Silicon regulates phosphate deficiency through involvement of auxin and nitric oxide in barley roots.

MAIN CONCLUSION: Silicon application mitigates phosphate deficiency in barley through an interplay with auxin and nitric oxide, enhancing growth, photosynthesis, and redox balance, highlighting the potential of silicon as a fertilizer for overcoming nutritional stresses. Silicon (Si) is reported to attenuate nutritional stresses in plants, but studies on the effect of Si application to plants grown under phosphate (Pi) deficiency are still very scarce, especially in barley. Therefore, the present work was undertaken to investigate the potential role of Si in mitigating the adverse impacts of Pi deficiency in barley Hordeum vulgare L. (var. BH902). Further, the involvement of two key regulatory signaling molecules--auxin and nitric oxide (NO)--in Si-induced tolerance against Pi deficiency in barley was tested. Morphological attributes, photosynthetic parameters, oxidative stress markers (O2·-, H2O2, and MDA), antioxidant system (enzymatic--APX, CAT, SOD, GR, DHAR, MDHAR as well as non-enzymatic--AsA and GSH), NO content, and proline metabolism were the key traits that were assessed under different treatments. The P deficiency distinctly declined growth of barley seedlings, which was due to enhancement in oxidative stress leading to inhibition of photosynthesis. These results were also in parallel with an enhancement in antioxidant activity, particularly SOD and CAT, and endogenous proline level and its biosynthetic enzyme (P5CS). The addition of Si exhibited beneficial effects on barley plants grown in Pi-deficient medium as reflected in increased growth, photosynthetic activity, and redox balance through the regulation of antioxidant machinery particularly ascorbate-glutathione cycle. We noticed that auxin and NO were also found to be independently participating in Si-mediated improvement of growth and other parameters in barley roots under Pi deficiency. Data of gene expression analysis for PHOSPHATE TRANSPORTER1 (HvPHT1) indicate that Si helps in increasing Pi uptake as per the need of Pi-deficient barley seedlings, and also auxin and NO both appear to help Si in accomplishing this task probably by inducing lateral root formation. These results are suggestive of possible application of Si as a fertilizer to correct the negative effects of nutritional stresses in plants. Further research at genetic level to understand Si-induced mechanisms for mitigating Pi deficiency can be helpful in the development of new varieties with improved tolerance against Pi deficiency, especially for cultivation in areas with Pi-deficient soils.

Cytokinin and indole-3-acetic acid crosstalk is indispensable for silicon mediated chromium stress tolerance in roots of wheat seedlings.

The rising heavy metal contamination of soils imposes toxic impacts on plants as well as other life forms. One such highly toxic and carcinogenic heavy metal is hexavalent chromium [Cr(VI)] that has been reported to prominently retard the plant growth. The present study investigated the potential of silicon (Si, 10 µM) to alleviate the toxicity of Cr(VI) (25 µM) on roots of wheat (Triticum aestivum L.) seedlings. Application of Si to Cr(VI)-stressed wheat seedlings improved their overall growth parameters. This study also reveals the involvement of two phytohormones, namely auxin and cytokinin and their crosstalk in Si-mediated mitigation of the toxic impacts of Cr(VI) in wheat seedlings. The application of cytokinin alone to wheat seedlings under Cr(VI) stress reduced the intensity of toxic effects of Cr(VI). In combination with Si, cytokinin application to Cr(VI)-stressed wheat seedlings significantly minimized the decrease induced by Cr(VI) in different parameters such as root-shoot length (10.8% and 13%, respectively), root-shoot fresh mass (11.3% and 10.1%, respectively), and total chlorophyll and carotenoids content (13.4% and 6.8%, respectively) with respect to the control. This treatment also maintained the regulation of proline metabolism (proline content, and P5CS and PDH activities), ascorbate-glutathione (AsA-GSH) cycle and nutrient homeostasis. The protective effect of Si and cytokinin against Cr(VI) stress was minimized upon supplementation of an inhibitor of polar auxin transport- 2,3,5-triiodobenzoic acid (TIBA) which suggested a potential involvement of auxin in Si and cytokinin-mediated mitigation of Cr(VI) toxicity. The exogenous addition of a natural auxin - indole-3-acetic acid (IAA) confirmed auxin is an active member of a signaling cascade along with cytokinin that aids in Si-mediated Cr(VI) toxicity alleviation as IAA application reversed the negative impacts of TIBA on wheat roots treated with Cr(VI), cytokinin and Si. The results of this research are also confirmed by the gene expression analysis conducted for nutrient transporters (Lsi1, CCaMK, MHX, SULT1 and ZIP1) and enzymes involved in the AsA-GSH cycle (APX, GR, DHAR and MDHAR). The overall results of this research indicate towards possible induction of a crosstalk between cytokinin and IAA upon Si supplementation which in turn stimulates physiological, biochemical and molecular changes to exhibit protective effects against Cr(VI) stress. Further, the information obtained suggests probable employment of Si, cytokinin and IAA alone or combined in agriculture to maintain plant productivity under Cr(VI) stress and data regarding expression of key genes can be used to develop new crop varieties with enhanced resistance against Cr(VI) stress together with its reduced load in seedlings.

