Protocol to produce plant-based hybrid nanovesicles from fresh turmeric and pepper using polyethylene glycol.

In addition to proteins, microRNAs, and lipids, plant-derived exosome-like nanovesicles (ENVs) are also enriched with host plant bioactives. Both curcumin and piperine are water insoluble, lack bioavailability, and are extracted by non-ecofriendly solvents. Herein, we present an eco-friendly protocol for co-isolating both curcumin and piperine in the form of hybrid ENVs. We describe steps for sample pre-processing, combined homogenization of plant materials, filtration, and differential centrifugation. We then detail procedures for polyethylene glycol-based fusion and precipitation of hybrid ENVs. For complete details on the use and execution of this protocol, please refer to Kumar et al.1.

HuR enhances FSTL1 transcript stability to promote invasion and metastasis of squamous cell carcinoma.

Squamous cell carcinoma (SCC) is a lethal malignancy with a high propensity for metastasis. Follistatin-like 1 (FSTL1), a pro-metastatic glycoprotein, is absent from healthy epithelia and aberrantly upregulated in SCC. The FSTL1 transcript encodes two alternative gene products whose dominance is post-transcriptionally regulated via a bistable switch. In healthy epithelia, FSTL1 mRNA is destabilized by binding of KH-type splicing regulatory protein (KSRP), and processed as a primary microRNA encoding miR-198. In SCC, KSRP downregulation terminates miR-198 processing, enabling FSTL1 translation. Here, we identify HuR (Human Antigen R) as an upstream regulator of FSTL1 and describe how downregulation of KSRP is permissive, but not sufficient, to promote sustained FSTL1 expression. Moreover, we demonstrate how the interplay between two RNA-binding proteins controls the translation of pro-oncogenic FSTL1. Increased expression of HuR in SCC outcompetes KSRP and enhances FSTL1 transcript stability, enabling persistent FSTL1 expression and activation of downstream metastatic pathways.

Low pH-Based Method to Increase the Yield of Plant-Derived Nanoparticles from Fresh Ginger Rhizomes.

Plant-derived nanoparticles (PDNPs) are naturally occurring exosome-like nanovesicles derived from dietary plants containing key plant bioactives. Ginger-derived PDNPs have a therapeutic effect on alcohol-induced liver injury, inflammatory bowel disease, and colon cancer. PDNPs are conventionally purified by differential ultracentrifugation, a technique not amenable for scale up. We have recently developed a polyethylene glycol (PEG) 6000-based method for cost-effective purification of ginger PDNPs, with comparable efficiency to differential ultracentrifugation (Sci. Rep. 2020, 10 (1), 4456.). Herein, we report a 4-5-fold higher ginger PDNP recovery when PEG precipitation was carried out in low pH conditions (pH 4 and 5). Low pH-derived ginger PDNPs were smaller in size without an overt change in zeta potential. The spontaneous intracellular entry and protection against oxidative stress in A431 cells were similar between ginger PDNPs purified under low, neutral, and alkaline pH. Low-pH purified ginger PDNPs had higher levels of total polyphenolic content compared to PDNPs purified under neutral and alkaline pH. Recently, ginger PDNP-derived microRNAs have been shown to exhibit cross-kingdom regulation by targeting human, gut microbiome, and viral transcripts. Using qRT-PCR, we also verified the presence of miRNAs that were predicted to target SARS-CoV-2 in ginger PDNPs purified under low pH. Thus, we have developed a method to purify ginger PDNPs in high yields by using low-pH conditions without affecting the major bioactive contents of PDNPs.

