Selenite selectively kills lung fibroblasts to treat bleomycin-induced pulmonary fibrosis.

BACKGROUND: Interstitial lung disease (ILD) treatment is a critical unmet need. Selenium is an essential trace element for human life and an antioxidant that activates glutathione, but the gap between its necessity and its toxicity is small and requires special attention. Whether selenium can be used in the treatment of ILD remains unclear. METHODS: We investigated the prophylactic and therapeutic effects of selenite, a selenium derivative, in ILD using a murine model of bleomycin-induced idiopathic pulmonary fibrosis (IPF). We further elucidated the underlying mechanism using in vitro cell models and examined their relevance in human tissue specimens. The therapeutic effect of selenite in bleomycin-administered mice was assessed by respiratory function and histochemical changes. Selenite-induced apoptosis and reactive oxygen species (ROS) production in murine lung fibroblasts were measured. RESULTS: Selenite, administered 1 day (inflammation phase) or 8 days (fibrotic phase) after bleomycin, prevented and treated deterioration of lung function and pulmonary fibrosis in mice. Mechanistically, selenite inhibited the proliferation and induced apoptosis of murine lung fibroblasts after bleomycin treatment both in vitro and in vivo. In addition, selenite upregulated glutathione reductase (GR) and thioredoxin reductase (TrxR) in murine lung fibroblasts, but not in lung epithelial cells, upon bleomycin treatment. GR and TrxR inhibition eliminates the therapeutic effects of selenite. Furthermore, we found that GR and TrxR were upregulated in the human lung fibroblasts of IPF patient samples. CONCLUSIONS: Selenite induces ROS production and apoptosis in murine lung fibroblasts through GR and TrxR upregulation, thereby providing a therapeutic effect in bleomycin-induced IPF.

Ex Vivo Evaluation of Combination Immunotherapy Using Tumor-Microenvironment-on-Chip.

Combination immunotherapy has emerged as a promising strategy to address the challenges associated with immune checkpoint inhibitor (ICI) therapy in breast cancer. The efficacy of combination immunotherapy hinges upon the intricate and dynamic nature of the tumor microenvironment (TME), characterized by cellular heterogeneity and molecular gradients. However, current methodologies for drug screening often fail to accurately replicate these complex conditions, resulting in limited predictive capacity for treatment outcomes. Here, a tumor-microenvironment-on-chip (TMoC), integrating a circulation system and ex vivo tissue culture with physiological oxygen and nutrient gradients, is described. This platform enables spatial infiltration of cytotoxic CD8+ T cells and their targeted attack on the tumor, while preserving the high complexity and heterogeneity of the TME. The TMoC is employed to assess the synergistic effect of five targeted therapy drugs and five chemotherapy drugs in combination with immunotherapy, demonstrating strong concordance between chip and animal model responses. The TMoC holds significant potential for advancing drug development and guiding clinical decision-making, as it offers valuable insights into the complex dynamics of the TME.

First Report of Pseudocercospora paraguayensis Causing Leaf Spots on Bixa orellana from Chinese Mainland.

