Prevailing practice patterns in keratoconus among Indian ophthalmologists.

Objective: The past few years have seen a rapid advancement in the management of keratoconus (KC). However, there is no prescribed standard of care for the management of KC. This study evaluated the prevailing practice patterns among Indian ophthalmologists in the diagnosis and treatment of KC via an online survey. Methods: This was a survey-based cross-sectional study in which a questionnaire (Supplement 1) was created. Questions pertaining to the practicing experience, setting of practice, and training background were asked in addition to the investigations done and decision making in KC management. Responses were collected via Survey Monkey (Survey Monkey, Palo Alto, California, USA) and statistical analysis performed using R software (4.1.3). Results: The survey was answered by 273 ophthalmologists. Pentacam was the most used topographer (195 users), followed by Orbscan (41 users), Sirius (34 users), and Galilei (3 users). The lowest limit of pachymetry for performing collagen crosslinking (CXL) was 400µ for most practitioners. More than half the respondents (50.55%) did not perform photorefractive keratectomy (PRK) or intracorneal ring segment (ICRS) implantation in a suitable patient. Accelerated 10-minute protocol (9 mW/cm2 for 10 minutes) was the most commonly (54.21%) used for CXL, followed by Dresden protocol (3 mW/cm2 for 30 minutes) (36.63%). When a patient was unsuitable for CXL, 55.31% surgeons advise contact lens (CL) trial, 35.16% surgeons advise keratoplasty, 26.74% surgeons perform stromal augmentation, and 7.69% surgeons advise spectacle correction. Corneal scar was the most common indication (49.45%) for performing keratoplasty. Conclusion: Topography remains the most used diagnostic modality for initial diagnosis. Optical coherence tomography and epithelial mapping are increasingly being used for early diagnosis of KC. Not all ophthalmologists were comfortable performing ICRS or PRK. When patients are unsuitable for CXL, CL trial remains the most frequently advised option followed by keratoplasty.

Functional interplay between antagonistic bacteria and Rhizoctonia solani in the tomato plant rhizosphere.

Microbial interactions with plant roots play an imperial role in tomato plant growth and defense against the Rhizoctonia solani. This study performed a field experiment with two antagonistic bacteria (Pseudomonas and Bacillus) inoculated in healthy and Rhizoctonia solani treated soil in tomato rhizosphere to understand the metabolic pattern and microbial function during plant disease suppression. In the present study, we assessed soil and microbial enzymes, bacterial and fungal cell forming unit (CFU), and carbon utilization profiling through Bio-Eco plates of rhizoplane samples. Antagonist bacteria and pathogen interaction significantly (p < 0.05) influenced the bacterial count, soil enzymes (chitinase and glucanase), and bacterial function (siderophore and chitinase production). These results indicated that these variables had an imperial role in disease suppression during plant development. Furthermore, the metabolic profiling showed that carbon source utilization enhanced under fruit development and ripening stages. These results suggested that carbon sources were essential in plant/pathogen/antagonist interaction. Substrates like ß-methyl-D-glucoside, D-mannitol, D-galacturonic acid, N-acetyl-D-glucosamine, and phenylethylamine strongly connect with the suppuration of root rot disease. These carbon sources may help to propagate a healthy microbial community to reduce the pathogen invasion in the plant root system, and these carbon sources can be stimulators of antagonists against pathogens in the future.

Cadmium-mediated morphological, biochemical and physiological tuning in three different Anabaena species.

Cyanobacteria are a natural inhabitant of paddy field and enhance the crop productivity in an eco-friendly manner. Cadmium (Cd) is a perilous trace metal element which not only limits the crop productivity but also inhibits the growth and nitrogen-fixing ability of these diazotrophs as well as the biodiversity of rice field semiaquatic agroecosystems. However, the impact of Cd toxicity in diazotrophic cyanobacteria is yet not adequately addressed. Therefore, in the present study, three diazotrophic cyanobacterial species, i.e., Anabaena sp. PCC7120, Anabaena L31, and Anabaena doliolum were subjected to their LC50 doses of Cd, and their physiological (PSII, Psi, respiration, energy status and nitrogen fixation rate), biochemical variables (such as antioxidant contents and antioxidant enzymes) together with morphological parameters were evaluated. The results of physiological variables suggested that the Cd exposure adversely affects the photosynthesis, respiration, and biological nitrogen fixation ability across three Anabaena species. The results of biochemical variables in terms of accumulation of antioxidants (glutathione, thiol, phytochelatin and proline) content as well as antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), catalase-peroxidase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) revealed that their inter-species stress tolerance behavior may be attributed to the differential accumulation of antioxidants as well as differential antioxidant enzyme activity in three species. Furthermore, the enhanced antioxidant enzymes activity such as GST, GR, CAT, and SOD in Anabaena L31 advocated significantly higher as compared to Anabaena PCC7120 and Anabaena doliolum. In conclusion, Cd-toxicity assessment regarding physiological, biochemical and morphological aspects across three species identified Anabaena L31 as Cd-resistant species than the other two tested species, i.e., Anabaena PCC7120 and Anabaena doliolum.

