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Objective: The identification of biomarkers for predicting chemoradiotherapy efficacy is essential to optimize personalized treatment. This study determined the effects of genetic variations in genes involved in apoptosis, pyroptosis, and ferroptosis on the prognosis of patients with locally advanced rectal cancer receiving postoperative chemoradiotherapy (CRT). Methods: The Sequenom MassARRAY was used to detect 217 genetic variations in 40 genes from 300 patients with rectal cancer who received postoperative CRT. The associations between genetic variations and overall survival (OS) were evaluated using hazard ratios (HRs) and 95% confidence intervals (CIs) computed using a Cox proportional regression model. Functional experiments were performed to determine the functions of the arachidonate 5-lipoxygenase (ALOX5) gene and the ALOX5 rs702365 variant. Results: We detected 16 genetic polymorphisms in CASP3, CASP7, TRAILR2, GSDME, CASP4, HO-1, ALOX5, GPX4, and NRF2 that were significantly associated with OS in the additive model (P < 0.05). There was a substantial cumulative effect of three genetic polymorphisms (CASP4 rs571407, ALOX5 rs2242332, and HO-1 rs17883419) on OS. Genetic variations in the CASP4 and ALOX5 gene haplotypes were associated with a higher OS. We demonstrated, for the first time, that rs702365 [G] > [C] represses ALOX5 transcription and corollary experiments suggested that ALOX5 may promote colon cancer cell growth by mediating an inflammatory response. Conclusions: Polymorphisms in genes regulating cell death may play essential roles in the prognosis of patients with rectal cancer who are treated with postoperative CRT and may serve as potential genetic biomarkers for individualized treatment.

Multidrug resistance (MDR), which is common in colorectal cancer (CRC), induces high mortality in patients. Due to its robust and selective apoptosis induction in some CRC cells with MDR, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is attractive as a novel tool for CRC therapy. However, TRAIL is limited by its poor tumor-homing ability and inefficient apoptosis induction in CRC cells expressing low levels of death receptor (DR). Here, the tumor-homing RGR peptide (CRGRRST) was fused to TRAIL to produce RGR-TRAIL. Compared with TRAIL, RGR-TRAIL showed greater cell binding and cytotoxicity in CRC cells. In addition, RGR-TRAIL exerted significantly enhanced tumor uptake and growth suppression in mice bearing CRC tumor xenografts. Notably, RGR-TRAIL eradicated all tumor xenografts of DR-overexpressing COLO205 cells. However, TRAIL only showed mild tumor growth suppression under the same conditions, indicating that RGR fusion significantly increased the antitumor effect of TRAIL in DR-overexpressing CRC cells by improving tumor homing. Nevertheless, RGR fusion did not significantly enhance the antitumor effect of TRAIL in HT29 cells expressing low levels of DR. We found that DR expression in HT29 cells was enhanced by epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT). Moreover, both the in vitro and in vivo antitumor effects of RGR-TRAIL were significantly improved by combination with PDT. HT29 tumor xenografts (∼20%) were even eradicated by combination therapy. These results indicate that it is valuable to further evaluate the combination therapy of RGR-TRAIL and tumor-targeted PDT for clinical therapy of CRC with MDR.

The self-growing bell-shaped structure of the nickel foam substrate have excellent binding ability with the NF substrate, which greatly improve the mass and charge transfer efficiency and provide the possibility of stabilizing the catalyst at large currents.

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRβ affibody, which targets platelet-derived growth factor receptor β (PDGFRβ), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRβ fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRβ fused to the IgBD. ZPDGFRβ-modified αPD-L1 bound to both PDGFRβ and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRβ+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRβ fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.

Fluconazole (FLC) tolerance increases the frequency of acquired FLC resistance, and a high FLC tolerance level is associated with persistent candidemia. Multiple functional proteins, such as calcineurin, heat shock protein 90 (Hsp90), and ADP ribosylation factor, are essential for the survival of C. albicans exposed to FLC, but how these factors increase the fungicidal activity of FLC remains to be determined.

Small-molecule induction can lead to the oriented migration of metal elements, which affords functional materials with synergistic components. In this study, phosphating nickel foam (NF)-supported octahedral WO

