The diagnostic accuracy of multiparametric magnetic resonance imaging before biopsy in the detection of prostate cancer.

OBJECTIVES: To determine the extent to which clinically significant prostate cancer (csPCa) can be detected in a routine National Health Service setting in men with no previous biopsy, when multiparametric magnetic resonance imaging (mpMRI) is introduced into the diagnostic pathway. PATIENTS AND METHODS: In all, 1 090 mpMRIs were performed between July 2013 and April 2016 in biopsy-naïve men with an abnormal prostate-specific antigen level and/or digital rectal examination. Data were collected from patient records at the Royal Devon and Exeter NHS Foundation Trust. mpMRI Prostate Imaging Reporting and Data System (PI-RADS) scores were compared to transperineal or transrectal ultrasonography (TRUS)-guided biopsy findings as the reference standard. csPCa was defined as Gleason score of ≥3+4. The diagnostic accuracy of mpMRI was also assessed. RESULTS: The mpMRI was interpretable in 1 023 men and 792 underwent biopsy, of which 106 were transperineal. The median number of cores taken in transperineal and TRUS-guided biopsy were 10 and 6, respectively. The detection rate of csPCa was 37%; csPCa rose from 15% of PI-RADS 1 and 2 to 86% of PI-RADS 5. The sensitivity, negative predictive value, specificity, and positive predictive value were 82%, 85%, 59% and 54%, respectively. The study is limited by its retrospective nature and lack of reporting of follow-up for 'missed cancers'. Men with low mpMRI PI-RADS were also less likely to undergo biopsy. Whilst this selection bias may overestimate the detection rate of csPCa, this reflects the shared decisions patients and clinicians make in day-to-day practice outside of research centres. CONCLUSION: In a routine clinical setting, the higher the mpMRI PI-RADS, the greater the detection rate of csPCa in biopsy-naïve men. A normal mpMRI does not exclude csPCa; however, mpMRI may have utility in informing shared-decision making on whether to proceed to biopsy and subsequent treatment.

Intravascular versus surface cooling for targeted temperature management after out-of-hospital cardiac arrest - an analysis of the TTM trial data.

BACKGROUND: Targeted temperature management is recommended after out-of-hospital cardiac arrest and may be achieved using a variety of cooling devices. This study was conducted to explore the performance and outcomes for intravascular versus surface devices for targeted temperature management after out-of-hospital cardiac arrest. METHOD: A retrospective analysis of data from the Targeted Temperature Management trial. N = 934. A total of 240 patients (26%) managed with intravascular versus 694 (74%) with surface devices. Devices were assessed for speed and precision during the induction, maintenance and rewarming phases in addition to adverse events. All-cause mortality, as well as a composite of poor neurological function or death, as evaluated by the Cerebral Performance Category and modified Rankin scale were analysed. RESULTS: For patients managed at 33 °C there was no difference between intravascular and surface groups in the median time taken to achieve target temperature (210 [interquartile range (IQR) 180] minutes vs. 240 [IQR 180] minutes, p = 0.58), maximum rate of cooling (1.0 [0.7] vs. 1.0 [0.9] °C/hr, p = 0.44), the number of patients who reached target temperature (within 4 hours (65% vs. 60%, p = 0.30); or ever (100% vs. 97%, p = 0.47), or episodes of overcooling (8% vs. 34%, p = 0.15). In the maintenance phase, cumulative temperature deviation (median 3.2 [IQR 5.0] °C hr vs. 9.3 [IQR 8.0] °C hr, p = <0.001), number of patients ever out of range (57.0% vs. 91.5%, p = 0.006) and median time out of range (1 [IQR 4.0] hours vs. 8.0 [IQR 9.0] hours, p = <0.001) were all significantly greater in the surface group although there was no difference in the occurrence of pyrexia. Adverse events were not different between intravascular and surface groups. There was no statistically significant difference in mortality (intravascular 46.3% vs. surface 50.0%; p = 0.32), Cerebral Performance Category scale 3-5 (49.0% vs. 54.3%; p = 0.18) or modified Rankin scale 4-6 (49.0% vs. 53.0%; p = 0.48). CONCLUSIONS: Intravascular and surface cooling was equally effective during induction of mild hypothermia. However, surface cooling was associated with less precision during the maintenance phase. There was no difference in adverse events, mortality or poor neurological outcomes between patients treated with intravascular and surface cooling devices. TRIAL REGISTRATION: TTM trial ClinicalTrials.gov number https://clinicaltrials.gov/ct2/show/NCT01020916 NCT01020916; 25 November 2009.

Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

In-Circuit High-Frequency Jet Ventilation for Radiological Tumor Ablation: A Case Series.

