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Introduction: A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern.
Methods: Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes.
Results: Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-g) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L_CD45RA+terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation.
Conclusions: Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients.
A firm link between endoplasmic reticulum (ER) stress and tumors has been wildly reported. Endoplasmic reticulum oxidoreductase 1 alpha (ERO1α), an ER-resident thiol oxidoreductase, is confirmed to be highly upregulated in various cancer types and associated with a significantly worse prognosis. Of importance, under ER stress, the functional interplay of ERO1α/PDI axis plays a pivotal role to orchestrate proper protein folding and other key processes. Multiple lines of evidence propose ERO1α as an attractive potential target for cancer treatment. However, the unavailability of specific inhibitor for ERO1α, its molecular inter-relatedness with closely related paralog ERO1β and the tightly regulated processes with other members of flavoenzyme family of enzymes, raises several concerns about its clinical translation. Herein, we have provided a detailed description of ERO1α in human cancers and its vulnerability towards the aforementioned concerns. Besides, we have discussed a few key considerations that may improve our understanding about ERO1α in tumors.
Trade of wild-caught animals is illegal for many taxa and in many countries. Common regulatory procedures involve documentation and marking techniques. However, these procedures are subject to fraud and thus should be complemented by routine genetic testing in order to authenticate the captive-bred origin of animals intended for trade. A suitable class of genetic markers are SNPSTRs that combine a short tandem repeat (STR) and single nucleotide polymorphisms (SNPs) within one amplicon. This combined marker type can be used for genetic identification and for parentage analyses and in addition, provides insight into haplotype history. As a proof of principle, this study establishes a set of 20 SNPSTR markers for Athene noctua, one of the most trafficked owls in CITES Appendix II. These markers can be coamplified in a single multiplex reaction. Based on population data, the percentage of observed and expected heterozygosities of the markers ranged from 0.400 to 1.000 and 0.545 to 0.850, respectively. A combined probability of identity of 5.3*10-23 was achieved with the whole set, and combined parentage exclusion probabilities reached over 99.99%, even if the genotype of one parent was missing. A direct comparison of an owl family and an unrelated owl demonstrated the applicability of the SNPSTR set in parentage testing. The established SNPSTR set thus proved to be highly useful for identifying individuals and analysing parentage to determine wild or captive origin. We propose to implement SNPSTR-based routine certification in wildlife trade as a way to reveal animal laundering and misdeclaration of wild-caught animals.
The French–Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau’s surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.
Nitrosamines have been identified as a probable human carcinogen and thus are of high concern in many manufacturing industries and various matrices (for example pharmaceutical, cosmetic and food products, workplace air or potable- and wastewater). This study aims to analyse nine nitrosamines relevant in the field of occupational safety using a gas chromatography-drift tube ion mobility spectrometry (GC-DT-IMS) system. To do this, single nitrosamine standards as well as a standard mix, each at 0.1 g/L, were introduced via liquid injection. A GC-DT-IMS method capable of separating the nitrosamine signals according to retention time (first dimension) and drift time (second dimension) in 10 min was developed. The system shows excellent selectivity as each nitrosamine gives two signals pertaining to monomer and dimer in the second dimension. For the first time, reduced ion mobility values for nitrosamines were determined, ranging from 1.18 to 2.03 cm2s−1V−1. The high selectivity of the GC-DT-IMS method could provide a definite advantage for monitoring nitrosamines in different manufacturing industries and consumer products.
The non-filarial and non-communicable disease podoconiosis affects around 4 million people and is characterized by severe leg lymphedema accompanied with painful intermittent acute inflammatory episodes, called acute dermatolymphangioadenitis (ADLA) attacks. Risk factors have been associated with the disease but the mechanisms of pathophysiology remain uncertain. Lymphedema can lead to skin lesions, which can serve as entry points for bacteria that may cause ADLA attacks leading to progression of the lymphedema. However, the microbiome of the skin of affected legs from podoconiosis individuals remains unclear. Thus, we analysed the skin microbiome of podoconiosis legs using next generation sequencing. We revealed a positive correlation between increasing lymphedema severity and non-commensal anaerobic bacteria, especially Anaerococcus provencensis, as well as a negative correlation with the presence of Corynebacterium, a constituent of normal skin flora. Disease symptoms were generally linked to higher microbial diversity and richness, which deviated from the normal composition of the skin. These findings show an association of distinct bacterial taxa with lymphedema stages, highlighting the important role of bacteria for the pathogenesis of podoconiosis and might enable a selection of better treatment regimens to manage ADLA attacks and disease progression.
Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cβ2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.
The development of whole-genome amplification (WGA) techniques has opened up new avenues for genetic analysis and genome research, in particular by facilitating the genome-wide analysis of few or even single copies of genomic DNA, such as from single cells (prokaryotic or eukaryotic) or virions. Using WGA, the few copies of genomic DNA obtained from such entities are unspecifically amplified using PCR or PCR-related processes in order to obtain higher DNA quantities that can then be successfully analysed further.
The epithelial sodium channel (ENaC) is a key regulator of sodium homeostasis that contributes to blood pressure control. ENaC open probability is adjusted by extracellular sodium ions, a mechanism referred to as sodium self-inhibition (SSI). With a growing number of identified ENaC gene variants associated with hypertension, there is an increasing demand for medium- to high-throughput assays allowing the detection of alterations in ENaC activity and SSI. We evaluated a commercially available automated two-electrode voltage-clamp (TEVC) system that records transmembrane currents of ENaC-expressing Xenopus oocytes in 96-well microtiter plates. We employed guinea pig, human and Xenopus laevis ENaC orthologs that display specific magnitudes of SSI. While demonstrating some limitations over traditional TEVC systems with customized perfusion chambers, the automated TEVC system was able to detect the established SSI characteristics of the employed ENaC orthologs. We were able to confirm a reduced SSI in a gene variant, leading to C479R substitution in the human α-ENaC subunit that has been reported in Liddle syndrome. In conclusion, automated TEVC in Xenopus oocytes can detect SSI of ENaC orthologs and variants associated with hypertension. For precise mechanistic and kinetic analyses of SSI, optimization for faster solution exchange rates is recommended.
Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.
ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species
(2023)
Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.
Pitfalls of using sequence databases for heterologous expression studies - a technical review
(2023)
Synthesis of DNA fragments based on gene sequences available in public resources has become an efficient and affordable method that gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterization of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: 1) Not every gene has yet been annotated; 2) Not all gene annotations are necessarily correct; 3) Transcripts may contain automated corrections; 4) There are mismatches between gene, mRNA, and protein sequences; and 5) Splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems. Abstract figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full of partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or that are available for refined search options using individual sequence information. The search results need to be scrutinised and compared to already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis are necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, e.g., eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G). This article is protected by copyright. All rights reserved.