In vitro evolution of ribonucleases from expanded genetic alphabets.

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid-base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

Reward insensitivity is associated with dopaminergic deficit in rapid eye movement sleep behaviour disorder.

Idiopathic rapid eye movement sleep behaviour disorder (iRBD) has now been established as an important marker of the prodromal stage of Parkinson's disease and related synucleinopathies. However, although dopamine transporter single photon emission computed tomography (SPECT) has been used to demonstrate the presence of nigro-striatal deficit in iRBD, quantifiable correlates of this are currently lacking. Sensitivity to rewarding stimuli is reduced in some people with Parkinson's disease, potentially contributing to aspects of the neuropsychiatric phenotype in these individuals. Furthermore, a role for dopaminergic degeneration is suggested by the fact that reward insensitivity can be improved by dopaminergic medications. Patients with iRBD present a unique opportunity to study the relationship between reward sensitivity and early dopaminergic deficit in the unmedicated state. Here, we investigate whether a non-invasive, objective measure of reward sensitivity might be a marker of dopaminergic status in prodromal Parkinson's disease by comparing with SPECT/CT measurement of dopaminergic loss in the basal ganglia. Striatal dopaminergic deficits in iRBD are associated with progression to Parkinsonian disorders. Therefore, identification of a clinically measurable correlate of this degenerative process might provide a basis for the development of novel risk stratification tools. Using a recently developed incentivized eye-tracking task, we quantified reward sensitivity in a cohort of 41 patients with iRBD and compared this with data from 40 patients with Parkinson's disease and 41 healthy controls. Patients with iRBD also underwent neuroimaging with dopamine transporter SPECT/CT. Overall, reward sensitivity, indexed by pupillary response to monetary incentives, was reduced in iRBD cases compared with controls and was not significantly different to that in patients with Parkinson's disease. However, in iRBD patients with normal dopamine transporter SPECT/CT imaging, reward sensitivity was not significantly different from healthy controls. Across all iRBD cases, a positive association was observed between reward sensitivity and dopaminergic SPECT/CT signal in the putamen. These findings demonstrate a direct relationship between dopaminergic deficit and reward sensitivity in patients with iRBD and suggest that measurement of pupillary responses could be of value in models of risk stratification and disease progression in these individuals.

Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

Deep learning-based time-of-flight (ToF) image enhancement of non-ToF PET scans.

PURPOSE: To improve the quantitative accuracy and diagnostic confidence of PET images reconstructed without time-of-flight (ToF) using deep learning models trained for ToF image enhancement (DL-ToF). METHODS: A total of 273 [18F]-FDG PET scans were used, including data from 6 centres equipped with GE Discovery MI ToF scanners. PET data were reconstructed using the block-sequential-regularised-expectation-maximisation (BSREM) algorithm with and without ToF. The images were then split into training (n = 208), validation (n = 15), and testing (n = 50) sets. Three DL-ToF models were trained to transform non-ToF BSREM images to their target ToF images with different levels of DL-ToF strength (low, medium, high). The models were objectively evaluated using the testing set based on standardised uptake value (SUV) in 139 identified lesions, and in normal regions of liver and lungs. Three radiologists subjectively rated the models using testing sets based on lesion detectability, diagnostic confidence, and image noise/quality. RESULTS: The non-ToF, DL-ToF low, medium, and high methods resulted in - 28 ± 18, - 28 ± 19, - 8 ± 22, and 1.7 ± 24% differences (mean; SD) in the SUVmax for the lesions in testing set, compared to ToF-BSREM image. In background lung VOIs, the SUVmean differences were 7 ± 15, 0.6 ± 12, 1 ± 13, and 1 ± 11% respectively. In normal liver, SUVmean differences were 4 ± 5, 0.7 ± 4, 0.8 ± 4, and 0.1 ± 4%. Visual inspection showed that our DL-ToF improved feature sharpness and convergence towards ToF reconstruction. Blinded clinical readings of testing sets for diagnostic confidence (scale 0-5) showed that non-ToF, DL-ToF low, medium, and high, and ToF images scored 3.0, 3.0, 4.1, 3.8, and 3.5 respectively. For this set of images, DL-ToF medium therefore scored highest for diagnostic confidence. CONCLUSION: Deep learning-based image enhancement models may provide converged ToF-equivalent image quality without ToF reconstruction. In clinical scoring DL-ToF-enhanced non-ToF images (medium and high) on average scored as high as, or higher than, ToF images. The model is generalisable and hence, could be applied to non-ToF images from BGO-based PET/CT scanners.

