[Effect of inactivated SARS-CoV-2 vaccine on the clinical outcomes of patients infected with the Omicron variant in Guangdong Province].

Objective: To evaluate the effect of inactivated SARS-CoV-2 vaccine on the clinical outcomes of patients infected with the Omicron variant. Methods: A total of 1 403 Omicron-infected patients admitted to 20 designated hospitals in Guangdong Province from January 1 to May 31, 2022, were selected as subjects in this study. A case-control study was conducted to collect the demographic data, underlying disease, vaccination status, last exposure date, gene sequencing of infected strains and clinical outcomes from the China Disease Prevention and Control Information System and Guangdong telemedicine platform. Pneumonia (common, severe and critical) and non-pneumonia (asymptomatic and mild) were selected as the case group and control group. The effect of inactivated SARS-CoV-2 vaccine on the clinical outcomes of patients infected with the Omicron variant was analyzed. Results: The median age [M (Q1, Q3)] of the subjects was 36 (27-47) years old, with males accounting for 52.25% (733 cases). The main outcome of the infection was non-pneumonia, accounting for 92.09% (1 292 cases), and the duration [M (Q1, Q3)] of the disease was 18 (14-22) days. There were 134 (9.55%), 39 (2.78%), 403 (28.72%), 437 (31.15%) and 390 (27.80%) cases with no or partial vaccination, within 90 days of primary vaccination, over 90 days of primary vaccination, within 90 days of booster vaccination and over 90 days of booster vaccination, respectively. Multivariate logistic regression analysis showed that after adjusting for gender, age, underlying disease, and location of the report, compared with those with no or partial vaccination, the risk of developing pneumonia was lower in those with over 90 days of primary vaccination, within 90 days of booster vaccination and over 90 days of booster vaccination [OR (95%CI) values were 0.52 (0.28-0.98), 0.39 (0.21-0.73) and 0.40 (0.21-0.77), respectively]. Cox proportional hazard regression model analysis showed that after adjusting for gender, age, underlying disease and location of the report, the duration of the disease was shorter in those who received booster vaccinated for more than 90 days compared with that in those who had no or partial vaccination [HR (95%CI): 1.26 (1.03-1.55)]. Conclusion: The inactivated SARS-CoV-2 vaccine affects the clinical outcomes of patients infected with the Omicron variant.

[Potential application of improved hard tissue section technique in the clinical pathological diagnosis of bone and bone marrow].

Objective: To discuss the application value of hard tissue section in the clinicopathology diagnosis. Methods: From March 2021 to December 2021, bone slices of 19 patients (1 patient with osteochondroma, 2 patients with chondrosarcoma, 4 patients with osteosarcoma, 2 patients with fibrous dysplasia, 2 patients with bone metastasis from thyroid papillary carcinoma, 2 patients with osteomyelitis, 4 patients with giant cell tumor of bone, 2 patients with Ewing sarcoma) and 16 hemopathy patients were collected from the Department of Pathology, Shanghai Sixth People's Hospital. Of the osteopathy patients, there were 14 male and 5 female with a median age of 31 (10-66) years. Meanwhile, there were 7 male and 9 female with a median age of 28 (16-65) years among these hemopathy patients. Thirty-five cases were treated with modified hard tissue slicing technique and paraffin embedding technique, respectively. The advantages and disadvantages of the two methods for clinical diagnosis of bone disease were compared by Hematoxylin-Eosin staining (H&E staining), immunohistochemical staining (IHC), fluorescence in situ hybridization (FISH) and Sanger sequencing. Results: The improved resin-embedded method showed better histological morphology and cell structure. Besides, the expression of Ki67, SATB2, CD34, SMA, CD68,MPO,CD4 and CD33 in immunohistochemical staining in bone tissues which were embedded in resin were more clear in the accurate positive localization than those using paraffin-embedded. MDM2 of FISH exhibited a higher fluorescence intensity and more accurate location. Meanwhile, both methods treated with Sanger sequencing met the requirements of DNA purity and mutation detection. Conclusion: The improved hard tissue section method is simple and short time-consuming, which is suitable for optimizing the clinical bone and bone marrow pathological diagnosis process.