Chemical profiling and in-vitro anti-inflammatory activity of bioactive fraction(s) from Trichodesma indicum (L.) R.Br. against LPS induced inflammation in RAW 264.7 murine macrophage cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichodesma indicum (L.) R. Br. (family: Boraginaceae) is a medicinal herb largely used to treat arthralgia, rheumatoid arthritis, wound healing, dysentery, etc. It's mechanism of anti-inflammatory activity has not been systematically analyzed yet. AIM OF THE STUDY: The present study was undertaken to examine the anti-inflammatory effects of successive solvent extracts (n-hexane extract (HE), ethyl acetate extract (EA), ethanol extract (EE), aqueous extract (AE) and fractions of HE) from the aerial parts of Trichodesma indicum (TI) against lipopolysaccharide (LPS) stimulated inflammatory reaction using mouse macrophage RAW 264.7 cells. MATERIALS AND METHODS: Cytotoxic effects of the extracts and fractions of TI were assessed by MTT assay. The effect of extracts and fractions on the production of nitric oxide (NO) in RAW 264.7 macrophages were measured using the Griess reagent method. IL - 6, IL - 1ß, TNF-α, iNOS and COX-2 gene expressions were examined by a qRT-PCR method. RESULTS: RAW 264.7 macrophages pretreated with HE, EA, EE and AE of TI showed a significant decrease in the production of proinflammatory cytokines and NO without exhibiting cytotoxicity. The potent HE was fractionated using flash chromatography into FA, FB, FC, FD and FE. Among the five fractions, FE displayed a stronger ability to reduce IL - 1ß, TNF-α, iNOS, COX2 and NO importantly no cytotoxicity was observed. The phytochemical compounds present in FE were further screened by Gas chromatography - Mass spectroscopy (GC-MS). GC-MS analysis revealed that 1,2-benzenedicarboxylic acid diisooctyl ester is the major compound in FE. Molecular docking analysis showed good inhibition of 1,2-benzenedicarboxylic acid diisooctyl ester against TLR-4, NIK and TACE. CONCLUSION: Our results suggested that 1,2-benzenedicarboxylic acid diisooctyl ester could be a potential candidate in alleviating inflammatory reactions in TI.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Edible plant-derived exosomal microRNAs: Exploiting a cross-kingdom regulatory mechanism for targeting SARS-CoV-2.

BACKGROUND: The current COVID-19 pandemic is caused by SARS-CoV-2 which belongs to coronaviridae family. Despite the global prevalence, there are currently no vaccines or drugs. Dietary plant derived exosome-like vesicles are known as edible nanoparticles (ENPs). ENPs are filled with microRNAs (miRNAs), in bioavailable form. Recently, cross-kingdom regulation of human transcripts by plant miRNAs have been demonstrated. However, ENP derived miRNAs targeting SARS-CoV-2 has not been described. STUDY DESIGN: Mature ENP-derived miRNA sequences were retrieved from small RNA sequencing datasets available in the literature. In silico target prediction was performed to identify miRNAs that could target SARS-CoV-2. ENPs were isolated from ginger and grapefruit plants and the expression of SARS-CoV-2 targeting miRNAs were confirmed by qRT-PCR. RESULTS: From a total of 260 ENP-derived miRNAs, we identified 22 miRNAs that could potentially target SARS-CoV-2 genome. 11 miRNAs showed absolute target specificity towards SARS-CoV-2 but not SARS-CoV. ENPs from soybean, ginger, hamimelon, grapefruit, tomato and pear possess multiple miRNAs targeting different regions within SARS-CoV-2. Interestingly, osa/cme miR-530b-5p specifically targeted the ribosomal slippage site between ORF1a and ORF1b. We validated the relative expression of six miRNAs (miR-5077, miR-6300, miR-156a, miR-169, miR-5059 and miR-166 m) in ginger and grapefruit ENPs by RT-PCR which showed differential enrichment of specific miRNAs in ginger and grapefruit ENPs. CONCLUSION: Since administration of ENPs leads to their accumulation into lung tissues in vivo, ENP derived miRNAs targeting SARS-CoV-2 genome has the potential to be developed as an alternative therapy.

A cost-effective polyethylene glycol-based method for the isolation of functional edible nanoparticles from ginger rhizomes.