Image enhancement of whole-body oncology [18F]-FDG PET scans using deep neural networks to reduce noise.

PURPOSE: To enhance the image quality of oncology [18F]-FDG PET scans acquired in shorter times and reconstructed by faster algorithms using deep neural networks. METHODS: List-mode data from 277 [18F]-FDG PET/CT scans, from six centres using GE Discovery PET/CT scanners, were split into ¾-, ½- and »-duration scans. Full-duration datasets were reconstructed using the convergent block sequential regularised expectation maximisation (BSREM) algorithm. Short-duration datasets were reconstructed with the faster OSEM algorithm. The 277 examinations were divided into training (n = 237), validation (n = 15) and testing (n = 25) sets. Three deep learning enhancement (DLE) models were trained to map full and partial-duration OSEM images into their target full-duration BSREM images. In addition to standardised uptake value (SUV) evaluations in lesions, liver and lungs, two experienced radiologists scored the quality of testing set images and BSREM in a blinded clinical reading (175 series). RESULTS: OSEM reconstructions demonstrated up to 22% difference in lesion SUVmax, for different scan durations, compared to full-duration BSREM. Application of the DLE models reduced this difference significantly for full-, ¾- and ½-duration scans, while simultaneously reducing the noise in the liver. The clinical reading showed that the standard DLE model with full- or ¾-duration scans provided an image quality substantially comparable to full-duration scans with BSREM reconstruction, yet in a shorter reconstruction time. CONCLUSION: Deep learning-based image enhancement models may allow a reduction in scan time (or injected activity) by up to 50%, and can decrease reconstruction time to a third, while maintaining image quality.

Dopaminergic imaging and clinical predictors for phenoconversion of REM sleep behaviour disorder.

This is an international multicentre study aimed at evaluating the combined value of dopaminergic neuroimaging and clinical features in predicting future phenoconversion of idiopathic REM sleep behaviour (iRBD) subjects to overt synucleinopathy. Nine centres sent 123I-FP-CIT-SPECT data of 344 iRBD patients and 256 controls for centralized analysis. 123I-FP-CIT-SPECT images were semiquantified using DaTQUANTTM, obtaining putamen and caudate specific to non-displaceable binding ratios (SBRs). The following clinical variables were also analysed: (i) Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale, motor section score; (ii) Mini-Mental State Examination score; (iii) constipation; and (iv) hyposmia. Kaplan-Meier survival analysis was performed to estimate conversion risk. Hazard ratios for each variable were calculated with Cox regression. A generalized logistic regression model was applied to identify the best combination of risk factors. Bayesian classifier was used to identify the baseline features predicting phenoconversion to parkinsonism or dementia. After quality check of the data, 263 iRBD patients (67.6 ± 7.3 years, 229 males) and 243 control subjects (67.2 ± 10.1 years, 110 males) were analysed. Fifty-two (20%) patients developed a synucleinopathy after average follow-up of 2 years. The best combination of risk factors was putamen dopaminergic dysfunction of the most affected hemisphere on imaging, defined as the lower value between either putamina (P < 0.000001), constipation, (P < 0.000001) and age over 70 years (P = 0.0002). Combined features obtained from the generalized logistic regression achieved a hazard ratio of 5.71 (95% confidence interval 2.85-11.43). Bayesian classifier suggested that patients with higher Mini-Mental State Examination score and lower caudate SBR asymmetry were more likely to develop parkinsonism, while patients with the opposite pattern were more likely to develop dementia. This study shows that iRBD patients older than 70 with constipation and reduced nigro-putaminal dopaminergic function are at high risk of short-term phenoconversion to an overt synucleinopathy, providing an effective stratification approach for future neuroprotective trials. Moreover, we provide cut-off values for the significant predictors of phenoconversion to be used in single subjects.

