Pyridoxine challenge reflects pediatric hypophosphatasia severity and thereby examines tissue-nonspecific alkaline phosphatase's role in vitamin B6 metabolism.

Alkaline phosphatase (ALP) is detected in most human tissues. However, ALP activity is routinely assayed using high concentrations of artificial colorimetric substrates in phosphate-free laboratory buffers at lethal pH. Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the ALP isoenzyme expressed in bone, liver, kidney, and elsewhere and is therefore designated "tissue-nonspecific" ALP (TNSALP). Consequently, HPP harbors clues concerning the biological function of this phosphohydrolase that is anchored onto the surface of cells. The biochemical signature of HPP features low serum ALP activity (hypophosphatasemia) together with elevated plasma levels of three natural substrates of TNSALP: i) phosphoethanolamine (PEA), a component of the linkage apparatus that binds ALPs and other proteins to the plasma membrane surface; ii) inorganic pyrophosphate (PPi), an inhibitor of bone and tooth mineralization; and iii) pyridoxal 5'-phosphate (PLP), the principal circulating vitameric form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving several hundred ALPL mutations underlies the remarkably broad-ranging expressivity of HPP featuring tooth loss often with muscle weakness and rickets or osteomalacia. Thus, HPP associates the "bone" isoform of TNSALP with biomineralization, whereas the physiological role of the "liver", "kidney", and other isoforms of TNSALP remains uncertain. Herein, to examine HPP's broad-ranging severity and the function of TNSALP, we administered an oral challenge of pyridoxine (PN) hydrochloride to 116 children with HPP. We assayed both pre- and post-challenge serum ALP activity and plasma levels of PLP, the B6 degradation product pyridoxic acid (PA), and the B6 vitamer pyridoxal (PL) that can enter cells. Responses were validated by PN challenge of 14 healthy adults and 19 children with metabolic bone diseases other than HPP. HPP severity was assessed using our HPP clinical nosology and patient height Z-scores. PN challenge of all study groups did not alter serum ALP activity in our clinical laboratory. In HPP, both the post-challenge PLP level and the PLP increment correlated (Ps < 0.0001) with the clinical nosology and height Z-scores (Rs = +0.6009 and + 0.4886, and Rs = -0.4846 and - 0.5002, respectively). In contrast, the plasma levels and increments of PA and PL from the PN challenge became less pronounced with HPP severity. We discuss how our findings suggest extraskeletal TNSALP primarily conditioned the PN challenge responses, and explain why they caution against overzealous B6 supplementation of HPP.

Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Phylogeny of North Americas largest cicada radiation redefines Tibicinoides and Okanagana (Hemiptera: Auchenorrhyncha: Cicadidae: Tibicininae).

Tibicinoides, with three small endemic California cicada species, has a confusing, intertwined systematic history with Okanagana that we unravel here. An ingroup including all species of Tibicinoides and the majority (84.7%) of Okanagana species were sampled for six gene regions, polarized with Clidophleps, Okanagodes, Subpsaltria, and Tibicina outgroups, and subjected to Bayesian phylogenetic analysis. Although the ingroup was monophyletic from all outgroups including Tibicina, Tibicinoides rendered Okanagana paraphyletic among two major ingroup clades. To bring classification into agreement with phylogeny, we redescribe and redefine Tibicinoides to include all Okanagana species with a hooked uncus in the male genitalia, all of which grouped with the type T. cupreosparsa (Uhler, 1889) in the first of these clades: T. boweni (Chatfield-Taylor & Cole, 2020) comb. n., T. catalina (Davis, 1936) comb. n., T. hesperia (Uhler, 1876) comb. n., T. mercedita (Davis, 1915), T. minuta (Davis, 1915), T. pallidula (Davis, 1917a) comb. n., T. pernix (Bliven, 1964) comb. n., T. rubrovenosa (Davis, 1915) comb. n., T. simulata (Davis, 1921) comb. n., T. striatipes (Haldeman, 1852) comb. n., T. uncinata (Van Duzee, 1915) comb. n., T. utahensis (Davis, 1919) comb. n., and T. vanduzeei (Distant, 1914) comb. n. Okanagana is redescribed and restricted to the species of the second major clade which contained the type O. rimosa (Say, 1830). We describe two new genera for morphologically distinct orphan lineages: Chlorocanta gen. nov. for C. viridis (Davis, 1918) comb. n. and Hewlettia gen. nov. for H. nigriviridis (Davis, 1921) comb. n. We recognize O. rubrobasalis Davis, 1926 stat. rev. as a species and relegate two former species to junior subjective synonyms: O. noveboracensis (Emmons, 1854) = O. canadensis (Provancher, 1889) and O. occidentalis (Walker in Lord, 1866) = O. lurida Davis, 1919. Tibicinoides and Okanagana together represent a rapid radiation that presents challenges to phylogenetic analysis including suboptimal outgroups and short internodes.

