Pembrolizumab combined with chemotherapy versus placebo combined with chemotherapy for HER2-negative advanced gastric cancer in China: a cost-effectiveness analysis.

OBJECTIVE: This study aims to conduct a cost-effectiveness analysis of pembrolizumab in combination with chemotherapy for HER2-negative advanced gastric cancer in China. METHODS: A partitioned survival approach model was constructed to simulate the progression of HER2-negative advanced gastric cancer and evaluate the outcomes of different treatment strategies. We calculated incremental cost-effectiveness ratios (ICER) to assess the cost associated with each quality-adjusted life-year (QALY) gained. One-way sensitivity analysis and probabilistic sensitivity analysis were performed to assess robustness and reliability. RESULTS: The analysis conducted in the base case demonstrated that the ICER associated with pembrolizumab was $177405.83/QALY gained in all population. In the subgroup analysis, it was found that individuals with a PD-L1 CPS ≥ 1 and those with a PD-L1 CPS ≥ 10 had ICERs of $152397.06/QALY and $109534.13/QALY, respectively. All ICER values for both the all population groups and the subgroups exceeded the WTP threshold in China. Our analysis shows the robustness of these results, as they remained consistent when input parameters were varied within a ± 25% range. CONCLUSION: The findings of this cost-effectiveness analysis suggest that pembrolizumab in combination with chemotherapy is not a cost-effective treatment option for HER2-negative advanced gastric cancer in China.

First Report of Coptis Wilt Disease Caused by Plectosphaerella cucumerina in China.

Coptis (Coptis chinensis) belongs to the Ranunculaceae family, the rhizomes used in traditional Chinese medicine. Since 2021, an uncommon stem and leaf wilt disease, with an average disease incidence of 70%~90%, has been observed in Guangdong and Guangxi provinces. The early wilt symptoms were observed on older leaves and stems, and the whole seedling wilted and died. The rhizome of the diseased seedlings changed in color, became necrotic, and rotted. Symptomatic roots and stems were surface-sterilized with 70% ethanol for 30 s, followed by 0.2% NaClO for 2-3 min, rinsed in sterile water three times, and then placed on potato dextrose agar (PDA) at 25℃for 14 days. Fungal growth was observed, and six isolates with similar morphology were obtained. The 14-day-old colonies on PDA were buff with few aerial hyphae and slimy surfaces. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were hyaline, smooth, ovoid, septate or aseptate, and 5.77 to 9.53 × 2.15 to 3.32 µm (n = 50). Three of the six isolates were subjected to further analysis. The genomic DNA of three isolates (CCF1-1, CCF1-2, CCF1-3) was extracted using Axygen MAG-FRAG- I-50 (Axygen Bio-Tek) for molecular identification. Partial sequences of the internal transcribed spacer of rDNA (ITS) and large subunit rDNA (LSU) were amplified using the primers ITS1/4 and LR5F/LROR, respectively (Vilgalys and Hester 1990). Their sequences were aligned by MEGA X (Kumar et al., 2018), and the sequences of each region showed 100% sequence similarity among our isolates. A BLAST search of ITS and LSU sequences (accession nos. ON377369, ON428244) showed that both regions had the highest nucleotide similarities (99.43 to 99.89%) to the Plectosphaerella cucumerina strains. Based on morphological and molecular analyses, the isolates were identified as P. cucumerina (Palm et al. 1995). The pathogenicity of our isolates CCF1-1, CCF1-2, CCF1-3 was tested on ten 2-month-old healthy seedlings of coptis, respectively. For the seedlings, 30 ml of fungal conidial suspension (1×106 conidia/ml) or sterile water, as control, were poured into their root area. Conidia suspension were prepared from 14-day-old cultures on PDA by eluting with sterilized water. The seedlings were incubated at 25°C and 75% relative humidity under a 12-h/12-h light/dark cycle. The test was repeated three times. After 20 days, only seedlings inoculated with P. cucumerina exhibited symptoms similar to those diseased seedlings in the field. The control seedling had no symptoms. The morphologically similar fungus was re-isolated from the tested seedlings, thus fulfilling Koch's postulates. Based on molecular, morphological, and pathogenic properties, P. cucumerina is the causal fungal pathogen of coptis wilt disease. Previously, P. cucumerina has been related to wilt disease in strawberry and Chinese cabbage (Yang et al. 2023; Gao et al. 2022), but to our knowledge, this is the first report of P. cucumerina causing wilt disease on coptis in China. Coptis wilt disease tends to occur in a warm and rainy environment, and strengthening the detection and quarantine of seedlings is the key to preventing the occurrence and spread of the disease.

Disease Occurrence and Climatic Factors Jointly Structure Pomelo Leaf Fungal Succession in Disturbed Agricultural Ecosystem.