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PM Objectives Making fluconazole (FLC) fungicidal in combination with adjuvants is a promising strategy to avoid the emergence of FLC resistance and eliminate the persistence and recurrence of fungal infections. To address this question, we combined in vitro screening of a library of FDA-approved drugs to identify compounds for making FLC fungicidal. Methods We performed a high-throughput screen of an FDA-approved compound library (HY-L022, MCE®), which contains 2372 drugs, to identify potentially novel FLC synergistic lethal adjuvants using broth microdilution and dose-matrix titration assays. The abilities of candidate drugs to turn FLC from fungistatic to fungicidal were further investigated by FLC disk diffusion assays carried out by four tested strains with different FLC tolerance levels (SC5314, SN152, cmp1-aid∆/cmp1-aid∆, and ADH1p-UPC2 strains). We determined the median lethal dose (LD50) of Candidate compounds by the Up-and-Down procedure (UDP) (OECD 425, 2008) via the intraperitoneal route in adult mice and used cyclosporine A and geldanamycin as control drugs to screen FLC synergistic lethal adjuvants with lower toxicity. Finally, we constructed heterozygous deletion mutants for ergosterol synthesis-related genes to identify the mechanism of action of the synergistic lethality of pitavastatin (calcium) (PIT) and FLC (Fig. 1a). Results We found that 200 compounds (≤100 μm) could make FLC (4 μg/ml) fungicidal and further confirmed that 30 compounds turned FLC (4 μg/ml) from fungistatic to fungicidal at a concentration lower than 12.5 μm by broth microdilution assays (Fig. 1b). We further identified that 12/30 compounds (≤3.125 μm) can make FLC fungicidal (≤4 μg/ml) using dose-matrix titration assays. Among these compounds, PIT can make FLC fungicidal at as low as 0.78 μm (Fig. 1c). In the FLC disk diffusion assay, we identified 8 compounds (5 μm) that were superior to or equivalent to the abilities of the control drugs to eliminate the FLC tolerance of four tested strains. It was worth noting that PIT could make FLC fungicidal against all four tested strains (Fig. 1d). The LD50 value of PIT is 103.6 mg/kg and the highest of the tested compounds. Spot assay results showed feeding exogenous 100 μm ergosterol counteracted the antifungal activity of PIT (16 μm) (Fig. 2a), but did not restore the growth defect of Tet-HMG1/hmg1∆ mutant, in which the HMG1 gene expression would be inhibited by tetracycline (Fig. 2b). This paradoxical phenotype suggests that Hmg1 is unlikely to be a target of PIT. We found four heterozygous deletion mutants increased susceptibility to PIT (Fig. 2c) and the growth defect of ERG8/erg8∆ and IDI1/idi1∆ mutants exposed to PIT can be restored by ergosterol (Fig. 2d), suggesting that Erg8 and Idi1 are potential targets of PIT. Conclusions Our study demonstrates that PIT has a better synergistic lethal effect with FLC by inhibiting ergosterol synthesis and higher safety and suggests that PIT may be repurposed as a promising adjuvant to make FLC fungicidal and enhance the efficacy of FLC in treating invasive fungal infections caused by pathogenic fungi with high FLC tolerance.

Synthesis of regular morphology catalysts with self-growing substrates is one of the effective methods to solve the problem of easy shedding of heterogeneous catalysts. In this study, Fe-doped Ni

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. Key points • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11136-x.

Chemotherapeutic multidrug resistance (MDR) is the major hindrance for clinical therapy of colorectal cancer (CRC). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with selective cytotoxicity might overcome MDR of CRC cells. Unfortunately, cross-resistance to TRAIL has been detected in many CRC cells, suggesting the need to combine TRAIL with sensitizers to combat refractory CRC. Our purpose is to explore the potential of combination therapy of TRAIL and tumor-cell targeted photodynamic therapy (PDT) in combating CRC with both chemotherapeutic MDR and TRAIL resistance. Methods: Tumor cell-targeted PDT was performed using a Ze-IR700 photosensitizer with high affinity for epidermal growth factor receptor (EGFR). The impact of PDT on the gene expression of CRC cells was revealed by RNA sequencing. The synergistic antitumor effect of long-acting TRAIL and PDT was evaluated in mice bearing tumor grafts of CRC cells with both chemotherapeutic MDR and TRAIL resistance. Results: Chemotherapeutic MDR and TRAIL resistance are common in CRC cells. Pretreatment of CRC cells with tumor cell-targeted PDT significantly (10-60 times) increased the sensitivity of these CRC cells to TRAIL by upregulating death receptors. Combination therapy, but not monotherapy, of long-acting TRAIL and PDT greatly induced apoptosis of CRC cells, thus efficiently eradicated large (~150 mm3) CRC tumor xenografts in mice. Conclusions: Tumor cell-targeted PDT extensively sensitizes CRC cells to TRAIL. Combination therapy of long-acting TRAIL and PDT is promising to combat CRC with both chemotherapeutic MDR and TRAIL resistance, which might be developed as a novel strategy for precision therapy of refractory CRC.

In order to improve the solubility of pipemidic acid (PPA) which belongs to quinolones and becomes an “old drug” due to its low aqueous solubility that restricts its application, two unreported pharmaceutical salts of PPA with catecholcarboxylic acid (CCA) (HPPA·C7H5O4·2(H2O)) and m-hydroxybenzoic acid (MHA) (HPPA·C7H5O3·C7H6O3·2(H2O)) have been designed, synthesized and characterized. A comprehensive analysis of the structure and Hirshfeld surface reveals that the CAHBs formed from the N atoms in the piperazine ring from PPA molecules with the carboxylic acid group in the coformers could have a stable crystal structure. As expected, the solubility experiments showed that the solubilities of the PPA–CCA and PPA–MHA were significantly improved compared with that of the PPA, which may be due to the increased hydration enthalpy resulting from the formation of CAHBs between PPA and coformers, which leads to increased solubility. Moreover, the antibacterial activities of PPA–CCA and PPA–MHA are also improved. At the same time, surprisingly, the permeability of PPA–CCA and PPA–MHA was also better than that of PPA. In addition, the hygroscopic stabilities of PPA–CCA and PPA–MHA were significantly improved compared with that of PPA. The reason may be due to that the hydrogen bonds between PPA and CCA weaken their attraction to external water molecules. Thus, the present work implies that CAHBs play a key role in adjusting the molecular packing via a cocrystallization strategy to improve the physicochemical properties of quinolones, which could offer a tremendous opportunity to recycle these “old” drugs and give them a new life.