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron-sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 µM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

Electrically stimulated auras as a potential biomarker of the epileptogenic zone.

Electrocortical stimulation mapping (ESM) is often performed in patients undergoing stereoelectroencephalography (SEEG) prior to epilepsy surgery, with the goal of identifying functional cortex and preserving it postoperatively. ESM may also evoke a patient's typical seizure semiology. The purpose of this study was to determine whether the sites at which typical auras are evoked during ESM are associated with other known clinical and electrophysiologic biomarkers of the epileptogenic zone: the seizure onset zone (SOZ), the early spread zone (ES), and high-frequency oscillations (HFOs). We found that the sites at which auras were provoked were not consistently associated with known biomarkers (p = 0.09). We conclude that evoked auras during ESM may reflect electrical spread rather than true epileptogenicity, and that a larger study is needed to assess their potential value as independent epileptic biomarkers.

A revision of the shield-back katydid genus Neduba (Orthoptera: Tettigoniidae: Tettigoniinae: Nedubini).

The Nearctic shield-back katydid genus Neduba is revised. Species boundaries were demarcated by molecular phylogenetic analysis, morphology, quantitative analysis of calling songs, and karyotypes. Nine previously described species are redescribed: N. carinata, N. castanea, N. convexa, N. diabolica, N. extincta, N. macneilli, N. propsti, N. sierranus, and N. steindachneri, and twelve new species are described: N. ambagiosa sp. n., N. arborea sp. n., N. cascadia sp. n., N. duplocantans sp. n., N. inversa sp. n., N. longiplutea sp. n., N. lucubrata sp. n., N. oblongata sp. n., N. prorocantans sp. n., N. radicata sp. n., N. radocantans sp. n., and N. sequoia sp. n. We chose a lectotype for N. steindachneri and transferred N. picturata from a junior synonym of N. diabolica to a junior synonym of N. steindachneri. Diversification in this relict group reflects cycles of allopatric isolation and secondary contact amidst the tumultuous, evolving geography of western North America. The taxonomy and phylogenies presented in this revision lay the groundwork for studies of speciation, biogeography, hybrid zones, and behavioral evolution. Given that one Neduba species is already extinct from human environmental disturbance, we suggest conservation priorities for the genus.

Thirteen new species of Chilecicada Sanborn, 2014 (Hemiptera: Auchenorrhyncha: Cicadidae: Tibicininae) expand the highly endemic cicada fauna of Chile.

The genus Chilecicada Sanborn, 2014 is shown to be a complex of closely related species rather than a monospecific genus. Chilecicada citatatemporaria Sanborn Cole n. sp., C. culenesensis Sanborn Cole n. sp., C. curacaviensis Sanborn Cole n. sp., C. impartemporaria Sanborn Cole n. sp., C. magna Sanborn Cole n. sp., C. mapuchensis Sanborn n. sp., C. oraria Sanborn Cole n. sp., C. parrajaraorum Sanborn n. sp., C. partemporaria Sanborn Cole n. sp., C. pehuenchesensis Sanborn Cole n. sp., C. trifascia Sanborn n. sp., C. trifasciunca Sanborn Cole n. sp., and C. viridicitata Sanborn Cole n. sp. are described as new. Chilecicada occidentis Walker, 1850 is re-described to facilitate separation of the new species from the only previously known species. Song and cytochrome oxidase I analysis available for most species support the separation of the new taxa from the type species of the genus. Known species distributions and a key to the species of the genus are also provided. The new species increases the known cicada diversity 61.9% to 34 species, 91.2% of which are endemic to Chile.

A new species of Okanagana from the Walker Lane region of Nevada and California (Hemiptera: Auchenorrhyncha: Cicadidae).