Diversity of Botryosphaeriaceae Species Associated with Chinese Hickory Tree (Carya cathayensis) Trunk Cankers.

Tree trunk cankers represent serious fungal diseases that pose significant threats to Chinese hickory trees (Carya cathayensis). To characterize the pathogen diversity associated with this disease, tissues were collected between 2016 and 2018 from the primary Chinese hickory plantation regions. A total of 97 cultures were isolated from trees in six towns (Longgang, Qingliangfeng, Changhua, Tuankou, Taiyang Town, and Lin'an urban area) within the Linan district, where 60% of Chinese hickory tree yields originate. The isolated cultures caused cankers on Chinese hickory tree branches, but infections did not occur on fruits or leaves under tested conditions. Combined morphological observations and phylogenetic analysis of multiple genes (ITS, ß-tubulin, and EF) indicated that five Botryosphaeriaceae species were recovered, including 89 isolates of Botryosphaeria dothidea, 4 isolates of Botryosphaeriaceae fabicerciana, 1 isolate of Botryosphaeriaceae qingyuanensis, 1 isolate of Botryosphaeriaceae corticis, and two isolates of Lasiodiplodia theobromae. B. dothidea was the most prevalent, and this is the first report of B. corticis, B. qingyuanensis, and L. theobromae infections in Chinese hickory trees. We investigated the mycelial growth, spore germination, and pathogenicity of these species at different temperatures. L. theobromae grew the fastest and B. cortices grew the slowest on potato dextrose agar. The optimum temperature of spore germination for all species was 30°C. L. theobromae was the most virulent species, followed by B. dothidea and B. qingyuanensis, then B. fabicerciana, and finally B. cortices. These new insights into fungal pathogen diversity provide critical new information to understand and manage tree trunk cankers of Chinese hickory.

NLRP3 inflammasome and endoplasmic reticulum stress in the epileptogenic zone in temporal lobe epilepsy: molecular insights into their interdependence.

AIM: Nod-like receptor protein 3 (NLRP3) inflammasome-mediated inflammation has emerged as a contributor to epileptogenesis. Endoplasmic reticulum stress (ERS) plays an important role in epilepsy-induced neurodegeneration. NLRP3 activation and ERS reactions share the same induction factors, suggesting that these processes may be interdependent. However, the correlation between NLRP3 and ERS in TLE has not been confirmed. METHODS: The expression patterns of NLRP3 inflammasome and ERS-related markers in the temporal neocortices of TLE patients were investigated by western blotting, immunohistochemistry and immunofluorescent labelling. Correlations between the protein levels of NLRP3 and the expression of ERS-related markers were assessed using Spearman's rank correlation test. To observe the relationship between the NLRP3 inflammasome and ERS, inhibitors were used in a status epilepticus (SE) model. RESULTS: Our results show that NLRP3 inflammasome components and ERS-related markers were upregulated in the temporal neocortices of TLE patients, and were mainly localized to neurons, astrocytes and microglia. We found a positive correlation between the protein levels of NLRP3 and the expression of ERS-related markers in the temporal neocortices of 20 TLE patients. Furthermore, after blocking the NLRP3 inflammasome with MCC950, the expression of ERS-related markers was markedly decreased in the hippocampi of SE mice. Moreover, TUDCA, a specific ERS inhibitor, also reduced the expression of NLRP3 components in the hippocampus under SE conditions. CONCLUSION: Taken together, our data reveal the interdependence of the NLRP3 inflammasome and ERS in the epileptogenic zone of TLE patients and in the hippocampi of mice in the early post-SE phase.

Genetic Diversity of Colletotrichum spp. Causing Strawberry Anthracnose in Zhejiang, China.

