Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1α.

Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

Dual-phase xenon time projection chambers for rare-event searches.

In the past decade, dual-phase xenon time projection chambers (Xe-TPCs) have emerged as some of the most powerful detectors in the fields of astroparticle physics and rare-event searches. Developed primarily towards the direct detection of dark matter particles, experiments presently operating deep underground have reached target masses at the multi-tonne scale, energy thresholds of approximately 1 keV and radioactivity-induced background rates similar to those from solar neutrinos. These unique properties, together with demonstrated stable operation over several years, allow for the exploration of new territory via high-sensitivity searches for a plethora of ultra-rare interactions. These include searches for particle dark matter, for second-order weak decays, and the observation of astrophysical neutrinos. We first review some properties of xenon as a radiation detection medium and the operation principles of dual-phase Xe-TPCs together with their energy calibration and resolution. We then discuss the status of currently running experiments and of proposed next-generation projects, describing some of the technological challenges. We end by looking at their sensitivity to dark matter candidates, to second-order weak decays and to solar and supernova neutrinos. Experiments based on dual-phase Xe-TPCs are difficult and, like all good experiments, they are constantly pushed to their limits. Together with many other endeavours in astroparticle physics and cosmology they will continue to push at the borders of the unknown, hopefully to reveal profound new knowledge about our cosmos. This article is part of the theme issue 'The particle-gravity frontier'.

Enhanced Performance of Artificial-Neural-Network-Based Equalization for Short-Haul Fiber-Optic Communications.

This work proposes an efficient and easy-to-implement single-layer artificial neural network (ANN)-based equalizer with improved compensation performance. The proposed equalizer is used for effectively mitigating the distortions induced in the short-haul fiber-optic communication systems based on intensity modulation and direct detection (IMDD). The compensation performance of the ANN equalizer is significantly improved, exploiting an introduced advanced training scheme. The efficiency and robustness of the proposed ANN equalizer are illustrated through 10- and 28-Gbaud short-reach optical-fiber communication systems. Compared to the efficient but computationally expensive maximum likelihood sequence estimator (MLSE), the proposed ANN equalizer not only significantly reduces its computational equalization cost and storage memory requirements, but it also outperforms its bit error rate performance.

Analyzing Peak-to-Average Power Ratio Characteristics in Multi-Channel Intensity Modulation and Direct Detection Flexible Transceivers Deploying Inverse Fast Fourier Transform/Fast Fourier Transform-Based Processing.

Cascaded inverse fast Fourier transform/fast Fourier transform (IFFT/FFT)-based multi-channel aggregation/de-aggregation offers a promising solution in constructing highly desirable flexible optical transceivers for considerably improving optical networks' elasticity, flexibility, and adaptability. However, the multi-channel aggregation operation unavoidably results in generated signals having high peak-to-average power ratios (PAPRs). To solve this technical challenge, this paper first explores the PAPR characteristics of the corresponding flexible transceivers in optical back-to-back (B2B) and 20 km intensity modulation and direct detection (IMDD) transmission systems, and then numerically investigates the feasibility and effectiveness of utilizing the conventional clipping techniques in reducing their PAPR reductions. The results show that the last IFFT operation size is the primary factor determining the PAPRs rather than the channel count and modulation format. For a given last IFFT operation size, the optimal clipping ratio can be identified, which is independent of channel count. With the identified optimal clipping ratio, when the channel count is >4, every two-channel increase in the channel count can only lead to <1.2 Gb/s decreases in the maximum aggregated signal transmission capacity.

Quantifying Microalgae Growth by the Optical Detection of Glucose in the NIR Waveband.

