Safety considerations for dietary supplement manufacturers in the United States.

Due to significant dietary supplement use in the US, product manufacturers must understand the importance of implementing a robust approach to establishing safety for all ingredients, including dietary ingredients, components, and finished dietary supplement products. Different regulatory pathways exist by which the safety of dietary ingredients can be established, and thus allowed to be marketed in a dietary supplement. For individual dietary ingredients, safety information may come from a variety of sources including history of safe use, presence of the ingredient in foods, and/or non-clinical and clinical data. On occasion safety data gaps are identified for a specific ingredient, particularly those of botanical origin. Modern toxicological methods and models can prove helpful in satisfying data gaps and are presented in this review. For finished dietary supplement products, issues potentially impacting safety to consider include claims, product labeling, overages, contaminants, residual solvents, heavy metals, packaging, and product stability. In addition, a safety assessment does not end once a product is marketed. It is important that manufacturers actively monitor and record the occurrence of adverse events reported in association with the use of their products, in accordance with the law. Herein, we provide a comprehensive overview of considerations for assessing dietary supplement safety.

Reduced Simian Immunodeficiency Virus Replication in Macrophages of Sooty Mangabeys Is Associated with Increased Expression of Host Restriction Factors.

UNLABELLED: Macrophages are target cells of HIV/SIV infection that may play a role in AIDS pathogenesis and contribute to the long-lived reservoir of latently infected cells during antiretroviral therapy (ART). In previous work, we and others have shown that during pathogenic SIV infection of rhesus macaques (RMs), rapid disease progression is associated with high levels of in vivo macrophage infection. In contrast, during nonpathogenic SIV infection of sooty mangabeys (SMs), neither spontaneous nor experimental CD4(+) T cell depletion results in substantial levels of in vivo macrophage infection. To test the hypothesis that SM macrophages are intrinsically more resistant to SIV infection than RM macrophages, we undertook an in vitro comparative assessment of monocyte-derived macrophages (MDMs) from both nonhuman primate species. Using the primary isolate SIVM949, which replicates well in lymphocytes from both RMs and SMs, we found that infection of RM macrophages resulted in persistent SIV-RNA production while SIV-RNA levels in SM macrophage cultures decreased 10- to 100-fold over a similar temporal course of in vitro infection. To explore potential mechanisms responsible for the lower levels of SIV replication and/or production in macrophages from SMs we comparatively assessed, in the two studied species, the expression of the SIV coreceptor as well as the expression of a number of host restriction factors. While previous studies showed that SM monocytes express lower levels of CCR5 (but not CD4) than RM monocytes, the level of CCR5 expression in MDMs was similar in the two species. Interestingly, we found that SM macrophages exhibited a significantly greater increase in the expression of tetherin (P = 0.003) and TRIM22 (P = 0.0006) in response to alpha interferon stimulation and increased expression of multiple host restriction factors in response to lipopolysaccharide stimulation and exposure to SIV. Overall, these findings confirm, in an in vitro infection system, that SM macrophages are relatively more resistant to SIV infection compared to RM macrophages, and suggest that a combination of entry and postentry restriction mechanisms may protect these cells from productive SIV infection. IMPORTANCE: This manuscript represents the first in vivo comparative analysis of monocyte-derived macrophages (MDMs) between rhesus macaques, i.e., experimental SIV hosts in which the infection is pathogenic and macrophages can be infected, and sooty mangabeys, i.e., natural SIV hosts in which the infection is nonpathogenic and macrophages are virtually never infected in vivo. This study demonstrates that mangabey-derived MDMs are more resistant to SIV infection in vitro compared to macaque-derived MDMs, and provides a potential explanation for this observation by showing increased expression of specific retrovirus restriction factors in mangabey-derived macrophages. Overall, this study is important as it contributes to our understanding of why SIV infection is nonpathogenic in sooty mangabeys while it is pathogenic in macaques, and is consistent with a pathogenic role for in vivo macrophage infection during pathogenic lentiviral infection.

Decreased HIV type 1 transcription in CCR5-Δ32 heterozygotes during suppressive antiretroviral therapy.

Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P=.035), RNA to DNA transcriptional ratios (P=.013), and frequency of detectable HIV 2-long terminal repeat circular DNA (P=.013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2=0.136; P=.002). Our findings suggest that curative strategies should further explore manipulation of CCR5.

Site-specific integrase-mediated transgenesis in mice via pronuclear injection.

Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

Selection pressure on HIV-1 envelope by broadly neutralizing antibodies to the conserved CD4-binding site.

The monoclonal antibody (MAb) VRC01 was isolated from a slowly progressing HIV-1-infected donor and was shown to neutralize diverse HIV-1 strains by binding to the conserved CD4 binding site (CD4bs) of gp120. To better understand the virologic factors associated with such antibody development, we characterized HIV-1 envelope (Env) variants from this donor and five other donors who developed broadly neutralizing antibodies. A total of 473 env sequences were obtained by single-genome amplification, and 100 representative env clones were expressed and tested for entry and neutralization sensitivity. While VRC01 neutralizes about 90% of the genetically diverse heterologous HIV-1 strains tested, only selective archival Env variants from the VRC01 donor were sensitive to VRC01 and all of the Env variants derived from the donor plasma were resistant, indicating strong antibody-based selection pressure. Despite their resistance to this broadly reactive MAb that partially mimics CD4, all Env variants required CD4 for entry. Three other CD4bs MAbs from the same donor were able to neutralize some VRC01 escape variants, suggesting that CD4bs antibodies continued to evolve in response to viral escape. We also observed a relatively high percentage of VRC01-resistant Env clones in the plasma of four of five additional broadly neutralizing donors, suggesting the presence of CD4bs-directed neutralizing antibodies in these donors. In total, these data indicate that the CD4bs-directed neutralizing antibodies exert ongoing selection pressure on the conserved CD4bs epitope of HIV-1 Env.

Circadian rhythms in Neurospora crassa: Downstream effectors.

The circadian rhythm in Neurospora crassa is exhibited as alternating areas of conidiating and non-conidiating mycelia growth. A significant role in this circadian rhythm is played by the frq (frequency) and wc (white-collar) genes, comprising the "FWC" oscillator. Strains lacking the FWC can be restored to rhythmicity, which has been attributed to a second oscillator, called the FLO (frq-less oscillator). This study reports additional conditions that allow this rhythmicity to occur. Rhythmicity was restored to mutants lacking either the frq, or wc-1, or wc-2 genes in D/D (constant darkness) or L/L (constant light) by the addition of low levels of menadione, a known stimulator of ROS (reactive oxygen species). Additional studies are reported on the rhythm effects from caffeine, a known cAMP phospho-diesterase inhibitor as well as the effects from mutations in the csp-1 gene, the rco-1 gene, and other genes. A theme ties all of these "downstream effects" together, i.e., they affect either components thought to be part of the conidiation process itself, or the RAS-cAMP-protein kinase pathway. Since mutations in these components unexpectedly had rhythm effects, this suggests that these components may be good candidates for some part of the frq-less oscillator.

Select host restriction factors are associated with HIV persistence during antiretroviral therapy.

OBJECTIVE: The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. DESIGN: We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. METHODS: We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-long terminal repeat (2-LTR) circle HIV-1 DNA and immunophenotypes of CD4 T cells in 72 HIV-1-infected individuals on suppressive ART (23 individuals initiated ART less than 1 year post-infection, and 49 individuals initiated ART greater than 1 year post-infection). Correlations were analysed using nonparametric tests. RESULTS: The enhanced expression of a few select host restriction factors, p21, schlafen 11 and PAF1, was strongly associated with reduced CD4 T-cell associated HIV RNA during ART (P < 0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4 T-cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in individuals who initiated ART during early versus chronic infection and may contribute to the reduced reservoir size observed in these individuals. CONCLUSION: Intrinsic immune responses modulate HIV persistence during suppressive ART and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo.

Biomimetic nanocomposites to control osteogenic differentiation of human mesenchymal stem cells.

The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano-hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell-culture medium. Nanocomposites comprising type I collagen and nano-hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone-formation technologies to the clinic.

Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing.

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.