In order to further explore the role of hydroxyl in the synthesis of pharmaceutical cocrystals and with a view to improving physicochemical properties of flavonoids, two dihydroflavonols in which 3-OH is exposed and cross-conjugated system is destroyed (dihydroquercetin (DHQ) and dihydromyricetin (DMY)) were selected to combine with 4,4′-bipyridine (BPY) to explore the role of 3-OH of dihydroflavonol in synthetic processes of pharmaceutical cocrystals of dihydroflavonol. As a result, two new pharmaceutical cocrystals of dihydroflavonol with BPY (2(C15H12O7)·3(C10H8N2), DHQ·BPY); and (C15H12O8·2.5(C10H8N2), DMY·BPY) have been synthesized and characterized. Structure analysis revealed that in DHQ·BPY the intermolecular hydrogen bond formed by 3-OH of DHQ with BPY participated in the formation of motifs R66(44) to form the cocrystal with helical structure. In DMY·BPY, the intermolecular hydrogen bond formed by 3-OH of DMY with BPY connects adjacent motifs R44(38) to form the cocrystal with layered structure. The solubility of DHQ·BPY is lower than that of pure DHQ, possibly because the helical structure of DHQ·BPY is tighter than that of nonplanar DHQ, which limits the entry of water molecules. Compared with nonplanar DMY, DMY·BPY with layered structure is more likely to allow water molecules to enter, which leads to the solubility of DMY·BPY being higher than that of pure DMY. The hygroscopic stabilities of DHQ·BPY and DMY·BPY are better than that of their corresponding parent APIs. It is possible that in DHQ·BPY and DMY·BPY, most of the hydroxyls in the A, B and C rings of DHQ and DMY are involved in the formation of intermolecular hydrogen bonds, which means the hydroxyls of DHQ·BPY and DMY·BPY are difficult to combine with moisture. This work shows that 3-OH of nonplanar dihydroflavonol can form hydrogen bonds with BPY after the destruction of the cross-conjugated system, and then form cocrystals with helical structure and layered structure, thereby having an effect on the properties of APIs. This successful study implies that the role of 3-OH in the self-assembly process of flavonoid pharmaceutical cocrystals and improving the physicochemical properties of flavonoid APIs should not be neglected in the future.

For improving solubility of inosine, its β-form has been designed and prepared from inosine powder upon induction by sulfosalicylic acid. A single crystal of β-form inosine has been grown. The structure of β-form inosine has been characterized by XRPD, NMR and single-crystal X-ray crystallography. The crystal structure of β-form inosine demonstrates the layered structure based on a primary asymmetric cyclic hydrogen-bonded tetramer R44(14) based on the hetero-nitrogen and the hydroxyl group of inosine molecules. The single crystal exhibits the spiral structure along b axis formed due to O–H···O and O–H···N hydrogen bonds. As a result, water solubility under 37°C of inosine β-form is higher than that of inosine powder, and β-form inosine is hygroscopically more stable at 98% RH with respect to inosine powder. The β-form of inosine demonstrates the improved in vitro activity against liver cancer cells compared to inosine powder.

A new cocrystal of baicalein, possessing three A-ring-hydroxyl groups and without B-ring-hydroxyl groups and 4,4′-bipyridine (BE·2BPY·H2O) has been prepared and characterized. The structural analyses and Hirshfeld surfaces show that baicalein is hydrogen-bonded to co-crystallized solvent water molecules featuring for example motifs that can be described with graph set R44(8) similar to those reported for the cocrystal of myricetin with 4,4′-bipyridine. A zipper-type architecture is observed in cocrystal BE·2BPY·H2O linking a baicalein, two 4,4′-bipyridine and a solvent water molecules via classical O–H⋯N and O–H⋯O and non-classical C–H⋯O hydrogen bonds into 2D sheets. Additionally, the cocrystal shows better solubility in 0.1 M HCl in comparison to pure baicalein. Cocrystal of baicalein with zipper-type architecture shows improved solubility compared to baicalein.

Keel bone damage (KBD) is more prevalent in alternative laying hen housing systems than in conventional cages, and its incidence differs from strain to strain. However, the information of KBD in Lindian chickens, a native Chinese strain, is limited. To investigate the effect of KBD on fearfulness and physiological indicators of stress in Lindian chickens and commercial laying hens, a total of two hundred 25-wk-old chickens (100 Hy-line Brown and 100 Lindian chickens) were studied for 7 wk. The birds were housed in furnished cages with 10 birds per cage for each strain. At 32-wk of age, the birds in each strain were divided into normal (NK), deviated (DK), and fractured (FK) hens according to the keel bone status. Ten birds in each keel bone status per strain were subsequently selected to collect blood for the determination of stress and fear-related indicators, including corticosterone, serotonin, interleukin-1β, and interleukin-6, and measure fear responses, including novel object test (NOT), human approach test (HAT), and tonic immobility (TI) test. The results showed that egg production was lower and the incidence of keel bone fractures was higher in Lindian chickens than in Hy-line Brown hens (P0.05). Lindian chickens showed a significantly increased whole blood serotonin content, NOT-latency, HAT-score, and TI induction times (P0.05) and decreased serum interleukin-6 content and TI-duration (P0.05) compared with Hy-line Brown hens. Additionally, FK hens had significantly elevated whole blood corticosterone, serum interleukin-1β and interleukin-6 levels, TI-duration, and NOT-latency (P0.05), and a reduced whole blood serotonin content (P0.05) compared with NK and DK hens. Our results indicated that KBD affected stress and fear responses, and this impact was mainly reflected by FK hens compared with NK and DK hens. We suggest that keel bone fractures are the main factor impairing hen welfare. Besides, the incidence of keel bone fractures and stress and fear responses of Lindian chickens are more severe than Hy-line Brown laying hens, indicating that the strain type can affect the health and welfare of laying hens.Keel bone damage (KBD) impairs production performance, welfare, and health in laying hens. This study aimed to compare the incidence of KBD and investigate the effects of KBD on stress and fear in two strains of laying hens. The results showed that commercial Hy-line Brown laying hens had high egg production and low incidence of KBD compared with Lindian chickens, a Chinese native breed. Besides, Lindian chickens had higher blood serotonin content and fear responses to human approach test and novel object test than Hy-line Brown laying hens. In addition, laying hens with keel bone fractures had elevated concentrations of blood corticosterone, interleukin-1β, and interleukin-6, and had a longer duration of tonic immobility and latency to approach a novel object, as well as reduced blood serotonin content compared with laying hens with normal and deviated keel bone. Overall, keel bone fractures caused stress and fear responses, impairing hen welfare; and behavioral and physiological responses in relation to stress and fear differed between strains of hens.

