Cinnamic acid, a natural plant compound, exhibits neuroprotection in a mouse model of Sandhoff disease via PPARα.

Tay-Sachs disease (TSD) and its severe form Sandhoff disease (SD) are autosomal recessive lysosomal storage metabolic disorders, which often result into excessive GM2 ganglioside accumulation predominantly in lysosomes of nerve cells. Although patients with these diseases appear normal at birth, the progressive accumulation of undegraded GM2 gangliosides in neurons leads to early death accompanied by manifestation of motor difficulties and gradual loss of behavioral skills. Unfortunately, there is still no effective treatment available for TSD/SD. The present study highlights the importance of cinnamic acid (CA), a naturally occurring aromatic fatty acid present in a number of plants, in inhibiting the disease process in a transgenic mouse model of SD. Oral administration of CA significantly attenuated glial activation and inflammation and reduced the accumulation of GM2 gangliosides/glycoconjugates in the cerebral cortex of Sandhoff mice. Besides, oral CA also improved behavioral performance and increased the survival of Sandhoff mice. While assessing the mechanism, we found that oral administration of CA increased the level of peroxisome proliferator-activated receptor α (PPARα) in the brain of Sandhoff mice and that oral CA remained unable to reduce glycoconjugates, improve behavior and increase survival in Sandhoff mice lacking PPARα. Our results indicate a beneficial function of CA that utilizes a PPARα-dependent mechanism to halt the progression of SD and thereby increase the longevity of Sandhoff mice.

Lipid-Lowering Drug Gemfibrozil Protects Mice from Tay-Sachs Disease via Peroxisome Proliferator-Activated Receptor α.

Tay-Sachs disease (TSD) is a progressive heritable neurodegenerative disorder characterized by the deficiency of the lysosomal ß-hexosaminidase enzyme (Hex-/-) and the storage of GM2 ganglioside, as well as other related glycoconjugates. Along with motor difficulties, TSD patients also manifest a gradual loss of skills and behavioral problems, followed by early death. Unfortunately, there is no cure for TSD; however, research on treatments and therapeutic approaches is ongoing. This study underlines the importance of gemfibrozil (GFB), an FDA-approved lipid-lowering drug, in inhibiting the disease process in a transgenic mouse model of Tay-Sachs. Oral administration of GFB significantly suppressed glial activation and inflammation, while also reducing the accumulation of GM2 gangliosides/glycoconjugates in the motor cortex of Tay-Sachs mice. Furthermore, oral GFB improved behavioral performance and increased the life expectancy of Tay-Sachs mice. While investigating the mechanism, we found that oral administration of GFB increased the level of peroxisome proliferator-activated receptor α (PPARα) in the brain of Tay-Sachs mice, and that GFB remained unable to reduce glycoconjugates and improve behavior and survival in Tay-Sachs mice lacking PPARα. Our results indicate a beneficial function of GFB that employs a PPARα-dependent mechanism to halt the progression of TSD and increase longevity in Tay-Sachs mice.

Tau fibrils induce glial inflammation and neuropathology via TLR2 in Alzheimer's disease-related mouse models.

Glial activation and inflammation coincide with neurofibrillary tangle (NFT) formation in neurons. However, the mechanism behind the interaction between tau fibrils and glia is poorly understood. Here, we found that tau preformed fibrils (PFFs) caused induction of inflammation in microglia by specifically activating the TLR2/MyD88, but not the TLR4/MyD88, pathway. Accordingly, the WT TLR2-interacting domain of MyD88 (wtTIDM) peptide inhibited tau PFF-induced activation of the TLR2/MyD88/NF-κB pathway, resulting in reduced inflammation. Nasal administration of wtTIDM in P301S tau-expressing PS19 mice was found to inhibit gliosis and inflammatory markers, as well as to reduce pathogenic tau in the hippocampus, resulting in improved cognitive behavior in PS19 mice. The inhibitory effect of wtTIDM on tau pathology was absent in PS19 mice lacking TLR2, reinforcing the essential involvement of TLR2 in wtTIDM-mediated effects in vivo. Studying the mechanism further, we found that the tau promoter harbored a potential NF-κB-binding site and that proinflammatory molecules increased transcription of tau in neurons via NF-κB. These results suggest that tau-induced neuroinflammation and neuropathology require TLR2 and that neuroinflammation directly upregulates tau in neurons via NF-κB, highlighting a direct connection between inflammation and tauopathy.