Modulation of ethylene and ROS-scavenging enzymes by multifarious plant growth-promoting endophytes in tomato (Solanum lycopersicum) plants to combat Xanthomonas -induced stress.

The purpose of the current study was to explore root endophytes- Priestia megaterium T3 and Bacillus cereus T4 from Moringa olefiera for the suppression of leaf spot disease in tomato plants challenged with Xanthomonas vesicatoria. Both strains had plant growth-stimulating characteristics including auxin production, solubilization of inorganic phosphate and zinc complexes, and production of ammonia, siderophore, as well as hydrolytic enzymes. An agar well diffusion and fluorescence viability assay have validated the antibacterial effect of the cell-free culture supernatant of strains T3 and T4. Liquid chromatography-mass spectrometry (LC-MS) profiling has identified the secondary metabolites in the cell-free supernatant of strains T3 and T4. The bio-priming of tomato seeds with a consortium of T3 and T4 strains has significantly declined ethylene (by 0.61-fold) and hydrogen peroxide (H2O2, 0.64-fold) concentration thus, maintaining a lower content of ROS-induced malondialdehyde (MDA, 0.91-fold) as compared to control counterparts. Consequently, the leaf spot disease severity was reduced by ∼70% in consortium-treated tomato plants in contrast to their pathogen-challenged control. The consortia (T3+T4) treatment has facilitated induced systemic resistance by enhancing enzymatic activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), catalase (CAT), and ascorbate oxidase (AO) to detoxify the excessive Xanthomonas-induced ROS accumulation in tomato plants. Conclusively, bacterial endophytes modulate X. vesicatoria-induced ROS response and ethylene levels in tomato plants. The current findings indicate that plant growth-promoting endophytic bacterial strains hold the potential to sustainably enhance plant growth and suppress bacterial leaf spot disease in tomato plants.

Zinc induced regulation of PCR1 gene for cadmium stress resistance in rice roots.

In this study, the interaction between zinc (Zn) and cadmium (Cd) was investigated in rice roots to evaluate how Zn can protect the plants from Cd stress. Rice seedlings were treated with Cd (100 µM) and Zn (100 µM) in different combinations (Cd alone, Zn alone, Zn+ Cd, Zn+ Cd+ L-NAME, Zn+ Cd+ L-NAME+ SNP). Rice roots treated with only Zn also displayed similar toxic effects, however when combined with Cd exhibited improved growth. Treating the plant with Zn along with Cd distinctly reduced Cd concentration in roots while increasing its own accumulation due to modulation in expression of Zinc-Regulated Transporter (ZRT)-/IRT-Like Protein (OsZIP1) and Plant Cadmium Resistance1 (OsPCR1). Cd reduced plant biomass, cell viability, pigments, photosynthesis and causing oxidative stress due to inhibition in ascorbate-glutathione cycle. L-NAME (NG-nitro L-arginine methyl ester), prominently suppressed the beneficial impacts of Zn against Cd stress, whereas the presence of a NO donor, sodium nitroprusside (SNP), significantly reversed this effect of L-NAME. Collectively, results point that NO signalling is essential for Zn- mediated cross-tolerance against Cd stress via by modulating uptake of Cd and Zn and expression of OsZIP1 and OsPCR1, and ROS homeostasis due to fine tuning of ascorbate-glutathione cycle which finally lessened oxidative stress in rice roots. The results of this study can be utilized to develop new varieties of rice through genetic modifications which will be of great significance for maintaining crop productivity in Cd-contaminated areas throughout the world.