For perennial plants, newly emerged organs are fresh hot spots for environmental microbes to occupy and assemble to form mature microbial communities. In the microbial community, some commensal fungi can play important roles in microbial succession, thus significantly improving host plant growth and disease resistance. However, their participating patterns in microbial assembly and succession remain largely unknown. In this study, we profiled the fungal community and found a similar fungal succession pattern of spring-emerged leaves from March to October in two pomelo orchards. Specifically, the fungal species, tracked on the old leaves, dominated the spring leaves after emergence and then decreased in relative abundance. This reduction in priority effects on the spring leaves was then followed by an increase in the number of observed species, Shannon and phylogenetic diversity indices, and the pathogen-associated fungal groups. In addition, we found that the temporal fungal succession on the spring leaves highly correlated with the disease occurrence in the orchards and with the temperature and precipitation variation from spring to summer. Of the pathogen-associated fungal groups, an increase in the relative abundance of Mycosphaerellaceae, hosting the causal agent of citrus greasy spot, correlated with the occurrence of the disease, while the relative abundance of Diaporthaceae, hosting the causal agent of melanose, was extremely low during the fungal succession. These results confirm that the two kinds of pathogen-associated fungal groups share different lifestyles on citrus, and also suggest that the study of temporal fungal succession in microbial communities can add to our understanding of the epidemiology of potential plant pathogens.

First Report of Neofusicoccum parvum Causing Fruit Rot on Psidium guajava in China.

Guava (Psidium guajava L.), a nutrient-rich and economically significant fruit, is extensively cultivated in southern China. In six continuous years (from 2018 to 2023), dark-purple rotted guava with sunken lesion were observed on guava trees (variety 'Zhenzhu', aged over 5 years) in Dongguan and Panyu districts, Guangdong Province. Annually, the incidence of fruit rot disease in the affected fields reached 30% to 50% and significantly reduced the yield and quality of harvest guava. The initial symptoms on the epicarp of the fruits were black, needle-like dots that rapidly spread, causing partial or complete fruit rot within two to three weeks. To identify the causative agent, six symptomatic fruits were collected from two different orchards. Samples of 0.5 cm³ were excised from the lesion margins of each fruit. These samples were surface-sterilized with 70% ethanol for 30 seconds, followed by 0.2% NaClO for 2-3 minutes, and rinsed in sterile water three times. The samples were then cultured on potato dextrose agar (PDA) at 25°C for five days. This process yielded eight fungal isolates with similar morphological. Initially, the colonies were white with dense aerial mycelium becoming dark gray after 4 to 5 days. The mycelia were septate and branched. No spores were observed on PDA. To induce spore formation, the isolates were cultured on water agar for 20 days. This process led to the production of hyaline, aseptate, ellipsoid conidia, which were thin-walled, smooth-surfaced, and measured 3.7-5.1 × 1.6-2.2 µm (n = 100). Three isolates, including at least one from each orchard (Np1, Np2, Np3), were selected for further analysis. Genomic DNA was extracted using Axygen MAG-FRAG-I-50 (Axygen Bio-Tek). The internal transcribed spacer of rDNAs (ITS), beta-tubulin (tub2), the nuclear ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α) gene regions were amplified using the primers ITS1/4, Bt2a/Bt2b, LR5/LR0R, and EF1-728F/EF1-986R (Golzar and Burgess 2011) and sequenced. Sequence analysis using MEGA 7.0 (Kumar et al., 2018) revealed 100% similarity among the isolates. BLAST searches of the ITS, tub2, LSU, and tef1-α sequences (accession nos. MN907637, MT537938, MT528156, MT537939) showed the highest nucleotide similarities (99.24 to 100%) to Neofusicoccum parvum strains (Crous et al. 2006). A phylogenetic tree was constructed with MEGA 7.0 based on the nucleotide sequence tub2 using the maximum likelihood method. Pathogenicity tests on 10 healthy guava fruits using mycelium-inoculated and control fruits confirmed the causative agent. The inoculated fruits, maintained at 25°C under a 12-h light/dark cycle, exhibited symptoms identical to the field infections within four to seven days, while control fruits remained symptomless. The fungus, reisolated from the inoculated fruits, was morphologically identical to the original isolates, fulfilling Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, N. parvum as the causal agent of the fruit rot disease on guava. Previously, N. parvum has been reported in association with fruit rot on Eriobotrya japonica and Juglans regia (Zhai et al. 2019; Chen et al. 2019). To our knowledge, this is the first report of N. parvum affecting guava in China.

Causal association of calcific aortic valve stenosis and atrial fibrillation: a Mendelian randomization study.