Muscle-building supplement ß-hydroxy ß-methylbutyrate binds to PPARα to improve hippocampal functions in mice.

This study underlines the importance of ß-hydroxy ß-methylbutyrate (HMB), a muscle-building supplement in human, in increasing mouse hippocampal plasticity. Detailed proteomic analyses reveal that HMB serves as a ligand of peroxisome proliferator-activated receptor α (PPARα), a nuclear hormone receptor involved in fat metabolism, via interaction with the Y314 residue. Accordingly, HMB is ineffective in increasing plasticity of PPARα-/- hippocampal neurons. While lentiviral establishment of full-length PPARα restores the plasticity-promoting effect of HMB in PPARα-/- hippocampal neurons, lentiviral transduction of Y314D-PPARα remains unable to do that, highlighting the importance of HMB's interaction with the Y314 residue. Additionally, oral HMB improves spatial learning and memory and reduces plaque load in 5X familial Alzheimer's disease (5XFAD) mice, but not in 5XFADΔPPARα mice (5XFAD lacking PPARα), indicating the involvement of PPARα in HMB-mediated neuroprotection in 5XFAD mice. These results delineate neuroprotective functions of HMB and suggest that this widely used supplement may be repurposed for AD.

Prenol, but Not Vitamin C, of Fruit Binds to SARS-CoV-2 Spike S1 to Inhibit Viral Entry: Implications for COVID-19.

Fruit consumption may be beneficial for fighting infection. Although vitamin C is the celebrity component of fruit, its role in COVID-19 is unclear. Because spike S1 of SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) on host cells to enter the cell and initiate COVID-19, using an α-screen-based assay, we screened vitamin C and other components of fruit for inhibiting the interaction between spike S1 and ACE2. We found that prenol, but neither vitamin C nor other major components of fruit (e.g., cyanidin and rutin), reduced the interaction between spike S1 and ACE2. Thermal shift assays indicated that prenol associated with spike S1, but not ACE2, and that vitamin C remained unable to do so. Although prenol inhibited the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus, into human ACE2-expressing HEK293 cells, vitamin C blocked the entry of pseudotyped vesicular stomatitis virus, not SARS-CoV-2, indicating the specificity of the effect. Prenol, but not vitamin C, decreased SARS-CoV-2 spike S1-induced activation of NF-κB and the expression of proinflammatory cytokines in human A549 lung cells. Moreover, prenol also decreased the expression of proinflammatory cytokines induced by spike S1 of N501Y, E484K, Omicron, and Delta variants of SARS-CoV-2. Finally, oral treatment with prenol reduced fever, decreased lung inflammation, enhanced heart function, and improved locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. These results suggest that prenol and prenol-containing fruits, but not vitamin C, may be more beneficial for fighting against COVID-19.

Cinnamein Inhibits the Induction of Nitric Oxide and Proinflammatory Cytokines in Macrophages, Microglia and Astrocytes.

Chronic inflammation driven by proinflammatory cytokines (TNFα, IL-1ß, IL-6, etc.), and nitric oxide (NO) plays an important role in the pathogenesis of several autoimmune, inflammatory as well as neurodegenerative disorders like rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. Therefore, identification of nontoxic anti-inflammatory drugs may be beneficial for these autoimmune, inflammatory and neurodegenerative disorders. Cinnamein, an ester derivative of cinnamic acid and benzyl alcohol, is used as a flavoring agent and for its antifungal and antibacterial properties. This study underlines the importance of cinnamein in inhibiting the induction of proinflammatory molecules in RAW 264.7 macrophages and primary mouse microglia and astrocytes. Stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and interferon γ (IFNγ) led to marked production of NO. However, cinnamein pretreatment significantly inhibited LPS- and IFNγ-induced production of NO in RAW 264.7 macrophages. Cinnamein also reduced the mRNA expression of inducible nitric oxide synthase (iNOS) and TNFα in RAW cells. Accordingly, LPS and viral double-stranded RNA mimic polyinosinic: polycytidylic acid (polyIC) stimulated the production of TNFα, IL-1ß and IL-6 in primary mouse microglia, which was inhibited by cinnamein pretreatment. Similarly, cinnamein also inhibited polyIC-induced production of TNFα and IL-6 in primary mouse astrocytes. These results suggest that cinnamein may be used to control inflammation in different autoimmune, inflammatory and neurodegenerative disorders.

Treadmill exercise reduces α-synuclein spreading via PPARα.