Assessing the role of ACC deaminase-producing bacteria in alleviating salinity stress and enhancing zinc uptake in plants by altering the root architecture of French bean (Phaseolus vulgaris) plants.

MAIN CONCLUSION: The consortium inoculation with strains R1 and R4 modified the root system to boost seedling growth, increase the zinc content of French bean pods, and reduce salinity stress. The present study demonstrated the effect of two 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing plant growth-promoting rhizobacteria (Pantoea agglomerans R1 and Pseudomonas fragi R4) alone and consortia on the root system development, French bean growth, and zinc content as well as salinity stress tolerance. Both the strains were characterized for ACC utilization activity (426.23 and 380.54 nmol α-ketobutyrate mg protein-1 h-1), indole acetic acid (IAA) production, phosphate solubilization, ammonia, hydrogen cyanide (HCN), and siderophore production. The strains exhibited zinc solubilization in both plate and broth assays with zinc oxide and zinc carbonate as zinc sources as validated by atomic absorption spectroscopy (AAS). Single or combined inoculations with the selected strains significantly modulated the architectural and morphological traits of the root system of French bean plants. Furthermore, the application of R1and R4 consortia has enhanced zinc content in roots (60.83 mg kg-1), shoots (15.41 mg kg-1), and pods (30.04 mg kg-1) of French bean plants grown in ZnCO3 amended soil. In another set of pot experiments, the consortium bacterization has significantly enhanced length as well as fresh and dry biomass of roots and shoots of the French bean plant under saline stress conditions. Additionally, inoculation with ACC-degrading rhizobacterial strains has increased chlorophyll and carotenoid contents, osmoprotectant content, and antioxidative enzyme (catalase and peroxidase) activity in comparison to their counterparts exposed to salt treatments only. Current findings suggested ACC deaminase-producing rhizobacterial strains hold the potential to improve root architecture which in turn promotes plant growth under salt-stressed conditions as well as enhances micronutrient concentration in host plants.

Bacterial community and root endodermis: a complementary relationship.

There are feedforward and feedback loops along the microbiota-root-shoot axis to maintain plant growth or defense under environmental stresses. Here, we highlight a reciprocal interaction between the endodermis and the plant-bacterial community, which stabilizes the diffusion barriers to maintain nutrient homeostasis under nutritional stress.

Miniature mass spectrometer-based point-of-care assay for measuring phosphatidylethanol in blood.

We demonstrate proof-of-concept for point-of-care assessment of long-term alcohol consumption by measuring phosphatidylethanol in blood/dried blood spots with nano-electrospray ionization and MS/MS using a miniature mass spectrometer. 'Abstinence', 'moderate', and 'chronic' consumption could be distinguished rapidly for both sample types, and quantitative performance was obtained with blood (LoQ-100 ng mL-1).

Nitric oxide overcomes copper and copper oxide nanoparticle-induced toxicity in Sorghum vulgare seedlings through regulation of ROS and proline metabolism.

This study aimed to investigate the phytotoxic effect of copper (Cu) and copper nanoparticles (CuONPs) and ameliorative potential of nitric oxide (NO) against these toxic materials in Sorghum vulgare Pers. seedlings. Data suggested that exposure of Cu and CuONPs significantly reduced growth, chlorophyll, carotenoids and protein in root and shoot, which coincided with increased Cu accumulation. However, addition of sodium nitroprusside (SNP, a donor of NO) lowered Cu and CuONPs mediated toxicity through restricting Cu accumulation and improving photosynthetic pigments and total soluble protein contents. Data further suggested that exposure of Cu and CuONPs significantly increased hydrogen peroxide (H2 O2 ), superoxide radicals (O2 •- ), and malondialdehyde (MDA) contents. Enhanced level of oxidative stress severely inhibited the enzymatic activities of glutathione reductase (GR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) but enhanced superoxide dismutase (SOD) and catalase (CAT) activity. However, addition of SNP positively regulated antioxidants enzymes activity, particularly the enzymes involved in the ascorbate-glutathione cycle to overcome Cu- and CuONPs-induced stress in Sorghum seedlings. Further, Cu and CuONPs enhanced accumulation of free proline through inducing Δ1 -pyrroline-5-carboxylate synthetase (P5CS) activity while lowering the proline dehydrogenase (PDH) activity. However, addition of SNP reversed these responses. Therefore, overall results revealed that SNP has enough potential of reducing the toxicity of Cu and CuONPs in Sorghum seedlings through regulation of proline metabolism and activity of enzymes of the ascorbate-glutathione cycle. These findings can be employed in developing new resistant varieties of Sorghum having enhanced tolerance against Cu or CuONP stress and improved productivity.

