Colonization potential of endophytes from halophytic plants growing in the "Runn of Kutch" salt marshes and their contribution to mitigating salt stress in tomato cultivation.

Increasing soil salinity depreciates the quantity of the crop produce. Looking at the tremendous potential of plant-associated microorganisms in salinity stress mitigation, it would be very useful in exploring and deciphering salt-tolerant microorganisms from halophytic plants and their utilization in cultivated plants. With this aim, in the present study, four halophytic plants were taken from Rann of Kutch, and bacterial endophytes were isolated from different plant organs. These endophytes were characterized by plant growth and health promotion features. The molecular identification was done based on 16 s rRNA sequence similarity. It was found that the endophytic bacteria isolated from 4 different halophytes found sharing phylogenetic relatedness. Four potential endophytes Alkalihalobacillus gibsonii 2H2, Achromobacter insuavis 2H18, Terribacillus halophilus 2H20, and Bacillus siamensis 4H1 were tested in tomato for salinity stress alleviation. Changes in the levels of antioxidants were analyzed. Total chlorophyll, total phenolics, malondialdehyde, and proline content indicated reduced damage in the plant system due to salinity by the application of endophytes. All the treatments exhibited low levels of electrolyte leakage. The accumulation of enzymatic reactive oxygen species scavengers was assessed from the levels of peroxidase, catalase, superoxide dismutase, phenylalanine ammonia-lyase, ascorbate peroxidase, and guiacol peroxidase. The NBT and DAB staining confirmed the findings. The reduction in the accumulation of Na+ ions in tomato leaves was visualized using Sodium Green probes under CSLM and found to be lowest in Terribacillus halophilus 2H20 and Bacillus siamensis 4H1 inoculated plants. The endophyte Terribacillus halophilus 2H20 was the most promising isolate. The colonization in tomato roots was confirmed using a cell tracker system. Results showed that the endophytes were found to have salinity stress mitigation traits. The efficiency could be further improved with the combination of other endophytes tested earlier.

Virulence and pathogenicity determinants in whole genome sequence of Fusarium udum causing wilt of pigeon pea.

The present study deals with whole genome analysis of Fusarium udum, a wilt causing pathogen of pigeon pea. The de novo assembly identified a total of 16,179 protein-coding genes, of which 11,892 genes (73.50%) were annotated using BlastP and 8,928 genes (55.18%) from KOG annotation. In addition, 5,134 unique InterPro domains were detected in the annotated genes. Apart from this, we also analyzed genome sequence for key pathogenic genes involved in virulence, and identified 1,060 genes (6.55%) as virulence genes as per the PHI-BASE database. The secretome profiling of these virulence genes indicated the presence of 1,439 secretory proteins. Of those, an annotation of 506 predicted secretory proteins through CAZyme database indicated maximum abundance of Glycosyl hydrolase (GH, 45%) family proteins followed by auxiliary activity (AA) family proteins. Interestingly, the presence of effectors for cell wall degradation, pectin degradation, and host cell death was found. The genome comprised approximately 895,132 bp of repetitive elements, which includes 128 long terminal repeats (LTRs), and 4,921 simple sequence repeats (SSRs) of 80,875 bp length. The comparative mining of effector genes among different Fusarium species revealed five common and two specific effectors in F. udum that are related to host cell death. Furthermore, wet lab experiment validated the presence of effector genes like SIX (for Secreted in Xylem). We conclude that deciphering the whole genome of F. udum would be instrumental in understanding evolution, virulence determinants, host-pathogen interaction, possible control strategies, ecological behavior, and many other complexities of the pathogen.

The Psychological Impact of Rhino-Orbital Mucormycosis During the Second Wave of COVID-19 Pandemic From South East Asian Country.

