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Advanced Science (Weinheim,... Aug 2022Tumor microenvironment crosstalk, in particular interactions between cancer cells, T cells, and myeloid-derived suppressor cells (MDSCs), mediates tumor initiation,...
Tumor microenvironment crosstalk, in particular interactions between cancer cells, T cells, and myeloid-derived suppressor cells (MDSCs), mediates tumor initiation, progression, and response to treatment. However, current patient-derived models such as tumor organoids and 2D cultures lack some essential niche cell types (e.g., MDSCs) and fail to model complex tumor-immune interactions. Here, the authors present the novel acoustically assembled patient-derived cell clusters (APCCs) that can preserve original tumor/immune cell compositions, model their interactions in 3D microenvironments, and test the treatment responses of primary tumors in a rapid, scalable, and user-friendly manner. By incorporating a large array of 3D acoustic trappings within the extracellular matrix, hundreds of APCCs can be assembled within a petri dish within 2 min. Moreover, the APCCs can preserve sensitive and short-lived (≈1 to 2-day lifespan in vivo) tumor-induced MDSCs and model their dynamic suppression of T cell tumor toxicity for up to 24 h. Finally, using the APCCs, the authors succesully model the combinational therapeutic effect of a multi-kinase inhibitor targeting MDSCs (cabozantinib) and an anti-PD-1 immune checkpoint inhibitor (pembrolizumab). The novel APCCs may hold promising potential in predicting treatment response for personalized cancer adjuvant therapy as well as screening novel cancer immunotherapy and combinational therapy.
Topics: Humans; Immune Checkpoint Inhibitors; Immunotherapy; Myeloid-Derived Suppressor Cells; Neoplasms; Tumor Microenvironment
PubMed: 35611994
DOI: 10.1002/advs.202201478 -
Physical Review. E May 2021Cell plating, the spreading out of a liquid suspension of cells on a surface followed by colony growth, is a common laboratory procedure in microbiology. Despite this,...
Cell plating, the spreading out of a liquid suspension of cells on a surface followed by colony growth, is a common laboratory procedure in microbiology. Despite this, the exact impact of its parameters on colony growth has not been extensively studied. A common protocol involves the shaking of glass beads within a Petri dish containing solid growth media. We investigated the effects of multiple parameters in this protocol: the number of beads, the shape of movement, and the number of movements. Standard suspensions of Escherichia coli were spread while varying these parameters to assess their impact on colony growth. Results were assessed by a variety of metrics: the number of colonies, the mean distance between closest colonies, and the variability and uniformity of their spatial distribution. Finally, we devised a mathematical model of shifting billiard to explain the heterogeneities in the observed spatial patterns. Exploring the parameters that affect the most fundamental techniques in microbiology allows us to better understand their function, giving us the ability to precisely control their outputs for our exact needs.
Topics: Culture Media; Escherichia coli; Movement
PubMed: 34134194
DOI: 10.1103/PhysRevE.103.052410 -
Plant Disease May 2022Rice ( L.) is the principle staple crops in the World and its production can be severely damaged by species. Several species including , , , , , have been recorded to...