The new-energy industry, which is a key area for coping with climate change, faces serious credit-financing difficulties. This study used a bilateral stochastic frontier model to measure the bargaining power of new-energy companies in China's credit-financing market. We then examined the main factors affecting the bargaining power of new-energy enterprises by comparing the results of the firms in different groups, including corporate-related factors, government policies, and financial development levels. The results showed that the bargaining power of both banks and enterprises had an important influence on the final financing price. In the bargaining process, new-energy companies were at a disadvantage, and financing prices were 17.2% higher than fair prices. The nature of state-owned enterprises hinders the improvement of the bargaining power of new-energy enterprises. Technology competence and relationships with banks are useful ways for new-energy enterprises to improve their bargaining power. In solving the financing difficulties in the credit market of new-energy enterprises, government subsidies are usually ineffective, while improving the financial development level is effective.

Tropospheric ozone threatens crop production in many parts of the world, especially in highly populated countries in economic transition. Crop models suggest substantial global yield losses for wheat, but typically such models fail to address differences in ozone responses between tolerant and sensitive genotypes. Therefore, the purpose of this study was to identify physiological traits contributing to yield losses or yield stability under ozone stress in 18 contrasting wheat cultivars that had been pre-selected from a larger wheat population with known ozone tolerance. Plants were exposed to season-long ozone fumigation in open-top chambers at an average ozone concentration of 70 ppb with three additional acute ozone episodes of around 150 ppb. Compared to control conditions, average yield loss was 18.7 percent, but large genotypic variation was observed ranging from 2.7 to 44.6 percent. Foliar chlorophyll content represented by normalized difference vegetation index and net CO

Chlorophyll content of the flag leaf is an important trait for drought resistance in wheat under drought stress. Understanding the regulatory mechanism of flag leaf chlorophyll content could accelerate breeding for drought resistance. In this study, we constructed a recombinant inbred line (RIL) population from a cross of drought-sensitive variety DH118 and drought-resistant variety Jinmai 919, and analyzed the chlorophyll contents of flag leaves in six experimental locations/years using the Wheat90K single-nucleotide polymorphism array. A total of 29 quantitative trait loci (QTLs) controlling flag leaf chlorophyll were detected with contributions to phenotypic variation ranging from 4.67 to 23.25%. Twelve QTLs were detected under irrigated conditions and 18 were detected under dryland (drought) conditions. Most of the QTLs detected under the different water regimes were different. Four major QTLs (Qchl.saw-3B.2, Qchl.saw-5A.2, Qchl.saw-5A.3, and Qchl.saw-5B.2) were detected in the RIL population. Qchl.saw-3B.2, possibly more suitable for marker-assisted selection of genotypes adapted to irrigated conditions, was validated by a tightly linked kompetitive allele specific PCR (KASP) marker in a doubled haploid population derived from a different cross. Qchl.saw-5A.3, a novel stably expressed QTL, was detected in the dryland environments and explained up to 23.25% of the phenotypic variation, and has potential for marker-assisted breeding of genotypes adapted to dryland conditions. The stable and major QTLs identified here add valuable information for understanding the genetic mechanism underlying chlorophyll content and provide a basis for molecular marker–assisted breeding.

As a barrier for plants to contact with the outside world, epidermal wax plays an important role in resisting biotic and abiotic stresses. In this study, we analyzed the effect of wax content on leaf permeability by measuring the wax loss rate in the leaf. To further clarify the wax composition of the wheat epidermis and its molecular regulation mechanism, we applied untargeted lipidomic and transcriptome analysis on the leaf epidermis wax of Jimai 22 low-wax mutant (waxless) and multi-wax mutant (waxy). Our research showed that the mutant waxy has a slow loss rate, which can maintain higher leaf water content. 31 lipid subclasses and 1,367 lipid molecules were identified. By analyzing the wax differences of the two mutants, we found that the main lipid components of leaf epidermis wax in Jimai 22 were WE (C19-C50), DG (C27-C53), MG (C31-C35), and OAHFA (C31-C52). Carbon chain length analysis showed that, in wheat epidermis wax, WE was dominated by C44 molecules, DG was mainly concentrated in C47, C45, C37, and C31 molecules, C48 played a leading role in OAHFA, and C35 and C31 played a major role in MG. Among them, DG, MG, and OAHFA were detected in wheat leaf wax for the first time, and they were closely related to stress resistance. Compared with the waxy, 6,840 DEGs were detected in the mutant waxless, 3,181 DEGs were upregulated, and 3,659 DEGs were downregulated. The metabolic pattern of main waxy components in the wheat epidermis was constructed according to KEGG metabolic pathway and 46 related genes were screened, including KSC, TER, FAR, WSD1, CER1, MAH1, ALDH7A1, CYP704B1, ACOT1_2_4, CYP86, MGLL, GPAT, ALDH, DPP1, dgkA, plsC, and E2.3.1.158 related genes. The screened wax-related genes were confirmed to be highly reliable by qRT-PCR. In addition, we found TER gene TraesCS6B03G1132900LC in wheat mutant waxless leaves for the first time, which inhibited the synthesis of long-chain acyl-CoA (n+2) by downregulating its expression. These results provide valuable reference information for further study of wheat epidermis wax heredity and molecular regulation.

