High-throughput profiling of point mutations across the HIV-1 genome

Background

The HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance.

Results

Here we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions – representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket.

Conclusion

The approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.

     

SIRT3 deficiency decreases oxidative metabolism capacity but increases lifespan in male mice under caloric restriction

Mitochondrial NAD⁺‐dependent protein deacetylase Sirtuin3 (SIRT3) has been proposed to mediate calorie restriction (CR)‐dependent metabolic regulation and lifespan extension. Here, we investigated the role of SIRT3 in CR‐mediated longevity, mitochondrial function, and aerobic fitness. We report that SIRT3 is required for whole‐body aerobic capacity but is dispensable for CR‐dependent lifespan extension. Under CR, loss of SIRT3 (Sirt3 ^−/− ) yielded a longer overall and maximum lifespan as compared to Sirt3 ^+/+ mice. This unexpected lifespan extension was associated with altered mitochondrial protein acetylation in oxidative metabolic pathways, reduced mitochondrial respiration, and reduced aerobic exercise capacity. Also, Sirt3 ^−/− CR mice exhibit lower spontaneous activity and a trend favoring fatty acid oxidation during the postprandial period. This study shows the uncoupling of lifespan and healthspan parameters (aerobic fitness and spontaneous activity) and provides new insights into SIRT3 function in CR adaptation, fuel utilization, and aging.     

Exposure to N-ethyl-N-nitrosourea in adult mice alters structural and functional integrity of neurogenic sites

Background

Previous studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis.

Methodology/Principal Findings

2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively.

Conclusions/Significance

The results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits.     

The possible role of L-carnitine on the skeletal muscle of ovariectomized rats

Low muscle strength is observed during the peri-and postmenopausal periods, when the secretion of ovarian hormones is drastically reduced. It is also a predictive of adverse health events as well as incident mobility limitation and disability. The objective of the present study is to study the biochemical and the histological changes in the skeletal muscle of premature menopause-induced rats and the possible protective role of L-carnitine. Ovariectomized (OVX) rats were gavaged with L-carnitine (100 mg/kg) daily for 60 days starting from the second post-operative day. Serum levels of estradiol and markers of skeletal muscle damage (creatine kinase and lactic dehydrogenase activities) were determined. Light and electron microscopic study of the quadriceps femoris muscle (QFM) specimens were done. OVX rats showed significant decrease in the serum estradiol level with significant increase the markers for skeletal muscle damage. Histopathological examination of the QFM showed degenerated myofibers, apoptotic changes and compensatory hypertrophy. Degenerated mitochondria, multiple lysosomes and lipid droplets among the damaged myofibrils were also noticed. L-carnitine administration to the OVX rats resulted in non-significant change in the serum estradiol level with significant attenuation of skeletal muscle damage either biochemically or histopathologically. In conclusion, L-carnitine administration recovered muscle degeneration after ovariectomy. This finding suggested that L-carnitine could be recommended in the management of post-menopausal myopathy.     

Stress-induced changes in gene interactions in human cells

Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.