Association of Plasmodium falciparum with Human Endothelial Cells in vitro.

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria.

Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron.

Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional folding at a rate constant similar to the folding of the active ribozyme, and folding of both P3-P7 and P1-P10 pseudoknots are inhibited by antisense oligonucleotides. We conclude that when RNA folding is coupled with transcription, formation of pseudoknot structures dominates the productive folding pathway and serves as a rate-limiting step in producing the self-splicing competent Candida intron.

Obtaining a faculty position in STEM at a research-intensive institution.

Progressing from postdoctoral training to a STEM faculty appointment at a Research Intensive Institution (RII) is a daunting transition, and may be especially challenging to those who have followed a less-than-conventional path or whose peers have lost interest in academic careers. This article describes how to prepare for and progress through the application process for institutions in the USA, which takes approximately 1 year, including what to expect at each step and recommendations for a successful transition. The odds of success for any individual application are low, making good preparation and careful planning the more important, as does managing expectations to avoid becoming discouraged early in the process. The rewards of landing the faculty appointment at an institution that matches your professional and personal needs and for which you are best suited more than exceeds the effort required to attain it.

Scientific Societies Advancing STEM Workforce Diversity: Lessons and Outcomes from the Minorities Affairs Committee of the American Society for Cell Biology.

Promoting diversity and inclusiveness in the STEM academic workforce remains a key challenge and national priority. Scientific societies can play a significant role in this process through the creation and implementation of programs to foster STEM academic workforce diversification, and by providing mentoring and skills development training that empower scientists from under-represented minority (URM) backgrounds to succeed in their communities of practice. In this article, we provide examples of challenges met by scientific societies in these areas and present data from the American Society for Cell Biology, highlighting the benefits received by trainees through long-term engagement with its programs. The success of these initiatives illustrates the impact of discipline-specific programming by scientific societies in supporting the development of URM scientists and an increasingly diverse and inclusive academic STEM community.

Scientific Societies Fostering Inclusive Scientific Environments through Travel Awards: Current Practices and Recommendations.

Diversity-focused committees continue to play essential roles in the efforts of professional scientific societies to foster inclusion and facilitate the professional development of underrepresented minority (URM) young scientists in their respective scientific disciplines. Until recently, the efforts of these committees have remained independent and disconnected from one another. Funding from the National Science Foundation has allowed several of these committees to come together and form the Alliance to Catalyze Change for Equity in STEM Success, herein referred to as ACCESS. The overall goal of this meta-organization is to create a community in which diversity-focused committees can interact, synergize, share their collective experiences, and have a unified voice on behalf of URM trainees in science, technology, engineering, and mathematics disciplines. In this Essay, we compare and contrast the broad approaches that scientific societies in ACCESS use to implement and assess their travel award programs for URM trainees. We also report a set of recommendations, including both short- and long-term outcomes assessment in populations of interest and specialized programmatic activities coupled to travel award programs.

Molecular characterization of two types of rDNA units in a single strain of Candida albicans.

Strains of the opportunistic fungal pathogen Candida albicans vary in the presence or absence of a self-splicing group I intron ribozyme (Ca.LSU) in the 25S rRNA gene on chromosome R. Strains of C. albicans typically either lack or contain this ribozyme. However, some strains have both intron-containing and intronless rRNA genes (rDNA). Pulsed-field gel electrophoresis analysis of undigested and restricted DNA showed at least six different karyotypes among eight independent colonies of such a heteroallelic strain. In each case, the variation was in chromosome R, and was due to changes in the number of rDNA units. In strains with only one type of rDNA, chromosome R also varied considerably. Polymerase chain reaction amplification spanning the rDNA unit demonstrated that intron-containing rDNA units are tandemly arrayed, and are immediately adjacent to intronless units in the same cluster. Both types of units were present in the rDNA clusters of both R chromosomes. Possible explanations of these results are loss of Ca.LSU group I intron through purifying selection and/or a relaxation of the commonly accepted concerted evolution of the rDNA units.

Novel multi-component nanopharmaceuticals derived from poly(ethylene) glycol, retro-inverso-Tat nonapeptide and saquinavir demonstrate combined anti-HIV effects.