This study underlines the importance of treadmill exercise in reducing α-synuclein (α-syn) spreading in the A53T brain and protecting nigral dopaminergic neurons. Preformed α-syn fibril (PFF) seeding in the internal capsule of young A53T α-syn mice leads to increased spreading of α-syn to substantia nigra and motor cortex and concomitant loss of nigral dopaminergic neurons. However, regular treadmill exercise decreases α-syn spreading in the brain and protects nigral dopaminergic neurons in PFF-seeded mice. Accordingly, treadmill exercise also mitigates α-synucleinopathy in aged A53T mice. While investigating this mechanism, we have observed that treadmill exercise induces the activation of peroxisome proliferator-activated receptor α (PPARα) in the brain to stimulate lysosomal biogenesis via TFEB. Accordingly, treadmill exercise remains unable to stimulate TFEB and reduce α-synucleinopathy in A53T mice lacking PPARα, and fenofibrate, a prototype PPARα agonist, reduces α-synucleinopathy. These results delineate a beneficial function of treadmill exercise in reducing α-syn spreading in the brain via PPARα.

Regression of Triple-Negative Breast Cancer in a Patient-Derived Xenograft Mouse Model by Monoclonal Antibodies against IL-12 p40 Monomer.

Although some therapies are available for regular breast cancers, there are very few options for triple-negative breast cancer (TNBC). Here, we demonstrated that serum level of IL-12p40 monomer (p40) was much higher in breast cancer patients than healthy controls. On the other hand, levels of IL-12, IL-23 and p40 homodimer (p402) were lower in serum of breast cancer patients as compared to healthy controls. Similarly, human TNBC cells produced greater level of p40 than p402. The level of p40 was also larger than p402 in serum of a patient-derived xenograft (PDX) mouse model. Accordingly, neutralization of p40 by p40 mAb induced death of human TNBC cells and tumor shrinkage in PDX mice. While investigating the mechanism, we found that neutralization of p40 led to upregulation of human CD4+IFNγ+ and CD8+IFNγ+ T cell populations, thereby increasing the level of human IFNγ and decreasing the level of human IL-10 in PDX mice. Finally, we demonstrated the infiltration of human cytotoxic T cells, switching of tumor-associated macrophage M2 (TAM2) to TAM1 and suppression of transforming growth factor ß (TGFß) in tumor tissues of p40 mAb-treated PDX mice. Our studies identify a possible new immunotherapy for TNBC in which p40 mAb inhibits tumor growth in PDX mice.

Development of Fe3O4 core-TiO2 shell nanocomposites and nanoconjugates as a foundation for neuroblastoma radiosensitization.

BACKGROUND: Neuroblastoma is the most common extracranial solid malignancy in childhood which, despite the current progress in radiotherapy and chemotherapy protocols, still has a high mortality rate in high risk tumors. Nanomedicine offers exciting and unexploited opportunities to overcome the shortcomings of conventional medicine. The photocatalytic properties of Fe3O4 core-TiO2 shell nanocomposites and their potential for cell specific targeting suggest that nanoconstructs produced using Fe3O4 core-TiO2 shell nanocomposites could be used to enhance radiation effects in neuroblastoma. In this study, we evaluated bare, metaiodobenzylguanidine (MIBG) and 3,4-Dihydroxyphenylacetic acid (DOPAC) coated Fe3O4@TiO2 as potential radiosensitizers for neuroblastoma in vitro. RESULTS: The uptake of bare and MIBG coated nanocomposites modestly sensitized neuroblastoma cells to ionizing radiation. Conversely, cells exposed to DOPAC coated nanocomposites exhibited a five-fold enhanced sensitivity to radiation, increased numbers of radiation induced DNA double-strand breaks, and apoptotic cell death. The addition of a peptide mimic of the epidermal growth factor (EGF) to nanoconjugates coated with MIBG altered their intracellular distribution. Cryo X-ray fluorescence microscopy tomography of frozen hydrated cells treated with these nanoconjugates revealed cytoplasmic as well as nuclear distribution of the nanoconstructs. CONCLUSIONS: The intracellular distribution pattern of different nanoconjugates used in this study was different for different nanoconjugate surface molecules. Cells exposed to DOPAC covered nanoconjugates showed the smallest nanoconjugate uptake, with the most prominent pattern of large intracellular aggregates. Interestingly, cells treated with this nanoconjugate also showed the most pronounced radiosensitization effect in combination with the external beam x-ray irradiation. Further studies are necessary to evaluate mechanistic basis for this increased radiosensitization effect. Preliminary studies with the nanoparticles carrying an EGF mimicking peptide showed that this approach to targeting could perhaps be combined with a different approach to radiosensitization - use of nanoconjugates in combination with the radioactive iodine. Much additional work will be necessary in order to evaluate possible benefits of targeted nanoconjugates carrying radionuclides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12645-021-00081-z.