AIM: The present study addressed overcoming the lacunae in the literature of psychiatric manifestations associated with rhino-orbital mucormycosis. The current study aimed to assess the symptoms of depression, anxiety, stress, coping measures, suicidal intent, and visual disability in patients of rhino-orbital mucormycosis (ROM) during the epidemic of the disease at the nodal tertiary care center in North India. METHODS: Fifty-four inpatients of laboratory-proven rhino-orbital mucor-mycosis (ROM) were included for an observational, cross-sectional study at nodal, designated COVID-19, and mucormycosis treating tertiary care hospital. Patients with Hindi Mini-Mental State Examination score <24, prior psychiatric illness, and severely ill requiring ventilator support were excluded. The psychological variables were assessed using Depression, Anxiety, and Stress Scale 21 (DASS 21), Beck's Suicide intent Scale, Coping Scale Questionnaire, and Visual disability scale (IND-VFQ33). Their socioeconomic status was assessed using the Modified Kuppuswamy Scale. RESULTS: Ninety percent of patients with ROM had diabetes mellitus. The majority (44%) of patients belonged to lower socioeconomic strata. Higher frequencies of severe depression (28%), extremely severe anxiety (26%), and mild stress (17%) were noted in the study participants. On the Tukey test, depression score was higher in patients of ROM compared to COVID (with ROM) (p-value= 0.016). On Tukey analysis, anxiety score was significantly higher in ROM patients compared to COVID (with ROM) patients (p-value = 0.018). Coping scores were significantly higher in COVID (with ROM) patients compared to ROM patients (p value = 0.035). Mild to moderate visual disability was noted in the study participants.  Conclusion: The current study reflects the association of higher depression and anxiety scores in cases with ROM that indicated higher mental health needs. Early assessment, early detection, and early intervention for psychological help, along with the multidisciplinary team, helped to improve the overall psychological outcome of the affected patients.

Evaluation of Osmotolerant Potential of Halomonas sulfidaeris MV-19 Isolated from a Mud Volcano.

Salinity is one of the major challenges for cultivation of crops in a sustainable way because it severely affects plant growth and yield. Keeping this challenge in view, in the current study, a salt-tolerant Halomonas MV-19 was isolated from an extreme niche of mud volcano of Andaman Nicobar Island, India and identified on the basis of standard morphological, biochemical, and physiological tests and identified as Halomonas sulfidaeris strain MV-19 by 16S rRNA gene sequencing. The bacterium can grow on nutrient agar and nutrient broth supplemented with 3.5 M (≥ 20%) sodium chloride (NaCl). Sugar utilization assay revealed that H. sulfidaeris MV-19 utilizes only three sugars (dextrose, fructose, and mannose) from among twenty four tested sugars. The best growth of H. sulfidaeris MV-19 was observed in nutrient broth supplemented with 8% NaCl. When the broth was supplemented with dextrose, fructose, and mannose, the H. sulfidaeris MV-19 grew maximally in nutrient broth supplemented with 8% NaCl and 5% fructose. This strain produced exopolysaccharides (EPS) in nutrient broth supplemented with 8% NaCl and sugars (dextrose, fructose, and mannose). The EPS production was increased by 350% (three and half time) after addition of 5% fructose in nutrient broth compare with the EPS production in nutrient broth without supplemented with sugars. H. sulfidaeris MV-19 strain can produce EPS, which can help aggregate soil particle and reduced osmotic potential in soil, thus, be useful in alleviation of salinity stress in different crops cultivated in saline soils. The findings of the current investigation are expected to contribute towards effective abiotic stress management.

Low Dose Atropine in Preventing the Progression of Childhood Myopia: A Randomised Controlled Trial.