Bixa orellana L. is a traditional Chinese medicine. In December 2019, a leaf spot disease was observed on B. orellana from a field in Zhanjiang (21°18'12''N, 110°17'22''E), China. Disease incidence was around 85% (n = 100 investigated plants from about 30 hectares). Initial leaf spots were circular, and the center of the lesions was grayish-white with a purple black border. The coalescence of individual spots eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces, and the surfaces were disinfected with 75% ethanol for 30 sec, and 2% sodium hypochlorite for 60 sec. The the samples were then rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Three representative isolates (BOPP-1, BOPP-2, and BOPP-3) were used for further study. The colonies of isolates on PDA were dark olive green with off-white aerial mycelia after 7 days at 28 °C. Conidia were solitary, smooth to verrucous, olive to light brown, slightly curved, narrowly obclavate, apex obtuse, base obconic-truncate, had 2-4 septa, and 30.4-55.5×2.0-3.5 µm in size.. These morphological characteristics showed did not differ from the description of Pseudocercospora paraguayensis (Crous et al. 1997). For molecular identification, the internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1) gene, and actin (ACT) gene were amplified using primer pairs ITS1/ITS4 (White et al., 1990), EF1/EF2 ( O'Donnell et al. 1998), and ACT-512F/ACT-783R (Carbone and Kohn, 1999) and sequenced from DNA extracted from the three isolates, respectively. Sequences were deposited in GenBank under accession no. MZ363823-MZ363825 (ITS), MZ614954-MZ614956 (TEF1), and MZ614951-MZ614953 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that the three isolates were nested within the clade containing the type specimen of P. paraguayensis (CBS 111286) but not within P. bixae (the type specimen CPC 25244). Pathogenicity was tested through in vivo experiments. Inoculation and control seedlings (n = 5, 1-month-old) were sprayed with a spore suspension (1 × 105 per ml) of P. paraguayensis and sterile distilled water (control), respectively, until run-off (Fang. 1998). The plants were grown in pots in a greenhouse at 28°C, with at approximately 80% RH. The test was performed three times. Symptoms similar to those in the field were observed on the inoculated plants after two weeks. The control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and comparison of ITS sequences with 100% identical to those of isolates. No original fungi were isolated from the control plants. A previous study reported that P. paraguayensis caused leaf spots on pistachio and eucalypts, and the fungus causing the leaf spots of B. orellana was redescribed as P. bixae (Crous et al. 2019). However, multilocus phylogenetic analyses differentiated P. paraguayensis from P. bixae. In the present study, P. paraguayensis was distinguished from P. bixae due to the absence of catenulate conidia and the presence of finely verruculose conidia (Crous et al. 2013). P. eucalypti as a synonyms was reported in Taiwan (www.MycoBank.org). The current study is the first to report P. paraguayensis causing leaf spots on B. orellana from Chinese Mainland. This finding will help to provide a scientific basis for the disease detection.

First Report of Mucor irregularis Causing Flower Rot on Selenicereus undatus in China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidence rate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with M. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relative humility approximately 80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregularis was reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report of M. irregularis causing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.

Cytotoxic T-lymphocyte antigen 4 polymorphisms and breast cancer susceptibility: Evidence from a meta-analysis.

BACKGROUND: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an immune checkpoint and regulates the immune function of T cells. However, previous findings regarding the association of CTLA-4 polymorphisms and breast cancer remain inconclusive. Therefore, we performed a meta-analysis to investigate the potential effects of five polymorphisms (-1722 T/C, -1661 A/G -318 C/T, +49 A/G, and CT60 A/G) in the CTLA-4 gene on breast cancer susceptibility. METHODS: Relevant literatures were systematically searched through electronic databases including PubMed, EMBASE, and Web of Science up to October 10, 2021. Available data were extracted and odds ratios (ORs) with 95% confidence intervals were used to estimate the pooling effect size. The Newcastle-Ottawa Scale was applied for assessing the quality of included studies. We conducted subgroup analyses based on ethnicity and control sources to explore levels of heterogeneity. Moreover, sensitivity analysis and publication bias were assessed. RESULTS: Finally, a total of 12 eligible studies regarding CTLA-4 polymorphisms and breast cancer were included. For overall analyses, only the +49 A/G polymorphism was significantly associated with breast cancer under allelic (OR = 1.19), dominant (OR = 1.27), and recessive (OR = 1.27) models. Ethnicity-based subgroup analysis found that the +49 A/G polymorphism has a significant risk (OR = 2.03) of breast cancer under the recessive model in the non-Asian population. Studies with hospital-based controls showed that the +49 A/G polymorphism has significant breast cancer risks under allelic (OR = 1.44), dominant (OR = 1.86), and recessive (OR = 1.60) models. In addition, those with population-based controls found that -1722 T/C polymorphism has a significant breast cancer risk under allelic (OR = 1.19) and dominant (OR = 1.26) models. CONCLUSION: This meta-analysis suggested that CTLA-4 + 49 A/G polymorphism may significantly associate with breast cancer susceptibility. Future studies containing various populations are helpful for evaluating the impacts of CTLA-4 polymorphisms on breast cancer susceptibility.