High-frequency jet ventilation (HFJV) can reduce organ movement that otherwise complicates percutaneous image-guided ablation (IGA) procedures. This study describes feasibility and safety of the technique in routine use. We describe our method for the use of HFJV and present 169 consecutive cases, including IGA of tumors of the lung, liver, kidney, and pancreas. Intended oncological treatment was delivered in all cases and HFJV used for the duration of treatment in all except one case. We describe the characteristics of patients, procedures, and adverse events. It is feasible to use HFJV as the routine standard of care for IGA.

Decreased degradation of internalized follicle-stimulating hormone caused by mutation of aspartic acid 6.30(550) in a protein kinase-CK2 consensus sequence in the third intracellular loop of human follicle-stimulating hormone receptor.

A naturally occurring mutation in follicle-stimulating hormone receptor (FSHR) gene has been reported: an amino acid change to glycine occurs at a conserved aspartic acid 550 (D550, D567, D6.30(567)). This residue is contained in a protein kinase-CK2 consensus site present in human FSHR (hFSHR) intracellular loop 3 (iL3). Because CK2 has been reported to play a role in trafficking of some receptors, the potential roles for CK2 and D550 in FSHR function were evaluated by generating a D550A mutation in the hFSHR. The hFSHR-D550A binds hormone similarly to WT-hFSHR when expressed in HEK293T cells. Western blot analyses showed lower levels of mature hFSHR-D550A. Maximal cAMP production of both hFSHR-D550A as well as the naturally occurring mutation hFSHR-D550G was diminished, but constitutive activity was not observed. Unexpectedly, when (125)I-hFSH bound to hFSHR-D550A or hFSHR-D550G, intracellular accumulation of radiolabeled FSH was observed. Both sucrose and dominant-negative dynamin blocked internalization of radiolabeled FSH and its commensurate intracellular accumulation. Accumulation of radiolabeled FSH in cells transfected with hFSHR-D550A is due to a defect in degradation of hFSH as measured in pulse chase studies, and confocal microscopy imaging revealed that FSH accumulated in large intracellular structures. CK2 kinase activity is not required for proper degradation of internalized FSH because inhibition of CK2 kinase activity in cells expressing hFSHR did not uncouple degradation of internalized radiolabeled FSH. Additionally, the CK2 consensus site in FSHR iL3 is not required for binding because CK2alpha coimmunoprecipitated with hFSHR-D550A. Thus, mutation of D550 uncouples the link between internalization and degradation of hFSH.

Follice-stimulating hormone receptor forms oligomers and shows evidence of carboxyl-terminal proteolytic processing.

FSH receptor (FSHR), a member of the G protein-coupled receptor superfamily, is present in the plasma membrane of ovarian granulosa cells and testicular Sertoli cells. FSH regulates normal ovarian follicle development and spermatogenesis through FSHR. The extracellular domain of FSHR is a weakly associated homodimer in the recently solved crystal structure of FSH in complex with the extracellular domain of FSHR. However, there is currently no biochemical data that demonstrate that FSHR exists as a dimer or higher-order oligomer in cell membranes. A fluorescence resonance energy transfer assay was used to determine whether full-length native FSHR is an oligomer. FSHR-specific monoclonal antibody or Fab fragments, labeled with two different fluorophores, allowed the study of nontagged receptor in situ. Unoccupied FSHR exhibited strong fluorescence resonance energy transfer profiles in situ. Complementary coimmunoprecipitation experiments of myc- or FLAG-tagged FSHR indicated that FSHR forms oligomers early in receptor biosynthesis. No effect of FSH treatment was observed. Thus, immature forms of FSHR, not yet fully processed, were observed to coimmunoprecipitate. An unexpected observation was made that the C-terminal epitope tags are removed from FSHR before arrival at the cell surface. These results provide the first evidence for oligomers of full-length FSHR in situ and for C-terminal proteolytic processing of FSHR and that both events take place during biosynthesis. This may explain how heterozygous mutations in the FSHR gene that affect receptor trafficking may be ameliorated by oligomer formation.

APPL1, APPL2, Akt2 and FOXO1a interact with FSHR in a potential signaling complex.

A number of signaling proteins have been demonstrated to interact with follicle stimulating hormone (FSH) receptor (FSHR), including APPL1, 14-3-3tau and Akt2. To further define the repertoire of proteins involved in FSH-induced signal transduction, several signaling and adapter proteins were examined for the ability to associate with FSHR. This report shows that, in addition to APPL1, FSHR interacts with FOXO1a and APPL2. Moreover, APPL1 and APPL2 associate with one another via the N-terminus of APPL1, presumably via the Bin-Amphiphysin-Rvs (BAR) domain. The interactions between FSHR and APPL2 and between FSHR and FOXO1a evidently are distinct since FOXO1a does not associate with either APPL1 or with APPL2. Though APPL1 and APPL2 show some similarity in primary sequence, APPL1 associates with Akt2, whereas APPL2 does not. This is the first documented difference in function between APPL1 and APPL2. These results suggest that FSHR, APPL1, APPL2, Akt2 and FOXO1a are organized into distinct scaffolding networks in the cell. Accordingly, the spatial organization of signaling and adapter proteins with FSHR likely facilitates and finely regulates the signal transduction induced by FSH.

