Novel development of Lecanicillium lecanii-based granules as a platform against malarial vector Anopheles culicifacies.

Mosquitoes are infectious vectors for a wide range of pathogens and parasites thereby transmitting several diseases including malaria, dengue, Zika, Japanese encephalitis and chikungunya which pose a major public health concern. Mostly synthetic insecticides are usually applied as a primary control strategy to manage vector-borne diseases. However excessive and non-judicious usage of such chemically derived insecticides has led to serious environmental and health issues owing to their biomagnification ability and increased toxicity towards non-target organisms. In this context, many such bioactive compounds originating from entomopathogenic microbes serve as an alternative strategy and environmentally benign tool for vector control. In the present paper, the entomopathogenic fungus, Lecanicillium lecanii (LL) was processed to make the granules. Developed 4% LL granules have been characterized using the technique of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The developed formulation was also subjected to an accelerated temperature study at 40 °C and was found to be stable for 3 months. Further, GCMS of the L. lecanii was also performed to screen the potential biomolecules present. The developed formulation was found to be lethal against Anopheles culicifacies with an LC50 value of 11.836 µg/mL. The findings from SEM and histopathology also substantiated the mortality effects. Further, the SEM EDX (energy dispersive X-ray) studies revealed that the treated larvae have lower nitrogen content which is correlated to a lower level of chitin whereas the control ones has higher chitin content and healthy membranes. The developed LL granule formulation exhibited high toxicity against Anopheles mosquitoes. The granule formulations can be used as an effective biocontrol strategy against malaria-causing mosquitoes.

Pomegranate Resembling Design of Starch Sago Beads Encapsulates Nanopyriproxyfen, Enabling Slow Release and Improved Bioactivity.

Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor to major hazards; further, disturbing non-target populations provoke a grave concern worldwide as they play essential roles in ecosystems. Pyriproxyfen is one of the most widely used pesticides; however, due to its probable toxicity, its global application in large amounts may result in water concentrations that exceed regulatory pollution thresholds. Herein, we describe nanopyriproxyfen-loaded sago beads (PPX-NCB) designed for the slow release of pyriproxyfen (PPX). Our design is inspired by the composite structure of sago beads, composed of several small beads resembling a pomegranate. The microscopic beads accumulate chitosan-PPX-nanomicelles cross-linked with tripolyphosphate via physical absorption, offering adequate room for water absorption and subsequent PPX release. PPX-NCB had distinct effects on the immature egg and larva of Anopheles stephensi, limiting embryonic development in the eggs while enhancing bioactivity. It affects the integument of larvae and alters the surface hydrocarbons of eggs and larvae. In addition, PPX-NCB demonstrates an improved safety profile in non-target Daphnia magna.

Analogous foliar uptake and leaf-to-root translocation of micelle nanoparticles in two dicot plants of diverse families.

Bio-inspired nanoparticles, including metallic, micelles, and polymeric, have been explored as a novel tool in the quest for effective and safe agrochemicals. Although nanoparticles (NPs) are being rapidly investigated for their usefulness in agricultural production and protection, little is known about the behaviour and interaction of oil-in-water micelle nanoparticles or nano-micelles (NM) with plants. We loaded a bio-based resin inherent of tree from the Pinaceae family as active material and produced stable nano-micelles using a natural emulsifier system. Here, we show that foliar-applied nano-micelle can translocate in two dicot plants belonging to diverse families (Coriandrum sativum -Apiaceae and Trigonella foenumgraecum -Fabaceae) via similar mode. Fluorescent-tagged NM (average diameter 11.20nm) showed strong signals and higher intensities as revealed by confocal imaging and exhibited significant adhesion in leaf compared to control. The NM subsequently translocates to other parts of the plants. As observed by SEM, the leaf surface anatomies revealed higher stomata densities and uptake of NM by guard cells; furthermore, larger extracellular spaces in mesophyll cells indicate a possible route of NM translocation. In addition, NM demonstrated improved wetting-spreading as illustrated by contact angle measurement. In a field bioassay, a single spray application of NM offered protection from aphid infestation for at least 9 days. There were no signs of phytotoxicity in plants post-application of NM. We conclude that pine resin-based nano-micelle provides an efficient, safe, and sustainable alternative for agricultural applications.