Anthracnose is a serious fungal disease that primarily infects strawberry roots and stolons during development. Here, 91 isolates from different areas of Zhejiang province, China, were collected. Morphological characteristics were analyzed, and a phylogenetic analysis based on multiple genes (actin, internal transcribed spacer, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase) was performed. We found that all of the Colletotrichum species causing strawberry anthracnose belonged to the Colletotrichum gloeosporioides complex. Among them, we identified 48 isolates of C. fructicola, 21 isolates of C. siamense, 13 isolates of C. gloeosporioides, and 9 isolates of C. aenigma. C. siamense was distributed in the central and eastern regions of Zhejiang province (Hangzhou, Jinhua, Shaoxing, Ningbo, and Taizhou). This is the first report of C. siamense causing strawberry anthracnose in Zhejiang province. C. fructicola was the most dominant species causing strawberry anthracnose in Zhejiang province. We identified the four species causing strawberry anthracnose in Zhejiang province, which will improve our understanding of the strawberry anthracnose epidemic and will benefit the development of future control measures.

A New Mutation Genotype of K218T in Myosin-5 Confers Resistance to Phenamacril in Rice Bakanae Disease in the Field.

In 2017 and 2018, a total of 294 Fusarium fujikuroi isolates were collected from bakanae-diseased rice plants in Jinhua, Shaoxing, and Jiaxing in Zhejiang Province, China. Phenamacril sensitivity of these isolates was determined by the 50% effective concentration value or minimum inhibitory concentration methods. Our results indicated that the phenamacril resistance frequency of F. fujikuroi increased from 18% in 2017 to 47% in 2018, and rice plants infected with F. fujikuroi-resistant isolates could not be protected effectively with 50 mg/liter of phenamacril. Phenamacril-resistant F. fujikuroi isolates obtained from rice fields showed stable resistance, because their fitness levels (i.e., mycelial growth, sporulation, and pathogenicity) were similar to the phenamacril-sensitive isolates. In addition to the point mutation at codon 219 in the myosin-5 gene that conferred resistance to phenamacril, our results also showed another point mutation at codon 218 (AAG→ACG) in myosin-5 that also conferred resistance to phenamacril. In this study, we found rapid development and persistence of diversified genotypes of phenamacril resistance, highlighting the importance of proper use of phenamacril in rice fields. Our results may also help researchers develop new fungicides or new control strategies using combinations of different fungicides in the control of phenamacril-resistant F. fujikuroi isolates.

[Study of local application of CpG-oligodeoxynucleotide combined with 4-1BB monoclonal antibody to treat hepatoma- bearing mice].

Objective: To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy. Methods: H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8(+)T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test. Results: Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm(3), (47.25 ± 0.93) mm(3))] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] (F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm(3), (1 380.01 ± 51.44) mm3] (F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8(+)T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8(+)T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ(2) = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05). Conclusion: 4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.

q-LAMP Assays for the Detection of Botryosphaeria dothidea Causing Chinese Hickory Canker in Trunk, Water, and Air Samples.

Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the ß-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea, which could be applied widely in epidemiology, forecast, and management of tree canker disease.

Shift of Sensitivity in Botrytis cinerea to Benzimidazole Fungicides in Strawberry Greenhouse Ascribing to the Rising-lowering of E198A Subpopulation and its Visual, On-site Monitoring by Loop-mediated Isothermal Amplification.

Grey mold disease results from Botrytis cinerea, a classical "high-risk" plant pathogenic fungus in meaning of resistance development to fungicides, and its management depends largely on the frequent applications of fungicides. The evolution of resistance to benzimidazole chemicals during 2008 and 2016 was monitored continuously in strawberry greenhouses located in Zhejiang province. Results showed that extensive applications of the mixture of carbendazim and diethofencarb caused the rapid spread of Ben MR subpopulation. The withdraw of this mixture lead to the sharply decrease of Ben MR and re-dominance of Ben HR isolates of B. cinerea with the E198A mutation in ß-tubulin gene. The LAMP primers, based on the E198A point mutation, were designed to detect the E198A genotype specifically. HNB (Hydroxynaphthol blue), a metalion indicator, acted as a visual LAMP reaction indicator that turned the violet colored into a sky-blue color. The detection limit of concentration of DNA was 100 × 10-2 ng/µL and this LAMP assay could be applied to detect the E198A genotype with 100% accuracy in strawberry greenhouses of three Province and was more rapid and easier to operate. In summary, we establish a simple and sensitive on-field LAMP assay which can be adopted to determine within 1.5 h whether the benzimidazoles or the mixture of a benzimidazole fungicide and diethofencarb is suitable for management of B. cinerea.