Large-Scale Single-Nucleus RNA Sequencing Compatible with Complex Archived Samples.

Transcriptome profiling at single-cell resolution allows us to identify and assess functional cell types and cellular states, including those within degenerating ocular tissues in retinitis pigmentosa. The technology is particularly valuable when studying tissues with high cellular heterogeneity, or when specific cell types are of interest. In this chapter, we introduce a detailed protocol of a medium-throughput single-nucleus RNA sequencing technique that utilizes frozen tissue as input sample. This protocol can be executed by any researcher with basic training in molecular biology techniques. With this protocol, a single experimenter can easily process two samples per day up to cDNA amplification, and library preparations can be done in batches of 8. Routinely we can obtain ~20 K nuclei per eye from 3 to 4 library preparations.

Glutathione peroxidase 4 expression predicts poor overall survival in patients with resected lung adenocarcinoma.

This study aimed to evaluate the protein expression of glutathione peroxidase 4 (GPX4) in resected non-small cell lung cancer (NSCLC). The clinical relevance and prognostic significance of GPX4 expression were analyzed. We reviewed patients with resected NSCLCs at Taipei Veterans General Hospital between September 2002 and January 2018. Available paraffin-embedded specimens were retrieved for immunohistochemistry (IHC) staining to detect GPX4 expression. The cutoff value for defining GPX4 positivity was determined according to the percentage of tumor stained in the microscopic field. The correlation between immune expression, clinicopathologic data, overall survival (OS), and disease-free survival (DFS) were analyzed. A total of 265 NSCLC specimens were retrieved for IHC staining. GPX4 expression positive was in 192 (72.5%) according to a cutoff value of 5%. GPX4 was a significant prognostic factor for OS and DFS on multivariate analysis at both 5% and 25% cutoff values. GPX4 expression was associated with poor OS and DFS, especially in lung adenocarcinoma (p = 0.008, and 0.027, respectively). In conclusions, IHC analysis revealed that GPX4 expression was associated with poor survival outcomes in patients with resected lung adenocarcinoma. Further research is needed to understand the role of GPX4 in tumorigenesis and the underlying mechanism responsible for survival outcomes in patients with resected lung adenocarcinoma.

Drainless thoracoscopic surgery should be avoided in primary spontaneous pneumothorax with pleural adhesion.

OBJECTIVES: Drainless thoracoscopic surgery, defined by omitting chest drain after surgery, has been demonstrated to be feasible in selected patients for pulmonary resection. However, drainless procedure for the treatment of primary spontaneous pneumothorax has raised concerns for its safety and thus has been less often reported. We aimed to share our preliminary experience regarding how to select patients with spontaneous pneumothorax for this procedure. METHODS: A retrospective study recruiting 303 consecutive patients with the diagnosis of spontaneous pneumothorax undergoing thoracoscopic surgery in our centre from August 2016 to June 2020 was done. After careful selection, the chest drain was omitted in selected patients who underwent non-intubated uniportal thoracoscopic surgery. Patients' clinical characteristics and perioperative outcomes were analysed. RESULTS: A total of 34 patients underwent drainless thoracoscopic surgery for the treatment of spontaneous pneumothorax. Pleural adhesion was noted in 9 patients during surgery, and all of them (100%) developed residual pneumothorax, among which intercostal drainage was required in 2 (22.2%) patients and ipsilateral pneumothorax recurred 3 years after surgery in 1 (11.1%) patient. Among the remaining 25 without pleural adhesion, 17 (68.0%) developed minor residual pneumothorax (P = 0.006), which all resolved spontaneously within 1-2 weeks, with no complications or recurrence during postoperative follow-up for at least 2 years. CONCLUSIONS: Drainless thoracoscopic surgery for the treatment of primary spontaneous pneumothorax is feasible but can be risky without careful patient selection. In our experience, the drainless procedure should be avoided in patients with identifiable pleural adhesion noted during surgery.

