Metabolic engineering for enhanced terpenoid production: Leveraging new horizons with an old technique.

Terpenoids are a vast class of plant specialized metabolites (PSMs) manufactured by plants and are involved in their interactions with environment. In addition, they add health benefits to human nutrition and are widely used as pharmaceutically active compounds. However, native plants produce a limited amount of terpenes restricting metabolite yield of terpene-related metabolites. Exponential growth in the plant metabolome data and the requirement of alternative approaches for producing the desired amount of terpenoids, has redirected plant biotechnology research to plant metabolic engineering, which requires in-depth knowledge and precise expertise about dynamic plant metabolic pathways and cellular physiology. Metabolic engineering is an assuring tool for enhancing the concentration of terpenes by adopting specific strategies such as overexpression of the key genes associated with the biosynthesis of targeted metabolites, controlling the modulation of transcription factors, downregulation of competitive pathways (RNAi), co-expression of the biosynthetic pathway genes in heterologous system and other combinatorial approaches. Microorganisms, fast-growing host plants (such as Nicotiana benthamiana), and cell suspension/callus cultures have provided better means for producing valuable terpenoids. Manipulation in the biosynthetic pathways responsible for synthesis of terpenoids can provide opportunities to enhance the content of desired terpenoids and open up new avenues to enhance their production. This review deliberates the worth of metabolic engineering in medicinal plants to resolve issues associated with terpenoid production at a commercial scale. However, to bring the revolution through metabolic engineering, further implementation of genome editing, elucidation of metabolic pathways using omics approaches, system biology approaches, and synthetic biology tactics are essentially needed.

A multiplex DNA probe-based method for simultaneous identification of adulteration in meat samples.

Meat adulteration and admixing are prevalent malpractices observed in processed and raw meat samples, where the consumption of adulterated meat has been associated with food allergies, financial losses, and consumer distrust. Meat authentication is pivotal to address these concerns. The meat authenticity can be determined through genetic, protein, and immunological markers and advanced detection methods. However, these methods often target a single species and lack the specificity to distinguish closely related species. Here, in the present study, we have developed a multiplex detection method based on the species-specific primers and probes, that can target four meat species in one reaction. The developed method amplifies the mitochondrial genomic regions of chicken, pork, sheep and goat using TaqMan multiplex probe-based RT-qPCR assay. Unique pairs of species-specific primers and probes that target specific mitochondrial DNA (mtDNA) regions of each species were designed and screened for specificity and sensitivity. The detection limit for species identification using the designed primers in real-time qPCR assays was 0.1 picogram per microliter (pg/µL) DNA detected in singleplex reaction and facilitates the simultaneous detection of closely related species, such as goat and sheep. Further, DNA-based probes were utilized in a multiplex real-time qPCR assay to identify chicken, pork, sheep and goat DNA in a single tube reaction. The multiplex assay was validated for raw and processed meat products, demonstrating its applications in ensuring the quality of meat products and safeguarding consumer interests.

Activated fibroblasts modify keratinocyte stem niche through TET1 and IL-6 to promote their rapid transformation in a mouse model of prenatal arsenic exposure.

Early life exposure to environmental pollutants such as arsenic (As) can increase the risk of cancers in the offspring. In an earlier study, we showed that only prenatal As exposure significantly increases epidermal stem cell proliferation and accelerates skin tumorigenesis in BALB/c mouse offspring. In the present work, we have examined the role of As-conditioned dermal fibroblasts (DFs) in creating pro-tumorigenic niches for Keratinocyte stem cells (KSCs) in the offspring. DFs isolated from prenatally exposed animals showed increased levels of activation markers (α-SMA, Fibronectin, Collagen IV), induction of ten-eleven translocation methylcytosine dioxygenase 1(TET1), and secreted high levels of niche modifying IL-6. This led to enhanced proliferation, migration, and survival of KSCs. Increased IL-6 production in As-conditioned fibroblast was driven through TET1 mediated 5-mC to 5-hmC conversion at -698/-526 and -856/-679 region on its promoter. IL-6 further acted through downstream activation of JAK2-STAT3 signaling, promoting epithelial-to-mesenchymal transition (EMT) in KSCs. Inhibition of pSTAT3 induced by IL-6 reduced the EMT process in KSCs resulting in a significant decrease in their proliferation, migration, and colony formation. Our results indicate that IL-6 produced by prenatally conditioned fibroblasts plays a major role in regulating the KSC niche and promoting skin tumor development in As-exposed offspring.

Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

Altered Igf2 imprint leads to accelerated adipogenesis and early onset of metabolic syndrome in male mice following gestational arsenic exposure.

Developmental exposure to environmental pollutants has been shown to promote adverse health outcomes in offspring. Exposure to heavy metals such as arsenic which also has endocrine-disrupting activity is being increasingly linked with cancers, diabetes, and lately with Metabolic Syndrome (MetS). In this work, we have assessed the effects of preconceptional plus gestational arsenic exposure on the developmental programming of MetS in offspring. In our study, only gestational arsenic exposure led to reduced birth weight, followed by catch-up growth, adiposity, elevated serum triglycerides levels, and hyperglycemia in male offspring. Significant adipocyte dysfunction was observed in offspring with increased hypertrophy, insulin resistance, and chronic inflammation in epididymal white adipose tissue. Adipose tissue regulates the metabolic health of individuals and its dysfunction resulted in elevated serum levels of metabolism-regulating adipokines (Leptin, Resistin) and pro-inflammatory cytokines (PAI-1, TNFα). The progenitor adipose-derived stem cells (AdSCs) from exposed progeny had increased proliferation and adipogenic potential with excess lipid accumulation. We also found increased activation of Akt, ERK1/2 & p38 MAPK molecules in arsenic-exposed AdSCs along with increased levels of phospho-Insulin-like growth factor-1 receptor (p-IGF1R) and its upstream activator Insulin-like growth factor-2 (IGF2). Overexpression of Igf2 was found to be due to arsenic-mediated DNA hypermethylation at the imprinting control region (ICR) located -2kb to -4.4 kb upstream of the H19 gene which caused a reduction in the conserved zinc finger protein (CTCF) occupancy. This further led to persistent activation of the MAPK signaling cascade and enhanced adipogenesis leading to the early onset of MetS in the offspring.

Development of an immunoassay for the detection of mycotoxins using xMAP technology and its evaluation in black tea samples.

Mycotoxins, a natural food contaminant, are secondary metabolites of fungi. Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are two major mycotoxins found in various food commodities. These mycotoxins are hepatotoxic, nephrotoxic, cytotoxic, mutagenic and carcinogenic, thus they are a public health concern and their monitoring in food commodities is necessary. There are several conventional techniques available for mycotoxin detection, such as HPLC, LCMS, and ELISA. However, extensive nature and huge cost allowances make it challenging to deploy these techniques for monitoring of mycotoxins in the large sample size. Therefore, a robust, responsive and high-throughput technique is required. Here, we aimed to develop a multiplexed Luminex suspension assay based on multi analyte profiling (xMAP) technology for the simultaneous detection of AFB1 and OTA in the black tea, which is found to be contaminated with these mycotoxins during the cultivation or processing steps. Limit of detection for AFB1 and OTA, was 0.06 ng/ml and 0.49 ng/ml, respectively without any cross-reactivity with other mycotoxins and this assay is suitable for simultaneous detection of AFB1 and OTA in the same sample. Collectively, based on the results, we suggest that the developed Luminex suspension assay is sensitive, accurate, rapid and suitable for high-throughput screening of multiple mycotoxins. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05848-3.

Chitosan/alginate nanogel potentiate berberine uptake and enhance oxidative stress mediated apoptotic cell death in HepG2 cells.

Biopolymer-based nanoscale drug delivery systems have become a promising approach to overcome the limitations associated with conventional chemotherapeutics used for cancer treatment. Herein, we reported to develop a hydrophilic nanogel (NG) composed of Chitosan (Chi) and sodium alginate (Alg) using the ion gelation method for delivering Berberine hydrochloride (BBR), an alkaloid obtained from Berberis aristata roots. The use of different nanocarriers for BBR delivery has been reported previously, but the bioavailability of these carriers was limited due to phagocytic uptake and poor systemic delivery. The developed NG showed enhanced stability and efficient entrapment of BBR ∼92 %, resulting in a significant increase in bioavailability. The pH-dependent release behavior demonstrated sustained and effective release of ∼86 %, ∼74 % and, ∼53 % BBR at pH 5.5, 6.6, and 7.4 respectively after 72h, indicating its potential as a drug carrier. Additionally, the cellular uptake of BBR was significantly higher ∼19 % in the BBR-NG (25 µM) than in bulk BBR (100 µM), leading to enhanced ROS generation, mitochondrial depolarisation, and inhibition of cell proliferation and colony formation in HepG2 cells. In summary, the results suggest that the Chi/Alg biopolymer-based nano-formulation could be an effective approach for delivering BBR and enhancing its cellular uptake, efficacy, and cytotoxicity.