Response rate and diagnostic accuracy of early PET-CT during neo-adjuvant therapies in oesophageal adenocarcinoma: A systematic review and meta-analysis.

PURPOSE: Only 25% of oesophageal adenocarcinoma (OAC) patients have a pathological response to neo-adjuvant therapy (NAT) before oesophagectomy. Early response assessment using PET imaging may help guide management of these patients. We performed a systematic review and meta-analysis to synthesise the evidence detailing response rate and diagnostic accuracy of early PET-CT assessment. METHODS: We systematically searched several databases including MEDLINE and Embase. Studies with mixed cohorts of histology, tumour location and a repeat PET-CT assessment after more than one cycle of NAT were excluded. Reference standard was pathological response defined by Becker or Mandard classifications. Primary outcome was metabolic response rate after one cycle of NAT defined by a reduction in maximum standardised uptake value (SUVmax) of 35%. Secondary outcome was diagnostic accuracy of treatment response prediction, defined as the sensitivity and specificity of early PET-CT using this threshold. Quality of evidence was also assessed. Random-effects meta-analysis pooled response rates and diagnostic accuracy. This study was registered with PROSPERO (CRD42019147034). RESULTS: Overall, 1341 articles were screened, and 6 studies were eligible for analysis. These studies reported data for 518 patients (aged 27-78 years; 452 [87.3%] were men) between 2005 and 2020. Pooled sensitivity of early metabolic response to predict pathological response was 77.2% (95% CI 53.2%-100%). Significant heterogeneity existed between studies (I2  = 80.6% (95% CI 38.9%-93.8%), P = .006). Pooled specificity was 75.0% (95% CI 68.2%-82.5%), however, no significant heterogeneity between studies existed (I2  = 0.0% (95% CI 0.0%-67.4%), P = .73). CONCLUSION: High-quality evidence is lacking, and few studies met the inclusion criteria of this systematic review. The sensitivity of PET using a SUVmax reduction threshold of 35% was suboptimal and varied widely. However, specificity was consistent across studies with a pooled value of 75.0%, suggesting early PET assessment is a better predictor of treatment resistance than of pathological response. Further research is required to define optimal PET-guided treatment decisions in OAC.

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

Temporal validation of metabolic nodal response of esophageal cancer to neoadjuvant chemotherapy as an independent predictor of unresectable disease, survival, and recurrence.