Rising tropospheric ozone concentrations can cause rice yield losses and necessitate the breeding of ozone-tolerant rice varieties. However, ozone tolerance should not compromise the resistance to important biotic stresses such as the rice blast disease. Therefore, we investigated the interactive effects of ozone and rice blast disease on nine different rice varieties in an experiment testing an ozone treatment, blast inoculation, and their interaction. Plants were exposed to an ozone concentration of 100 ppb for 7 h per day or ambient air throughout the growth period. Half of the plants were simultaneously infected with rice blast inoculum. Grain yield was significantly reduced in the blast treatment (17%) and ozone treatment (37%), while the combination of both stresses did not further decrease grain yields compared to ozone alone. Similar trends occurred for physiological traits such as vegetation indices, normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), Lichtenthaler index 2 (Lic2), and anthocyanin reflectance index 1 (ARI1), as well as stomatal conductance and lipid peroxidation. Ozone exposure mitigated the formation of visible blast symptoms, while blast inoculation did not significantly affect visible ozone symptoms. Although different genotypes showed contrasting responses to the two types of stresses, no systematic pattern was observed regarding synergies or trade-offs under the two types of stresses. Therefore, we conclude that despite the similarities in physiological stress responses to ozone and blast, the tolerance to these stresses does not appear to be genetically linked in rice.

Purpose To evaluate the role of programmed death-ligand 1 (PD-L1) and mammalian target of rapamycin (mTOR) signaling pathway in locally advanced rectal cancer (LARC). Methods Between February 2012 and February 2018, 103 patients with LARC treated by neoadjuvant chemoradiotherapy (neoCRT) and total mesorectal excision (TME) were included. PD-L1, mTOR and p-mTOR of pair-matched pre-neoCRT biopsies and post-neoCRT surgical tissue were evaluated by immunohistochemistry. Results The mean combined positive score (CPS), tumor proportion score (TPS) and immune cell score (IC) of pre-neoCRT were 2.24 (0–70), 1.87 (0–70) and 0.67 (0–10), respectively. The mean CPS, TPS and IC of post-neoCRT were 2.19 (0–80), 1.38 (0–80) and 1.60 (0–20), respectively. Significant difference was observed in terms of IC between pre-neoCRT and post-neoCRT (p = 0.010). The 5-year disease-free survival (DFS) rate of the whole group was 62.4%. Multivariate analysis by Cox model indicated that pre-neoCRT TPS [hazard ratio (HR) 1.052, 95% confidence interval (CI) 1.020–1.086, p = 0.001] and post-neoCRT CPS (HR 0.733, 95% CI 0.555–0.967, p = 0.028) were associated with DFS. In the 89 patients without pathological complete response, p-mTOR and IC were upregulated after neoCRT. Conclusions For patients with LARC treated by neoCRT and TME, p-mTOR and IC were upregulated after neoCRT. Pre-neoCRT TPS and post-neoCRT CPS were independent prognostic predictors of DFS.

Higenamine (HG), a cardioactive component of some foods and medicines, has been listed in the doping category by the International Olympic Committee, which may lead to misuse by athletes. We report the development of a gas chromatography-mass spectrometry (GC–MS) method for determination of HG in various matrix samples (biological samples, different forms of Chinese patent medicine, Chinese herbal medicine) based on acylation derivatization of HG by heptafluorobutyric anhydride. Under optimal conditions, the linearity of HG in the range of 5–200 ng mL−1 was acceptable (R2 > 0.999), and the limit of detection (LOD) and limit of quantitation (LOQ) for HG was 1.52 ng mL−1 and 5 ng mL−1, respectively. Low, medium, and high concentrations (25, 100 and 160 ng mL−1) of HG were added to plasma, urine, oral liquid, capsule, watered bolus, honeyed bolus and Chinese herbal medicine samples, with recovery ranging from 82.70 to 109.80%, intra-day and inter-day precisions were both less than 3.39%. The results indicated that the method had sufficient sensitivity for analysis of biological samples, and Chinese patent and herbal medicine. Keywords: Higenamine, Gas chromatography-mass spectrometry, Derivatization, Heptafluorobutyric anhydride, Multi-matrix

Liver fibrosis usually progresses to liver cirrhosis and even hepatocellular carcinoma. Since activated hepatic stellate cells (aHSCs) are responsible for liver fibrosis, reducing the quantity of aHSCs was considered the essential strategy for clinical antihepatofibrotic therapy. Due to the overexpression of TRAIL receptor 2 (DR5) in aHSCs, human TNF-related apoptosis-inducing ligand (hTRAIL) that could induce aHSCs apoptosis might be feasible for antihepatofibrotic therapy. However, the in vivo aHSCs-apoptosis-induction of hTRAIL is limited by its poor cell-targeting and a short half-life. In this study, we found that platelet-derived growth factor receptor β (PDGFRβ) was co-expressed with DR5 in aHSCs. And the ZPDGFRβ affibody with high affinity for PDGFRβ could bind aHSCs and, thus, accumulate in the fibrotic liver. ZPDGFRβ was fused to hTRAIL to produce the fusion protein Z-hTRAIL. Compared to hTRAIL, Z-hTRAIL showed greater in vitro cell binding and apoptosis-induction in aHSCs. In addition, Z-hTRAIL induced apoptosis of aHSCs but spared other normal liver cells. In vivo, Z-hTRAIL accumulated preferentially in fibrotic livers and exerted greater effects than hTRAIL in inducing aHSCs apoptosis and reducing extracellular matrix (ECM) deposition. These results demonstrated that the antihepatofibrotic effect of hTRAIL was improved by PDGFRβ-targeted delivery. To enhance its pharmacokinetics, Z-hTRAIL was modified with 10 kDa polyethylene glycol (PEG), which significantly (30–40 times) prolonged its half-life. The PEGylated long-acting Z-hTRAIL was more potent than the native Z-hTRAIL in regressing liver fibrosis. These results suggest that the aHSC-targeting and long-acting Z-hTRAIL might serve as a novel tool for antihepatofibrotic therapy.

