The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

We recently characterised a low-activity form of E. coli transketolase, TKlow, which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TKhigh, in the presence of Mg2+. We observed previously that partial oxidation was responsible for increased TKhigh activity, while low-activity TKlow was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the β-hydroxypyruvate (HPA) donor substrate to wild-type transketolase and a variant, S385Y/D469T/R520Q, that is active towards aromatic aldehydes. Transketolase HPA affinity again revealed the two distinct forms of transketolase at a TKhigh:TKlow ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TKlow was comparable to the substrate-inhibition dissociation constant, KiHPA, determined previously. We provide evidence that KiHPA is a convolution of binding to the low-activity TKlow-TKlow dimer, and the TKlow subunit of the partially-active TKhigh-TKlow mixed dimer, where HPA binding to the TKlow subunit of the mixed dimer results in inhibition of the active TKhigh subunit. Heat-activation of transketolase was similarly investigated and found to convert the TKlow subunit of the mixed dimer to have TKhigh-like properties, but without oxidation

Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations

Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form

What's in the Gift? Towards a Molecular Dissection of Nuptial Feeding in a Cricket.

Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Nuptial gifts produced by males and transferred to females during copulation are common in insects. Yet, their precise composition and subsequent physiological effects on the female recipient remain unresolved. Male decorated crickets Gryllodes sigillatus transfer a spermatophore to the female during copulation that is composed of an edible gift, the spermatophylax, and the ampulla that contains the ejaculate. After transfer of the spermatophore, the female detaches the spermatophylax and starts to eat it while sperm from the ampulla are evacuated into the female reproductive tract. When the female has finished consuming the spermatophylax, she detaches the ampulla and terminates sperm transfer. Hence, one simple function of the spermatophylax is to ensure complete sperm transfer by distracting the female from prematurely removing the ampulla. However, the majority of orally active components of the spermatophylax itself and their subsequent effects on female behavior have not been identified. Here, we report the first analysis of the proteome of the G. sigillatus spermatophylax and the transcriptome of the male accessory glands that make these proteins. The accessory gland transcriptome was assembled into 17,691 transcripts whilst about 30 proteins were detected within the mature spermatophylax itself. Of these 30 proteins, 18 were encoded by accessory gland encoded messages. Most spermatophylax proteins show no similarity to proteins with known biological functions and are therefore largely novel. A spermatophylax protein shows similarity to protease inhibitors suggesting that it may protect the biologically active components from digestion within the gut of the female recipient. Another protein shares similarity with previously characterized insect polypeptide growth factors suggesting that it may play a role in altering female reproductive physiology concurrent with fertilization. Characterization of the spermatophylax proteome provides the first step in identifying the genes encoding these proteins in males and in understanding their biological functions in the female recipient.Max Planck GesellschaftNational Science FoundationBBSRRoyal Societ

Joining the dots: Conditional pass and programmatic assessment enhances recognition of problems with professionalism and factors hampering student progress

<p>Abstract</p> <p>Background</p> <p>Programmatic assessment that looks across a whole year may contribute to better decisions compared with those made from isolated assessments alone. The aim of this study is to describe and evaluate a programmatic system to handle student assessment results that is aligned not only with learning and remediation, but also with defensibility. The key components are standards based assessments, use of "Conditional Pass", and regular progress meetings.</p> <p>Methods</p> <p>The new assessment system is described. The evaluation is based on years 4-6 of a 6-year medical course. The types of concerns staff had about students were clustered into themes alongside any interventions and outcomes for the students concerned. The likelihoods of passing the year according to type of problem were compared before and after phasing in of the new assessment system.</p> <p>Results</p> <p>The new system was phased in over four years. In the fourth year of implementation 701 students had 3539 assessment results, of which 4.1% were Conditional Pass. More in-depth analysis for 1516 results available from 447 students revealed the odds ratio (95% confidence intervals) for failure was highest for students with problems identified in more than one part of the course (18.8 (7.7-46.2) p < 0.0001) or with problems with professionalism (17.2 (9.1-33.3) p < 0.0001). The odds ratio for failure was lowest for problems with assignments (0.7 (0.1-5.2) NS). Compared with the previous system, more students failed the year under the new system on the basis of performance during the year (20 or 4.5% compared with four or 1.1% under the previous system (p < 0.01)).</p> <p>Conclusions</p> <p>The new system detects more students in difficulty and has resulted in less "failure to fail". The requirement to state conditions required to pass has contributed to a paper trail that should improve defensibility. Most importantly it has helped detect and act on some of the more difficult areas to assess such as professionalism.</p

Bilateral pedicle stress fracture in the lumbar spine of a sedentary office worker