A case of relapsing anti-GBM disease secondary to alemtuzumab therapy.

We report the first case of relapsing anti-GBM disease secondary to alemtuzumab in a 24-year-old female with relapsing-remitting multiple sclerosis. Initial anti-GBM disease was detected 10 months after alemtuzumab was given and was diagnosed by demonstrating high anti-GBM antibody titers and with a confirmatory kidney biopsy. The patient presented with a rapidly progressive glomerulonephritis with no pulmonary involvement. After appropriate treatment, the patient went into remission with undetectable anti-GBM antibodies. However, 20 months later, the patient re-presented with relapsing anti-GBM disease. Despite aggressive treatment, the patient became dialysis-dependent.

Graphene oxide nanosheets augment silk fibroin aerogels for enhanced water stability and oil adsorption.

Nanocomposite aerogels exhibit high porosity and large interfacial surface areas, enabling enhanced chemical transport and reactivity. Such mesoporous architectures can be prepared by freeze-casting naturally-derived biopolymers such as silk fibroin, but often form mechanically weak structures that degrade in water, which limits their performance under ambient conditions. Adding 2D material fillers such as graphene oxide (GO) or transition metal carbides (e.g. MXene) could potentially reinforce these aerogels via stronger intermolecular interactions with the polymeric binder. Here, we show that freeze-casting of GO nanosheets with silk fibroin results in a highly water-stable, mechanically robust aerogel, with considerably enhanced properties relative to silk-only or silk-MXene aerogels. These silk-GO aerogels exhibit high contact angles with water and are highly water stable. Moreover, aerogels can adsorb up 25-35 times their mass in oil, and can be used robustly for selective oil separation from water. This increased stability may occur due to strengthened intermolecular interactions such as hydrogen bonding, despite the random coil and α-helix conformation of silk fibroin, which is typically more soluble in water. Finally, we show these aerogels can be prepared at scale by freeze-casting on a copper mesh. Ultimately, we envision that these multicomponent aerogels could be widely utilized for molecular separations and environmental sensing, as well as for thermal insulation and electrical conductivity.

Plant salinity stress, sensing, and its mitigation through WRKY.

Salinity or salt stress has deleterious effects on plant growth and development. It imposes osmotic, ionic, and secondary stresses, including oxidative stress on the plants and is responsible for the reduction of overall crop productivity and therefore challenges global food security. Plants respond to salinity, by triggering homoeostatic mechanisms that counter salt-triggered disturbances in the physiology and biochemistry of plants. This involves the activation of many signaling components such as SOS pathway, ABA pathway, and ROS and osmotic stress signaling. These biochemical responses are accompanied by transcriptional modulation of stress-responsive genes, which is mostly mediated by salt-induced transcription factor (TF) activity. Among the TFs, the multifaceted significance of WRKY proteins has been realized in many diverse avenues of plants' life including regulation of plant stress response. Therefore, in this review, we aimed to highlight the significance of salinity in a global perspective, the mechanism of salt sensing in plants, and the contribution of WRKYs in the modulation of plants' response to salinity stress. This review will be a substantial tool to investigate this problem in different perspectives, targeting WRKY and offering directions to better manage salinity stress in the field to ensure food security.

Performance enhancement in clustering cooperative spectrum sensing for cognitive radio network using metaheuristic algorithm.

