Magnetic Resonance Imaging Follow-up of Targeted Biopsy-negative Prostate Lesions.

BACKGROUND: The optimal radiological follow-up of prostate lesions negative on magnetic resonance imaging (MRI)-targeted biopsy (MRI-TB) is yet to be optimised. OBJECTIVE: To present medium-term radiological and clinical follow-up of biopsy-negative lesions. DESIGN, SETTING, AND PARTICIPANTS: The records for men who underwent multiparametric MRI at the UCLH one-stop clinic for suspected prostate cancer between September 2017 and March 2020 were reviewed (n = 1199). Patients with Likert 4 or 5 lesions were considered (n = 495), and those with a subsequent negative MRI-TB comprised the final study population (n = 91). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Baseline and follow-up MRI and biopsy data (including prostate-specific antigen [PSA], prostate volume, radiological scores, and presence of any noncancerous pathology) were extracted from reports. The last follow-up date was the date of the last test or review in clinic. RESULTS AND LIMITATIONS: Median follow-up was 1.8 yr (656 d, interquartile range [IQR] 359-1008). At baseline, the median age was 65.4 yr (IQR 60.7-70.0), median PSA was 7.1 ng/ml (IQR 4.7-10.0), median prostate volume was 54 ml (IQR 39.5-75.0), and median PSA density (PSAD) was 0.13 ng/ml2 (IQR 0.09-0.18). Eighty-six men (95%) had Likert 4 lesions, while the remaining five (5%) had Likert 5 lesions. Only 21 men (23%) had a single lesion; most had at least two. Atrophy was the most prevalent pathology on MRI-TB, present in 64 men (74%), and followed by acute inflammation in 42 (46%), prostatic intraepithelial neoplasia in 33 (36%), chronic inflammation in 18 (20%), atypia in 13 (14%), and granulomatous inflammation in three (3%). Fifty-eight men had a second MRI study (median 376 d, IQR 361-412). At the second MRI, median PSAD decreased to 0.11 ng/ml2 (IQR 0.08-0.18). A Likert 4 or 5 score persisted only in five men (9%); 40 men (69%) were scored Likert 3, while the remaining 13 (22%) were scored Likert 2 (no lesion). Of 45 men with a Likert ≥3 score, most only had one lesion at the second MRI (28 men; 62%). Of six men with repeat MRI-TB during the study period, two were subsequently diagnosed with prostate cancer and both had persistent Likert 4 scores (at baseline and at least one follow-up MRI). CONCLUSIONS: Most biopsy-negative MRI lesions in the prostate resolve over time, but any persistent lesions should be closely monitored. PATIENT SUMMARY: Lesions in the prostate detected via magnetic resonance imaging (MRI) scans that are negative for cancer on biopsy usually resolve. Repeat MRI can indicate persistent lesions that might need a second biopsy.

Author Correction: Genetic alterations in the 3q26.31-32 locus confer an aggressive prostate cancer phenotype.

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

Genetic alterations in the 3q26.31-32 locus confer an aggressive prostate cancer phenotype.

Large-scale genetic aberrations that underpin prostate cancer development and progression, such as copy-number alterations (CNAs), have been described but the consequences of specific changes in many identified loci is limited. Germline SNPs in the 3q26.31 locus are associated with aggressive prostate cancer, and is the location of NAALADL2, a gene overexpressed in aggressive disease. The closest gene to NAALADL2 is TBL1XR1, which is implicated in tumour development and progression. Using publicly-available cancer genomic data we report that NAALADL2 and TBL1XR1 gains/amplifications are more prevalent in aggressive sub-types of prostate cancer when compared to primary cohorts. In primary disease, gains/amplifications occurred in 15.99% (95% CI: 13.02-18.95) and 14.96% (95% CI: 12.08-17.84%) for NAALADL2 and TBL1XR1 respectively, increasing in frequency in higher Gleason grade and stage tumours. Gains/amplifications result in transcriptional changes and the development of a pro-proliferative and aggressive phenotype. These results support a pivotal role for copy-number gains in this genetic region.

Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains.

Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD-CTD to the nucleosome.

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones.

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.

Vertebrate nucleoplasmin and NASP: egg histone storage proteins with multiple chaperone activities.

Recent reviews have focused on the structure and function of histone chaperones involved in different aspects of somatic cell chromatin metabolism. One of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by egg histone storage chaperones. These include members of the nucleoplasmin (NPM2/NPM3), which are preferentially associated with histones H2A-H2B in the egg and the nuclear autoantigenic sperm protein (NASP) families. Interestingly, in addition to binding and providing storage to H3/H4 in the egg and in somatic cells, NASP has been shown to be a unique genuine chaperone for histone H1. This review revolves around the structural and functional roles of these two families of chaperones whose activity is modulated by their own post-translational modifications (PTMs), particularly phosphorylation. Beyond their important role in the remodeling of paternal chromatin in the early stages of embryogenesis, NPM and NASP members can interact with a plethora of proteins in addition to histones in somatic cells and play a critical role in processes of functional cell alteration, such as in cancer. Despite their common presence in the egg, these two histone chaperones appear to be evolutionarily unrelated. In contrast to members of the NPM family, which share a common monophyletic evolutionary origin, the different types of NASP appear to have evolved recurrently within different taxa.

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease.

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.

The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function.

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1-171, contains two amphipathic domains spanning residues 24-41 and 66-83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1₋171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1₋171 undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1₋171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional.

Nucleoplasmin binds histone H2A-H2B dimers through its distal face.

