Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability.     

The apical annuli of Toxoplasma gondii are composed of coiled‐coil and signalling proteins embedded in the inner membrane complex sutures

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1–5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super‐resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring‐like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled‐coil and signalling proteins assembled in a pore‐like structure crossing the IMC barrier maintained during internal budding.     

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.     

Phospholipid signaling during stramenopile development

Development of sessile organisms requires adaptation to an ever-changing environment. In order to respond quickly to these challenges, complex signaling mechanisms have evolved to facilitate cellular modifications. The importance of phospholipid-based signaling pathways in plants, as well as animals, has recently been gaining attention. Both the PLD and PLC pathways produce the signaling molecule PA, which modulates MTs, F-actin and endomembrane trafficking. We have examined the roles of the PLD signaling pathway during development of the marine brown alga Silvetia compressa. Zygotes were treated with 1- and 2-butanol, both of which activate the PLD enzyme. However, only 1-butanol competes with water as a transphosphatidylation substrate, at the expense of PA production. Interestingly, we found that 1- and 2-butanol both disrupted MT organization and thereby cell division, with 1-butanol being more potent. These findings question whether the effects of butyl alcohol treatment are due to lowered PA levels or activation of the PLD enzyme. Additionally, preliminary results show that inhibition of DAGK results in loss of centrosomal MTs and formation of cortical MT cages that are strikingly similar to those formed following 1-butanol treatment. These data suggest that perturbation of the PLD or PLC pathway leads to cortical stabilization and/or nucleation of MT arrays.Key words: actin, brown algae, cytoskeleton, development, endomembrane, microtubule, phosphatidic acid, phospholipase C, phospholipase D, stramenopile     

Biogeographical venom variation in the Indian spectacled cobra (Naja naja) underscores the pressing need for pan-India efficacious snakebite therapy

BackgroundSnake venom composition is dictated by various ecological and environmental factors, and can exhibit dramatic variation across geographically disparate populations of the same species. This molecular diversity can undermine the efficacy of snakebite treatments, as antivenoms produced against venom from one population may fail to neutralise others. India is the world’s snakebite hotspot, with 58,000 fatalities and 140,000 morbidities occurring annually. Spectacled cobra (Naja naja) and Russell’s viper (Daboia russelii) are known to cause the majority of these envenomations, in part due to their near country-wide distributions. However, the impact of differing ecologies and environment on their venom compositions has not been comprehensively studied.MethodsHere, we used a multi-disciplinary approach consisting of venom proteomics, biochemical and pharmacological analyses, and in vivo research to comparatively analyse N. naja venoms across a broad region (>6000 km; seven populations) covering India’s six distinct biogeographical zones.FindingsBy generating the most comprehensive pan-Indian proteomic and toxicity profiles to date, we unveil considerable differences in the composition, pharmacological effects and potencies of geographically-distinct venoms from this species and, through the use of immunological assays and preclinical experiments, demonstrate alarming repercussions on antivenom therapy. We find that commercially-available antivenom fails to effectively neutralise envenomations by the pan-Indian populations of N. naja, including a complete lack of neutralisation against the desert Naja population.ConclusionOur findings highlight the significant influence of ecology and environment on snake venom composition and potency, and stress the pressing need to innovate pan-India effective antivenoms to safeguard the lives, limbs and livelihoods of the country’s 200,000 annual snakebite victims.     

Characterization of Leukocyte-platelet Rich Fibrin,A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.     

Effects of class-specific,synthetic, and natural proteinase inhibitors on life-history traits of the cotton bollworm Helicoverpa armigera

Herbivorous insects have more difficulty obtaining proteins from their food than do predators and parasites. The scarcity of proteins in their diet requires herbivores to feed voraciously, thus heavily damaging their host plants. Plants respond to herbivory by producing defense compounds, which reduce insect growth, retard development, and increase mortality. Herbivores use both pre- and postdigestive response mechanisms to detect and avoid plant defense compounds. Proteinase inhibitors (PIs) are one example of plant compounds produced as a direct defense against herbivory. Many insects can adapt to PIs when these are incorporated into artificial diets. However, little is known about the effect of PIs on diet choice and feeding behavior. We monitored the diet choice, life-history traits, and gut proteinase activity of Helicoverpa armigera larvae using diets supplemented with synthetic and natural PIs. In choice experiments, both neonates and fourth-instar larvae preferred the control diet over PI-supplemented diets, to varying degrees. Larvae that fed on PI-supplemented diets weighed less than those that fed on the control diet and produced smaller pupae. Trypsin-specific PIs had a stronger effect on mean larval weight than did other PIs. A reduction of trypsin activity but not of chymotrypsin activity was observed in larvae fed on PI-supplemented diets. Therefore, behavioral avoidance of feeding on plant parts high in PIs could be an adaptation to minimize the impact of this plant's defensive strategy.