Microalgae have become a popular area of research over the past few decades due to their enormous benefits to various sectors, such as pharmaceuticals, biofuels, and food and feed. Nevertheless, the benefits of microalgae cannot be fully exploited without the optimization of their upstream production. The growth of microalgae is commonly measured based on the optical density of the sample. However, the presence of debris in the culture and the optical absorption of the intercellular components affect the accuracy of this measurement. As a solution, this paper introduces the direct optical detection of glucose molecules at 940-960 nm to accurately measure the growth of microalgae. In addition, this paper also discusses the effects of the presence of glucose on the absorption of free water molecules in the culture. The potential of the optical detection of glucose as a complement to the commonly used optical density measurement at 680 nm is discussed in this paper. Lastly, a few recommendations for future works are presented to further verify the credibility of glucose detection for the accurate determination of microalgae's growth.

Time Taken to Detect and Respond to Polio Outbreaks in Africa and the Potential Impact of Direct Molecular Detection and Nanopore Sequencing.

BACKGROUND: Detection of poliovirus outbreaks relies on a complex laboratory algorithm of cell-culture, polymerase chain reaction (PCR), and sequencing to distinguish wild-type and vaccine-derived polioviruses (VDPV) from Sabin-like strains. We investigated the potential for direct molecular detection and nanopore sequencing (DDNS) to accelerate poliovirus detection. METHODS: We analyzed laboratory data for time required to analyze and sequence serotype-2 VDPV (VDPV2) in stool collected from children with acute flaccid paralysis in Africa (May 2016-February 2020). Impact of delayed detection on VDPV2 outbreak size was assessed through negative binomial regression. RESULTS: VDPV2 confirmation in 525 stools required a median of 49 days from paralysis onset (10th-90th percentile, 29-74), comprising collection and transport (median, 16 days), cell-culture (7 days), intratypic differentiation quantitative reverse transcription PCR (3 days), and sequencing, including shipping if required (15 days). New VDPV2 outbreaks were confirmed a median of 35 days (27-60) after paralysis onset, which we estimate could be reduced to 16 days by DDNS (9-37). Because longer delays in confirmation and response were positively associated with more cases (P < .001), we estimate that DDNS could reduce the number of VDPV2 cases before a response by 28% (95% credible interval, 12%-42%). CONCLUSIONS: DDNS could accelerate poliovirus outbreak response, reducing their size and the cost of eradication.

Direct detection of dark matter-APPEC committee report.

This report provides an extensive review of the experimental programme of direct detection searches of particle dark matter. It focuses mostly on European efforts, both current and planned, but does it within a broader context of a worldwide activity in the field. It aims at identifying the virtues, opportunities and challenges associated with the different experimental approaches and search techniques. It presents scientific and technological synergies, both existing and emerging, with some other areas of particle physics, notably collider and neutrino programmes, and beyond. It addresses the issue of infrastructure in light of the growing needs and challenges of the different experimental searches. Finally, the report makes a number of recommendations from the perspective of a long-term future of the field. They are introduced, along with some justification, in the opening overview and recommendations section and are next summarised at the end of the report. Overall, we recommend that the direct search for dark matter particle interactions with a detector target should be given top priority in astroparticle physics, and in all particle physics, and beyond, as a positive measurement will provide the most unambiguous confirmation of the particle nature of dark matter in the Universe.

Three-Dimensional Division of Visible Light Communication Irradiation Area.

In this article, we divide the irradiated area of visible light communication (VLC) into three parts, according to the influence of diffuse reflection, the irradiance half angle at the source and the communication distance on VLC. We present a volume ratio method to quantitatively analyze each divided part. In this work, based on the Lambertian reflection model of the VLC system in line-of-sight channels, five factors affecting the VLC performance are compared and discussed. A VLC system of a single white-light-emitting diode in a 10 m line-of-sight channel indoors is designed by using the intensity modulation and direct detection technique, and a three-dimensional model of the irradiated area is established.By comparing the distribution of the bit error rate (BER) of the optical signal at different lampshade heights, the volume ratio method is used to calculate the volume percentage of the three irradiated areas. The experimental results show that area II with a volume ratio greater than 50% is the best signal receiving area when compared with areas I and III, having a volume ratio in the range 20∼30%.

Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods.

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

Elemental mapping of labelled biological specimens at intermediate energy loss in an energy-filtered TEM acquired using a direct detection device.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal-to-noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.

