Predictive genetic testing for Huntington's disease: Exploring participant experiences of uncertainty and ambivalence between clinic appointments.

Ambivalence and uncertainty are key themes throughout the psychology of healthcare literature. This is especially so for individuals at risk of Huntington's disease (HD) deliberating the decision to undergo genetic testing because there is currently no treatment that modifies disease progression. A better understanding of the experience of making a decision about genetic prediction will help practitioners support and guide individuals through this process. Our aim was to capture participants' experiences of uncertainty and ambivalence in between their genetic counseling appointments. We explored these issues through the experiences of nine participants who were referred for predictive HD testing at four regional genetics services in England and Wales. Data consisted of recordings of clinic consultations, diaries, and an in-depth interview conducted at the end of the testing process. Data were analyzed thematically. Four themes were identified representing four possible futures, each future dependent on the decision to undergo testing and the result of that test. Our results showed that participants, as well as attending more to a future that represents their current situation of not having undergone predictive testing, also attended more to a distant future where a positive predictive result is received and symptoms have started. Participants attended less to the two futures that were more immediate once testing was undertaken (a future where a positive result is received and symptoms have not started and a future where a negative result is received). The use of diaries gave us a unique insight into these participants' experiences of ambivalence and uncertainty, psychological distress, and the emotional burden experienced. These findings help inform discussions within the clinic appointment as well as encourage researchers to consider diary use as a method of exploring what happens for individuals outside of clinical encounters.

Genomics and justice: mitigating the potential harms and inequities that arise from the implementation of genomics in medicine.

Advances in human genetics raise many social and ethical issues. The application of genomic technologies to healthcare has raised many questions at the level of the individual and the family, about conflicts of interest among professionals, and about the limitations of genomic testing. In this paper, we attend to broader questions of social justice, such as how the implementation of genomics within healthcare could exacerbate pre-existing inequities or the discrimination against social groups. By anticipating these potential problems, we hope to minimise their impact. We group the issues to address into six categories: (i) access to healthcare in general, not specific to genetics. This ranges from healthcare insurance to personal behaviours. (ii) data management and societal discrimination against groups on the basis of genetics. (iii) epigenetics research recognises how early life exposure to stress, including malnutrition and social deprivation, can lead to ill health in adult life and further social disadvantage. (iv) psychiatric genomics and the genetics of IQ may address important questions of therapeutics but could also be used to disadvantage specific social or ethnic groups. (v) complex diseases are influenced by many factors, including genetic polymorphisms of individually small effect. A focus on these polygenic influences distracts from environmental factors that are more open to effective interventions. (vi) population genomic screening aims to support couples making decisions about reproduction. However, this remains a highly contentious area. We need to maintain a careful balance of the competing social and ethical tensions as the technology continues to develop.

Thai Norms for Name, Image, and Category Agreement, Object Familiarity, Visual Complexity, Manipulability, and Age of Acquisition for 480 Color Photographic Objects.

Normative databases containing psycholinguistic variables are commonly used to aid stimulus selection for investigations into language and other cognitive processes. Norms exist for many languages, but not for Thai. The aim of the present research, therefore, was to obtain Thai normative data for the BOSS, a set of 480 high resolution color photographic images of real objects (Brodeur et al. in PLoS ONE 5(5), 2010.  https://doi.org/10.1371/journal.pone.0010773 ). Norms were provided by 584 Thai university students on eight dimensions: name agreement, object familiarity, visual complexity, category agreement, image agreement, two types of manipulability (graspability and mimeability), and age of acquisition. The results revealed comparatively similar levels of name agreement to Brodeur et al. especially when unfamiliar items were factored out. The pattern of intercorrelations among the Thai psycholinguistic norms was comparable to previous studies and our cross-linguistic correlations were robust for the same set of pictures in English and French. Conjointly, the findings extend the relevancy of the BOSS to Thailand, supporting this photographic resource for investigations of language and other cognitive processes in monolingual, multilingual, and brain-impaired populations.

