Pain control and neonatal outcomes in 211 women under epidural anesthesia during childbirth at high altitude in Qinghai, China.

Background: High altitudes are characterized by low-pressure oxygen deprivation. This is further exacerbated with increasing altitude. High altitudes can be associated with reduced oxygenation, which in turn, can affect labor, as well as maternal and fetal outcomes. Epidural anesthesia can significantly relieve labor pain. This study aimed to assess the effects of elevation gradient changes at high altitude on the analgesic effect of epidural anesthesia, labor duration, and neonatal outcomes. Methods: We divided 211 women who received epidural anesthesia into groups according to varying elevation of their residence (76 in Xining City, mean altitude 2,200 m; 63 in Haibei Prefecture, mean altitude 3,655 m; and 72 in Yushu Prefecture, mean altitude 4,493 m). The analgesic effect was assessed using a visual analog scale (VAS). Labor duration was objectively recorded. The neonatal outcome was assessed using Apgar scores and fetal umbilical artery blood pH. Results: VAS scores among the three groups did not differ significantly (p > 0.05). The neonatal Apgar scores in descending order were: Xining group > Haibei group > Yushu group (p < 0.05). The stage of labor was similar among the three groups (p > 0.05). Fetal umbilical artery blood pH in descending order were: Xining group > Haibei group > Yushu group (p < 0.05). Conclusion: Elevation gradient changes in highland areas did not affect the efficacy of epidural anesthesia or labor duration. However, neonatal outcomes were affected.

SLC25A28 Overexpression Promotes Adipogenesis by Reducing ATGL.

Adipose tissue dysfunction is seen among obese and type 2 diabetic individuals. Adipocyte proliferation and hypertrophy are the root causes of adipose tissue expansion. Solute carrier family 25 member 28 (SLC25A28) is an iron transporter in the inner mitochondrial membrane. This study is aimed at validating the involvement of SLC25A28 in adipose accumulation by tail vein injection of adenovirus (Ad)-SLC25A28 and Ad-green fluorescent protein viral particles into C57BL/6J mice. After 16 weeks, the body weight of the mice was measured. Subsequently, morphological analysis was performed to establish a high-fat diet (HFD)-induced model. SLC25A28 overexpression accelerated lipid accumulation in white and brown adipose tissue (BAT), enhanced body weight, reduced serum triglyceride (TG), and impaired serum glucose tolerance. The protein expression level of lipogenesis, lipolysis, and serum adipose secretion hormone was evaluated by western blotting. The results showed that adipose TG lipase (ATGL) protein expression was reduced significantly in white and BAT after overexpression SLC25A28 compared to the control group. Moreover, SLC25A28 overexpression inhibited the BAT formation by downregulating UCP-1 and the mitochondrial biosynthesis marker PGC-1α. Serum adiponectin protein expression was unregulated, which was consistent with the expression in inguinal white adipose tissue (iWAT). Remarkably, serum fibroblast growth factor (FGF21) protein expression was negatively related to the expansion of adipose tissue after administrated by Ad-SLC25A28. Data from the current study indicate that SLC25A28 overexpression promotes diet-induced obesity and accelerates lipid accumulation by regulating hormone secretion and inhibiting lipolysis in adipose tissue.

NatD epigenetically activates FOXA2 expression to promote breast cancer progression by facilitating MMP14 expression.

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation of histone H4 (Nt-Ac-H4), but its role in breast cancer metastasis remains unknown. Here, we show that depletion of NatD directly represses the expression of FOXA2, and is accompanied by a significant reduction in Nt-Ac-H4 enrichment at the FOXA2 promoter. We show that NatD is commonly upregulated in primary breast cancer tissues, where its expression level correlates with FOXA2 expression, enhanced invasiveness, and poor clinical outcomes. Furthermore, we show that FOXA2 promotes the migration and invasion of breast cancer cells by activating MMP14 expression. MMP14 is also upregulated in breast cancer tissues, where its expression level correlates with FOXA2 expression and poor clinical prognosis. Our study shows that the NatD-FOXA2-MMP14 axis functions as a key signaling pathway to promote the migratory and invasive capabilities of breast cancer cells, suggesting that NatD is a critical epigenetic modulator of cell invasion during breast cancer progression.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Dietary supplementation of VA enhances growth, feed utilization, glucose and lipid metabolism, appetite, and antioxidant capacity of Chinese perch (Siniperca chuatsi).