Direct detection of Strongyloides infection via molecular and antigen detection methods.

Laboratory diagnosis of Strongyloides infections can be grouped into direct and indirect detection methods, and a combination of the two methods is often needed to reach an accurate and timely diagnosis. This review focuses on non-conventional direct detection via molecular and antigen detection assays. Conventional PCR is the most commonly used molecular diagnostic for Strongyloides. Real-time PCR is accurate and highly sensitive for quantitative and qualitative analysis. Meanwhile, PCR-RFLP can efficiently distinguish human and dog isolates of S. stercoralis, S. fuelleborni (from monkey), and S. ratti (from rodent). Loop-mediated isothermal amplification (LAMP) amplifies DNA isothermally with high specificity, efficiency, and rapidity, and has potential for point-of-care (POC) translation. As for antigen detection assay, coproantigen detection ELISAs for strongyloidiasis traditionally relied on raising rabbit polyclonal antibodies against the parasite antigens for use as capture or detection reagents. Subsequently, hybridoma technology using animals has enabled the discovery of monoclonal antibodies specific to Strongyloides antigens and was utilised to develop antigen detection assays. In recent times, phage display technology has facilitated the discovery of scFv antibody against Strongyloides protein that can accelerate the development of such assays. Improvements in both direct detection methods are being made. Strongyloides molecular diagnostics is moving from the detection of a single infection to the simultaneous detection of soil-transmitted helminths. Meanwhile, antigen detection assays can also be multiplexed and aptamers can be used as antigen binders. In the near future, these two direct detection methods may be more widely used as diagnostic tools for strongyloidiasis.

Simulating 50 keV X-ray Photon Detection in Silicon with a Down-Conversion Layer.

Simulation results are presented that explore an innovative, new design for X-ray detection in the 20-50 keV range that is an alternative to traditional direct and indirect detection methods. Typical indirect detection using a scintillator must trade-off between absorption efficiency and spatial resolution. With a high-Z layer that down-converts incident photons on top of a silicon detector, this design has increased absorption efficiency without sacrificing spatial resolution. Simulation results elucidate the relationship between the thickness of each layer and the number of photoelectrons generated. Further, the physics behind the production of electron-hole pairs in the silicon layer is studied via a second model to shed more light on the detector's functionality. Together, the two models provide a greater understanding of this detector and reveal the potential of this novel form of X-ray detection.

DFT-Spread Spectrally Overlapped Hybrid OFDM-Digital Filter Multiple Access IMDD PONs.

A novel transmission technique-namely, a DFT-spread spectrally overlapped hybrid OFDM-digital filter multiple access (DFMA) PON based on intensity modulation and direct detection (IMDD)-is here proposed by employing the discrete Fourier transform (DFT)-spread technique in each optical network unit (ONU) and the optical line terminal (OLT). Detailed numerical simulations are carried out to identify optimal ONU transceiver parameters and explore their maximum achievable upstream transmission performances on the IMDD PON systems. The results show that the DFT-spread technique in the proposed PON is effective in enhancing the upstream transmission performance to its maximum potential, whilst still maintaining all of the salient features associated with previously reported PONs. Compared with previously reported PONs excluding DFT-spread, a significant peak-to-average power ratio (PAPR) reduction of over 2 dB is achieved, leading to a 1 dB reduction in the optimal signal clipping ratio (CR). As a direct consequence of the PAPR reduction, the proposed PON has excellent tolerance to reduced digital-to-analogue converter/analogue-to-digital converter (DAC/ADC) bit resolution, and can therefore ensure the utilization of a minimum DAC/ADC resolution of only 6 bits at the forward error correction (FEC) limit (1 × 10-3). In addition, the proposed PON can improve the upstream power budget by >1.4 dB and increase the aggregate upstream signal transmission rate by up to 10% without degrading nonlinearity tolerances.

