Project description:Comparison of chitosan-treated B. cereus ATCC 14579 cells with non-treated B. cereus ATCC 14579 cells. 2 chitosans with similar molecular weight (Mw) but different degrees of acetylation (Fa) were used: chitosan B (Mw: 28.4 kDa, Fa: 0.16) (samples 1-3) and chitosan A (Mw: 36.0 kDa, Fa: 0.01) (samples 4-6).
Project description:Fifty healthcare workers (HCW) who had received Mycobacterium-w (Mw) and at least one dose of ChAdOx1 nCoV-19 vaccine subsequently (Mw+ChAdOx1 group) were monitored for symptomatic COVID-19, during a major outbreak with the delta variant of SARS-CoV-2 (April-June, 2021) in India, along with 201 HCW receiving both doses of the vaccine without Mw (ChAdOx1 group). Bulk RNA-Seq analysis was carried out on 4 subjects enrolled from each group.
Project description:Tumor-derived microparticles (TMPs), a subtype of extracellular vesicles, hold great promise in tumor immunotherapies and vaccines, and have demonstrated significant potential as drug delivery systems for clinical tumor treatment. In our previous study, we utilized TMPs released from parental tumor cells upon ultraviolet irradiation (UV-TMPs) to deliver methotrexate (MTX), effectively managing malignant pleural effusion (MPE) in advanced lung cancer cases. However, concerns remain regarding the potential risks associated with nucleic acid incorporation, alongside the limited antitumor efficacy of UV-TMPs alone. Here, we introduce a microwave (MW)-assisted method for preparing TMPs, termed MW-TMPs. Brief exposure to short-wavelength MW radiation triggered various forms of programmed cell death, including apoptosis, necroptosis, and pyroptosis, promoting the release of TMPs. These resulting MW-TMPs contained fewer nucleic acids and exhibited superior antitumor activity in vivo compared to UV-TMPs. Mechanistically, MW-TMPs induced immunogenic cell death (ICD) by shuttling HMGB1 and enabled dual targeting of tumor cells by natural killer (NK) and T cells, while reprogramming of suppressive tumor immune microenvironments in LLC lung adenocarcinoma (LUAD) mouse models. Furthermore, similar effective tumor inhibition was observed in clinical MPE samples and zebrafish PDX models, suggesting the potential of MW-TMPs for clinical cancer immunotherapy. In further application studies, MW-TMPs successfully encapsulated the chemotherapeutic drug MTX, demonstrating enhanced antitumor efficacy both in vitro and in vivo, thereby establishing their potential as an innovative drug delivery platform. Moreover, combination immunotherapy with PD-L1 blockade confirmed a synergistic antitumor effect. This MW-TMP development strategy has improved upon traditional UV-TMP methods in terms of simplicity, efficiency, safety, and efficacy.
Project description:Kertinocyte cultures grown in 60 mm petri dishes were placed 186 mm from the solar simulator source (Solar-simulated ultraviolet radiation 1600W Xenon short arc lamp with an Oriel Air Mass 1 Direct Filter, (AM1:D:B; model 81074) and KG2 Short Pass Filter. irradiance 9.84 mW/cm² for UVA (98.3%), 0.174 mW/cm² for UVB (1.7%) and 10 mW/cm² (0.017 mW/cm² erythemally-weighted) for the total UVR irradiance) and received a dose of either 0, 10, 20 and 150 kJ/m2 of unweighted ultraviolet radiation and 0, 10 and 150 kJ/m2 of unweighted ultraviolet radiation with SPF 15 sunscreen filtration (Homosalate 3%, Octisalate 4%, Avobenzone 2%, Titanium dioxide 0.66%) (2 mg/cm2 sandwiched between two 5x5 inch quartz plates) and were temperature controlled at 37oC using a customized water-bath. Six and Twenty-four hours post-exposure cells were harvested and RNA was extracted and subjected to microarray analysis.
Project description:Pristine groundwater is a highly stable environment with microbes adapted to dark, oligotrophic conditions. Input events like heavy rainfalls can introduce excess particulate organic matter including surface-derived microbes into the groundwater, hereby creating a disturbance to the groundwater microbiome. Some of the translocated bacteria are not able to thrive in groundwater and will form necromass. Here, we investigated the effects of necromass addition to the microbial community in fractured bedrock groundwater, using groundwater mesocosms as model systems. We followed the uptake of 13C-labeled necromass by the bacterial and eukaryotic groundwater community quantitatively and over time by employing a combined protein and DNA stable isotope probing approach. Necromass was rapidly depleted in the mesocosms within four days, accompanied by a strong decrease of Shannon diversity and an increase of bacterial 16S rRNA gene copy numbers by one order of magnitude. Species of Flavobacterium, Massilia, Rheinheimera, Rhodoferax and Undibacterium dominated the microbial community within two days and were identified as key players in necromass degradation, based on a 13C incorporation of > 90% in their peptides. Their proteomes showed various uptake and transport related proteins, and many proteins involved in metabolizing amino acids. After four and eight days of incubation, autotrophic and mixotrophic groundwater species of Nitrosomonas, Limnohabitans, Paucibacter and Acidovorax increased in abundance, with a 13C incorporation between 0.5 and 23%. Our data point towards a very fast and exclusive uptake of labeled necromass by a few specialists followed by a concerted action of groundwater microorganisms, including autotrophs presumably fueled by released, reduced nitrogen and sulfur compounds generated during necromass degradation.
