Cells Deploy a Two-Pronged Strategy to Rectify Misfolded Proinsulin Aggregates.

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.

How host ER membrane chaperones and morphogenic proteins support virus infection.

The multi-functional endoplasmic reticulum (ER) is exploited by viruses to cause infection. Morphologically, this organelle is a highly interconnected membranous network consisting of sheets and tubules whose levels are dynamic, changing in response to cellular conditions. Functionally, the ER is responsible for protein synthesis, folding, secretion and degradation, as well as Ca2+ homeostasis and lipid biosynthesis, with each event catalyzed by defined ER factors. Strikingly, these ER host factors are hijacked by viruses to support different infection steps, including entry, translation, replication, assembly and egress. Although the full repertoire of these ER factors that are hijacked is unknown, recent studies have uncovered several ER membrane machineries that are exploited by viruses - ranging from polyomavirus to flavivirus and coronavirus - to facilitate different steps of their life cycle. These discoveries should provide better understanding of virus infection mechanisms, potentially leading to the development of more effective anti-viral therapies.

Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a ß-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Hypochlorous acid as a disinfectant for high-risk HPV: Insight into the mechanism of action.

Medical instruments that are not autoclavable but may become contaminated with high-risk human papillomaviruses (HPVs) during use must be thoroughly disinfected to avoid the possibility of iatrogenic transmission of infection. There is an expectation that prolonged soaking of instruments in the United States Food and Drug Administration-cleared chemical disinfectant solutions will result in high-level decontamination, but HPV16 and HPV18 are known to be resistant to commonly used formulations. However, they are susceptible to a variety of oxidative agents, including those based on chlorine. Here, we tested the efficacy of homogeneous hypochlorous acid (HOCl) solutions against mature infectious virions of HPV16 and HPV18 dried onto butadiene styrene coupons and ultrasonic probes. Both viruses were inactivated to >4 log reduction value (LRV) after 15 s on coupons and 5 min on ultrasonic probes. Morphologic changes became evident within those contact times by transmission electron microscopy when HPV16 virus-like particles were exposed to HOCl under identical conditions. Mass spectrometry analysis of trypsin-digested products of L1 capsid proteins exposed to HOCl showed that mostly conserved residues were modified by oxidation and that these changes rapidly lead to instability of the protein demonstrable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Modifications to these residues may contribute to rapid virus inactivation. The use of homogeneous HOCl solutions for HPV decontamination provides a highly effective means of assuring the safety of nonautoclavable medical instruments.

Testing the Utility of Alternative Metrics of Branch Support to Address the Ancient Evolutionary Radiation of Tunas, Stromateoids, and Allies (Teleostei: Pelagiaria).

The use of high-throughput sequencing technologies to produce genome-scale data sets was expected to settle some long-standing controversies across the Tree of Life, particularly in areas where short branches occur at deep timescales. Instead, these data sets have often yielded many well-supported but conflicting topologies, and highly variable gene-tree distributions. A variety of branch-support metrics beyond the nonparametric bootstrap are now available to assess how robust a phylogenetic hypothesis may be, as well as new methods to quantify gene-tree discordance. We applied multiple branch-support metrics to a study of an ancient group of marine fishes (Teleostei: Pelagiaria) whose interfamilial relationships have proven difficult to resolve due to a rapid accumulation of lineages very early in its history. We analyzed hundreds of loci including published ultraconserved elements and newly generated exonic data along with their flanking regions to represent all 16 extant families for more than 150 out of 284 valid species in the group. Branch support was typically lower at inter- than intra-familial relationships regardless of the type of marker used. Several nodes that were highly supported with bootstrap had a very low site and gene-tree concordance, revealing underlying conflict. Despite this conflict, we were able to identify four consistent interfamilial clades, each comprised of two or three families. Combining exons with their flanking regions also produced increased branch lengths at the deep branches of the pelagiarian tree. Our results demonstrate the limitations of employing current metrics of branch support and species-tree estimation when assessing the confidence of ancient evolutionary radiations and emphasize the necessity to embrace alternative measurements to explore phylogenetic uncertainty and discordance in phylogenomic data sets.[Concatenation; exons; introns; phylogenomics; species-tree methods; target capture.].

Influence of Solvent in Crystal Engineering: A Significant Change to the Order-Disorder Transition in Ferrocene.