The aim of this study was to investigate the effect of dietary vitamin A on juvenile Chinese perch (Siniperca chuatsi). Chinese perch were fed with five experimental diets containing 0, 20, 40, 60, and 80 mg VA·kg-1 for 8 weeks. Results showed that dietary vitamin A significantly influenced the fish's growth, feed utilization, glucose and lipid metabolism, appetite, and antioxidant capacity. Vitamin A-supplemented groups had higher weight gain rate (WGR) and specific growth rate (SGR) compared to the control group. Feed conversion ratio (FCR) was also lower in the vitamin A-supplemented groups. Dietary vitamin A had no significant effect on the survival rate (SR). Compared to the control group, fish fed with vitamin A had increased feed intake (FI), and the expression of appetite-promoting genes (npy and agrp) was significantly higher in the 40 mg VA·kg-1 group. Vitamin A also enhanced the utilization of dietary protein by Chinese perch. The serum glucose content of the fish fed with 40 mg VA·kg-1 diet was significantly higher than that of the control group and 20 mg VA·kg-1 diet, indicating that the promoting effect of VA on gluconeogenesis was greater than that on glycolysis. Additionally, dietary vitamin A increased the expression of lipid metabolism-related genes (hl and fas) and antioxidant genes (nrf2 and gpx) in the fish. These results suggest that the optimal vitamin A requirement of juvenile Chinese perch bream was estimated to be 37.32 mg VA·kg-1 based on broken-line regression analysis of WGR. In conclusion, this study provides valuable insights into the potential benefits of dietary vitamin A on the growth, metabolism, and antioxidant capacity of Chinese perch.

Dietary soybean lecithin promoted growth performance and feeding in juvenile Chinese perch (Siniperca chuatsi) could be by optimizing glucolipid metabolism.

To explore the potential benefits of dietary phospholipids (PLs) in fish glucose metabolism and to promote feed culture of Chinese perch (Siniperca chuatsi), we set up six diets to feed Chinese perch (initial mean body weight 37.01 ± 0.20 g) for 86 days, including: Control diet (CT), 1% (SL1), 2% (SL2), 3% (SL3), 4% (SL4) soybean lecithin (SL) and 2% (KO2) krill oil (KO) supplemental diets (in triplicate, 20 fish each). Our study found that the SL2 significantly improved the weight gain rate and special growth rate, but the KO2 did not. In addition, the SL2 diet significantly improved feed intake, which is consistent with the mRNA levels of appetite-related genes (npy, agrp, leptin A). Additionally, in the CT and SL-added groups, leptin A expression levels were nearly synchronized with serum glucose levels. Besides, the SL2 significantly upregulated expression levels of glut2, gk, cs, fas and downregulated g6pase in the liver, suggesting that it may enhance glucose uptake, aerobic oxidation, and conversion to fatty acids. The SL2 also maintained the hepatic crude lipid content unchanged compared to the CT, possibly by significantly down-regulating the mRNA level of hepatic lipase gene (hl), and by elevating serum low-density lipoprotein (LDL) level and intraperitoneal fat ratio in significance. Moreover, the serum high-density lipoprotein levels were significantly increased by PL supplementation, and the SL2 further significantly increased serum total cholesterol and LDL levels, suggesting that dietary PLs promote lipid absorption and transport. Furthermore, dietary SL at 1% level could enhance non-specific immune capacity, with serum total protein level being markedly higher than that in the CT group. In conclusion, it is speculated that the promotion of glucose utilization and appetite by 2% dietary SL could be linked. We suggest a 1.91% supplementation of SL in the diet for the best growth performance in juvenile Chinese perch.

