Elevated plasma dihydroorotate in Miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine.

BACKGROUND: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis. METHODS: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems. RESULTS: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO. CONCLUSION: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening.

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches.

UNLABELLED: By combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution of Bacteroidales ectosymbionts of the protist Barbulanympha from the hindguts of the wood-eating cockroach Cryptocercus punctulatus In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of the Barbulanympha ectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis of C. punctulatus hindgut symbionts exposed in vivo to (15)N2 supports the hypothesis that Barbulanympha ectosymbionts are capable of nitrogen fixation. This genomic and in vivo functional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions. IMPORTANCE: The ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protist Barbulanympha from the hindgut of the wood-eating cockroach Cryptocercus punctulatus In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

UNLABELLED: Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. CONCLUSION: Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts.

Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of (15)N-enriched ions from metabolically labeled (15)N-sphingolipids in the plasma membrane, using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids--both in living cells and during fixation of living cells--exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous (15)N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long-range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2.

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Expanded newborn screening in New South Wales: missed cases.

There have been few reports of cases missed by expanded newborn screening. Tandem mass spectrometry was introduced in New South Wales, Australia in 1998 to screen for selected disorders of amino acid, organic acid and fatty acid metabolism. Of 1,500,000 babies screened by 2012, 1:2700 were diagnosed with a target disorder. Fifteen affected babies were missed by testing, and presented clinically or in family studies. In three cases (cobalamin C defect, very-long-chain acyl-CoA dehydrogenase deficiency and glutaric aciduria type 1), this led to modification of analyte cut-off values or protocols during the first 3 years. Two patients with intermittent MSUD, two with ß-ketothiolase deficiency, two with citrin deficiency, two siblings with arginosuccinic aciduria, two siblings with homocystinuria, and one with cobalamin C defect had analyte values and ratios below the action limits which could not have been detected without unacceptable false-positive rates. A laboratory interpretation error led to missing one case of cobalamin C defect. Reference ranges, regularly reviewed, were not altered. For citrin deficiency, while relevant metabolites are detectable by tandem mass spectrometry, our cut-off values do not specifically screen for that disorder. Most of the missed cases are doing well and with no acute presentations although eight of 15 are likely to have been somewhat adversely affected by a late diagnosis. Analyte ratio and cut-off value optimisations are important, but for some disorders occasional missed cases may have to be tolerated to maintain an acceptable specificity, and avoid harm from screening.

Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice.

Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.

Morphology and molecular phylogeny of Staurojoenina mulleri sp. nov. (Trichonymphida, Parabasalia) from the hindgut of the kalotermitid Neotermes jouteli.

Staurojoenina is a large and structurally complex genus of hypermastigont parabasalians found in the hindgut of lower termites. Although several species of Staurojoenina have been described worldwide, all Staurojoenina observed to date in different species of North American termites have been treated as the same species, S. assimilis. Here, we characterize Staurojoenina from the North American termite Neotermes jouteli using light microscopy, scanning electron microscopy, and phylogenetic analysis of small subunit ribosomal RNA, and compare it with S. assimilis from its type host, Incisitermes minor. The basic morphological characteristics of the N. jouteli symbiont, including its abundant bacterial epibionts, are similar as far as they may be compared with existing data from S. assimilis, although not consistently identical. In contrast, we find that they are extremely distantly related at the molecular level, sharing a pairwise similarity of SSU rRNA genes comparable to that seen between different genera or even families of other parabasalians. Based on their evolutionary distance and habitat in different termite genera, we consider the N. jouteli Staurojoenina to be distinct from S. assimilis, and describe a new species, Staurojoenina mulleri, in honor of the pioneering parabasalian researcher, Miklos Muller.

Correlated SEM, FIB-SEM, TEM, and NanoSIMS imaging of microbes from the hindgut of a lower termite: methods for in situ functional and ecological studies of uncultivable microbes.

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.

An unusual case of neck pain.

The complaint of nontraumatic neck pain in a pediatric patient without fever or any other symptoms is unusual and can be very challenging. We present the case of a 4-year-old boy with imaging consistent with a rare diagnosis. This report discusses this diagnosis as well as the utility of advanced imaging and laboratory evaluations in the presentation of pediatric neck pain.

Human Variome Project country nodes: documenting genetic information within a country.

The Human Variome Project (http://www.humanvariomeproject.org) is an international effort aiming to systematically collect and share information on all human genetic variation. The two main pillars of this effort are gene/disease-specific databases and a network of Human Variome Project Country Nodes. The latter are nationwide efforts to document the genomic variation reported within a specific population. The development and successful operation of the Human Variome Project Country Nodes are of utmost importance to the success of Human Variome Project's aims and goals because they not only allow the genetic burden of disease to be quantified in different countries, but also provide diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research, respectively. Here, we report the discussions and recommendations that resulted from the inaugural meeting of the International Confederation of Countries Advisory Council, held on 12th December 2011, during the 2011 Human Variome Project Beijing Meeting. We discuss the steps necessary to maximize the impact of the Country Node effort for developing regional and country-specific clinical genetics resources and summarize a few well-coordinated genetic data collection initiatives that would serve as paradigms for similar projects.

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry.

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.

Induction and prevention of severe hyperammonemia in the spfash mouse model of ornithine transcarbamylase deficiency using shRNA and rAAV-mediated gene delivery.

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.

Genotype-phenotype correlations in CPT1A deficiency detected by newborn screening in Pacific populations.

