Performance evaluation of IVC systems

An expert Working Group was set up in December 2000 to develop recommendations for users and industry on the evaluation of proper function and operation of individually ventilated cage (IVC) systems. The full report of their recommendations is in two parts--'Part 1: Test Instructions' and 'Part 2: Evaluation Criteria'--both of which have been published in full on the Laboratory Animals Ltd website. They can be found at http://www.lal.org.uk/IVC/index.html. Evaluation of and feedback on the recommendations to further refine their use and scientific basis is encouraged. This Summary Report provides a brief overview of the background to the development of the full report and the issues it addresses.     

Rhodopsin photoproducts in 2D crystals

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

MALDI‐TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and nano‐electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe‐IMAC column chromatography and subjected to LC‐MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI‐TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC‐MS/MS systems was comparable to that of using alternative proteases such as Asp‐N, Arg‐C, chymotrypsin, Glu‐C and Lys‐C on just one LC‐MS/MS instrument. Notably, MALDI‐TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (～20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC‐MALDI MS/MS can be a useful complement to LC‐nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.     

Molecular studies in 37 Silver-Russell syndrome patients: frequency and etiology of uniparental disomy

We report studies on the etiology of uniparental disomy (UPD) in Silver-Russell syndrome (SRS) patients. Thirty-seven SRS families were typed with short tandem repeat markers from chromosomes 2, 7, 9, 14, and 16. UPD for these chromosomes has either been described in association with growth retardation or has been observed in confined placental mosaicism, a mechanism that may result in UPD. Maternal UPD7 was detected in three SRS patients, accounting for approximately 10% of the tested SRS patients. These results agree with previously published studies. The allelic distribution in one of the three families indicates complete isodisomy, whereas allelic patterns in the other two families are consistent with partial and complete heterodisomy, respectively, suggesting that, in the latter cases, UPD originates from maternal meiosis, whereas in the first case, it seems to be of mitotic origin. STR typing for UPD of chromosomes 2, 9, 14, and 16 showed no abnormalities. Our results demonstrate the necessity of screening SRS patients for UPD7, although the effect of UPD7 cannot be correlated with the SRS phenotype as yet. An association between UPD for the other investigated chromosomes and SRS seems to be negligible. Received: 13 February 1997 / Accepted: 13 May 1997     

Can transgenerational plasticity contribute to the invasion success of annual plant species?

Adaptive transgenerational plasticity (TGP), i.e., significantly higher fitness when maternal and offspring conditions match, might contribute to the population growth of non-native species in highly variable environments. However, comparative studies that directly test this hypothesis are lacking. Therefore, we performed a reciprocal split-brood experiment to compare TGP in response to N and water availability in single populations of two invasive (Amaranthus retroflexus, Galinsoga parviflora) and two congeneric non-invasive introduced species (Amaranthus albus, Galinsoga ciliata). We hypothesized that the transgenerational effect is adaptive: (1) in invasive species compared with non-invasive adventives, and (2) in stressful conditions compared with resource-rich environments. The phenotypic variation among offspring was generated, in large part, by our experimental treatments in the maternal generation; therefore, we demonstrated a direct TGP effect on the offspring’s adult fitness. We found evidence, for the first time, that invasive and non-invasive adventive species differ regarding the expression of TGP in the adult stage, as adaptive responses were found exclusively in the invasive species. The manifestation of TGP was more explicit under resource-rich conditions; therefore, it might contribute to the population dynamics of non-native species in resource-rich sites rather than to their ecological tolerance spectra.     