Unusual case of acute posteromedial papillary muscle rupture after acute anterior myocardial infarction.

Papillary muscle rupture is now a rare complication of acute myocardial infarction. Posteromedial papillary muscle rupture is more common than anterolateral papillary muscle rupture. The posteromedial papillary muscle is usually supplied from a branch of the right coronary artery. We present a case of posteromedial papillary muscle rupture due to an isolated left anterior descending artery lesion. This was diagnosed on the fifth day post infarction. The patient progressed to mitral valve replacement and coronary artery bypass grafting to the left anterior descending artery. We believe this unusual arterial supply to the posteromedial papillary muscle is due to an apex forming left anterior descending artery coupled with an apically located posteromedial papillary muscle.

Publisher Correction: Rapid Thermal Processing to Enhance Steel Toughness.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dissociating word stem completion and cued recall as a function of divided attention at retrieval.

The aim of this study was to investigate the widely held, but largely untested, view that implicit memory (repetition priming) reflects an automatic form of retrieval. Specifically, in Experiment 1 we explored whether a secondary task (syllable monitoring), performed during retrieval, would disrupt performance on explicit (cued recall) and implicit (stem completion) memory tasks equally. Surprisingly, despite substantial memory and secondary costs to cued recall when performed with a syllable-monitoring task, the same manipulation had no effect on stem completion priming or on secondary task performance. In Experiment 2 we demonstrated that even when using a particularly demanding version of the stem completion task that incurred secondary task costs, the corresponding disruption to implicit memory performance was minimal. Collectively, the results are consistent with the view that implicit memory retrieval requires little or no processing capacity and is not seemingly susceptible to the effects of dividing attention at retrieval.

Rapid Thermal Processing to Enhance Steel Toughness.

Quenching and Tempering (Q&T) has been utilized for decades to alter steel mechanical properties, particularly strength and toughness. While tempering typically increases toughness, a well-established phenomenon called tempered martensite embrittlement (TME) is known to occur during conventional Q&T. Here we show that short-time, rapid tempering can overcome TME to produce unprecedented property combinations that cannot be attained by conventional Q&T. Toughness is enhanced over 43% at a strength level of 1.7 GPa and strength is improved over 0.5 GPa at an impact toughness of 30 J. We also show that hardness and the tempering parameter (TP), developed by Holloman and Jaffe in 1945 and ubiquitous within the field, is insufficient for characterizing measured strengths, toughnesses, and microstructural conditions after rapid processing. Rapid tempering by energy-saving manufacturing processes like induction heating creates the opportunity for new Q&T steels for energy, defense, and transportation applications.

Points to consider for laboratories reporting results from diagnostic genomic sequencing.

Although NGS technologies are well-embedded in the clinical setting for identification of genetic causes of disease, guidelines issued by professional bodies are inconsistent regarding some aspects of reporting results. Most recommendations do not give detailed guidance about whether variants of uncertain significance (VUS) should be reported by laboratory personnel to clinicians, and give conflicting messages regarding whether unsolicited findings (UF) should be reported. There are also differences both in their recommendations regarding whether actively searching for secondary findings (SF) is appropriate, and in the extent to which they address the duty (or lack thereof) to reanalyse variants when new information arises. An interdisciplinary working group considered the current guidelines, their own experiences, and data from a recent qualitative study to develop a set of points to consider for laboratories reporting results from diagnostic NGS. These points to consider fall under six categories: (i) Testing approaches and technologies used, (ii) Approaches for VUS; (iii) Approaches for reporting UF, (iv) Approaches regarding SF; (v) Reanalysis of data & re-contact; and vi) Minors. While it is unclear whether uniformity in reporting across all laboratories is desirable, we hope these points to consider will be useful to diagnostic laboratories as they develop their processes for making decisions about reporting VUS and UF from NGS in the diagnostic context.

Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients.

MECP2 mutations are identifiable in approximately 80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.

Essential tryptophan residues in the function of cellulase from Schizophyllum commune.