PURPOSE: To study the efficacy of low dose atropine (0.01%) eye drops in preventing myopia progression in children by comparing the mean change in spherical equivalent (diopter) and axial length (mm) over a period of one year to a control group and study its effect on near vision, pupil size, keratometry and pachymetry. METHODS: 200 eyes of 100 myopic children were randomized into two groups based on a computer-generated random number table. The treatment group was administered 0.01% atropine eye drop once at bedtime and control group was administered a placebo. The follow up was done 3-monthly for 12 months by assessing the mean change in spherical equivalent and mean change in axial length. Other parameters like near vision, pupil size, keratometry and pachymetry were assessed at each follow up. RESULT: The study was age and sex matched. The mean change in spherical equivalent refraction and axial length was significantly lower in the treatment group (0.31 ± 0.55 D; 0.11 ± 0.22 mm) than the placebo group (0.80 ± 1.65 D; 0.23 ± 0.44 D) (p-value: 0.003). Less steepening of the corneal curvature was observed in the treatment group (0.16 ± 0.28 D vs 0.29 ± 0.3 D; p < 0.001) and the mean change in pachymetry was comparable between the groups (0.00 ± 0.01) (p-value 0.489). No significant change was seen in near vision (96% of the eyes with atropine had no change in near vision; 2% of the eyes had a change of near vision by one line (p-value 0.500); 2% had a change by 3 lines (p-value: 0.07) or pupil size following treatment. CONCLUSION: The use of 0.01% atropine eye drop reduced the progression of myopia over the study period of one year with no significant changes in near vision, pupil size. No patient reported any systemic and local side effects with administration of 0.01% atropine eye drop.

Endophytic Bacillus subtilis antagonize soil-borne fungal pathogens and suppress wilt complex disease in chickpea plants (Cicer arietinum L.).

The present study aimed to identify potential endophytic bacteria antagonistic against three soil-borne fungal pathogens, Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum f.sp. ciceri causing root rot, collar rot, and fungal wilt diseases in chickpea plants, respectively. A total of 255 bacterial endophytes were isolated from the leaves, stems, and roots of seven different crop plants (chickpea, tomato, wheat, berseem, mustard, potato, and green pea). The dual culture-based screening for antifungal properties indicated that three endophytic isolates had strong inhibition (>50%) against all three pathogens tested. Based on morphological, biochemical, and molecular characterization, the selected isolates (TRO4, CLO5, and PLO3) were identified as different strains of Bacillus subtilis. The bacterial endophytes (TRO4 and CLO5) were positive for plant growth promoting (PGP) traits viz., ammonia, siderophore, and indole-3-acetic acid (IAA) production. The bio-efficacy of the endophytes (TRO4, CLO5, and PLO3) was tested by an in planta trial in chickpea pre-challenged with R. solani, S. rolfsii, and F. oxysporum f.sp. ciceri. The B. subtilis strains TRO4 and CLO5 were found to be effective in reducing percent disease incidence (p ≤ 0.05) and enhancing plant growth parameters. The different root parameters viz. root length (mm), surface area (cm2), root diameter (mm), and root volume (cm3) were significantly (p ≤ 0.05) increased in TRO4 and CLO5 inoculated chickpea plants. Confocal Scanning Laser Microscopy showed heavy colonization of bacteria in the roots of endophyte-inoculated chickpea plants. The inoculation of endophytic Bacillus subtilis strains TRO4 and CLO5 in chickpea plants through seed biopriming reduced the accumulation of superoxide, enhanced the plant defense enzymes, and induced the expression of Pathogenesis-Related (PR) genes. Semi-quantitative analysis of defense-related genes showed differential activation of PR genes (60srp and IFR) by endophyte inoculation. The results of the present study reveal the antagonistic potential of B. subtilis strains TRO4 and CLO5 against three major soil-borne fungal pathogens and their ability to suppress wilt complex disease in chickpea plants. This is the first report on the simultaneous suppression of three major soil-borne fungal pathogens causing wilt complex in chickpea plants by endophytic B. subtilis strains.

Endophytic Burkholderia: Multifunctional roles in plant growth promotion and stress tolerance.