Endoscopic Screening for Second Primary Tumors of the Esophagus Among Head and Neck Cancer Patients.

Malignancies of the head and neck (HN) region and esophagus are among the most common cancers worldwide. Due to exposure to common carcinogens and the theory of field cancerization, HN cancer patients have a high risk of developing second primary tumors (SPTs). In our review of 28 studies with 51,454 HN cancer patients, the prevalence of SPTs was 12%. The HN area is the most common site of SPTs, followed by the lungs and esophagus, and 13% of HN cancer patients have been reported to have esophageal high-grade dysplasia or invasive carcinoma. The prognosis of HN cancer patients with concomitant esophageal SPTs is poor, and therefore identifying esophageal SPTs as early as possible is of paramount importance for risk stratification and to guide the treatment strategy. Image-enhanced endoscopy, especially using narrow-band imaging endoscopy and Lugol's chromoendoscopy, has been shown to improve the diagnostic performance in detecting esophageal neoplasms at an early stage. Moreover, the early detection and minimally invasive endoscopic treatment of early esophageal neoplasm has been shown to improve the prognosis. Well-designed prospective studies are warranted to establish appropriate treatment and surveillance programs for HN cancer patients with esophageal SPTs.

Molecular target therapeutics of EGF-TKI and downstream signaling pathways in non-small cell lung cancers.

Lung carcinoma (LC) is the third most common cancer diagnosis and accounted for the most cancer-related mortality worldwide in 2018. Based on the type of cells from which it originates, LC is commonly classified into non-small cell lung cancers (NSCLC) and small cell lung cancers (SCLC). NSCLC account for the majority of LC and can be further categories into adenocarcinoma, large cell carcinoma, and squamous cell carcinoma. Accurate classification of LC is critical for its adequate treatment and therapeutic outcome. Since NSCLC express more epidermal growth factor receptor (EGFR) with activation mutations, targeted therapy EGFR-tyrosine kinase inhibitors (TKIs) have been considered as primary option of NSCLC patients with activation EGFR mutation. In this review, we present the genetic alterations, reported mutations in EGFR, and TKIs treatment in NSCLC patients with an emphasis on the downstream signaling pathways in NSCLC progression. Among the signaling pathways identified, mitogen activation protein kinase (MAPK), known also as extracellular signal-regulated protein kinase (Erk) pathway, is the most investigated among the related pathways. EGFR activation leads to the autophosphorylation of its kinase domain and subsequent activation of Ras, phosphorylation of Raf and MEK1/2, and the activation of ERK1/2. Phosphatidylinositol 3-kinase (PI3K)/Akt is another signal pathway that regulates cell cycle and has been linked to NSCLC progression. Currently, three generations of EGFR TKIs have been developed as a first-line treatment of NSCLC patients with EGFR activation and mutation in which these treatment options will be further discussed in this review. The Supplementary Appendix for this article is available at http://links.lww.com/JCMA/A138.

First Report of Seedling Rot of Castor (Ricinus communis) caused by Choanephora cucurbitarum in China.