High photobiont diversity in the common European soil crust lichen Psora decipiens

The genetic diversity of green algal photobionts (chlorobionts) in soil crust forming lichens was studied as part of the SCIN-project (Soil Crust InterNational). A total of 64 lichen samples were collected from four different sites along latitudinal and altitudinal gradients in Europe (Tabernas/Spain; Hochtor-Großglockner/Austria; Gynge Alvar/Sweden; Ruine Homburg/Germany). The dominant lichen species at all four sites was Psora decipiens, often occurring with Buellia elegans, Fulgensia bracteata, F. fulgens and Peltigera rufescens. Genetic identification of chlorobionts was carried out using the nuclear marker (nrITS) and a chloroplast marker (psbL-J). We found P. decipiens to be associated with several different species of Trebouxia and Asterochloris, although previously described to only have Asterochloris sp. The phylogenetic analyses revealed a high chlorobiont diversity with 12 well supported clades, including Trebouxia asymmetrica, T. jamesii, T. impressa and other, as yet taxonomically unidentified clades (Trebouxia sp. URa1-4, T. sp. URa6, T. sp. URa7-13). Additionally, five clades of Asterochloris were identified (A. magna, A. sp. URa14 -17). Most of the chlorobiont species appeared to be cosmopolitan, but five clades were unevenly distributed between the sampling sites with only Trebouxia being found in the warm and dry Spanish habitats and combinations of Trebouxia and Asterochloris in the cooler and more humid habitats. The wide range of chlorobiont species might contribute to the observed domination of P. decipiens at all four research sites of the SCIN project which range from a desert in Spain to an alpine site in the Alps of Austria.     

Complex temporal decay: a complete analysis

Relaxation dynamics is universal in science and engineering; its study serves to parameterize a system's response and to help identify a microscopic model of the processes involved. When measured data for a phenomenon cannot be fitted using one exponential, the choice of an alternative function to describe the decay becomes nontrivial. Here, we contrast two different, but fundamentally related approaches to fitting nontrivial decay curves; exponential decomposition and the gamma probability density function. Copyright © 2013 John Wiley & Sons, Ltd.     

Neutralization-Sensitive R5-Tropic Simian-Human Immunodeficiency Virus SHIV-2873Nip,Which Carries env Isolated from an Infant with a Recent HIV Clade C Infection