Utilization of the castor seed cake (biowaste) for mosquito vector control.

The present work is related to the utilization of castor (Ricinus communis) seed cake, biowaste produced during the oil extraction of castor seeds, as efficient mosquitocidal composition against Aedes aegypti and Anopheles culicifacies. The efficacy of coil formulations was evaluated in the Peet Grady chamber and resulted in 90% and 100% knocked down and mortality against A. aegypti and A. Culicifacies, respectively. Further heavy metals' (Cr, Pb, Co, As, Cd, Cu, Mn, and Zn) analysis of the coil was performed using Inductively Coupled Plasma mass spectrometry and was compared with commercially available mosquito repellent coil. Heavy metal analysis revealed that commercial repellent coil had a higher content of heavy metals than the castor seed cake coil. Finding of the present research study indicates that castor seed cake coil has the potential to be used in mosquito vector control. Castor seed cake coil formulation will also open up avenues in future for sustainable utilization of the biowaste.

ACE2, TMPRSS2, and L-SIGN Expression in Placentae From HIV-Positive Pregnancies Exposed to Antiretroviral Therapy-Implications for SARS-CoV-2 Placental Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding receptor ACE2 and the spike protein priming protease TMPRSS2 are coexpressed in human placentae. It is unknown whether their expression is altered in the context of HIV infection and antiretroviral therapy (ART). METHODS: We compared mRNA levels of SARS-CoV-2 cell-entry mediators ACE2, TMPRSS2, and L-SIGN by quantitative polymerase chain reaction in 105 placentae: 45 from pregnant women with HIV (WHIV) on protease inhibitor (PI)-based ART, 17 from WHIV on non-PI-based ART, and 43 from HIV-uninfected women. RESULTS: ACE2 levels were lower, while L-SIGN levels were higher, in placentae from WHIV on PI-based ART compared to those on non-PI-based ART and to HIV-uninfected women. TMPRSS2 levels were similar between groups. Black race was significantly associated with lower expression of ACE2 and higher expression of L-SIGN. ACE2 levels were significantly higher in placentae of female fetuses. CONCLUSIONS: We identified pregnant women of black race and WHIV on PI-based ART to have relatively lower expression of placental ACE2 than those of white race and HIV-uninfected women. This may potentially contribute to altered susceptibility to COVID-19 in these women, favorably by reduced viral entry or detrimentally by loss of ACE2 protection against hyperinflammation.

Exploring the impact of HIV infection and antiretroviral therapy on placenta morphology.

INTRODUCTION: Women living with HIV experience more adverse birth outcomes; the mechanisms are not fully understood. We examined placenta morphology and associations with birth outcomes in a Canadian cohort of women living with HIV (HIV+) on antiretroviral therapy (ART) from conception and HIV-uninfected (HIV-) women. METHODS: Term placentas from 94 women (40 HIV-, 54 HIV+) were studied. Trimmed placenta weight was collected. Placenta digital photos were used to compute morphometric parameters. Regression models investigated associations between log-transformed placenta parameters and birth outcomes. RESULTS: We observed a trend towards lower placenta weight and smaller placenta area in the HIV+ group, both of which were significantly associated with small for gestational age births. HIV+ serostatus was associated with 6-fold (95%CI 2-20) greater odds of having placenta area in the lowest quartile (<236 cm2). Cord marginality (distance from the edge) was significantly lower in the HIV+ group (p = 0.004), with 35% of placenta having an abnormal (marginal or velamentous) cord insertion vs. 12.5% in the HIV- group (p = 0.01). Velamentous cord insertion was seen in 13% of placentas in the HIV+ vs. 0% in HIV- group (p = 0.02). A significant correlation between cord marginality and placenta thickness was observed in the HIV- group, with a more marginal cord being associated with a thicker placenta. This correlation was not observed in the HIV+ group. HIV+ placentas exposed to protease inhibitors were significantly less circular compared to the HIV- group (p = 0.03). CONCLUSION: Our data suggest that HIV/ART exposure affects placenta morphology and is associated with higher rates of abnormal cord insertion.

