First Report of Cercospora chevalieri Causing A Leaf Spot Disease on Amorphophallus konjac in China.

Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 µm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 µm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 µm, with an average of 130 × 6 µm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar（PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch's postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant was inoculated with mycelial plugs, and one leaf was inoculated with a sterile PDA plug. These plants were enclosed in plastic bags for 72 h. Only leaves inoculated with mycelium plugs produced brown lesions, which appeared after 10 to 14 days on inoculated leaves. Control plants treated with sterile PDA plugs remained asymptomatic. This experiment was repeated twice with the same results. C. chevalieri was reisolated from infected leaves and identified based on morphology and Sanger sequencing of the ITS region. To our knowledge, this is the first report of C. chevalieri causing leaf spot on A. konjac and the first report of this species from China (Braun et al. 2014), which provides key information for diagnosis and management of this disease.

Computational identification of novel potential genetic pathogenesis and otherwise biomarkers in acute liver allograft rejection.

Acute cellular rejection (ACR) is a prevalent postoperative complication following liver transplantation (LT), exhibiting an increasing incidence of morbidity and mortality. However, the molecular mechanisms of ACR following LT remain unclear. To explore the genetic pathogenesis and identify biomarkers of ACR following LT, three relevant Gene Expression Omnibus (GEO) datasets consisting of data on ACR or non-ACR patients after LT were comprehensively investigated by computational analysis. A total of 349 upregulated and 260 downregulated differentially expressed genes (DEGs) and eight hub genes (ISG15, HELZ2, HNRNPK, TIAL1, SKIV2L2, PABPC1, SIRT1, and PPARA) were identified. Notably, HNRNPK, TIAL1, and PABPC1 exhibited the highest predictive potential for ACR with AUCs of 0.706, 0.798, and 0.801, respectively. KEGG analysis of hub genes revealed that ACR following LT was predominately associated with ferroptosis, protein processing in the endoplasmic reticulum, complement and coagulation pathways, and RIG-I/NOD/Toll-like receptor signaling pathway. According to the immune cell infiltration analysis, γδT cells, NK cells, Tregs, and M1/M2-like macrophages had the highest levels of infiltration. Compared to SIRT1, ISG15 was positively correlated with γδT cells and M1-like macrophages but negatively correlated with NK cells, CD4+ memory T cells, and Tregs. In conclusion, this study identified eight hub genes and their potential pathways, as well as the immune cells involved in ACR following LT with the greatest levels of infiltration. These findings provide a new direction for future research on the underlying mechanism of ACR following LT.

Host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system.

Cophylogeny has been identified between gut bacteria and their animal host and is highly relevant to host health, but little research has extended to gut bacteriophages. Here we use bee model to investigate host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system. Through metagenomic sequencing upon different bee species, the gut phageome revealed a more variable composition than the gut bacteriome. Nevertheless, the bacteriome and the phageome showed a significant association of their dissimilarity matrices, indicating a reciprocal interaction between the two kinds of communities. Most of the gut phages were host generalist at the viral cluster level but host specialist at the viral OTU level. While the dominant gut bacteria Gilliamella and Snodgrassella exhibited matched phylogeny with bee hosts, most of their phages showed a diminished level of cophylogeny. The evolutionary rates of the bee, the gut bacteria and the gut phages showed a remarkably increasing trend, including synonymous and non-synonymous substitution and gene content variation. For all of the three codiversified tripartite members, however, their genes under positive selection and genes involving gain/loss during evolution simultaneously enriched the functions into metabolism of nutrients, therefore highlighting the tripartite coevolution that results in an enhanced ecological fitness for the whole holobiont.

Thermodynamics and explainable machine learning assist in interpreting biodegradability of dissolved organic matter in sludge anaerobic digestion with thermal hydrolysis.