OBJECTIVES: We recently described metabolic nodal stage (mN) and response (mNR) of cancer of the esophagus and gastro-esophageal junction (GEJ) to neoadjuvant chemotherapy (NAC) using 18F-FDG PET-CT as new markers of disease progression, recurrence, and death. We aimed to validate our findings. METHODS: Our validation cohort comprised all patients consecutive to our discovery cohort, staged before and after NAC using PET-CT from 2014 to 2017. Multivariate binary logistic and Cox regression were performed. RESULTS: Fifty-one of the 200 patients had FDG-avid nodes after NAC (25.5%; i.e., lack of complete mNR), and were more likely to progress during NAC to incurable disease on PET-CT or at surgery: odds ratio 3.84 (1.46-10.1; p = 0.006). In 176 patients undergoing successful resection, patients without complete mNR had a worse prognosis: disease-free survival hazard ratio 2.46 (1.34-4.50); p = 0.004. These associations were independent of primary tumor metabolic, pathological response, and stage. In a hybrid pathological/metabolic nodal stage, avid nodal metastases conferred a worse prognosis than non-avid metastases. Lack of complete mNR predicted recurrence or death at 1 and 2 years: positive predictive values 44.4% (31.7-57.8) and 74.1% (56.6-86.3) respectively. CONCLUSIONS: This study provides temporal validation for mNR as a new and independent predictive and prognostic marker of esophageal and GEJ cancer treated with NAC and surgery, although external validation is required to assess generalizability. mNR may provide surrogate information regarding the phenotype of metastatic cancer clones beyond the mere presence of nodal metastases, and might be used to better inform patients, risk stratify, and personalize management, including adjuvant therapy. KEY POINTS: • We previously described metabolic nodal response (mNR) of esophageal cancer to neoadjuvant chemotherapy using 18 F-FDG PET-CT as a predictor of unresectable disease, early recurrence, and death. • We report the first validation of these findings. In an immediately consecutive cohort, we found consistent proportions of patients with and without mNR, and associations with abandoned resection, early recurrence, and death. • This supports mNR as a new and actionable biomarker in esophageal cancer. Although external validation is required, mNR may provide surrogate information about the chemosensitivity of metastatic subclones, and the means to predict treatment success, guide personalized therapy, and follow-up.

Time-series hyperpolarized xenon-129 MRI of lobar lung ventilation of COPD in comparison to V/Q-SPECT/CT and CT.

PURPOSE: To derive lobar ventilation in patients with chronic obstructive pulmonary disease (COPD) using a rapid time-series hyperpolarized xenon-129 (HPX) magnetic resonance imaging (MRI) technique and compare this to ventilation/perfusion single-photon emission computed tomography (V/Q-SPECT), correlating the results with high-resolution computed tomography (CT) and pulmonary function tests (PFTs). MATERIALS AND METHODS: Twelve COPD subjects (GOLD stages I-IV) participated in this study and underwent HPX-MRI, V/Q-SPECT/CT, high-resolution CT, and PFTs. HPX-MRI was performed using a novel time-series spiral k-space sampling approach. Relative percentage ventilations were calculated for individual lobe for comparison to the relative SPECT lobar ventilation and perfusion. The absolute HPX-MRI percentage ventilation in each lobe was compared to the absolute CT percentage emphysema score calculated using a signal threshold method. Pearson's correlation and linear regression tests were performed to compare each imaging modality. RESULTS: Strong correlations were found between the relative lobar percentage ventilation with HPX-MRI and percentage ventilation SPECT (r = 0.644; p < 0.001) and percentage perfusion SPECT (r = 0.767; p < 0.001). The absolute CT percentage emphysema and HPX percentage ventilation correlation was also statistically significant (r = 0.695, p < 0.001). The whole lung HPX percentage ventilation correlated with the PFT measurements (FEV1 with r = - 0.886, p < 0.001*, and FEV1/FVC with r = - 0.861, p < 0.001*) better than the whole lung CT percentage emphysema score (FEV1 with r = - 0.635, p = 0.027; and FEV1/FVC with r = - 0.652, p = 0.021). CONCLUSION: Lobar ventilation with HPX-MRI showed a strong correlation with lobar ventilation and perfusion measurements derived from SPECT/CT, and is better than the emphysema score obtained with high-resolution CT. KEY POINTS: • The ventilation hyperpolarized xenon-129 MRI correlates well with ventilation and perfusion with SPECT/CT with the advantage of higher temporal and spatial resolution. • The hyperpolarized xenon-129 MRI correlates with the PFT measurements better than the high-resolution CT with the advantage of avoiding the use of ionizing radiation.

Routinely staging gastric cancer with 18F-FDG PET-CT detects additional metastases and predicts early recurrence and death after surgery.