A case of bilateral pedicle fracture in the lumbar spine of a sedentary office worker is being presented. No such case has been reported in the literature previously. Bilateral pedicle fracture is a rare entity. Few cases have been reported in literature. All the reported cases had some underlying causative factors like degenerative spine disease, previous spinal surgery or stress-related activities, e.g. athletes. Our case is a 36-year-old sedentary office worker with none of the factors mentioned. We present a case of a 36-year-old sedentary worker with long-standing low backache. There were no root tension signs. Plain radiographs were inconclusive. The patient had a CT scan. The CT scan revealed long-standing defects in the pedicles of L2 vertebra with pseudoarthrosis. Infiltration with anaesthetic relieved the symptoms. Our patient was managed conservatively with spine rehabilitation physiotherapy program. Pedicle fracture can develop due to abnormal stresses in the pedicle either because of previous spinal surgery or spondylitic changes in the spine. Bilateral pedicle fracture in the absence of these conditions is extremely rare

Survival of the Systems

This is the author accepted manuscript. The final version is available from cell Press via the DOI in this recordSince Darwin, individuals and more recently genes have been the focus of evolutionary thinking. The idea that selection operates on non-reproducing, higher-level systems including ecosystems or societies has met with scepticism. But research emphasising that natural selection can be based solely on differential persistence invites reconsideration of their evolution. Self-perpetuating feedback cycles involving biotic as well as abiotic components are critical to determining persistence. Evolution of autocatalytic networks of molecules is well studied, but the principles hold for any ‘self-perpetuating’ system. Ecosystem examples include coral reefs, rainforests and savannahs. Societal examples include agricultural systems, dominant belief systems and economies. Persistence-based selection of feedbacks can help us understand how ecological and societal systems survive or fail in a changing world

A Randomized Trial of Intravenous Iron Supplementation and Exercise on Exercise Capacity in Iron-Deficient Nonanemic Patients With CKD

Introduction: Patients with chronic kidney disease (CKD) are often iron deficient, even when not anemic. This trial evaluated whether iron supplementation enhances exercise capacity of nonanemic patients with CKD who have iron-deficiency. Methods: Prospective, multicenter double-blind randomized controlled trial of nondialysis patients with CKD and iron-deficiency but without anemia (Hemoglobin [Hb] >110 g/l). Patients were assigned 1:1 to intravenous (IV) iron therapy, or placebo. An 8-week exercise program commenced at week 4. The primary outcome was the mean between-group difference in 6-minute walk test (6MWT) at 4 weeks. Secondary outcomes included 6MWT at 12 weeks, transferrin saturation (TSAT), serum ferritin (SF), Hb, renal function, muscle strength, functional capacity, quality of life, and adverse events at baseline, 4 weeks, and at 12 weeks. Mean between-group differences were analyzed using analysis of covariance models. Results: Among 75 randomized patients, mean (SD) age for iron therapy (n = 37) versus placebo (n = 38) was 54 (16) versus 61 (12) years; estimated glomerular filtration rate (eGFR) (34 [12] vs. 35 [11] ml/min per 1.73 m2], TSAT (23 [12] vs. 21 [6])%; SF (57 [64] vs. 62 [33]) μg/l; Hb (122.4 [9.2] vs. 127 [13.2] g/l); 6MWT (384 [95] vs. 469 [142] meters) at baseline, respectively. No significant mean between-group difference was observed in 6MWT distance at 4 weeks. There were significant increases in SF and TSAT at 4 and 12 weeks (P < 0.02), and Hb at 12 weeks (P = 0.009). There were no between-group differences in other secondary outcomes and no adverse events attributable to iron therapy. Conclusion: This trial did not demonstrate beneficial effects of IV iron therapy on exercise capacity at 4 weeks. A larger study is needed to confirm if IV iron is beneficial in nondialysis patients with CKD who are iron-deficient

Developmental disruption to the cortical transcriptome and synaptosome in a model of SETD1A loss-of-function.

This is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this record Data Availability: Transcriptomic data from RNA sequencing is available from the Gene Expression Omnibus (GEO) with identifier GSE199428. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (80) partner repository with the dataset identifier PXD032742.Large-scale genomic studies of schizophrenia implicate genes involved in the epigenetic regulation of transcription by histone methylation and genes encoding components of the synapse. However, the interactions between these pathways in conferring risk to psychiatric illness are unknown. Loss-of-function (LoF) mutations in the gene encoding histone methyltransferase, SETD1A, confer substantial risk to schizophrenia. Among several roles, SETD1A is thought to be involved in the development and function of neuronal circuits. Here, we employed a multi-omics approach to study the effects of heterozygous Setd1a LoF on gene expression and synaptic composition in mouse cortex across five developmental timepoints from embryonic day 14 to postnatal day 70. Using RNA sequencing, we observed that Setd1a LoF resulted in the consistent downregulation of genes enriched for mitochondrial pathways. This effect extended to the synaptosome, in which we found age-specific disruption to both mitochondrial and synaptic proteins. Using large-scale patient genomics data, we observed no enrichment for genetic association with schizophrenia within differentially expressed transcripts or proteins, suggesting they derive from a distinct mechanism of risk from that implicated by genomic studies. This study highlights biological pathways through which SETD1A loss-of-function may confer risk to schizophrenia. Further work is required to determine whether the effects observed in this model reflect human pathology.Medical Research CouncilWellcome Trus