Decoding the role of hypothetical protein All3255 of Anabaena PCC7120 in heavy metal stress management in Escherichia coli.

Cadmium is a non-essential toxic heavy metal for organisms, including plants and cyanobacteria. Cadmium resistance transporters involved in resistance of cells against various toxicants such as drugs and effluxes cytotoxic compounds from cells. However, cadmium resistance-associated protein (CadD) has never been reported from a diazotrophic cyanobacterium Anabaena sp. To test whether the hypothetical protein All3255 of Anabaena sp. PCC7120 a homolog of cadmium resistance-associated protein (CadD) involved in cadmium or heavy metal resistance or not, cloning and heterologous expression analysis of all3255 performed in Escherichia coli BL21 (DE3). Our results revealed that the strain transformed with pGEX-5X-2 + all3255 showed resistant towards not only to cadmium but also other heavy metals such as nickel, copper, zinc, lead and cobalt in addition to arsenic than those of transformed with empty vector (pGEX-5X-2). Furthermore, the results of metal accumulation analysis of these cells unveil a lower accumulation of tested heavy metals in all3255-overexpressing E. coli cells than those transformed with empty vector. This study strongly supports the role of All3255 of Anabaena sp. PCC7120 as a CadD efflux pump of heavy metals in E.coli.

In silico characterization and transcriptomic analysis of nif family genes from Anabaena sp. PCC7120.

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.

Alr2954 of Anabaena sp. PCC 7120 with ADP-ribose pyrophosphatase activity bestows abiotic stress tolerance in Escherichia coli.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 µmol min-1 mg protein-1, and K cat/K m 9.438 × 104 µM-1 min-1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

Overexpression of AhpC enhances stress tolerance and N2-fixation in Anabaena by upregulating stress responsive genes.

The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40-60% compared to a 33-40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena.

In silico and wet-lab study revealed cadmium is the potent inhibitor of HupL in Anabaena sp. PC C 7120.

The hupL of Anabaena sp. PCC 7120 encodes the large subunit of uptake hydrogenase found in all diazotrophic cyanobacteria and boosts up the nitrogen-fixing potential by catalyzing the removal of the molecular hydrogen produced as a by-product of dinitrogen fixation. Bioinformatics analysis revealed that HupL from Anabaena sp. PCC7120 is a 60.2 kDa, thermostable, glycine-rich protein having highest structural similarity with NiFeSe hydrogenase of Desulfomicrobium baculatumis. Toxicity of selected abiotic stresses like arsenic, cadmium, copper, and salt with HupL was further reconciled by wet-lab approaches like qRT-PCR, hydrogenase and nitrogenase activity assay as hydrogenases unintendedly affect the nitrogenase activity in Anabaena. Down-regulated transcript along with highly inhibited hydrogenase and nitrogenase activities under cadmium stress revealed that cadmium is a potent inhibitor of hydrogenases in Anabaena which indirectly affects its nitrogen-fixing capabilities

Role of initial cell density of algal bioassay of toxic chemicals.

A variety of toxicants such as, metal ions, pesticides, dyes, etc. are continuously being introduced anthropogenically in the environment and adversely affect to the biotic component of the ecosystem. Therefore, the assessment of negative effects of these toxicants is required. However, toxicity assessment anticipated by chemical analysis are extremely poor, therefore the application of the living systems for the same is an excellent approach. Concentration of toxicant as well as cell density both influenced the result of the algal toxicity assay. Here, Scenedesmus sp, a very fast growing green microalgae was selected for study the effects of initial cell densities on the toxicity of Cu(II), Cd(II), Zn(II), paraquat and 2,4-D. Results demonstrated concentration dependent decrease in biomass and specific growth rate of Scenedesmus sp. on exposure of abovesaid toxicants. Paraquat and 2,4-D emerged as extremely toxic to the test alga which reflected from the lowest EC value and very steep decline in biomass was evident with increasing concentration of paraquat and 2,4-D in the medium. Result also demonstrated that initial cell density is a very important parameter than specific growth rate for algal bioassay of various toxicants. Present study clearly illustrated that the use of smaller cell density is always recommended for assaying toxicity of chemicals in algal assays.

UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species.

Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

A novel alkyl hydroperoxidase (AhpD) of Anabaena PCC7120 confers abiotic stress tolerance in Escherichia coli.

In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

Enhanced Photosynthesis and Carbon Metabolism Favor Arsenic Tolerance in Artemisia annua, a Medicinal Plant as Revealed by Homology-Based Proteomics.

