Biofortification revisited: Addressing the role of beneficial soil microbes for enhancing trace elements concentration in staple crops.

Trace element deficiency is a pervasive issue contributing to malnutrition on a global scale. The primary cause of this hidden hunger is related to low dietary intake of essential trace elements, which is highly prevalent in numerous regions across the world. To address deficiency diseases in humans, fortification of staple crops with vital trace elements has emerged as a viable solution. Current methods for fortifying crops encompass chemical amendments, genetic breeding, and transgenic approaches, yet these approaches possess certain limitations, constraining their agricultural application. In contrast, fortifying staple crops through the utilization of soil-beneficial microbes has emerged as a promising and economically feasible approach to enhance trace element content in crops. A specific subset of these beneficial soil microbes, referred to as plant growth-promoting microbes, have demonstrated their ability to influence the interactions between plants, soil, and minerals. These microbes facilitate the transport of essential soil minerals, such as zinc, iron, and selenium, into plants, offering the potential for the development of tailored bioinoculants that can enhance the nutritional quality of cereals, pulses, and vegetable crops. Nevertheless, further research efforts are necessary to comprehensively understand the molecular mechanisms underlying the uptake, transport, and augmentation of trace element concentrations in staple crops. By delving deeper into these mechanisms, customized bioinoculants of soil-beneficial microbes can be developed to serve as highly effective strategies in combating trace element deficiency and promoting global nutritional well-being.

Identification of a vicilin-like major allergen from Prosopis juliflora exhibiting cross- reactivity with legume food allergens.

BACKGROUND: Prosopis juliflora is a clinically relevant allergic sensitizer worldwide and shares cross-reactivity with allergens from several tree pollen and food. The present study aims to purify and immunobiochemically characterize a major allergen from Prosopis pollen. The allergen was further investigated for its cross-reactivity with legume allergens. METHODS: Prosopis extract was fractionated by Q Sepharose and Superdex 75 gel filtration column to purify the allergen. Specific IgE against purified protein was estimated via ELISA and immunoblot. The protein was subjected to mass spectrometric analysis. Glycan characterization was performed by Schiff staining and lectin binding assay followed by deglycosylation studies. The functional activity of the purified protein was evaluated by the basophil activation test. Cross-reactivity was assessed by inhibition studies with legume extracts. RESULTS: A 35 kDa protein was purified and showed 75% IgE reactivity with the patients' sera by ELISA and immunoblot. Glycan characterization of protein demonstrated the presence of terminal glucose and mannose residues. A reduction of 40% and 27% in IgE binding was observed upon chemical and enzymatic deglycosylation of the protein, respectively. The glycoprotein allergen upregulates the expression of CD203c on basophils which was significantly reduced upon deglycosylation, signifying its biological ability to activate the effector cells. The identified protein shared significant homology with Lup an 1 from the lupine bean. Immunoblot inhibition studies of the purified allergen with legume extracts underlined high cross-reactive potential. Complete inhibition was observed with peanut and common bean, while up to 70% inhibition was demonstrated with soy, black gram, chickpea, and lima bean. CONCLUSION: A 35 kDa vicilin-like major allergen was isolated from P. juliflora. The protein possesses glycan moieties crucial for IgE binding and basophil activation. Furthermore, the purified protein shows homology with Lup an 1 and exhibits cross-reactivity with common edible legume proteins.

Development of Bacillus safensis-based liquid bioformulation to augment growth, stevioside content, and nutrient uptake in Stevia rebaudiana.

