Synthesis and biological evaluation of nitrogen-containing benzophenone analogues as TNF-α and IL-6 inhibitors with antioxidant activity

A series of nitrogen-containing benzophenone analogues were synthesized by Mannich reaction and evaluated for the inhibition of pro-inflammatory cytokines, TNF-α and IL-6. DPPH (1,1-diphenyl-2-picryl hydrazine) radical scavenging activity and its kinetics were studied to determine the antioxidant potential of the test samples. All the synthesized compounds exhibited promising activity against IL-6 in a range of 81–89%, 09–42% at 10 and 1 μM, respectively, concentration. Exceptionally, the compound 20e was observed to be an effective inhibitor of TNF-α (54%) and IL-6 (97%), (47%) at 10 and 1 μM concentrations with minimum toxicity (22%) against CCK-8 cells. With few exceptions, all other compounds were found to be excellent inhibitors of IL-6 and moderate to excellent inhibitors of TNF-α, however the toxicity profiles of these compounds need to be ameliorated in further optimization studies. Amongst the tested compounds, 16a, 17g, 18f, 18g, 19g and 20e were found to possess significant antioxidant activity.     

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans

Invasion of epithelial cells is a major virulence determinant of Candida albicans ; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans . Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx^−/− neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.Oxidative stress is an etiological factor in many neurological diseases, including Alzheimer''s disease, Parkinson''s disease, and Huntington''s disease. One type of macromolecule damaged by reactive oxygen species is DNA, and oxidative damage to DNA has been suggested to be a significant factor in these and other neurological conditions (2). In particular, a number of rare hereditary neurodegenerative disorders have provided direct support for the notion that unrepaired DNA damage causes neural dysfunction. Not least of these are the recessive spinocerebellar ataxias, a number of which are associated with mutations in DNA damage response proteins (17). The archetypal DNA damage-associated spinocerebellar ataxia is ataxia-telangiectasia (A-T), in which mutations in ATM protein result in defects in the detection and signaling of DNA double-strand breaks (DSBs) (3). A-T-like disorder is a related disease that exhibits neurological features similar to those of A-T, resulting from mutation of Mre11, a component of the MRN complex that operates in conjunction with ATM during DSB detection and signaling (28).Two additional spinocerebellar ataxias are spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) and ataxia oculomotor apraxia 1 (AOA1), in which the TDP1 and aprataxin proteins are mutated, respectively (9, 19, 27). Both TDP1 and aprataxin are components of the DNA strand break repair machinery (recently reviewed in references 6 and 24). Whereas SCAN1 is currently limited to nine individuals from a single family, AOA1 is one of the commonest recessive spinocerebellar ataxias. Aprataxin is a member of the histidine triad superfamily of nucleotide hydrolases/transferases and has been reported to remove phosphate and phosphoglycolate moieties from the 3′ termini of DNA strand breaks (26). Aprataxin can also remove AMP from a variety of ligands in vitro, including adenosine polyphosphates, AMP-lysine, AMP-NH₂ (adenine monophosphoramidate), and adenylated DNA in which AMP is covalently attached to the 5′ terminus of a DNA single-strand break (SSB) or DSB (1, 16, 23, 25). To date, aprataxin activity is greatest on AMP-DNA, suggesting that this may be the physiological substrate of this enzyme.In vitro, DNA strand breaks with 5′-AMP termini can arise from premature DNA ligase activity. DNA ligases adenylate 5′ termini at DNA breaks to enable nucleophilic attack of the resulting pyrophosphate bonds by 3′-hydroxyl termini, thereby resealing the breaks. However, DNA adenylation by DNA ligases can occur prematurely, before a 3′-hydroxyl terminus is available. Aprataxin reverses these premature DNA adenylation events, in vitro at least, effectively “resetting” the DNA ligation reaction to the beginning (1). Whether or not 5′-AMP arises in DNA in vivo or is a physiological substrate of aprataxin, however, is unknown. Moreover, attempts to measure DNA strand break repair rates in vivo are conflicting and have failed to identify a consistent defect in DNA SSB repair (SSBR) or DSB repair (DSBR) in AOA1 cells (14, 15, 20). It is thus not clear whether or not defects in DNA strand break repair can account for this neurodegenerative disease.Here we have resolved the discrepancy between the requirements for aprataxin in vitro and in vivo by identifying the stage at which SSBR reactions fail in vitro and by carefully analyzing chromosomal SSBR rates in vivo. We show that short-patch SSBR reactions are defective in AOA1 cell extracts at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks, and that this defect can be rescued in AOA1 extracts independently of aprataxin by addition of recombinant DNA ligase. We also find that treatment with aphidicolin, an inhibitor of DNA polymerase δ (Pol δ) and Pol ɛ, unveils a measurable defect in chromosomal SSBR in Aptx^−/− primary neural astrocytes, suggesting that the adenylated nicks that arise from the short-patch repair defect can be channeled into long-patch repair in vivo. These data demonstrate that aprataxin participates in chromosomal SSBR and suggest that this process arrests in AOA1, at oxidative SSBs, due to insufficient levels of nonadenylated DNA ligase.     