Dissolved organic matter (DOM) is essential in biological treatment, yet its specific roles remain incompletely understood. This study introduces a machine learning (ML) framework to interpret DOM biodegradability in the anaerobic digestion (AD) of sludge, incorporating a thermodynamic indicator (λ). Ensemble models such as Xgboost and LightGBM achieved high accuracy (training: 0.90-0.98; testing: 0.75-0.85). The explainability of the ML models revealed that the features λ, measured m/z, nitrogen to carbon ratio (N/C), hydrogen to carbon ratio (H/C), and nominal oxidation state of carbon (NOSC) were significant formula features determining biodegradability. Shapley values further indicated that the biodegradable DOM were mostly formulas with λ lower than 0.03, measured m/z value higher than 600 Da, and N/C ratios higher than 0.2. This study suggests that a strategy based on ML and its explainability, considering formula features, particularly thermodynamic indicators, provides a novel approach for understanding and estimating the biodegradation of DOM.

Efficacy and safety of fexuprazan in patients with symptoms and signs of laryngopharyngeal reflux disease: a randomized clinical trial.

PURPOSE: Laryngopharyngeal reflux disease (LPRD) is mainly treated with proton pump inhibitors (PPI) such as esomeprazole, which have shortcomings like delayed absorption and increased osteoporosis. Fexuprazan is a novel potent potassium-competitive acid blocker that inhibits gastric acid secretion with rapid onset and long duration of action. To assess the efficacy and safety of fexuprazan compared to esomeprazole in patients with LPRD. METHODS: This prospective, randomized, double-blinded, multicenter, active-controlled trial was conducted in nine otolaryngologic clinics. Patients with reflux symptom index (RSI) ≥ 13 and reflux finding score (RFS) ≥ 7 were randomly assigned to the fexuprazan or esomeprazole groups, and received fexuprazan 40-mg or esomeprazole 40-mg once daily for 8 weeks. The outcomes were (1) mean change, change rate, and valid rate in RSI, RFS, and LPR-related questionnaires; and (2) adverse events. RESULTS: A total of 136 patients (fexuprazan n = 68, esomeprazole n = 68) were followed up for ≥ 1 month. Each parameter significantly improved after 4 and 8 weeks in each group, with no significant differences between the two groups. For those with severe symptoms (RSI ≥ 18), the fexuprazan group (n = 32) showed more improvement in the mean change and change rate in the RSI than esomeprazole group (n = 31) after 4 weeks (p = .036 and .045, respectively). This phenomenon was especially observed in hoarseness and troublesome cough. CONCLUSION: Fexuprazan improved symptoms and signs without no serious adverse events in patients with LPRD. In patients with severe symptoms, fexuprazan resulted in a faster symptom improvement than PPI. TRIAL REGISTRATION: KCT0007251, https://cris.nih.go.kr/cris/search/detailSearch.do?seq=22100 .

Immunogenicity of a novel inactivated canine adenovirus type 2 variant vaccine for dogs.

Purpose: The immunogenicity of vaccines containing the canine adenovirus (CAdV) type 2 (CAdV-2) variant has not yet been reported. We prepared a novel inactivated CAdV-2 variant vaccine using the CAV2232-41 strain, and evaluated its safety and immunogenicity in raccoon dogs. Materials and Methods: The growth kinetics of CAV2232-41 were determined using Madin-Darby Canine Kidney (MDCK) cells. The nucleotide sequences of CAV2232 and CAV2232-41 were determined by next-generation sequencing. To generate the CAdV-2 variant vaccine, CAV2232-41 propagated in the MDCK cells was inactivated with 0.1% formaldehyde. Two vaccines were prepared by blending inactivated CAV2232-41 with Cabopol and Rehydragel adjuvants. Safety and immunogenicity of the CAV2232C and CAV2232R vaccines were evaluated in guinea pigs. Safety and immunogenicity of the CAV2232C vaccine were also evaluated in raccoon dogs. The virus neutralizing antibody (VNA) titer against CAV2232-41 was measured in sera collected from immunized guinea pigs and raccoon dogs. Results: CAV2232-41 showed the highest viral titer on days 4-6 post-inoculation and had a deletion in the E3 gene, which was confirmed as a CAdV-2 variant. Guinea pigs inoculated with CAV2232C showed slightly higher VNA titers than those inoculated with CAV2232R 2 weeks after booster vaccination. Raccoon dogs immunized with the CAV2232C vaccine developed high mean VNA titers, while non-vaccinated raccoon dogs were antibody-negative. Conclusion: The CAV2232C vaccine is safe and induces a protective VNA titer in raccoon dogs.