The application of chemical fertilizers to enhance crop production is a major concern due to associated environmental pollution and health hazards. Hence, there is an urgent need to develop an eco-friendly solution to improve crop production and promote sustainable agriculture simultaneously. Stevia rebaudiana is an important medicinal crop being substitute for sugar, superior flavor outline, extensive medicinal properties, and also of agronomic interest. In the present study, bacterium STJP isolated from the rhizospheric soil of S. rebaudiana and identified as Bacillus safensis on the basis of 16S rRNA gene sequencing, showed good amount of zinc (4.4 mg/L) and potassium (5.4 mg/L) solubilization. Paneer-whey (a dairy waste) based bioformulation (P-WBF) was developed utilizing isolate B. safensis STJP (accession number NAIMCC TB-2833) and inspected for the quality and ability to enhance the growth, nutrients uptake, and stevioside content in S. rebaudiana. The application of P-WBF displayed a significantly higher concentration (153.12%) of stevioside in S. rebaudiana as compared to control. P-WBF treated Stevia plants showed significantly higher fresh and dry weight as well (as compared to control). Further, enhancement of phosphorous, nitrogen, potassium, and zinc uptake in plant tissue was also recorded by application of P-WBF. This study suggests the use of P-WBF based biofertilizer using B. safensis STJP to increase stevioside content in Stevia plant by a nutrient(s) linked mechanism. This novel approach can also be beneficial for utilization of a dairy waste in preparation of bioformulation and, for enhancement of crop yield by an ecofriendly manner leading to sustainable agriculture.

Proteomic profiling of the weed feverfew, a neglected pollen allergen source.

Feverfew (Parthenium hysterophorus), an invasive weed from the Asteraceae family, has been reported as allergen source. Despite its relevance, knowledge of allergens is restricted to a partial sequence of a hydroxyproline-rich glycoprotein. We aimed to obtain the entire sequence for recombinant production and characterize feverfew pollen using proteomics and immunological assays. Par h 1, a defensin-proline fusion allergen was obtained by cDNA cloning and recombinantly produced in E. coli. Using two complementary proteomic strategies, a total of 258 proteins were identified in feverfew pollen among those 47 proteins belonging to allergenic families. Feverfew sensitized patients' sera from India revealed IgE reactivity with a pectate lyase, PR-1 protein and thioredoxin in immonoblot. In ELISA, recombinant Par h 1 was recognized by 60 and 40% of Austrian and Indian sera, respectively. Inhibition assays demonstrated the presence of IgE cross-reactive Par h 1, pectate lyase, lipid-transfer protein, profilin and polcalcin in feverfew pollen. This study reveals significant data on the allergenic composition of feverfew pollen and makes recombinant Par h 1 available for cross-reactivity studies. Feverfew might become a global player in weed pollen allergy and inclusion of standardized extracts in routine allergy diagnosis is suggested in exposed populations.

Intranasal administration of a combination of choline chloride, vitamin C, and selenium attenuates the allergic effect in a mouse model of airway disease.

Respiratory allergic disease is an inflammatory condition accompanied by oxidative stress. Supplementation of an anti-inflammatory agent with antioxidants may have a therapeutic effect. In this study, the effects of choline chloride in combination with antioxidants were evaluated via the intranasal route in a mouse model of allergic airway disease. Balb/c mice were sensitized on days 0, 7, and 14 and challenged on days 25-30 with cockroach extract (CE) and with a booster challenge on day 38. They were treated with choline chloride (ChCl; 1mg/kg), vitamin C (Vit C; 308.33 mg/kg), and selenium (Se; 1mg/kg) alone or in combination via the intranasal route on days 31, 33, 35, 37, and 39. The mice were sacrificed on day 40 to collect blood, bronchoalveolar lavage fluid, lungs, and spleen. Mice immunized with CE showed a significant increase in airway hyperresponsiveness (AHR), lung inflammation, Th2 cytokines, and the oxidative stress markers intracellular reactive oxygen species and 8-isoprostanes compared to the phosphate-buffered saline control group. A significant decrease was observed in these parameters with all the treatments (p<0.01). The highest decrease was noticed in the ChCl+Vit C+Se-treated group, with AHR decreased to the normal level. This group also showed the highest decrease in airway inflammation (p<0.001), IL-4 and IL-5 (p<0.001), IgE and IgG1 (p<0.001), NF-κB (p<0.001), and 8-isoprostane levels (p<0.001). Glutathione peroxidase activity, which was decreased significantly in CE-immunized mice, was restored to normal levels in this group (p<0.001). IL-10 level was decreased in CE-immunized mice and was restored to normal by combination treatment. The combination treatment induced FOXP3(+) cells in splenocyte culture, responsible for the upregulation of IL-10. In conclusion, the combination of choline chloride, vitamin C, and selenium via the intranasal route reduces AHR, inflammation, and oxidative stress, probably by causing IL-10 production by FOXP3(+) cells, and possesses therapeutic potential against allergic airway disease.