Ethylene Renders Silver Nanoparticles Stress Tolerance in Rice Seedlings by Regulating Endogenous Nitric Oxide Accumulation.

Developments in the field of nanotechnology over the past few years have increased the prevalence of silver nanoparticles (AgNPs) in the environment, resulting in increased exposure of plants to AgNPs. Recently, various studies have reported the effect of AgNPs on plant growth at different concentrations. However, identifying the mechanisms and signaling molecules involved in plant responses against AgNPs stress is crucial to find an effective way to deal with the phytotoxic impacts of AgNPs on plant growth and development. Therefore, this study was envisaged to investigate the participation of ethylene in mediating the activation of AgNPs stress tolerance in rice (Oryza sativa L.) through a switch that regulates endogenous nitric oxide (NO) accumulation. Treatment of AgNPs alone hampered the growth of rice seedlings due to severe oxidative stress as a result of decline in sulfur assimilation, glutathione (GSH) biosynthesis and alteration in the redox status of GSH. These results are also accompanied by the higher endogenous NO level. However, addition of ethephon (a donor of ethylene) reversed the AgNP-induced effects. Though the application of silicon nanoparticles (SiNPs) alone promoted the growth of rice seedlings but, interestingly their application in combination with AgNPs enhanced the AgNP-induced toxicity in the seedlings through the same routes as exhibited in the case of AgNPs alone treatment. Interestingly, addition of ethephon reversed the negative effects of SiNPs under AgNPs stress. These results suggest that ethylene might act as a switch to regulate the level of endogenous NO, which in turn could be associated with AgNPs stress tolerance in rice. Furthermore, the results also indicated that addition of l-NG-nitro arginine methyl ester (l-NAME) (an inhibitor of endogenous NO synthesis) also reversed the toxic effects of SiNPs together with AgNPs, further suggesting that the low level of endogenous NO was associated with AgNPs stress tolerance. Overall, the results indicate that the low level of endogenous NO triggers AgNPs stress tolerance, while high level leads to AgNPs toxicity by regulating sulfur assimilation, GSH biosynthesis, redox status of GSH and oxidative stress markers. The results revealed that ethylene might act as a switch for regulating AgNPs stress in rice seedlings by controlling endogenous NO accumulation.

Silicon nanoforms in crop improvement and stress management.

Although, silicon - the second most abundant element in the earth crust could not supersede carbon (C) in the competition of being the building block of life during evolution, yet its presence has been reported in some life forms. In case of the plants, silicon has been reported widely to promote the plant growth under normal as well as stressful situations. Nanoform of silicon is now being explored for its potential to improve plant productivity and its tolerance against various stresses. Silicon nanoparticles (SiNPs) in the form of nanofertilizers, nanoherbicides, nanopesticides, nanosensors and targeted delivery systems, find great utilization in the field of agriculture. However, the mechanisms underlying their uptake by plants need to be deciphered in detail. Silicon nanoformss are reported to enhance plant growth, majorly by improving photosynthesis rate, elevating nutrient uptake and mitigating reactive oxygen species (ROS)-induced oxidative stress. Various studies have reported their ability to provide tolerance against a range of stresses by upregulating plant defense responses. Moreover, they are proclaimed not to have any detrimental impacts on environment yet. This review includes the up-to-date information in context of the eminent role of silicon nanoforms in crop improvement and stress management, supplemented with suggestions for future research in this field.

Decoding the Plant Growth Promotion and Antagonistic Potential of Bacterial Endophytes From Ocimum sanctum Linn. Against Root Rot Pathogen Fusarium oxysporum in Pisum sativum.