Calcific aortic valve stenosis (CAVS) is associated with an increased risk of atrial fibrillation (AF) in observational studies, but whether these associations are causal has not been determined. This study aimed to explore the potential causal relationship between CAVS and AF via Mendelian randomization (MR). Genetic variants from the genome-wide association study (GWAS) summary data of the European population for CAVS were used to investigate the association with AF. The inverse variance weighted (IVW) approach was used to obtain the primary causal inference, and several sensitivity analysis approaches, such as the MR‒Egger and weighted median (WM), were performed to assess the robustness of the results. A total of nineteen valid and independent genetic SNPs associated with CAVS were obtained from the GWAS database. Genetically predicted CAVS (OR: 1.105; 95% CI: 1.072-1.139; p = 8.60E-11) was associated with an increased risk of AF. Similar results were discovered in the sensitivity analyses by using MR Egger and weighted median approaches. An MR design was used to reduce confounding variables and the potential for reverse causality bias. The results provide genetic evidence that CAVS considerably increased the risk of AF.

First-line nivolumab plus ipilimumab for unresectable MPM in China: a cost-effectiveness analysis.

BACKGROUND: The regimen of nivolumab plus ipilimumab (NI) has been recommended by the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology-Malignant Pleural Mesothelioma (Version 1.2022) and Chinese Guidelines for the Clinical Diagnosis and Treatment of Malignant Pleural Mesothelioma (2021 edition) as the first-line treatment for Malignant Pleural Mesothelioma (MPM). But whether immunotherapy has a financial advantage over conventional chemotherapy (pemetrexed plus cisplatin/carboplatin, C) is uncertain. METHODS: Based on survival and safety data from the CheckMate 743 clinical trial (NCT02899299), a partitioned survival model was constructed using TreeAge Pro2022 software. The model cycle was set to 1 month and the study period was 10 years. The output indicators included total cost, quality-adjusted life year (QALY) and incremental cost-effectiveness ratio (ICER). One-way and probabilistic sensitivity analyses were used to assess the robustness of the results, considering only direct medical costs. RESULTS AND DISCUSSION: The ICER for group NI versus Group C was $375,656/QALY in all randomized patients, $327,943/QALY in patients with epithelioid histology, and $115,495/QALY in patients with non-epithelioid histology. The ICERs of all three different populations all exceeded the willingness-to-pay threshold (three times the per capita gross domestic product of China in 2021). The results of univariate sensitivity analysis showed that the price of pemetrexed and nivolumab had great influence on the analysis results. The results of the probabilistic sensitivity analysis show that the probability of the NI scheme being more economical in all three different populations was 0. WHAT IS NEW AND CONCLUSION: From the perspective of the Chinese healthcare system, in patients with unresectable MPM, NI has no economic advantage over C.

Synergistic Effect of Combined Walnut Peptide and Ginseng Extracts on Memory Improvement in C57BL/6 Mice and Potential Mechanism Exploration.

This work aimed to investigate whether there are synergistic effects between walnut peptide (WNP) and ginseng extracts (GSE) treatments to ameliorate the memory impairment caused by scopolamine (SCOP). The Morris water maze trial, hippocampal neuron morphology, neurotransmitters, and synaptic ultrastructure were examined, along with brain-derived neurotrophic factor (BDNF)-related signaling pathway proteins. The results of the Morris water maze trial demonstrated that the combined administration of WNP and GSE effectively alleviated memory impairment in C57BL/6 rats caused by SCOP. Improvement in the morphology of hippocampal neurons, dendritic spines, and synaptic plasticity and upregulation of neurotransmitters AChE, ACh, ChAT, Glu, DA, and 5-HT supported the memory improvement effects of WNP + GSE. In addition, compared with the model group, WNP + GSE significantly enhanced the protein levels of VAChT, Trx-1, and the CREB/BDNF/TrkB pathway in hippocampal and PC12 cells induced by SCOP (p < 0.05). Notably, WNP + GSE boosted memory via multiple pathways, not only the BDNF/TrkB/CREB target.

First-line nivolumab plus chemotherapy versus chemotherapy alone for advanced gastric cancer, gastroesophageal junction cancer, and esophageal adenocarcinoma: a cost-effectiveness analysis.

Background: The CheckMate-649 trial compared nivolumab plus chemotherapy (NC) with chemotherapy alone as first-line treatment for advanced gastric cancer (GC), gastroesophageal junction cancer (GEJC), and esophageal adenocarcinoma (EAC) and showed significant benefits to progression-free survival and overall survival. This study evaluated the lifetime cost-effectiveness of NC versus chemotherapy alone in patients with GC/GEJC/EAC from the perspective of the US payers. Methods: A 10-year partitioned survival model was constructed to evaluate the cost-effectiveness of NC and chemotherapy alone and measured the health achievements in quality-adjusted life-years (QALYs), incremental cost-effectiveness ratios (ICERs), and life-years. Health states and transition probabilities were modeled from the survival data from the CheckMate-649 clinical trial (NCT02872116). Only direct medical costs were considered. One-way and probabilistic sensitivity analyses were conducted to assess the robustness of the results. Results: On comparing the chemotherapy, we found that NC incurred substantial health costs, resulting in ICERs of $240,635.39/QALY, $434,182.32/QALY, and $386,715.63/QALY for the model of patients with programmed cell death-ligand 1 (PD-L1) combined positive score (CPS) ⩾5, PD-L1 CPS ⩾1, and all-treated patients, respectively. All ICERs were significantly higher than the willingness-to-pay threshold value of $150,000/QALY. The main influencing factors were the cost of nivolumab, the utility value of the progression-free disease, and the discount rate. Conclusion: Compared with chemotherapy alone, NC may not be a cost-effective option for treating advanced GC, GEJC, and EAC in the United States.