With our consistent aim to further expand the solid-state landscape via molecular recognition and regulate solubility of tetrahydroberberine (THB), charge-assisted hydrogen bond (CAHB) recognitions and solubility regulation of pharmaceutical salts/cocrystals of THB are comprehensively investigated through exploring four pharmaceutical salts/cocrystals of THB. Four pharmaceutical salts/cocrystals of THB with oxalic acid, malonic acid, maleic acid and fumaric acid were obtained with solution evaporation approach. Comprehensive crystal structures and Hirshfeld surface analyses reveal CAHB recognitions between THBs and coformers are dominant intermolecular interactions to stabilize crystal packing. As expected, solubilities of four pharmaceutical salts/cocrystals of THB have been improved by dicarboxylic acids. Importantly, the dicarboxylic acid with higher solubility can modulate solubility of THB to become higher. The enthalpy of hydration for four pharmaceutical salts/cocrystals is greater than that for neutral pure THB when CAHBs (with electrostatic interactions) exist, which effectively improve solubility of THB. Solvent molecules can disrupt interactions of pharmaceutical salts/cocrystals by solvent···solute interactions when dicarboxylic acids are highly soluble thereby leading to enhancement of dissolution. Research results inspirationally enlightens that the solubility of insoluble APIs could be effectively regulated by utilizing gradient solubility coformers with simultaneously forming CAHBs between active pharmaceutical ingredients and coformers.

Yi Zhong,1,* Tao Su,1,* Qiuxiao Shi,1 Yanru Feng,1 Ze Tao,1 Qiuxia Huang,1 Lan Li,1 Liqiang Hu,1 Shengfu Li,1 Hong Tan,2 Shan Liu,3 Hao Yang1 1Key Lab of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China; 2Department of General Surgery, Chengdu Integrated TCM & Western Medicine Hospital (Chengdu First People’s Hospital), Chengdu 610041, People’s Republic of China; 3Department of Laboratory Medicine, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shan LiuDepartment of Laboratory Medicine, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, No. 32, West Second Section First Ring Road, Chengdu, Sichuan 610072, People’s Republic of ChinaTel +86 28 6138 2518Email shanliusyy@163.comHao YangKey Lab of Transplant Engineering and Immunology, MOH, West China Hospital, No. 1, Keyuan 4th Road, Hi-tech Zone, Chengdu 610041, People’s Republic of ChinaTel +86 28 85164031Email yanghao@scu.edu.cnBackground: Nanoparticles exhibit great promise for improving the solubility and tissue-specific distribution of chemotherapeutic agents; however, the passive and highly variable enhanced permeability and retention (EPR) effects observed in tumors frequently leads to insufficient delivery of nanodrugs into tumors. The tumor-penetrating peptide iRGD can actively enhance tumor-selective delivery of nanoparticles into tumors by binding to integrin and interacting with tissue-penetrating receptor neuropilin-1.Materials and methods: To improve colorectal cancer treatment, in this study, we prepared a paclitaxel (PTX)-loaded PLGA nanoparticle (PLGA-PTX) and evaluated its tumor-targeting and antitumor activity by co-administration with iRGD.Results: Compared to free PTX, encapsulated PTX retained preferential cytotoxicity toward various colorectal cancer cells while effectively sparing healthy cells. PLGA-PTX treatment resulted in cell cycle arrest at the G2/M phase and apoptosis, leading to inhibition of cancer cell migration and invasion. PLGA-PTX combined with iRGD displayed little enhancement of cytotoxicity in vitro. Despite this, iRGD receptors integrin and neuropilin-1 were found to be primarily overexpressed on abundant tumor vessels in mice bearing colorectal tumors. Consequently, co-administration of nanoparticles with iRGD promoted the selective delivery of nanoparticles into tumor tissues in vivo. Additionally, the combined regimen enhanced the antitumor effects compared to those of each individual reagent.Conclusion: Our findings suggest that PLGA nanoparticles combined with the iRGD peptide provide a promising drug delivery strategy for facilitating active drug accumulation into tumors, given that iRGD receptors are overexpressed on tumor vessels. This co-administration system lacking covalent conjugation provides a more convenient means to combine various therapeutic agents with iRGD to achieve personalized nanotherapy.Keywords: tumor penetrating peptide, nanoparticle, iRGD, paclitaxel, tumor vasculature, colorectal cancer

Objective The objective of the study was to evaluate whether any association exists between systemic inflammation score (SIS) and adverse events (AEs) and survival of locally advanced rectal cancer patients treated with total mesorectal excision (TME) followed by adjuvant chemoradiotherapy. Patients and methods All of the 109 rectal cancer patients recruited between May 2008 and June 2015 were treated with TME followed by adjuvant chemoradiotherapy. The prognostic ability of SIS for overall survival (OS) was calculated by the receiver operating characteristic (ROC) curves. Results According to the classification of the SIS, 22 (20.2%), 59 (54.1%) and 28 (25.7%) patients were classified as a score of 2, 1 and 0, respectively. With an area under the curve (AUC) of 0.616, the SIS score of 1 was defined as the optimal cut-off value. Therefore, we divided the patients into the SIS-low group (SIS score of 1 or 0, n=87) and SIS-high group (SIS score of 2, n=22). Multivariate analysis indicated that SIS was associated with OS (HR 0.390, 95% CI 0.186-0.817, P=0.012). The 5-year OS rate in patients without adjuvant chemotherapy was lower than the patients with adjuvant chemotherapy (53.3% vs 75.8%, P=0.010). Multivariate analysis showed that adjuvant chemotherapy was associated with OS (HR 0.217, 95% CI 0.089-0.529, P=0.001). A marginal statistically significant difference was observed in terms of leukopenia during adjuvant chemoradiotherapy between the SIS-low group and the SIS-high group (P=0.05). Conclusion These results suggest that SIS might serve as an independent biomarker for predicting AEs and prognosis in locally advanced rectal cancer treated with TME followed by adjuvant chemoradiotherapy. Strengthening treatment may be administered to locally advanced rectal cancer with high SIS score obtained before adjuvant chemoradiotherapy.