OBJECTIVES: Fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) is typically considered to have minimal yield in gastric cancer, and so is not consistently recommended by international guidelines. However, its yield is considerable in esophageal and junctional cancer, identifying unsuspected metastases and risk-stratifying patients using metabolic nodal stage (mN). We aimed to determine the contemporary utility of routine 18F-FDG PET-CT in gastric cancer. METHODS: We routinely stage patients with non-junctional gastric cancer with PET-CT, provided initial CT does not demonstrate unequivocal metastases. We performed a retrospective study of all such patients staged in our institution from January 2007 to July 2016. Our primary endpoint was detection of incurable disease. Our secondary endpoint was disease-free survival following gastrectomy. Decision theory, economic, and predictive models were generated. RESULTS: The primary tumor was FDG-avid in 225/279 patients (80.6%). Seventy-two (25.8%) had FDG-avid nodes (resectable by D2 lymphadenectomy). This was not influenced by the Lauren classification. Unsuspected metastases were identified in 20 patients (7.2%). In 13 (4.7%), these would not have been otherwise identified. Decision theory and economic modeling supported routine PET-CT. Patients with FDG-avid nodes were more likely to have incurable disease (51.4% versus 15.5%; p < 0.001), and a worse prognosis if not: multivariate hazard ratio 2.19 (1.23-3.91; p = 0.008). Prognosis worsened with mN stage. CONCLUSIONS: PET-CT appears useful when used routinely for non-junctional gastric cancer, and should be considered in international recommendations. Any extra costs appear small and offset by avoiding futile investigations and radical treatment. mN stage identifies patients at risk of early recurrence and death. KEY POINTS: • PET-CT is typically not considered useful when staging gastric cancer. We describe a retrospective study of 279 patients routinely staged with PET-CT in the absence of metastases on CT. • The primary tumor was avid in 80% of patients. Twenty-five percent had resectable avid nodes. PET-CT identified previously unsuspected metastases in 7% of patients, which would likely not have been identified by conventional staging without PET-CT in 5%. These patients were much more likely to have avid nodes. • Beyond avoiding futile investigations and radical treatment in this 5%, we found patients with FDG-avid nodes (metabolic nodal stage, mN) to have a worse disease-free survival after gastrectomy.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

BACKGROUND: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. METHODS: We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). RESULTS: The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. CONCLUSIONS: Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

Apathy in rapid eye movement sleep behaviour disorder is associated with serotonin depletion in the dorsal raphe nucleus.

Apathy is a common and under-recognized disorder that often emerges in the prodromal phase of Parkinsonian diseases. The mechanism by which this occurs is not known, but recent evidence from patients with established Parkinson's disease suggests that serotonergic dysfunction may play a role. The integrity of the raphe serotonergic system can be assessed alongside dopaminergic basal ganglia imaging using the radioligand 123I-ioflupane, which binds both serotonin and dopamine transporters. To investigate the relative roles of these neurotransmitters in prodromal parkinsonism, we imaged patients with idiopathic rapid eye movement sleep behaviour disorder, the majority of whom will develop a parkinsonian disorder in future. Forty-three patients underwent brain imaging with 123I-ioflupane single photon emission computed tomography and structural MRI. Apathy was quantified using the Lille Apathy Rating Scale. Other clinical parkinsonian features were assessed using standard measures. A negative correlation was observed between apathy severity and serotonergic 123I-ioflupane signal in the dorsal raphe nucleus (r = -0.55, P < 0.001). There was no significant correlation between apathy severity and basal ganglia dopaminergic signal, nor between dorsal raphe signal and other neuropsychiatric scores. This specific association between apathy and raphe 123I-ioflupane signal suggests that the serotonergic system might represent a target for the treatment of apathy.

Effect of a Bayesian Penalized Likelihood PET Reconstruction Compared With Ordered Subset Expectation Maximization on Clinical Image Quality Over a Wide Range of Patient Weights.

