Fighting the storm: could novel anti-TNFα and anti-IL-6 C. sativa cultivars tame cytokine storm in COVID-19?

The main aspects of severe COVID-19 disease pathogenesis include hyper-induction of proinflammatory cytokines, also known as 'cytokine storm', that precedes acute respiratory distress syndrome (ARDS) and often leads to death. COVID-19 patients often suffer from lung fibrosis, a serious and untreatable condition. There remains no effective treatment for these complications. Out of all cytokines, TNFα and IL-6 play crucial roles in cytokine storm pathogenesis and are likely responsible for the escalation in disease severity. These cytokines also partake in the molecular pathogenesis of fibrosis. Therefore, new approaches are urgently needed, that can efficiently and swiftly downregulate TNFα, IL-6, and the inflammatory cytokine cascade, in order to curb inflammation and prevent fibrosis, and lead to disease remission. Cannabis sativa has been proposed to modulate gene expression and inflammation and is under investigation for several potential therapeutic applications against autoinflammatory diseases and cancer. Here, we hypothesized that the extracts of novel C. sativa cultivars may be used to downregulate the expression of pro-inflammatory cytokines and pathways involved in inflammation and fibrosis. Initially, to analyze the anti-inflammatory effects of novel C. sativa cultivars, we used a well-established full thickness human 3D skin artificial EpiDermFTTM tissue model, whereby tissues were exposed to UV to induce inflammation and then treated with extracts of seven new cannabis cultivars. We noted that out of seven studied extracts of novel C. sativa cultivars, three (#4, #8 and #14) were the most effective, causing profound and concerted down-regulation of COX2, TNFα, IL-6, CCL2, and other cytokines and pathways related to inflammation and fibrosis. These data were further confirmed in the WI-38 lung fibroblast cell line model. Most importantly, one of the tested extracts had no effect at all, and one exerted effect that may be deleterious, signifying that careful cannabis cultivar selection must be based on thorough pre-clinical studies. The observed pronounced inhibition of TNFα and IL-6 is the most important finding, because these molecules are currently considered to be the main targets in COVID-19 cytokine storm and ARDS pathogenesis. Novel anti-TNFα and anti-IL-6 cannabis extracts can be useful additions to the current anti-inflammatory regimens to treat COVID-19, as well as various rheumatological diseases and conditions, and 'inflammaging' - the inflammatory underpinning of aging and frailty.

Depletion of eukaryotic initiation factor 5B (eIF5B) reprograms the cellular transcriptome and leads to activation of endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK).

During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.

Bioinformatics Analysis of Small RNA Transcriptomes: The Detailed Workflow.

Next-generation sequencing became a method of choice for the investigation of small RNA transcriptomes in plants and animals. Although a technical side of sequencing itself is becoming routine, and experimental costs are affordable, data analysis still remains a challenge, especially for researchers with limited computational experience. Here, we present a detailed description of a computational workflow designed to take raw sequencing reads as input, to obtain small RNA predictions, and to detect the differentially expressed microRNAs as a result. The exact commands and pieces of code are provided and hopefully can be adapted and used by other researchers to facilitate the study of small RNA regulation.

Liver irradiation causes distal bystander effects in the rat brain and affects animal behaviour.

Radiation therapy can not only produce effects on targeted organs, but can also influence shielded bystander organs, such as the brain in targeted liver irradiation. The brain is sensitive to radiation exposure, and irradiation causes significant neuro-cognitive deficits, including deficits in attention, concentration, memory, and executive and visuospatial functions. The mechanisms of their occurrence are not understood, although they may be related to the bystander effects.We analyzed the induction, mechanisms, and behavioural repercussions of bystander effects in the brain upon liver irradiation in a well-established rat model.Here, we show for the first time that bystander effects occur in the prefrontal cortex and hippocampus regions upon liver irradiation, where they manifest as altered gene expression and somewhat increased levels of γH2AX. We also report that bystander effects in the brain are associated with neuroanatomical and behavioural changes, and are more pronounced in females than in males.

Persistent gene expression changes in NAc, mPFC, and OFC associated with previous nicotine or amphetamine exposure.

Highly addictive drugs like nicotine and amphetamine not only change an individual's behaviour in the short and long-term, they also induce persistent changes in neuronal excitability and morphology. Although research has started to examine the epigenetic changes that occur immediately after drug exposure, there has been little investigation into the persistent modifications to the epigenome that likely moderate the stable maintenance of the neurological changes. Male Long-Evans rats were administered amphetamine, nicotine, or saline for 14 consecutive days, given a 14 day withdrawal period, and then sacrificed. DNA from the mPFC, OFC, and nucleus accumbens (NAc) was used for global DNA methylation analysis and RNA from the same brain regions was used for gene expression analysis. Following the two-week withdrawal period, exposure to amphetamine or nicotine was associated with a decrease in global DNA methylation in each brain region examined. Previous exposure to nicotine was associated with changes in expression of 16 genes (NAc:6, mPFC:5, OFC:5) whereas exposure to amphetamine was associated with changes in expression of 25 genes (NAc:13, OFC:8, mPFC:4). The persistent epigenetic changes associated with exposure to amphetamine and nicotine were region and drug dependent, and differ from the latent epigenetic changes that occur immediately after drug exposure. The changes in DNA methylation are consistent with the gene expression results and provide further support to the notion that DNA methylation is the key regulatory mechanism for experience dependent changes.