Effect of thermal processing and γ-irradiation on allergenicity of legume proteins.

Legumes are implicated in IgE mediated food allergy in different countries. The present study aimed to investigate the effect of different processing methods on allergenicity of legume proteins. The extracts were processed by boiling, γ-irradiation or by combination of both. The changes in soluble protein content, specific IgE binding and allergenic potential of legume proteins were assessed. Thermal processing resulted in a 3- to 4-fold reduction in soluble protein. Specific IgE binding was reduced 74±6.5%, 83±11.6% and 62±7.2% in the soluble protein of kidney bean, black gram and peanut, respectively, after boiling (p<0.01) whereas there was 34±5.2%, 74±15.6% and 44±11.1% IgE binding reduction in the insoluble protein fraction of respective legumes. Boiling followed by γ-irradiation reduced IgE binding significantly (p<0.05). Biopotency of soluble protein of kidney bean, black gram and peanut was reduced 7-, 3- and 26-folds (p<0.001), respectively, and that of insoluble protein decreased 6-, 4- and 8-folds (p<0.001), respectively, after boiling. Combination treatment was effective in reducing the potency of both soluble and insoluble protein significantly as compared to boiling alone (p<0.001). However, γ-irradiation alone did not bring any change in allergenicity. In conclusion, boiling followed by γ-irradiation is effective in attenuating allergenicity of legume proteins.

Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo).

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients' sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14-16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues 'DDYDNLQL' showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

Choline attenuates immune inflammation and suppresses oxidative stress in patients with asthma.

Asthma is a chronic immune inflammatory disease characterized by variable airflow obstruction and increased bronchial hyperreactivity (BHR). Therapeutic interventions reduce airway inflammation and relieve symptoms but associated with potential side effects that limit their usefulness. The present study was undertaken to assess the effect of choline on immune inflammation and BHR in asthma subjects. The patients of asthma (n=76) were recruited and treated with choline supplement (1500 mg twice) or standard pharmacotherapy for 6 months in two groups. The patients were evaluated by clinical, immunologic and biochemical parameters. The treatment with choline showed significant reduction in symptom/drug score and improvement in PC(20) FEV1 compared to baseline or standard pharmacotherapy (p<0.01). Choline therapy significantly reduced IL-4, IL-5 and TNF-alpha level as compared to baseline or standard pharmacotherapy after 6 months (p<0.01). Blood eosinophil count and total IgE levels were reduced in both the treatment groups. Cysteinyl leukotriene and leukotriene B4 were suppressed significantly by choline treatment (p<0.01). This was accompanied by decreased 8-isoprostanes, a biomarker for oxidative stress after choline treatment (p<0.01). Choline therapy modulates immune inflammation and suppresses oxidative stress in asthma patients. It can be used as an adjunct therapy for asthma patients.

Acute toxicity assessment of choline by inhalation, intraperitoneal and oral routes in Balb/c mice.

Studies suggest that choline has potential to be used as a dietary supplement and a drug for immune inflammatory diseases like asthma and rhinitis. But there are apprehensions regarding adverse effects of choline when given orally in high doses. To address this knowledge gap, toxicity assessment of choline chloride was carried out by intranasal (i.n.), oral and intraperitoneal (i.p.) routes in Balb/c mice for 28days. Body weight, food and water consumption of mice were recorded daily. Hematology and clinical chemistry were assessed to check hepatocellular functions and morphological alterations of the cells. Splenocyte counts were analysed for evaluating cellular immunity. Liver function test was performed by assaying different enzyme systems in serum such as, urea, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Body weight, food and water consumption did not differ between mice treated with choline and the saline control group. Hematologic and biochemical variables were not affected with any increase in serum toxicity marker enzymes indicating normal liver functioning. Choline administration did not affect total cholesterol and high density lipoprotein levels as compared to their respective controls. Urea and blood urea nitrogen levels in choline treated mice were not different than controls. Creatinine level was, however, higher than control in i.p. treatment group, but other parameters were normal. In conclusion, the repeated consumption of choline chloride via i.n. and oral or i.p. routes did not cause toxicity in mice in the toxicological endpoints examined.

