Neither the HIV Protease Inhibitor Lopinavir-Ritonavir nor the Antimicrobial Trimethoprim-Sulfamethoxazole Prevent Malaria Relapse in Plasmodium cynomolgi-Infected Non-Human Primates

Plasmodium vivax malaria causes significant morbidity and mortality worldwide, and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax relapses. HIV and P. vivax malaria geographically overlap in many areas of the world, including South America and Asia. Despite the increasing body of knowledge regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no data regarding the effects of these treatments on P. vivax''s hypnozoite form and clinical relapses of malaria. We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remains untested. Separately, although other antifolates have been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole, commonly used in HIV infection and exposure management, has not been evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we investigated the antimalarial activity of these drugs on Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested. Because HIV and malaria geographically overlap, and more patients are being managed for HIV infection and exposure, understanding HIV drug impact on malaria infection is important.     

Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site

An unusually thick (~1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related to Leptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within the Archaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating with Acidimicrobium were detected. Some of these were closely related to “Ferromicrobium acidophilus,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.     

Effect of clinician information sessions on diagnostic testing for Chagas disease

BackgroundChagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.Methodology/Principal findingsWe conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).Conclusion/SignificanceIn this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.     

QTL mapping and phenotypic variation for root architectural traits in maize (Zea mays L.)

Key message

QTL were identified for root architectural traits in maize.

Abstract

Root architectural traits, including the number, length, orientation, and branching of the principal root classes, influence plant function by determining the spatial and temporal domains of soil exploration. To characterize phenotypic patterns and their genetic control, three recombinant inbred populations of maize were grown for 28 days in solid media in a greenhouse and evaluated for 21 root architectural traits, including length, number, diameter, and branching of seminal, primary and nodal roots, dry weight of embryonic and nodal systems, and diameter of the nodal root system. Significant phenotypic variation was observed for all traits. Strong correlations were observed among traits in the same root class, particularly for the length of the main root axis and the length of lateral roots. In a principal component analysis, relationships among traits differed slightly for the three families, though vectors grouped together for traits within a given root class, indicating opportunities for more efficient phenotyping. Allometric analysis showed that trajectories of growth for specific traits differ in the three populations. In total, 15 quantitative trait loci (QTL) were identified. QTL are reported for length in multiple root classes, diameter and number of seminal roots, and dry weight of the embryonic and nodal root systems. Phenotypic variation explained by individual QTL ranged from 0.44 % (number of seminal roots, NyH population) to 13.5 % (shoot dry weight, OhW population). Identification of QTL for root architectural traits may be useful for developing genotypes that are better suited to specific soil environments.     

Dynamic viral populations in hypersaline systems as revealed by metagenomic assembly

Viruses of the Bacteria and Archaea play important roles in microbial evolution and ecology, and yet viral dynamics in natural systems remain poorly understood. Here, we created de novo assemblies from 6.4 Gbp of metagenomic sequence from eight community viral concentrate samples, collected from 12 h to 3 years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Through extensive manual assembly curation, we reconstructed 7 complete and 28 partial novel genomes of viruses and virus-like entities (VLEs, which could be viruses or plasmids). We tracked these 35 populations across the eight samples and found that they are generally stable on the timescale of days and transient on the timescale of years, with some exceptions. Cross-detection of the 35 LT populations in three previously described haloviral metagenomes was limited to a few genes, and most previously sequenced haloviruses were not detected in our samples, though 3 were detected upon reducing our detection threshold from 90% to 75% nucleotide identity. Similar results were obtained when we applied our methods to haloviral metagenomic data previously reported from San Diego, CA: 10 contigs that we assembled from that system exhibited a variety of detection patterns on a timescale of weeks to 1 month but were generally not detected in LT. Our results suggest that most haloviral populations have a limited or, possibly, a temporally variable global distribution. This study provides high-resolution insight into viral biogeography and dynamics and it places "snapshot" viral metagenomes, collected at a single time and location, in context.     

Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses

Recent experimental evidence has demonstrated that bacteria acquire resistance to viruses by incorporation of short transcribed nucleotide sequences into regions of clustered regularly interspaced short palindromic repeats (CRISPR). We have analysed community genomic data from acidophilic microbial biofilms and discovered that evolution of the CRISPR regions in two distinct Leptospirillum group II bacteria occurs fast enough to promote individuality in otherwise nearly clonal populations. Comparative genomics strongly indicates very recent lateral transfer of the CRISPR locus between these populations, followed by significant loss of spacer sequences and locus expansion by unidirectional heterogeneous addition of new spacer sequences. Diversification of the CRISPR region is inferred to be a population-level response to the rapidly changing selective pressure of phage predation. Results reinforce the importance of phage–host interactions in shaping microbial ecology and evolution over very short time scales.     

Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G₁ phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.     

Sporicidal efficacy of pH-adjusted bleach for control of bioburden on production facility surfaces

pH-adjusted bleach was one of the agents used to disinfect contaminated public buildings in the USA following the 2001 bioterrorist attack with Bacillus anthracis spores. A USEPA fact sheet describes the preparation of pH-adjusted bleach by combining diluted sodium hypochlorite (NaOCl) with a controlled amount of 5 % acetic acid. This paper reports a modification of this procedure to qualify the use of pH-adjusted bleach for routine disinfection of cleanroom surfaces in pharmaceutical manufacturing facilities whenever a short contact time is desirable or there is a need for enhanced germicidal or sporicidal activity. Adjustment of pH was obtained reproducibly with either acetic acid or HCl, confirming the feasibility of developing standard procedures for the controlled addition of acid to diluted NaOCl solutions without compromising operator safety and convenience. Efficacy testing using spores from an in-house isolate of Bacillus pumilus confirmed that NaOCl solutions in the pH 5–8 range have much greater sporicidal activity on surfaces than do unadjusted alkaline solutions (pH > 11). With a contact time of 0.5 min, the log₁₀ reduction in spore viable counts was >5.4 for the five representative surfaces tested relative to untreated controls. Solutions of pH-adjusted NaOCl are known to be less stable than unadjusted alkaline solutions. Stability studies were performed by monitoring sporicidal efficacy, level of free available chlorine (FAC), and pH. Testing included several NaOCl concentrations and adjustment to different starting pHs. The efficacy of pH-adjusted solutions persisted in open containers for at least 12 h even though some FAC degradation occurred. In addition, solutions of 0.29 or 0.50 % NaOCl stored at room temperature protected from light retained efficacy for at least 4 weeks, indicating that short-term storage of solutions is possible following pH adjustment. The inorganic chemical degradation of pH-adjusted NaOCl solutions generates chlorate ion, an undesirable by-product. A comparison of chemical stability for 0.12, 0.25, and 0.50 % NaOCl solutions adjusted to different initial pHs indicated that the least chlorate formation occurred with 0.12 % NaOCl.     

Lactoferrin promotes bone growth

We have demonstrated bovine or human lactoferrin to be an anabolic factor in skeletal tissue. In vitro, lactoferrin stimulates the proliferation of bone forming cells, osteoblasts, and cartilage cells at physiological concentrations (above 0.1 microg/ml). The magnitude of this effect exceeds that observed in response to other skeletal growth factors such as IGF-1 and TGFbeta. DNA synthesis is also stimulated in a bone organ culture system likely reflecting the proliferation of cells of the osteoblast lineage. Lactoferrin is also a potent osteoblast survival factor. In TUNEL and DNA fragmentation assays, lactoferrin decreased apoptosis, induced by serum withdrawal, by up to 70%. In addition, lactoferrin has powerful effects on bone resorbing cells, osteoclasts, decreasing osteoclast development at concentrations > 1 microg/ml in a murine bone marrow culture system. However, lactoferrin did not alter bone resorption in calvarial organ culture, suggesting that it does not influence mature osteoclast function. In vivo, local injection of lactoferrin in adult mice resulted in increased calvarial bone growth, with significant increases in bone area and dynamic histomorphometric indices of bone formation after only 5 injections. Taken together, these data demonstrate that the naturally-occurring glycoprotein lactoferrin is anabolic to bone in vivo, an effect which is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis. Lactoferrin may therefore have a physiological role in bone growth, and a potential therapeutic role in osteoporosis.