Project description:Comparison of chitosan-treated B. cereus ATCC 14579 cells with non-treated B. cereus ATCC 14579 cells. 2 chitosans with similar molecular weight (Mw) but different degrees of acetylation (Fa) were used: chitosan B (Mw: 28.4 kDa, Fa: 0.16) (samples 1-3) and chitosan A (Mw: 36.0 kDa, Fa: 0.01) (samples 4-6). One-condition design comparision of treated vs. non-treated control. 3 biological replicates, including a dye swap.
Project description:Tumor-derived microparticles (TMPs), a subtype of extracellular vesicles, hold great promise in tumor immunotherapies and vaccines, and have demonstrated significant potential as drug delivery systems for clinical tumor treatment. However, concerns remain regarding the potential risks associated with nucleic acid incorporation, alongside the limited antitumor efficacy of ultraviolet irradiation (UV-TMPs) alone. Here, we introduce a microwave (MW)-assisted method for preparing TMPs, termed MW-TMPs. Brief exposure to short-wavelength MW radiation triggered various forms of programmed cell death, including apoptosis, necroptosis, and pyroptosis, promoting the release of TMPs. These resulting MW-TMPs contained fewer nucleic acids and exhibited superior antitumor activity in vivo compared to UV-TMPs. Mechanistically, MW-TMPs induced immunogenic cell death (ICD) by shuttling HMGB1 and enabled dual targeting of tumor cells by natural killer (NK) and T cells, while reprogramming of suppressive tumor immune microenvironments in LLC lung adenocarcinoma (LUAD) mouse models. When the intervention to LLC mouse models was completed, cell suspensions were prepared by tissue homogenate of fresh subcutaneous tumors. The single-cell suspension was added to the single-cell sorting honeycomb plate and excessive magnetic beads with barcodes were put into the plate. During the phase of cell lysis, the RNA was labeled as bounding to the barcode. Subsequently, reverse transcription of RNA and cDNA synthesis were carried out to obtain cDNA library for subsequent sequencing. Paired-end sequencing was conducted in the Nova seq6000 sequencing platform to get the transcriptome results. BD Rhapsody Analysis pipeline was applied to identify the UMI sequence and the cell tag sequence alignment to the reference genome (GRCm39).
Project description:Whole transcriptome Differential Gene Expression (DGE) analysis was carried out on four biological replicates of both Mw (0.1 ml Mw administrated intradermally in each arm) and Control group at 6 months following exposure to Mycobacterium-w. Sequencing was done through Direct cDNA Sequencing (oxford nanopore technologies, Oxford, UK) using RNA isolated from Peripheral blood mononuclear cells (PBMC) by Trizol method. Native barcoding and adaptor ligation was done according to the manufacturer’s instructions. Ligated cDNA was loaded on the flow cell (R9.4.1) in MinION and were sequenced specifying 72 hours protocol. MinKNOW (v21.06.10, Microsoft Windows OS based) was used to generate FAST5 files. FAST5 files were base-called with CPU based Guppy basecaller (v.5.0.11) (ONT) to generate FASTQ files. DGE analysis was done using “pipeline-transcriptome-de” (https://github.com/nanoporetech/pipeline-transcriptome-de) pipeline. DGE analysis confirmed that upregulation of ANK pathway was evident at 6 months in the Mw group. Apart from upregulation of KLRC2 and B3GAT1 and downregulation of KLRC1, the key transcription factor in the ANK pathway, BCL11b, was persistently upregulated. Downregulation of EAT-2 and PLZF further corroborated the classic gene expression signature of ANK cells. Moreover, increased expression of AT-rich interaction domain 5B (ARID5B), as demonstrated in the Mw group plays an important role in enhanced metabolism in ANK cells as well as increased IFN-γ release and survival. DGE analysis also revealed an enhancement of ANK mediated ADCC pathway, with significant upregulation of CD247 along with downregulation of FCER1G, which is a typical signature of ANK-ADCC. Both CD247 and FCER1G are adapter molecules for FCGRIIIA (CD16) with CD247 possessing 3 ITAMs against one ITAM of FCER1G, increasing the ADCC several folds. It is possible that Mw induced augmentation of NK-ADCC might potentiate the efficacy of SARS-CoV2 vaccines as well.