Impregnation of pectin-cedarwood essential oil nanocapsules onto mini cotton bag improves larvicidal performances.

The use pesticide is one of the indispensable means to combat mosquito borne diseases. However, the repeated use of synthetic pesticides has induced resistance in the vector pest along with undesirable impact on the environment. The biodegradability, non-persistent and user's safety are the root cause to prefer plant-derived pesticides to synthetic ones. The botanical based pesticides tend to degrade rapidly under the influence of several environmental factors. For the feasible application as pesticides, the plant products are formulated either as liquid or as purely solid. Despite well-established formulation technology in pesticide delivery, their handling trouble is being ignored. There is difficulty in liquid formulation of pesticide products, as they are prone to splashing and spillage, resulting in contamination, wastage and direct exposure to skin; whereas a solid formulation tends to produce dust. In the present work, cedarwood (Cedrus deodara) essential oil embedded pectin nanocapsules were produced. The nanocapsules were characterized according to their morphology, size, encapsulation efficiency and thermal stability. Furthermore, the nanocapsules were impregnated onto mini cotton tea bags to be employed as RTU (ready to use) formulation for treating the breeding sites of mosquitoes. The larvicidal activity of the bags treated with pectin-cedar wood nanocapsules was assessed against malaria vector, Anopheles culicifacies and 98% mortality was recorded till 4 weeks, this suggests its potential and hassle free applications in controlling mosquito vector.