The genus Burkholderia has proven potential in improving plant performance. In recent decades, a huge diversity of Burkholderia spp. have been reported with diverse capabilities of plant symbiosis which could be harnessed to enhance plant growth and development. Colonization of endophytic Burkholderia spp. have been extensively studied through techniques like advanced microscopy, fluorescent labelling, PCR based assays, etc., and found to be systemically distributed in plants. Thus, use of these biostimulant microbes holds the promise of improving quality and quantity of crops. The endophytic Burkholderia spp. have been found to support plant functions along with boosting nutrient availability, especially under stress. Endophytic Burkholderia spp. improve plant survival against deadly pathogens via mechanisms like competition, induced systemic resistance, and antibiosis. At the same time, they are reported to extend plant tolerance towards multiple abiotic stresses especially drought, salinity, and cold. Several attempts have been made to decipher the potential of Burkholderia spp. by genome mining, and these bacteria have been found to harbour genes for plant symbiosis and for providing multiple benefits to host plants. Characteristics specific for host recognition and nutrient acquisition were confirmed in endophytic Burkholderia by genomics and proteomics-based studies. This could pave the way for harnessing Burkholderia spp. for biotechnological applications like biotransformation, phytoremediation, insecticidal activity, antimicrobials, etc. All these make Burkholderia spp. a promising microbial agent in improving plant performance under multiple adversities. Thus, the present review highlights critical roles of endophytic Burkholderia spp., their colonization, alleviation of biotic and abiotic stresses, biotechnological applications and genomic insights.

Linking Soil Microbial Diversity to Modern Agriculture Practices: A Review.

Agriculture is a multifarious interface between plants and associated microorganisms. In contemporary agriculture, emphasis is being given to environmentally friendly approaches, particularly in developing countries, to enhance sustainability of the system with the least negative effects on produce quality and quantity. Modern agricultural practices such as extensive tillage, the use of harmful agrochemicals, mono-cropping, etc. have been found to influence soil microbial community structure and soil sustainability. On the other hand, the question of feeding the ever-growing global population while ensuring system sustainability largely remains unanswered. Agriculturally important microorganisms are envisaged to play important roles in various measures to raise a healthy and remunerative crop, including integrated nutrient management, as well as disease and pest management to cut down agrochemicals without compromising the agricultural production. These beneficial microorganisms seem to have every potential to provide an alternative opportunity to overcome the ill effects of various components of traditional agriculture being practiced by and large. Despite an increased awareness of the importance of organically produced food, farmers in developing countries still tend to apply inorganic chemical fertilizers and toxic chemical pesticides beyond the recommended doses. Nutrient uptake enhancement, biocontrol of pests and diseases using microbial inoculants may replace/reduce agrochemicals in agricultural production system. The present review aims to examine and discuss the shift in microbial population structure due to current agricultural practices and focuses on the development of a sustainable agricultural system employing the tremendous untapped potential of the microbial world.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow.

Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug-producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mimicking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides "what to grow" (the endophytes) and "what not to grow" (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%-10%), ethanol (70%-90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X-100, Tween 80, and Tween 20. This review comprehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length.

Inter-Genera Colonization of Ocimum tenuiflorum Endophytes in Tomato and Their Complementary Effects on Na+/K+ Balance, Oxidative Stress Regulation, and Root Architecture Under Elevated Soil Salinity.

Endophytic bacilli of ethano-botanical plant Ocimum tenuiflorum were screened for salt stress-alleviating traits in tomato. Four promising O. tenuiflorum endophytes (Bacillus safensis BTL5, Bacillus haynesii GTR8, Bacillus paralicheniformis GTR11, and Bacillus altitudinis GTS16) were used in this study. Confocal scanning laser microscopic studies revealed the inter-genera colonization of O. tenuiflorum endophytes in tomato plants, giving insights for widening the applicability of potential endophytes to other crops. Furthermore, in a pot trial under 150 mM NaCl concentration, the inoculated endophytes contributed in reducing salt toxicity and improving recovery from salt-induced oxidative stress by different mechanisms. Reduction in reactive oxygen species (ROS) (sub-cellular H2O2 and superoxide) accumulation was observed besides lowering programmed cell death and increasing chlorophyll content. Endophyte inoculation supplemented the plant antioxidant enzyme system via the modulation of enzymatic antioxidants, viz., peroxidase, ascorbate peroxidase, superoxide dismutase, and catalase, apart from increasing proline and total phenolics. Antioxidants like proline have dual roles of antioxidants and osmoregulation, which might also have contributed to improved water relation under elevated salinity. Root architecture, viz., root length, projection area, surface area, average diameter, tips, forks, crossings, and the number of links, was improved upon inoculation, indicating healthy root growth and enhanced nutrient flow and water homeostasis. Regulation of Na+/K+ balance and water homeostasis in the plants were also evident from the modulation in the expression of abiotic stress-responsive genes, viz., LKT1, NHX1, SOS1, LePIP2, SlERF16, and SlWRKY39. Shoot tissues staining with light-excitable Na+ indicator Sodium GreenTM Tetra (tetramethylammonium) salt showed low sodium transport and accumulation in endophyte-inoculated plants. All four endophytes exhibited different mechanisms for stress alleviation and indicated complementary effects on plant growth. Furthermore, this could be harnessed in the form of a consortium for salt stress alleviation. The present study established inter-genera colonization of O. tenuiflorum endophytes in tomato and revealed its potential in maintaining Na+/K+ balance, reducing ROS, and improving root architecture under elevated salinity.