The present study demonstrates plant growth promotion and induction of systemic resistance in pea (Pisum sativum) plant against Fusarium oxysporum f.sp. pisi by two bacterial endophytes, Pseudomonas aeruginosa OS_12 and Aneurinibacillus aneurinilyticus OS_25 isolated from leaves of Ocimum sanctum Linn. The endophytes were evaluated for their antagonistic potential against three phytopathogens Rhizoctonia solani, F. oxysporum f. sp. pisi, and Pythium aphanidermatum by dual culture assay. Maximum inhibition of F. oxysporum f. sp. pisi was observed by strains OS_12 and OS_25 among all root rot pathogens. Scanning electron microscopy of dual culture indicated hyphal distortion and destruction in the case of F. oxysporum f. sp. pisi. Further, volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry (GC-MS). The GC-MS detected eight bioactive compounds from hexane extracts for instance, Dodecanoic acid, Tetra decanoic acid, L-ascorbic acid, Trans-13-Octadecanoic acid, Octadecanoic acid. Both the endophytes exhibited multifarious plant growth promoting traits such as indole acetic production (30-33 µg IAA ml-1), phosphate solubilization, and siderophore and ammonia production. Pot trials were conducted to assess the efficacy of endophytes in field conditions. A significant reduction in disease mortality rate and enhancement of growth parameters was observed in pea plants treated with consortium of endophytes OS_12 and OS_25 challenged with F. oxysporum f.sp. pisi infection. The endophytic strains elicited induced systemic resistance (ISR) in pathogen challenged pea plants by enhancing activities of Phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), ascorbate oxidase (AO), catalase (CAT) and total phenolic content. The endophytes reduced the oxidative stress as revealed by decrease in malondialdehyde (MDA) content and subsequently, lipid peroxidation in host plant leaves. Robust root colonization of pea seedlings by endophytes was observed by scanning electron microscopy (SEM) and fluorescence microscopy. Thus, plant growth promoting endophytic P. aeruginosa and A. aneurinilyticus can be further exploited through bio-formulations for sustainable protection of crops against root rot diseases as bio-control agents.

Miniature mass spectrometer-based point-of-care assay for cabotegravir and rilpivirine in whole blood.

HIV prevention and treatment with injectable cabotegravir and/or rilpivirine administered once every 4 to 8 weeks is an attractive alternative to daily therapy. Prescribed dosage and drug concentrations in plasma are based on patient data collected in clinical trials, but actual patients are expected to exhibit more variability in drug concentrations, which is important to quantify. Here, we demonstrate the first quantitative point-of-care assay with a miniature mass spectrometer to assess these drug concentrations in whole blood. Quantitative performance is obtained using paper spray ionization in combination with tandem mass spectrometry (MS/MS) in the clinically relevant concentration range of both drugs. Limits of quantitation (LoQs) of cabotegravir and rilpivirine are measured to be 750 ng/mL and 20 ng/mL, respectively. The assay turnaround time is < 4 min, and strong linear relationships are established between MS/MS responses and concentration, with percentage of relative standard deviations (RSDs) that are <15% at concentrations above the LoQs. The speed, portability, low power consumption, and specificity offered by the miniature instrument should make it an appropriate platform for measuring drug concentrations in a walk-in clinic using small volumes of patient blood.

Evaluation of Pseudomonas sp. for its multifarious plant growth promoting potential and its ability to alleviate biotic and abiotic stress in tomato (Solanum lycopersicum) plants.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is one of the most beneficial traits of plant growth promoting (PGP) rhizobacteria responsible for protecting the plants from detrimental effects of abiotic and biotic stress. The strain S3 with ACC deaminase activity (724.56 nmol α-ketobutyrate mg-1 protein hr-1) was isolated from rhizospheric soil of turmeric (Curcuma longa), a medicinal plant, growing in Motihari district of Indian state, Bihar. The halotolerant strain S3, exhibited optimum growth at 8% (w/v) NaCl. It also exhibited multiple PGP traits such as indole acetic acid production (37.71 µg mL-1), phosphate solubilization (69.68 mg L-1), siderophore, hydrocyanic acid (HCN) and ammonia production as well as revealed antagonism against Rhizoctonia solani. The potential of isolated strain to alleviate salinity stress in tomato plants was investigated through pots trials by inoculating strain S3 through-seed bacterization, soil drenching, root dipping as well as seed treatment + soil drenching. The strain S3 inoculated through seed treatment and soil drenching method led to improved morphological attributes (root/shoot length, root/shoot fresh weight and root/shoot dry weight), photosynthetic pigment content, increased accumulation of osmolytes (proline and total soluble sugar), enhanced activities of antioxidants (Catalase and Peroxidase) and phenolic content in salt stressed tomato plants. The biochemical characterisation, FAMEs analysis and 16S rRNA gene sequencing revealed that strain S3 belongs to the genus Pseudomonas. The overall findings of the study revealed that Pseudomonas sp. strain S3 can be explored as an effective plant growth promoter which stimulate growth and improve resilience in tomato plants under saline condition.