Cost-effectiveness analysis of pembrolizumab plus chemotherapy for patients with recurrent or metastatic cervical cancer in China.

OBJECTIVE: To evaluate the cost-effectiveness of adding pembrolizumab to various therapy combinations in patients with recurrent or metastatic cervical cancer from the Chinese perspective. MATERIALS AND METHODS: The clinical data for our model was taken from the KEYNOTE-826 trial. The direct costs and utilities were collected from local price databases or previously published literature. A three-state partitioned survival model was designed to simulate the disease process of patients with recurrent or metastatic cervical cancer. All costs were estimated in US dollars, with an annual RMB exchange rate of $1 to 6.45 Yuan in 2021. The willingness to pay threshold (WTP) was set at US$37,663.26/QALY. One-way sensitivity analyses and probabilistic sensitivity analyses were performed to evaluate the influence of variables on the model parameters. RESULTS: For patients with a programmed death-ligand 1 combined positive score greater than 1,compared to the chemotherapy group, pembrolizumab plus chemotherapy contributed an incremental 1.12 Quality-adjusted Life Years (QALYs) with an incremental cost of US$71,884.42, resulting in an incremental cost-effectiveness ratio (ICER) of US$64,338.19, which is beyond the willingness-to-pay (WTP) threshold of China. According to sensitivity analyses, the ICERs were most sensitive to the utility of progressive disease and the cost of pembrolizumab. However, those parameters had no significant impact on the model's outcomes. CONCLUSIONS: The addition of pembrolizumab to various therapy combinations chemotherapy is exorbitant and may not be cost-effective for patients with recurrent or metastatic cervical cancer in China.

The root enrichment of bacteria is consistent across different stress-resistant plant species.

Bacteria, inhabiting around and in plant roots, confer many beneficial traits to promote plant growth and health. The secretion of root exudates modulates the nutritional state of the rhizosphere and root area, further selecting specific bacteria taxa and shaping the bacteria communities. Many studies of the rhizosphere effects have demonstrated that selection by the plant rhizosphere consistently enriches a set of bacteria taxa, and this is conserved across different plant species. Root selection effects are considered to be stronger than the rhizosphere selection effects, yet studies are limited. Here, we focus on the root selection effects across a group of 11 stress-resistant plant species. We found that the root selection consistently reduced the alpha diversity (represented by total number of observed species, Shannon's diversity, and phylogenetic diversity) and altered the structure and composition of bacteria communities. Furthermore, root selection tended to enrich for clusters of bacteria genera including Pantoea, Akkermansia, Blautia, Acinetobacter, Burkholderia-Paraburkholderia, Novosphingobium, Massilia, Pseudomonas, Chryseobacterium, and Stenotrophomonas. Our study offers some basic knowledge for understanding the microbial ecology of the plant root, and suggests that several bacteria genera are of interest for future studies.

Analysis of the fungicidal efficacy, environmental fate, and safety of the application of a mefentrifluconazole and pyraclostrobin mixture to control mango anthracnose.

BACKGROUND: Mango anthracnose is among the most severe diseases impacting mango yields and quality. While this disease can be effectively controlled through chemical means, it is vital that appropriate field efficacy and fate determination studies be conducted when applying pesticides to crops in order to appropriately gauge the ecological and health risks associated with the use of these agents. RESULTS: GAP field trials were conducted to explore the efficacy, dissipation, and terminal residues associated with the application of mefentrifluconazole and pyraclostrobin to mango crops in six locations throughout China. These analyses revealed that three applications of mefentrifluconazole [160 mg active ingredient (a.i.) kg-1 ] in combination with pyraclostrobin mixture achieved satisfactory disease control efficacy. To simultaneously detect mefentrifluconazole and pyraclostrobin residues on mangoes, a 'quick, easy, cheap, effective, rugged and safe' (QuEChERS) high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)-based approach was established. The initial mefentrifluconazole and pyraclostrobin concentrations ranged from 0.18 to 0.34 mg kg-1 , and these two compounds exhibited respective half-lives of 5.6 to 10.8 days and 5.5 to 9.0 days. At 21 days following foliage application, the terminal mefentrifluconazole and pyraclostrobin residue concentrations were 0.02-0.04 and 0.01-0.04 mg kg-1 , with these concentrations being below the maximum residue limit (MRL) established for pyraclostrobin. Both short-term [acute reference dose percent (ARfD%) 0.78-2.36% and 2.0-6.08%] and chronic [acceptable daily intake percent (ADI%) 0.08-0.47% and 0.09-0.55%] dietary intake risk assessments for mefentrifluconazole and pyraclostrobin indicated that these terminal residue concentrations are acceptable for the general population. CONCLUSION: Mefentrifluconazole and pyraclostrobin in mango was rapidly degraded following first-order kinetics models. The dietary risk of mefentrifluconazole and pyraclostrobin through mango was negligible to consumers. The application of a 400 g L-1 mefentrifluconazole-pyraclostrobin suspension concentrate mixture represents a highly efficacious fungicidal approach to controlling mango anthracnose that exhibits significant potential for development as it is easily degraded and associated with low residual concentrations after application. © 2022 Society of Chemical Industry.