Castor (Ricinus communis L.) oil is used in the manufacture of cosmetics, lubricants, plastics, pharmaceuticals, and soaps and is grown in more than 40 countries with India and China leading in oil production(Tunaru et al. 2012). In June 2021, a seedling rot disease was observed on castor cv. Zibi-5 in a plant nursery in Zhanjiang (21°17' N, 110°18' E), China. Initial symptoms on leaves and stems were water-soaked and dark green lesions that resulted in rapid rotting. Disease incidence was 25% and resulted in seedling death. White fungal mycelia developed on the rotting plant tissues. Leaves and stems were collected from 10 diseased plants, surface disinfected in 0.5% sodium hypochlorite and 75% ethyl alcohol, and tissue pieces placed in plates of potato dextrose agar (PDA) which were maintained at 28℃. Hyphal tips from fungal mycelia that developed in the PDA plates were selected to establish pure cultures and three representative fungal isolates, designated RCC-1, RCC-2, and RCC-3, were selected for further study. The fungal isolates produced sporangiophores that were smooth, hyaline, aseptate, and apically swollen. Sporangiophores bore monosporous sporangiola that were broadly ellipsoidal, longitudinally coarsely striate, brown to dark brown, and measured 6.2 to 14.8 x 10.5 to 26.5 um (n=30). Sporangia contained few to many spores that were spherical, brown, and measured 59 to 150 um in diameter (n=20). Sporangiospores were ellipsoid, striate, and brown with multiple hyaline polar appendages and measured 6.6 to 12.3 x 10.6 to 25.5 um (n=30) in size. Based on these morphological characteristics, the fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk, 1984). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and NL1/LR3 (Walther et al. 2013). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376748-OL376750, and LSU, OL763430-OL763432. BLAST analysis of these sequences revealed a 100% to 99% identity with the sequences (ITS, MG650194; 573/573, 573/573, and 573/573; LSU, AF157181; 673/676, 673/676, and 673/676) of C. cucurbitarum in GenBank. Pathogenicity tests, to fulfill Koch's postulates, were performed in a greenhouse with a temperature range of 24℃ to 30℃ and 80% relative humidity. Thirty-day-old cv. Zibi-5 castor plants were grown in pots and used for inoculation tests. Ten plants were inoculated by placing agar plugs with mycelia of fungal isolate RCC-1 on leaves or stems. Ten control plants were inoculated with agar plugs only and the test was repeated three times in total. Five days after inoculation, all plants, with either leaf or stem inoculations, became infected and began rotting. Symptom progression was consistent with that observed in the nursery and all control plants remained healthy. C. cucurbitarum was successfully reisolated from all inoculated plants and identified by morphological characteristics and by sequence analysis. This fungus is known to cause serious damage on a wide range of hosts (Liu et al. 2019) and previously was reported on castor in India (Shaw 1984) and Papua New Guinea (Peregrin and Ahmad 1982). We observed that the pathogen grows very rapidly and causes serious damage to castor seedlings, warranting further investigation on the epidemiology and control of this disease.

Factors Associated with Nodal Upstaging in Clinical T1a-bN0M0 Non-Small Cell Lung Cancers.

Nodal upstaging of lung adenocarcinoma occurs when unexpected pathological lymph node metastasis is found after surgical intervention, and may be associated with a worse prognosis. In this study, we aimed to determine the predictive factors of nodal upstaging in cT1a-bN0M0 primary lung adenocarcinoma. We retrospectively reviewed a prospective database (January 2011 to May 2017) at National Taiwan University Hospital and identified patients with cT1a-bN0M0 (solid part tumor diameter ≤ 2 cm) lung adenocarcinoma who underwent video-assisted thoracoscopic lobectomy. Logistic regression models and survival analysis were used to examine and compare the predictive factors of nodal upstaging. A total of 352 patients were included. Among them, 28 (7.8%) patients had nodal upstaging. Abnormal preoperative serum carcinoembryonic antigen (CEA) levels, solid part tumor diameter ≥ 1.3 cm, and consolidation-tumor (C/T) ratio ≥ 0.50 on chest computed tomography (CT) were significant predictive factors associated with nodal upstaging, and patients with nodal upstaging tended to have worse survival. Standard lobectomy is recommended for patients with these predictive factors. If neither of the predictive factors are positive, a less invasive procedure may be a reasonable alternative. Further studies are needed to verify these data.

First Report of Colletotrichum siamense Causing Anthracnose in Osmanthus fragrans in China.