Activation of PPARα enhances astroglial uptake and degradation of ß-amyloid.

Astrocytes are a type of glial cell that are activated in the brain tissue of patients with Alzheimer's disease to induce the accumulation of amyloid (Aß). We previously found that a combination of low-dose gemfibrozil (GFB; a drug approved to treat high cholesterol) and retinoic acid (RA; a vitamin A derivative) induces lysosomal bio-genesis through peroxisome proliferator­activated receptor α (PPARα)­mediated transcription of the gene encoding transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy. Here, we found that the same combination (GFB-RA) enhanced the uptake of Aß from the extracellular space and its subsequent degradation in astrocytes through a PPARα-dependent pathway. GFB-RA stimulated the abundance of both low-density lipoprotein receptor (LDLR) and TFEB in astrocytes through PPARα. LDLR was critical for Aß uptake, whereas TFEB was critical for its degradation. GFB-RA treatment also increased autophagic flux and lysosomal activity in astrocytes. Consistent with these effects and in a manner dependent on astroglial PPARα, oral administration of GFB-RA switched astroglial activation to a neuroprotective state, lowered Aß burden in the brain, and improved spatial learning and memory in the 5XFAD mouse model of Alzheimer's disease. These findings uncover a new function of PPARα in stimulating astroglial uptake and degradation of Aß and suggest possible repurposing of GFB-RA combination therapy for AD.

Eugenol, a Component of Holy Basil (Tulsi) and Common Spice Clove, Inhibits the Interaction Between SARS-CoV-2 Spike S1 and ACE2 to Induce Therapeutic Responses.

Spike S1 of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells to enter the cell and initiate COVID-19. Since ACE2 is a favorable enzyme, we were interested in finding a molecule capable of binding spike S1, but not ACE2, and inhibiting the interaction between spike S1 and ACE2. Holy basil (Tulsi) has a long history as a medicine for different human disorders. Therefore, we screened different components of Tulsi leaf and found that eugenol, but not other major components (e.g. ursolic acid, oleanolic acid and ß-caryophylline), inhibited the interaction between spike S1 and ACE2 in an AlphaScreen-based assay. By in silico analysis and thermal shift assay, we also observed that eugenol associated with spike S1, but not ACE2. Accordingly, eugenol strongly suppressed the entry of pseudotyped SARS-CoV-2, but not vesicular stomatitis virus (VSV), into human ACE2-expressing HEK293 cells. Eugenol also reduced SARS-CoV-2 spike S1-induced activation of NF-κB and the expression of IL-6, IL-1ß and TNFα in human A549 lung cells. Moreover, oral treatment with eugenol reduced lung inflammation, decreased fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1, but not ACE2, by eugenol may be beneficial for COVID-19 treatment.

ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy.

COVID-19 is an infectious respiratory illness caused by the virus strain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and until now, there is no effective therapy against COVID-19. Since SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) for entering into host cells, to target COVID-19 from therapeutic angle, we engineered a hexapeptide corresponding to the ACE2-interacting domain of SARS-CoV-2 (AIDS) that inhibits the association between receptor-binding domain-containing spike S1 and ACE-2. Accordingly, wild type (wt), but not mutated (m), AIDS peptide inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 in human lungs cells. Interestingly, intranasal intoxication of C57/BL6 mice with recombinant SARS-CoV-2 spike S1 led to fever, increase in IL-6 in lungs, infiltration of neutrophils into the lungs, arrhythmias, and impairment in locomotor activities, mimicking some of the important symptoms of COVID-19. However, intranasal treatment with wtAIDS, but not mAIDS, peptide reduced fever, protected lungs, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of ACE2-to-SARS-CoV-2 interaction by wtAIDS may be beneficial for COVID-19.

Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Improves Cognitive Functions in Mice after Controlled Cortical Impact Injury.

