Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus

The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5′ extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5′ ends at position −141 and 3′ ends adjacent to the mature 5′ end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3′ ends and unprocessed 5′ ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome

Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ∼20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5′ OH on the 3′ product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei

9G4 Autoreactivity Is Increased in HIV-Infected Patients and Correlates with HIV Broadly Neutralizing Serum Activity

The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4−IgD− memory B cell population, the 9G4+IgD− memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward “IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies

Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3′-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism

Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform

The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform

Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform

The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform

Exploration of Kalanchoe Daigremontiana Phytochemicals for Antimicrobial Properties

Kalanchoe daigremontiana is a tropical plant traditionally used to treat infections, decrease inflammation, and promote wound healing. Prior work on a related species, Kalanchoe pinnata, has demonstrated significant inhibition of fungal and bacterial pathogens. This project aims to test effectiveness of organic extracts of K. daigremontiana against a range of human pathogens. We have cultivated K. daigremontiania under controlled conditions (soil, pH, water, light). At the three- and six-month time points in their growth, the aerial parts of the plants were dried, macerated, and exhaustively extracted using organic solvents of increasing polarity. Each solvent extract was evaporated under reduced pressure, and the resulting biomass was weighed. Samples at the nine- and 12-month points will also be acquired. Extracts will be tested against several human pathogens (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans) using a disk diffusion assay. Factors for future consideration are modifying the sample acquisition time and the use of abiotic stress factors to upregulate secondary metabolite production

Lifespan extending properties of Kalanchoe daigremontiana plant extracts in Saccharomyces cerevisiae

Kalanchoe daigremontiana, commonly known as mother of thousands and the alligator plant, is a traditional remedy that has been used in various countries to treat inflammation and related disorders. The long-standing use of this plant makes it a promising candidate for the discovery of new anti-inflammatory and anti-aging compounds. Varying concentrations of K. daigremontiana extract (water (H2O), hexane (Hex), methanol (MeOH), chloroform (CHCl3) and ethyl acetate (EtOAc)) were tested on Saccharomyces cerevisiae and the yeast were tolerant of all extract concentrations examined. The extracts were then tested for lifespan altering effects in yeast utilizing the growth-based lifespan measuring DeaD (Death of Daughters) assay. It was found that each extract under multiple concentrations increased replicative lifespan. Furthermore, extracts from the EtOAc extract were able to overcome the powerful lifespan shortening effects of the sirtuin inhibitor nicotinamide. The increase in yeast lifespan with the addition of these extracts, indicates the value of K. daigremontiana in the study of lifespan and the potential for the development of drugs to combat age-related disease, inflammation and to extend healthspan/ lifespan

Exploration of Kalanchoe Daigremontiana Phytochemicals for Antimicrobial Properties

Kalanchoe daigremontiana is a tropical plant traditionally used to treat infections, decrease inflammation, and promote wound healing. Prior work on a related species, Kalanchoe pinnata, has demonstrated significant inhibition of fungal and bacterial pathogens. This project aims to test effectiveness of organic extracts of K. daigremontiana against a range of human pathogens. We have cultivated K. daigremontiania under controlled conditions (soil, pH, water, light). At the three- and six-month time points in their growth, the aerial parts of the plants were dried, macerated, and exhaustively extracted using organic solvents of increasing polarity. Each solvent extract was evaporated under reduced pressure, and the resulting biomass was weighed. Samples at the nine- and 12-month points will also be acquired. Extracts will be tested against several human pathogens (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Candida albicans) using a disk diffusion assay. Factors for future consideration are modifying the sample acquisition time and the use of abiotic stress factors to upregulate secondary metabolite production