We present the structure of a novel solvate adduct formed by dissolving ferrocene, FeCp2, in hexafluorobenzene, C6F6. This adduct demonstrates the remarkably strong interactions between the five-membered aromatic rings of FeCp2 and the six-membered aromatic ring of C6F6. These molecular interactions are sufficiently strong and anisotropic to change the temperature of the order-disorder transition of the ferrocene molecule from below ca. 164 K to RT. No solvate adduct could be formed between benzene and FeCp2. These observations will be of particular relevance to the crystal engineering community, whose goal is the design of solids with bespoke properties.

Decreases in Mixed Venous Blood O2 Saturation in Cardiac Surgery Patients Following Extubation.

BACKGROUND: Decreases in mixed venous O2 saturation (SvO2) have been reported to occur in postcardiac surgery patients during weaning from mechanical ventilation. Our aim was to establish whether the physiological mechanism responsible for this phenomenon was a decrease in systemic O2 delivery (DO2) or an increase in global O2 consumption (V˙ O 2). METHODS: We studied 21 mechanically ventilated, postoperative cardiac patients for 30 minutes before and 60 minutes after extubation. We monitored continuously arterial O2 saturation by pulse oximetry (SaO2) and central venous O2 saturation (ScvO2) with an oximetry catheter. Mixed venous O2 saturation (SvO2) and cardiac output were also measured continuously with an oximetry pulmonary artery catheter. Systemic O2 delivery and V˙ O 2 were calculated according to accepted formulae. RESULTS: Immediately following extubation, ScvO2 and SvO2 decreased rapidly (P < .01). Systemic O2 consumption increased from 65 (57) mL·min-1 to 194 (66) mL·min-1 (P < .05) with no changes in DO2. Consequently, systemic O2 extraction rose from 38% (8%) to 45% (9%; P < .01). Preoperative left ventricular ejection fraction correlated with the decline in SvO2 postextubation. All patients weaned successfully. CONCLUSIONS: Decreases in SvO2 after discontinuation of ventilatory support in postcardiac surgery patients occur as V˙ O 2 increases in response to greater energy requirements by muscles of ventilation that are not initially matched by increases in DO2.

Retraction mechanics of Finochietto-style self-retaining thoracic retractors.

OBJECTIVES: Analyze the mechanics of Finochietto-style retractors, including the responses of thoracic tissues during thoracotomy, with an emphasis on tissue trauma and means for its reduction. METHODS: Mechanical analyses of the retractor were performed, including analysis of deformation under load and kinematics of the crank mechanism. Thoracotomies in a porcine model were performed in anesthetized animals (7) and fresh cadavers (17) using an instrumented retractor. RESULTS: Mechanical analyses revealed that arm motion is a non-linear function of handle rotation, that deformation of the retractor under load concentrates force at one edge of the retractor blade, and that the retractor behaves like a spring, deforming under the load of retraction and continuing to force open the incision long after crank rotation stops. Experimental thoracotomies included retractions ranging from 50 to 112 mm over 30 to 370 s, generating maximum forces of 118 to 470 N (12-50 kgf). Tissue ruptures occurred in 12 of the 24 retractions. These ruptures all occurred at retraction distances wider than 30 mm and at forces greater than 122.5 N. Significant tissue ruptures were observed for nearly all retractions at higher retraction rates (exceeding ½ rotation of the crank per 10 s). CONCLUSIONS: The Finochietto-style retractor can generate large forces and some aspects of its design increase the probability of tissue trauma.

The Effects of Cupping on Hamstring Flexibility in College Soccer Players.

Context: College soccer players suffer from hamstring injuries due to inflexibility and repetitive motions involving intense hamstring lengthening and contraction during sport. Although it is a popular intervention for muscular injury, there exists limited evidence of the effects of therapeutic cupping on hamstring flexibility. Objective: To determine the effect of cupping therapy on hamstring flexibility in college soccer players. Design: Cohort design. Setting: Athletic training clinic. Patients: A total of 25, asymptomatic, National Collegiate Athletic Association Division III soccer players (10 males and 15 females; age = 19.4 [1.30] y, height = 175.1 [8.2] cm, and mass = 69.5 [6.6] kg). Intervention(s): A 7-minute therapeutic cupping treatment was delivered to the treatment group. Four 2-in cups were fixed atop trigger point locations within the hamstring muscle bellies of participants' dominant legs. Control group participants received no intervention between pretest and posttest measurements. Main Outcome Measures: Pretest and posttest measurements of hamstring flexibility, using a passive straight leg raise, were performed on both groups. Passive straight leg raise measurements were conducted by blinded examiners using a digital inclinometer. An independent samples t test was used to analyze changes in hamstring flexibility from pretreatment to posttreatment with P values set a priori at .05. Results: An independent samples t test demonstrated no significant difference in change in hamstring flexibility between participants in the treatment group and those in the control group (t23 = -.961, P = .35). Conclusions: The findings of this study demonstrated no statistically significant changes in hamstring flexibility following a cupping treatment.

