Pharmacogenomics for Prader-Willi syndrome: caregiver interest and planned utilization.

Aim: The study aim was to determine caregiver interest and planned utilization of pharmacogenomic (PGx) results for their child with Prader-Willi syndrome. Methods: Caregivers consented to PGx testing for their child and completed a survey before receiving results. Results: Of all caregivers (n = 48), 93.8% were highly interested in their child's upcoming PGx results. Most (97.9%) planned to share results with their child's medical providers. However, only 47.9% of caregivers were confident providers would utilize the PGx results. Conclusion: Caregivers are interested in utilizing PGx but are uncertain providers will use these results in their child's care. More information about provider comfort with PGx utilization is needed to understand how PGx education would benefit providers and ultimately patients with PGx results.

IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains.

Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

Structural and antigenic characterization of a computationally-optimized H5 hemagglutinin influenza vaccine.

Influenza A virus is a leading cause of death worldwide. Viruses of the H5 subtype have the potential to induce high mortality, and no vaccines are currently available to protect against H5 influenza viruses in the event of an outbreak. Experimental vaccination with one clade 2 virus does not protect against other subclades. The computationally optimized broadly reactive (COBRA) methodology was previously used to generate a H5 hemagglutinin (HA) antigen (COBRA2) that elicited increased serological breadth against multiple clade 2 H5N1 influenza viruses. In this report, we structurally and antigenically characterized the COBRA2 HA antigen. We examined the biochemical characteristics of the COBRA2 protein and determined the protein is correctly cleaved, properly folded into a trimeric structure, and antigenically correct by probing with HA head- and stem-specific monoclonal antibodies (mAbs). We further probed the antigenicity by examining binding of a panel of H5 mouse mAbs to the COBRA2 antigen, as well as several other HA antigens. We determined the X-ray crystal structure of the COBRA2 HA antigen to 2.8 Šand the protein was observed to be in the expected trimeric form. The COBRA2 HA was structurally similar to the naturally occurring H5 HA antigens and suggests the protein folds similar to known HA structures. Overall, our data allow us to formulate a hypothesis on the mechanism of increased breadth due to vaccination with the COBRA2 HA antigen, which is that the protein incorporates antigenic sites from numerous HA antigens, and elicits mAbs with limited breadth, but with diversity in targeted antigenic sites.

A Potent Neutralizing Site III-Specific Human Antibody Neutralizes Human Metapneumovirus In Vivo.

Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV; however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs, termed MPV196, MPV201, MPV314, and MPV364, are described. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro Binding studies with monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity for monomeric hMPV F compared to the other three mAbs, yet binding experiments with trimeric hMPV F showed limited differences in binding affinity, suggesting that MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, both of which cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests that it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope yet is unique from previously discovered site III mAbs, as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo and found that it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development and provide insights into hMPV vaccine development.

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

The biosynthesis of UDP-d-FucNAc-4N-(2)-oxoglutarate (UDP-Yelosamine) in Bacillus cereus ATCC 14579: Pat and Pyl, an aminotransferase and an ATP-dependent Grasp protein that ligates 2-oxoglutarate to UDP-4-amino-sugars.

Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.

In-microbe formation of nucleotide sugars in engineered Escherichia coli.

Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated "in-microbe", reactions occur within 2 to 4 h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5 µg/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP-2-acetamido-2-deoxy-glucuronic acid (UDP-GlcNAcA) and UDP-2-acetamido-2-deoxy-xylose (UDP-XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP-galacturonic acid (UDP-GalA) and UDP-galactose (UDP-Gal). ¹³C- and ¹5N-labeled NDP-sugars are formed using [¹³C] glucose as the carbon source and with [¹5N]NH4Cl as the nitrogen source.