High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Source, fate and management of recreational fishing marine debris.

Marine debris, directly and indirectly, threatens marine habitat and biota. Fishing activity is generally recognised as a contributor to marine debris, but the relative input from recreational fishing remains unassessed. Here we provide the first comprehensive literature review of recreational fishing marine debris (RFMD) on a global scale. A systematic literature review identified 70 studies related to RFMD, and plastic and metal respectively were the dominant debris materials found. Nearshore coastal areas and reefs, acted as both sources and sinks of RFMD and a diverse suite of potential impacts such as ghost fishing and entanglement were identified at local scales. Overall, research of RFMD is lacking globally, however, its role in marine debris input is likely underestimated. We recommend more research on the volumes and risks, using a standardised classification approach. Where intervention is required, we suggest cooperative approaches between the sector and authorities.

Home First: Exploring the Impact of Community-based Home Care for Older Adults and Their Family Caregivers.

Given the rapidly expanding older adult population, finding health care approaches that support older adults to age in their choice of place, with an accompanying philosophical re-orientation of health services, is becoming more urgent. We studied the Home Care Home First - Quick Response Project to understand how clients over age 75 and their family caregivers perceived the enhanced community-based services delivered through Home First. Using interpretive description as the methodological design, we explored the experiences of eight older adults and 11 family caregivers; all older adults were enrolled in Home First due to a significant change in their health status. We identified four themes: growing older in chosen places with support, philosophy of care, processes of Home First, and the significance of Home First for clients. Overall, clients and family caregivers responded positively to the Home First services. Clients valued their independence and growing older in places they had specifically chosen.

Distinct pathways of viral spread in the host determined by reovirus S1 gene segment.

The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways.

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

Accumulation of unintegrated circular viral DNA in monocytes and growth-arrested T cells following infection with HIV-1.

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.

Serological detection of attenuated HIV-1 variants with nef gene deletions.

OBJECTIVE: To investigate whether members of a transfusion-linked cohort (the Sydney Bloodbank Cohort) infected with a nef-deleted strain of HIV-1 could be differentiated from individuals infected with wild-type strains of HIV-1 by characterizing the Nef antibody response of cohort members. DESIGN: Retrospective and prospective analysis of the nef gene sequence and the antibody response to Nef peptides in HIV-infected subjects. METHODS: Plasma was obtained from all individuals of the Sydney cohort, and from a variety of HIV-1-infected and uninfected controls. Antibodies recognizing full-length recombinant HIV-1NL43 Nef protein and synthetic peptide analogues were assessed by enzyme-linked immunosorbent assay. RESULTS: All 34 individuals infected with wild-type HIV-1 had antibodies reacting with full-length Nef protein as well as with a series of synthetic peptides (6-23-mers) spanning most of the Nef protein of HIV-1NL43. Although the HIV-1 quasispecies infecting the Sydney cohort had a consensus deletion of the nef gene corresponding to amino-acids 165-206, HIV-1 strains from individual members of the cohort had additional deletions comprising up to 80% of the nef gene. Members of the cohort had antibodies to peptides homologous to all regions of the Nef protein tested, except for a single peptide (amino-acids 162-177) that lies within the consensus nef deletion for the cohort quasispecies. CONCLUSION: These data show that nef-deleted strains of HIV-1 can be detected serologically. In the Sydney cohort, detection of antibodies to all regions of Nef tested, except that corresponding to amino-acids 162-177, suggests that observed deletions outside this domain occurred after this virus had infected these subjects and stimulated an immune response. A Nef peptide serological assay may be useful for identifying further examples of individuals infected with nef-deleted, attenuated HIV-1 quasispecies and for assessing the evolution of those variants in vivo.

Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies.

OBJECTIVE: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. DESIGN: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. METHODS: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. RESULTS: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. CONCLUSIONS: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.

Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies.

An enzyme immunoassay (EIA) utilizing a synthetic peptide analogue of HIV gp41 (amino acids 579-599, RILAVERYLKDQQLLGIWGCS) as antigen was compared with two commercial assays (Genetic Systems, Abbott ENVACORE) for the ability to detect antibodies in the early stages of infection. Two panels, consisting of 96 sera from 15 people and 140 sera from 44 people seroconverting to HIV, were examined. In the first group the synthetic peptide assay (gp41 EIA) detected antibodies before the Genetic Systems EIA in seven out of 15 people and concurrently in the remaining eight. With the second panel the Abbott ENVACORE assay detected antibodies before the gp41 EIA in two out of 44 people while the gp41 EIA detected antibodies first in six out of 44. In the remaining 36 people antibodies were detected simultaneously by the two tests. The gp41 EIA usually detected anti-HIV antibodies before or concurrently with the two commercial assays examined suggesting that the epitope cluster represented by this peptide is recognized early in infection.

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma.

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Molecular and genetic aspects of the pathogenesis of viral infections of the central nervous system.