Confocal microscopy multi-focus image fusion method based on axial information guidance.

Aiming at the problems of poor anti-interference of existing pixel-level fusion rules and low efficiency of transform domain fusion rules, this study proposes a confocal microscopic multi-focus image fusion method (IGCM) based on differential confocal axial information guidance. Unlike traditional multi-focus image fusion (MFIF) methods, IGCM uses height information rather than grayscale or frequency to determine clear areas. First, the differential confocal axial measurement curve is calibrated to determine the suitable scan step u. Second, the image set required for fusion is constructed by performing a hierarchical scan of the measurement samples. Then, multiple differential image pairs are constructed using the step size u and the set of images, and the extraction area of the current reference image is decided based on the height obtained from the differential image. Finally, the regions determined by each reference image are extracted and the duplicated pixels are averaged to obtain the MFIF image. The results were that IGCM improves the interference immunity based on pixel-level image fusion compared to the maximum peak fusion method. Compared with other MFIFs, IGCM has excellent fusion efficiency while ensuring fusion clarity, which can meet the application scenario of real-time fusion and offers a new approach to panoramic depth images for confocal devices.

Dietary supplementation of pyridoxine can enhance the growth performance and improve the protein, lipid utilization efficiency of mandarin fish (Siniperca chuatsi).

This study aimed to assess the effect of pyridoxine supplementation in the mandarin fish diet on growth performance, protein and lipid metabolism, and liver and intestinal histology. Mandarin fish were fed six diets with different levels of pyridoxine (2.67 mg/kg (control), 4.41 mg/kg, 6.57 mg/kg, 10.25 mg/kg, 17.93 mg/kg, 33.12 mg/kg diet) for 8 weeks, and samples were collected for analysis. The findings demonstrated that feeding mandarin fish a diet with 6.57 mg/kg pyridoxine led to a significant increase in weight gain rate (WGR), protein efficiency ratio (PER), whole-body crude protein, whole-body crude lipid, serum protein, cholesterol (CHO), triacylglycerol (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and alkaline phosphatase (ALP), as well as significantly lower serum glucose (GLU) and feed conversion ratio (FCR), compared to the control group (P < 0.05). Furthermore, we found a significant upregulation of the relative expression of genes associated with hepatic lipid oxidation and synthesis (hl, lpl, pparα, cpt1, cs, srebp1, and fas) and proteolysis (ast, alt, and gdh) in fish fed a diet containing 6.57 mg/kg pyridoxine (P < 0.05). Regarding the histological analysis, we observed a notable decrease in the quantity of intestinal mucus-secreting cells when the fish fed a diet containing 10.25 mg/kg pyridoxine (P < 0.05). These findings suggest that dietary pyridoxine supplementation promotes mandarin fish growth by improving the efficiency of protein and lipid utilization. Additionally, we used a broken-line regression analysis to estimate the optimal dietary pyridoxine requirement for mandarin fish in the range of 6.17-6.41 mg/kg based on WGR, FCR, and PER.

Differential confocal over-range determination method based on an information theory.

The existing differential confocal axial three-dimensional (3D) measurement method cannot determine whether the surface height of the sample in the field of view is within its effective measurement range. Therefore, in this paper, we propose a differential confocal over-range determination method (IT-ORDM) based on an information theory to determine whether the surface height information of the sample to be examined is within the effective measurement range of the differential confocal axial measurement. First, the IT-ORDM finds the boundary position of the axial effective measurement range by the differential confocal axial light intensity response curve. Then the effective intensity measurement ranges of the pre-focus axial response curve (ARC) and the post-focus ARC are determined by the correspondence between the boundary position and the ARC. Finally, the intersection operation of the pre-focus image of effective measurement and the post-focus image of effective measurement is used to realize the extraction of the effective measurement area of the differential confocal image. The experimental results show that the IT-ORDM can effectively determine and restore the 3D shape of the measured sample surface at the reference plane position in the multi-stage sample experiments.

The SMARCA4R1157W mutation facilitates chromatin remodeling and confers PRMT1/SMARCA4 inhibitors sensitivity in colorectal cancer.