Diversity analysis of ACC deaminase producing bacteria associated with rhizosphere of coconut tree (Cocos nucifera L.) grown in Lakshadweep islands of India and their ability to promote plant growth under saline conditions.

ACC deaminase producing Plant growth promoting rhizobacteria (PGPR) offers a great promise for ameliorating the negative impacts of salinity stress manifested on plants. In this context, 28 rhizospheric bacteria associated with ACC deaminase potential (198-1069 nmol α-ketobutyrate mg protein-1 h-1) were isolated from 5 different islands of Lakshadweep, union territory, India- Agatti, Kavaratti, Bangaram, Kadmat, and Thinnakara islands using DF-minimal medium. The diversity of cultivable ACC deaminase producing bacteria was analysed by PCR-RFLP (Restriction Fragment Length Polymorphism) method using three endonucleases AluI, MspI and HaeIII which led to the grouping of these isolates into six clusters at 80 % similarity index. Subsequently, isolates were functionally characterized for various PGP traits such that indole-3-acetic acid (IAA) production (∼10-80 µg mL-1); 16 isolates had phosphate solubilizing potential ranging from ∼19 to 88 P mg L-1 ; siderophore and ammonia production abilities were observed in 5 and 24 isolates, respectively while two strains tolerated up to 8% NaCl. Phylogenetic analysis of 16S rRNA gene sequences of representative strain from each cluster revealed that twenty-eight ACC deaminase producing PGPR belong to eight distinct genera: Pseudomonas, Bacillus, Azospirillum, Azotobacter, Escherichia, Paenibacillus, Burkholderia, and Klebsiella. Two isolates, CO1 (Pseudomonas putida) and CO8 (Bacillus paramycoides) were evaluated for plant growth promoting effects on French bean (Phaseolus vulgaris) under salinity (100 mM NaCl) stress. Both the selected isolates in consortium form significantly increased the root length, shoot length, root fresh and dry weight, shoot fresh and dry weight of French bean seedlings exposed to salinity stress, compared to non-inoculated control plants. The co-inoculation with selected strains CO1 and CO8 has significantly improved chlorophyll concentration, relative water content, membrane stability index, gas exchange parameters including net photosynthesis rate (PN), stomatal conductance (gs), transpiration rate (E) and water use efficiency of French bean plants by ∼100 %, ∼85 %, ∼40 %, ∼198 %, ∼80 %, ∼70 % and ∼75 %, respectively under saline conditions in comparison with non-inoculated plants. Moreover, the consortium treated French bean plants showed lower levels of stress-induced ethylene by 38 %, electrolyte leakage and Malondialdehyde (MDA) content by ∼15 % under salt stress compared to non-inoculated ones. This study unveiled the potential of halotolerant strains, Pseudomonas putida and Bacillus paramycoides as French bean biofertilizers in mitigating the adverse effects of salinity in plant growth in sustainable agriculture.

Correction to: Direct quantitation of tenofovir diphosphate in human blood with mass spectrometry for adherence monitoring.

Unfortunately, after online publication, a formatting mistake was found in Table 1 (Chemical structures and MRM transitions used for quantitation). The original article has been corrected.

Direct quantitation of tenofovir diphosphate in human blood with mass spectrometry for adherence monitoring.

