO)3]+ intermediate needs to be prepared first before the product required to be purified by HPLC. Compared with the method of direct labeling of [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA, the preparation process of [99mTc]Tc-10 was tedious, time-consuming, and not suitable for clinical application.In 2019, Lodhi et al. developed a folate receptor tracer Tc-CN5FA and [99mTc]Tc-CNPFA complexes were obtained by adding saline to blank kits containing 0.06 mg of SnCl2·2H2O, 1 mg of sodium citrate, 1 mg of l-cysteine, 5 mg of mannitol, 10 µg of CN5FA (compound 6) or CNPFA (compound 7), and freshly washed Na99mTcO4 solution (37–370 MBq) in turn, adjusting the pH of the solution to approximately 8.0 with NaOH (0.1 M) and reacting for 20 min at 100 °C.The [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA stayed at the origin (Rf = 0–0.1), while 99mTcO4− (Rf = 0.9–1.0) and 99mTcO2·nH2O (Rf = 0.8–1.0) were moved to the front. Radio-HPLC was analyzed by a mobile phase system consisting of 0.1% TFA water (solvent A) and 0.1% TFA acetonitrile (solvent B) with a gradient of 0−2 min 10% B, 2−5 min 10%−90% B, 10−20 min 90% B, and 20−25 min 90%−10% B (system B) at a flow rate of 1 mL/min. All experiments were performed in triplicate.The complexes were determined by ITLC/SG of the ACD system and radio-HPLC. The ITLC/SG trip was cut into 10 equal parts, and radioactive counting of ITLC strips was determined by a γ-counter. ITLC results of both complexes were as follows: in ACD solution, [99mTc]Tc-CN5FA and [99mTc]Tc-CNPFA in vitro was evaluated by measuring the RCP of the complexes. Stability in saline was achieved by holding the solution at room temperature for 4 h. Stability in the serum was achieved by mixing 100 μL of [99mTc]Tc-CN5FA or [99mTc]Tc-CNPFA with 200 μL of mouse serum at 37 °C for 4 h. After precipitation using acetonitrile, centrifugation, and concentration, the solutions were analyzed by radio-HPLC.The stability of Tc-CN5FA and [99mTc]Tc-CNPFA, to target FR without further purification. In vitro and in vivo studies demonstrated the high affinity and specificity of these probes for targeting FR. As they can be prepared as a kit formulation, they are promising candidates as tumor imaging agents to target folate receptors.In this work, we successfully developed two novel molecular probes, [


Background: To determine the respiratory outcomes in patients following COVID-19-related hospitalization.Methods: Systematic review and meta-analysis of the literature.Results: Forced vital capacity : 0–3 months post discharge: 96.1, 95% CI [82.1–110.0]; 3–6 months post discharge: 99.9, 95% CI ; >6 months post discharge: 97.4, 95% CI [76.8–118.0]. Diffusing capacity of the lungs for carbon monoxide : 0–3 months post discharge: 83.9, 95% CI [68.9–98.9]; 3–6 months post discharge: 91.2, 95% CI [74.8–107.7]; >6 months post discharge: 97.3, 95% CI [76.7–117.9]. Percentage of patients with FVC less than 80% of predicted: 0–3 months post discharge: 10%, 95% CI [6–14%]; 3–6 months post discharge: 10%, 95% CI [2–18%]; >6 months post discharge: 13%, 95% CI [8–18%]. Percentage of patients with DLCO less than 80% of predicted: 0–3 months post discharge: 48%, 95% CI [41–56%]; 3–6 months post discharge: 33%, 95% CI [23–44%]; >6 months post discharge: 43%, 95% CI [22–65%].Conclusion: The meta-analysis confirms a high prevalence of persistent lung diffusion impairment in patients following COVID-19-related hospitalization. Routine respiratory follow-up is thus strongly recommended. In thisWe included randomised controlled trials (RCTs) and observational studies  of patients with a confirmed diagnosis of COVID-19. The studies included aimed to determine the respiratory outcomes, in particular forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO), in patients following COVID-19-related hospitalisation. The selected studies had to follow the ATS / ERS clinical guidelines. The included literatures should be published before May 15, 2021.Study’s subjects who were not infected with COVID-19. Studies didn’t report the time of hospital discharge or the time was calculated