Genomic studies have demonstrated a high frequency of genetic alterations in components of the SWI/SNF complex including the core subunit SMARCA4. However, the mechanisms of tumorigenesis driven by SMARCA4 mutations, particularly in colorectal cancer (CRC), remain largely unknown. In this study, we identified a specific, hotspot mutation in SMARCA4 (c. 3721C>T) which results in a conversion from arginine to tryptophan at residue 1157 (R1157W) in human CRC tissues associated with higher-grade tumors and controls CRC progression. Mechanistically, we found that the SMARCA4R1157W mutation facilitated its recruitment to PRMT1-mediated H4R3me2a (asymmetric dimethylation of Arg 3 in histone H4) and enhanced the ATPase activity of SWI/SNF complex to remodel chromatin in CRC cells. We further showed that the SMARCA4R1157W mutant reinforced the transcriptional expression of EGFR and TNS4 to promote the proliferation of CRC cells and patient-derived tumor organoids. Importantly, we demonstrated that SMARCA4R1157W CRC cells and mutant cell-derived xenografts were more sensitive to the combined inhibition of PRMT1 and SMARCA4 which act synergistically to suppress cell proliferation. Together, our findings show that SMARCA4-R1157W is a critical activating mutation, which accelerates CRC progression through facilitating chromatin recruitment and remodeling. Our results suggest a potential precision therapeutic strategy for the treatment of CRC patients carrying the SMARCA4R1157W mutation.

Biomimetic supramolecular polyurethane with sliding polyrotaxane and disulfide bonds for strain sensors with wide sensing range and self-healing capability.

To prolong the service life of flexible electronic materials, polymeric matrixes with excellent self-healing capability and integrated mechanical properties are highly desirable, but the balance between the self-healing capability and mechanical properties is a grand challenge. Here, polyrotaxanes as sliding crosslinkers and dynamic disulfide bonds are incorporated into the main chains of polyurethane (PU) via one-pot synthesis, which endows the PU with polydisperse hard/soft segments, high density of self-healing points and energy dissipation. Based on this judicious molecular design, the PU elastomers exhibit exceptional mechanical properties, such as high stretchability (1167 % with a tensile strength of 3.49 MPa), high fracture energy (20,775 J m-2) and high puncture energy (200.70 mJ). Moreover, due to the presence of dynamic reversible hydrogen and disulfide bonds, the elastomer could achieve stress and strain repair efficiencies of 93.98 % and 99.21 % at 100 ℃ within 1 h, respectively. The above-mentioned superiorities enable the bioinspired strain sensors to possess a large sensing range (∼596 %), high sensitivity (∼79.98), short response time (∼128 ms), along with excellent reliability and self-healing ability. Besides, the strain sensor exhibits remarkable recyclability and prominent reprocessability, which nicely solves the pollution by discarded electronics.

The value of Copeptin, myocardial fatty acid-binding protein and myocardial markers in the early diagnosis of acute myocardial infarction.

OBJECTIVE: To evaluate and detect the value of Copeptin, myocardial fatty acid binding protein (H-FABP) and myocardial markers in the early diagnosis of acute myocardial infarction (AMI). METHOD: A retrospective analysis was carried out in 153 patients with chest pain who came to Xianyang Hospital of Yan'an University from August 2019 to April 2022, of whom 87 patients were finally diagnosed with AMI. Cardiac troponin I (cTnI), Copeptin, and H-FABP levels were measured immediately at the patient's visit. Receiver operating characteristic (ROC) curve was drawn to evaluate and compare the value of Copeptin, H-FABP and cTnI in early diagnosis of AMI and their joint effect in improving the accuracy of early diagnosis of AMI. RESULTS: (1) The levels of Copeptin, H-FABP and cTnI in AMI patients were evidently higher than those in non-AMI patients with chest pain. (2) The diagnostic sensitivity and specificity of Copeptin were 82.89% and 64.37%, respectively. Those of cTnI were 78.95% and 64.37% respectively, and those of H-FABP were 85.53% and 75.86%, respectively. The AUC size under the ROC curve was H-FABP > hopeptin > cTnI. (3) Joint detection of Copeptin, H-FABP and cTnI was better than mono-detection in early diagnosis of AMI. CONCLUSION: H-FABP has high accuracy in detecting early AMI, which is better than cTnI and Copeptin, but the joint detection of the three is of the highest value.