MiR-421 expression independently predicts unfavorable overall survival in patients with esophageal adenocarcinoma.

OBJECTIVE: This study aimed to examine the prognostic value of miR-421 in terms of overall survival (OS) and recurrence free survival (RFS) in ESAD and its potential regulatory network. PATIENTS AND METHODS: An in-silico analysis was conducted using data from large databases, including The Cancer Genome Atlas-Esophageal Carcinoma (TCGA-ESCA), Starbase 3.0 and GeneMANIA. RESULTS: Both esophageal adenocarcinoma (ESAD) and esophageal squamous cell carcinoma (ESCC) tissues had significantly upregulate miR-421 expression, compared with adjacent normal tissues. Upregulated miR-421 expression was associated with shorter OS, but not RFS in ESAD. In patients with ESCC, no difference in miR-421 expression was observed regards to OS or RFS status. Univariate and multivariate analysis showed that high miR-421 expression was independently associated with shorter OS (HR: 2.77, 95%CI: 1.41-5.46, p<0.01), after adjustment of histological grade and pathological stages. The predicted regulatory network of miR-421 in ESAD includes both tumor suppressors and oncogenes, which makes the role of miR-421 quite mysterious in this cancer. CONCLUSIONS: MiR-421 expression might serve as a valuable prognostic biomarker in patients with ESAD. But future molecular studies are required to explore the exact regulatory effect of it.

Molecular Detection of QoI Resistance in Colletotrichum gloeosporioides Causing Strawberry Anthracnose Based on Loop-Mediated Isothermal Amplification Assay.

Anthracnose is one of the most common diseases in strawberry plants. Colletotrichum gloeosporioides is the major cause of anthracnose in China, including Zhejiang Province. Early, specific, reliable, and time-saving detection is urgently needed to prevent the further spread of C. gloeosporioides, guiding farmers to utilize chemicals to control anthracnose. In this study, we showed that the high resistance to pyraclostrobin, caused by a point mutation at codon 143 (GGT→GCT) in the cytochrome b gene of C. gloeosporioides was prevalent in the strawberry growing regions, and we developed a loop-mediated isothermal amplification (LAMP) assay as a detection method. Primer sets S0 and S4 could be used to specifically detect C. gloeosporioides isolates and the G143A mutations, respectively. A detection limit of 10-2 ng (10 pg), which is at least 10-fold more sensitive than conventional polymerase chain reaction, was achieved by the LAMP assay. Here, we utilized lateral-flow devices (LFDs), nitrocellulose membranes that can absorb nucleic acids, to acquire the total genomic DNA of strawberry plants within 2 min. The LFD membranes were used as DNA templates for the LAMP assays to accurately detect strawberry plants infected with C. gloeosporioides. This diagnostic method for strawberry anthracnose was accomplished within 1 h, including the sample preparation and LAMP assays. Collectively, we developed a sensitive and practical method for monitoring C. gloeosporioides and its quinone outside inhibitor-resistant mutants. The LAMP assay for detection of C. gloeosporioides in strawberry plants has great potential for rapid strawberry anthracnose surveillance and will provide farmers with advice on preventing C gloeosporioides at the early stages of strawberry development.

One-step loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Fusarium fujikuroi in bakanae disease through NRPS31, an important gene in the gibberellic acid bio-synthesis.