Purpose The aim of the present study was to evaluate the prognostic value of magnetic resonance imaging (MRI)‒determined lymph nodal necrosis (LNN) in nasopharyngeal carcinoma (NPC) and explore the feasibility of an N-classification system based on the 8th edition of the American Joint Committee on Cancer (AJCC) system. Materials and methods The MRI scans of 616 patients with newly diagnosed stage T1-4N1-3M0 NPC who were treated with definitive intensity-modulated radiotherapy (IMRT) were reviewed. Results Multivariate analysis showed that LNN was an independent negative prognostic predictor of distant metastasis free survival (hazard ratio, 1.634; 95% confidence interval, 1.023 to 2.609; p=0.040) and overall survival (hazard ratio, 2.154; 95% confidence interval, 1.282 to 3.620; p=0.004). Patients of classification N1 disease with LNN were reclassified as classification N2, and classification N2 disease with LNN as classification N3 in the proposed N-classification system. Correlation with death and distant failure was significant, and the total difference between N1 and N3 was wider with the proposed system. Conclusion MRI-determined LNN is an independent negative prognostic factor for NPC. The proposed N classification system is powerfully predictive.

In this paper, new two-photon fluorescent nucleic acid probes, 3-(N-methyl -2- vinyl quinolinium iodide)-9-ethylcarbazole (M2EQC) and 3,6-di-(N-methyl -2- vinyl quinolinium iodide)-9-ethylcarbazole (DM2EQC), have been designed and synthesized. The two compounds are fluorescent DNA probes with long wavelength emission (670 and 650 nm, respectively) irradiated by near-infrared light. The new probes exhibit an obvious improvement in the two-photon absorption action cross-section (222.68 GM and 290.72 GM, respectively). They show the same nuclear localization level as Hoechst 33342 when staining 3T3 cells together. The bioluminescence imaging effect of the compound’s stained cells excited by the 800 nm laser was better than that at 400 nm. The probes also show three-dimensional imaging capability of a single nucleus and lower cytotoxicity, besides the lower fluorescence bleaching effect and higher optical stability than the reported two-photon nucleic acid probe. The fluorescence decay rate was 0.023%/min and 0.020%/min, respectively. The quantum chemical calculation results reveal the reason for spectra changing and we speculate the mechanism of the probes binding with DNA.

Glyphosate (GLY) is the most widely used broad-spectrum, non-selective herbicide in the world, whose main degradation product is aminomethyl phosphonic acid (AMPA). Because of long-term and large-scale use, residual GLY and AMPA in the environment pose great environmental and human health threats. The purpose of this study is to evaluate the effects and mechanism of residual low-concentrations of GLY and AMPA in the environment on the development of zebrafish embryos. Zebrafish embryos were exposed to 0, 1, 10, 100, and 700 ng·mL−1 GLY and AMPA for 72 h (from 2 to 74 h post-fertilization). With increasing exposure dose, heart rates of both embryos and larvae showed a rising trend and obvious arrhythmia appeared. Defects in cardiac development and function of zebrafish juveniles may be related to altered transcription levels of cardiac development genes (TBX5, NKX2.5, BMP4) and apoptosis genes (Bcl-2, Bax). In addition, pericardial edema and bone deformation of zebrafish embryos may be caused by inhibition of Na+/K+-ATPase and Ca2+-ATPase after exposure to GLY and AMPA. The present results demonstrated that at typical environmental residual concentrations of GLY and AMPA had similar developmental toxicity in zebrafish embryos.

Ascorbate (Vitamin C) fulfills various functions in plant photosynthesis and abiotic stress tolerance. The four key enzymes involved in the ascorbate-turnover pathway are ascorbate peroxidase, ascorbate oxidase, monodehydroascorbate reductase, and dehydroascorbate reductase. Several reports have shown the pivotal roles of these enzymes in plant development and stress tolerance. Therefore, reliable and rapid assay protocols are required for researchers to investigate their enzymatic activities during plant development and stress responses. Previously published methods for analyzing these enzymatic activities rely on cuvette spectrophotometers, which can only handle one sample per test, leading to a prolonged investigation. In this protocol, we employed a 96-well microplate reader to analyze at least eight samples with two technical replicates simultaneously. We analyzed two rice (Oryza sativa L.) genotypes with distinct ascorbate oxidase and dehydroascorbate reductase activities to demonstrate the assay process, including plant growth, sample preparation, reaction setup, and data analysis. Our protocol provides a high throughput method for investigating ascorbate turnover-related enzymatic activities in plants.