Polyhedral Oligomeric Silsesquioxane Encountering Tannic Acid: A Mild and Efficient Strategy for Interface Modification on Carbon Fiber Composites.

Designing and controlling the interfacial chemistry and microstructure of the carbon fiber is an important step in the surface modification and preparation of high-performance composites. To address this issue, a tannic acid (TA)/polyhedral oligomeric silsesquioxane (POSS) hybrid microstructure, similar to the topological structure, is designed on the fiber surface by one-pot synthesis under mild conditions. Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) show that the functionality and surface roughness of the fiber are significantly broadened. Correspondingly, the tensile strength (TS) of CF-TA/POSS100 and interlaminar shear strength (ILSS) of CF-TA/POSS100-based composites increased by 18 and 34%, respectively. Following that, a failure mechanism study is conducted to demonstrate the interphase structure containing TA/POSS, which is quite critical in optimizing the mechanical performance of the multiscale composites. Moreover, the strategy for the use of TA for constructing a robust coating to replace the traditional modification without affecting the fiber intrinsic strength is an improved design and provides a new idea for the development of high-performance composites.

[Analysis of animal models of stable chronic obstructive pulmonary disease based on clinical features of traditional Chinese medicine and western medicine].

This study analyzed the advantages and disadvantages of existing animal models in China and abroad and their goodness of fit based on the clinical characteristics and diagnostic criteria of stable chronic obstructive pulmonary disease(COPD) in traditional Chinese medicine(TCM) and western medicine, followed by the collation and summarization of model evaluation methodologies. The results showed that the existing animal models of stable COPD were mainly modeled via smoke exposure or the combination of multiple methods like smoke exposure plus lipopolysaccharide or protease or bacterial infection. These animal models generally failed to simulate the clinical characteristics of TCM, and their goodness of fit in western medicine was higher than that in TCM. There is a lack of research on the animal models of stable COPD and the disease-syndrome combination models. Although the modeling is guided by the pathogenesis or mechanism of diseased humans, the established models were still not identical with the actual clinical situations. In-depth research is needed to develop quantitative standards for stable COPD models.

STEM multiplication nano-moiré method with large field of view and high sensitivity.

Strain is one of the important factors that determine the photoelectric and mechanical properties of semiconductor materials and devices. In this paper, the scanning transmission electron microscopy multiplication nano-moiré method is proposed to increase the measurement range and sensitivity for strain field. The formation principle, condition, and measurement range of positive and negative multiplication moiré fringes (PMMFs and NMMFs) are analysed in detail here. PMMF generally refers to the multiplication of field of view, NMMF generally refers to the multiplication of displacement measurement sensitivity. Based on the principle of multiplication nano-moiré, Theoretical formulas of the fringe spacing and strain field are derived. Compared with geometric phase analysis of deformation measurements based on high-resolution atom images, both the range of field of view and the sensitivity of displacement measurements of the multiplication moiré method are significantly improved. Most importantly, the area of field of view of the PMMF method is increased by about two orders of magnitude, which is close to micrometre-scale with strain measurement sensitivity of 2 × 10-5. In addition, In order to improve the quality of moiré fringe and the accuracy of strain measurement, the secondary moiré method is developed.The strain laws at the interface of the InP/InGaAs superlattice materials are characterised using the developed method.

Design, synthesis and biological evaluation of 2-methyl-(1,1'-biphenyl)-pyrimidine conjugates.