Edible nanoparticles (ENPs) are nano-sized vesicles derived from edible plants. These ENPs are loaded with plant derived microRNAs, protein, lipids and phytochemicals. Recently, ginger derived ENPs was shown to prevent inflammatory bowel diseases and colon cancer, in vivo, highlighting their therapeutic potential. Conventionally, differential centrifugation with an ultra-centrifugation step is employed to purify these ENPs which imposes limitation on the cost-effectiveness of their purification. Herein, we developed polyethylene glycol-6000 (PEG6000) based ginger ENP purification (PEG-ENPs) method, which eliminates the need for expensive ultracentrifugation. Using different PEG6000 concentrations, we could recover between 60% to 90% of ENPs compared to ultracentrifugation method. PEG-ENPs exhibit near identical size and zeta potential similar to ultra-ENPs. The biochemical composition of PEG-ENPs, such as proteins, lipids, small RNAs and bioactive content is comparable to that of ultra-ENPs. In addition, similar to ultra-ENPs, PEG-ENPs are efficiently taken up by the murine macrophages and protects cells from hydrogen peroxide induced oxidative stress. Since PEG has been approved as food additive, the PEG method described here will provide a cost-effective alternative to purify ENPs, which can be directly used as a dietary supplement in therapeutic formulations.

Belinostat resolves skin barrier defects in atopic dermatitis by targeting the dysregulated miR-335:SOX6 axis.

BACKGROUND: Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Skin barrier defects contribute to disease initiation and development; however, underlying mechanisms remain elusive. OBJECTIVE: To understand the underlying cause of barrier defect, we investigated aberrant expression of specific microRNAs (miRNAs) in AD. Delineating the molecular mechanism of dysregulated miRNA network, we focused on identification of specific drugs that can modulate miRNA expression and repair the defective barrier in AD. METHODS: A screen for differentially expressed miRNAs between healthy skin and AD lesional skin resulted in the identification of miR-335 as the most consistently downregulated miRNA in AD. Using in silico prediction combined with experimental validation, we characterized downstream miR-335 targets and elucidated the molecular pathways by which this microRNA maintains epidermal homeostasis in healthy skin. RESULTS: miR-335 was identified as a potent inducer of keratinocyte differentiation; it exerts this effect by directly repressing SOX6. By recruiting SMARCA complex components, SOX6 suppresses epidermal differentiation and epigenetically silences critical genes involved in keratinocyte differentiation. In AD lesional skin, miR-335 expression is aberrantly lost. SOX6 is abnormally expressed throughout the epidermis, where it impairs skin barrier development. We demonstrate that miR-335 is epigenetically regulated by histone deacetylases; a screen for suitable histone deacetylase inhibitors identified belinostat as a candidate drug that can restore epidermal miR-335 expression and rescue the defective skin barrier in AD. CONCLUSION: Belinostat is of clinical significance not only as a candidate drug for AD treatment, but also as a potential means of stopping the atopic march and further progression of this systemic allergic disease.

Dietary non-coding RNAs from plants: Fairy tale or treasure?

The past two decades have witnessed soaring interest in the field of non-coding RNAs, largely attributed by its regulatory role in controlling two third of human transcriptional output. Though, there are several classes of non-coding RNAs found in nature, microRNAs takes the central stage because of their pleiotropic roles. In particular, extracellular microRNAs are gaining traction due to their relative stability and bio availability. Extracellular microRNAs has been shown to occur in all living organisms, including dietary plants. Some of the recent reports suggest that these dietary microRNAs pass through the gut, enter systemic circulation and exert biological effects on animal physiology. However, evidences against this hypothesis are also presented in literature and hence this area has been strongly debated. In this review, I will briefly summarise the evidences accumulated for and against this hypothesis and discuss potential implications of such findings in human health.

Carcinoma Cells Reprogram a Wound-healing Switch to Promote Metastasis.

Wound healing is a dynamic event where barrier disruption is transient and miR-198/FSTL1 molecular switch orchestrate wound re-epithelialization. However, epithelial carcinomas maintain a prolonged wound-healing phase to promote malignant transformation. Delineating the molecular mechanism we demonstrate, how epidermal growth factor (EGF) hijacks the wound-healing switch to promote metastasis of carcinoma.