Rice bakanae disease caused by Fusarium fujikuroi is one of the most famous seed borne diseases. If infected seeds are used, this disease will occur with serious impacts. Thus, a simple, reliable, specific and sensitive method for surveillance is urgently needed to screen infected seeds and seedlings at early developmental stages. In this study, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect F. fujikuroi in contaminated rice seeds and seedlings for diagnosis of bakanae disease. NRPS31 gene plays an important role in the gibberellic acid (GA) bio-synthesis of F. fujikuroi, and is not present in any other sequenced fungal genome, and thus was adopted as the target for LAMP primer design. The LAMP assay enables the fast detection of as little as 1 fg of pure genomic F. fujikuroi DNA within 60 minutes. Further tests indicated that the LAMP assay was more sensitive and faster than the traditional isolation method for F. fujikuroi detection in rice seeds and seedlings. Our results show that this LAMP assay is a useful and convenient tool for detecting F. fujikuroi, and it can be applied widely in seed quarantine of bakanae disease, providing valid data for disease prevention.

[Analysis of 833 times measured hepatic venous pressure gradient].

Objective: To summarize and analyze the clinical data of hepatic venous pressure gradient (HVPG) and to explore the application value of HVPG in the diagnosis, evaluation and clinical treatment of portal hypertension in cirrhosis. Methods: The patient data of HVPG measurement performed in Shandong Provincial Hospital from April 2010 to November 2017 were collected. Results: A total of 633 patients with 833 times of HVPG measurements were included. There was significant difference in HVPG between patients with different etiologies, different Child-pugh grades and different degrees of decompensated cirrhosis. Conclusion: The HVPG test is suitable for the diagnosis and evaluation of portal hypertension. The HVPG of patients with different severity of liver cirrhosis can guide the choice of the treatment plan, and the HVPG measurement should also be strictly standardized and quality control.

LncRNA FER1L4 suppressed cancer cell growth and invasion in esophageal squamous cell carcinoma.

OBJECTIVE: To investigate the regulatory effect of long non-coding ribonucleic acid (lncRNA) FER1L4 on biological behaviors of esophageal squamous cell carcinoma (ESCC) cells, such as proliferation and invasion. PATIENTS AND METHODS: The expressions of FER1L4 were detected in 42 pairs of ESCC tissues and corresponding para-carcinoma tissues and 5 kinds of ESCC cell lines via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Polyethyleneimine (PEI) and liposomes were used for FER1L4 expression or interference elimination assays, respectively. The proliferation and invasion of ESCC cells were detected via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), apoptosis assay, cell cycle assay, and transwell chamber. RESULTS: Results of qRT-PCR showed that, compared with that in normal tissues, FER1L4 was lowly expressed in ESCC tissues. Overexpression of FER1L4 could inhibit cell proliferation and invasion, promote apoptosis and increase the cell cycle distribution in G0/G1 phase. Knockout of FER1L4 could promote the proliferation and invasion of ESCC cells, inhibit apoptosis and decrease the cell cycle distribution in G0/G1 phase. CONCLUSIONS: FER1L4 is involved in the occurrence and development of ESCC and plays a key role as a tumor suppressor gene in ESCC.

Attenuating effect of melatonin on lipopolysaccharide-induced chicken small intestine inflammation.