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a long chain fatty acid oxidation disorder, typically presenting with hypoketotic hypoglycaemia and liver dysfunction during fasting and intercurrent illness. Classical CPT1A deficiency is a rare disease, although a milder 'Arctic variant' (p.P479L) is common in the Inuit population. Since the introduction of expanded metabolic screening (EMS), the newborn screening programmes of Hawai'i and New Zealand (NZ) have detected a significant increase in the incidence of CPT1A deficiency. We report 22 individuals of Micronesian descent (12 in NZ and 10 in Hawai'i), homozygous for a CPT1A c.100T>C (p.S34P) variant detected by EMS or ascertained following diagnosis of a family member. No individuals with the Micronesian variant presented clinically with metabolic decompensation prior to diagnosis or during follow-up. Three asymptomatic homozygous adults were detected following the diagnosis of their children by EMS. CPT1A activity in cultured skin fibroblasts showed residual enzyme activity of 26% of normal controls. Secondly, we report three individuals from two unrelated Niuean families who presented clinically with symptoms of classic CPT1A deficiency, prior to the introduction of EMS. All were found to be homozygous for a CPT1A c.2122A>C (p.S708R) variant. CPT1A activity in fibroblasts of all three individuals was severely reduced at 4% of normal controls. Migration pressure, in part due to climate change may lead to increased frequency of presentation of Pacific peoples to regional metabolic services around the world. Knowledge of genotype-phenotype correlations in these populations will therefore inform counselling and treatment of those detected by newborn screening.

Maternal riboflavin deficiency, resulting in transient neonatal-onset glutaric aciduria Type 2, is caused by a microdeletion in the riboflavin transporter gene GPR172B.

Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) molecules, required in biological oxidation-reduction reactions. We previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl-CoA dehydrogenation deficiency (MADD), which was corrected by riboflavin supplementation. The mother was then found to be persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. Two recently-identified riboflavin transporters G protein-coupled receptor 172B (GPR172B or RFT1) and riboflavin transporter 2 (C20orf54 or RFT2) were screened for mutations. Two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were found in GPR172B. In vitro functional studies of both missense variations showed that riboflavin transport was unaffected by these variations. Quantitative real-time PCR revealed a de novo deletion in GPR172B spanning exons 2 and 3 in one allele from the mother. We postulate that haploinsufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient riboflavin-responsive disease seen in her newborn infant. This is the first report of a genetic defect in riboflavin transport in humans.

Molecular and morphological analysis of the family Calonymphidae with a description of Calonympha chia sp. nov., Snyderella kirbyi sp. nov., Snyderella swezyae sp. nov. and Snyderella yamini sp. nov.

Calonymphids are a group of multinucleate, multiflagellate protists belonging to the order Cristamonadida (Parabasalia) that are found exclusively in the hindgut of termites from the family Kalotermitidae. Despite their impressive morphological complexity and diversity, few species have been formally described and fewer still have been characterized at the molecular level. In this study, four novel species of calonymphids were isolated and characterized: Calonympha chia and Snyderella yamini spp. nov., from Neotermes castaneus and Calcaritermes nearcticus from Florida, USA, and Snyderella kirbyi and Snyderella swezyae, spp. nov., from Calcaritermes nigriceps and Cryptotermes cylindroceps from Colombia. Each of these species was distinguished from its congeners by residing in a distinct host and by differences at the molecular level. Phylogenetic analyses of small subunit (SSU) rDNA indicated that the genera Calonympha and Stephanonympha were probably not monophyletic, though the genus Snyderella, previously only represented by one sequence in molecular analyses, appeared with these new data to be monophyletic. This was in keeping with the traditional evolutionary view of the group in which the morphology of the genus Snyderella is considered to be derived, while that of the genus Stephanonympha is ancestral and therefore probably plesiomorphic.

Symbiosis, morphology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus.

Anaerobic cellulolytic flagellate protists of the hindguts of lower termites and the wood-feeding cockroach Cryptocercus are essential to their host's ability to digest lignocellulose. Many have bacteria associated with their surfaces and within cytoplasmic vesicles-likely important symbioses as suggested by molecular and other data. Some of the most striking examples of these symbioses are in the parabasalid family Hoplonymphidae, but little or no data exist on the structural aspects of their symbioses, their relationships with bacteria through different life-cycle stages, or their diversity and phylogenetic relationships in Cryptocercus. We investigated these areas in the hoplonymphid genera Barbulanympha and Urinympha from Cryptocercus punctulatus using light and electron microscopy, and analysis of small subunit rRNA. Microscopy reveals variation in density of bacterial surface symbionts related to life-cycle stage, a glyococalyx possibly important in bacterial adhesion and/or metabolite exchange, and putative viruses associated with bacterial surface symbionts. Patterning of surface bacteria suggests protists emerging from the resistant (dormant) stage are colonized by a small population of bacterial cells, which then divide to cover their surface. Additionally, cytoplasmic protrusions from the protist are covered by bacteria. Phylogenetic analysis rejects the monophyly of Hoplonymphidae, suggesting multiple origins or losses of these bacterial symbioses.

Perinatal hypophosphatasia presenting as neonatal epileptic encephalopathy with abnormal neurotransmitter metabolism secondary to reduced co-factor pyridoxal-5'-phosphate availability.

We describe two neonates presenting with perinatal hypophosphatasia and severe epileptic encephalopathy resulting in death. Both had increased levels of urinary vanillactate, indicating functional deficiency of aromatic amino acid decarboxylase, a pyridoxal-5-phosphate (PLP)-dependent enzyme required for dopamine and serotonin biosynthesis. Clinical findings and results of subsequent metabolic investigations were consistent with secondary pyridoxine-deficient encephalopathy. These patients highlight the importance of tissue non-specific alkaline phosphatase in the neuronal PLP-dependent metabolism of neurotransmitters. In addition, the disturbance of PLP metabolism appears to underlie the predominant neurological presentation in our patients. We recommend the measurement of serum alkaline phosphatase (ALP) during the assessment of perinatal seizures.