Predicting avian diversity based on land use and cover on a national scale.

This study explores the relationship between landscape features and avian diversity in South Korea, examining both taxonomic and functional diversity. The Korean Peninsula serves as a pivotal habitat for resident bird species and a migratory pathway in the East Asia-Pacific flyway. Using a national dataset with block sizes ranging from 3.5 to 4.5 kilometers per side, we found that less urbanized open plains exhibit higher taxonomic diversity, while coastal regions with diverse water bird populations show higher functional diversity. These findings underscore the significance of conserving the existing land types and qualities in specific regions to substantially impact bird distribution and regional biodiversity. Remarkably, closed forests display diversity patterns akin to urban/built-up areas, despite their disparate land use characteristics. The stability of bird diversity indices across different land use types enables us to predict bird diversity indices based on the particular land use and land cover configurations. This study emphasizes the complementary nature of functional biodiversity indices in comprehending bird distribution patterns alongside taxonomic diversity indices.

CD47 and IFT57 Are Colinear Genes That Are Highly Coexpressed in Most Cancers and Exhibit Parallel Cancer-Specific Correlations with Survival.

An association between high CD47 expression and poor cancer survival has been attributed to its function on malignant cells to inhibit phagocytic clearance. However, CD47 mRNA expression in some cancers lacks correlation or correlates with improved survival. IFT57 encodes an essential primary cilium component and is colinear with CD47 across amniote genomes, suggesting coregulation of these genes. Analysis of The Cancer Genome Atlas datasets identified IFT57 as a top coexpressed gene with CD47 among 1156 human cancer cell lines and in most tumor types. The primary cilium also regulates cancer pathogenesis, and correlations between IFT57 mRNA and survival paralleled those for CD47 in thyroid and lung carcinomas, melanoma, and glioma. CD47 ranked first for coexpression with IFT57 mRNA in papillary thyroid carcinomas, and higher expression of both genes correlated with significantly improved overall survival. CD47 and IFT57 mRNAs were coordinately regulated in thyroid carcinoma cell lines. Transcriptome analysis following knockdown of CD47 or IFT57 in thyroid carcinoma cells identified the cytoskeletal regulator CRACD as a specific target of IFT57. CRACD mRNA expression inversely correlated with IFT57 mRNA and with survival in low-grade gliomas, lung adenocarcinomas, and papillary thyroid carcinomas, suggesting that IFT57 rather than CD47 regulates survival in these cancers.

A High-Carbohydrate Diet Induces Cognitive Impairment and Promotes Amyloid Burden and Tau Phosphorylation via PI3K/Akt/GSK-3ß Pathway in db/db Mice.

BACKGROUND: Cognitive impairment is a prevalent complication of type 2 diabetes, influenced significantly by various dietary patterns. High-carbohydrate diets (HCDs) are commonly consumed nowadays; however, the specific impact of HCDs on cognitive function in diabetes remains unclear. METHODS: The objective of this study was to investigate whether an HCD has effects on cognition in diabetes. Eight-week-old diabetic (db/db) mice and wild-type (WT) mice underwent a twelve-week dietary intervention, including a normal diet (ND), an HCD, or a high-fat diet (HFD). Following this, behavioral tests were conducted, and related hippocampal pathology was evaluated. RESULTS: Our results demonstrated that an HCD exacerbated cognitive decline in db/db mice compared to an ND. Additionally, an HCD increased amyloid-ß burden and expression of ß-site APP cleaving enzyme-1. An HCD was also found to promote the phosphorylation of tau protein via the PI3K/Akt/GSK-3ß pathway. Furthermore, an HCD markedly induced neuroinflammation and increased the quantity of microglia and astrocytes. However, these damages induced by an HCD were less severe than those caused by an HFD. CONCLUSIONS: Collectively, our findings indicate that a high intake of carbohydrates can have an adverse impact on cognitive function in diabetes.