First Report of Curvularia lunata Causing Leaf Spot on Shatangju in China.

Shatangju (Citrus reticulata Blanco) is an economically important citrus cultivar in China. It has a unique flavor and is very popular among consumers. In May 2018, we observed leaf spots on ~20% of Shatangju trees in a 7 ha orchard in Huaiji country, Guangdong, China. Small maroon spots initially developed on the lower leaf surfaces of the symptomatic trees, which then expanded and coalesced into larger lesions. The lesions became visible from the upper leaves, with light gray centers and dark brown margins surrounded by yellow halos. The infected leaves would finally become wilted. To isolate the pathogen, we cut 0.5 × 0.5 cm2 squares from the lesion margins of the symptomatic leaves, disinfested them using 1% NaClO for 20 s, and then with 70% ethanol for 1 min, rinsed them in sterile distilled water three times, and inoculated them onto three potato dextrose agar (PDA) plates (5 squares/plate), which were kept under 25 °C in the dark. Colonies with similar appearance were observed on all the plates and subcultured via the single-spore method (Ho et al. 1997). The mycelia of the subcultures gradually turned from white to black. The colonies were morphologically close to the fungal species Curvularia lunata (Wakker) Boedijn (Ellis 1971). At 8 days after inoculation, the conidia were 18.9-25.1 µm × 8.1-11.6 µm in size (n=50), fusiform or geniculate, smooth-walled, dark-brown, 3-septate, and with a slightly curved second cell and an expanded third cell from the pore end. Three randomly selected isolates from different plates were applied in further analysis. The internal transcribed spacer (ITS: MZ026467) region, translation elongation factor (EF-1α: MZ042646), large subunit ribosomal RNA (LSU: MZ026469), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: OK086974) segments were amplified and sequenced using primers ITS1/ITS4, EF-1/EF-2, LR5F/ LROR, and gpd1/gpd2, respectively (White et al. 1990; O'Donnell 1998; Marin-Felix 2020). The four segments were identical among the three isolates, which shared the highest nucleotide identities (100% on ITS, LSU, and GAPDH, and 99.8% on EF-1α) with C. lunata strains in the GenBank database. Based on the ITS and GAPDH sequences, phylogenetic analysis using Maximum-likelihood and Bayesian inference methods by W-IQ-TREE (accessed on 09/01/2021) (Trifinopoulos et al., 2016) and MrBayes v3.2.7a (Ronquist et al., 2012) both supported that the isolates belong to C. lunata. For pathogenicity tests, we sprayed the conidial suspension (1×106 conidia/ml) of each of the three isolates on three healthy three-month-old Shatangju seedlings, which were kept under 27 °C and ~100% humidity in plastic bags in the greenhouse. Another three seedlings were sprayed with sterile water and kept under the same conditions as negative controls. Three days after inoculation, small maroon dots started appearing on the inoculated leaves, and the symptoms, same as those observed in the field, developed afterward. No symptoms appeared on the negative controls. C. lunata was reisolated from the infected leaves of all plants inoculated with the three isolates but not from the negative controls. This pathogen has been reported to cause diseases in Pennisetum hydridum and Capsicum frutescens in China (Xu et al. 2018; Pei et al. 2017). But to our knowledge, this is the first report of C. lunata causing leaf spots on citrus plants in the world.

Cost-effectiveness of pembrolizumab for second-line treatment of esophageal squamous cell carcinoma patients with a combined positive score of 10 or more.

Aim: This study aimed to evaluate the cost-effectiveness of pembrolizumab compared with that of chemotherapy in the second-line treatment of locally advanced or metastatic esophageal squamous cell carcinoma (ESCC) patients with a combined positive score ≥10. Methods: A Markov model was established to compare the lifetime costs and quality-adjusted life years (QALYs) of different treatment options. Sensitivity analysis was performed to test the stability of the model. Results: The increased utility and cost of pembrolizumab were 0.442 QALYs and US$11,826.79 compared with those of chemotherapy. The incremental cost-effectiveness ratio was US$26,757.45/QALY, which was less than the threshold of three-times the GDP per capita. The prices of paclitaxel and pembrolizumab were the most important influencing factors. Conclusion: Pembrolizumab is a cost-effective second-line treatment of ESCC.

