Detection of minor variants in Mycobacterium tuberculosis whole genome sequencing data.

The study of genetic minority variants is fundamental to the understanding of complex processes such as evolution, fitness, transmission, virulence, heteroresistance and drug tolerance in Mycobacterium tuberculosis (Mtb). We evaluated the performance of the variant calling tool LoFreq to detect de novo as well as drug resistance conferring minor variants in both in silico and clinical Mtb next generation sequencing (NGS) data. The in silico simulations demonstrated that LoFreq is a conservative variant caller with very high precision (≥96.7%) over the entire range of depth of coverage tested (30x to1000x), independent of the type and frequency of the minor variant. Sensitivity increased with increasing depth of coverage and increasing frequency of the variant, and was higher for calling insertion and deletion (indel) variants than for single nucleotide polymorphisms (SNP). The variant frequency limit of detection was 0.5% and 3% for indel and SNP minor variants, respectively. For serial isolates from a patient with DR-TB; LoFreq successfully identified all minor Mtb variants in the Rv0678 gene (allele frequency as low as 3.22% according to targeted deep sequencing) in whole genome sequencing data (median coverage of 62X). In conclusion, LoFreq can successfully detect minor variant populations in Mtb NGS data, thus limiting the need for filtering of possible false positive variants due to sequencing error. The observed performance statistics can be used to determine the limit of detection in existing whole genome sequencing Mtb data and guide the required depth of future studies that aim to investigate the presence of minor variants.

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities.

The next-generation, short-read sequencing technologies that generate comprehensive, whole-genome data with single nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance, and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long-read ONT sequencing data with potential applications for M. tuberculosis. Only eight studies presented results of ONT sequencing of M. tuberculosis, of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data are available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

Urban-rural disparities in old-age mortality vary systematically with age: evidence from Germany and England & Wales.

OBJECTIVES: Population aging - which tends to be more pronounced in rural than in urban areas - poses important challenges for facilitating equal opportunities for aging well and 'aging in place.' Unmet health care needs among the older rural population may result in poorer health and higher mortality, but the scientific evidence of a systematic rural mortality disadvantage at older ages is scarce. We argue that systematic urban-rural mortality differences by age may be found if the confounding effect of life expectancy is considered. STUDY DESIGN: Nationwide population-based study. METHODS: We draw on age- and sex-specific data for the population aged 60+ years in NUTS-3 regions in Germany (2016-2018) and LAU-1 regions in England & Wales (2017-2019). To account for the confounding effect of life expectancy, we compare age-specific mortality only across urban and rural regions with similar life expectancy levels. We quantify statistical uncertainty with bootstrapping. RESULTS: The results show a remarkable shift from higher mortality in urban regions to higher mortality in rural regions with increasing age, when controlling for the confounding effect of life expectancy. That is, the urban mortality disadvantage is strongest for the population aged 60-79 years, whereas the pattern shifts toward a rural mortality disadvantage for the population aged 80 years and older. This pattern is present at all levels of life expectancy, for both sexes and in both countries. CONCLUSION: The shift from urban to rural excess mortality over age suggests that regions may vary in their capability to respond to arising health issues across older ages. This systematic mortality disadvantage is of high public health relevance and should be considered in designing policies to reduce regional mortality disparities.

Mechanisms of Drug-Induced Tolerance in Mycobacterium tuberculosis.

Successful treatment of tuberculosis (TB) can be hampered by Mycobacterium tuberculosis populations that are temporarily able to survive antibiotic pressure in the absence of drug resistance-conferring mutations, a phenomenon termed drug tolerance. We summarize findings on M. tuberculosis tolerance published in the past 20 years. Key M. tuberculosis responses to drug pressure are reduced growth rates, metabolic shifting, and the promotion of efflux pump activity. Metabolic shifts upon drug pressure mainly occur in M. tuberculosis's lipid metabolism and redox homeostasis, with reduced tricarboxylic acid cycle activity in favor of lipid anabolism. Increased lipid anabolism plays a role in cell wall thickening, which reduces sensitivity to most TB drugs. In addition to these general mechanisms, drug-specific mechanisms have been described. Upon isoniazid exposure, M. tuberculosis reprograms several pathways associated with mycolic acid biosynthesis. Upon rifampicin exposure, M. tuberculosis upregulates the expression of its drug target rpoB Upon bedaquiline exposure, ATP synthesis is stimulated, and the transcription factors Rv0324 and Rv0880 are activated. A better understanding of M. tuberculosis's responses to drug pressure will be important for the development of novel agents that prevent the development of drug tolerance following treatment initiation. Such agents could then contribute to novel TB treatment-shortening strategies.