Smart cities are a leading concept on the current global urbanization agenda. Despite such widespread acclaim, with respect to their actual development, planners face many operational challenges; one such obstacle involves a lack of inclusion for diverse actors in existing research methods. Collaboration in the planning process can be enhanced by formulating an empirical research framework for evaluating stakeholder opinions and potential conflicts. This paper seeks to isolate the stakeholder’s perspective as an assessment factor for smart city development. The researchers utilize a Q methodology to extract stakeholder attitudes and subjective opinions in the case of the Smart Gusu project in China. Conclusions drawn from this research will contribute toward the broader subjective landscape of knowledge involving our ongoing understanding of smart cities and their implications toward evolving urbanization strategies. By continuing to develop empirical tools for mapping conflicts within the expanding field of smart cities, this chapter aims to contribute to the efficiency of associated and future projects.

Ascorbate (Vitamin C) fulfills various functions in plant photosynthesis and abiotic stress tolerance. The four key enzymes involved in the ascorbate-turnover pathway are ascorbate peroxidase, ascorbate oxidase, monodehydroascorbate reductase, and dehydroascorbate reductase. Several reports have shown the pivotal roles of these enzymes in plant development and stress tolerance. Therefore, reliable and rapid assay protocols are required for researchers to investigate their enzymatic activities during plant development and stress responses. Previously published methods for analyzing these enzymatic activities rely on cuvette spectrophotometers, which can only handle one sample per test, leading to a prolonged investigation. In this protocol, we employed a 96-well microplate reader to analyze at least eight samples with two technical replicates simultaneously. We analyzed two rice (Oryza sativa L.) genotypes with distinct ascorbate oxidase and dehydroascorbate reductase activities to demonstrate the assay process, including plant growth, sample preparation, reaction setup, and data analysis. Our protocol provides a high throughput method for investigating ascorbate turnover-related enzymatic activities in plants.

Colorectal cancer (CRC) is one of the most common types of malignancy and the third most commonly diagnosed form of cancer worldwide, ranking as the fourth leading cause of cancer-associated mortality. MicroRNA (miR)-576-5p has been reported to be highly expressed in patients with CRC; however, its biological role remains unclear. The present study aimed therefore to investigate the biological role and underlying mechanism of miR-576-5p in CRC cell line SW480. The viability of SW480 cells following transfection with miR-576-5p mimic or inhibitor was analyzed using MTT assay. Wound healing and Transwell assays were performed to determine the cell migratory and invasive abilities, respectively. A dual luciferase reporter assay was used to verify the predicted binding site between miR-576-5p and Wnt5a. Reverse transcription-quantitative PCR and western blotting were used to analyze the expression levels of miR-576-5p, E-cadherin, N-cadherin, vimentin, Snail1, Wnt5a, β-catenin, c-myc, cyclin D1 and p/t-c-Jun. Using bioinformatics analysis, high expression of miR-576-5p was found not only in tumor tissues, compared with the normal tissue, but also in CRC cells, compared with NCM460 cells. Furthermore, the inhibition of miR-576-5p expression significantly decreased the cell viability and the migratory and invasive abilities of SW480 cells, and suppressed the epithelial-to-mesenchymal transition (EMT). In addition, miR-576-5p could interact with Wnt5a and regulate the expression level of Wnt5a in order to influence the activity of Wnt/β-catenin signaling. The results from rescue experiments further demonstrated that the effect of miR-576-5p overexpression on cell metastasis and EMT was reversed by Wnt5a overexpression or treatment with XAV-939, which is an inhibitor of the Wnt/β-catenin signaling pathway. In conclusion, the findings from the present study suggested that inhibition of miR-576-5p may suppress SW480 cell metastasis and EMT by targeting Wnt5a and regulating the Wnt5a-mediated Wnt/β-catenin signaling pathway, providing a potential therapeutic target for the treatment of CRC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive antitumor drug candidate for precision cancer therapy due to its superior selective cytotoxicity in a variety of tumor cells. However, the clinical application of TRAIL in cancer therapy has been limited by its poor tumor-homing capacities and short half-life. Herein, we designed a tridomain TRAIL variant, Z-ABD-TRAIL, by sequentially fusing the platelet-derived growth factor receptor beta (PDGFRβ)-specific affibody Z

We report three new types of two-photon-absorption compounds, which contain a carbazole center core linked to two quinolinium units. Their action two-photon absorption cross-sections are 287.13, 267.75, and 236.25 GM. Under near-infrared excitation, their fluorescence emissions are observed at approximately 650 nm. The new compounds can stain deoxyribonucleic acid (DNA) with the same level of nuclear localization as that of Hoechst 33342. Under continuous irradiation with a near-infrared laser, the fluorescence decays of the three new compounds were less than those of 4,6-diamidino-2-phenylindole; the average fluorescence decay rates were 0.014, 0.015, and 0.014% s-1. They are expected to become new two-photon fluorescent probes of DNA.

Background Development of wheat cultivars with multiple disease resistance and high quality are major objectives in modern wheat breeding programs. Gene stacking is an efficient approach to achieve this target. In this study, we pyramided yellow rust resistance gene (Yr26), powdery mildew resistance gene (ML91260) and high-molecular-weight glutenin subunits Dx5 + Dy10 into the dwarf mutant of an elite wheat cultivar, Xiaoyan22. Results Six pyramided wheat lines were obtained by molecular marker-assisted selection (MAS) and field evaluation of disease resistance. The desirable agronomic traits of pyramided lines, their identity with the original cultivar Xiaoyan22 except for plant height, tiller number and disease resistance, was achieved in this study. Meanwhile, the yield of pyramided lines is higher than Xiaoyan22 in the field test. In addition, analysis of flour quality indicated that the dough stability time of pyramided lines was longer than that of Xiaoyan22. Conclusions Six pyramided wheat lines with two disease resistance and high quality were achieved in this study. It is feasible to improve multiple agronomic traits simultaneously by rational application of MAS.