Rice ( L.) is the principle staple crops in the World and its production can be severely damaged by species. Several species including , , , , , have been recorded to cause rice seedling root rot in Taiwan (List of Plant Diseases in Taiwan edited by Tzean et al., 2019). During the survey of rice seedling diseases, we identified a new species of that causes seedling root rot on rice in commercial nursery trays in two nursery fields in 2019 in Taichung, Taiwan. Stunting and root rot symptom were found on the affected plants and up to 20% seedlings in a nursery tray showed similar symptoms. To isolate the pathogen, symptomatic roots were surface sterilized with 75% ethanol for 1 min and rinsed in sterile water. The margin of lesion was cut off, placed on 1.5% water agar and incubated at 28 ℃. After 24 h, the hyphal tips of a white colony growing from the diseased region were transferred to potato dextrose agar (PDA) medium. Koch's postulates were fulfilled by inoculating the germinated rice seeds with mycelia. Rice seeds of var. Tainan11 (TN11) were treated with 75% ethanol and then 1.2% NaOCl for 15 min. The sterilized seeds were soaked in sterile water under dark condition for 3 days and the water was replaced every day. Five of the pre-germinated seeds with 2~5 mm embryonic shoot were placed in a sterile petri-dish and inoculated with 3-ml mycelial suspension (OD = 0.045) prepared by blending the mycelia of a 3-days PDA culture using an Oster 10 speed blender 6640 (Oster, USA). The seeds-mycelia were then covered with sterilized soil mixture of Akadama soil and rice husk (1:1, volume to volume) and incubated in a growth chamber at 28 ℃. Seven days post-inoculation, the inoculated seedlings showed stunting with short and necrotic roots (Fig. S1). The pathogen was reisolated from the diseased seedlings and identified with morphology and molecular methods. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia and zoospore (Chamswarng and Cook 1985). Globose oogonia with multiple antheridia (1-5 per oogonium), inflated filamentous sporangia, vesicle with abundant zoospores, main hypha with up to 6.57 μm wide and mature aplerotic oospores with diameter 24.35-30.81 μm (average= 27.22 μm; n=20) were observed (Fig. S1) that are similar to the descriptions for (van der Plaats-Niterink 1981). Genomic DNA was extracted with CTAB method (Wang and White 1997) and the sequences of the internal transcribed spacer (ITS) region and gene region of β-tubulin () and cytochrome c oxidase subunit II ( II) were amplified with published primers (Villa et al., 2006). The obtained sequences were submitted to GenBank (accession nos: OL701302 (ITS), OL763269 (tub), and OL763270 (cox II); Fig. S2). Phylogenetic relationships between this pathogen and other 55 isolates, including the type species of (ATCC11101), were conducted with the concatenated sequences of tub and cox II and analyzed by Bayesian interference (Fig. S3). Based on the tree built with and II sequences, this pathogen was identified as that has not been reported in rice and other plants in Taiwan. It was observed in laboratory assays that this pathogen caused significant root-rot symptoms on several major rice varieties grown in Taiwan, including TN11, Tainung67 and Kaoshiung139. It may potentially cause severe crop loss in rice production, especially in nurseries. This identification provides important information on rice disease management.
PubMed: 35596245
DOI: 10.1094/PDIS-01-22-0092-PDN -
ELife Nov 2022Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the...
Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more , by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.
Topics: Animals; Rabbits; Bordetella bronchiseptica; Bordetella Infections; Helminths; Respiratory Tract Infections; Respiratory System
PubMed: 36346138
DOI: 10.7554/eLife.70347 -
Frontiers in Endocrinology 2023Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the...
Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24 h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.
Topics: Animals; Male; Leydig Cells; Ducks; Testosterone; Testis; Cells, Cultured
PubMed: 37347106
DOI: 10.3389/fendo.2023.1195618 -
Journal of Cancer 2021Gastric cancer is one of the most common malignant tumors in the world. IGHG1 is a differentially expressed protein screened out in gastric cancer in the early stage of...
Gastric cancer is one of the most common malignant tumors in the world. IGHG1 is a differentially expressed protein screened out in gastric cancer in the early stage of the subject group. This topic explores the expression of IGHG1 in gastric cancer and the effect of IGHG1 on the proliferation, migration, invasion and EMT of gastric cancer SGC7901 cells and its mechanism of action. Twenty cases of gastric cancer were purified by laser Capture Microdissection. The isotopic tags for relative and absolute quantification was used to label the proteins, and then analyzed and identified them by quantitative proteomics. Immunohistochemical staining method was used to detect the expression of IGHG1 protein in gastric cancer tissues. Western blot was used to detect the expression of IGHG1 in gastric cancer cells. The MTT and Petri dish clone formation experiment analyzed the effect of low expression of IGHG1 on the proliferation of SGC7901 cells. Scratch test and Transwell migration and invasion test to observe the effect of low expression of IGHG1 on the migration and invasion of SGC7901 cells. Western blot was used to detect the effect of low expression of IGHG1 on the expression of EMT-related proteins. 243 proteins related to gastric mucosal lesions were preliminarily identified. We found that IGHG1 is highly expressed in gastric cancer tissues compared with normal control tissues. IGHG1 promotes the proliferation, migration and invasion of gastric cancer cells. Compared with the control group, the expression of EMT-related proteins Vimentin, N-cadherin, TGF-β, P-SMAD3 was decreased and the expression of E-cadherin was increased after IGHG1 low expression. IGHG1 induces EMT in SGC7901 cells by regulating the TGF-β/SMAD3 signaling pathway.