Restructuring the Cellular Responses: Connecting Microbial Intervention With Ecological Fitness and Adaptiveness to the Maize (Zea mays L.) Grown in Saline-Sodic Soil.

Salt stress hampers plant growth and development. It is now becoming one of the most important threats to agricultural productivity. Rhizosphere microorganisms play key roles in modulating cellular responses and enable plant tolerant to salt stress, but the detailed mechanisms of how this occurs need in-depth investigation. The present study elucidated that the microbe-mediated restructuring of the cellular responses leads to ecological fitness and adaptiveness to the maize (Zea mays L.) grown in saline-sodic soil. In the present study, effects of seed biopriming with B. safensis MF-01, B. altitudinis MF-15, and B. velezensis MF-08 singly and in consortium on different growth parameters were recorded. Soil biochemical and enzymatic analyses were performed. The activity and gene expression of High-Affinity K+ Transporter (ZmHKT-1), Sodium/Hydrogen exchanger 1 (zmNHX1), and antioxidant enzymes (ZmAPX1.2, ZmBADH-1, ZmCAT, ZmMPK5, ZmMPK7, and ZmCPK11) were studied. The expression of genes related to lateral root development (ZmHO-1, ZmGSL-1, and ZmGSL-3) and root architecture were also carried out. Seeds bioprimed with consortium of all three strains have been shown to confer increased seed germination (23.34-26.31%) and vigor indices (vigor index I: 38.71-53.68% and vigor index II: 74.11-82.43%) as compared to untreated control plant grown in saline-sodic soil at 30 days of sowing. Results indicated that plants treated with consortium of three strains induced early production of adventitious roots (tips: 4889.29, forks: 7951.57, and crossings: 2296.45) in maize compared to plants primed with single strains and untreated control (tips: 2019.25, forks: 3021.45, and crossings: 388.36), which was further confirmed by assessing the transcript level of ZmHO-1 (7.20 folds), ZmGSL-1 (4.50 folds), and ZmGSL-3 (12.00 folds) genes using the qPCR approach. The uptake and translocation of Na+, K+, and Ca2+ significantly varied in the plants treated with bioagents alone or in consortium. qRT-PCR analysis also revealed that the ZmHKT-1 and zmNHX1 expression levels varied significantly in the maize root upon inoculation and showed a 6- to 11-fold increase in the plants bioprimed with all the three strains in combination. Further, the activity and gene expression levels of antioxidant enzymes were significantly higher in the leaves of maize subjected seed biopriming with bioagents individually or in combination (3.50- to 12.00-fold). Our research indicated that ZmHKT-1 and zmNHX1 expression could effectively enhance salt tolerance by maintaining an optimal Na+/K+ balance and increasing the antioxidant activity that keeps reactive oxygen species at a low accumulation level. Interestingly, up-regulation of ZmHKT-1, NHX1, ZmHO-1, ZmGSL-1, and ZmGSL-3 and genes encoding antioxidants regulates the cellular responses that could effectively enhance the adaptiveness and ultimately leads to better plant growth and grain production in the maize crop grown in saline-sodic soil.

