Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic ...resolution structures along the reaction coordinate of an inverting α‐mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α‐D‐mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.
Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use ...as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar “epoxide” mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (K D 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.
Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence‐based mannosidase families have evolved ingenious strategies to overcome the ...stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X‐ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole‐type inhibitors are energetically poised to report faithfully on mannosidase transition‐state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β‐mannanases from families GH26 and GH113. Isofagomine‐type inhibitors are poor mimics of transition‐state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar‐shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active‐site residues involved in substrate recognition.
Shipshape inhibitors: Quantum mechanical calculations of the free‐energy landscape (see figure) of the glycosidase transition‐state mimics isofagomine and mannoimidazole reveals that only the latter is energetically poised to report upon the mannosidase transition‐state conformation. X‐ray structures of β‐mannanases from different families reveal they both adopt a boat conformation, thus allowing unification of the enzymatic conformational itinerary of a range of diverse α‐ and β‐mannosidases.
Abstract
Background:
Vaginal cancer is a rare malignancy that poses a challenge to treat and cure, as surgical excision requires life-changing procedures because of the proximity and involvement of ...rectum, bladder and anus. We report in this case study the successful delivery of stereotactic ablative radiotherapy (SABR) for a patient with vaginal cancer after previous radiotherapy.
Methods:
A 71-year-old white female who presented with dyspareunia and irritative urinary symptoms proven by biopsy was our candidate patient. Subsequent PET/CT revealed a hypermetabolic 3 cm lesion at the 12–1 o’clock position in the distal vagina involving the clitoris. The patient was initially treated with volumetric-modulated arc therapy (VMAT) with simultaneous integrated boost technique to the involved nodes, and later upon recurrence treated with SABR using 30 Gy in six fractions.
Findings:
To our knowledge, this is the first report of a vaginal cylinder used to physically distance organs at risk from the treatment target and also as a localising device with image guidance for the delivery of SABR using an external beam.
A dominant human gut microbe, the well studied symbiont Bacteroides thetaiotaomicron (Bt), is a glyco‐specialist that harbors a large repertoire of genes devoted to carbohydrate processing. Despite ...strong similarities among them, many of the encoded enzymes have evolved distinct substrate specificities, and through the clustering of cognate genes within operons termed polysaccharide‐utilization loci (PULs) enable the fulfilment of complex biological roles. Structural analyses of two glycoside hydrolase family 92 α‐mannosidases, BT3130 and BT3965, together with mechanistically relevant complexes at 1.8–2.5 Å resolution reveal conservation of the global enzyme fold and core catalytic apparatus despite different linkage specificities. Structure comparison shows that Bt differentiates the activity of these enzymes through evolution of a highly variable substrate‐binding region immediately adjacent to the active site. These observations unveil a genetic/biochemical mechanism through which polysaccharide‐processing bacteria can evolve new and specific biochemical activities from otherwise highly similar gene products.
Analysing two sequence‐related bacterial glycoside hydrolase family 92 mannosidases with distinct functions, a structural basis for their varied specificities is revealed.
Abstract
Background
The aim of our study is to determine the incidence, timing, and risk factors for cerebral vasculopathy after cranial proton and photon radiation for pediatric brain tumors.
...Methods
We performed a single-institution retrospective review of a cohort of children treated with proton radiation for brain tumors. MRA and/or MRI were reviewed for evidence of cerebral vascular stenosis and infarcts. Twenty-one similar studies (17 photon, 4 proton) were identified by systematic literature review.
Results
For 81 patients with median follow-up of 3 years, the rates of overall and severe vasculopathy were 9.9% and 6.2% respectively, occurring a median of 2 years post radiation. Dose to optic chiasm greater than 45 Gy and suprasellar location were significant risk factors. Results were consistent with 4 prior proton studies (752 patients) that reported incidence of 5% to 6.7%, 1.5 to 3 years post radiation. With significantly longer follow-up (3.7-19 years), 9 studies (1108 patients) with traditional photon radiation reported a higher rate (6.3%-20%) and longer time to vasculopathy (2-28 years). Significant risk factors were neurofibromatosis type 1 (NF-1; rate 7.6%-60%) and suprasellar tumors (9%-20%). In 10 studies with photon radiation (1708 patients), the stroke rate was 2% to 18.8% (2.3-24 years post radiation).
Conclusions
Childhood brain tumor survivors need screening for vasculopathy after cranial radiation, especially with higher dose to optic chiasm, NF-1, and suprasellar tumors. Prospective studies are needed to identify risk groups, and ideal modality and timing, for screening of this toxicity.
To review institutional outcomes for patients treated with external-beam radiotherapy (EBRT) for orbital pseudotumor.
This is a single-institution retrospective review of 20 orbits in 16 patients ...diagnosed with orbital pseudotumor that received EBRT at the University of Oklahoma, Department of Radiation Oncology. Treated patients had a median follow-up of 16.5 months.
Fifteen patients (93.7%) were initially treated with corticosteroids. Eight had recurrence after steroid cessation, six were unable to taper corticosteroids completely or partially, and one experienced progression of symptoms despite corticosteroid therapy. Fourteen patients (87.5%) initially responded to radiotherapy indicated by clinical improvement of preradiation symptoms and/or tapering of corticosteroid dose. Mean EBRT dose was 20 Gy (range, 14-30 Gy). Thirteen patients (81.2%) continued to improve after radiation therapy. Patient outcomes were complete cessation of corticosteroid therapy in nine patients (56.3%) and reduced corticosteroid dose in four patients (25%). Radiotherapy did not achieve long-term control for three patients (18.7%), who still required preradiation corticosteroid dosages. Three patients received retreatment(s) of four orbits, of which two patients achieved long-term symptom control without corticosteroid dependence. One patient received retreatment to an orbit three times, achieving long-term control without corticosteroid dependence. No significant late effects have been observed in retreated patients.
Radiotherapy is an effective treatment for acute symptomatic improvement and long-term control of orbital pseudotumor. Orbital retreatment can be of clinical benefit, without apparent increase in morbidity, when initial irradiation fails to achieve complete response.
The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays ...vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment.
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•The unique proteome of the photoreceptor outer segment plasma membrane is identified.•TMEM67, TMEM237, and embigin are novel unique components of this membrane.•Embigin in this membrane is associated with the monocarboxylate transporter MCT1.•The photoreceptor outer segment likely facilitates lactate transport.
The plasma membrane which envelopes the light-sensitive outer segment organelle of vertebrate photoreceptor cells plays diverse roles in supporting photoreceptor function and health. Protein correlation profiling of this membrane revealed a surprisingly small number of unique protein components. Among them are TMEM67 and TMEM237, whose mutations are associated with various syndromic ciliopathies, and embigin found to be associated with the monocarboxylate transporter MCT1. The MCT1–embigin complex likely facilitates lactate transport through this cellular compartment.
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