Osmanthus fragrans Lin. is widely cultivated in China. Its flower is precious spices. It is also a garden ornamental plant. In March 2021, anthracnose-type lesions were observed on the leaves of O. fragrans in a public garden in Zhanjiang, Guangdong Province, China (21˚17'47''N, 110˚18'58''E). Disease incidence was around 50% (n = 100 investigated plants from about 30 hectares). The early symptoms were yellow spots on the edge or tip of the leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec . Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 ℃. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Thirty-two isolates of Colletotrichum ssp. were obtained (isolation frequency = 32/4×10 = 80%). Three representative single-spore isolates (OFC-1, OFC-2, and OFC-3) were used for further study. Colonies on PDA were white to gray with cottony mycelia in 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 10.5 to 17.5 µm × 3.5 to 5.0 µm (n = 50). Appressoria were oval to irregular in shape and dark brown in color, and they measured 6.5 to 8.5 µm × 4.5 to 7.5 µm (n = 20). Morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences of them deposited in GenBank under nos. MZ047368-MZ047370 (ITS), MZ126925-MZ126927 (GAPDH), MZ126895-MZ126897 (CHS-1), and MZ126835-MZ126837 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, CHS-1, and ACT that clustered the isolates with C. siamense (the type strain MFLU 090230), while distanced the isolates with C. gloeosporioides (the type strain CBS 112999). The pathogenicity was tested through in vivo experiments. In group 1, the inoculation and control plants (n = 5, 3-month-old) were sprayed with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water, respectively, until run-off. In group 2, the unwounded leaflets were inoculated with mycelial plugs of the isolates or agar plugs (as control). Three plugs were for each leaflet ( n = 5). The plants were grown in pots in a greenhouse at 25°C to 28°C, with relative humidities approximately 80%. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy. The pathogen re-isolated from all the inoculated leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C. gloeosporioides has been reported to cause leaf spot on O. fragrans in Jiangxi Province of China (Tanget al., 2018), but not by C. siamense. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on O. fragrans. Thus, this work provides a foundation for controlling anthracnose in O. fragrans in the future.

Heart Rate Variability Reactivity to Food Image Stimuli is Associated with Body Mass Index.

Appetitive control is driven by the hedonic response to food and affected by several factors. Heart rate variability (HRV) signals have been used to index autonomic activity and arousal levels towards visual stimuli. The current research aimed to examine the influence of body mass index (BMI), disordered eating behaviors, and sex on the HRV reactivity to food in a nonclinical sample. Thirty-eight healthy male and sixty-one healthy female participants completed questionnaires assessing disordered eating symptoms. HRV was recorded when the participants received visual stimuli of high-calorie food, neutral and negative emotional signals. Generalized estimating equation models were used to investigate the associations between HRV, BMI, disordered eating behaviors, and sex across the three stimulus types. Male participants demonstrated a higher ratio of low-frequency power to high-frequency power (LF/HF) than females across all the stimulus types. An increase in LF/HF reactivity to food signals was observed in all the study subjects. The moderation effect of BMI on LF/HF in response to food signals was also observed. Our study suggests that body weight may play a role in the interaction between sympathetic activity and food stimuli; however, how the interaction between sympathetic activity and food stimuli contributes to diet control warrants further investigation.

Early Predictors of De Novo and Subthreshold Late-Onset ADHD in a Child and Adolescent Cohort.

Objective: The study aimed to identify early childhood risk factors for de novo and subthreshold late-onset ADHD. Method: ADHD symptoms were assessed in 9,875 participants from the Twins Early Development Study (TEDS) using the Conners' Parent Rating Scale at ages 8, 12, 14, and 16 years, along with other childhood characteristics and adolescent outcomes. Multinomial logistic regressions were implemented to identify early childhood predictors of late-onset ADHD and childhood-onset persistent ADHD, with non-ADHD controls as the reference category. Results: Male sex, increased childhood conduct problems, and low socioeconomic status predicted de novo late-onset ADHD. Additional risk factors predicted subthreshold late-onset ADHD and childhood-onset persistent ADHD. Late-onset ADHD symptoms were also accompanied by increased co-occurring behavioral and emotional problems. Conclusion: Findings of different childhood predictors between subthreshold and de novo late-onset ADHD suggest further investigation into time-varying environmental and biological factors driving psychopathological changes is warranted to fully characterize late-onset ADHD.