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here, we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information can help to understand the function of NP as a histone chaperone.

Pharmacokinetic assessment of the uptake of 16beta-18F-fluoro-5alpha-dihydrotestosterone (FDHT) in prostate tumors as measured by PET.

UNLABELLED: The aim of this study was to develop a clinically applicable noninvasive method to quantify changes in androgen receptor (AR) levels based on (18)F-16beta-fluoro-5alpha-dihydrotestosterone ((18)F-FDHT) PET in prostate cancer patients undergoing therapy. METHODS: Thirteen patients underwent dynamic (18)F-FDHT PET over a selected tumor. Concurrent venous blood samples were acquired for blood metabolite analysis. A second cohort of 25 patients injected with (18)F-FDHT underwent dynamic PET of the heart. These data were used to generate a population-based input function, essential for pharmacokinetic modeling. Linear compartmental pharmacokinetic models of increasing complexity were tested on the tumor tissue data. Four suitable models were applied and compared using the Bayesian information criterion (BIC). Model 1 consisted of an instantaneously equilibrating space, followed by a unidirectional trap. Models 2a and 2b contained a reversible space between the instantaneously equilibrating space and the trap, into which metabolites were excluded (2a) or allowed (2b). Model 3 built on model 2b with the addition of a second reversible space preceding the unidirectional trap and from which metabolites were excluded. RESULTS: The half-life of the (18)F-FDHT in blood was between 6 and 7 min. As a consequence, the uptake of (18)F-FDHT in prostate cancer lesions reached a plateau within 20 min as the blood-borne activity was consumed. Radiolabeled metabolites were shown not to bind to ARs in in vitro studies with CWR22 cells. Model 1 produced reasonable and robust fits for all datasets and was judged best by the BIC for 16 of 26 tumor scans. Models 2a, 2b, and 3 were judged best in 7, 2, and 1 cases, respectively. CONCLUSION: Our study explores the clinical potential of using (18)F-FDHT PET to estimate free AR concentration. This process involved the estimation of a net uptake parameter such as the k(trap) of model 1 that could serve as a surrogate measure of AR expression in metastatic prostate cancer. Our initial studies suggest that a simple body mass-normalized standardized uptake value correlates reasonably well to model-based k(trap) estimates, which we surmise may be proportional to AR expression. Validation studies to test this hypothesis are underway.

H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies.

Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.

Leishmania donovani peroxin 14 undergoes a marked conformational change following association with peroxin 5.

The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.

sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly.

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of alpha-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N- and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.

Divergent modes of glycan recognition by a new family of carbohydrate-binding modules.

The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.

Nucleoplasmin-mediated unfolding of chromatin involves the displacement of linker-associated chromatin proteins.

We have previously characterized the interaction of nucleoplasmin with core histones and studied the possible involvement of this chaperone molecule in transcription. Here we study the interaction of nucleoplasmin with chromatin. We show that highly phosphorylated Xenopus laevis egg nucleoplasmin can unfold sperm and somatic chromatin in a way that involves the removal of chromosomal proteins from linker DNA regions without a stable interaction with the nucleosome. The complexes between egg nucleoplasmin and both somatic and sperm-specific linker proteins have been hydrodynamically characterized using sedimentation equilibrium in the analytical ultracentrifuge. The results are discussed within the context of the possible implication of nucleoplasmin in processes such as transcription and replication licensing which take place after egg fertilization at the onset of development.

The anthracycline antibiotics: antitumor drugs that alter chromatin structure.

Anthracycline antibiotics are an important group of antitumor drugs widely used in cancer chemotherapy. However, despite the increasing interest in these chemotherapeutic agents, their mechanism of action is not yet completely understood. Here, we review what is currently known about the molecular mechanisms involved with special emphasis on the interaction of these drugs with chromatin and its constitutive components: DNA and histones. The evidence suggests that one very important component of the activity of these drugs is the result of these manifold interactions that lead to a chromatin unfolding and aggregation. This chromatin structural disruption is likely to interfere with the metabolic processes of DNA (replication and transcription) and it may play an important role in the apoptosis undergone by the cells upon treatment with these drugs.

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome.

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Binding of antitumor antibiotic daunomycin to histones in chromatin and in solution.

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.

Alpha-glucan recognition by a new family of carbohydrate-binding modules found primarily in bacterial pathogens.

TmPul13, a family 13 glycoside hydrolase from Thermotoga maritima, is a four-module protein having pullulanase activity; the three N-terminal modules are of unknown function while the large C-terminal module is likely the catalytic module. Dissection of the functions of the three unknown modules revealed that the 100 amino acid module at the extreme N-terminus of TmPul13 comprises a new family of carbohydrate-binding modules (CBM) that a bioinformatic analysis shows are most frequently found in pullulanase-like sequences from bacterial pathogens. Detailed binding studies of this isolated CBM, here called TmCBM41, reveals a preference for alpha-(1,4)-linked glucans, but occasional alpha-(1,6)-linked glucose residues, such as those found in pullulan, are tolerated. UV difference, isothermal titration calorimetry, and analytical ultracentrifugation binding studies suggest that maltooligosaccharides longer than four glucose residues are able to bind two TmCBM41 molecules per oligosaccharide when sugar concentrations are below the CBM concentration. This is explained in terms of an equilibrium expression involving the formation of both a 1 to 1 sugar to CBM complex and a 1 to 2 sugar to CBM complex (i.e., a CBM dimer ligated by an oligosaccharide). The presence of an alpha-(1-6) linkage in the oligosaccharide appears to prevent this phenomenon.