The tryptophan residues of the cellulase (EC 3.2.1.4; 1,4-beta-D-glucan 4-glucanohydrolase) from Schizophyllum commune were oxidized by N-bromosuccinimide in both the presence and absence of substrates and inhibitors of the enzyme. In the absence of protective ligands, eight of the twelve tryptophan residues in the cellulase were susceptible to modification with concomitant inactivation of the enzyme. The binding of the substrates, CM-cellulose, methyl cellulose, cellohexaose or lichenan and the competitive inhibitor, cellobiose, protected one tryptophan residue from oxidation but did not prevent the inactivation. Characterization of the oxidized enzyme derivatives by ultraviolet difference absorption and by fluorescence spectroscopy indicated that two tryptophan residues are essential in the mechanism of cellulase catalysis. One residue appears to be directly involved in the binding of substrate, while the second residue is proposed to constitute an integral part of a catalytically sound active centre.

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups.

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.

The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85.

Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.

Inactivation of Bacillus cereus 569/H beta-lactamase I by 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone and its N-methyl derivative.

6-beta-(Trifluoromethane sulfonyl)amidopenicillanic acid sulfone and its N-methyl derivative were found to be potent inhibitors of Bacillus cereus 569/H beta-lactamase I. The rate of the inactivation of the enzyme by both compounds was found to increase with the decreasing pH of the reaction medium. The reaction of the enzyme with 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone was found to be irreversible at the pH values investigated. In contrast, the reaction with the N-methyl derivative at neutral pH was consistent with the partitioning of the acyl enzyme intermediate in three pathways which included (a) deacylation to yield active enzyme, (b) conversion to a transiently inhibited species, and (c) conversion to an irreversibly inactive form. The amino acid composition of the chromophoric peptide obtained from the enzyme inactivated by either of the compounds was consistent with the occurrence of an initial acylation of serine-70 of the protein.

ALK positive inflammatory myofibroblastic tumour of the pineal region.

Inflammatory myofibroblastic tumours (IMTs) are an uncommon spindle cell neoplasm with a dense inflammatory infiltrate, usually encountered in children. IMTs of the central nervous system are extremely rare. This report describes the case of an IMT in a 61 year old man, in the pineal region. The tumour was completely excised, and immunohistochemistry demonstrated anaplastic lymphoma kinase 1 expression. There was no tumour recurrence during 18 months of follow-up. Our case extends both the age range and sites of occurrence of this rare tumour.

A study of X chromosome activity in two incontinentia pigmenti families with probable linkage to Xq28.

Linkage analysis was carried out in two British families with incontinentia pigmenti (IP). Both showed exclusion at several markers in Xp and proximal Xq and showed probable linkage to the DXS52 and F8C loci in Xq28. This suggests that in these families the disease locus is IP2. Using a method based on the androgen receptor gene, and confirming the results where possible at the PGK-1 and DXS255 loci, it was shown that in affected females the maternally inherited X chromosome, where it could be identified, is inactive in the majority of cells.

Identification of the catalytic nucleophile in the cellulase from Schizophyllum commune and assignment of the enzyme to Family 5, subtype 5 of the glycosidases.

Differential chemical modification of the cellulase from Schizophyllum commune with [N-methyl-3H]1-ethyl-3(4-azonia-4,4-dimethylpentyl)-carbodiimide in the presence and absence of substrate identified an active site glutamate residue within the peptide: Leu-Gln-Ala-Ala-Thr-Glu-Trp-Leu-(Lys). This Glu residue is proposed to participate in binding of substrate as amino acid sequence homology studies combined with mechanism-based inhibition of the cellulase with 4',5'-epoxypentyl-beta-D-cellobioside identified a neighboring Glu residue, which conforms to the Glu-X-Gly motif of Family 5 glycosidases, as the catalytic nucleophile. These data allow the assignment of the S. commune cellulase to Family 5, subtype 5 of the glycosidases.

Inhibition of membrane-bound lytic transglycosylase B by NAG-thiazoline.

The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The beta-hexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, Kd values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.