from diagnosis of COVID-19. Studies did not report FVC (% of predicted) or DLCO (% of predicted) or FVC <80% of predicted or DLCO <80% of predicted. Animal experiments, medical records, case reports, famous medical experience and review were excluded.Firstly, according to the literature inclusion criteria, two researchers independently searched at Pubmed, ScienceDirect, Embase and Web of Science. Secondly, two researchers selected the literature and extracted the data independently in accordance with the standard data extraction table. When it came to divergences, a third researcher did the judgement. After the discussion, researchers reached a consensus. Finally, after the extraction and input of the data, two independent researchers did the subsequent analysis.According to the inclusion criteria, we assessed the design of research, patients, and outcome indicators. First author, published year, number of cases, nationality, ages, body mass index (BMI), smoking status, respiratory comorbidities, time of assessment and, index quantity of FVC, % of predicted, DLCO, % of predicted; FVC <80% of predicted and DLCO <80% of predicted were extracted from eligible studies.The studies with randomised controlled trials were evaluated by Newcastle-Ottawa Scale . As for 2 statistic and classified as low (I2 < 25%), moderate (I2 25–50%), and high (I2 > 50%) (We used package “meta (version 4.18-0)” in R 4.0.1 and R studio to perform meta-analysis of the following pulmonary function tests (PFTs) indexes . Patients were divided into three groups: less than 3 months (0–3 months), more than or equal to 3 months and less than 6 months (3–6 months), and more than or equal to 6 months (≥6 months). We re-calculated the median  to mean ± standard deviation (SD) for FVC (% of predicted) and DLCO (% of predicted) in several studies. Statistical heterogeneity was measured through the I2 > 50%) . SubgrouA total of 1,123 articles was retrieved from databases via the retrieval methods. Duplicate literatures were excluded through titles and abstracts. By reading the full text, we excluded 1,110 papers and conference abstracts with incomplete or no specific research method. Finally, 13 papers published in English were included , with a . reported all the indexes of the patients after the 3, 6 and 12 months following COVID-19-related hospitalisation , eight studies reported DLCO (% of predicted), six reported FVC <80% of predicted, and nine reported DLCO <80% of predicted. lisation . For tholisation .p = 0.93; p = 0.54; p = 0.94; Publication bias refers to the fact that research results with statistical significance are more likely to be reported and published than those without statistical significance and invalid results . We exam2 = 0%), and the overall value of FVC (% of predicted) in all studies was 97.7 (95% CI [88.6–106.9]) (Nine studies with 11 groups of data showed the results of FVC (% of predicted). Based on the time of patients discharged from hospital, we divided them into three groups: 0–3 months, 3–6 months and ≥6 months. FVC (% of predicted) in 0–3 months, 3–6 months and ≥6 months post discharge were 96.1 (95% CI [82.1–110.0]), 99.9 (95% CI [84.8–115.0]) and 97.4 (95% CI [76.8–118.0]), respectively. In this study, heterogeneity was extremely low (I–106.9]) .2 = 0%) using a fixed effect model (Eight studies with 10 groups of data showed the results of DLCO (% of predicted). DLCO (% of predicted) in 0–3 months, 3–6 months and ≥6 months post discharge were 83.9 (95% CI [68.9–98.9]), 91.2 (95% CI [74.8–107.7]) and 97.3 (95% CI [76.7–117.9]), respectively. Heterogeneity was considered low  with I2 = 6% , 10% (95% CI [2–18%]) and 13% , 33% (95% CI [23–44%]) and 43% (95% CI [22–65%]), respectively .Post-acute COVID-19 syndrome, also known as long COVID, encompasses a wide range of physical and mental health symptoms that persist after recovery from acute SARS-CoV-2 infections . Systeman = 83)  . In thisn = 83) . Low DLCn = 83) . Our stun = 83) , althougIn general, the heterogeneity of the studies included in the meta-analysis was low. However, the heterogeneity of DLCO less than 