60-nt DNA Direct Detection without Pretreatment by Surface-Enhanced Raman Scattering with Polycationic Modified Ag Microcrystal Derived from AgCl Cube.

Direct detection of long-strand DNA by surface-enhanced Raman scattering (SERS) is a valuable method for diagnosis of hereditary diseases, but it is currently limited to less than 25-nt DNA strand in pure water, which makes this approach unsuitable for many real-life applications. Here, we report a 60-nt DNA label-free detection strategy without pretreatment by SERS with polyquaternium-modified Ag microcrystals derived from an AgCl cube. Through the reduction-induced decomposition, the size of the about 3 × 3 × 3 µm3 AgCl cube is reduced to Ag, and the surface is distributed with the uniform size of 63 nm silver nanoparticles, providing a large area of a robust and highly electromagnetic enhancement region. The modified polycationic molecule enhances the non-specific electrostatic interaction with the phosphate group, thereby anchoring DNA strands firmly to the SERS enhanced region intactly. As a result, the single-base recognition ability of this strategy reaches 60-nt and is successfully applied to detect thalassemia-related mutation genes.

Diagnostic options for human polyomaviruses in clinical practice.

The members of the viral family Polyomavirae are widespread in the human population. According to serological studies, almost all adults are infected with at least one of this group of viruses. The primary infection usually occurs in childhood without any clinical signs, and after the primary infection, the viruses establish a persistent infection accompanied by occasional reactivation and shedding of the virus. These viruses often reactivate in immunosuppressed individuals, but only in a minority of these patients, the reactivation results in disease development. This biological property of human polyomaviruses makes laboratory diagnosis considerably difficult. The paper provides an overview of methods for diagnosing human polyomaviruses, which are commonly used for screening, and methods that are still validated by research but have the potential to improve detection and to identify patients at risk of developing diseases associated with polyomavirus infection.

Evidence Review for Centers for Disease Control and Prevention Guidance Development on Laboratory Testing to Detect Treponema pallidum Infection (Syphilis).

The articles in this supplement address key questions on syphilis diagnostics, provide reference tables of test performances, and discuss optimal specimens and knowledge gaps. Laboratory-developed genetic direct detection tests could be most useful at the point of care and add to the currently available serologic methods of nontreponemal and treponemal tests.

Performance of PCR/Electrospray Ionization-Mass Spectrometry on Whole Blood for Detection of Bloodstream Microorganisms in Patients with Suspected Sepsis.

Blood culture (BC) often fails to detect bloodstream microorganisms in sepsis. However, molecular diagnostics hold great potential. The molecular method PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect DNA from hundreds of different microorganisms in whole blood. The aim of the present study was to evaluate the performance of this method in a multicenter study including 16 teaching hospitals in the United States (n = 13) and Europe (n = 3). First, on testing of 2,754 contrived whole blood samples, with or without spiked microorganisms, PCR/ESI-MS produced 99.1% true-positive and 97.2% true-negative results. Second, among 1,460 patients with suspected sepsis (sepsis-2 definition), BC and PCR/ESI-MS on whole blood were positive in 14.6% and 25.6% of cases, respectively, with the following result combinations: BC positive and PCR/ESI-MS negative, 4.3%; BC positive and PCR/ESI-MS positive, 10.3%; BC negative and PCR/ESI-MS positive, 15.3%; and BC negative and PCR/ESI-MS negative, 70.1%. Compared with BC, PCR/ESI-MS showed the following sensitivities (coagulase-negative staphylococci not included): Gram-positive bacteria, 58%; Gram-negative bacteria, 78%; and Candida species, 83%. The specificities were >94% for all individual species. Patients who had received prior antimicrobial medications (n = 603) had significantly higher PCR/ESI-MS positivity rates than patients without prior antimicrobial treatment-31% versus 22% (P < 0.0001)-with pronounced differences for Gram-negative bacteria and Candida species. In conclusion, PCR/ESI-MS showed excellent performance on contrived samples. On clinical samples, it showed high specificities, moderately high sensitivities for Gram-negative bacteria and Candida species, and elevated positivity rates during antimicrobial treatment. These promising results encourage further development of molecular diagnostics to be used with whole blood for detection of bloodstream microorganisms in sepsis.

