Development of an Antioxidant-Rich Sugar-Free Plantain Candy and Assessment of Its Shelf Life in a Flexible Laminate.

Research background: Candy is a popular confection worldwide, and it would be beneficial to society if it were converted into a source of antioxidant molecules to eliminate its adverse health effects. The amount of antioxidants available even in fruit candies is questionable due to the high thermal processing losses they undergo and the presence of various food additives. Plantains (Musa paradisiaca) are less known as good sources of biotherapeutic antioxidants, namely l-tryptophan, serotonin and melatonin, and consumption of this highly nutritious fruit is limited to underdeveloped and developing countries. The objectives of this study are: to develop a functional antioxidant-rich sugar-free plantain-based candy with valuable contents of the mentioned biomolecules in synergy; and to ensure its extended shelf life without compromising its physicochemical properties and functionality by wrapping it with a suitable packaging laminate. Experimental approach: To accomplish the first objective, lyophilized plantain powder, sorbitol and mannitol were used as base materials with minimal additives under minimal processing conditions to reduce processing loss. Sensory, proximate, physicochemical and phytochemical properties, including the antioxidant synergy among the mentioned biomolecules of the developed candies were evaluated. For the second objective, the candies were enclosed in two different flexible packaging laminates and the optimal packaging was determined based on the microbiological safety and sensory appeal of the wrapped candies. Subsequently, the above-mentioned properties of the packaged (in the most suitable laminate) candies were evaluated at regular time intervals during storage for assessment of their shelf life. Results and conclusions: The candy had a characteristic flavour of plantain, uniform dark brown colour, rich mouthfeel, pleasant aroma, moderately hard texture and moderate sweetness, along with high antioxidant activity and considerable content of l-tryptophan, serotonin and melatonin (present as a synergistic consortium). During storage of the packaged candy under ambient conditions, it remained microbiologically safe for up to 56 days, and also maintained sensory attributes, antioxidant activity and synergy compared to the control candy. Novelty and scientific contribution: This newly developed semi-hard sugar-free candy with high antioxidant content, containing three important antioxidants, namely l-tryptophan, serotonin and melatonin, could be a good source of biotherapeutic molecules and a substitute for commercial candies consumed globally.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

The plant pathogenic bacterium Candidatus Liberibacter solanacearum induces calcium-regulated autophagy in midgut cells of its insect vector Bactericera trigonica.

Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica. The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

Crossing the Gateless Barriers: Factors Involved in the Movement of Circulative Bacteria Within Their Insect Vectors.

Plant bacterial pathogens transmitted by hemipteran vectors pose a large threat to the agricultural industry worldwide. Although virus-vector relationships have been widely investigated, a significant gap exists in our understanding of the molecular interactions between circulative bacteria and their insect vectors, mainly leafhoppers and psyllids. In this review, we will describe how these bacterial pathogens adhere, invade, and proliferate inside their insect vectors. We will also highlight the different transmission routes and molecular factors of phloem-limited bacteria that maintain an effective relationship with the insect host. Understanding the pathogen-vector relationship at the molecular level will help in the management of vector-borne bacterial diseases.

Pathophysiology Associated with Diabetes-induced Tauopathy and Development of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia that affects the elderly around the world. Chronic type 2 diabetes (T2DM) has been proven to be closely related to neurodegeneration, especially AD. T2DM is characterized by the cell's failure to take up insulin as well as chronic hyperglycemia. In the central nervous system, insulin plays vital regulatory roles, while in chronic hyperglycemia, it leads to the formation and accumulation of advanced glycation end products (AGEs). Inflammation plays a crucial role in development of insulin resistance in AD and T2DM. The microtubule-related protein tau is involved in the pathogenesis of several neurological diseases known as tauopathies, and is found to be abnormally hyperphosphorylated in AD and accumulated in neurons. Chronic neuroinflammation causes the breakdown of the blood-brain barrier (BBB) observed in tauopathies. The development of pro-inflammatory signaling molecules, such as cytokines, chemokines from glial cells, neurons and endothelial cells, decides the structural integrity of BBB and immune cell migration into the brain. This review highlights the use of antidiabetic compounds as promising therapeutics for AD, and also describes several new pathological molecular mechanisms associated with diabetes that increase AD pathogenesis.

Blood-based biomarkers for diagnosis, prognosis, and severity prediction of COVID-19: Opportunities and challenges.