Small nucleolar RNA interference in Trypanosoma brucei: mechanism and utilization for elucidating the function of snoRNAs

Expression of dsRNA complementary to small nucleolar RNAs (snoRNAs) in Trypanosoma brucei results in snoRNA silencing, termed snoRNAi. Here, we demonstrate that snoRNAi requires the nuclear TbDCL2 protein, but not TbDCL1, which is involved in RNA interference (RNAi) in the cytoplasm. snoRNAi depends on Argonaute1 (Slicer), and on TbDCL2, suggesting that snoRNA dicing and slicing takes place in the nucleus, and further suggesting that AGO1 is active in nuclear silencing. snoRNAi was next utilized to elucidate the function of an abundant snoRNA, TB11Cs2C2 (92 nt), present in a cluster together with the spliced leader associated RNA (SLA1) and snR30, which are both H/ACA RNAs with special nuclear functions. Using AMT-UV cross-linking and RNaseH cleavage, we provide evidence for the interaction of TB11Cs2C2 with the small rRNAs, srRNA-2 and srRNA-6, which are part of the large subunit (LSU) rRNA. snoRNAi of TB11Cs2C2 resulted in defects in generating srRNA-2 and LSUβ rRNA. This is the first snoRNA described so far to engage in trypanosome-specific processing events.     

Eriocaulon balakrishnanii (Eriocaulaceae), a new species from the Western Ghats of India

A new species, Eriocaulon balakrishnanii (Eriocaulaceae) from the Western Ghats of Karnataka State in India is described and illustrated. This species is allied to E. robusto-brownianum and E. lanceolatum but differs in a number of characters from both.     

Phylogenetic community patterns suggest Central Indian tropical dry forests are structured by montane climate refuges

Aim

We used an eco-phylogenetic approach to investigate the diversity and assembly patterns of tropical dry forests (TDFs) in Central India. We aimed at informing conservation and restoration practices in these anthropogenically disturbed forests by identifying potential habitats of conservation significance and elements of regional biodiversity most vulnerable to human impact and climate change.

Location

Tropical dry forests of Madhya Pradesh, Central India.

Methods

We analysed the species richness, stem density, basal area and phylogenetic structure (standardized effect size of MNTD, MPD, PD and community evolutionary distinctiveness cED) of 117 tree species assemblages distributed across a ~230 to ~940 m elevational gradient. We examined how these community measures and taxonomic (Sørensen) and phylogenetic (UniFrac) beta diversity varied with elevation, precipitation, temperature and climatic stress.

Results

Species richness, phylogenetic diversity, stem density and basal area were positively correlated with elevation, with high-elevation plots exhibiting cooler temperatures, higher precipitation and lower stress. High-elevation assemblages also trended towards greater phylogenetic dispersion, which diminished at lower elevations and in drier, more stressful plots. Phylogenetic turnover was observed across the elevation gradient, and species evolutionary distinctiveness increased at lower elevations and under harsher abiotic conditions.

Main Conclusions

Harsher abiotic conditions at low elevations may act as a selective filter on plant lineages, leading to phylogenetically clustered low-diversity assemblages. These assemblages contained more evolutionarily distinct species that may contribute disproportionately to biodiversity. Conversely, milder abiotic conditions at high elevations may serve as refuges for drought-sensitive species, resulting in more diverse assemblages. Conservation practices that prioritize both high- and low-elevation habitats could promote the persistence of evolutionarily distinct species and areas of high biodiversity within the Central Indian landscape. Establishing connectivity between these habitats may provide a range of climatic conditions for species to retreat to or persist within as climates change.     

Heterogeneous diacylglycerol acyltransferase expression enhances lipids and PUFA in Chlorella species

Algae have been explored for renewable energy, nutraceuticals, and value-added products. However, low lipid yield is a significant impediment to its commercial viability. Genetic engineering can improve the fatty acid profile of algae without compromising its growth. This study introduced the diacylglycerol acyltransferase (BnDGAT) gene from Brassica napus into Chlorella sorokiniana-I, a fast-growing and thermotolerant natural strain isolated from wastewater, which increased its intracellular lipid accumulation. Hygromycin-resistant cells were selected, and enhanced green florescence protein fluorescence was used to distinguish pure transgenic cell lines from mixed cultures. Compared to the wild type, BnDGAT expression in transgenic C. sorokiniana-I caused a threefold increase in non-polar lipid and a twofold increase in polyunsaturated fatty acids. Nile red staining reaffirmed the presence of higher intracellular lipid bodies in transgenic cells. There was a substantial alteration in the fatty acid profile of transgenic alga expressing BnDGAT. The non-essential omega 9 (C18: 1) fatty acid decreased (5%–7% from 18%), while alpha-linolenic acid, an essential omega 3 fatty acid (C18: 3), was increased (23%–24% from 11%). This study substantiates a valuable strategy for enhancing essential omega-3 fatty acids and neutral lipids to improve its nutritional value for animal feed. The increased lipid productivity should reduce the cost of producing fatty acid methyl esters (FAME). Improved FAME quality should address the clouding issues in cold regions.     

Computational analysis of looping of a large family of highly bent DNA by LacI

Sequence-dependent intrinsic curvature of DNA influences looping by regulatory proteins such as LacI and NtrC. Curvature can enhance stability and control shape, as observed in LacI loops formed with three designed sequences with operators bracketing an A-tract bend. We explore geometric, topological, and energetic effects of curvature with an analysis of a family of highly bent sequences, using the elastic rod model from previous work. A unifying straight-helical-straight representation uses two phasing parameters to describe sequences composed of two straight segments that flank a common helically supercoiled segment. We exercise the rod model over this two-dimensional space of phasing parameters to evaluate looping behaviors. This design space is found to comprise two subspaces that prefer parallel versus anti-parallel binding topologies. The energetic cost of looping varies from 4 to 12 kT. Molecules can be designed to yield distinct binding topologies as well as hyperstable or hypostable loops and potentially loops that can switch conformations. Loop switching could be a mechanism for control of gene expression. Model predictions for linking numbers and sizes of LacI-DNA loops can be tested using multiple experimental approaches, which coupled with theory could address whether proteins or DNA provide the observed flexibility of protein-DNA loops.