A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.     

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.     

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.     

Next-generation sequencing for diagnosis of rare diseases in the neonatal intensive care unit

Background:Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU.Methods:We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children’s Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype–phenotype correlations.Results:Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys–Drash syndrome.Interpretation:This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU.A rare disease is defined by a prevalence of less than 1 in 2000 individuals.¹ However, when considered in aggregate, 1%–2% of Canadians will manifest a rare disease in their lifetime.^2,3 These disorders can present in the newborn period, and a third of these young children will succumb to the disease in their first year of life.^3–5 Newborns who present with rare diseases typically require admission to a neonatal intensive care unit (NICU), where the standard of care includes exhaustive consultations and investigations to determine a molecular diagnosis. Reaching such a diagnosis is a challenge, given the considerable clinical and genetic heterogeneity associated with rare diseases; diagnosis is also confounded by the early stage of presentation, which is further accentuated in premature newborns. As a result, traditional genetic or metabolic investigations can be lengthy and expensive, and they often fail to arrive at a diagnosis in a timely manner.⁶The current approach during a medical genetics consultation begins with a clinical assessment, followed by diagnostic testing that usually includes sequential testing of one or more candidate genes or panels of candidate genes. This step often requires approval for out-of-country testing, as only a limited number of gene tests are available for clinical testing in Canada. If the result of the first test is negative, the clinician may consider testing the next most likely candidate gene, frequently with diminishing returns. This approach can take months or years and can be a frustrating process for the patient, family and clinicians providing care.⁷ The inability to arrive at a timely and efficient diagnosis represents a substantial lost opportunity, as a diagnosis can limit or even halt further invasive, and at times futile, investigations for the neonate. Importantly, an accurate diagnosis informs prognosis and may guide management decisions.The advent of next-generation sequencing has greatly advanced the ability to rapidly identify the novel genes responsible for disease.⁸ Whole-exome sequencing (sequencing of the coding portion of the genome) is beginning to be used on a clinical basis in tertiary care centres.^9,10 In these initial clinical cohort studies, a molecular diagnosis was provided by whole-exome sequencing for about 25% of families. The proportion increased to 31% when the patient’s parents were also analyzed.⁹ Another study used retrospective whole-genome sequencing to make a diagnosis in 57% of 35 children from the intensive care setting.¹¹Although whole-exome and whole-genome sequencing are powerful tools, important conditions are required for translation of these methods to the clinic or hospital setting. The availability of high-throughput sequencers, complex and costly infrastructure, and personnel with bioinformatics expertise are prerequisites. These resources may not be broadly available within some health care systems, and other strategies may be more relevant and effective.Another attractive alternative is analysis based on next-generation sequencing that focuses only on the clinically relevant genes with known associated clinical phenotypes.¹² This strategy offers several advantages over whole-exome or whole-genome sequencing — interpretation of variants may be more straight-forward, a higher depth of coverage can be readily achieved, and less infrastructure and fewer personnel are required — all of which contribute to a more rapid return of results.For this pilot study, we evaluated the performance of a targeted next-generation sequencing panel that included 4813 “disease-relevant” genes in a cohort of newborns with rare disease in the NICU and assessed the effectiveness of this method to accurately diagnose these critically ill babies.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Intensified Downstream Processing of Monoclonal Antibodies Using Membrane Technology

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention toward implementing membrane technologies. With the industry moving toward continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies are reviewed. The recent progress toward developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal are focused upon.     

Range‐wide genetic structure of a cooperative mouse in a semi‐arid zone: Evidence for panmixia

Landscape topography and the mobility of individuals will have fundamental impacts on a species’ population structure, for example by enhancing or reducing gene flow and therefore influencing the effective size and genetic diversity of the population. However, social organization will also influence population genetic structure. For example, species that live and breed in cooperative groups may experience high levels of inbreeding and strong genetic drift. The western pebble‐mound mouse (Pseudomys chapmani), which occupies a highly heterogeneous, semi‐arid landscape in Australia, is an enigmatic social mammal that has the intriguing behaviour of working cooperatively in groups to build permanent pebble mounds above a subterranean burrow system. Here, we used both nuclear (microsatellite) and mitochondrial (mtDNA) markers to analyse the range‐wide population structure of western pebble‐mound mice sourced from multiple social groups. We observed high levels of genetic diversity at the broad scale, very weak genetic differentiation at a finer scale and low levels of inbreeding. Our genetic analyses suggest that the western pebble‐mound mouse population is both panmictic and highly viable. We conclude that high genetic connectivity across the complex landscape is a consequence of the species’ ability to permeate their environment, which may be enhanced by “boom‐bust” population dynamics driven by the semi‐arid climate. More broadly, our results highlight the importance of sampling strategies to infer social structure and demonstrate that sociality is an important component of population genetic structure.     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 2

The continued SAR investigation of 2-amino-8-alkoxy quinolines as melanin concentrating hormone receptor-1 (MCHr1) antagonists is reported. Prior hit-to-lead efforts resulted in the identification of 1 as a robust MCHr1 antagonist. Further delineation of the structural parameters essential for MCHr1-binding affinity of this class of nontraditional GPCR ligands resulted in the identification of compounds such as 33, 34 and 37, which demonstrate single digit nanomolar antagonism of MCHr1-mediated Ca(2+) release. The synthesis and biological evaluation of these compounds are reported.