The reasons for high morbidity and mortality with Corona virus disease (COVID-19) disease remain unanswered with extremes of manifestation and uncertainty of modes of transmission for which biomarkers are urgently needed for early prediction of severity and prompt treatment. We have reviewed publications from PubMed (years 2019-2021) analysing the biochemical, immune-inflammatory, nucleic acid, and cellular biomarkers that predict infection, disease progression in COVID-19 with emphasis on organ-specific damage. Our analysis of 65 biomarkers assessing the impact of SCoV-2 infection on five organs (lung, liver, cardiac, kidney, and neural) reported that increased levels of CRP, TNF-α, ferritin, IL-6, D-dimer, Procalcitonin, Fibrinogen to Albumin Ratio (FAR), and decrease platelet count (PC), lymphocyte count, leukocyte count, and CD4+/CD8 + ratio shows promising association in the early diagnosis, prediction of prognosis and severity disease and also correlates with cytokine storm a cardinal feature of COVID-19 progression. In the above scenario, this review has put forth the most promising biomarkers for COVID diagnosis and prognosis based on the reported literature. In recent year's chemically synthesized antibody-like biomolecules, aptamers were also used in the diagnosis of COVID-19 which could be preferably used for diagnosis over antibodies. Biomarkers including increase in free DNA and Fibrinogen-to-Albumin Ratio, CRP, PCT, and Ferritin along with a consequential decrease of CD3+ T, CD4+ T, CD8+ T, NK cells with corresponding increase in CD4+/CD8+ ratio following SARS CoV-2 infection has been consistently correlated with disease severity. Despite the two waves of COVID-19 pandemic, currently there is no standard clinical practice guideline for evaluating the severity of the devastating pandemic of COVID-19, hence these biomarkers will have immense relevance for the third and subsequent wave of COVID-19 and related pandemic.

Interaction of Liberibacter Solanacearum with Host Psyllid Vitellogenin and Its Association with Autophagy.

Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major constraint for carrot production in Israel. Unveiling the molecular interactions between the psyllid vector and CLso can facilitate the development of nonchemical approaches for controlling the disease caused by CLso. Bacterial surface proteins are often known to be involved in adhesion and virulence; however, interactions of CLso with carrot psyllid proteins that have a role in the transmission process has remained unexplored. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast two-hybrid system assay. We identified psyllid vitellogenin (Vg) to interact with both OmpA and flagellin of CLso. As Vg and autophagy are often tightly linked, we also studied the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy. Induced Vg expression significantly suppressed autophagy-related gene expression and the levels of CLso significantly increased, resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter. IMPORTANCE Pathogen transmission by vectors involves multiple levels of interactions, and for the transmission of liberibacter species by psyllid vectors, much of these interactions are yet to be explored. Candidatus Liberibacter solanacearum (CLso) haplotype D inflicts severe economic losses to the carrot industry. Understanding the specific interactions at different stages of infection is hence fundamental and could lead to the development of better management strategies to disrupt the transmission of the bacteria to new host plants. Here, we show that two liberibacter membrane proteins interact with psyllid vitellogenin and also induce autophagy. Altering vitellogenin expression directly influences autophagy and CLso abundance in the psyllid vector. Although the exact mechanism underlying this interaction remains unclear, this study highlights the importance of immune responses in the transmission of this disease agent.

The Actin Cytoskeleton Mediates Transmission of "Candidatus Liberibacter solanacearum" by the Carrot Psyllid.

Several vector-borne plant pathogens have evolved mechanisms to exploit and to hijack vector host cellular, molecular, and defense mechanisms for their transmission. In the past few years, Liberibacter species, which are transmitted by several psyllid vectors, have become an economically important group of pathogens that have devastated the citrus industry and caused tremendous losses to many other important crops worldwide. The molecular mechanisms underlying the interactions of Liberibacter species with their psyllid vectors are poorly studied. "Candidatus Liberibacter solanacearum," which is associated with important vegetable diseases, is transmitted by the carrot psyllid Bactericera trigonica in a persistent manner. Here, we elucidated the role of the B. trigonica Arp2/3 protein complex, which plays a major role in regulation of the actin cytoskeleton, in the transmission of "Ca Liberibacter solanacearum." "Ca Liberibacter solanacearum" colocalized with ArpC2, a key protein in this complex, and this colocalization was strongly associated with actin filaments. Silencing of the psyllid ArpC2 disrupted the colocalization and the dynamics of F-actin. Silencing of RhoGAP21 and Cdc42, which act in the signaling cascade leading to upregulation of Arp2/3 and F-actin bundling, showed similar results. On the other hand, silencing of ArpC5, another component of the complex, did not induce any significant effects on F-actin formation. Finally, ArpC2 silencing caused a 73.4% reduction in "Ca Liberibacter solanacearum" transmission by psyllids, strongly suggesting that transmission of "Ca Liberibacter solanacearum" by B. trigonica is cytoskeleton dependent and "Ca Liberibacter solanacearum" interacts with ArpC2 to exploit the intracellular actin nucleation process for transmission. Targeting this unique interaction could lead to the development of a novel strategy for the management of Liberibacter-associated diseases.IMPORTANCE Plant diseases caused by vector-borne pathogens are responsible for tremendous losses and threaten some of the most important agricultural crops. A good example is the citrus greening disease, which is caused by bacteria of the genus Liberibacter and is transmitted by psyllids; it has devastated the citrus industry in the United States, China, and Brazil. Here, we show that psyllid-transmitted "Candidatus Liberibacter solanacearum" employs the actin cytoskeleton of psyllid gut cells, specifically the ArpC2 protein in the Arp2/3 complex of this system, for movement and transmission in the vector. Silencing of ArpC2 dramatically influenced the interaction of "Ca Liberibacter solanacearum" with the cytoskeleton and decreased the bacterial transmission to plants. This system could be targeted to develop a novel approach for the control of Liberibacter-associated diseases.