Vicryl Mesh Coverage Reduced Recurrence After Bullectomy for Primary Spontaneous Pneumothorax.

BACKGROUND: Although thoracoscopic stapled bullectomy is a standard procedure for primary spontaneous pneumothorax (PSP), the postoperative recurrence rate is high. We investigated whether using a Vicryl (Ethicon, Somerville, NJ) mesh to cover the staple line after bullectomy reduces the postoperative recurrence rate. METHODS: Our single-blind, parallel-group, prospective, randomized controlled trial at 2 medical centers in Taiwan studied patients with PSP who were aged 15 to 50 years and required thoracoscopic bullectomy. On the day of operation, patients were randomly assigned (1:1) to receive Vicryl mesh (mesh group) or not (control group) after thoracoscopic bullectomy with linear stapling and mechanical apical pleural abrasion. Randomization was achieved using computer-generated random numbers in sealed envelopes. Our primary end point was the pneumothorax recurrence rate within 1 year after the operation (clinicaltrials.gov number, NCT01848860.) RESULTS: Between June 2013 and March 2016, 102 patients were assigned to the mesh group and 102 to the control group. Within 1 year after operation, recurrent pneumothorax was diagnosed in 3 patients (2.9%) in the mesh group compared with 16 (15.7%) in the control group (P = .005). The short-term postoperative results and hospitalization duration were comparable between the groups. CONCLUSIONS: For thoracoscopic bullectomy with linear stapling and mechanical apical pleural abrasion, the use of a Vicryl mesh to cover the staple line is effective for reducing the postoperative recurrence of pneumothorax. Vicryl mesh coverage can be considered an optimal adjunct to the standard surgical procedure for PSP.

Are there causal relationships between attention-deficit/hyperactivity disorder and body mass index? Evidence from multiple genetically informed designs.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) and body mass index (BMI) are associated. However, it remains unclear whether this association reflects causal relationships in either direction or confounding. Here, we implemented genetically informed methods to examine bidirectional causality and potential confounding. METHODS: Three genetically informed methods were employed: (i) cross-lagged twin-differences analyses to assess bidirectional effects of ADHD symptoms and BMI at ages 8, 12, 14 and 16 years in 2386 pairs of monozygotic twins from the Twins Early Development Study (TEDS); (ii) within- and between-family ADHD and BMI polygenic score (PS) analyses in 3320 pairs of dizygotic TEDS twins; and (iii) two-sample bidirectional Mendelian randomization (MR) using summary statistics from genome-wide association studies (GWAS) on ADHD (N = 55,374) and BMI (N = 806,834). RESULTS: Mixed results were obtained across the three methods. Twin-difference analyses provided little support for cross-lagged associations between ADHD symptoms and BMI over time. PS analyses were consistent with bidirectional relationships between ADHD and BMI, with plausible time-varying effects from childhood to adolescence. MR findings also suggested bidirectional causal effects between ADHD and BMI. Multivariable MR indicated the presence of substantial confounding in bidirectional relationships. CONCLUSIONS: The three methods converged to highlight multiple sources of confounding in the association between ADHD and BMI. PS and MR analyses suggested plausible causal relationships in both directions. Possible explanations for mixed causal findings across methods are discussed.

Nanodiamond-based microRNA delivery system promotes pluripotent stem cells toward myocardiogenic reprogramming.