Trichoderma harzianum- and Methyl Jasmonate-Induced Resistance to Bipolaris sorokiniana Through Enhanced Phenylpropanoid Activities in Bread Wheat (Triticum aestivum L.).

The aim of the present study was to evaluate the impact of Trichoderma harzianum UBSTH-501- and methyl jasmonate-induced systemic resistance and their integration on the spot blotch pathogen, Bipolaris sorokiniana through enhanced phenylpropanoid activities in bread wheat (Triticum aestivum L.). It was found that the application of MeJA (>100 mg L-1) inhibits the germination of B. sorokiniana spores under controlled laboratory conditions. To assess the effect of MeJA (150 mg L-1) in combination with the biocontrol agent T. harzianum UBSTH-501 in vivo, a green house experiment was conducted. For this, biocontrol agent T. harzianum UBSTH-501 was applied as seed treatment, whereas MeJA (150 mg L-1) was applied 5 days prior to pathogen inoculation. Results indicated that application of MeJA (150 mg L-1) did not affect the root colonization of wheat by T. harzianum UBSTH-501 in the rhizosphere. The combined application of T. harzianum UBSTH-501 and MeJA also enhanced indole acetic acid production in the rhizosphere (4.92 µg g-1 of soil) which in turn helps in plant growth and development. Further, the combined application found to enhance the activities of defense related enzymes viz. catalase (5.92 EU min-1 g-1 fresh wt.), ascorbate peroxidase [µmol ascorbate oxidized (mg prot)-1 min-1], phenylalanine ammonia lyase (102.25 µmol cinnamic acid h-1 mg-1 fresh wt.) and peroxidase (6.95 Unit mg-1 min-1 fresh wt.) significantly in the plants under treatment which was further confirmed by assessing the transcript level of PAL and peroxidase genes using semi-quantitative PCR approach. The results showed manifold increase in salicylic acid (SA) along with enhanced accumulation of total free phenolics, ferulic acid, caffeic acid, coumaric acid, and chlorogenic acid in the leaves of the plants treated with the biocontrol agent alone or in combination with MeJA. A significant decrease in the disease severity (17.46%) and area under disease progress curve (630.32) were also observed in the treatments with biocontrol agent and MeJA in combination as compared to B. sorokiniana alone treated plant (56.95% and 945.50, respectively). Up-regulation of phenylpropanoid cascades in response to exogenous application of MeJA and the biocontrol agent was observed. It was depicted from the results that PAL is the primary route for lignin production in wheat which reduces cell wall disruption and tissue disintegration and increases suberization and lignification of the plant cell as seen by Scanning Electron microphotographs. These results clearly indicated that exogenous application of MeJA with T. harzianum inducing JA- and/or SA-dependent defense signaling after pathogen challenge may increase the resistance to spot blotch by stimulating enzymatic activities and the accumulation of phenolic compounds in a cooperative manner. This study apparently provides the evidence of biochemical cross-talk and physiological responses in wheat following MeJA and biocontrol agent treatment during the bio-trophic infection.

Presenile cataract and its risk factors: A case control study.

PURPOSE: Early onset opacification of the lens (cataract) has been observed to be on the rise globally. This study was conducted to determine the various types of presenile cataract and to determine the probable risk factors associated with the occurrence of presenile cataract. MATERIALS AND METHODS: Patients in the age group of 18-40 years attending the Out-Patient Clinic of the Ophthalmology Department who were found to have presenile cataract were recruited as cases. An equal number of consecutive patients of the same age group were included in the control group. Those who gave consent to participate in the study were interviewed through a preformed questionnaire and underwent a complete ocular examination and set of blood investigations. The type of cataract was noted, data obtained were compiled, and examination and investigations done were documented and analyzed using frequency distribution and Chi-squared test. RESULTS: In total, 90 cases and 90 controls that fulfilled the inclusion criteria were recruited. Most common type of cataract was found to be posterior subcapsular cataract. Presenile cataract was observed to be significantly associated with tobacco intake (P = 0.035), hypercholesterolemia (P = 0.002), fuel exposure (P = 0.004), and lower socioeconomic status (P = <0.001). CONCLUSIONS: Tobacco chewing, hypercholesterolemia, and excessive fuel exposure are risk factors for early development of cataract.