Genome-wide identification of the WRKY gene family in blueberry (Vaccinium spp.) and expression analysis under abiotic stress.

Introduction: The WRKY transcription factor (TF) family is one of the largest TF families in plants and is widely involved in responses to both biotic and abiotic stresses. Methods: To clarify the function of the WRKY family in blueberries, this study identified the WRKY genes within the blueberry genome and systematically analyzed gene characteristics, phylogenetic evolution, promoter cis-elements, expression patterns, and subcellular localization of the encoded products. Results: In this study, 57 VcWRKY genes were identified, and all encoding products had a complete WRKY heptapeptide structure and zinc-finger motif. The VcWRKY genes were divided into three subgroups (I-III) by phylogenetic analysis. Group II was divided into five subgroups: IIa, IIb, IIc, IId, and IIe. 57 VcWRKY genes were distributed unevenly across 32 chromosomes. The amino acids ranged from 172 to 841, and molecular weights varied from 19.75 to 92.28 kD. Intra-group syntenic analysis identified 12 pairs of duplicate segments. Furthermore, 34 cis-element recognition sites were identified in the promoter regions of VcWRKY genes, primarily comprising phytohormone-responsive and light-responsive elements. Comparative syntenic maps were generated to investigate the evolutionary relationships of VcWRKY genes, revealing the closest homology to dicotyledonous WRKY gene families. VcWRKY genes were predominantly expressed in the fruit flesh and roots of blueberries. Gene expression analysis showed that the responses of VcWRKY genes to stress treatments were more strongly in leaves than in roots. Notably, VcWRKY13 and VcWRKY25 exhibited significant upregulation under salt stress, alkali stress, and saline-alkali stress, and VcWRKY1 and VcWRKY13 showed notable induction under drought stress. Subcellular localization analysis confirmed that VcWRKY13 and VcWRKY25 function within the nucleus. Conclusion: These findings establish a foundation for further investigation into the functions and regulatory mechanisms of VcWRKY genes and provide guidance for selecting stress-tolerant genes in the development of blueberry cultivars.

Orchestrating NK and T cells via tri-specific nano-antibodies for synergistic antitumor immunity.

The functions of natural killer (NK) and T cells in innate and adaptive immunity, as well as their functions in tumor eradication, are complementary and intertwined. Here we show that utilization of multi-specific antibodies or nano-antibodies capable of simultaneously targeting both NK and T cells could be a valuable approach in cancer immunotherapy. Here, we introduce a tri-specific Nano-Antibody (Tri-NAb), generated by immobilizing three types of monoclonal antibodies (mAbs), using an optimized albumin/polyester composite nanoparticle conjugated with anti-Fc antibody. This Tri-NAb, targeting PDL1, 4-1BB, and NKG2A (or TIGIT) simultaneously, effectively binds to NK and CD8+ T cells, triggering their activation and proliferation, while facilitating their interaction with tumor cells, thereby inducing efficient tumor killing. Importantly, the antitumor efficacy of Tri-NAb is validated in multiple models, including patient-derived tumor organoids and humanized mice, highlighting the translational potential of NK and T cell co-targeting.

Graft-to-recipient weight ratio and risk of systemic inflammatory response syndrome early after liver transplantation in children.

BACKGROUND: Systemic inflammatory responses soon after liver transplantation in children can lead to complications and poor outcomes, so here we examined potential risk factors of such responses. METHODS: Data were retrospectively analyzed for 69 children who underwent liver transplantation at a single center between July 2017 and November 2019 through follow-up lasting up to one years. Numerous clinicodemographic factors were compared between those who suffered early systemic inflammatory response syndrome (SIRS) or not. RESULTS: Of the 69 patients in our analysis, early SIRS occurred in 35 [50.7%, 95% confidence interval (CI), 38.6-62.8%]. Those patients showed significantly higher graft-to-recipient weight ratio (3.69 ± 1.26 vs. 3.12 ± 0.99%, P = 0.042) and lower survival rate at one year (85.7% vs. 100%, P = 0.023). Multivariate analysis found graft-to-recipient weight ratio > 4% to be an independent risk factor for early SIRS [odds ratio (OR) 3.8, 95% CI 1.08-13.371, P = 0.037], and a cut-off value of 4.04% predicted the syndrome in our patients, and area under the receiver operating characteristic curve of 0.656 (95% CI 0.525-0.788, P = 0.026). CONCLUSIONS: Graft-to-recipient weight ratio > 4% may predict higher risk of SIRS soon after liver transplantation in children.