Small molecule inhibitors of biphenyl structure as core backbone have shown a significant effect on PD-1/PD-L1 axis, and 2-amino-pyrimidine structure is a promising privileged scaffold in medicinal chemistry and drug discovery. We designed by combination principles and synthesized 27 novel compounds with N-((2-methyl-[1,1'-biphenyl]-3-yl)methyl)pyrimidin-2-amine as a basic skeletal structure, and their anti-cancer activity was evaluated. Among compounds, 15a-d and 16b displayed strong anti-cancer effects on 9 tested cancer cell lines, in particular, the 16b did the highest inhibitive activity, but against HepG2 cells, and possessed the lowest IC50 value of 2.08 µΜ towards HT-29 cells.

Exosomes as drug carriers for cancer therapy and challenges regarding exosome uptake.

With the development of biomedicine, exosomes are rapidly developing as a new therapy for tumors. As biological carriers, exosomes possess biological activity and can transport their contents between cells. The contents are natural or artificially loaded with biomolecules or chemical drugs. Exosomes deliver biomolecules or chemical drugs into the pathological sites of recipient, which can effectively inhibit the progression of tumors. However, the treatments of tumors through the delivery of exosomes are not sufficiently accurate or efficient, and various challenges need to be overcome. Exosomes from different cell sources possess different characteristics, as well as different specificity for various cells. In the future, for the promotion and application of exosomes, it is of great significance to understand how to select appropriate exosomes loaded with biomolecules or chemical drugs for different tumors types, and how to deliver exosomes to recipient cells accurately and efficiently. This review introduces the application and challenges of exosomes as delivery carriers in tumors.

Knockdown of TGF-ß1 expression in human umbilical cord mesenchymal stem cells reverts their exosome-mediated EMT promoting effect on lung cancer cells.

The effect of mesenchymal stem cells (MSCs) on lung cancer cells is controversial, and the underlying mechanisms remain unclear, which harms the utilization of MSCs in tumor therapy. In this study, we found that human umbilical cord MSC-conditioned medium (MSC-CM) promotes EMT, invasion, and migration, yet inhibits proliferation and promotes apoptosis of lung cancer cells. The EMT-promoting effect of MSCs was mediated by exosomes derived from MSCs (MSC-exo) and eliminated by inhibiting exosome release. Moreover, silencing TGF-ß1 expression in MSCs can revert the EMT-promoting effect and enhance the anti-proliferative and pro-apoptotic effect of MSCs on lung cancer cells via MSC-exo. Further investigation found that Smad2/3, Akt/GSK-3ß/ß-catenin, NF-κB, ERK, JNK, and p38 MAPK in TGF-ß1 signaling pathways could be activated by MSC-exo in lung cancer cells, while silencing TGF-ß1 expression in MSCs may deactivate these pathways. These findings suggest a method by which MSCs may be safely employed in lung cancer therapy.

Determinants of Virulence and In Vitro Development Colocalize on a Genetic Map of Setosphaeria turcica.

Generating effective and stable strategies for resistance breeding requires an understanding of the genetics of host-pathogen interactions and the implications for pathogen dynamics and evolution. Setosphaeria turcica causes northern leaf blight (NLB), an important disease of maize for which major resistance genes have been deployed. Little is known about the evolutionary dynamics of avirulence (AVR) genes in S. turcica. To test the hypothesis that there is a genetic association between avirulence and in vitro development traits, we (i) created a genetic map of S. turcica, (ii) located candidate AVRHt1 and AVRHt2 regions, and (iii) identified genetic regions associated with several in vitro development traits. A cross was generated between a race 1 and a race 23N strain, and 221 progeny were isolated. Genotyping by sequencing was used to score 2,078 single-nucleotide polymorphism markers. A genetic map spanning 1,981 centimorgans was constructed, consisting of 21 linkage groups. Genetic mapping extended prior evidence for the location and identity of the AVRHt1 gene and identified a region of interest for AVRHt2. The genetic location of AVRHt2 colocalized with loci influencing radial growth and mycelial abundance. Our data suggest a trade-off between virulence on Ht1 and Ht2 and the pathogen's vegetative growth rate. In addition, in-depth analysis of the genotypic data suggests the presence of significant duplication in the genome of S. turcica.

A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya.

Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.