256 research outputs found
A structural snapshot of base-pair opening in DNA
The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine
Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants
Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues
Thio-linked UDP-peptide conjugates as O-GlcNAc transferase inhibitors
O-GlcNAc
transferase (OGT) is an essential glycosyltransferase
that installs the O-GlcNAc post-translational modification on the
nucleocytoplasmic proteome. We report the development of S-linked
UDP–peptide conjugates as potent bisubstrate OGT inhibitors.
These compounds were assembled in a modular fashion by photoinitiated
thiol–ene conjugation of allyl-UDP and optimal acceptor peptides
in which the acceptor serine was replaced with cysteine. The conjugate
VTPVCÂ(S-propyl-UDP)ÂTA (<i>K</i><sub>i</sub> = 1.3 ÎĽM)
inhibits the OGT activity in HeLa cell lysates. Linear fusions of
this conjugate with cell penetrating peptides were explored as prototypes
of cell-penetrant OGT inhibitors. A crystal structure of human OGT
with the inhibitor revealed mimicry of the interactions seen in the
pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative
of the inhibitor works as a high affinity probe in a fluorescence
polarimetry hOGT assay
Analysis of the LKB1-STRAD-MO25 complex
Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25
Prognostic significance of MRI-detected extramural venous invasion according to grade and response to neo-adjuvant treatment in locally advanced rectal cancer A national cohort study after radiologic training and reassessment
Background: Detection of grade 3–4 extra mural venous invasion (mrEMVI) on magnetic resonance imaging (MRI) is associated with an increased distant metastases (DM)-rate. This study aimed to determine the impact of different grades of mrEMVI and their disappearance after neoadjuvant therapy.Methods: A Dutch national retrospective cross-sectional study was conducted, including patients who underwent resection for rectal cancer in 2016 from 60/69 hospitals performing rectal surgery. Patients with a cT3-4 tumour ≤8 cm from the anorectal junction were selected and their MRI-scans were reassessed by trained abdominal radiologists. Positive mrEMVI grades (3 and 4) were analyzed in regard to 4-year local recurrence (LR), DM, disease-free survival (DFS) and overall survival (OS). Results: The 1213 included patients had a median follow-up of 48 months (IQR 30–54). Positive mrEMVI was present in 324 patients (27%); 161 had grade 3 and 163 had grade 4. A higher mrEMVI stage (grade 4 vs grade 3 vs no mrEMVI) increased LR-risk (21% vs 18% vs 7%, <0.001) and DM-risk (49% vs 30% vs 21%, p < 0.001) and decreased DFS (42% vs 55% vs 69%, p < 0.001) and OS (62% vs 76% vs 81%, p < 0.001), which remained independently associated in multivariable analysis. When mrEMVI had disappeared on restaging MRI, DM-rate was comparable to initial absence of mrEMVI (both 26%), whereas LR-rate remained high (22% vs 9%, p = 0.006). Conclusion: The negative oncological impact of mrEMVI on recurrence and survival rates was dependent on grading. Disappearance of mrEMVI on restaging MRI decreased the risk of DM, but not of LR.</p
Prognostic significance of MRI-detected extramural venous invasion according to grade and response to neo-adjuvant treatment in locally advanced rectal cancer A national cohort study after radiologic training and reassessment
Background: Detection of grade 3–4 extra mural venous invasion (mrEMVI) on magnetic resonance imaging (MRI) is associated with an increased distant metastases (DM)-rate. This study aimed to determine the impact of different grades of mrEMVI and their disappearance after neoadjuvant therapy.Methods: A Dutch national retrospective cross-sectional study was conducted, including patients who underwent resection for rectal cancer in 2016 from 60/69 hospitals performing rectal surgery. Patients with a cT3-4 tumour ≤8 cm from the anorectal junction were selected and their MRI-scans were reassessed by trained abdominal radiologists. Positive mrEMVI grades (3 and 4) were analyzed in regard to 4-year local recurrence (LR), DM, disease-free survival (DFS) and overall survival (OS). Results: The 1213 included patients had a median follow-up of 48 months (IQR 30–54). Positive mrEMVI was present in 324 patients (27%); 161 had grade 3 and 163 had grade 4. A higher mrEMVI stage (grade 4 vs grade 3 vs no mrEMVI) increased LR-risk (21% vs 18% vs 7%, <0.001) and DM-risk (49% vs 30% vs 21%, p < 0.001) and decreased DFS (42% vs 55% vs 69%, p < 0.001) and OS (62% vs 76% vs 81%, p < 0.001), which remained independently associated in multivariable analysis. When mrEMVI had disappeared on restaging MRI, DM-rate was comparable to initial absence of mrEMVI (both 26%), whereas LR-rate remained high (22% vs 9%, p = 0.006). Conclusion: The negative oncological impact of mrEMVI on recurrence and survival rates was dependent on grading. Disappearance of mrEMVI on restaging MRI decreased the risk of DM, but not of LR.</p