Glioma-Stem-Cell-Derived Exosomes Remodeled Glioma-Associated Macrophage via NEAT1/miR-125a/STAT3 Pathway.

Glioblastoma (GBM), as the most common primary brain tumor, usually results in an extremely poor prognosis, in which glioma stem cells (GSCs) and their immunosuppressive microenvironment prominently intervene in the resistance to radiotherapy and chemotherapy that directly leads to tumor recurrence and shortened survival time. The specific mechanism through which exosomes generated from GSCs support the creation of an immunosuppressive microenvironment remains unknown, while it is acknowledged to be engaged in intercellular communication and the regulation of the glioma immunosuppressive microenvironment. The elevated expression of LncRNA-NEAT1 was found in glioma cells after radiotherapy, chemotherapy, and DNA damage stimulation, and NEAT1 could promote the malignant biological activities of GSCs. Emerging evidence suggests that lncRNAs may reply to external stimuli or DNA damage by playing a role in modulating different aspects of tumor biology. Our study demonstrated a promotive role of the carried NEAT1 by GSC-derived exosomes in the polarization of M2-like macrophages. Further experiments demonstrated the mediative role of miR-125a and its target gene STAT3 in NEAT1-induced polarization of M2-like macrophages that promote glioma progression. Our findings elucidate the mechanism by which GSCs influence the polarization of M2-like macrophages through exosomes, which may contribute to the formation of immunosuppressive microenvironments. Taken together, our study reveals the miR-125a-STAT3 pathway through which exosomal NEAT1 from treatment-resistant GSCs contributes to M2-like macrophage polarization, indicating the potential of exosomal NEAT1 for treating glioma.

Spirobipyridine Ligand Enabled Iridium-Catalyzed Site-Selective C-H Activation via Non-Covalent Interactions.

The selective borylation of specific C-H bonds in organic synthesis remains a formidable challenge. In this study, we present a novel spirobipyridine ligand that features a binaphthyl backbone. This ligand facilitates the iridium-catalyzed selective C-H borylation of benzene derivatives. The ligand is designed with "side-arm-wall" substituents that allow vicinal di- or multi-substituted benzene derivatives to approach metal center and effectively block other reactive sites by non-covalent interactions with substrates. The effectiveness of this strategy is demonstrated by the successful selective distal C-H activation of various alkaloids and its broad compatibility with functional groups.

Evaluating the effectiveness of mitigation measures in environmental impact assessments: A comprehensive review of development projects in Korea.

Rapid urbanization and development projects in Korea have posed significant threats to biodiversity; thus, effective mitigation measures are required to preserve natural habitats. Nevertheless, the factors underlying variations in mitigation measure effectiveness according to the disturbance level and surrounding environmental conditions have not been clarified. This study evaluated the effectiveness of mitigation measures implemented in environmental impact assessments (EIAs) of development projects in Korea, with a focus on their effectiveness with respect to the disturbance level and surrounding environmental conditions. A review of 288 EIA reports from selected projects that implemented all 10 mitigation measures classified according to the Wildlife Conservation Comprehensive Plan was conducted. Using the biodiversity tipping point framework, the effects of mitigation measures on biodiversity were categorized into four levels and analyzed. Analysis of variance and redundancy analysis were then performed to discern the variance in mitigation measure effectiveness in terms of the disturbance level, surrounding environment, and species. The results revealed significant variations in the effectiveness of mitigation measures depending on the surrounding environment and disturbance level. Linear projects exhibited a clear impact on various species as the disturbance level increased, whereas area-based projects did not exhibit such pronounced effects. All species demonstrated a negative relationship with development duration, development area, and distance from urban centers. Notably, avian and amphibian species showed a strong negative correlation with the digital elevation model while reptiles and mammals exhibited a strong positive relationship with pre-development biodiversity and distance from protected areas, respectively. Mitigation measures play a key role in alleviating the adverse effects of development projects; therefore, our findings indicate the need for spatially tailored mitigation plans to augment their effectiveness.