The Genome of Banana Leaf Blight Pathogen Fusarium sacchari str. FS66 Harbors Widespread Gene Transfer From Fusarium oxysporum.

Fusarium species have been identified as pathogens causing many different plant diseases, and here we report an emerging banana leaf blight (BLB) caused by F. sacchari (Fs) discovered in Guangdong, China. From the symptomatic tissues collected in the field, a fungal isolate was obtained, which induced similar symptoms on healthy banana seedlings after inoculation. Koch's postulates were fulfilled after the re-isolation of the pathogen. Phylogenetic analysis on two gene segments and the whole genome sequence identified the pathogen belonging to Fs and named as Fs str. FS66. A 45.74 Mb genome of FS66 was acquired through de novo assembly using long-read sequencing data, and its contig N50 (1.97 Mb) is more than 10-fold larger than the previously available genome in the species. Based on transcriptome sequencing and ab initio gene annotation, a total of 14,486 protein-encoding genes and 418 non-coding RNAs were predicted. A total of 48 metabolite biosynthetic gene clusters including the fusaric acid biosynthesis gene cluster were predicted in silico in the FS66 genome. Comparison between FS66 and other 11 Fusarium genomes identified tens to hundreds of genes specifically gained and lost in FS66, including some previously correlated with Fusarium pathogenicity. The FS66 genome also harbors widespread gene transfer on the core chromosomes putatively from F. oxysporum species complex (FOSC), including 30 involved in Fusarium pathogenicity/virulence. This study not only reports the BLB caused by Fs, but also provides important information and clues for further understanding of the genome evolution among pathogenic Fusarium species.

First Report of Anthracnose Fruit Rot Caused by Colletotrichum fioriniae on Litchi in China.

Anthracnose fruit rot of litchi (Litchi chinensis Sonn.), caused by Colletotrichum spp., has been mainly associated with the C. acutatum species complex and C. gloeosporioides species complex (Farr and Rossman 2020). In June 2010, isolates of the C. acutatum species complex were isolated together with the C. gloeosporioides species complex from anthracnose lesions on litchi fruits (cv. Nuomici) obtained from a litchi orchard in Shenzhen (N 22.36°, E 113.58°), China. The symptoms typically appeared as brown lesions up to 25 mm in diameter, causing total fruit rot and sometimes fruit cracking. Based on the number of isolates we collected, the C. acutatum species complex appears less frequently on infected fruit compared to the C. gloeosporioides species complex. Since only the C. gloeosporioides species complex has been reported in China (Qi 2000; Ann et al. 2004), we focused on the C. acutatum species complex in this study. Pure cultures of fungal isolates were obtained by single-spore isolation. The isolate GBLZ10CO-001 was used for morphological characterization, molecular and phylogenetic analysis, and pathogenicity testing. Colonies were cultured on potato dextrose agar (PDA) at 25 ℃ for 7 days, circular, raised, cottony, gray or pale orange, with reverse carmine, and 39.6 to 44.7 mm in diameter. Conidia were 13.5 to 19 × 4 to 6 µm (mean ± SD = 15.9 ± 1.1 × 5.2 ± 0.3 µm, n = 50) in size, hyaline, smooth-walled, aseptate, straight, fusiform to cylindrical with both ends acute. Appressoria were 5.5 to 13.5 × 4.5 to 7.5 µm (mean ± SD = 7.6 ± 1.6 × 6.0 ± 0.7 µm, n = 50) in size, subglobose to elliptical, sometimes clavate or irregular, smooth-walled, with entire edge, sometimes undulate, pale to medium brown. These morphological characteristics were consistent with the descriptions of several Colletotrichum species belonging to the C. acutatum species complex, including C. fioriniae (Shivas and Tan 2009; Damm et al. 2012). For molecular identification, genomic DNA was extracted and the ribosomal internal transcribed spacer (ITS), partial sequences of the ß-tubulin (TUB2), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and histone3 (HIS3) genes were amplified and sequenced using the primer pairs ITS4/ITS5, T1/Bt2b, ACT512F/ACT783R, GDF1/GDR1, CHS-79F/CHS-354R, and CYLH3F/CYLH3R, respectively (White et al. 1990; Damm et al. 2012). The resulting sequences were submitted to GenBank (ITS: MN527186, TUB2: MT740310, ACT: MN532321, GAPDH: MN532427, CHS-1: MT740311, HIS3: MT740312). BLAST searches showed 98.70%-100% identity to the sequences of the C. fioriniae ex-holotype culture CBS 128517. The phylogram reconstructed from the combined dataset using MrBayes 3.2.6 (Ronquist et al. 2012) showed that isolate GBLZ10CO-001 clustered with C. fioriniae with high posterior probability. Koch's postulates were performed in the field to confirm pathogenicity. Isolate GBLZ10CO-001 was grown on PDA (25 ℃ for 7 days) to produce conidia. In June 2014, litchi fruits (cv. Nuomici) were sprayed with conidial suspensions (106 conidia/ml), with sterile water as blank controls, and each treatment inoculated at least 15 fruits. Inoculated fruits were covered by an adhesive-bonded fabric bag until the trial ended. After 31 days, typical symptoms were observed, while control fruits remained asymptomatic. The fungus was re-isolated from diseased fruits and identified as C. fioriniae according to the methods described above. To our knowledge, this is the first report of anthracnose fruit rot on litchi caused by C. fioriniae, one species of the C. acutatum species complex, in China. For the difficulty in distinguishing anthracnose caused by C. fioriniae from the C. gloeosporioides species complex just by the symptoms, and mixed infection usually occurring in the field, further investigations are required to reliably assess the potential threat posed by C. fioriniae for litchi production in China.