Inadequate adherence to chronic medications is a far-reaching problem with financial and human health consequences. By a wide margin, non-adherence is the leading cause of therapeutic failures of HIV pre-exposure chemoprophylaxis (PrEP) and antiretroviral therapy (ART). It has been proven that HIV infection can be prevented by daily dosing of emtricitabine and tenofovir disoproxil fumarate. Measurement of intracellular tenofovir diphosphate in red blood cells has been established as an effective way to assess cumulative adherence, however, the LC-MS-based analytical method developed for the purpose is both complicated and expensive. Here, we report a simple method for adherence monitoring based on direct MS quantification of intracellular tenofovir diphosphate in human whole blood. The method requires only microliters of whole blood, employs special membranes to perform plasma separation and concomitant desalting during blood collection, and uses nanoelectrospray on a triple quadrupole instrument. Quantitative performance in this proof-of-concept study includes RSDs of < 15% and successful analysis of a small number of patient samples with medium to high adherence levels. The results correlate with those of a validated LC-MS/MS method, and an R2 value of 0.9962 is achieved. This methodology has promise for extension to point-of-care testing using miniature mass spectrometers. Graphical abstract.

ACC Deaminase Producing Bacteria With Multifarious Plant Growth Promoting Traits Alleviates Salinity Stress in French Bean (Phaseolus vulgaris) Plants.

Plant growth promoting rhizobacteria (PGPR) with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity has the potential to promote plant growth and development under adverse environmental conditions. In the present study, rhizobacterial strains were isolated from Garlic (Allium sativum) rhizosphere and were screened in vitro ACC deaminase activity in DF salt minimal media supplemented with 3 mM ACC. Out of six isolates, two could degrade ACC into α-ketobutyrate, exhibiting ACC deaminase activity producing more than ∼1500 nmol of α-ketobutyrate mg protein-1 h-1, and assessed for other plant growth promoting (PGP) functions including indole acetic acid production (greater than ∼30 µg/ml), siderophore, Ammonia, Hydrogen cyanide production and inorganic Ca3(PO4)2 (∼85 mg/L) and ZnSO4 solubilization. Besides facilitating multifarious PGP activities, these two isolates augmented in vitro stress tolerance in response to 6% w/v NaCl salt stress and drought stress (-0.73 Mpa). The strains ACC02 and ACC06 were identified Aneurinibacillus aneurinilyticus and Paenibacillus sp., respectively on the basis of 16S rDNA gene sequence analysis and were evaluated for growth promoting potential in French bean seedlings under non-saline and salinity stress conditions through pot experiments. The seed bacterization by ACC02 and ACC06 revealed that treatment of plants with bacterial isolates in the form of consortia significantly declined (∼60%) stress stimulated ethylene levels and its associated growth inhibition by virtue of their ACC deaminase activity. The consortia treatment alleviated the negative effects of salinity stress and increased root length (110%), root fresh weight (∼45%), shoot length (60%), shoot fresh weight (255%), root biomass (220%), shoot biomass (425%), and total chlorophyll content (∼57%) of French bean seedlings subjected to salinity stress.

Unravelling the potential of microbes isolated from rhizospheric soil of chickpea (Cicer arietinum) as plant growth promoter.

In the present study, the Cicer arietinum (chickpea) rhizosphere bacterial strains Azotobacter chroococcum (AU-1), Bacillus subtilis (AU-2), Pseudomonas aeruginosa (AU-3) and Bacillus pumilis (AU-4) were isolated and characterized for plant growth-promoting traits with an aim of developing bio-fertilizing agent to improve growth and yield of chickpea plants under normal conditions. The ACC degrading potential of strains AU-1, AU-2, AU-3, and AU-4 was in the range of 600-1700 nmol α-ketobutyrate per mg of cellular protein per hour, respectively. These four rhizobacteria exhibited Indole acetic acid production approximately between 20 and 35.34 µg/ml. The phosphate solubilization potential was in the range of 78-87.64 mg Soluble P/L with maximum solubilization displayed by strains P. aeruginosa and B. pumilis. All the growth-promoting isolates displayed Fe-chelating siderophore and ammonia production while no isolate was able to produce hydrocyanic acid. Besides evaluating the presence of multifaceted in vitro plant growth-promoting traits, these four rhizobacterial isolates were halotolerant as well as water stress (drought) tolerant of up to - 1.2 Mpa of PEG 6000. The optimum pH and temperature for their growth were found to be pH 7 and 30 °C temperature. Under normal conditions, inoculation with formulated bacterial consortia significantly improved the (P ≤ 0.05) germination index, plant height, leaf area index, stem diameter, and chlorophyll content by ~ 50%, 100%, 63%, 185%, and 63%, respectively, as compared to uninoculated chickpea plants. The consortia of halotolerant and drought tolerant bacterial strains were shown to exert a positive impact on the growth of chickpea plants under normal conditions.