Accuracy Rates of Ancestry Estimation by Forensic Anthropologists Using Identified Forensic Cases.

A common task in forensic anthropology involves the estimation of the ancestry of a decedent by comparing their skeletal morphology and measurements to skeletons of individuals from known geographic groups. However, the accuracy rates of ancestry estimation methods in actual forensic casework have rarely been studied. This article uses 99 forensic cases with identified skeletal remains to develop accuracy rates for ancestry estimations conducted by forensic anthropologists. The overall rate of correct ancestry estimation from these cases is 90.9%, which is comparable to most research-derived rates and those reported by individual practitioners. Statistical tests showed no significant difference in accuracy rates depending on examiner education level or on the estimated or identified ancestry. More recent cases showed a significantly higher accuracy rate. The incorporation of metric analyses into the ancestry estimate in these cases led to a higher accuracy rate.

Loss of the LIM domain protein Lmo4 in the mammary gland during pregnancy impedes lobuloalveolar development.

LMO4, a member of the LIM-only family of zinc-finger proteins, is overexpressed in a significant proportion of breast carcinomas and acts as a negative regulator of mammary epithelial differentiation. To delineate cell types within the developing mouse mammary gland that express Lmo4, we analysed different stages of mammopoiesis by immunohistochemistry. Lmo4 was found to be highly expressed in the proliferating cap cells of the terminal end bud and in the ductal and alveolar luminal cells of the mature mammary gland but was negligible or low in myoepithelial cells. To assess the physiological role of Lmo4 in the mammary gland, we generated conditionally targeted mice lacking Lmo4 in the mammary epithelium during pregnancy. Acute loss of Lmo4 in late pregnancy impaired lobuloalveolar development, accompanied by a two-fold reduction in the percentage of BrdU-positive cells. In contrast, germline loss of Lmo4 did not alter lobuloalveolar development arising from transplanted mammary anlagen, implying the existence of a compensatory mechanism in these knockout mice. Thus, the use of a conditional targeting strategy has revealed that Lmo4 is required for proper development of the mammary gland during pregnancy and indicated that Lmo4 acts as a positive regulator of alveolar epithelial proliferation.

Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage.

This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display.

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.

Accuracy Rates of Sex Estimation by Forensic Anthropologists through Comparison with DNA Typing Results in Forensic Casework.

A common task in forensic anthropology involves the estimation of the biological sex of a decedent by exploiting the sexual dimorphism between males and females. Estimation methods are often based on analysis of skeletal collections of known sex and most include a research-based accuracy rate. However, the accuracy rates of sex estimation methods in actual forensic casework have rarely been studied. This article uses sex determinations based on DNA results from 360 forensic cases to develop accuracy rates for sex estimations conducted by forensic anthropologists. The overall rate of correct sex estimation from these cases is 94.7% with increasing accuracy rates as more skeletal material is available for analysis and as the education level and certification of the examiner increases. Nine of 19 incorrect assessments resulted from cases in which one skeletal element was available, suggesting that the use of an "undetermined" result may be more appropriate for these cases.

Tables for the metric evaluation of pair-matching of human skeletal elements.

A common task in forensic anthropology involves pair-matching of left and right skeletal elements. This can be achieved through visual pair-matching by evaluating similarities in morphology, and through osteometric sorting, a quantitative technique. To simplify the process of osteometric sorting, this article explains the use of a statistic (M), which captures the amount of size variation found between homologous bones from single individuals. A database of skeletal measurements for all major paired postcranial bones is used to calculate values of M from a variety of sources. The maximum value and the 90th and 95th percentiles of M are provided in tabular format, and values of M from forensic cases can be compared to these tables as an objective means for determining whether homologous bones could have originated from the same individual. This simple technique can be combined with visual pair-matching to be particularly effective in cases involving commingling of skeletons.

Validation of X-ray fluorescence spectrometry for determining osseous or dental origin of unknown material.