Traumatic brain injury (TBI) is a major health concern, sometimes leading to long-term neurological disability, especially in children, young adults and war veterans. Although research investigators and clinicians have applied different treatment strategies or neurosurgical procedures to solve this health issue, we are still in need of an effective therapy to halt the pathogenesis of brain injury. Earlier, we reported that sodium benzoate (NaB), a metabolite of cinnamon and a Food and Drug Administration-approved drug against urea cycle disorders and glycine encephalopathy, protects neurons in animal models of Parkinson's disease and Alzheimer's disease. This study was undertaken to examine the therapeutic efficacy of NaB in a controlled cortical impact (CCI)-induced preclinical mouse model of TBI. Oral treatment with NaB, but not sodium formate (NaFO), was found to decrease the activation of microglia and astrocytes and to inhibit the expression of inducible nitric oxide synthase (iNOS) in the hippocampus and cortex of CCI-insulted mice. Further, administration of NaB also reduced the vascular damage and decreased the size of the lesion cavity in the brain of CCI-induced mice. Importantly, NaB-treated mice showed significant improvements in memory and locomotor functions as well as displaying a substantial reduction in depression-like behaviors. These results delineate a novel neuroprotective property of NaB, highlighting its possible therapeutic importance in TBI.

Reduction of Lewy Body Pathology by Oral Cinnamon.

α-Synucleinopathies in a broader sense comprise of several neurodegenerative disorders that primarily include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These disorders are well characterized by the accumulation of aggregated insoluble α-synuclein (α-syn) protein known as Lewy bodies. Till date no effective cure is available to reduce the burden of Lewy body. The present investigation underlines the importance of a naturally used spice and flavoring agent viz. cinnamon in reducing α-syn deposits in transgenic mice expressing mutant A53T human α-syn. Upon oral administration, cinnamon markedly reduced the level of insoluble α-syn in nigra, hippocampus and brain stem of A53T mice. We also demonstrated that sodium benzoate (NaB), a metabolite of cinnamon, a widely used food additive and a FDA-approved drug for glycine encephalopathy, was also capable of reducing α-syn deposits in A53T mice. In addition, both cinnamon and NaB treatments showed improvement in their motor and cognitive functions. Glial activation plays an important role in the pathogenesis of various neurodegenerative disorders including PD, DLB and MSA, and we found suppression of microglial and astroglial activation in the nigra of A53T mice upon cinnamon treatment. Moreover, neuroprotective proteins like DJ-1 and Parkin are known to reduce the formation of Lewy bodies in the CNS. Accordingly, we observed upregulation and/or normalization of DJ-1 and Parkin in the nigra of A53T mice by treatment with cinnamon and NaB. Together, these results highlight a new therapeutic property of cinnamon and suggest that cinnamon and NaB may be used to halt the progression of α-synucleinopathies.Graphical Abstract.

Upregulation of BDNF and hippocampal functions by a hippocampal ligand of PPARα.

Discovery strategies commonly focus on the identification of chemical libraries or natural products, but the modulation of endogenous ligands offers a much better therapeutic strategy due to their low adverse potential. Recently, we found that hexadecanamide (Hex) is present in hippocampal nuclei of normal mice as an endogenous ligand of PPARα. This study underlines the importance of Hex in inducing the expression of brain-derived neurotrophic factor (BDNF) from hippocampal neurons via PPARα. The level of Hex was lower in the hippocampi of 5XFAD mice as compared with that in non-Tg mice. Oral administration of Hex increased the level of this molecule in the hippocampus to stimulate BDNF and its downstream plasticity-associated molecules, promote synaptic functions in the hippocampus, and improve memory and learning in 5XFAD mice. However, oral Hex remained unable to stimulate hippocampal plasticity and improve cognitive behaviors in 5XFADPparα-null and 5XFADPparα-ΔHippo mice, indicating an essential role of hippocampal PPARα in Hex-mediated improvement in hippocampal functions. This is the first demonstration to our knowledge of protection of hippocampal functions by oral administration of a hippocampus-based drug, suggesting that Hex may be explored for therapeutic intervention in AD.

Enrichment of apoplastic fluid with therapeutic recombinant protein for efficient biofarming.

OBJECTIVE: For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study. RESULTS: Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts. CONCLUSION: Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:726-736, 2017.

Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells.

The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5' AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

Comparative analysis of synthetic DNA promoters for high-level gene expression in plants.

MAIN CONCLUSION: We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter. We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (-329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using ß-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (-341 to +5) and CmpS (-349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.

Overexpression of the synthetic chimeric native-T-phylloplanin-GFP genes optimized for monocot and dicot plants renders enhanced resistance to blue mold disease in tobacco (N. tabacum L.).

To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco.

KEY MESSAGE: Phylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen. ABSTRACT: Tobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene--as a fusion with the GFP gene--targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.