Molecular mechanisms of radiation resistance in doxorubicin-resistant breast adenocarcinoma cells.

A positive response to breast cancer treatment is largely dependent on the successful combination of anticancer treatment modalities, such as chemotherapy and radiation therapy. Unfortunately, chemotherapy resistance occurs frequently. Furthermore, drug­resistant tumors can become unresponsive to other antitumor therapies, and they often fail to respond to radiation therapy. The molecular structures underlying the radiation responses of chemoresistant cells and tumors are not well understood. We analyzed the effect of ionizing radiation on MCF-7 human breast adenocarcinoma cells and their doxorubicin­resistant variant, MCF-7/DOX. The results demonstrated that drug­resistant MCF-7/DOX cells were less susceptible to radiation-induced DNA damage and apoptosis. This was proven through gene expression profiling, lower levels of γH2AX foci upon irradiation, and altered levels of DNA repair proteins, including pATM, KU70 and RAD51. Additionally, MCF-7/DOX drug­resistant cells harbored DNA polymerases with significantly low fidelity. In summary, our study revealed that drug-resistant MCF-7/DOX cells have high DNA repair potential and low-fidelity DNA polymerases, seemingly sacrificing specificity and efficiency to gain higher survival potential. In the long run, this may lead to an increased probability of mutation accumulation and further the development of an even more pronounced resistance phenotype. Therefore, this study provides a roadmap for the analysis of the roles of the DNA repair function and effectiveness, and apoptosis in response to radiation, chemotherapy and combinations of both treatment modalities.

Epigenetic bystander-like effects of stroke in somatic organs.

Clinical evidence suggests that stroke may lead to damage of somatic organs. This communication of damage is well- established in the case of exposure to genotoxic agents is termed a bystander effect. Genotoxic stress-induced bystander effects are epigenetically mediated. Here we investigated whether stroke causes epigenetic bystander-like effects in the liver, kidney and heart. We found a significant increase in the levels of H3K3 acetylation and H3K4 trimethylation, as well as a decrease in the H3K9 trimethylation in the kidney tissue of stroked rats. Furthermore, here we for the first time show changes in the gene and microRNA expression profile in the kidney tissues of stroked rats, as compared to intact control animals. Interestingly, the observed changes were somewhat similar to those reported earlier in kidney injury, inflammation, and acute renal failure. Our data explain the recent epidemiological evidence for the increased incidence of acute kidney injury post-stroke and provide an important roadmap for the future analysis of the mechanisms and cellular repercussions of the stroke-induced bystander-like effects in distal somatic organs.

Parental enrichment and offspring development: modifications to brain, behavior and the epigenome.

Environmental enrichment has been shown to have profound effects on the healthy adult brain and as a remedial tool for brains compromised by injury, disease, or negative experience. Based upon these findings and evidence from the prenatal stress literature, we ventured an exploratory study to examine the effects of parental enrichment on offspring development. Using Long Evans rats, paternal enrichment was achieved by housing sires in enriched environments for 28 days prior to mating with a control female. For the maternal enrichment paradigm, female rats were also housed in enriched environments for 28 days (7 days prior to conception and for the duration of pregnancy). Increased size, multiple levels for exploration, an abundance of stimulating toys, and numerous cagemates for social interaction were characteristic of the enriched environments. Offspring were assessed using two early behavioral tests and then sacrificed at postnatal day 21 (P21). Brain tissue from the frontal cortex and hippocampus was harvested for global DNA methylation analysis. Parental enrichment, preconceptionally and prenatally, altered offspring behavior on the negative geotaxis task and openfield exploratory behavior task. Paternal enrichment significantly decreased offspring brain weight at P21. Additionally, both environmental enrichment paradigms significantly decreased global methylation levels in the hippocampus and frontal cortex of male and female offspring. This study demonstrates that positive prenatal experiences; preconceptionally in fathers and prenatally in mothers, have the ability to significantly alter offspring developmental trajectories.

Prenatal bystander stress alters brain, behavior, and the epigenome of developing rat offspring.

The prenatal environment, including prenatal stress, has been extensively studied in laboratory animals and humans. However, studies of the prenatal environment usually directly stress pregnant females, but stress may come 'indirectly', through stress to a cage-mate. The current study used indirect prenatal bystander stress and investigated the effects on the gross morphology, pre-weaning behavior, and epigenome of rat offspring. Pregnant Long-Evans rats were housed with another female rat that underwent elevated platform stress from gestational days 12 to 16. We found that ultrasonic vocalizations of female cage-mates were disrupted following the stress procedure. After birth, offspring were tested on two behavioral tasks and sacrificed at postnatal day 21 (p21). Frontal cortex and hippocampal tissue was used to measure global DNA methylation and gene expression changes. At p21, bystander-stressed female offspring exhibited increased body weight. Offspring behavior on the negative geotaxis task was altered by prenatal bystander stress, and locomotor behavior was reduced in female offspring. Global DNA methylation increased in the frontal cortex and hippocampus of bystander-stressed offspring. Microarray analysis revealed significant gene expression level changes in 558 different genes, of which only 10 exhibited overlap between males and females or brain areas. These alterations in gene expression were associated with overrepresentation of 36 biological processes and 34 canonical pathways. Prenatal stress thus does not have to be experienced by the mother herself to influence offspring brain development. Furthermore, this type of 'indirect' prenatal stress alters offspring DNA methylation patterns, gene expression profiles, and behavior.