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

STUDY QUESTION: Does HIV protease inhibitor (PI)-based combination antiretroviral therapy (cART) initiated at periconception affect key events in early pregnancy, i.e. decidualization and spiral artery remodeling? SUMMARY ANSWER: Two PIs, lopinavir and darunavir, currently offered as cART options in HIV-positive pregnancies were evaluated, and we found that lopinavir-based cART, but not darunavir-based cART, impaired uterine decidualization and spiral artery remodeling in both human ex vivo and mouse in vivo experimental models. WHAT IS KNOWN ALREADY: Early initiation of cART is recommended for pregnant women living with HIV. However, poor birth outcomes are frequently observed in HIV-positive pregnancies exposed to PI-based cART, especially when it is initiated prior to conception. The correlation between early initiation of PI-cART and adverse birth outcomes is poorly understood, due to lack of data on the specific effects of PI-cART on the early stages of pregnancy involving uterine decidualization and spiral artery remodeling. STUDY DESIGN, SIZE, DURATION: Lopinavir and darunavir were evaluated in clinically relevant combinations using an ex vivo human first-trimester placenta-decidua explant model, an in vitro human primary decidual cell culture system, and an in vivo mouse pregnancy model. The first-trimester (gestational age, 6-8 weeks) human placenta-decidua tissue was obtained from 11 to 15 healthy women undergoing elective termination of pregnancy. C57Bl/6 female mice (four/treatment group) were administered either lopinavir-cART, darunavir-cART or water by oral gavage once daily starting on the day of plug detection until sacrifice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human: Spiral artery remodeling was assessed by immunohistochemical analysis of first-trimester placenta-decidua explant co-culture system. Trophoblast migration was measured using a placental explant culture. A primary decidual cell culture was used to evaluate the viability of immune cell populations by flow cytometry. Soluble factors, including biomarkers of decidualization and angiogenesis, were quantified by ELISA and Luminex assay using decidua-conditioned media. Mouse: In the mouse pregnancy model, gestational day 6.5 or 9.5 implantation sites were used to assess decidualization, spiral artery remodeling and uterine natural killer (uNK) cell numbers by immunohistochemistry. Transcription factor STAT3 was assayed by immunohistochemistry in both human decidua and mouse implantation sites. MAIN RESULTS AND THE ROLE OF CHANCE: Lopinavir-cART, but not darunavir-cART, impaired uterine decidualization and spiral artery remodeling in both experimental models. Lopinavir-cART treatment was also associated with selective depletion of uNK cells, reduced trophoblast migration and defective placentation. The lopinavir-associated decidualization defects were attributed to a decrease in expression of transcription factor STAT3, known to regulate decidualization. Our results suggest that periconceptional initiation of lopinavir-cART, but not darunavir-cART, causes defective maturation of the uterine endometrium, leading to impairments in spiral artery remodeling and placentation, thus contributing to the poor birth outcomes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human first-trimester placenta/decidua samples could only be obtained from healthy females undergoing elective termination of pregnancy. As biopsy is the only way to obtain first-trimester decidua from pregnant women living with HIV on PI-cART, ethics approval and participant consent are difficult to obtain. Furthermore, our animal model is limited to the study of cART and does not include HIV. HIV infection is also associated with immune dysregulation, inflammation, alterations in angiogenic factors and complement activation, all of which could influence decidual and placental vascular remodeling and modify any cART effects. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide mechanistic insight with direct clinical implications, rationalizing why the highest adverse birth outcomes are reported in HIV-positive pregnancies exposed to lopinavir-cART from conception. We demonstrate that dysregulation of decidualization is the mechanism through which lopinavir-cART, but not darunavir-cART, use in early pregnancy leads to poor birth outcomes. Although lopinavir is no longer a first-line regimen in pregnancy, it remains an alternate regimen and is often the only PI available in low resource settings. Our results highlight the need for reconsidering current guidelines recommending lopinavir use in pregnancy and indicate that lopinavir should be avoided especially in the first trimester, whereas darunavir is safe to use and should be the preferred PI in pregnancy.Further, in current times of the COVID-19 pandemic, lopinavir is among the top drug candidates which are being repurposed for inclusion in clinical trials world-over, to assess their therapeutic potential against the dangerous respiratory disease. Current trials are also testing the efficacy of lopinavir given prophylactically to protect health care workers and people with potential exposures. Given the current extraordinary numbers, these might include women with early pregnancies, who may or may not be cognizant of their gestational status. This is a matter of concern as it could mean that women with early pregnancies might be exposed to this drug, which can cause decidualization defects. Our findings provide evidence of safety concerns surrounding lopinavir use in pregnancy, that women of reproductive age considering participation in such trials should be made aware of, so they can make a fully informed decision. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (PJT-148684 and MOP-130398 to L.S.). C.D. received support from CIHR Foundation (FDN143262 to Stephen Lye). S.K. received a TGHRI postdoctoral fellowship. The authors declare that there are no conflicts of interest. L.S. reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank.

Sulfonated inhibitors of the RNA editing ligases validate the essential role of the MRP1/2 proteins in kinetoplastid RNA editing.

The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.

Chitosan-acrylate nanogel for durable anti mosquito finishing of cotton fabric and its dermal toxicity profiling on Swiss albino mice.

Chitosan nanocapsules, containing lemongrass (Cymbopogon citratus) oil (LGO), have been developed in gel form in which acrylate (Ac) was incorporated as a thickener and fabric binder. The gel was impregnated on fabric to achieve long-lasting and wash-durable mosquito repellency. The interaction between cotton fibers and gel was investigated by FTIR and XRD. Wash durability of gel was compared with chitosan nanocapsules without acrylate (LGO-encap) using SEM and GC-MS. The SEM analyses revealed that acrylate containing nanocapsules retains on fabric after a series of washing. The GC-MS results indicated that the relative amount of deducible oil components from fabric was found to be higher after the series of washing in acrylate containing nanocapsules (LGO-encap-Ac), which further points to the improved wash durability and retention of capsules on fabric. The bio-efficacy results of post-fifteen washing turned out was 75% of repellency against mosquitoes with the use of acrylate; while in nanocapsules without acrylate, only 51% of repellency was achieved. Furthermore, the 36 days repeated application of nanogel on Swiss albino mice did not show any signs of dermal toxicity. The formulation is, thus, suitable to impregnate dress of the military personals and individuals who have to perform field duty and where risk of mosquito bites is probably more.