Okanagana boweni sp. n. is described from the western margin of the Great Basin of North America. The new species is diagnosed from allopatric O. simulata Davis and sympatric O. utahensis Davis using morphological, bioacoustical, and molecular characters. The distribution of this new species coincides with the Walker Lane region that lies along the border of California and Nevada, USA. Based on geography, bioacoustics, morphology, and molecular phylogenetics, we hypothesize that O. boweni sp. n. is the allopatric sister species of O. simulata.

Intraindividual relative deficits in visual memory to lateralize seizure onset in temporal lobe epilepsy.

It is well established that presurgical neuropsychological assessment can assist in lateralizing and localizing focal epileptogenic regions. However, unlike verbal memory impairment, which is a robust and reliable finding in patients with left temporal lobe epilepsy (LTLE), nonverbal memory deficits are less consistently found among patients with right TLE (RTLE). This study aimed to determine whether memory assessment for spatial location in addition to visual content would differentiate patients with RTLE and LTLE. We compared performances between patients with 25 RTLE and 37 patients with LTLE on the Wechsler Advanced Clinical Solutions-Faces (ACS-F) subscales (Faces I, Faces II, Content, and Spatial), verbal-visual memory asymmetry scores, and intelligence quotient (IQ)-visual memory difference scores. Results revealed no significant differences between patients with RTLE and LTLE for any ACS-F memory score. By contrast, groups demonstrated significant differences in memory asymmetry scores (p = .007) and IQ difference scores (p = .006). Thus, visual memory scores in isolation failed to differentiate groups with RTLE and LTLE; however, within-patient differences between visual memory and other cognitive abilities successfully differentiated the groups. These results highlight the importance of using an intraindividual model of neuropsychological assessment to identify relative weaknesses potentially associated with the epileptogenic region.

Multilocus phylogeny of Gryllus field crickets (Orthoptera: Gryllidae: Gryllinae) utilizing anchored hybrid enrichment.

We present the first comprehensive molecular phylogeny of Gryllus field cricket species found in the United States and Canada, select additional named Gryllus species found in Mexico and the Bahamas, plus the European field cricket G. campestris Linnaeus and the Afro-Eurasian cricket G. bimaculatus De Geer. Acheta, Teleogryllus, and Nigrogryllus were used as outgroups. Anchored hybrid enrichment was used to generate 492,531 base pairs of DNA sequence from 563 loci. RAxML analysis of concatenated sequence data and Astral analysis of gene trees gave broadly congruent results, especially for older branches and overall tree structure. The North American Gryllus are monophyletic with respect to the two Old World taxa; certain sub-groups show rapid recent divergence. This is the first Anchored Hybrid Enrichment study of an insect group done for closely related species within a single genus, and the results illustrate the challenges of reconstructing the evolutionary history of young rapidly diverged taxa when both incomplete lineage sorting and probable hybridization are at play. Because Gryllus field crickets have been used extensively as a model system in evolutionary ecology, behavior, neuro-physiology, speciation, and life-history and life-cycle evolution, these results will help inform, interpret, and guide future research in these areas.

How Bioinformatic Tools Guide Experiments To Resolve the Chaos of Apparently Unlimited Metabolic Variation.

Hexuronic acids, oxidation products of common sugars, are widespread in eukaryotic cells. Galacturonic acid is the main carbohydrate component of pectin found in plant cell walls and glucuronic acid is a component of proteoglycans in animals. However, despite their importance as carbohydrate substrates, metabolism of hexuronic acids has long remained a poorly studied corner of the bacterial metabolic map. In the current issue of Journal of Bacteriology, Bouvier and coworkers present a detailed analysis of genes involved in hexuronate utilization in various proteobacteria and report the verification of their bioinformatics predictions by carefully designed experiments (J. T. Bouvier et al., J Bacteriol 201:e00431-18, 2019, https://doi.org/10.1128/JB.00431-18). This study provides a solid basis for understanding hexuronate metabolism and its regulation in other bacterial phyla.

Assessment of Naming in Non-native English Speakers with Epilepsy.

