Massive Sorghum Collection Genotyped with SSR Markers to Enhance Use of Global Genetic Resources

Large ex situ collections require approaches for sampling manageable amounts of germplasm for in-depth characterization and use. We present here a large diversity survey in sorghum with 3367 accessions and 41 reference nuclear SSR markers. Of 19 alleles on average per locus, the largest numbers of alleles were concentrated in central and eastern Africa. Cultivated sorghum appeared structured according to geographic regions and race within region. A total of 13 groups of variable size were distinguished. The peripheral groups in western Africa, southern Africa and eastern Asia were the most homogeneous and clearly differentiated. Except for Kafir, there was little correspondence between races and marker-based groups. Bicolor, Caudatum, Durra and Guinea types were each dispersed in three groups or more. Races should therefore better be referred to as morphotypes. Wild and weedy accessions were very diverse and scattered among cultivated samples, reinforcing the idea that large gene-flow exists between the different compartments. Our study provides an entry to global sorghum germplasm collections. Our reference marker kit can serve to aggregate additional studies and enhance international collaboration. We propose a core reference set in order to facilitate integrated phenotyping experiments towards refined functional understanding of sorghum diversity.     

Galleria mellonella as a model for studying Enterococcus faecium host persistence

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleria mellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process.     

The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires the combined activation of histone deposition with the firing of replication origins. We report here the direct interaction of chromatin assembly factor 1 (CAF1), a key factor involved in histone deposition, with the replication kinase Cdc7-Dbf4. We isolated a complex containing both the largest subunit of CAF1 (p150) and the Cdc7-Dbf4 kinase specifically in S phase and thus prove the existence of this interaction in vivo. We then show that the Cdc7-Dbf4 kinase efficiently phosphorylates p150. This event induces a change in p150 oligomerization state, which promotes binding to proliferating cell nuclear antigen (PCNA). Conversely, CAF1 recruitment is reduced in a PCNA/DNA loading assay using Cdc7-depleted extracts. Our data define p150 as a new target for this kinase with implications for the coordination between DNA replication and CAF1 functions.     

Chronic High-Fat Diet Impairs Collecting Lymphatic Vessel Function in Mice

Lymphatic vessels play an essential role in intestinal lipid uptake, and impairment of lymphatic vessel function leads to enhanced adipose tissue accumulation in patients with lymphedema and in genetic mouse models of lymphatic dysfunction. However, the effects of obesity on lymphatic function have been poorly studied. We investigated if and how adipose tissue accumulation influences lymphatic function. Using a lymphatic specific tracer, we performed in vivo near-infrared (NIR) imaging to assess the function of collecting lymphatic vessels in mice fed normal chow or high-fat diet (HFD). Histological and whole mount analyses were performed to investigate the morphological changes in initial and the collecting lymphatic vessels. HFD was associated with impaired collecting lymphatic vessel function, as evidenced by reduced frequency of contractions and diminished response to mechanostimulation. Moreover, we found a significant negative correlation between collecting lymphatic vessel function and body weight. Whole mount analyses showed an enlargement of contractile collecting lymphatic vessels of the hind limb. In K14-VEGF-C mice, HFD resulted in a reduced spreading of the tracer within dermal lymphatic vessels. These findings indicate that adipose tissue expansion due to HFD leads to a functional impairment of the lymphatic vasculature, predominantly in collecting lymphatic vessels.     

Sensitivity of Five Rapid HIV Tests on Oral Fluid or Finger-Stick Whole Blood: A Real-Time Comparison in a Healthcare Setting

Background

Health authorities in several countries recently recommended the expansion of human immunodeficiency virus (HIV) antibody testing, including the use of rapid tests. Several HIV rapid tests are now licensed in Europe but their sensitivity on total blood and/or oral fluid in routine healthcare settings is not known.