Feasibility of two-step approach for screening NTRK fusion in two major subtypes of non-small cell lung cancer within a large cohort.

Our objective is to investigate a cost-effective approach to screen for NTRK fusion in the major subtypes of non-small cell lung cancer (NSCLC). Evaluate the concordance between immunohistochemistry (IHC) and next-generation sequencing (NGS), as well as between fluorescence in situ hybridization (FISH) and NGS, to detect any discrepancies in methodological consistency between lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC). Analyze the factors influencing IHC results. A cohort of 1654 patients with NSCLC underwent screening for NTRK fusion using whole slide IHC. The positive cases were analyzed by both FISH and NGS. Totally, 57 tested positive for pan-TRK, with positivity rates of 0.68% (10/1467) for LADC and 29.01% (47/162) for LSCC. FISH showed separate NTRK1 and NTRK3 rearrangements in two pan-TRK-positive LADCs, while all LSCCs tested negative. NGS confirmed functional NTRK fusion in two FISH-positive cases: one involving TPM3-NTRK1 and the other involving SQSTM1-NTRK3. A non-functional fusion of NTRK2-XRCC1 was detected in LSCC, while FISH was negative. According to our approach, the prevalence of NTRK fusion in NSCLC is 0.12%. The concordance rate between IHC and RNA-based NGS was 20% (2/10) in LADC and 0% (0/162) in LSCC. When the positive criteria increased over 50% of tumor cells showing strong staining, the concordance would be 100% (2/2). A concordance rate of 100% (2/2) was observed between FISH and RNA-based NGS in LADC. The expression of pan-TRK was significantly correlated with the tumor proportion score (TPS) of PD-L1 (p < 0.05) and transcript per million (TPM) values of NTRK2 (p < 0.05). We recommend using IHC with strict criteria to screen NTRK fusion in LADC rather than LSCC, confirmed by RNA-based NGS directly. When the NGS results are inconclusive, FISH validation is necessary.

Validation of an Analytical Method of 3',4',5-Trihydroxy-3-Methoxy-6,7-Methylenedioxyflavone 4'-Glucuronide for Standardization of Spinacia oleracea.

Spinach (Spinacia oleracea) is one of the most famous vegetables worldwide, rich in essential metabolites for various health benefits. It is a valuable plant source that has the potential to be a nutraceutical. This study aimed to evaluate the single characteristic marker compound to establish the validation of HPLC-DAD methods applied to the development of a nutraceutical using spinach samples. Six metabolites (1-6) were identified from the spinach samples such as freeze-dried spinach (FDS) and spinach extract concentrate (SEC) by LC-Q-TOF/MS analysis. Among the six metabolites, 3',4',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4'-glucuronide (TMG) was selected as a marker compound due to its highest abundance and high selectivity. The specificity, accuracy, linearity, precision, repeatability, limit of detection (LOD), and limit of quantification (LOQ) of TMG in the spinach samples (FDS and SEC) were validated according to AOAC international guideline. The specificity was confirmed by monitoring the well separation of the marker compound from other compounds of spinach samples in the base peak intensity (BPI) and ultraviolet (UV) chromatogram. The calibration curve of TMG (15.625~500 µg/mL) had reasonable linearity (R2 = 0.999) considered with LOD and LOQ values, respectively. Recovery rate of TMG was 93-101% for FDS and 90-95% for SEC. The precision was less than 3 and 6% in the intraday and interday. As a result, the HPLC-DAD validation method of TMG in the spinach samples (FDS and SEC) was first established with AOAC and KFDA regulations for approving functional ingredients in functional foods.