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum.

Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.IMPORTANCE How a nonenveloped virus transports across a biological membrane to cause infection remains mysterious. One enigmatic step is whether host cytosolic components are co-opted to transport the viral particle into the cytosol. During ER-to-cytosol membrane transport of the nonenveloped polyomavirus SV40, a decisive infection step, a cytosolic complex composed of Hsc70-SGTA-Hsp105 was previously shown to associate with the ER membrane. SGTA and Hsp105 have been shown to extract SV40 from the ER and transport the virus into the cytosol. We demonstrate here a critical role of Hsc70 in SV40 ER-to-cytosol penetration and reveal how SGTA controls Hsc70 to impact this process.

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid.

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.

Quantifying photothermal heating at plasmonic nanoparticles by scanning electrochemical microscopy.

Photothermal heating at metal nanoparticles results from the non-radiative decay of localized surface plasmons. The local heat generation enhances the mass transport rate of redox molecules and causes a shift in their formal potential, both of which can impact an electrochemical process at the nanoparticle interface. Here we present a methodology for probing the surface temperature at a plasmonic nanoparticle substrate using scanning electrochemical microscopy (SECM). Light is used to excite a plasmonic substrate electrode, while an ultramicroelectrode tip is positioned close to the substrate to read out both the mass transfer rate and concentration profile of the redox molecules. The measured mass transfer rate and the shift in the equilibrium potential provide a quantitative value of the temperature increase at the substrate surface, which is verified by simulations using a mass transfer model coupled with heat dissipation. The developed SECM approach is suitable for probing heat generation at a variety of both plasmonic and non-plasmonic nanostructures.

A young man with recurrent paralysis.

Hypokalemia can cause reactions from mild muscular cramping to life-threatening paralysis and cardiac dysrhythmias. This article describes a patient whose unusual, recurrent muscular symptoms and electrolyte abnormalities were eventually identified as Gitelman syndrome, a rare genetic disorder resulting in severe refractory hypokalemia.

A phase II trial of bevacizumab and everolimus as treatment for patients with refractory, progressive intracranial meningioma.

Meningiomas that progress after standard therapies are challenging with limited effective chemotherapy options. This phase II trial evaluated the efficacy of everolimus plus bevacizumab in patients with recurrent, progressive meningioma after treatment with surgical resection and local radiotherapy when appropriate. Patients with recurrent meningioma (WHO grade I, II, or III) following standard treatments with surgical resection and radiotherapy received bevacizumab (10 mg/kg IV days 1 and 15) and everolimus (10 mg PO daily) each 28 day cycle. Evaluation of response occurred every 2 cycles. The primary endpoint was progression-free survival (PFS). Secondary endpoints included response rate, overall survival and safety. Seventeen patients with a median age of 59 years (29-84) received study treatment. WHO grades at study entry included: I, 5 (29 %); II, 7 (41 %); III, 4 (24 %); unknown, 1 (6 %). Patients received a median of 8 cycles (1-37); all patients are off study treatment. A best response of SD was observed in 15 patients (88 %), and 6 patients had SD for >12 months. Overall median PFS was 22 months (95 % CI 4.5-26.8) and was greater for patients with WHO grade II and III compared to grade I tumors (22.0 months vs 17.5 months). Four patients discontinued treatment due to toxicity (proteinuria, 2; colitis, 1, thrombocytopenia, 1). However, other grade 3 toxicity was uncommon, and no patient had grade 4 toxicity. The combination of everolimus and bevacizumab was well-tolerated, and produced stable disease in 88 % of patients; the median duration of disease stabilization of 10 months (2-29). The median PFS from this prospective trial was similar to previous retrospective reports of bevacizumab in the treatment of recurrent meningioma.

Effects of a Responsiveness-Focused Intervention in Family Child Care Homes on Children's Executive Function.

Caregiver responsiveness has been theorized and found to support children's early executive function (EF) development. This study examined the effects of an intervention that targeted family child care provider responsiveness on children's EF. Family child care providers were randomly assigned to one of two intervention groups or a control group. An intervention group that received a responsiveness-focused online professional development course and another intervention group that received this online course plus weekly mentoring were collapsed into one group because they did not differ on any of the outcome variables. Children (N = 141) ranged in age from 2.5 to 5 years (mean age = 3.58 years; 52% female). At pretest and posttest, children completed delay inhibition tasks (gift delay-wrap, gift delay-bow) and conflict EF tasks (bear/dragon, dimensional change card sort), and parents reported on the children's level of attention problems. Although there were no main effects of the intervention on children's EF, there were significant interactions between intervention status and child age for delay inhibition and attention problems. The youngest children improved in delay inhibition and attention problems if they were in the intervention rather than the control group, whereas older children did not. These results suggest that improving family child care provider responsive behaviors may facilitate the development of certain EF skills in young preschool-age children.