Design, Synthesis, and Anti-RNA Virus Activity of 6'-Fluorinated-Aristeromycin Analogues.

The 6'-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6'-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N6-methyladenosine analogues 2a-e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6',6'-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6'-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.

Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.

An efficient, mild and metal free l-proline catalyzed construction of fused pyrimidines under microwave conditions in water.

One-pot condensation of 4-hydroxy coumarins, aldehydes and urea/thiourea to build C-C and C-N bonds is described. Fused pyrimidines have been synthesized under mild reaction conditions using l-proline. The protocol has been performed rapidly and efficiently in water under metal free conditions. Heterocyclic derivatives have been synthesized using the present methodology and avoid the use of hazardous solvents over conventional organic solvents. A proposed mechanism could be established for three component reactions. The present study reveals the first case in which l-proline has been explored as a homogeneous catalyst in the synthesis of fused pyrimidines in water under microwave irradiation. This synthesis involves simple workup and acceptable efficiency. The most notable feature of this protocol is the ability of the catalyst to influence asymmetric induction in the reaction.

Evaluation of intravitreal bevacizumab as monotherapy and in combination with macular grid laser photocoagulation in diffuse diabetic macular edema.

BACKGROUND AND OBJECTIVE: The objective of the study was to compare the efficacy of intravitreal bevacizumab (IVB) as monotherapy versus combination with modified macular grid (MMG) laser photocoagulation in the primary treatment of diffuse diabetic macular edema (DDME). MATERIALS AND METHODS: A prospective randomized trial was carried out in sixty eyes with DDME after Institutional Ethical clearance. Group A received three doses of IVB at a 1-month interval. Group B received MMG in addition to IVB. Complete examination including best-corrected visual acuity (BCVA) (Snellen's), central macular thickness (CMT) using spectral domain-optical coherence tomography was carried out at 0, 1, 4, 8, 12, and 24 weeks. RESULTS: mean CMT at baseline, 12 and 24 weeks in Group A was 401 (±76), 280 (±49), and 307 (±46) and in Group B, 405 (±73), 237 (±33), and 242 (±45), respectively. Group B had significantly greater reductions (P < 0.001) from 12 weeks onward. BCVA and contrast sensitivity showed improvements with no significant difference. CONCLUSION: Combined therapy has more advantage in primary DDME by reducing CMT on longer follow-up.

Design, synthesis, ADME prediction and pharmacological evaluation of novel benzimidazole-1,2,3-triazole-sulfonamide hybrids as antimicrobial and antiproliferative agents.