PubMed: 33995624
DOI: 10.7150/jca.56056 -
Biology Open May 2023Head and neck cancer (HNC) differs at anatomical sites and hypopharyngeal cancer (HPC) is a type of HNC. The non-surgical treatment option for advanced cases of HPC is...
Head and neck cancer (HNC) differs at anatomical sites and hypopharyngeal cancer (HPC) is a type of HNC. The non-surgical treatment option for advanced cases of HPC is radiotherapy (RT) with or without chemotherapy but survival is poor. Thus, new treatment approaches in combination with RT are essential. Yet, obtaining post-RT treated tumour specimens and lack of animal models with identical anatomical sites are the major translational research barriers. To overcome these barriers, for the first time, we have developed a tumour-stroma based in vitro three-dimensional (3D)-tumouroid co-culture model of HPC by growing FaDu and HS-5 cells together to mimic the complex tumour-microenvironment in a Petri dish. Before growing the cells together, imaging flow cytometry revealed distinct epithelial and non-epithelial characteristics of the cells. Growth rate of the 3D-tumouroid co-culture was significantly higher compared to the tumouroid monoculture of FaDu. Histology and morphometric analysis were done for the characterisation as well as the development of hypoxia was measured by CAIX immunostaining in this 3D-tumouroid co-culture. Taken together, this innovative in vitro 3D model of HPC resembles many features of the original tumour. The wider application of this pre-clinical research tool is in understanding newer combination (e.g. immunotherapy) treatment approaches with RT in HPC and beyond.
Topics: Animals; Coculture Techniques; Hypopharyngeal Neoplasms; Bioengineering; Tumor Microenvironment
PubMed: 37194999
DOI: 10.1242/bio.059949 -
ELife Jun 2020Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and...
Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.
Topics: Bacteria; Bacteriological Techniques; Drug Resistance, Bacterial; Gastrointestinal Microbiome; High-Throughput Screening Assays
PubMed: 32553109
DOI: 10.7554/eLife.56998 -
Plant Disease Jul 2022Palm grass (Setaria palmifolia) has been used as an ornamental plant and vegetable crop (Wu, 2009; Plarre, 1995). In June 2019, 2-10 mm severe leaf lesions with gray...
Palm grass (Setaria palmifolia) has been used as an ornamental plant and vegetable crop (Wu, 2009; Plarre, 1995). In June 2019, 2-10 mm severe leaf lesions with gray centers and brown-yellow edges were observed on the leaves of palm grass in Liuyang city (28°43'N, 114°12'E), Hunan province, China (Fig. 1A). Disease incidence on leaves was 20 - 40%. The infected leaves were collected and disinfected with 75% alcohol for 30 sec and 1% sodium hypochlorite for 1 min, followed by three rinses in sterilized ddH2O, dried on sterilized filter paper, and incubated on water agar for 48 h under continuous fluorescent light at 26℃. Then, typical pyriform and 2-septate conidia (23.97 - 30.37 × 7.42 - 9.98 μm, N = 30) appeared at the lesions (Fig. 1B). Four single-spore were captured, and then grew on oatmeal tomato agar for seven days under continuous fluorescent light at 26℃ to obtain four isolates (LY-ZY-7a, -7b, -9b and -9c) and produce conidia for inoculation tests. The colony morphology of LY-ZY-7b on OTA was gray and floccose, and the growth rate was 6.15 - 6.31 mm/d at 26 °C (Fig. 1C). Spores of LY-ZY-7b were washed off with sterilized ddH2O plus 0.025% Tween-20 to make spore suspensions. For scratch inoculation, 10 μL spore suspension (1 × 105 spores/mL) was inoculated on the wound scratched with a sterilized pin along the vein (3 mm × 3 mm) on palm grass middle leaf of 4-week-old seedlings. The inoculated leaves were sealed in a 15-cm Petri dish. For spray inoculation, 20 mL spore suspension (5 × 104 spores/mL) was made and sprayed on ten healthy palm grasses of 4-week-old seedlings. Plants used as negative controls were sprayed with sterilized ddH2O plus 0.025% Tween-20 (Liu et al. 2022; Zhang et al. 2014). After inoculation, all plants were put into transparent boxes to maintain > 95% humidity and