Enriched melatonin (MEL) has been found in the mammalian intestine and has been recently demonstrated to alleviate rodent colitis. In this study, the effect of MEL on lipopolysaccharide (LPS)-induced intestinal inflammations was investigated in new chicken hatchlings. The chicks were fed with a diet supplemented with MEL (12.5 mg/day) from D1 to D10. Meanwhile, the chicks in the LPS or MEL + LPS groups were injected with LPS (10 mg/kg BW, i.p.) at D10. LPS treatment for 6 h increased the expression of IL-6, IL-4, caspase-3 mRNAs and TUNEL-positive cell populations, but decreased populations of the goblet and PCNA+ cells, IgA production and the expression of MUC2 mRNA in the duodenum. Compared with the LPS group, MEL pre-feeding alleviated duodenal inflammation and decreased the expression of TNF-α mRNAs by 23.6% (P = 0.004), IL-6 mRNAs by 69.4% (P = 0.001), IL-4 mRNAs by 4.1% (P = 0.824) and caspase-3 mRNAs by 45.8% (P < 0.001). Conversely, MEL pre-feeding attenuated the LPS-induced changes of IgA production by 161.6% (P = 0.013) and PCNA+ cell populations by 172.1% (P < 0.001) in the duodenum. TLR4 mRNA was also up-regulated by LPS treatment but down-regulated by MEL pre-feeding. In conclusion, dietary MEL could attenuate LPS-induced chick duodenal inflammation by down-regulating the expression of inflammatory cytokines, promoting epithelial cell proliferation, improving the immunological barrier and inhibiting epithelial apoptosis via the mediation of TLR4.

Culture and characterization of chicken small intestinal crypts.

The integrity and normal function of the small intestinal epithelium depends critically on the rapid renewal of epithelial cells from basal stem cells. The intensive proliferation that fuels this self-renewal process is confined to the intestinal crypts. Establishment of suitable protocols for crypt isolation and culture is pivotal for the studies of intestinal self-renewal mechanisms. In this study, chicken small intestinal crypts were isolated, purified, and further cultured in a Matrigel 3-D culture system. The growth factor concentration assay on the fourth d of culture showed that Group C (50 ng/mL epidermal growth factor (EGF), 100 ng/mL Noggin, and 500 ng/mL R-spondin 1) supplement in culture medium could significantly enlarge the diameter of organoids when compared with Group A (5 ng/mL EGF, 10 ng/mL Noggin, 50 ng/mL, and R-spondin 1) and Group B (10 ng/mL EGF, 20 ng/mL Noggin, and 100 ng/mL R-spondin 1) by 188.4% (P = 0.026) and 176.9% (P = 0.034), respectively. Transmission electron microscopy, neutral red staining, and 5-ethynyl-2΄-deoxyuridine incorporation demonstrated the integrated structure, high viability, and proliferative activity in cultured chicken intestinal organoids. In addition, intestinal stem cell marker genes (Olfm4, Znrf3, Hopx, and Lgr5) also could be detected in cultured intestinal organoids. Furthermore, CHIR99021 (a glycogen synthase kinase 3ß inhibitor) could enhance the expression of Olfm4, Znrf3, Hopx, and Lgr5 by 750% (P = 0.001), 467% (P < 0.001), 450% (P < 0.001), and 333% (P = 0.008), respectively, indicating the responsiveness of the cultured chicken intestinal organoids to exogenous stimulus. This study modified a murine culture model and optimized it to provide a chicken intestinal organoid model for use as a physiological or pathological research platform in vitro.

Rapid on-site evaluation of the development of resistance to quinone outside inhibitors in Botrytis cinerea.

Botrytis cinerea, a typical "high-risk" pathogenic fungus that rapidly develops resistance to fungicides, affects more than 1,000 species of 586 plant genera native to most continents and causes great economic losses. Therefore, a rapid and sensitive assay of fungicide resistance development in B. cinerea populations is crucial for scientific management. In this study, we established a Loop-mediated isothermal amplification (LAMP) system for the monitoring and evaluation of the risk of development of B. cinerea resistance to QoI fungicides; the method uses two LAMP assays. The first assay detects G143A mutants of B. cinerea, which are highly resistance to QoI fungicides. BCbi143/144 introns in B. cinerea are then detected by the second assay. HNB acts as a visual LAMP reaction indicator. The optimum reaction conditions of the LAMP assays were 61 °C for 50 min, and the detection limit of the LAMP assays was 100 × 10-4 ng/µl. We directly pre-treated the field samples by using All-DNA-Fast-Out to extract DNA within ten minutes, then performed the LAMP assay to achieve one-step rapid detection. In conclusion, we established a rapid and sensitive LAMP assay system for resistance risk assessment and for monitoring QoI-resistance of B. cinerea in the field.