80% of predicted was higher, which may be caused by different ethnic groups, ages, disease severity, therapies and other factors. In general, the models we used were robust and reliable.There are several limitations in this study. Firstly, age, sex ratio, nationality and disease severity of the patients included in the study are quite different, which may cause great heterogeneity and affect the final research results. Secondly, we only selected four indicators of lung function, so we cannot investigate the relationship between other indicators and discharge time. To be consistent and comparable with our earlier publication , we excl


Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers  Microglia are central nervous system (CNS) resident, macrophage-like cells whose activities are highly relevant in neuroimmune interactions, especially after immune insult or trauma to the brain ,2. In paTo date, the regulatory mechanisms underlying the control of microglial behaviours during inflammation are not fully understood . MultiplThe neuropeptides PACAP and VIP have gained more interest within the CNS due to their wide range of neuroprotective functions as well as their widespread CNS localisation ,34,35,36In addition to their well-known neuro- and glio-protective functions, at nanomolar concentrations, PACAP and VIP are also able to modulate immunological function ,44,45,46Each peptide has been shown to inhibit pro-inflammatory cytokines and neurotoxic factors secreted by activated microglia in a model of PD and brain injury ,48. AddiWhile many of the protective activities of PACAP and VIP on neurons and glial cells have been identified ,36,49, tIL-1β, IL-6, Itgam, AIF1, Adgre1, CD68 and NOS2) and the anti-inflammatory gene IL-10 at increasing concentrations of LPS . When LPS was used at a concentration of 0.1 μg/mL, there was only a significant increase in IL-1β  = 78.95, * p < 0.05), Itgam  = 5.373, * p < 0.05), Adgre1  = 18.54, ** p < 0.01) and CD68  = 16.54, * p < 0.05) , IL-6  = 245.2, *** p < 0.001), Itgam (** p < 0.01), AIF1  = 5.925, * p < 0.05), Adgre1 (*** p < 0.001), CD68 (*** p < 0.001) and NOS2  = 67.85, *** p < 0.001) (see p > 0.05). At the highest concentration tested (10 μg/mL LPS), IL-1β (*** p < 0.001), IL-6 (*** p < 0.001), IL-10  = 3.827, * p < 0.05), Adgre1 (*** p < 0.001), CD68 (** p < 0.01) and NOS2 (*** p < 0.001) were all increased significantly when compared to controls (Itgam and AIF1 (p > 0.05).In order to establish the most suitable concentration of LPS for triggering a robust inflammatory response in the BV2 microglial cell line, we conducted real-time quantitative polymerase chain reaction (qPCR) to measure the gene expression of a panel of pro-inflammatory mediators ( < 0.05) A,D,F,G, 001) see A,D–H, whcontrols A–C,F–H. IL-1β  = 196, *** p < 0.001), IL-6  = 16.87, *** p < 0.001), CD68  = 16.99, *** p < 0.001) and NOS2 transcripts  = 262.6, *** p < 0.001) .Once it had been established that 1 μg/mL LPS was the most effective concentration to trigger inflammation in our cell model, we sought to determine the temporal response of BV2 cells to treatment. We conducted a time-course analysis of the gene expression profiles of the markers used in the main experiments at five different time points . We found that after 6 h exposure to LPS, there were significant increases in < 0.001) A,B,G,H.  changes C–F, (p >IL-1β (*** p < 0.001), IL-6 (*** p < 0.001), IL-10  = 7.316, *** p < 0.001), Adgre1  = 27.15, *** p < 0.001), AIF1  = 8.547, * p < 0.05), CD68 (*** p < 0.001) and NOS2 (*** p < 0.001)  A–C,E–H.IL-1β (*** p < 0.001), IL-6 (** p < 0.01), Adgre1 (*** p < 0.001), CD68 (** p < 0.01) and NOS2 (*** p < 0.001) when compared with control , IL-6 (*** p < 0.001), AIF1 (*** p < 0.001), Adgre1 (*** p < 0.001) and NOS2 genes (*** p < 0.001) were all significantly upregulated   C,D,G.Adcyap1) mRNA expression by either 6- or 8-fold  = 57.91, *** p < 0.001) , VPAC1 receptor (Vipr1) or VPAC2 receptor (Vipr2)  A but didAIF1/Iba1  = 24.89, *** p < 0.001 vs. control, NOS2 mRNA levels  = 240.8, *** p < 0.001, p < 0.01 and iNOS: F  = 5.666, ** p < 0.01, AIF1  