Tauroursodeoxycholic acid (TUDCA) enhanced intracytoplasmic sperm injection (ICSI) embryo developmental competence by ameliorating endoplasmic reticulum (ER) stress and inhibiting apoptosis.

PURPOSE: The objective of this study was to examine the effect of tauroursodeoxycholic acid (TUDCA) on intracytoplasmic sperm injection (ICSI) embryos by evaluating endoplasmic reticulum (ER) stress, apoptosis, and embryo developmental competence in vitro and in vivo. METHODS: ER stress-associated genes and apoptosis-associated genes were measured and apoptosis index was analyzed. Embryo developmental competence was assessed in vitro and in vivo via the inner cell mass (ICM)/trophectoderm (TE) index, pregnancy and implantation rates, and birth rate. RESULTS: The relative mRNA and protein expression of binding immunoglobulin protein (BIP) was significantly higher in the ICSI embryo group without TUDCA treatment (ICSI-C) than in the in vitro fertilization (IVF) group and in the ICSI embryo group with TUDCA treatment (200 µM) (ICSI-T), while TUDCA ameliorated ER stress in ICSI embryos. Embryos in the ICSI-C group showed a higher apoptosis index than those in the IVF group and ICSI-T group, and there was no significant difference between the IVF group and ICSI-T group. TUDCA can significantly improve ICSI embryo developmental competence in vitro and in vivo based on the ICM/TE index, pregnancy and implantation rates, and birth rate. CONCLUSION: ICSI embryos manifested high ER stress and high apoptosis, while TUDCA ameliorated ER stress and reduced apoptosis in ICSI embryos. TUDCA can significantly improve the developmental competence of ICSI embryos in vitro and in vivo. This study provides a new idea for improving the efficiency of ICSI, and it will also have a positive effect on the development of assisted reproduction technologies for humans and other animals.

High-glucose concentrations change DNA methylation levels in human IVM oocytes.

STUDY QUESTION: What are the effects of high-glucose concentrations on DNA methylation of human oocytes? SUMMARY ANSWER: High-glucose concentrations altered DNA methylation levels of Peg3 and Adiponectin in human in vitro maturation oocytes. WHAT IS KNOWN ALREADY: Maternal diabetes has a detrimental influence on oocyte quality including epigenetic modifications, as shown in non-human mammalian species. STUDY DESIGN, SIZE, DURATION: Immature metaphase I (MI) stage oocytes of good quality were retrieved from patients who had normal ovarian potential and who underwent ICSI in the Reproductive Medicine Center of People's Hospital of Zhengzhou University. MI oocytes were cultured in medium with different glucose concentrations (control, 10 mM and 15 mM) in vitro and 48 h later, oocytes with first polar body extrusion were collected to check the DNA methylation levels. PARTICIPANTS/MATERIALS, SETTING, METHODS: MI oocytes underwent in vitro maturation (IVM) at 37°C with 5% mixed gas for 48 h. Then the mature oocytes were treated with bisulfite buffer. Target sequences were amplified using nested or half-nested PCR and the DNA methylation status was tested using combined bisulfite restriction analysis (COBRA) and bisulfite sequencing (BS). MAIN RESULTS AND THE ROLE OF CHANCE: High-glucose concentrations significantly decreased the first polar body extrusion rate. Compared to controls, the DNA methylation levels of Peg3 in human IVM oocytes were significantly higher in 10 mM (P < 0.001) and 15 mM (P < 0.001) concentrations of glucose. But the DNA methylation level of H19 was not affected by high-glucose concentrations in human IVM oocytes. We also found that there was a decrease in DNA methylation levels in the promoter of adiponectin in human IVM oocytes between controls and oocytes exposed to 10 mM glucose (P = 0.028). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: It is not clear whether the alterations are beneficial or not for the embryo development and offspring health. The effects of high-glucose concentrations on the whole process of oocyte maturation are still not elucidated. Another issue is that the number of oocytes used in this study was limited. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that the effects of high-glucose concentration on DNA methylation of human oocytes have been elucidated. Our result indicates that in humans, the high risk of chronic diseases in offspring from diabetic mothers may originate from abnormal DNA modifications in oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the fund of National Natural Science Foundation of China (81401198) and Doctor Foundation of Qingdao Agricultural University (1116008).The authors declare that there are no potential conflicts of interest relevant to this article.