covered with black plastic bags for one day. Then, the boxes containing the plants were placed in a growth chamber at 26°C (12 h light / 12 h darkness photoperiod). After six days, typical blast-type lesions with brown-yellow edges were visible on the leaves. Control plants did not show symptoms (Fig. 1D, 1E). Microscopical examination showed that the conidia and conidiophore recovered from the lesion of the inoculated plants have the same morphology as those recovered from natural infected tissues (Fig. 1F, 1G). The colony morphology of the pathogen isolated from the artificially inoculated tissue was consistent with that of isolate LY-ZY-7b (Fig. 1C). The spore suspension (5 × 104 spores/mL) of isolate LY-ZY-7b and one rice-infecting strain P131 (Yang et al., 2010) was made and sprayed onto 4-week-old seedlings of three rice cultivars. But unfortunately, isolate LY-ZY-7b could not cause any disease lesions on the tested rice cultivars, whereas strain P131 produced many typical blast lesions on rice leaves (Fig. 1H). Then, the fungal genetic identity of four isolates (LY-ZY-7a, -7b, -9b, and -9c) was confirmed by comparison of the sequence obtained from partial DNA of Actin (ACT), ITS, and RPB1 loci from our isolates and those previously published by Klaubauf et al. 2014. The nucleotide sequences of ACT, ITS, and RPB1 were submitted to GenBank ON228695-ON228697 (ACT), ON210978-ON210980 (ITS), ON228698-ON228701 (RPB1). A phylogenetic tree deduced from a maximum likelihood analysis based on combined ACT-ITS-RPB1 sequence data of Pyricularia showed that these four isolates (LY-ZY-7a, -7b, -9b, and -9c) clustered together on Pyricularia oryzae, with a high bootstrap support value (Fig. 2). Based on morphological characteristics and molecular phylogeny, these four isolates were identified as P. oryzae (Klaubauf et al. 2014; Qi et al. 2019). To our knowledge, this is the first report of blast disease on palm grass caused by P. oryzae in China, which will help develop disease management strategies against palm grass blast. Moreover, as a host of P. oryzae, palm grass might contribute as an inoculum source for blast diseases on cereal crops (such as rice, wheat, and barley) caused by P. oryzae in the field.
PubMed: 35815958
DOI: 10.1094/PDIS-05-22-1077-PDN -
International Journal of Clinical... 2022The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files Autoclave, Glass-bead sterilizer,...
AIM AND OBJECTIVE
The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser.
MATERIALS AND METHODS
Fifty-two k-files of size #25 and length 21 mm were taken for the study. All the 52 files were presterilized in an endodontic instrument box autoclave. spore suspension was prepared and all the presterilized files were contaminated with stearothermophilus spore suspension in a sterile Petri dish under vacuum hood safety. Later, the test files were randomly divided into four groups of 13 each and subjected to four different methods of sterilization- Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Files were then be placed in thioglycollate media containing test tubes and incubated in an incubator at 55°C and checked for turbidity at 3 days and 21 days.
RESULT
The result revealed that there was no Turbidity present in test tubes on both the 3rd and 21st day for autoclave. In all the remaining sterilization procedures there was some amount of turbidity present. In terms of sterilization provided autoclave provides complete sterilization and glutaraldehyde solution is the least effective.The specificity of was then confirmed with a sugar test viz., starch hydrolysis test which gave a positive result confirming the presence of .
CONCLUSION
We can conclude that autoclave is the perfect process of sterilization providing 100% sterility and although Glass-bead didn't provide 100% sterility, it can be used as an alternative if autoclave is not available.
HOW TO CITE THIS ARTICLE
Ameer B, Khatib MS, Peerzade SM, Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An Study. Int J Clin Pediatr Dent 2022;15(2):149-152.
PubMed: 37457218
DOI: 10.5005/jp-journals-10005-2346