Integration of Colloidal PbS/CdS Quantum Dots with Plasmonic Antennas and Superconducting Detectors on a Silicon Nitride Photonic Platform.

Single-photon sources and detectors are indispensable building blocks for integrated quantum photonics, a research field that is seeing ever increasing interest for numerous applications. In this work, we implemented essential components for a quantum key distribution transceiver on a single photonic chip. Plasmonic antennas on top of silicon nitride waveguides provide Purcell enhancement with a concurrent increase of the count rate, speeding up the microsecond radiative lifetime of IR-emitting colloidal PbS/CdS quantum dots (QDs). The use of low-fluorescence silicon nitride, with a waveguide loss smaller than 1 dB/cm, made it possible to implement high extinction ratio optical filters and low insertion loss spectrometers. Waveguide-coupled superconducting nanowire single-photon detectors allow for low time-jitter single-photon detection. To showcase the performance of the components, we demonstrate on-chip lifetime spectroscopy of PbS/CdS QDs. The method developed in this paper is predicted to scale down to single QDs, and newly developed emitters can be readily integrated on the chip-based platform.

Superconducting nanowire single photon detectors operating at temperature from 4 to 7 K.

We experimentally investigate the performance of NbTiN superconducting nanowire single photon detectors above the base temperature of a conventional Gifford-McMahon cryocooler (2.5 K). By tailoring design and thickness (8 - 13 nm) of the detectors, high performance, high operating temperature, single-photon detection from the visible to telecom wavelengths are demonstrated. At 4.3 K, a detection efficiency of 82 % at 785 nm wavelength and a timing jitter of 30 ± 0.3 ps are achieved. In addition, for 1550 nm and similar operating temperature we measured a detection efficiency as high as 64 %. Finally, we show that at temperatures up to 7 K, unity internal efficiency is maintained for the visible spectrum. Our work is particularly important to allow for the large scale implementation of superconducting single photon detectors in combination with heat sources such as free-space optical windows, cryogenic electronics, microwave sources and active optical components for complex quantum optical experiments and bio-imaging.

Controlled integration of selected detectors and emitters in photonic integrated circuits.

Integration of superconducting nanowire single-photon detectors and quantum sources with photonic waveguides is crucial for realizing advanced quantum integrated circuits. However, scalability is hindered by stringent requirements on high-performance detectors. Here we overcome the yield limitation by controlled coupling of photonic channels to pre-selected detectors based on measuring critical current, timing resolution, and detection efficiency. As a proof of concept of our approach, we demonstrate a hybrid on-chip full-transceiver consisting of a deterministically integrated detector coupled to a selected nanowire quantum dot through a filtering circuit made of a silicon nitride waveguide and a ring resonator filter, delivering 100 dB suppression of the excitation laser. In addition, we perform extensive testing of the detectors before and after integration in the photonic circuit and show that the high performance of the superconducting nanowire detectors, including timing jitter down to 23 ± 3 ps, is maintained. Our approach is fully compatible with wafer-level automated testing in a cleanroom environment.

Quantum detector tomography of a time-multiplexed superconducting nanowire single-photon detector at telecom wavelengths.

Superconducting nanowire single-photon detectors (SNSPDs) are widely used in telecom wavelength optical quantum information science applications. Quantum detector tomography allows the positive-operator-valued measure (POVM) of a single-photon detector to be determined. We use an all-fiber telecom wavelength detector tomography test bed to measure detector characteristics with respect to photon flux and polarization, and hence determine the POVM. We study the SNSPD both as a binary detector and in an 8-bin, fiber based, Time-Multiplexed (TM) configuration at repetition rates up to 4 MHz. The corresponding POVMs provide an accurate picture of the photon number resolving capability of the TM-SNSPD.

Kilometer-range, high resolution depth imaging via 1560 nm wavelength single-photon detection.

This paper highlights a significant advance in time-of-flight depth imaging: by using a scanning transceiver which incorporated a free-running, low noise superconducting nanowire single-photon detector, we were able to obtain centimeter resolution depth images of low-signature objects in daylight at stand-off distances of the order of one kilometer at the relatively eye-safe wavelength of 1560 nm. The detector used had an efficiency of 18% at 1 kHz dark count rate, and the overall system jitter was ~100 ps. The depth images were acquired by illuminating the scene with an optical output power level of less than 250 µW average, and using per-pixel dwell times in the millisecond regime.

On-chip single plasmon detection.