and NOS2 gene expression (### p < 0.001 vs. LPS for both peptides), as well as the expression of the translated Iba1  and iNOS proteins (# p < 0.05 vs. LPS for both peptides)  A,B,D.p < 0.01 vs. Ctrl, p < 0.001 for both PACAP and VIP, To complement our biochemical analyses, we performed immunocytochemistry. BV2 microglia were grown on coverslips under each of the experimental conditions  and stained using an antibody to Iba1 . Iba1 exIL-1β , IL-6 , Itgam , Adgre1  and CD68  compared to untreated groups. Of note, the mRNA expression levels of the anti-inflammatory cytokine IL-10 remained unchanged (p > 0.05).In these experiments, we evaluated whether PACAP or VIP reduced the heightened levels of pro-inflammatory cytokines in BV2 microglial cells following exposure to LPS. Cells were exposed to LPS and treated as described above. Additional drug-treatment controls were also included. As shown in IL-1β (### p < 0.001), IL-6 (### p < 0.001), Itgam (### p < 0.001), Adgre1 (# p < 0.05) and CD68 (### p < 0.001) when compared with the LPS-only group (IL-10 mRNA expression levels (p > 0.05).PACAP treatment of cells exposed to LPS significantly decreased the mRNA expression levels of ly group A,B,D–F. IL-1β (### p < 0.001), IL-6 (### p < 0.001), Itgam (### p < 0.001) and CD68 mRNAs (### p < 0.001) (IL-10 gene expression (p > 0.05). Adgre1 transcript levels were also reduced, although this decrease was not statistically significant (p > 0.05).VIP treatment of cells exposed to LPS produced similar patterns of change to those seen following PACAP treatment. Specifically, there were significant reductions in < 0.001) A,B,D,F. NOS2/iNOS induced by PACAP or VIP was paralleled by reductions in the level of nitrites secreted by the cells, the relative abundance of NO was measured in the culture media using the Griess method [p < 0.001) , although it did not return to control levels .To determine whether the downregulation of s method . As show< 0.001) . TreatmeWhen BV2 cells were challenged with LPS and co-treated with either PACAP or VIP, we observed gross morphological differences in the cells between the two treatments. To systematically describe these differences, we conducted morphometric analyses of untreated cells, or cells exposed for 24 h to either LPS alone or in combination with PACAP or VIP.2, max size = 924 μm2). This large heterogeneity of cell sizes across treatment groups prompted us to conduct a distribution analysis aimed at determining if each treatment resulted in a distinct pattern of cell distribution as a function of their size. As shown in 2; (ii) mid-sized cells = 200–400 μm2; and (iii) large cells = > 400 μm2) whose patterns of changes were distinctly affected across our treatment groups. We detected a strong correlation between cell surface area and the length of cellular processes  vs. 20/440 (4.55%) in controls, When LPS-exposed BV2 cells were co-treated with PACAP, we observed a major shift towards a phenotype characterized by small BV2 cells (238/440 (54.09%) vs. 53/440 (12.05%), BV2 cells that were exposed to LPS and co-treated with VIP showed a distinct redistribution of cells, based on their cell surface areas A,B. In cOnce we had established that PACAP and VIP each differed in their abilities to segregate cell subpopulations within a distinct cell size-based category, we sought to determine whether changes in cell size might also relate to changes in cell morphology. Therefore, stereological analyses were undertaken in BV2 cells, treated as indicated above. In physiological conditions, BV2 cells exhibit three distinct morphologies: rounded, bipolar/spindle and multipolar shapes.p < 0.01). Specifically, within the LPS+VIP group, we found that cells exhibiting a bipolar/spindle shape were the most frequently represented cell population (53%), both in comparison with control groups  or LPS-treated cells   A.p > 0.05)  were significantly different across treatment groups ( > 0.05) A.2), irrespective of treatment. Conversely, analyses of bipolar/spindle-shaped cells showed that between 60–70% of these cells were mid-sized. However, in VIP-stimulated