Cancer: the dark side of wound healing.

Complex multicellular organisms have evolved sophisticated mechanisms to rapidly resolve epithelial injuries. Epithelial integrity is critical to maintaining internal homeostasis. An epithelial breach represents the potential for pathogen ingress and fluid loss, both of which may have severe consequences if not limited. The mammalian wound healing response involves a finely tuned, self-limiting series of cellular and molecular events orchestrated by the transient activation of specific signalling pathways. Accurate regulation of these events is essential; failure to initiate key steps at the right time delays healing and leads to chronic wounds, while aberrant initiation of wound healing processes may produce cell behaviours that promote cancer progression. In this review, we discuss how wound healing pathways co-opted in cancer lose their stringent regulation and become compromised in their reversibility. We hypothesize on how the commandeering of wound healing 'master regulators' is involved in this process, and also highlight the implications of these findings in the treatment of both chronic wounds and cancer.

C/EBPß mediates RNA polymerase III-driven transcription of oncomiR-138 in malignant gliomas.

MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein ß (C/EBPß) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPß via an embedded 'C/EBPß responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPß and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.

Long noncoding RNA EGFR-AS1 mediates epidermal growth factor receptor addiction and modulates treatment response in squamous cell carcinoma.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

EGF hijacks miR-198/FSTL1 wound-healing switch and steers a two-pronged pathway toward metastasis.

Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

Molecular interplay of pro-inflammatory transcription factors and non-coding RNAs in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma is the sixth most common cancer in the developing world. The aggressive nature of esophageal squamous cell carcinoma, its tendency for relapse, and the poor survival prospects of patients diagnosed at advanced stages, represent a pressing need for the development of new therapies for this disease. Chronic inflammation is known to have a causal link to cancer pre-disposition. Nuclear factor kappa B and signal transducer and activator of transcription 3 are transcription factors which regulate immunity and inflammation and are emerging as key regulators of tumor initiation, progression, and metastasis. Although these pro-inflammatory factors in esophageal squamous cell carcinoma have been well-characterized with reference to protein-coding targets, their functional interactions with non-coding RNAs have only recently been gaining attention. Non-coding RNAs, especially microRNAs and long non-coding RNAs demonstrate potential as biomarkers and alternative therapeutic targets. In this review, we summarize the recent literature and concepts on non-coding RNAs that are regulated by/regulate nuclear factor kappa B and signal transducer and activator of transcription 3 in esophageal cancer progression. We also discuss how these recent discoveries can pave way for future therapeutic options to treat esophageal squamous cell carcinoma.

HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome.

BACKGROUND: Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies. RESULTS: We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer. CONCLUSIONS: Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas.

'See-saw' expression of microRNA-198 and FSTL1 from a single transcript in wound healing.

Post-transcriptional switches are flexible effectors of dynamic changes in gene expression. Here we report a new post-transcriptional switch that dictates the spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. Expression of primate-specific exonic microRNA-198 (miR-198), located in the 3'-untranslated region of follistatin-like 1 (FSTL1) messenger RNA, switches to expression of the linked open reading frame of FSTL1 upon wounding in a human ex vivo organ culture system. We show that binding of a KH-type splicing regulatory protein (KSRP, also known as KHSRP) to the primary transcript determines the fate of the transcript and is essential for the processing of miR-198: transforming growth factor-ß signalling switches off miR-198 expression by downregulating KSRP, and promotes FSTL1 protein expression. We also show that FSTL1 expression promotes keratinocyte migration, whereas miR-198 expression has the opposite effect by targeting and inhibiting DIAPH1, PLAU and LAMC2. A clear inverse correlation between the expression pattern of FSTL1 (pro-migratory) and miR-198 (anti-migratory) highlights the importance of this regulatory switch in controlling context-specific gene expression to orchestrate wound re-epithelialization. The deleterious effect of failure of this switch is apparent in non-healing chronic diabetic ulcers, in which expression of miR-198 persists, FSTL1 is absent, and keratinocyte migration, re-epithelialization and wound healing all fail to occur.