Surface plasmon polaritons (plasmons) have the potential to interface electronic and optical devices. They could prove extremely useful for integrated quantum information processing. Here we demonstrate on-chip electrical detection of single plasmons propagating along gold waveguides. The plasmons are excited using the single-photon emission of an optically emitting quantum dot. After propagating for several micrometers, the plasmons are coupled to a superconducting detector in the near-field. Correlation measurements prove that single plasmons are being detected.

Linewidth narrowing and Purcell enhancement in photonic crystal cavities on an Er-doped silicon nitride platform.

Light emission at 1.54 microm from an Er-doped amorphous silicon nitride layer coupled to photonic crystal resonators at cryogenic and room temperatures and under varying optical pump powers has been studied. The results demonstrate that small mode volume, high quality factor resonators enhance Er absorption and emission rates at the cavity resonance. Time resolved measurements give 11- to 17-fold Purcell enhancement of spontaneous emission at cryogenic temperatures, and 2.4-fold enhancement at room temperature. Resonances exhibit linewidth narrowing with pump power, signifying absorption bleaching and partial inversion of the Er ions cryogenic temperatures. We estimate that 31% of Er ions are excited at the highest pump power.

Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

Experimental realization and characterization of an electronic Lieb lattice.

Geometry, whether on the atomic or nanoscale, is a key factor for the electronic band structure of materials. Some specific geometries give rise to novel and potentially useful electronic bands. For example, a honeycomb lattice leads to Dirac-type bands where the charge carriers behave as massless particles [1]. Theoretical predictions are triggering the exploration of novel 2D geometries [2-10], such as graphynes, Kagomé and the Lieb lattice. The latter is the 2D analogue of the 3D lattice exhibited by perovskites [2]; it is a square-depleted lattice, which is characterised by a band structure featuring Dirac cones intersected by a flat band. Whereas photonic and cold-atom Lieb lattices have been demonstrated [11-17], an electronic equivalent in 2D is difficult to realize in an existing material. Here, we report an electronic Lieb lattice formed by the surface state electrons of Cu(111) confined by an array of CO molecules positioned with a scanning tunneling microscope (STM). Using scanning tunneling microscopy, spectroscopy and wave-function mapping, we confirm the predicted characteristic electronic structure of the Lieb lattice. The experimental findings are corroborated by muffin-tin and tight-binding calculations. At higher energies, second-order electronic patterns are observed, which are equivalent to a super-Lieb lattice.

Morphological evidence of an activated cytotoxic T-cell infiltrate in EBV-positive gastric carcinoma preventing lymph node metastases.

Recently, we showed that Epstein-Barr virus (EBV)-positive gastric carcinoma (GC) forms a distinct clinicopathologic entity with a better prognosis due to lower incidence of lymph node metastases (LN+). Here we investigated whether in EBV-positive GC more pronounced activation of cellular immune responses is associated with absence of (micro)metastases. Twenty EBV-positive primary tumors (PT) (9 LN+) were matched with 28 EBV-negative GC (11 LN+) for T- and N-stage, gender, and age. The PT (n = 28) and its LNs were analyzed by EBER RNA in situ hybridization and by immunohistochemistry for MHC class I and II expression, for CD3, CD8, CD4, CD20, CD56, CD83, and Granzyme B (GzB) expression. In LN metastases of EBV-positive GC, the EBV genome is maintained, excluding tumor escape by virus deletion. All GC express MHC class I independently of EBV status. In comparison with EBV-negative GC, EBV-positive GC have higher expression of MHC class II on the tumor cells (P = 0.029) and a more extensive infiltrate (P < 0.0001) of activated GzB+ CD8+ T cells (P = 0.028), which is most abundant in those EBV-positive tumors that do not metastasize (P < 0.0001). In addition, in EBV-positive GC without metastases, the infiltrate contains higher numbers of mature dendritic cells (DC) (P = 0.018). At present, the antigenic target has to be determined. These data support the notion that local triggering of cellular immune responses in EBV-positive GC prevents lymph node metastasis formation.

Capacitive readout and gating of superconducting single photon detectors.

We propose and develop a readout scheme for superconducting single-photon detectors based on an integrated circuit, relaxing the need for large bandwidth amplification and resulting in voltage steps proportional to the number of detected photons. We also demonstrate time gating, to filter scattered light in time and reduce dark counts. This could lead to a higher signal-to-noise ratio. The gate pulse is generated on the detection of a photon created by a spontaneous parametric down-conversion source, heralding the presence of a second photon. These two schemes could find applications within advanced multi-array imaging detection systems.