Spectrum sensing describes, whether the spectrum is occupied or empty. Main objective of cognitive radio network (CRN) is to increase probability of detection (Pd) and reduce probability of error (Pe) for energy consumption. To reduce energy consumption, probability of detection should be increased. In cooperative spectrum sensing (CSS), all secondary users (SU) transmit their data to fusion center (FC) for final measurement according to the status of primary user (PU). Cluster should be used to overcome this problem and improve performance. In the clustering technique, all SUs are grouped into clusters on the basis of their similarity. In cluster technique, SU transfers their data to cluster head (CH) and CH transfers their combined data to FC. This paper proposes the detection performance optimization of CRN with a machine learning-based metaheuristic algorithm using clustering CSS technique. This article presents a hybrid support vector machine (SVM) and Red Deer Algorithm (RDA) algorithm named Hybrid SVM-RDA to identify spectrum gaps. Algorithm proposed in this work outperforms the computational complexity, an issue reported with various conventional cluster techniques. The proposed algorithm increases the probability of detection (up to 99%) and decreases the probability of error (up to 1%) at different parameters.

Analysis of gut bacteriome of in utero arsenic-exposed mice using 16S rRNA-based metagenomic approach.

Introduction: Approximately 200 million people worldwide are affected by arsenic toxicity emanating from the consumption of drinking water containing inorganic arsenic above the prescribed maximum contaminant level. The current investigation deals with the role of prenatal arsenic exposure in modulating the gut microbial community and functional pathways of the host. Method: 16S rRNA-based next-generation sequencing was carried out to understand the effects of in utero 0.04 mg/kg (LD) and 0.4 mg/kg (HD) of arsenic exposure. This was carried out from gestational day 15 (GD-15) until the birth of pups to understand the alterations in bacterial diversity. Results: The study focused on gestational exposure to arsenic and the altered gut microbial community at phyla and genus levels, along with diversity indices. A significant decrease in firmicutes was observed in the gut microbiome of mice treated with arsenic. Functional analysis revealed that a shift in genes involved in crucial pathways such as insulin signaling and non-alcoholic fatty liver disease pathways may lead to metabolic diseases in the host. Discussion: The present investigation may hypothesize that in utero arsenic exposure can perturb the gut bacterial composition significantly as well as the functional pathways of the gestationally treated pups. This research paves the way to further investigate the probable mechanistic insights in the field of maternal exposure environments, which may play a key role in epigenetic modulations in developing various disease endpoints in the progeny.

Increasing Chemotherapeutic Efficacy Using pH-Modulating and Doxorubicin-Releasing Injectable Chitosan-Poly(ethylene glycol) Hydrogels.

Modulation of pH is crucial to maintaining the chemical homeostasis of biological environments. The irregular metabolic pathways exhibited by cancer cells result in the production of acidic byproducts that are excreted and accumulate in the extracellular tumor microenvironment, reducing the pH. As a consequence of the lower pH in tumors, cancer cells increase the expression of metastatic phenotypes and chemotherapeutic resistance. A significant limitation in current cancer therapies is the inability to locally deliver chemotherapeutics, leading to significant damage to healthy cells in systemic administration. To overcome these challenges, we present an injectable chitosan-poly(ethylene glycol) hydrogel that is dual-loaded with doxorubicin and sodium bicarbonate providing alkaline buffering of extracellular acidity and simultaneous chemotherapeutic delivery to increase chemotherapeutic efficacy. We conducted in vitro studies of weak base chemotherapeutic and alkaline buffer release from the hydrogel. The release of doxorubicin from hydrogels increased in a low-pH environment and was dependent on the encapsulated sodium bicarbonate concentration. We investigated the influence of pH on the doxorubicin efficacy and viability of MCF-7 and MDA-MB-231 breast cancer cell lines. The results show a 2- to 3-fold increase in IC50 values from neutral pH to low pH, showing decreased cancer cell viability at neutral pH as compared to acidic pH. The IC50 results were shown to correlate with a decrease in intracellular uptake of doxorubicin at low pH. The proposed hydrogels were confirmed to be nontoxic to healthy MCF-10A mammary epithelial cells. Rheological studies were performed to verify that the dual-loaded hydrogels were injectable. The mechanical and release properties of the hydrogels were maintained after extended storage. The chemotherapeutic activity of doxorubicin was evaluated in the presence of the proposed pH-regulating hydrogels. The findings suggest a promising nontoxic, biodegradable hydrogel buffer delivery system that can achieve two simultaneous important goals of local acidosis neutralization and chemotherapeutic release.

Metabolic engineering in hairy roots: An outlook on production of plant secondary metabolites.