Viral pathogenesis can be defined in terms of a series of successive interactions between a virus and its target host. In order for a virus to injure a target organ such as the central nervous system (CNS), it must first enter the host animal, replicate in some primary site near its place of entry, spread from this site to the CNS and infect and injure specific populations of cells within the CNS. At each of these steps, the virus must avoid or overcome a variety of immunological and nonimmunological host defenses. It has recently become possible to begin to identify the role of specific viral genes and the proteins they encode at specific steps in the pathogenesis cycle. This review focuses on current knowledge concerning the molecular and genetic basis for the pathogenesis of viral infections of the CNS. Emphasis is placed on recent research with a wide variety of neurotropic viruses including reoviruses, bunyaviruses, lymphocytic choriomeningitis virus, rabies virus, polio virus, herpes viruses, lentiviruses, and the unconventional agents responsible for disease such as scrapie.

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.

N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.

Kinetics of HIV-1 replication and intracellular accumulation of particles in HTLV-I-transformed cells.

The replication kinetics of HIV were examined in HTLV-I-transformed MT-2 cells. The duration of the initial replication cycle was 20 hours, determined by the first detection of infectious progeny virus, development of syncytia, and production of viral RNA and protein. A phase of exponential virus production followed until 62 h postinfection. Cell death occurred in the final phase of infection during which infectious virus production remained constant even though viral RNA and protein production increased at an exponential rate. Accumulations of HIV particles were observed within cytoplasmic vacuoles of infected MT-2 cells. Although cell lysates contained high titers of infectious virus, our data show that an increasing proportion of particles produced late in infection were not infectious.

Synthetic peptide analogs of intercellular adhesion molecule 1 (ICAM-1) inhibit HIV-1 replication in MT-2 cells.

On the basis of reports demonstrating possible roles for leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), the ligand for LFA-1, in human immunodeficiency virus type 1 (HIV-1) infection, we have explored the involvement of the ICAM-1 molecule by using selected synthetic peptides derived from the protein sequence. Replication was assessed in MT-2 cells, highly susceptible to HIV infection, in the presence of four synthetic peptides derived from the ICAM-1 amino acid sequence. This cell type was chosen for the ability to form marked syncytia on infection with cell-free virus. Under the conditions used, minimal or no cytotoxicity was observed with the peptides up to concentrations of 50 micrograms/ml. A peptide corresponding to a unique region of ICAM-1, JF9 [ICAM-1(367-394, A-378)], had little effect on virus replication despite its ability to inhibit cell-cell adhesion. In contrast, an N-terminal peptide, JF7B [ICAM-1(1-23)], consistently inhibited virus replication in MT-2 cells in a dose-dependent manner, as measured by cell-free reverse transcriptase (RT) activity (up to 70% inhibition), soluble virus antigen production (up to 60% inhibition), and syncytium formation (virtually complete inhibition up to 6 days post infection). Testing of W-CAM-1 antibody, and anti-ICAM-1 antibody that inhibits cell-cell adhesion, revealed no significant inhibitory effects on RT activity, virus antigen production, and syncytium formation in HIV-1-infected MT-2 cells at a level that markedly inhibited cell-cell adhesion (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of fusion peptide homologs in blocking cell lysis and HIV-induced fusion.

Contrary to earlier reports, we have found that tri- and hexapeptides analogous or homologous with segments of the 23-residue N-terminal fusion sequence (FS) of the viral transmembrane glycoprotein gp41 (residues 517-539) did not significantly inhibit HIV-1-induced syncytium formation, using an uninfected cell-infected cell fusion assay. In contrast, we found that the high molecular weight apolipoprotein A-1 and a 23-residue analog of the FS, with the phenylalanine residues at positions 524 and 527 replaced with alanine residues, were effective inhibitors. Although the tripeptides were ineffective as inhibitors of syncytium formation, we found a number of them inhibited red cell lysis induced by the synthetic peptide AVGIGALFLGFLGAAGSTMGARS (based on the HIV-1 gp41 FS). This effect was also seen with apolipoprotein A-1. The Ala524,527 analog of the fusion sequence could not be tested in this system because it was hemolytic. We concluded that the smaller peptides were effective inhibitors of hemolysis because they interfered with pore formation by the fusion sequence peptide, either by disrupting the pores or by preventing the peptide from adopting the alpha-helical conformation found in the pores. On the other hand, membrane fusion, which is a prelude to syncytium formation, has been shown to require the fusion sequence in the beta-strand conformation. We argue that small peptides would be unable to block interaction between such strands, although larger molecules, such as apolipoprotein A-1 and the Ala524,527 analog, would be able to do so and thus inhibit fusion. It seems, therefore, that a successful drug directed against the FS-cell membrane interaction stage of syncytium formation would need to be of relatively high molecular weight and complexity.

Quantitation of infectious human immunodeficiency virus using a modified plaque forming assay.

This report describes a modified plaque forming assay which has been used for quantitation of infectious human immunodeficiency virus (HIV) in cell culture supernatant fluid. The number of infectious units determined by the plaque assay correlates closely with the 50% infectious dose determined by a conventional assay in cell culture. In contrast, particle associated reverse transcriptase (RT) activity correlates poorly with the amount of infectious HIV present in culture supernatant fluids.