BACKGROUND: Nitrogen heterocyclic rings and sulfonamides have attracted attention of several researchers. RESULTS: A series of regioselective imidazole-based mono- and bis-1,4-disubstituted-1,2,3-triazole-sulfonamide conjugates 4a-f and 6a-f were designed and synthesized. The first step in the synthesis was a regioselective propargylation in the presence of the appropriate basic catalyst (Et3N and/or K2CO3) to afford the corresponding mono-2 and bis-propargylated imidazoles 5. Second, the ligation of the terminal C≡C bond of mono-2 and/or bis alkynes 5 to the azide building blocks of sulfa drugs 3a-f using optimized conditions for a Huisgen copper (I)-catalysed 1,3-dipolar cycloaddition reaction yielded targeted 1,2,3-triazole hybrids 4a-f and 6a-f. The newly synthesized compounds were screened for their in vitro antimicrobial and antiproliferative activities. Among the synthesized compounds, compound 6a emerged as the most potent antimicrobial agent with MIC values ranging between 32 and 64 µg/mL. All synthesized molecules were evaluated against three aggressive human cancer cell lines, PC-3, HepG2, and HEK293, and revealed sufficient antiproliferative activities with IC50 values in the micromolar range (55-106 µM). Furthermore, we conducted a receptor-based electrostatic analysis of their electronic, steric and hydrophobic properties, and the results were in good agreement with the experimental results. In silico  ADMET prediction studies also supported the experimental biological results and indicated that all compounds are nonmutagenic and noncarcinogenic. CONCLUSION: In summary, we have successfully synthesized novel targeted benzimidazole-1,2,3-triazole-sulfonamide hybrids through 1,3-dipolar cycloaddition reactions between the mono- or bis-alkynes based on imidazole and the appropriate sulfonamide azide under the optimized Cu(I) click conditions. The structures of newly synthesized sulfonamide hybrids were confirmed by means of spectroscopic analysis. All newly synthesized compounds were evaluated for their antimicrobial and antiproliferative activities. Our results showed that the benzimidazole-1,2,3-triazole-sulfonamide hybrids inhibited microbial and fungal strains within MIC values from 32 to 64 µg/mL. The antiproliferative evaluation of the synthesized compounds showed sufficient antiproliferative activities with IC50 values in the micromolar range (55-106 µM). In conclusion, compound 6a has remarkable antimicrobial activity. Pharmacophore elucidation of the compounds was performed based on in silico ADMET evaluation of the tested compounds. Screening results of drug-likeness rules showed that all compounds follow the accepted rules, meet the criteria of drug-likeness and follow Lipinski's rule of five. In addition, the toxicity results showed that all compounds are nonmutagenic and noncarcinogenic.

Design, Synthesis and Anticancer Screening of Novel Benzothiazole-Piperazine-1,2,3-Triazole Hybrids.

A library of novel regioselective 1,4-di and 1,4,5-trisubstituted-1,2,3-triazole based benzothiazole-piperazine conjugates were designed and synthesized using the click synthesis approach in the presence and absence of the Cu(I) catalyst. Some of these 1,2,3-triazole hybrids possess in their structures different heterocyclic scaffold including 1,2,4-triazole, benzothiazole, isatin and/or benzimidazole. The newly designed 1,2,3-triazole hybrids were assessed for their antiproliferative inhibition potency against four selected human cancer cell lines (MCF7, T47D, HCT116 and Caco2). The majority of the synthesized compounds demonstrated moderate to potent activity against all the cancer cell lines examined. Further, we have established a structure activity relationship with respect to the in silico analysis of ADME (adsorption, distribution, metabolism and excretion) analysis and found good agreement with in vitro activity.

Orally active, species-independent novel A3 adenosine receptor antagonist protects against kidney injury in db/db mice.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease, and the current pharmacological treatment for DKD is limited to renin-angiotensin system (RAS) inhibitors. Adenosine is detectable in the kidney and is significantly elevated in response to cellular damage. While all 4 known subtypes of adenosine receptors, namely, A1AR, A2aAR, A2bAR, and A3AR, are expressed in the kidney, our previous study has demonstrated that a novel, orally active, species-independent, and selective A3AR antagonist, LJ-1888, ameliorates unilateral ureteral obstruction-induced tubulointerstitial fibrosis. The present study examined the protective effects of LJ-2698, which has higher affinity and selectivity for A3AR than LJ-1888, on DKD. In experiment I, dose-dependent effects of LJ-2698 were examined by orally administering 1.5, 5, or 10 mg/kg for 12 weeks to 8-week-old db/db mice. In experiment II, the effects of LJ-2698 (10 mg/kg) were compared to those of losartan (1.5 mg/kg), which is a standard treatment for patients with DKD. LJ-2698 effectively prevented kidney injuries such as albuminuria, glomerular hypertrophy, tubular injury, podocyte injury, fibrosis, inflammation, and oxidative stress in diabetic mice as much as losartan. In addition, inhibition of lipid accumulation along with increases in PGC1α, a master regulator of mitochondrial biogenesis, were demonstrated in diabetic mice treated with either LJ-2698 or losartan. These results suggest that LJ-2698, a selective A3AR antagonist, may become a novel therapeutic agent against DKD.