Analysis of IgE binding proteins of mesquite (Prosopis juliflora) pollen and cross-reactivity with predominant tree pollens.

Pollen from the mesquite tree, Prosopis juliflora, is an important source of respiratory allergy in tropical countries. Our aim was to partially characterize the IgE binding proteins of P. juliflora pollen extract and study cross-reactivity with prevalent tree pollen allergens. Intradermal tests with P. juliflora and five other tree pollen extracts were performed on respiratory allergy patients from Bikaner (arid) and Delhi (semi arid). Prosopis extract elicited positive skin reactions in 71/220 of the patients. Sera were collected from 38 of these 71 patients and all demonstrated elevated specific IgE to P. juliflora. Immunoblotting with pooled patients' sera demonstrated 16 IgE binding components, with components of 24, 26, 29, 31, 35, 52, 58, 66 and 95 kDa recognized by more than 80% of individual patients' sera. P. juliflora extract is allergenically potent requiring 73 ng of self-protein for 50% inhibition of IgE binding in ELISA inhibition. Cross-inhibition assays showed close relationship among P. juliflora, Ailanthus excelsa, Cassia siamea and Salvadora persica. IgE binding components of 14, 41, 52 and 66 kDa were shared allergens whereas 26 and 29 kDa were specific to P. juliflora. The findings suggest that purification of cross-reactive allergens will be helpful for diagnosis and immunotherapy of tree pollen allergic patients.

Recombinant glutathione-S-transferase a major allergen from Alternaria alternata for clinical use in allergy patients.

Recombinant proteins are used for vaccines, therapy and diagnosis of many diseases. Biological activity of these may differ from native counterpart and needs investigation. The present study aimed to compare recombinant (r) and native (n) glutathione-S-transferase (GST) from Alternaria alternata. Glutathione-S-transferase sequence showed an ORF of 696bp encoding 26-kDa protein with N-terminus conserved domain. Secondary structure of both forms was comparable with melting temperature of 57 and 59 degrees C, respectively. rGST and nGST showed similar enzymatic activity, allergenicity and potency by ELISA inhibition. Histamine release was comparable in 14/17 patients for both the GSTs. rGST and nGST induced proliferation in PBMC at different concentration. Cell supernatant revealed higher IL-4 and IL-5 levels with low levels of IFN-gamma. In summary, recombinant and native GST demonstrated similar physio-chemical, biological and immunological properties and induced comparable cell mediated and humoral response to be used for diagnosis and specific immunotherapy for the fungal allergy cases.

Standardizing Imperata cylindrica--source material for quality allergen preparations.

BACKGROUND: Tropical countries experience wide variations in daytime temperature and relative humidity. This affects the quality of the source material used for allergen extracts. The present study was undertaken to standardize the processing and preservation conditions of Imperata cylindrica grass pollen. METHODS: I. cylindrica (Ic) inflorescence were freeze-dried, pollens sieved out and stored at -70 degrees C (IcA). Alternatively, the inflorescence were dried at room temperature and then at 37 degrees C, pollens sieved out and stored at 4 degrees C (IcB). The extracts prepared in PBS were analyzed in vivo by skin tests and in vitro by immunochemical methods. RESULTS: Reduced SDS-PAGE revealed 37 protein bands in IcA extract and 23 in IcB extract. IgE immunoblot with a pool of sera from Ic hypersensitive patients showed 30 allergenic bands in IcA and 14 in IcB. Immunoblot using anti-Ic rabbit sera revealed 33 antigenic bands in IcA and 22 in IcB. In both blots, the IcA extract exhibited sharp bands and the IcB extract exhibited diffuse bands. ELISA, ELISA inhibition and skin test procedures showed that IcA extracts had a higher potency than IcB extracts. CONCLUSIONS: Extracts prepared from -70 degrees C processed and preserved pollens (IcA) are allergenically more potent and contain a greater number of major and minor allergens than IcB extracts.