cells, this percentage increased to 80.19% , treatment with either LPS alone or in combination with PACAP or VIP triggered a redistribution of the percentage of cells of a given size category . Specifically, we found that about 90% of all rounded BV2 cells were small sized . Cell motility was partially recovered at 30 h (p > 0.05 vs. control); however, the percentage of the wound area was still significantly larger than in controls at 36 h , 24 h (### p < 0.001) and 36 h (## p < 0.01)  A,B.p < 0.05 vs. LPS), with further increases recorded at later time points   A,B.uPA mRNA expression at all doses tested (*** p < 0.001) , a plasminogen-cleaving enzyme known to be involved in cell migration . Dose–re< 0.001) C. PACAP In the present study, we demonstrate that both PACAP and VIP exert potent immunosuppressive activities in BV2 microglial cells exposed to lipopolysaccharide (LPS), an inflammatory mimetic. By exposing healthy BV2 murine microglia cells to LPS, we attempt to replicate in vitro the functions of resident microglial cell populations in response to stimulation by PAMPs or DAMPs. We show that the peptides downregulated the expression of a range of pro-inflammatory mediators following LPS induction whilst also altering gene expression for PACAP/VIP and their receptors. Additionally, morphological and morphometric evaluations of this cell line revealed patterns of phenotypic redistributions of cell subpopulations that were distinct for cells treated with either peptide during an LPS challenge. We report for the first time that PACAP- or VIP-induced BV2 phenotypes are characterized by distinct effects on cell motility and the expression of uPA, an enzyme involved in regulating this biological function. When taken together, the data from this study provide a better understanding of the mechanisms through which these naturally occurring peptides may dampen inflammation or affect cell surveillance functions by microglia.Adcyap1 expression in cells treated with VIP was an unexpected finding and given the well-known neuroprotective effect of the encoded peptide, we suggest a strong translational potential [Investigating the effects that LPS and peptide addition have on the PACAP/VIP family is pivotal to uncover individual peptide functions. In the literature, studies of PACAP-deficient mice have demonstrated that PACAP plays critical roles in the endogenous cytoprotective machinery. In these knockout phenotypes, mice show increased vulnerability to stressors in the nervous system, and they exhibit distinct morphological and behavioural abnormalities ,63,64. Iotential ,66,67.Vip mRNAs in response to immune challenge with LPS. The changes showed a marked recovery of expression following LPS + PACAP/VIP treatments in comparison to both resting and LPS only  mice demonstrate that endogenous PACAP and the activation of PAC1 receptor exerts anti-inflammatory effects, as the mice exhibited higher levels of IL-6 [Adcyapr1r1 gene expression after treatment with LPS + PACAP/VIP in comparison with LPS only. Vipr1 and Vipr2, also known as VPAC1 and VPAC2 receptors, respectively, have equally high affinity for both PACAP and VIP peptides. Earlier studies show that by using a VPAC1 receptor agonist on LPS-treated cells, it reduced TNF levels and IL-6 in vitro, and also that VIP treatment was able to inhibit pro-inflammatory cytokine production from monocytes, mainly through the VPAC1 receptor [Vipr1 gene expression is upregulated post-LPS + PACAP/VIP treatment, and levels are increased in comparison with control and LPS only , the main enzyme capable of synthesizing nitric oxide (NO), so we anticipated that we would not observe high expression of the gene in the control group . The amose of NO ,76,77. ANO [NOS2 ,79. ThisNO [NOS2 ,81. In o and VIP . This re and VIP . These oIL-1β, IL-6 and Itgam, defines the activation state of the cells. These inflammatory cytokines allow for the initiation of inflammatory cascade, and thus result in damage. In the present study, the levels of pro-inflammatory cytokines, including IL-1β, IL-6, Itgam, Adgre1 and CD68 were significantly