Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Human papillomavirus 16 E6 and E7 oncoprotein expression alters microRNA expression in extracellular vesicles.

Extracellular vesicles released by cancer cells are mediators of intercellular communication that have been reported to contribute to carcinogenesis. Since they are readily detected in bodily fluids, they may also be used as cancer biomarkers. The E6/E7 oncoproteins drive human papillomavirus (HPV)-associated cancers, which account for approximately 5% of all human cancers worldwide. Here, we investigate how HPV16 E6/E7 oncogene expression in primary human epithelial cells alters miR expression in extracellular vesicles and compare these to changes in intracellular miR expression. Examining a panel of 68 cancer related miRs revealed that many miRs had similar expression patterns in cells and in extracellular vesicles, whereas some other miRs had different expression patterns and may be selectively packaged into extracellular vesicles. Interestingly, the set of miRs that may be selectively packaged in HPV16 E6/E7 extracellular vesicles is predicted to inhibit necrosis and apoptosis.

Human papillomavirus molecular biology.

Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses.

Perturbation of DROSHA and DICER expression by human papillomavirus 16 oncoproteins.

Many tumors, including cervical carcinoma, show dysregulated expression of the microRNA processing machinery, specifically DROSHA and DICER. Some cervical cancers exhibit chromosome 5p amplifications and DROSHA is the most significantly upregulated transcript and is observed in all tumors with 5p gain. DROSHA and DICER mRNA levels, however, are higher in HPV positive cancer lines than in an HPV negative cervical carcinoma line. We show that high-risk HPV E6/E7 expression in HPV negative C33A cervical carcinoma cells and primary human epithelial cell causes increased expression of DROSHA and DICER mRNA and protein. Most importantly, many DROSHA regulated microRNAs are dysregulated in HPV16 E6/E7 expressing cells. These results suggest that increased DROSHA levels contribute to HPV16 E6/E7 dysregulation of cellular microRNA expression.

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins.

The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. IMPORTANCE: High-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development.

Macacine Herpesvirus 1 in Long-Tailed Macaques, Malaysia, 2009-2011.

Macacine herpesvirus 1 (MaHV1; B virus) naturally infects macaques (Macaca spp.) and can cause fatal encephalitis in humans. In Peninsular Malaysia, wild macaques are abundant, and translocation is used to mitigate human-macaque conflict. Most adult macaques are infected with MaHV1, although the risk for transmission to persons who handle them during capture and translocation is unknown. We investigated MaHV1 shedding among 392 long-tailed macaques (M. fascicularis) after capture and translocation by the Department of Wildlife and National Parks in Peninsular Malaysia, during 2009-2011. For detection of MaHV1 DNA, PCR was performed on urogenital and oropharyngeal swab samples. Overall, 39% of macaques were shedding MaHV1 DNA; rates of DNA detection did not differ between sample types. This study demonstrates that MaHV1 was shed by a substantial proportion of macaques after capture and transport and suggests that persons handling macaques under these circumstances might be at risk for exposure to MaHV1.

Discovery of herpes B virus-encoded microRNAs.

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with approximately 80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs.

Single-walled carbon nanotubes in the intact organism: near-IR imaging and biocompatibility studies in Drosophila.

The ability of near-infrared fluorescence imaging to detect single-walled carbon nanotubes (SWNTs) in organisms and biological tissues has been explored using Drosophila melanogaster (fruit flies). Drosophila larvae were raised on food containing approximately 10 ppm of disaggregated SWNTs. Their viability and growth were not reduced by nanotube ingestion. Near-IR nanotube fluorescence was imaged from intact living larvae, and individual nanotubes in dissected tissue specimens were imaged, structurally identified, and counted to estimate a biodistribution.