Improving the clinical relevance of a mouse pregnancy model of antiretroviral toxicity; a pharmacokinetic dosing-optimization study of current HIV antiretroviral regimens.

Animal models can be useful tools for the study of HIV antiretroviral (ARV) safety/toxicity in pregnancy and the mechanisms that underlie ARV-associated adverse events. The utility and translatability of animal model-based ARV safety/toxicity data is improved if ARVs are tested in clinically relevant concentrations. The objective of this work was to improve the clinical relevance of our mouse pregnancy model of ARV toxicity, by determining the doses of currently prescribed ARV regimens that would yield human therapeutic plasma concentrations. Pregnant mice were administered increasing doses of ARV combinations by oral gavage, followed by measurement of drug concentrations in the maternal plasma and amniotic fluid. Concentrations of ten different ARVs in maternal plasma and amniotic fluid samples of pregnant mice are presented, with dosing optimization to yield human pregnancy-relevant plasma drug concentrations. We have proposed optimal dosing for different regimen component drugs to achieve human therapeutic plasma levels, so that a clinically relevant standard dosing is established. A review of related ARV pharmacokinetic studies in (pregnant/non-pregnant) rodents and human pregnancy is also shown. We hope these data will inform and encourage the use of mouse pregnancy models in the study of ARV safety/toxicity.

Phage Morons Play an Important Role in Pseudomonas aeruginosa Phenotypes.

The viruses that infect bacteria, known as phages, play a critical role in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria but also from the formation of lysogens. In this state, the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell, since increasing the survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons," as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa, PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed an inhibition of the production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons.IMPORTANCE Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons." In this study, we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviors. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases.

Larvicidal activity of Ricinus communis extract against mosquitoes.

BACKGROUND & OBJECTIVES: Vector control strategies play significant role in reducing the transmission of malaria, dengue and other vector-borne diseases. The control of vector population using synthetic insecticides has resulted in development of insecticide resistance and negative effects on humans and environment. The present investigation evaluated the larvicidal potential of methanol, dichloromethane and hexane extracts of leaves and seeds of Ricinus communis (castor) plant against the early IV instar larvae of the dengue vector, Aedes aegypti, and malaria vector, Anopheles culicifacies. METHODS: Plant extracts were screened for their efficacy against Ae. aegypti and An. culicifacies using WHO standard larval susceptibility test method. Dose response bioassay was performed to get lethal concentrations. Further, gas chromatography-mass spectroscopy (GC-MS) analysis was carried out to identify the bioactive chemical constituents of the extracts of R. communis. Toxicity of the extracts towards non-target organism, Poecilia reticulata was also evaluated. RESULTS: The leaf and seed extracts of R. communis showed significant mortality against the larvae of Ae. aegypti and An. culicifacies at concentrations of 31.25, 62.5, 125, 250, 500 ppm; and 2, 4, 8, 16, 32, 64 ppm, respectively. At 24 h of the exposure period, the larvicidal activities were highest for the methanol extract of seeds with LC50 15.52 and 9.37 ppm and LC90 45.24 and 31.1 ppm for Ae. aegypti and An. culicifacies, respectively. The methanol extract of seeds and leaves was found to be safe towards non-target organism, P. reticulata. The GC-MS profile showed that seed extracts were having higher concentration of stigmasterol (7.5%), ß-sitosterol (11.48%), methyl linoleate (2.5%), vitamin E (11.93%), and ricinoleic acid (34%) than the leaf extracts. INTERPRETATION & CONCLUSION: The seed extract of R. communis has better larvicidal activity than the leaf extract and can be used as an effective larvicide against mosquitoes. The non-toxicity of the extracts towards P. reticulata further suggests that these plant extracts could be used along with predatory fishes in integrated vector control approaches.

The interaction of a Trypanosoma brucei KH-domain protein with a ribonuclease is implicated in ribosome processing.