A new kind of membrane-tethered eukaryotic transcription factor that shares an auto-proteolytic processing mechanism with bacteriophage tail-spike proteins.

MrfA, a transcription factor that regulates Dictyostelium prestalk cell differentiation, is an orthologue of the metazoan myelin gene regulatory factor (MRF) proteins. We show that the MRFs contain a predicted transmembrane domain, suggesting that they are synthesised as membrane-tethered proteins that are then proteolytically released. We confirm this for MrfA but report a radically different mode of processing from that of paradigmatic tethered transcriptional regulators, which are cleaved within the transmembrane domain by a dedicated protease. Instead, an auto-proteolytic cleavage mechanism, previously only described for the intramolecular chaperone domains of bacteriophage tail-spike proteins, processes MrfA and, by implication, the metazoan MRF proteins. We also present evidence that the auto-proteolysis of MrfA occurs rapidly and constitutively in the ER and that its specific role in prestalk cell differentiation is conferred by the regulated nuclear translocation of the liberated fragment.

Parenting predictors of cognitive skills and emotion knowledge in socioeconomically disadvantaged preschoolers.

This study examined the concurrent and longitudinal associations of parental responsiveness and inferential language input with cognitive skills and emotion knowledge among socioeconomically disadvantaged preschoolers. Parents and 2- to 4-year-old children (mean age=3.21 years, N=284) participated in a parent-child free play session, and children completed cognitive (language, early literacy, early mathematics) and emotion knowledge assessments. Approximately 1 year later, children completed the same assessment battery. Parental responsiveness was coded from the videotaped parent-child free play sessions, and parental inferential language input was coded from transcripts of a subset of 127 of these sessions. All analyses controlled for child age, gender, and parental education, and longitudinal analyses controlled for initial skill level. Parental responsiveness significantly predicted all concurrent cognitive skills as well as literacy, math, and emotion knowledge 1 year later. Parental inferential language input was significantly positively associated with children's concurrent emotion knowledge. In longitudinal analyses, an interaction was found such that for children with stronger initial language skills, higher levels of parental inferential language input facilitated greater vocabulary development, whereas for children with weaker initial language skills, there was no association between parental inferential language input and change in children's vocabulary skills. These findings further our understanding of the roles of parental responsiveness and inferential language input in promoting children's school readiness skills.

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

Longitudinal mediators of achievement in mathematics and reading in typical and atypical development.

Longitudinal studies of neurodevelopmental disorders that are diagnosed at or before birth and are associated with specific learning difficulties at school-age provide one method for investigating developmental precursors of later-emerging academic disabilities. Spina bifida myelomeningocele (SBM) is a neurodevelopmental disorder associated with particular problems in mathematics, in contrast to well-developed word reading. Children with SBM (n=30) and typically developing children (n=35) were used to determine whether cognitive abilities measured at 36 and 60 months of age mediated the effect of group on mathematical and reading achievement outcomes at 8.5 and 9.5 years of age. A series of multiple mediator models showed that: visual-spatial working memory at 36 months and phonological awareness at 60 months partially mediated the effect of group on math calculations, phonological awareness partially mediated the effect of group on small addition and subtraction problems on a test of math fluency, and visual-spatial working memory mediated the effect of group on a test of math problem solving. Groups did not differ on word reading, and phonological awareness was the only mediator for reading fluency and reading comprehension. The findings are discussed with reference to theories of mathematical development and disability and with respect to both common and differing cognitive correlates of math and reading.

Protocol for building a reference standard sequence library for DNA-based seafood identification.

With the recent adoption of a DNA sequencing-based method for the species identification for seafood products by the U.S. Food and Drug Administration (FDA), a library of standard sequences derived from reference specimens with authoritative taxonomic authentication was required. Provided here are details of how the FDA and its collaborators are building this reference standard sequence library that will be used to confirm the accurate labeling of seafood products sold in interstate commerce in the United States. As an example data set from this library, information for 117 fish reference standards, representing 94 species from 43 families in 15 orders, collected over a 4-year period from the Gulf of Mexico, U.S., that are now stored at the Smithsonian Museum Support Center in Suitland, MD, are provided.