Methods and Findings

200 adults with documented HIV-1 (n = 194) or HIV-2 infection (n = 6) were prospectively screened with five HIV rapid tests using either oral fluid (OF) or finger-stick whole blood (FSB). The OraQuick Advance rapid HIV1/2® was first applied to OF and then to FSB, while the other tests were applied to FSB, in the following order: Vikia HIV 1/2®, Determine HIV 1–2®, Determine® HIV-1/2 Ag/Ab Combo® and INSTI HIV-1/HIV-2®. Tests negative on FSB were repeated on paired serum samples. Twenty randomly selected HIV-seronegative subjects served as controls, and the results were read blindly. Most patients had HIV-1 subtype B infection (63.3%) and most were on antiretroviral therapy (68.5%). Sensitivity was 86.5%, 94.5%, 98.5%, 94.9%, 95.8% and 99% respectively, with OraQuick OF, OraQuick FSB, Vikia, Determine, Determine Ag/Ab Combo and INSTI (p<0.0001). OraQuick was less sensitive on OF than on FSB (p = 0.008). Among the six patients with three or more negative tests, two had recent HIV infection and four patients on antiretroviral therapy had undetectable plasma viral load. When patients positive in all the tests were compared with patients who had at least one negative test, only a plasma HIV RNA level <200 cp/ml was significantly associated with a false-negative result (p = 0.009). When the 33 rapid tests negative on FSB were repeated on serum, all but six (5 negative with OraQuick, 1 with INSTI) were positive. The sensitivity of OraQuick, Determine and Determine Ag/Ab Combo was significantly better on serum than on FSB (97.5%, p = 0.04; 100%, p = 0.004; and 100%, p = 0.02, respectively).

Conclusion

When evaluated in a healthcare setting, rapid HIV tests were less sensitive on oral fluid than on finger-stick whole blood and less sensitive on finger-stick whole blood than on serum.     

Functionality of Sortase A in Lactococcus lactis

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.The GRAS (generally recognized as safe) status of lactic acid bacteria (LAB) has catalyzed a myriad of promising applications using these bacteria as a vehicle for in situ delivery of bioactive proteins such as antigens or digestive enzymes in the gastrointestinal tract of the human host (4, 26). In the context of therapeutic applications of LAB, a major fundamental goal is to determine whether they can be engineered to deliver bioactive proteins to the right bacterial and host locations. We previously designed a protein-targeting system in LAB that addressed proteins to the desired bacterial site (i.e., cytoplasm, cell wall, or external medium), as validated using a model protein reporter and various antigens (14, 15). Studies investigating the use of LAB as vaccine delivery vehicles suggested that the cell-wall-anchored protein form may possess superior ability to induce a strong immune response (3, 14). Among the various surface display systems described in Gram-positive bacteria (13), a dedicated surface protein anchoring system catalyzed by sortases was first described and characterized in Staphylococcus aureus (29). It covalently anchors proteins via their C-terminal cell wall anchor (CWA) domain to the bacterial peptidoglycan. SrtA-like sortases process proteins bearing an LPXTG C-terminal motif and are considered to be the housekeeping sortase that anchors most proteins harboring a sorting signal (32). Other sortases were subsequently shown to anchor proteins bearing the same or other motifs (11, 16).Surprisingly, while the roles of sortases and LPXTG proteins are well documented in pathogens, few reports have examined these functions in other bacteria. A report suggests a relationship between sortase activity and adhesion of the LAB Lactobacillus salivarius, although direct involvement of sortase was not demonstrated (47). Recently, sortase activity was correlated to assembly of pili and adhesion properties in Lactobacillus rhamnosus (21). To further characterize sortase in LAB, we chose an industrially important member of this bacterial group, Lactococcus lactis, to study sortase A functionality in anchoring its putative substrates on the cell wall.     

Effective production of the biodiesel additive STBE by a continuous flow process

A new fuel additive, namely solketal tert-butyl ether (STBE), was developed and optimized under continuous flow conditions using a Corning? Advanced-Flow? glass reactor. STBE was obtained in two steps from glycerol, a renewable building-block produced in large amount in the processing of biodiesel. The advantages of the highly engineered Corning glass reactor included high mixing and heat-exchange efficiency, chemical resistance under corrosive flow conditions and a small hold-up. A robust, continuous, green and safe industrial-scale process is described.