More than Proton Detection-New Avenues for NMR Spectroscopy of RNA.

Ribonucleic acid oligonucleotides (RNAs) play pivotal roles in cellular function (riboswitches), chemical biology applications (SELEX-derived aptamers), cell biology and biomedical applications (transcriptomics). Furthermore, a growing number of RNA forms (long non-coding RNAs, circular RNAs) but also RNA modifications are identified, showing the ever increasing functional diversity of RNAs. To describe and understand this functional diversity, structural studies of RNA are increasingly important. However, they are often more challenging than protein structural studies as RNAs are substantially more dynamic and their function is often linked to their structural transitions between alternative conformations. NMR is a prime technique to characterize these structural dynamics with atomic resolution. To extend the NMR size limitation and to characterize large RNAs and their complexes above 200 nucleotides, new NMR techniques have been developed. This Minireview reports on the development of NMR methods that utilize detection on low-γ nuclei (heteronuclei like 13 C or 15 N with lower gyromagnetic ratio than 1 H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1 H coherences, detected by 13 C NMR spectroscopy. Furthermore, 13 C and 15 N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-γ nuclei detection experiments in RNA.

Spectrally efficient optical orthogonal frequency division multiplexing.

This paper charts the development of spectrally efficient forms of optical orthogonal frequency division multiplexing (OFDM) that are suited for intensity-modulated direct detection systems, such as wireless optical communications. The journey begins with systems using a DC-bias to ensure that no parts of the signal that modulates the optical source are negative in value, as negative optical intensity is unphysical. As the DC-part of the optical signal carries no information, it is wasteful in energy; thus asymmetrically clipped optical OFDM was developed, removing any negative-going peaks below the mean. Unfortunately, the clipping causes second-order distortion and intermodulation, so some subcarriers appear to be unusable, halving spectral efficiency; this is similar for unipolar and flipped optical OFDM. Thus, a considerable effort has been made to regain spectral efficiency, using layered techniques where the clipping distortion is mostly cancelled at the receiver, from a knowledge of one unpolluted layer, enabling one or more extra 'layers/paths/depths' to be received on the previously unusable subcarriers. Importantly, for a given optical power and high-order modulation, layered methods offer the best spectral efficiencies and need the lowest signal-to-noise ratios, especially if diversity combining is used. Thus, they could be important for high-bandwidth optical fibre systems. Efficient methods of generating all layers simultaneously, using fast Fourier transforms with their partial calculations extracted, are discussed, as are experimental demonstrations in both wireless and short-haul communications links. A musical analogy is also provided, which may point to how orchestral and rock music is deciphered in the brain. This article is part of the theme issue 'Optical wireless communication'.

Optical spatial modulation design.

Visible light communication (VLC) systems are inherently signal-to-noise ratio (SNR) limited due to link budget constraints. One favourable method to overcome this limitation is to focus on the pre-log factors of the channel capacity. Multiple-input multiple-output (MIMO) techniques are therefore a promising avenue of research. However, inter-channel interference in MIMO limits the achievable capacity. Spatial modulation (SM) avoids this limitation. Furthermore, the performance of MIMO systems in VLC is limited by the similarities among spatial channels. This limitation becomes particularly severe in intensity modulation/direct detection (IM/DD) systems because of the lack of phase information. The motivation of this paper is to propose a system that results in a multi-channel transmission system that enables reliable multi-user optical MIMO SM transmission without the need for a precoder, power allocation algorithm or additional optics at the receiver. A general bit error performance model for the SM system is developed for an arbitrary number of light-emitting diodes (LEDs) in conjunction with pulse amplitude modulation. Based on this model, an LED array structure is designed to result in spatially separated multiple channels by manipulating the transmitter geometry. This article is part of the theme issue 'Optical wireless communication'.