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus.

Amorphophallus is a perennial monocotyledonous herbaceous plant native to the southwestern region of China, widely used in various fields such as food processing, biomedicine and chemical agriculture. However, Amorphophallus is a typical thermolabile plant, and the continuous high temperature in summer have seriously affected the growth, development and economic yield of Amorphophallus in recent years. Calmodulin (CaM), a Ca2+ sensor ubiquitous in eukaryotes, is the most important multifunctional receptor protein in plant cells, which affects plant stress resistance by participating in the activities of a variety of signaling molecules. In this study, the key gene AaCaM3 for the Ca2+-CaM regulatory pathway was obtained from A. albus, the sequence analysis confirmed that it is a typical calmodulin. The qRT-PCR results demonstrated that with the passage of heat treatment time, the expression of AaCaM3 was significantly upregulated in A. albus leaves. Subcellular localization analysis revealed that AaCaM3 localized on the cytoplasm and nucleus. Meanwhile, heterologous transformation experiments have shown that AaCaM3 can significantly improve the heat tolerance of Arabidopsis under heat stress. The promoter region of AaCaM3 was sequenced 1,338 bp by FPNI-PCR and GUS staining assay showed that the promoter of AaCaM3 was a high-temperature inducible promoter. Yeast one-hybrid analysis and Luciferase activity reporting system analysis showed that the AaCaM3 promoter may interact with AaHSFA1, AaHSFA2c, AaHSP70, AaDREB2a and AaDREB2b. In conclusion, this study provides new ideas for further improving the signal transduction network of high-temperature stress in Amorphophallus.

Occurrence and characterization of viruses infecting Amorphophallus in Yunnan, China.

Viral diseases are becoming an important problem in Amorphophallus production due to the propagation of seed corms and their trade across regions. In this study, combined-High-Throughput Sequencing, RT-PCR, electron microscopy, and mechanical inoculation were used to analyze virus-like infected Amorphophallus samples in Yunnan province to investigate the distribution, molecular characterization, and diversity and evolution of Amorphophallus-infecting viruses including three isolates of dasheen mosaic virus and three orthotospoviruses: mulberry vein banding associated virus (MVBaV), tomato zonate spot virus (TZSV) and impatiens necrotic spot virus (INSV). The results showed that DsMV is the dominant virus infecting Amorphophallus, mixed infections with DsMV and MVBaV to Amorphophallus were quite common in Yunnan province, China. This is the first report on infection of Amorphophallus with MVBaV, TZSV, and impatiens necrotic spot virus (INSV) in China. This work will help to develop an effective integrated management strategy to control the spread of Amorphophallus viral diseases.

Plasma EBV quantification is associated with the efficacy of immune checkpoint blockade and disease monitoring in patients with primary pulmonary lymphoepithelioma-like carcinoma.

Objectives: Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is a subtype of lung carcinoma associated with the Epstein-Barr virus (EBV). The clinical predictive biomarkers of immune checkpoint blockade (ICB) in PLELC require further investigation. Methods: We prospectively analysed EBV levels in the blood and immune tumor biomarkers of 31 patients with ICB-treated PLELC. Viral EBNA-1 and BamHI-W DNA fragments in the plasma were quantified in parallel using quantitative polymerase chain reaction. Results: Progression-free survival (PFS) was significantly longer in EBNA-1 high or BamHI-W high groups. A longer PFS was also observed in patients with both high plasma EBNA-1 or BamHI-W and PD-L1 ≥ 1%. Intriguingly, the tumor mutational burden was inversely correlated with EBNA-1 and BamHI-W. Plasma EBV load was negatively associated with intratumoral CD8+ immune cell infiltration. Dynamic changes in plasma EBV DNA level were in accordance with the changes in tumor volume. An increase in EBV DNA levels during treatment indicated molecular progression that preceded the imaging progression by several months. Conclusions: Plasma EBV DNA could be a useful and easy-to-use biomarker for predicting the clinical activity of ICB in PLELC and could serve to monitor disease progression earlier than computed tomography imaging.