Forensic anthropological examinations typically involve the analysis of human skeletal remains, but in cases where samples are very small and/or physically compromised, it may first be necessary to determine whether the material is even osseous or dental in origin. X-ray fluorescence spectrometry (XRF) is a technique that reveals the elemental composition of materials and is hypothesized to have utility in such cases. XRF analysis was conducted on a variety of tissues and materials in unaltered and altered (damaged) states. With few exceptions, osseous and dental tissues in unaltered and altered conditions contained characteristic levels of calcium and phosphorus, while other materials did not. Materials could be accurately identified as osseous or dental in origin based on the calcium and phosphorus levels identified by XRF, and we therefore conclude that XRF analysis is a valid and effective means of determining osseous or dental origin of unknown material.

The adapter protein APPL1 links FSH receptor to inositol 1,4,5-trisphosphate production and is implicated in intracellular Ca(2+) mobilization.

FSH binds to its receptor (FSHR) on target cells in the ovary and testis, to regulate oogenesis and spermatogenesis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and inositol 1,4,5-trisphosphate-mediated calcium signaling pathways. The adapter protein APPL1 (Adapter protein containing Pleckstrin homology domain, Phosphotyrosine binding domain and Leucine zipper motif), which has been linked to an assortment of other signaling proteins, was previously identified as an interacting protein with FSHR. Thus, alanine substitution mutations in the first intracellular loop of FSHR were generated to determine which residues are essential for FSHR-APPL1 interaction. Three amino acids were essential; when any one of them was altered, APPL1 association with FSHR mutants was abrogated. Two of the mutants (L377A and F382A) that displayed poor cell-surface expression were not studied further. Substitution of FSHR-K376A did not affect FSH binding or agonist-stimulated cAMP production in either transiently transfected human embryonic kidney cells or virally transduced human granulosa cells (KGN). In the KGN line, as well as primary cultures of rat granulosa cells transduced with wild type or mutant receptor, FSH-mediated progesterone or estradiol production was not affected by the mutation. However, in human embryonic kidney cells inositol 1,4,5-trisphosphate production was curtailed and KGN cells transduced with FSHR-K376A evidenced reduced Ca(2+) mobilization from intracellular stores after FSH treatment.

A negative allosteric modulator demonstrates biased antagonism of the follicle stimulating hormone receptor.

High quality gamete production in males and females requires the pituitary gonadotropin follicle stimulating hormone (FSH). In this report a novel chemical class of small molecule inhibitors of FSH receptor (FSHR) is described. ADX61623, a negative allosteric modulator (NAM), increased the affinity of interaction between (125)I-hFSH and human FSHR (hFSHR) five fold. This form of FSHR occupied simultaneously by FSH and ADX61623 was inactive for cAMP and progesterone production in primary cultures of rat granulosa cells. In contrast, ADX61623 did not block estrogen production. This demonstrates for the first time, biased antagonism at the FSHR. To determine if ADX61623 blocked FSH induction of follicle development in vivo, a bioassay to measure follicular development and oocyte production in immature female rats was validated. ADX61623 was not completely effective in blocking FSH induced follicular development in vivo at doses up to 100mg/kg as oocyte production and ovarian weight gain were only moderately reduced. These data illustrate that FSHR couples to multiple signaling pathways in vivo. Suppression of one pool of FSHR uncouples Gαs and cAMP production, and decreases progesterone production. Occupancy of another pool of FSHR sensitizes granulosa cells to FSH induced estradiol production. Therefore, ADX61623 is a useful tool to investigate further the mechanism of the FSHR signaling dichotomy. This may lead to a greater understanding of the signaling infrastructure which enables estrogen biosynthesis and may prove useful in treating estrogen dependent disease.

Emerging roles for the FSH receptor adapter protein APPL1 and overlap of a putative 14-3-3τ interaction domain with a canonical G-protein interaction site.

The interaction of cytoplasmic proteins with intracellular domains of membrane receptors can occur at several opportunities, including: during biosynthesis, while in membrane residency and during internalization and recycling following ligand binding. Since the initial discovery that it interacts with the FSH receptor (FSHR) together with additional members of a potential signaling complex, APPL1 has been shown to interact with a variety of membrane receptors. Recent subcellular localizations of APPL1 place it in dynamic and varied venues in the cell, including at the cell membrane, the nucleus and the early endosomes. Another adapter protein family the 14-3-3 proteins, are largely recognized as binding to phosphorylation sites but recent work demonstrated that in the case of FSHR, the 14-3-3 site overlaps with the canonical G-protein binding site. G-proteins appear to sample the environment and exchange between the membrane and intracellular locales and this binding could be mediated by or modulated by receptor interactions at the 14-3-3 binding site. Observations that multiple proteins can interact with cytoplasmic domains of GPCRs leads to the inescapable conclusion that either the interactions occur via orderly replacement or exchange, or that receptors are simultaneously occupied by a variety of adapters and effectors or even that oligomers of dimeric GPCRs provide for platforms that can simultaneously interact with effectors and adaptors.