BACKGROUND: Current anti-AIDS therapeutic agents and treatment regimens can provide a dramatically improved quality of life for HIV-positive people, many of whom have no detectable viral load for prolonged periods of time. Despite this, curing AIDS remains an elusive goal, partially due to the occurrence of drug resistance. Since the development of resistance is linked to, among other things, fluctuating drug levels, our long-term goal has been to develop nanotechnology-based drug delivery systems that can improve therapy by more precisely controlling drug concentrations in target cells. The theme of the current study is to investigate the value of combining AIDS drugs and modifiers of cellular uptake into macromolecular conjugates having novel pharmacological properties. RESULTS: Bioconjugates were prepared from different combinations of the approved drug, saquinavir, the antiviral agent, R.I.CK-Tat9, the polymeric carrier, poly(ethylene) glycol and the cell uptake enhancer, biotin. Anti-HIV activities were measured in MT-2 cells, an HTLV-1-transformed human lymphoid cell line, infected with HIV-1 strain Vbu 3, while parallel studies were performed in uninfected cells to determine cellular toxicity. For example, R.I.CK-Tat9 was 60 times more potent than L-Tat9 while the addition of biotin resulted in a prodrug that was 2850 times more potent than L-Tat9. Flow cytometry and confocal microscopy studies suggest that variations in intracellular uptake and intracellular localization, as well as synergistic inhibitory effects of SQV and Tat peptides, contributed to the unexpected and substantial differences in antiviral activity. CONCLUSION: Our results demonstrate that highly potent nanoscale multi-drug conjugates with low non-specific toxicity can be produced by combining moieties with anti-HIV agents for different targets onto macromolecules having improved delivery properties.

Concerted folding of a Candida ribozyme into the catalytically active structure posterior to a rapid RNA compaction.

Folding of the major population of Tetrahymena intron RNA into the catalytically active structure is trapped in a slow pathway. In this report, folding of Candida albicans intron was investigated using the trans-acting Ca.L-11 ribozyme as a model. We demonstrated that both the catalytic activity (k(obs)) and compact folding equilibrium of Ca.L-11 are strongly dependent on Mg(2+) at physiological concentrations, with both showing an Mg(2+) Hill coefficient of 3. Formation of the compact structure of Ca.L-11 is shown to occur very rapidly, on a subsecond time scale similar to that of RNase T1 cleavage. Most of the ribozyme RNA population folds into the catalytically active structure with a rate constant of 2 min(-1) at 10 mM Mg(2+); neither slower kinetics nor obvious Mg(2+) inhibition is observed. These results suggest that folding of the Ca.L-11 ribozyme is initiated by a rapid magnesium-dependent RNA compaction, which is followed by a slower searching for the native contacts to form the catalytically active structure without interference from the long-lived trapped states. This model thus provides an ideal system to address a range of interesting aspects of RNA folding, such as conformational searching, ion binding and the role of productive intermediates.

Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding.

The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 microM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.

A hydrogel prepared by in situ cross-linking of a thiol-containing poly(ethylene glycol)-based copolymer: a new biomaterial for protein drug delivery.

A new poly(ethylene glycol)-based copolymer containing multiple thiol (-SH) groups was cross-linked in situ to form a polymer hydrogel under mild conditions. No organic solvent, elevated temperature, or harsh pH is required in the formulation or patient administration processes, making it particularly useful for delivery of fragile therapeutics, such as proteins. The in vitro release of fluorescein-labeled bovine serum albumin and the in vivo release of the model proteins, erythropoietin, RANTES and three PEG-conjugated RANTES derivatives showed sustained release for 2-4 weeks and demonstrated prolonged biological activity of the released proteins in animals.

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA.

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.

The role of professional identity in graduate school success for under-represented minority students.

We examined the relationship between sense of professional identity and academic success among under-represented minority graduate students in a biomedical doctoral program. We found that a sense of professional identity is related to science success among under-represented minority students, but not for non-underrepresented minority students. Sense of professional identity appears to be linked to the use of mentors and to development as a scientist and member of the professional community, rather than someone who simply performs laboratory experiments.

Partnered research experiences for junior faculty at minority-serving institutions enhance professional success.

Scientific workforce diversity is critical to ensuring the realization of our national research goals and minority-serving institutions play a vital role in preparing undergraduate students for science careers. This paper summarizes the outcomes of supporting career training and research practices by faculty from teaching-intensive, minority-serving institutions. Support of these faculty members is predicted to lead to: 1) increases in the numbers of refereed publications, 2) increases in federal grant funding, and 3) a positive impact on professional activities and curricular practices at their home institutions that support student training. The results presented show increased productivity is evident as early as 1 yr following completion of the program, with participants being more independently productive than their matched peers in key areas that serve as measures of academic success. These outcomes are consistent with the goals of the Visiting Professorship Program to enhance scientific practices impacting undergraduate student training. Furthermore, the outcomes demonstrate the benefits of training support for research activities at minority-serving institutions that can lead to increased engagement of students from diverse backgrounds. The practices and results presented demonstrate a successful generalizable approach for stimulating junior faculty development and can serve as a basis for long-term faculty career development strategies that support scientific workforce diversity.

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Optimizing size and copy number for PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarrier uptake by macrophages.