Impact of the new rectal cancer definition on multimodality treatment and interhospital variability:Results from a nationwide cross-sectional study
Aim: This study aimed to determine the consequences of the new definition of rectal cancer for decision-making in multidisciplinary team meetings (MDT). The new definition of rectal cancer, the lower border of the tumour is located below the sigmoid take-off (STO), was implemented in the Dutch guideline in 2019 after an international Delphi consensus meeting to reduce interhospital variations. Method: All patients with rectal cancer according to the local MDT, who underwent resection in 2016 in the Netherlands were eligible for this nationwide collaborative cross-sectional study. MRI-images were rereviewed, and the tumours were classified as above or on/below the STO. Results: This study registered 3107 of the eligible 3178 patients (98%), of which 2784 patients had an evaluable MRI. In 314 patients, the tumour was located above the STO (11%), with interhospital variation between 0% and 36%. Based on TN-stage, 175 reclassified patients with colon cancer (6%) would have received different treatment (e.g., omitting neoadjuvant radiotherapy, candidate for adjuvant chemotherapy). Tumour location above the STO was independently associated with lower risk of 4-year locoregional recurrence (HR 0.529; p = 0.030) and higher 4-year overall survival (HR 0.732; p = 0.037) compared to location under the STO. Conclusion: By using the STO, 11% of the prior MDT-based diagnosis of rectal cancer were redefined as sigmoid cancer, with potential implications for multimodality treatment and prognostic value. Given the substantial interhospital variation in proportion of redefined cancers, the use of the STO will contribute to standardisation and comparability of outcomes in both daily practice and trial settings.</p
Fungal chitinases: diversity, mechanistic properties and biotechnological potential
Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review
Behavioural and psychological symptoms in the older population without dementia - relationship with socio-demographics, health and cognition
<p>Abstract</p> <p>Background</p> <p>Behavioural and psychological symptoms are associated with dementia, but are also present in a significant number of the older population without dementia. Here we explore the distribution of behavioural and psychological symptoms in the population without dementia, and their relationship with domains and severity of health and cognitive impairment.</p> <p>Methods</p> <p>The Medical Research Council Cognitive Function and Ageing Study is a two-phase longitudinal study of ageing representative of the population aged 65 and over of England and Wales. A subsample of 1781 participants without a study diagnosis of dementia was included in this study. Information on symptoms including depression, apathy, anxiety, feelings of persecution, hallucination, agitated behaviour, elation, irritability, sleep problems, wandering, confabulation and misidentification, cognitive function, health related factors and socio-demographic information was extracted from interviews with participants and knowledgeable informants. Participants were classified according to the Mini-Mental State Examination and by criteria for subtypes of mild cognitive impairment (MCI). The prevalence of behavioural and psychological symptoms and associations with cognitive function, health and socio-demographics was examined. Co-occurrence of symptoms was tested using factor analysis.</p> <p>Results</p> <p>Most symptoms were reported more frequently in those with more severe cognitive impairment. Subjective memory complaints were the strongest independent predictor of reported symptoms, and most were reported more often in those classified as having MCI than in those with cognitive impairments that did not meet the MCI criteria. The pattern of co-occurrence of symptoms is similar to that seen in dementia.</p> <p>Conclusions</p> <p>Our results highlight that behavioural and psychological symptoms are prevalent in the cognitively impaired older population, and partly explain the variation observed in previous cohorts of individuals with MCI. Behavioural and psychological symptoms offer a target for intervention and so are an important consideration in the assessment of cognitively impaired older people.</p
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