Plants are one of the vital sources of secondary metabolites. These secondary metabolites have diverse roles in human welfare, including therapeutic implication. Nevertheless, secondary metabolite yields obtained through the exploitation of natural plant populations is insufficient to meet the commercial demand due to their accumulation in low volumes. Besides, in-planta synthesis of these important metabolites is directly linked with the age and growing conditions of the plant. Such limitations have paved the way for the exploration of alternative production methodologies. Hairy root cultures, induced after the interaction of plants with Rhizobium rhizogenes (Agrobacterium rhizogenes), are a practical solution for producing valuable secondary metabolite at low cost and without the influence of seasonal, geographic or climatic variations. Hairy root cultures also offer the opportunity to get combined with other yield enhancements strategies (precursor feeding, elicitation and metabolic engineering) to further stimulate and/or enhance their production potential. Applications of metabolic engineering in exploiting hairy root cultures attracted the interest of several research groups as a means of yield enhancement. Currently, several engineering approaches like overexpression and silencing of pathway genes, and transcription factor overexpression are used to boost metabolite production, along with the contextual success of genome editing. This review attempts to cover metabolic engineering in hairy roots for the production of secondary metabolites, with a primary emphasis on alkaloids, and discusses prospects for taking this research forward to meet desired production demands.

Innovative Spectrum Handoff Process Using a Machine Learning-Based Metaheuristic Algorithm.

A cognitive radio network (CRN) is an intelligent network that can detect unoccupied spectrum space without interfering with the primary user (PU). Spectrum scarcity arises due to the stable channel allocation, which the CRN handles. Spectrum handoff management is a critical problem that must be addressed in the CRN to ensure indefinite connection and profitable use of unallocated spectrum space for secondary users (SUs). Spectrum handoff (SHO) has some disadvantages, i.e., communication delay and power consumption. To overcome these drawbacks, a reduction in handoff should be a priority. This study proposes the use of dynamic spectrum access (DSA) to check for available channels for SU during handoff using a metaheuristic algorithm depending on machine learning. The simulation results show that the proposed "support vector machine-based red deer algorithm" (SVM-RDA) is resilient and has low complexity. The suggested algorithm's experimental setup offers several handoffs, unsuccessful handoffs, handoff delay, throughput, signal-to-noise ratio (SNR), SU bandwidth, and total spectrum bandwidth. This study provides an improved system performance during SHO. The inferred technique anticipates handoff delay and minimizes the handoff numbers. The results show that the recommended method is better at making predictions with fewer handoffs compared to the other three.

Nuclear factor I-C overexpression promotes monocytic development and cell survival in acute myeloid leukemia.

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML.

The nuclear effector ArPEC25 from the necrotrophic fungus Ascochyta rabiei targets the chickpea transcription factor CaßLIM1a and negatively modulates lignin biosynthesis, increasing host susceptibility.

Fungal pathogens deploy a barrage of secreted effectors to subvert host immunity, often by evading, disrupting, or altering key components of transcription, defense signaling, and metabolic pathways. However, the underlying mechanisms of effectors and their host targets are largely unexplored in necrotrophic fungal pathogens. Here, we describe the effector protein Ascochyta rabiei PEXEL-like Effector Candidate 25 (ArPEC25), which is secreted by the necrotroph A. rabiei, the causal agent of Ascochyta blight disease in chickpea (Cicer arietinum), and is indispensable for virulence. After entering host cells, ArPEC25 localizes to the nucleus and targets the host LIM transcription factor CaßLIM1a. CaßLIM1a is a transcriptional regulator of CaPAL1, which encodes phenylalanine ammonia lyase (PAL), the regulatory, gatekeeping enzyme of the phenylpropanoid pathway. ArPEC25 inhibits the transactivation of CaßLIM1a by interfering with its DNA-binding ability, resulting in negative regulation of the phenylpropanoid pathway and decreased levels of intermediates of lignin biosynthesis, thereby suppressing lignin production. Our findings illustrate the role of fungal effectors in enhancing virulence by targeting a key defense pathway that leads to the biosynthesis of various secondary metabolites and antifungal compounds. This study provides a template for the study of less explored necrotrophic effectors and their host target functions.

Development and Clinical Validation of RT-LAMP-Based Lateral-Flow Devices and Electrochemical Sensor for Detecting Multigene Targets in SARS-CoV-2.

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL-1 for N, ORF1ab, E genes and 100 agµL-1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL-1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL-1 and log 0.067 µA/pg µL-1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.