increased with LPS, and these levels were restored following treatments with the peptides , whose pattern of changes were affected by LPS exposure and, importantly, that were found to be affected by PACAP or VIP treatment , associated with peculiar changes in morphological features , supplemented with either 1% or 10% heat-inactivated foetal bovine serum (FBS) and 1% penicillin (100 IU/mL)/streptomycin (100 μg/mL) . Cells were incubated at 37 °C in a humidified atmosphere with 5% COCells were subjected to different treatments: (1) control, (2) 100 nM PACAP , (3) 100 nM VIP , (4) 1 μg/mL LPS , (5) LPS + PACAP and (6) LPS + VIP. Thereafter, cells were harvested at 6, 12, 24 or 48 h for downstream molecular analyses.n = 3–6) and isolated using 1mL TRIsureTM reagent , 0.2 mL chloroform and precipitated with 0.5 mL isopropanol . The pellet was washed with 75% ethanol and air dried by inversion of tubes. Total RNA (1 μg) was diluted in milliQ H2O to a final volume of 11 μL. A total of 9 μL of Tetro cDNA synthesis mix  was added to each 11 μL aliquot, each containing: 1 μL oligo (dT)18 primers, 1 μL random hexamer primers mix, 1 μL 10 mM dNTP mix, 4 μL 5× RT buffer, 1 μL RNase inhibitor and 1 μL reverse transcriptase. A volume of 20 μL of each sample were then incubated at 25 °C for 10 min, followed by 45 °C for 30 min. The reaction was terminated by incubation of samples at 85 °C for 5 min.Total RNA extracts from harvested cells were subjected under certain treatments  were utilised, and up to 4 genes were tested per run. Each PCR reaction mix was prepared in a final volume of 7 μL, to which we added 3 μL of cDNA  and conducted using the CFX96 Touch™ Real-Time PCR Detection System . Each PCR reaction mix contained 5 μL SensiFAST SYBR No-ROX kit , 0.8 μL 5 μM forward primer, 0.8 μL 5 μM reverse primer and 0.4 μL milliQ H2O. PCR was performed using the following settings:Aliquots of cDNA were amplified using specific primers shown in −ΔΔCt was used to calculate fold changes. Baseline measurements for untreated controls were set to 1.(1) 95 °C for 2 min, (2) 60 °C for 10 s, (3) 72 °C for 10 s, (4) plate read, (5) repeat step 2 for 45 cycles, (6) 65 °C for 35 s, (7) plate read and (8) repeat step 6 × 60 times using the PCR instrument. After the protocol was completed, the Ct values were exported and analysed. Relative changes in gene expression were computed using the comparative Ct method . The forProtein lysate was homogenised in RIPA buffer , which was supplemented with cOmplete™ULTRA protease inhibitor cocktail . Lysates were then sonicated twice at 50% power for 10 s using an ultrasonic probe, followed by centrifugation prior to protein quantification. Protein concentrations were determined using the Bicinchoninic Acid (BCA) Assay Kit .® TGX Stain-Free™ protein gels (15 wells) . Samples were prepared by 3.75 μL of 4× Laemmli buffer  and β-mercaptoethanol  mix (Laemmli buffer 20:1 with β-mercaptoethanol) to a final volume of 15 μL. Samples were then denatured at 70 °C for 10 min using the T100™ thermal cycler.Protein samples (15 μg) were separated by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% Mini-PROTEAN®20 (TBST) 1× three times before blocking with 5% blocking buffer containing 5% skim milk in TBST 1× for 1 h at room temperature at 55–60 rpm. The membranes were probed with anti-rabbit primary antibodies recognizing either Iba1, iNOS or GAPDH (used as loading control) overnight at 4 °C under gentle oscillation (55 rpm)  were resolved on gels and transferred to a PVDF membrane using the semi-dry approach . Once proteins were transferred, the membrane was removed from the apparatus and briefly washed in tris-buffered saline containing Tween®20) for 30 min. Blocking serum was replaced with appropriately diluted primary antibody (in PBS)  and incubated at 4 °C overnight with gentle agitation. Cells were rinsed with PBS for 3 changes for 5 min each. A volume of 1 mL of appropriately diluted fluorescein-conjugated sondary antibody (in PBS)  was applied to the wells and incubated at room temperature for 1 h protected from light. Following the sondary antibody