OBJECTIVE: A study was performed to compare background liver signal-to-noise ratio (SNR) and visually assessed image quality of clinical PET/CT studies from the same PET acquisition data reconstructed by Bayesian penalized likelihood (BPL) and ordered subset expectation maximization (OSEM) over a range of patient weights. MATERIALS AND METHODS: The effect of a BPL PET reconstruction algorithm on liver SNR and visually assessed image quality over a range of patient weights (41-196 kg; n = 108) was retrospectively compared with standard-of-care OSEM reconstruction on the same PET acquisition data after IV administration of 18F-FDG (4 MBq/kg). RESULTS: BPL showed no significant change (p > 0.05) in liver SNR with increasing weight and body mass index (BMI), whereas OSEM showed increasing noise with increasing weight and BMI. The liver SNR was significantly higher using BPL than a standard OSEM reconstruction (p < 0.0002 for all BMI groups). Visually assessed image quality declined at a greater rate with increasing weight and BMI in the OSEM images than with BPL images. CONCLUSION: BPL provides a more consistent visually assessed image quality and liver background SNR than does OSEM, with the greatest benefit for the heaviest patients.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen.

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.

An expressed retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility.

Genetic association studies of prostate and other cancers have identified a major risk locus at chromosome 8q24. Several independent risk variants at this locus alter transcriptional regulatory elements, but an affected gene and mechanism for cancer predisposition have remained elusive. The retrogene POU5F1B within the locus has a preserved open reading frame encoding a homolog of the master embryonic stem cell transcription factor Oct4. We find that 8q24 risk alleles are expression quantitative trait loci correlated with reduced expression of POU5F1B in prostate tissue and that predicted deleterious POU5F1B missense variants are also associated with risk of transformation. POU5F1 is known to be self-regulated by the encoded Oct4 transcription factor. We further observe that POU5F1 expression is directly correlated with POU5F1B expression. Our results suggest that a pathway critical to self-renewal of embryonic stem cells may also have a role in the origin of cancer.

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

BACKGROUND: Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. METHODS: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. RESULTS: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. CONCLUSIONS: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

In vitro selection with artificial expanded genetic information systems.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

Novel penalised likelihood reconstruction of PET in the assessment of histologically verified small pulmonary nodules.

OBJECTIVES: Investigate the effect of a novel Bayesian penalised likelihood (BPL) reconstruction algorithm on analysis of pulmonary nodules examined with 18F-FDG PET/CT, and to determine its effect on small, sub-10-mm nodules. METHODS: 18F-FDG PET/CTs performed for nodule evaluation in 104 patients (121 nodules) were retrospectively reconstructed using the new algorithm, and compared to time-of-flight ordered subset expectation maximisation (OSEM) reconstruction. Nodule and background parameters were analysed semi-quantitatively and visually. RESULTS: BPL compared to OSEM resulted in statistically significant increases in nodule SUVmax (mean 5.3 to 8.1, p < 0.00001), signal-to-background (mean 3.6 to 5.3, p < 0.00001) and signal-to-noise (mean 24 to 41, p < 0.00001). Mean percentage increase in SUVmax (%ΔSUVmax) was significantly higher in nodules ≤10 mm (n = 31, mean 73%) compared to >10 mm (n = 90, mean 42 %) (p = 0.025). Increase in signal-to-noise was higher in nodules ≤10 mm (224%, mean 12 to 27) compared to >10 mm (165%, mean 28 to 46). When applying optimum SUVmax thresholds for detecting malignancy, the sensitivity and accuracy increased using BPL, with the greatest improvements in nodules ≤10 mm. CONCLUSION: BPL results in a significant increase in signal-to-background and signal-to-noise compared to OSEM. When semi-quantitative analyses to diagnose malignancy are applied, higher SUVmax thresholds may be warranted owing to the SUVmax increase compared to OSEM. KEY POINTS: • Novel Bayesian penalised likelihood PET reconstruction was applied for lung nodule evaluation. • This was compared to current standard of care OSEM reconstruction. • The novel reconstruction generated significant increases in lung nodule signal-to-background and signal-to-noise. • These increases were highest in small, sub-10-mm pulmonary nodules. • Higher SUV max thresholds may be warranted when using semi-quantitative analyses to diagnose malignancy.