Human immunodeficiency virus clade C (HIV-C) accounts for >56% of all HIV infections worldwide. To investigate vaccine safety and efficacy in nonhuman primates, a pathogenic, R5-tropic, neutralization-sensitive simian-human immunodeficiency virus (SHIV) carrying HIV-C env would be desirable. We have constructed SHIV-2873Ni, an R5-tropic SHIV carrying a primary pediatric HIV-C env gene isolated from a 2-month-old Zambian infant, who died within 1 year of birth. SHIV-2873Ni was constructed using SHIV-1157ipd3N4 (R. J. Song, A. L. Chenine, R. A. Rasmussen, C. R. Ruprecht, S. Mirshahidi, R. D. Grisson, W. Xu, J. B. Whitney, L. M. Goins, H. Ong, P. L. Li, E. Shai-Kobiler, T. Wang, C. M. McCann, H. Zhang, C. Wood, C. Kankasa, W. E. Secor, H. M. McClure, E. Strobert, J. G. Else, and R. M. Ruprecht. J. Virol. 80:8729-8738, 2006) as the backbone, since the latter contains additional NF-κB sites in the long terminal repeats to enhance viral replicative capacity. The parental virus, SHIV-2873Ni, was serially passaged through five rhesus monkeys (RMs); SHIV-2873Nip, the resulting passaged virus, was reisolated from the fourth recipient about 1 year postinoculation. SHIV-2873Nip was replication competent in RM peripheral blood mononuclear cells of all random donors tested and was exclusively R5 tropic, and its env gene clustered with HIV-C by phylogenetic analysis; its high sensitivity to neutralization led to classification as a tier 1 virus. Indian-origin RMs were inoculated by different mucosal routes, resulting in high peak viral RNA loads. Signs of virus-induced disease include depletion of gut CD4⁺ T lymphocytes, loss of memory T cells in blood, and thrombocytopenia that resulted in fatal cerebral hemorrhage. SHIV-2873Nip is a highly replication-competent, mucosally transmissible, pathogenic R5-tropic virus that will be useful to study viral pathogenesis and to assess the efficacy of immunogens targeting HIV-C Env.Currently, 33 million people are living with human immunodeficiency virus (HIV)/AIDS (www.unaids.org), and the majority of them live in sub-Saharan Africa and South and Southeast Asia, including China and India, where HIV subtype C (HIV-C) circulates in >90% of the HIV-infected population (UNAIDS) (50). This distribution makes HIV-C the most prevalent subtype in the global pandemic, accounting for >56% of all HIV infections worldwide (www.unaids.org). Globally, HIV is one of the leading causes of childhood morbidity and mortality. Children account for 20% of all HIV-related deaths, 7% of individuals living with HIV, and 16% of new infections annually (reviewed in references 26, 29, and 38). In sub-Saharan Africa, HIV-C is responsible for approximately 50% of all infections, and a significant number of infections are in infants and children. HIV transmission from infected mothers to their infants is the primary mode of infection in children and can occur in utero, intrapartum, or postnatally through breast milk. The use of antiretroviral drugs has successfully reduced the rate of HIV infection in infants in the developed world to approximately 1%; nevertheless, such regimens have only recently become available in many of the developing nations where mother-to-child transmission of HIV is most significant (reviewed in references 26 and 38).Simian-human immunodeficiency viruses (SHIVs) are chimeric viruses that contain HIV envelope genes in the simian immunodeficiency virus (SIV) backbone. They have been used in a wide range of studies investigating lentiviral pathogenesis, antiviral immunity, virus-host interactions, mucosal transmission, and vaccine and drug efficacy (20). However, the majority of current SHIV strains utilize envelope genes derived from HIV clade B strains, which represent fewer than 10% of all global infections. Therefore, the available SHIV chimeras do not reflect the genetic diversity of the HIV epidemic, which is dominated by non-B clades, especially by HIV-C. Only a few studies have focused on developing anti-clade C Env vaccines (25, 27, 44, 49), with one efficacy study in primate models (44). To investigate lentiviral pathogenesis as well as anti-HIV-C vaccine safety and efficacy in nonhuman primate models, a pathogenic, CCR5-restricted, clade C SHIV (SHIV-C) would be very useful.Previously, we have generated an R5-tropic SHIV-C, SHIV-1157i (6, 51), which carries env from a 6-month-old Zambian infant born to an HIV-positive mother. During prospective long-term follow-up, this infant turned out to be a long-term nonprogressor who has remained asymptomatic at 8 years of age (61). The rhesus monkey (RM)-adapted strain, SHIV-1157ip, was pathogenic and has caused AIDS in several monkeys thus far, but with a relatively low rate of disease progression. AIDS developed in RMs between 127 and 300 weeks postinoculation (17a). A late virus was reisolated and engineered to contain extra NF-κB sites in the long terminal repeats (LTRs) (51); follow-up times for monkeys infected with this late form are not yet sufficient to assess development to AIDS, although signs of disease have developed. A possible explanation is that the env gene used to construct the original SHIV-1157i is an important determinant of the disease progression rate. The fact that the env gene was derived from a long-term nonprogressor may be linked to the relatively slow disease progression we observed in RMs infected with SHIV carrying the corresponding env gene.We sought to test whether constructing an R5-tropic SHIV with an env gene derived from a rapid progressor would give rise to a more virulent R5-tropic SHIV-C. Although HIV- or SIV-infected individuals with either typical rates of disease progression or long-term nonprogression have been studied extensively, few reports were focused on the virologic and immunologic characteristics of patients with rapid disease progression (9, 22). Patients who progress to AIDS within 1 to 2 years from the time of infection have been identified among infants and adults (7, 13, 34, 35, 46), with a higher frequency in infant populations. These patients demonstrate rapid loss of CD4⁺ T cells and lack potent cellular and humoral immune responses.Here we report the construction of SHIV-2873Ni, a chimera that carries env of an R5-tropic HIV-C strain isolated from a rapid progressor, a 2-month-old Zambian baby who died of AIDS-related disease within 1 year of birth. SHIV-2873Ni was serially passaged through five RMs; SHIV-2873Nip, the passaged virus, was reisolated and characterized from the fourth recipient about 1 year postinoculation when signs of disease were manifest. The RM-adapted virus caused T-cell depletion within a few months postinoculation.     

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background

HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models.

Methodology/Principal Findings

We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4⁺ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM.

Conclusions/Significance

SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.