Curing HIV-1 infection has remained elusive because of low and fluctuating drug levels arising from poor absorption, the development of viral reservoirs and sanctuary sites, toxicity, and patient nonadherence. The present study addresses the issue of insufficient drug exposure in macrophages. Viral reservoir sites such as macrophages are believed to be responsible for the viral rebound effect observed upon the discontinuation of anti-HIV drug therapy. In our proposed model, a drug can be covalently attached to a nanocarrier in order to facilitate the delivery of therapeutic agents to the site(s) of infection. As an initial step, we propose the covalent attachment of several copies of N-formyl-Met-Leu-Phe (fMLF), a known chemo-attractant for macrophages. In this article, one or more copies of fMLF were conjugated to multifunctional commercially available or novel, peptide-based PEG nanocarriers in which the structure was varied by appending PEGs with average molecular weights of 5, 20, and 40 kDa. U937 cell-specific binding and cellular uptake were analyzed. The results of uptake studies indicate that (i) uptake is energy dependent and mediated by a fMLF receptor, (ii) appending only 2 copies of the targeting ligand to the multifunctional nanocarrier appears sufficient for binding in vitro, and (iii) of the three configurations studied, the nanocarrier with a molecular weight of about 20 kDa, corresponding to a size of 20-60 nm, demonstrated the highest uptake. The results of the current studies demonstrate the feasibility of targeting macrophages and the suitability of using these synthetically versatile peptide--backbone PEG nanocarriers. The convenience, flexibility and possible limitations of this nanocarrier approach are discussed.

Peritoneal macrophage uptake, pharmacokinetics and biodistribution of macrophage-targeted PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarriers for improving HIV drug delivery.

PURPOSE: To assess in vivo macrophage targeting potential of PEG-fMLF nanocarriers and to investigate their biodistribution, peritoneal macrophage uptake, and pharmacokinetics. METHODS: Multiple copies of fMLF were conjugated to purchased and novel (branched, peptide-based) PEG nanocarriers. Peritoneal macrophage uptake was evaluated in mice 4 hours after IP administration of fluorescence-labeled PEG-fMLF nanocarriers. Pharmacokinetics and biodistribution were determined in rats after IV administration of tritiated PEG-fMLF nanocarriers. RESULTS: Attachment of one, two, or four fMLF copies increased uptake in macrophages by 3.8-, 11.3-, and 23.6-fold compared to PEG without fMLF. Pharmacokinetic properties and tissue distribution also differed between nanocarriers with and without fMLF. Attachment of fMLF residues increased the t(1/2) of PEG(5K) by threefold but decreased the t(1/2) of PEG(20K) by 40%. Attachment of fMLF increased accumulation of nanocarriers into macrophages of liver, kidneys and spleen. However, on a molar basis, penetration was equivalent suggesting nanocarrier size and targeting moieties are important determinants. CONCLUSIONS: These results demonstrate the feasibility for targeting macrophages, a primary HIV reservoir site. However, these studies also suggest that balancing peripheral tissue penetration (a size-dependent phenomenon) versus target cell uptake specificity remains a challenge to overcome.

Multiple-peptide conjugates for binding beta-amyloid plaques of Alzheimer's disease.

Formation of beta-amyloid plaques in Alzheimer's disease is initiated by intermolecular contact of the 5-amino acid sequence, KLVFF, in beta-amyloid peptides ranging in size from 40 to 43 residues. Through optimization of binding avidity using structure/function studies, we have found that the retro-inverso peptide, ffvlk, binds artificial fibrils made from Abeta(1)(-)(40) with moderate affinity (K(d) = 5 x 10(-)(7) M). Conjugates having two copies of this peptide, whether connected by a long poly(ethylene glycol) (PEG) spacer or just two amino acids, display about 100-fold greater affinity for fibrils. Placing six copies of ffvlk on a branched PEG resulted in a 10 000-fold greater affinity (K(d) = 1 x 10(-)(10) M) than the monomer peptide. This increased affinity was accompanied by more effective inhibition of the thioflavin T fluorescence signal, which correlates with neurotoxicity of plaques and fibrils. We propose that conjugates bearing several copies of ffvlk may be useful as diagnostic and therapeutic agents for Alzheimer's disease.

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay.

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.

Conjugates bearing multiple formyl-methionyl peptides display enhanced binding to but not activation of phagocytic cells.

N-Formyl-methionyl peptides can specifically bind to surface receptors on phagocytic cells. A single copy of N-formyl-methionine-leucine-phenylalanine (fMLF) covalently linked to a poly(ethylene glycol)-based polymer displayed reduced binding avidity (K(d) = 190 nM) for differentiated HL-60 cells relative to free fMLF (K(d) = 28 nM). Increasing the number of fMLF residues (up to eight) attached to a single polymer results in enhanced avidity for these cells (K(d) = 0.18 nM), which appears to be independent of whether the polymer backbone is linear or branched. However, no conjugate showed enhanced ability to activate phagocytic cells, relative to the free peptide (EC(50) = 5 nM), as measured by transient stimulation of release of calcium ions from intracellular stores into the cytoplasm. A polymer bearing four fMLF and four digoxigenin residues showed specific enhancement in binding to differentiated HL-60 cells and mouse peritoneal macrophages in situ relative to a polymer lacking fMLF; no such enhancement was seen in binding to receptor-negative lymphocytic Jurkat cells. These results suggest that multiple fMLF residues linked to a drug-delivery polymer can be used to target appended drugs to phagocytic cells with relatively little toxicity due to cellular activation.

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells.

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.