incubation, cells were washed with PBS. A total of 0.3 μg/mL DAPI stain  was applied to the cells for 1 min and discarded. Cells were washed again with PBS and then coverslips were mounted onto one-frosted ended microscope slides  using mounting medium . Clear nail polish was applied to the edges of the coverslips to ensure that they remain sure and sealed. Colours of antibody staining were observed with the Delta Vision Elite deconvolution microscope. Relative fluorescence was determined using the open-source software ImageJ. Nuclei were defined using the DAPI channel (435–485 nm emission) using a 420 nm longpass filter, whereas Iba1 immunosignal was captured using the Fluorescein Iso-thiocyanate (FITC) channel and collected through a 525/50 nm bandpass filter. To avoid the possibility of DAPI spectral bleed-through into Alexa fluor 488 dye, fluorescent signals were captured and recorded on separate channels using sequential imaging and merged afterwards. Regions of Interest (ROIs) used to quantify the integrated intensity of fluorescent images were selected by applying a macro with a preset threshold level and gamma, brightness and contrast values, which were kept constant throughout the experiments. Background values were extrapolated from at least three separate fixed areas taken from the same photomicrograph. To calculate relative fluorescence intensity, we applied the following formula:BV2 microglial cells were grown on cover slips pre-coated in poly-l-lysine  and fixed with 4% paraformaldehyde (PFA) . Then, cells were permeabilised in PBS containing 0.25% Triton X-100  for 10 min and then rinsed with PBS. Thereafter, fixed cells were treated with blocking solution  and 0.1% Tween5) were seeded in 25 cm2 flasks and starved in 1% FGM for 24 h, prior to initiating the six treatments for 12 h. Following treatments, culture media was replaced with freshly prepared media not containing any treatment and cells were incubated for a further 24 h to allow accumulation of NO. Twenty-four hours later, the supernatant was aspirated and collected. Griess reagent  was prepared by dissolving 1 g Griess reagent in 25 mL milliQ H2O (1:25 w/vol) in an autoclaved bottle shielded from the light. Under sterile conditions, 100 μL of conditioned media was added into a 96-well plate, using at least 6 replicates from each group. Each experiment was repeated at least twice. An amount of 100 μL of Griess reagent was added to each well containing 100 ul of conditioned media from each group. The plate was incubated at room temperature for 15 min, protected from light. Absorbance using a 540 nm filter was measured by the Tecan infinite M1000 Pro ELISA reader which reflected nitrite levels. Changes in NO were normalized to that of untreated cells and are expressed as percentage of controls.Levels of nitric oxide (NO) in culture supernatants were quantified by the utilisation of the Griess reaction. Briefly, BV2 cells (5 × 105) were seeded in 25 cm2 flasks and starved in 1% FGM for 24 h, prior to initiating treatments: control, LPS only, LPS + 100 nM PACAP and LPS + 100 nM VIP for 24 h. Images were then captured using a Nikon Eclipse TS2 inverted microscope and subjected to morphological assortment and analyses (magnification 20×). Four experimenters blind to the cell culture conditions were assigned random images to analyse. After measuring the average diameter and length of cellular processes, along with soma surface area  and assessing the gross morphology , cells were then grouped into three different categories: (a) small, (b) mid-sized, and (c) large cells , where cells were labelled and measured individually.BV2 cells . All data were tested for normality with the Kolmogorov–Smirnov test. All variables were normally distributed. Datasets are reported as mean ± standard error of mean (SEM). One-way or two-way repeated measures (RM) analysis of variance (ANOVA) followed by post hoc tests (as appropriate) were utilised to assess which groups were statically different. Result for all statistical tests were considered significant when p ≤ 0.05.Data were analysed using GraphPad Prism