Azithromycin resistance mechanisms in typhoidal salmonellae in India: A 25 years analysis.

Background & objectives: : Azithromycin has been in use as an alternate treatment option for enteric fever even when the guidelines on the susceptibility testing were not available. There is lack of data on susceptibility and mechanisms of resistance of azithromycin in Salmonella Typhi and S. Paratyphi A. The aim of the present study was to determine the azithromycin susceptibility and resistance mechanisms in typhoidal salmonellae isolates archived in a tertiary care centre in north India for a period of 25 years. Methods: : Azithromycin susceptibility was determined in 602 isolates of S. Typhi (469) and S. Paratyphi A (133) available as archived collection isolated during 1993 to 2016, by disc diffusion and E-test method.PCR was done for ereA, ermA, ermB, ermC, mefA, mphA and msrA genes from plasmid and genomic DNA and sequencing was done to detect mutations in acrR, rplD and rplV genes. Results: : Azithromycin susceptibility was seen in 437/469 [93.2%; 95% confidence interval (CI), 90.5 to 95.1%] isolates of S. Typhi. Amongst 133 isolates of S. Paratyphi A studied, minimum inhibitory concentration (MIC) of ≤16 mg/l was found in 102 (76.7%; 95% CI, 68.8 to 83.0). MIC value ranged between 1.5 and 32 mg/l with an increasing trend in MIC50and MIC90with time. Mutations were found in acrR in one and rplV in two isolates of S. Typhi. No acquired mechanism for macrolide resistance was found. Interpretation & conclusions: : Azithromycin could be considered as a promising agent against typhoid fever on the basis of MIC distribution in India. However, due to emergence of resistance in some parts, there is a need for continuous surveillance of antimicrobial susceptibility and resistance mechanisms. There is also a need to determine the breakpoints for S. Paratyphi A.

Changing trends of culture-positive typhoid fever and antimicrobial susceptibility in a tertiary care North Indian Hospital over the last decade.

PURPOSE: The present study was undertaken to analyse the trend in prevalence of culture-positive typhoid fever during the last decade and to determine antimicrobial susceptibility profile of Salmonella Typhi and Salmonella Paratyphi A isolated from patients of enteric fever presenting to our hospital. METHODS: All the culture-positive enteric fever cases during 2005-2016 presenting to our Hospital were included in the study. Antimicrobial susceptibility was done against chloramphenicol, amoxicillin, co-trimoxazole, ciprofloxacin, ofloxacin, levofloxacin, pefloxacin, ceftriaxone and azithromycin as per corresponding CLSI guidelines for each year. We also analysed the proportion of culture positivity during 1993-2016 in light of the antibiotic consumption data from published literature. RESULTS: A total of 1066 strains-S. Typhi (772) and S. Paratyphi A (294) were isolated from the blood cultures during the study. A maximum number of cases were found in July-September. Antimicrobial susceptibility for chloramphenicol, amoxicillin and co-trimoxazole was found to be 87.9%, 75.5%, 87.3% for S. Typhi and 94.2%, 90.1% and 94.2% for S. Paratyphi A, respectively. Ciprofloxacin, ofloxacin and levofloxacin susceptibility were 71.3%, 70.8% and 70.9% for S. Typhi and 58.1%, 57.4% and 57.1% for S. Paratyphi A, respectively. Azithromycin susceptibility was 98.9% in S. Typhi. Although susceptibility to ceftriaxone and cefixime was 100% in our isolates, there is a continuous increase in ceftriaxone minimum inhibitory concentration (MIC)50and MIC90values over the time. The proportion of blood culture-positive cases during 1993-2016 ranged from a minimum of 0.0006 in 2014 to a maximum of 0.0087 in 1999. CONCLUSION: We found that the most common etiological agent of enteric fever is S. Typhi causing the majority of cases from July to October in our region. MIC to ceftriaxone in typhoidal salmonellae is creeping towards resistance and more data are needed to understand the azithromycin susceptibility.