Ribosomal RNA maturation is best understood in yeast. While substantial efforts have been made to explore parts of these essential pathways in animals, the similarities and uniquenesses of rRNA maturation factors in non-Opisthokonts remain largely unexplored. Eukaryotic ribosome synthesis requires the coordinated activities of hundreds of Assembly Factors (AFs) that transiently associate with pre-ribosomes, many of which are essential. Pno1 and Nob1 are two of six AFs that are required for the cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA in yeast where it has been almost exclusively analyzed. Specifically, Nob1 ribonucleolytic activity generates the mature 3'-end of 18S rRNA. We identified putative Pno1 and Nob1 homologues in the protist Trypanosoma brucei, named TbPNO1 and TbNOB1, and set out to explore their rRNA maturation role further as they are both essential for normal growth. TbPNO1 is a nuclear protein with limited cytosolic localization relative to its yeast homologue. Like in yeast, it interacts directly with TbNOB1, with indications of associations with a larger AF-containing complex. Interestingly, in the absence of TbPNO1, TbNOB1 exhibits non-specific degradation activity on RNA substrates, and its cleavage activity becomes specific only in the presence of TbPNO1, suggesting that TbPNO1-TbNOB1 interaction is essential for regulation and site-specificity of TbNOB1 activity. These results highlight a conserved role of the TbPNO1-TbNOB1 complex in 18S rRNA maturation across eukaryotes; yet reveal a novel role of their interaction in regulation of TbNOB1 enzymatic activity.

HNH proteins are a widespread component of phage DNA packaging machines.

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.

The oligonucleotide binding (OB)-fold domain of KREPA4 is essential for stable incorporation into editosomes.

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.

Inhibitors of RNA editing as potential chemotherapeutics against trypanosomatid pathogens.

The related trypanosomatid pathogens, Trypanosoma brucei spp., Trypanosoma cruzi and Leishmania spp. cause devastating diseases in humans and animals and continue to pose a major challenge in drug development. Mitochondrial RNA editing, catalyzed by multi-protein complexes known as editosomes, has provided an opportunity for development of efficient and specific chemotherapeutic targets against trypanosomatid pathogens. This review will discuss both methods for discovery of RNA editing inhibitors, as well as inhibitors against the T. brucei editosome that were recently discovered through creative virtual and high throughput screening methods. In addition, the use of these inhibitors as agents that can block or perturb one or more steps of the RNA editing process will be discussed. These inhibitors can potentially be used to study the dynamic processing and assembly of the editosome proteins. A thorough understanding of the mechanisms and specificities of these new inhibitors is needed in order to contribute to both the functional studies of an essential gene expression mechanism and to the possibility of future drug development against the trypanosomatid pathogens.

Naphthalene-based RNA editing inhibitor blocks RNA editing activities and editosome assembly in Trypanosoma brucei.

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.

Serum and organ-associated anti-hemoglobin humoral autoreactivity: association with anti-Sm responses and inflammation.

The release of hemoglobin (Hb) occurs in some infectious and autoimmune diseases characterized by inflammation. As levels of haptoglobin (Hp) fall, free Hb can cause pathology. Humoral autoreactivity to human Hb was demonstrated in the sera of systemic lupus erythematosus (SLE), leishmania and malaria patients. Serum anti-murine Hb antibody levels in lupus-prone mice also exhibited an age-dependent increase, with progressive organ sequestration; significant isotypic correlation was observed with anti-dsDNA antibodies. A suggestive link between anti-Hb and anti-Sm responses was observed: Human lupus sera expressing anti-Sm antibody reactivity preferentially contained heightened levels of anti-Hb autoantibodies, and immunization of lupus-prone mice with Sm led to enhanced anti-murine Hb reactivity. Human and murine anti-Hb monoclonal antibodies were generated, some of which were preferentially reactive toward disease-associated methemoglobin. Epitope-mapping studies revealed evidence of intra-molecular cross-reactivity. One such autoantibody synergized with Hb to enhance the secretion of pro-inflammatory cytokines while eliciting the increased production of monocyte migratory signals from endothelial cells. Preferential usage of specific variable region gene segments was not observed, although somatic mutations were documented. These studies reveal that, while the etiology, specificity and sequences of anti-Hb autoreactive antibodies can vary, they occur quite frequently and can have inflammatory consequences.