OBJECTIVES: Naming assessment is a core component of neuropsychological evaluation, particularly in the surgical work up for patients with pharmacologically refractory epilepsy. Specifically, naming deficits are typically associated with left, but not right hemisphere epilepsy, thereby assisting with lateralization of seizure onset. We sought to determine whether bilingual (English as second language, ESL) and monolingual epilepsy patients with comparable education, intelligence, and objective vocabulary performance would perform similarly on standard naming measures, and whether ESL patients would demonstrate laterality effects in naming, similar to that observed in monolingual patients. METHODS: Participants were 242 adults with epilepsy (186 native, 56 ESL) who underwent neuropsychological evaluation and obtained normal range or higher scores on the Wechsler Adult Intelligence Scale (R/III/IV) Vocabulary subtest (scaled score≥8). Groups were compared on demographic factors and language performances (i.e., Boston Naming Test, Auditory & Visual Naming Test, word reading, fluency). RESULTS: Groups did not differ with respect to age, education, FSIQ, vocabulary, reading, or verbal fluency. However, ESL speakers earned poorer scores than native English speakers on all naming measures. Moreover, among ESL participants with unilateral epilepsy, a significant proportion of right hemisphere patients scored below cutoff for impairment. This contrasted with the more typical finding among native English speakers, whereby a significant proportion of left patients demonstrated naming impairment. CONCLUSIONS: These results underscore the complexity of verbal assessment in bilinguals, suggesting that naming performances by ESL individuals, even those considered proficient, with strong performances on other English verbal measures, cannot be interpreted by the same standards applied for native speakers. (JINS, 2018, 24, 1057-1063).

Reestablishing a host-affiliate relationship: migratory fish reintroduction increases native mussel recruitment.

Co-extirpation among host-affiliate species is thought to be a leading cause of biodiversity loss worldwide. Freshwater mussels (Unionida) are at risk globally and face many threats to survival, including limited access to viable host fish required to complete their life history. We examine the relationship between the common eastern elliptio mussel (Elliptio complanata) and its migratory host fish the American eel (Anguilla rostrata), whose distribution in the Chesapeake Bay watershed is limited, in part, by dams. We examined population demographics of E. complanata across locations in the Chesapeake Bay watershed, primarily in the Susquehanna River in the absence of American eels, and conducted experimental restocking of eels to assess potential impacts on mussel recruitment. Compared to surveys completed ~20 yr prior, E. complanata could be experiencing declines at several historically abundant sites. These sites also had extremely limited evidence of recruitment. Restoration of host fish improved recruitment, but results were not equivalent between stocking sites, indicating that host reintroduction alone may not be fully effective in reestablishing mussel populations. One site where eels were introduced (Pine Creek, Tioga County, Pennsylvania, USA) experienced an increase from 0 juveniles found during quantitative surveys prior to eel stocking to 151 (21% of individuals collected during quantitative surveys) E. complanata juveniles found four years after stocking. A second site (Buffalo Creek, Union County, Pennsylvania) experienced a more moderate increase from 2 to 7 juveniles found during 2010 and 2014 quantitative surveys, respectively. Continued examination of other potential interacting factors affecting recruitment, including water quality or habitat conditions, is necessary to target favorable sites for successful restoration.

Development of a generic ß-lactamase screening system for improved signal peptides for periplasmic targeting of recombinant proteins in Escherichia coli.

Targeting of recombinant proteins to the Escherichia coli periplasm is a desirable industrial processing tool to allow formation of disulphide bonds, aid folding and simplify recovery. Proteins are targeted across the inner membrane to the periplasm by an N-terminal signal peptide. The sequence of the signal peptide determines its functionality, but there is no method to predict signal peptide function for specific recombinant proteins, so multiple signal peptides must be screened for their ability to translocate each recombinant protein, limiting throughput. We present a screening system for optimising signal peptides for translocation of a single chain variable (scFv) antibody fragment employing TEM1 ß-lactamase (Bla) as a C-terminal reporter of periplasmic localisation. The Pectobacterium carotovorum PelB signal peptide was selected as the starting point for a mutagenic screen. ß-lactamase was fused to the C-terminal of scFv and ß-lactamase activity was correlated against scFv translocation. Signal peptide libraries were generated and screened for ß-lactamase activity, which correlated well to scFv::Bla production, although only some high activity clones had improved periplasmic translocation of scFv::Bla. Selected signal peptides were investigated in fed-batch fermentations for production and translocation of scFv::Bla and scFv without the Bla fusion. Improved signal peptides increased periplasmic scFv activity by ~40%.