This paper provides the first proteomic evidence of arsenic (As) tolerance and interactive regulatory network between primary and secondary metabolism in the medicinal plant, Artemisia annua. While chlorophyll fluorescence and photosynthetic rate depicted mild inhibition, there was a significant enhancement in PSI activity, whole chain, ATP, and NADPH contents in 100 µ M As treatments compared to the control plants. However, a decrease in the above variables was recorded under 150 µ M treatments. Proteomic decoding of the survival strategy of A. annua under As stress using 2-DE followed by MALDI-MS/MS revealed a total of 46 differentially expressed protein spots. In contrast to other plants where As inhibits photosynthesis, A. annua showed appreciable photosynthetic CO2 assimilation and allocation of carbon resources at 100 µ M As concentration. While an increased accumulation of ATP synthase, ferredoxin-NADP(H) oxidoreductase, and FeS-rieske proteins supported the operation of cyclic electron transport, mdr ABC transporter protein and pcs gene might be involved in As detoxification. The most interesting observation was an increased accumulation of LEAFY like novel protein conceivably responsible for an early onset of flowering in A. annua under As stress. This study not only affirmed the role of energy metabolism proteins but also identified potential candidates responsible for As tolerance in plants.

Comparative proteomics unveils cross species variations in Anabaena under salt stress.

The present study compares protein diversity within three Anabaena species (Anabaena doliolum, Anabaena sp.PCC 7120 and Anabaena L31). 2-DE based analysis of 256 protein spots in control and 1, 3, 5, and 7days of salt treatment resulted into 96 proteins arching across fourteen functional categories were assigned to biochemical pathways using KOBAS 2.0. While 52.34% of the evaluated protein spots were common across three species, the remaining 47.66% fraction mainly comprised of the hypothetical and unknown proteins. PSORTb, CDD, Motifscan and Pfam revealed function and subcellular localization for 27 of the 31 hypothetical and unknown proteins. The differences in high salt tolerance (LC50) of A. doliolum over A. L31 was reflected by (i) many fold accumulation (as spot volumes) of Alr3090, Alr0803, peptidyl prolyl cis-trans isomerase and modulator of DNA gyrase proteins, and (ii) a better photosynthesis and energy homeostasis as indicated through photosystem activity, respiration, ATP and NADPH contents. Some common noteworthy salt effects include (i) photosystem damage, (ii) DNA damage repair, (iii) upregulated protein synthesis, (iv) enhanced sulphur metabolism, and (v) upregulated pentose phosphate pathway. 34 of the identified protein spots are novel entries to the Anabaena salt proteome. This study reveals the existence of separate strategies even within species to combat stress. BIOLOGICAL SIGNIFICANCE: This study for the first time enumerates protein diversity in three Anabaena species employing their presence/absence and relative abundance. Proteomics integrated with physiology and bioinformatics deciphers differential salt tolerance among the studied species and is the first of its kind to predict the function of hypothetical and unknown proteins. Salt-induced proteomic alterations clearly demonstrate significant metabolic shifts and existence of separate molecular phenome among the species investigated. This may be responsible for niche specificity limiting their application as biofertilizer. Of the 96 identified proteins, a large chunk are new entries to the Anabaena salt proteome while some protein genes may be used as potential candidates for engineering salt tolerant cyanobacteria.

Salt and UV-B induced changes in Anabaena PCC 7120: physiological, proteomic and bioinformatic perspectives.

This study examines response of Anabaena sp. PCC 7120 to salt and UV-B stress by combining physiological, biochemical, proteomics and bioinformatics approaches. Sixty five significantly altered protein spots corresponding to 51 protein genes identified using MALDI-TOF MS/MS were divided into nine functional categories. Based on relative abundance, these proteins were grouped into four major sets. Of these, 27 and 5 proteins were up- and downregulated, respectively, both under salt and UV-B while 8 and 11 proteins showed accumulation in salt and UV-B applied singly. Some responses common to salt and UV-B included (i) enhanced expression of FeSOD, alr3090 and accumulation of MDA indicating oxidative stress, (ii) accumulation of PDH, G6P isomerase, FBPaldolase, TK, GAPDH and PGK suggesting enhanced glycolysis, (iii) upregulation of 6-PGD, 6PGL and NADPH levels signifying operation of pentose phosphate pathway, (iv) upregulation of Dps, NDK and alr3199 indicating DNA damage, and (v) accumulation of proteins of ribosome assembly, transcriptional and translational processing. In contrast, enhanced expression of RUBISCO, increased glycolate oxidase activity and ammonium content under salt signify the difference. Salt was found to be more damaging than UV-B probably due to a cumulative effect of ionic, osmotic and oxidative damage. A group of proteins having common expression represent decreased toxicity of salt and UV-B when applied in combination.

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses.

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl(2), H(2)O(2), UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl(2) demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.