Rapid Screening of Antimicrobial Synthetic Peptides

Increasing resistance to conventional antibiotics among microorganisms is one of the leading problems of medicine nowadays. Antimicrobial peptides are compounds exhibiting both antibacterial and antifungal activities. However, it is difficult to predict whether a designed new compound would exhibit any biological activity. Moreover, purification of the peptides is one of the most time-consuming and expensive steps of the synthesis that sometimes leads to unnecessary loss of solvents and reagents. In our study we have developed a thin-layer chromatography (TLC) direct bioautography technique for rapid determination of antimicrobial activity of peptides without the necessity of high-performance liquid chromatography purification. In this assay, crude peptides were applied and separated on a TLC plate. Then, pre-prepared plates were dipped into microbial suspension and incubated under optimum conditions for bacteria and fungi as well. The activity of the tested compounds was visualized by spraying the TLC plates with a cell viability reagent, resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide). Effectiveness of this assay was compared with minimal inhibitory concentration results obtained by broth microdilution assay. Interestingly, so far such a screening method has not been applied for this group of compounds.     

Transferrins,haptoglobins, and ceruloplasmins among tribal groups of Madagascar

Transferrin phenotypes of 403 Malagasy were determined by autoradiography after electrophoresis of plasma or serum. Three phenotypes, Tf C, Tf CD, and Tf D, were found. The frequency of Tf^D was 0.051 in the total sample, 0.041 in tribes of the Madagascar plateau, and 0.075 in tribes of the coastal and lowland regions. Haptoglobin phenotypes of 402 Malagasy were determined. The frequency of Hp¹ was 0.41 in the total sample, 0.38 in tribes of the plateau, and 0.49 in coastal-lowland tribes. There is a significant difference in the frequency of Hp¹ between the tribes of the plateau and those of the coastal and lowland regions. This confirms earlier observations of significant differences in frequencies of haptoglobins and hemoglobin S. Plasma or serum samples from 405 Malagasy were examined by electrophoresis to determine ceruloplasmin phenotypes. Seven distinct ceruloplasmin bands were observed. These were A, B, Galveston, Bridgeport, New Haven, Tananarive, and X, the last two described for the first time. Ten different phenotypes composed of one or two of the bands were observed. The frequency of the allele for ceruloplasmin B (CpB) was 0.78 in the total sample, 0.73 in tribes of the plateau, and 0.92 in coastal and lowland tribes. There is a significant difference in the frequency of this allele in the two major tribal groups, and this provides additional evidence of genetic differences between the peoples of the plateau and those of the coast and lowland regions of Madagascar.     

X-linked,polymorphic genetic variation of thyroxin-binding globulin (TBG) in baboons and screening of additional primates

X-linked polymorphic variation of thyroxin-binding globulin (TBG) is observed in several human groups. Isoelectric focusing of plasma samples labeled in vitro with [¹²⁵I]thyroxin, followed by autoradiography, also reveals genetically determined polymorphic electrophoretic variation in baboon TBG. The protein detected by this method in baboon plasma is immunologically similar to human TBG and is distinct from the other thyroxin-binding proteins, albumin and prealbumin. The isoelectric patterns of human and baboon TBG are very similar and both have an isoelectric range of pH 4.1 to 4.5. The baboon TBG polymorphism is inherited in a two-allele X-linked fashion, with a frequency of 72% for the common allele and 28% for the slow allele. A survey of seven other primate species including African green monkey, bonnet macaque, chimpanzee, crab-eating macaque, gorilla, rhesus monkey, and spider monkey revealed no polymorphic variation in TBG, although isoelectric patterns were similar to the human and baboon patterns. In addition, samples from pregnant chimpanzees demonstrate a pronounced quantitative anodal shift in relative band densities, a shift also observed in pregnant humans. This shift was not observed in samples from pregnant baboons. TBG should prove to be a useful X-linked genetic marker in baboons and provides a model of serum protein changes in pregnancy, at least in humans and chimpanzees.This research was supported by NIH Grant 2R01-EY-02388 and a Biomedical Research Support Grant from the Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston.     

High rates of energy expenditure and water flux in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea

We measured water flux and energy expenditure in free-ranging Point Reyes mountain beavers Aplodontia rufa phaea by using the doubly labeled water method. Previous laboratory investigations have suggested weak urinary concentrating ability, high rates of water flux, and low basal metabolic rates in this species. However, free-ranging measurements from hygric mammals are rare, and it is not known how these features interact in the environment. Rates of water flux (210+/-32 mL d(-1)) and field metabolic rates (1,488+/-486 kJ d(-1)) were 159% and 265%, respectively, of values predicted by allometric equations for similar-sized herbivores. Mountain beavers can likely meet their water needs through metabolic water production and preformed water in food and thus remain in water balance without access to free water. Arginine-vasopressin levels were strongly correlated with rates of water flux and plasma urea : creatinine ratios, suggesting an important role for this hormone in regulating urinary water loss in mountain beavers. High field metabolic rates may result from cool burrow temperatures that are well below lower critical temperatures measured in previous laboratory studies and suggest that thermoregulation costs may strongly influence field energetics and water flux in semifossorial mammals.     

Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.     

From foraging to farming in the southern Levant: the development of Epipalaeolithic and Pre-pottery Neolithic plant management strategies

This paper reviews the archaeobotanical record of the transition from foraging to farming in the southern Levant. The concise presentation of the published botanical evidence follows a critical assessment of: (a) the nature of Epipalaeolithic plant management strategies, (b) the place of the southern Levant in the polycentric development of Near Eastern plant cultivation and domestication, and (c) region-specific pathways for the emergence of domesticated crop “packages”. Some inferences are drawn and suggestions are made concerning the potential contribution of archaeobotanical research to questions of broader archaeological significance about socio-economic change in the southern Levant during the Pre-pottery Neolithic.     

Therapeutic Memory T Cells Require Costimulation for Effective Clearance of a Persistent Viral Infection

Persistent viral infections are a major health concern worldwide. During persistent infection, overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection, and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously, we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However, the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8⁺ and CD4⁺ memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers, illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen.Persistent viruses, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, cause major health problems worldwide and are extraordinarily difficult to clear following the establishment of persistence. Given the challenges associated with clearing persistent infections, it is important to develop and mechanistically understand therapeutic strategies that successfully achieve viral eradication without inducing permanent damage in the host. Studies using the lymphocytic choriomeningitis virus (LCMV) model system have convincingly demonstrated that a systemic persistent viral infection can be completely purged from a murine host by using a therapeutic approach referred to as adoptive immunotherapy (1, 15, 22, 29, 30). Remarkably, total body control of multiple persistent viral infections in both the mouse (1, 15, 22, 29, 30) and humans (8, 14, 24, 26, 31) can be achieved using adoptive immunotherapy. When mice are persistently infected at birth or in utero with LCMV (referred to as carrier mice), the virus establishes systemic persistence (6). Adult LCMV carrier mice are tolerant to the virus at the T-cell level and thus are unable to eradicate the pathogen (23), which provides an excellent model to study immunotherapeutic regimens. Immunocytotherapy relies on the adoptive transfer of virus-specific memory CD8 and CD4 T cells from LCMV-immune donor mice into recipient carrier mice (1, 15, 22, 29, 30). Following the therapeutic administration of memory cells, LCMV is purged from most peripheral tissues of carrier mice in 14 days, whereas more than 100 days are required to clear virus from the central nervous system (CNS) and kidneys (1, 15, 22). Furthermore, successful viral clearance requires antiviral “memory” but not “effector” T cells (11). Thus, in addition to its proven therapeutic relevance, this model also provides a paradigm to understand factors that regulate memory T cells following secondary exposure to pathogens in vivo.The mechanisms leading to activation of naïve T cells have been well described and involve recognition of major histocompatibility complex (MHC) peptide through the T-cell receptor (TCR) as well as costimulation (e.g., CD80 and CD86 interactions) (4, 25, 27). On the other hand, the factors that govern the activation and secondary expansion of memory CD8⁺ and CD4⁺ T cells are less clearly defined, particularly in an in vivo therapeutic setting. When memory T cells reencounter cognate antigen, they respond rapidly by producing cytokines and dividing. Previous studies indicated that there was no role for dendritic cells or costimulation (4, 27) in the reactivation of memory T cells; however, three recent studies have shown that dendritic cells (DCs) stimulate memory T-cell activity upon antigen rechallenge (2, 33) and during adoptive immunotherapy (15). Because MHC class I antigen (MHC-I) is expressed on nearly all cell types but costimulatory molecules are not, these three studies strongly suggested that DCs were influencing memory T cells with costimulatory pathways thought only to be required during priming. Indeed, when the issue was reexamined, it was revealed that memory CD8⁺ and CD4⁺ T cells require CD28-CD80/CD86 costimulation to be fully reactivated upon secondary exposure to antigen (3, 7, 21).Because therapeutically administered memory T cells require effective interactions with the host hematopoietic system (10), in particular dendritic cells (15), to achieve successful viral clearance, we set out to address several unanswered questions. First, is costimulation required for the immunotherapeutic clearance of an established persistent viral infection? This is a particularly important question because the requirements imposed on therapeutically administered memory T cells, which encounter immediate and overwhelmingly high levels of virus, heightened antigenic stimulation, and a unique inflammatory milieu, are likely to be different than those faced by endogenous memory T cells following pathogen rechallenge in an otherwise-quiescent environment. The second question we set out to address in this study was whether costimulation blockade could modulate the activities of an immunotherapeutic regimen consisting of memory T cells. This question is of great importance in a clinical setting where pathogen-specific memory T cells can induce severe tissue pathology through the release of effector molecules (12). Thus, it is critical to have a strategy to limit the magnitude of an undesirable response without impeding viral clearance.