BACKGROUND: Gene therapy is the advanced therapeutics for supplying or replacing the genetic material in patients with inherited disorders. Recent clinical studies have made some progress in a wide range of applications, including monogenic disorders, neurodegenerative diseases, malignant tumors, and congenital diseases. Heart diseases, especially myocardial ischemia, remain one of the leading causes of mortality worldwide and usually result in irreparable cardiomyocyte damage and severe heart failure. METHODS: Most advances in induced pluripotent stem cell (iPSC) technologies for promoting regenerative medicine and stem cell research. However, the driver molecules of myocardial-lineage differentiation and the functional reconstruction capacity of iPSC-derived cardiomyocytes are still an open question. Nanomedicine-based gene delivery provided a crucial platform to carry on the biogenomic materials for equipping functionalities and engineering the living organ environment. Nanodiamond (ND), a carbon-based nanomaterial, has been discovered and shown the high biocompatible and less toxicity for transporting protein, drug, and genomic plasmids. RESULTS: Here, we applied ND as a gene delivery vehicle to carry microRNA (miR-181a), and then transfected into iPS to promote cardiomyocyte-lineage differentiation. Notably, miR-181a plays a key role in iPS-derived cardiomyocyte differentiation which directly targets Hox-A11, leading to elevated MyoD expression and enhanced cardiomyocyte differentiation. CONCLUSION: Our study demonstrated that miR-181a promotes iPSC differentiation into functional cardiomyocytes. Delivery of NANO-DIAMOND-miR-181a may host clinical potential to enhance the differentiation and recovery of the cardiogenic function in injured cardiomyocytes.

Generation of osimertinib-resistant cells from epidermal growth factor receptor L858R/T790M mutant non-small cell lung carcinoma cell line.

BACKGROUND: Lung cancer contributes to high cancer mortality worldwide with 80% of total cases diagnosed as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain serves as a druggable target in NSCLC patients with exon 19 deletion and L858R mutation. However, patients eventually succumbed to resistance to first- and second-generation EGFR-TK inhibitors through activation of T790M mutation. Third-generation EGFR-TKI, Osimertinib exhibits high efficacy in patients with exon 19 deletion/L858R/T790M mutation but they experienced acquired resistance thereafter. Available treatment options in NSCLC patients remains a challenge due to unknown molecular heterogeneity responsible for acquired resistance to EGFR-TKI. In this study, we aim to generate Osimertinib-resistant (OR) cells from H1975 carrying L858R/T790M double mutation which can be used as a model to elucidate mechanism of resistance. METHODS: OR cells were established via stepwise-dose escalation and limiting single-cell dilution method. We then evaluated Osimertinib resistance potential via cell viability assay. Proteins expression related to EGFR-signalling, epithelial to mesenchymal transition (EMT), and autophagy were analyzed via western blot. RESULTS: OR cell lines exhibited increased drug resistance potential compared to H1975. Distinguishable mesenchymal-like features were observed in OR cells. Protein expression analysis revealed EGFR-independent signaling involved in the derived OR cells as well as EMT and autophagy activity. CONCLUSION: We generated OR cell lines in-vitro as evidenced by increased drug resistance potential, increased mesenchymal features, and enhanced autophagy activity. Development of Osimertinib resistance cells may serve as in-vitro model facilitating discovery of molecular aberration present during acquired mechanism of resistance.

Using multiple-steps bioinformatic analysis to predict the potential microRNA targets by cardiogenic HoxA11.

BACKGROUND: In this study, our major aim is to using multiple-steps bioinformatic analysis to predict cardiogenic genes with targeting mRNA profiling for predicting cardiogenic HoxA11 gene. METHODS: We first analyzed the microarray data with bioinformatic measurement, including combining with panel module 1 (mouse embryonic stem cells), panel module 2 (mouse induced pluripotent stem cells), and panel module 3 (gene list form literature of heart development). A literature-based comparison of the two microarrays and a software-based (Targetscan program, www.targetscan.org) comparative analysis of the two datasets. Furthermore, we select the common central pathways and potential candidate genes involved in the cardiomyocyte-lineaged differentiation and development. RESULTS: Schematic presentation of a putative miR181a target site in Hox-A11 3'UTR. The bioinformatic result showed that potential interacted cardiogenic targets of Tbx5, Tbx20, Mal2c, Nkx2.5, cTNT, Cx43, MHC, and MCK in different treatment groups of pluripotent stem cells by using a literature-based comparison of the two microarrays and a software-based gene-lineage system. CONCLUSION: Our findings support that mir181a is an up-stream regulating microRNA to target the 3'UTR of HoxA11 mRNA during the process of cardiomyocyte differentiation.