Ameliorative role of ferulic acid against diabetes associated oxidative stress induced spleen damage.

Spleen, a secondary lymphoid organ, is the site of initiation of most of the immune responses. The present study is centered on the ameliorative role of ferulic acid against diabetic complications in the spleen of male Wistar rats. Induction of diabetes by STZ (at a dose of 50 mg kg-1 body wt, i.p.) reduced the spleen size, plasma insulin level, enhanced the blood glucose level and disrupted the intracellular antioxidant machineries along with the depletion of splenic white pulp. Induction of oxidative stress mediated inflammation and apoptosis (upregulation in the levels of inflammatory cytokines, translocation of NF ĸB in the nucleus, alteration in Bax/Bcl-2 ratio, release of cytochrome c from mitochondria, activation of caspase-9 and 3, PARP cleavage and DNA fragmentation) were evidenced from immunoblot analyses, DNA fragmentation and TUNEL assay. However, ferulic acid administration post diabetes induction, (at a dose of 50 mg kg-1 body wt, orally for eight weeks) could reverse such adverse effects. Therefore, ferulic acid, as a potential therapeutic agent may hold promise in evading oxidative stress mediated diabetic splenotoxicity in rats.

Prophylactic role of taurine and its derivatives against diabetes mellitus and its related complications.

Taurine is a conditionally essential amino acid present in the body in free form. Mammalian taurine is synthesized in the pancreas via the cysteine sulfinic acid pathway. Anti-oxidation and anti-inflammation are two main properties through which it exerts its therapeutic effects. Many studies have shown its excellent therapeutic potential against diabetes mellitus and related complications like diabetic neuropathy, retinopathy, nephropathy, hematological dysfunctions, reproductive dysfunctions, liver and pancreas related complications etc. Not only taurine, a number of its derivatives have also been reported to be important in ameliorating diabetic complications. The present review has been aimed to describe the importance of taurine and its derivatives against diabetic metabolic syndrome and related complications.

Perspectives of the Nrf-2 signaling pathway in cancer progression and therapy.

The Nuclear factor erythroid2-related factor2 (Nrf2), a master regulator of redox homoeostasis, is a key transcription factor regulating a wide array of genes for antioxidant and detoxification enzymes. It protects organs from various kinds of toxic insults. On the other hand, activation of Nrf2 is also correlated with cancer progression and chemoresistance. Downregulation of Nrf2 activity has attracted an increasing amount of attention as it may provide an alternative cancer therapy. In this review, we examine recent studies on roles of Nrf2 in several pathophysiological conditions emphasising cancer. We discuss elaborately the current knowledge on Nrf2 regulation including KEAP1-dependent and KEAP1-independent cascades. KEAP1/Nrf2 system is a master regulator of cellular response against a variety of environmental stresses. We also highlight several tightly controlled regulations of Nrf2 by numerous proteins, small molecules, toxic metals, etc. In addition, we evaluate the possible therapeutic approaches of increasing chemosensitivity via modulating Nrf2 signaling.

Overexpression of biologically safe Rorippa indica defensin enhances aphid tolerance in Brassica juncea.

MAIN CONCLUSION: Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71-2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest-L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

Functional characterization of Rorippa indica defensin and its efficacy against Lipaphis erysimi.

Rorippa indica, a wild crucifer, has been previously reported as the first identified plant in the germplasm of Brassicaceae known to be tolerant towards the mustard aphid Lipaphis erysimi Kaltenbach. We herein report the full-length cloning, expression, purification and characterization of a novel R. indica defensin (RiD) and its efficacy against L. erysimi. Structural analysis through homology modeling of RiD showed longer α-helix and 3rd ß-sheet as compared to Brassica juncea defensin (BjD). Recombinant RiD and BjD was purified for studying its efficacy against L. erysimi. In the artificial diet based insect bioassay, the LC50 value of RiD against L. erysimi was found to be 9.099 ± 0.621 µg/mL which is far lower than that of BjD (43.51 ± 0.526 µg/mL). This indicates the possibility of RiD having different interacting partner and having better efficacy against L. erysimi over BjD. In the transient localization studies, RiD signal peptide directed the RiD: yellow fluorescent protein (YFP) fusion protein to the apoplastic regions which indicates that it might play a very important role in inhibiting nutrient uptake by aphids which follow mainly extracellular route to pierce through the cells. Hence, the present study has a significant implication for the future pest management program of B. juncea through the development of aphid tolerant transgenic plants.