Significantly lengthened telomere in granulosa cells from women with polycystic ovarian syndrome (PCOS).

PURPOSE: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy among women at reproductive age. However, its etiology remains poorly understood. Recent studies indicated that telomere length was related to PCOS. However, the association between telomere length and PCOS has only been shown in leucocytes and remained controversial across different studies. To clarify the association between telomere length and PCOS, the current study interrogated telomere length not only in leucocytes, but also in follicular granulosa cells, which is essential for folliculogenesis and steroidogenesis. METHODS: Seventy-five patients with PCOS and 81 controls with mechanical infertility undergoing their first in vitro fertilization cycle were enrolled. Their peripheral blood and granulosa cells were collected on the oocyte retrieval day. Telomere length of both leucocytes in the blood and granulosa cells was assayed by quantitative polymerase chain reaction. RESULTS: No significant difference was found in the leucocyte telomere length between controls and PCOS patients (0.99 ± 0.44 vs. 1.00 ± 0.38, p = 0.93). Interestingly, when comparing telomere length in granulosa cells between controls and PCOS subjects, significantly lengthened telomere length was found in PCOS subjects (1.00 ± 0.37 vs. 1.57±0.67, p < 0.0001). After adjustments for age and body mass index, the p value remained significant (p < 0.0001). CONCLUSIONS: This finding reinforced the association between telomere abnormalities and PCOS. Given the importance of telomere length in cellular proliferation, our findings provided novel insights into the pathophysiology of PCOS that abnormalities in telomere length possibly disturb folliculogenesis and subsequently result in PCOS.

A novel functional variant in Wilms' Tumor 1 (WT1) is associated with idiopathic non-obstructive azoospermia.

Idiopathic nonobstructive azoospermia (INOA) is one of the most severe forms of male infertility, yet its pathophysiology remains unclear. WT1 (Wilms' tumor 1) regulates the polarity of Sertoli cells, thereby playing a critical, indirect role in spermatogenesis. Here, we evaluated WT1 gene variation associates with INOA by assessing its promoter and coding regions in 200 patients diagnosed with INOA and 200 proven-fertile men. Three novel variants in the WT1 coding region were detected only in INOA patients, including two synonymous variants and one missense variant, p.Phe435Leu (p.F435L), which was predicted to be deleterious to protein function. The results of dual luciferase reporter showed that the WT1 p.F435L variant decreases transcription of COL4A1 and WNT4 promoters through a dominant-negative effect. Furthermore, chromatin immunoprecipitation assays revealed that COL4A1 and WNT4 promoter is directly bound by wild-type WT1 protein, but not the p.F435L WT1 variant. Thus, we identified a novel functional variant of WT1 functionally associated with INOA. Mol. Reprod. Dev. 84: 222-228, 2017. © 2017 Wiley Periodicals, Inc.

Patients with polycystic ovary syndrome have successful embryo arrest.

In this retrospective study, we investigate the relationship between embryo arrest and polycystic ovary syndrome (PCOS) during in vitro fertilization-embryo transfer (IVF-ET). In this study, 667 subjects were enrolled, including 330 patients with PCOS and 337 subjects without PCOS. The subjects underwent in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) cycles at the Reproductive Medical Centre of Henan Provincial Hospital from January 2009 to December 2012. Four protocols were used to stimulate the ovaries, including long protocol, super-long down-regulation protocol, short protocol and antagonist protocol. Oocytes were retrieved using transvaginal ultrasound guidance. Pronuclei were checked on the next morning after IVF/ICSI. Cleavage stage embryo was assessed after 62-66 hours. Women with PCOS had significantly elevated body mass index, basal luteinizing hormone, estradiol and testosterone compared with normal women. Basal Follicle stimulating hormone level in PCOS patients was lower compared with that in control group. After IVF-ET, PCOS patients had more available oocytes than subjects in control group. PCOS patients had slightly lower fertilization rate than the controls in IVF cycles, but in ICSI cycles, fertilization rate in PCOS patients was significantly higher than that in controls. For either IVF or ICSI, the embryo arrest rate was not changed by PCOS. Moreover, there was no significant difference in embryo arrest rate between both groups adopting different stimulation protocols. Interestingly, embryo arrest rate was not correlated with testosterone for patients in PCOS group. The data indicated that patients with PCOS had successful early embryo arrest during IVF-ET.