The desire to analyse limited amounts of biological material, historic samples and rare cell populations has collectively driven the need for efficient methods for whole genome sequencing (WGS) of limited amounts of poor quality DNA. Most protocols are designed to recover double-stranded DNA (dsDNA) by ligating sequencing adaptors to dsDNA with or without subsequent polymerase chain reaction amplification of the library. While this is sufficient for many applications, limited DNA requires a method that can recover both single-stranded DNA (ssDNA) and dsDNA. Here, we present a WGS library preparation method, called ‘degraded DNA adaptor tagging’ (DDAT), adapted from a protocol designed for whole genome bisulfite sequencing. This method uses two rounds of random primer extension to recover both ssDNA and dsDNA. We show that by using DDAT we can generate WGS data from formalin-fixed paraffin-embedded (FFPE) samples using as little as 2 ng of highly degraded DNA input. Furthermore, DDAT WGS data quality was higher for all FFPE samples tested compared to data produced using a standard WGS library preparation method. Therefore, the DDAT method has potential to unlock WGS data from DNA previously considered impossible to sequence, broadening opportunities to understand the role of genetics in health and disease. Whole genome sequencing (WGS) has radically changed medical diagnostics and research and is a rapidly evolving technology platform . IlluminThere are numerous WGS library preparation methods available to researchers, and these differ in their price, preparation time and recommended input material . Most liIn this study, we modified an existing method for DNA methylation analysis ,8 to cirFFPE blocks anonymized to the researchers were provided by collaborators at University College London Hospitals Biobank  and Oxford University Hospitals .® 3.0 fluorometer (Life Technologies) and quality was estimated using a multiplex polymerase chain reaction (PCR)-based assay as previously described  according to the manufacturer’s protocol. DNA was quantified using the Qubitescribed .® Multiplex Oligos for Illumina® (NEB). Samples were amplified for 10 PCR cycles before purification of library using a 1:0.8 ratio of DNA to beads and elution in 15 μl of water. The library was quantified using the Qubit® 3.0 fluorometer, 2200 TapeStation  and KAPA Library Quantification Kit (Roche).To remove damaged bases, 2 ng of good or poor quality FFPE DNA and 10 ng of very poor quality DNA were combined with 5 U of SMUG1, 1 U Fpg, 1× NEB buffer 1 and 0.1 μg/ml bovine serum albumin (NEB) in 10 μl and incubated for 1 h at 37°C. (This enzyme digestion step was excluded in the pilot experiment.) First strand synthesis was performed immediately afterwards by combining the 10 μl reaction with 1× blue buffer, 400 nM dNTPs and 4 μM oligo 1 (5′- CTACACGACGCTCTTCCGATCTNNNNNNNNN-3′) in 49 μl. Samples were heated to 95°C for 1 min and immediately cooled on ice. 50 U of Klenow  fragment was added to each sample and the tubes were incubated at 4°C for 5 min before slow ramping (4°C/min) to 37°C (i.e. 8 min for the ramping step), and then held at 37°C for 90 min. After this step, samples can be stored overnight at −20°C if required. The remaining primers were digested with 20 U of exonuclease I (NEB) at 37°C for 1 h in 100 μl before purification using AMPure XP beads (Beckman). For purification, 80 μl AMPure XP beads were added directly to the samples and incubated for 10 min at room temperature. After collecting beads on a magnet, we performed 2 × 200 μl 80% ethanol washes on the magnet. Beads were dried for 6–10 min being vigilant not to allow beads to overdry and crack. DNA was eluted in 38 μl of water before adding components for second strand synthesis  to the PCR tube still containing the beads. Samples were heated at 98°C for 2 min and then incubated on ice before 50 U of Klenow (3′ → 5′ exo-) was added and incubated using the same conditions as for first strand synthesis. To purify the second strand synthesis reaction, an aliquot of AMPure XP beads was centrifuged and the supernatant collected. After addition of 50 μl of water to the sample, 80 μl of bead buffer was added and mixed to resuspend the beads still within the tube and the DNA was purified as described earlier. After the final drying step, beads were resuspended in 33 μl of water and incubated for 10 min to elute the DNA. The beads were collected using a magnetic rack and the 33 μl of purified DNA transferred to a new PCR tube before adding the components for the final library PCR amplification  and the indexed custom reverse primer based on the Illumina TruSeq sequence (5′-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′). For the pilot experiment, the library was dual indexed using NEBNext® FFPE DNA Repair Mix, according the manufacturer’s protocol . Library preparation was performed using the NEBNext® Ultra II™ DNA Library Prep Kit for Illumina® according to the manufacturer’s protocol for FFPE samples  and 10 cycles of library amplification, during which the library was indexed using custom PE1.0 (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) and indexed reverse primer . For the pilot experiment, the library was dual indexed using NEBNext® Multiplex Oligos for Illumina® (NEB). The library was purified and quantified using the same methods as described for DDAT. We chose the NEBNext Ultra II DNA Library Prep Kit for comparison as although many WGS library preparation reagents are now commercially available, this kit is very widely used in the community, and as such is a relevant comparison for our DDAT methodology.Good quality FFPE DNA was sonicated using the Covaris M220 focused ultrasonicator to an average fragment size of 300 bp; poor and very poor quality samples did not require further fragmentation . DNA wasFor each sample, the paired-end sequence reads were initially quality checked with FastQC v0.11.5 to investigate base quality scores, sequence length distributions and additional features of the data. The reads were then aligned to the reference human genome hg19 (for the pilot experiment) and hg38 (for the samples in Significance testing was performed using Prism (v.5.04) and one-way ANOVA with Bonferroni post-hoc tests as specified in the figure legend. Where applicable, data are plotted as mean ± standard error of the mean (SEM).We first performed a pilot experiment to compare WGS data generated using the DDAT and the NEBNext Ultra II (‘standard’) library preparation methods, using a representative FFPE colorectal cancer DNA sample. We anticipated that the DDAT method would be of greatest benefit for FFPE DNA that was substantially degraded, as these samples contain more ssDNA that is inaccessible using the standard method. We therefore used poor quality FFPE DNA for our first test of the DDAT method  gave higher library yields compared to the standard method for all samples Figure , using eAfter sequencing the samples and aligning the reads to the human genome, we assessed the alignment metrics Table . In geneIn agreement with our pilot experiment, the samples prepared using the DDAT method had higher genomic coverage than those prepared using the standard method Figure . For ther2 = 0.87 versus without r2 = 0.87; poor sample with repair r2 = 0.75 versus without r2 = 0.72; very poor sample with repair r2 = 0.88 versus without r2 = 0.88), demonstrating that the coverage is not affected when artefacts are removed.To quantify whether removing the damaged DNA bases using enzymes SMUG1 and Fpg decreased the number of sequencing artefacts in the DDAT method, we calculated the ratio of C>T/A>G transitions within each dataset Figure  16). Th. Th16). In summary, the DDAT library preparation method increases the library yield and quality of WGS data when compared to a standard method. Therefore, application of DDAT to sequencing of degraded FFPE samples is expected to recover a larger fraction of the starting DNA material than standard methods. This increases library yield, allowing for fewer PCR cycles prior to sequencing and therefore fewer PCR duplicates in the sequencing data and a 2- to 3-fold increase in genomic coverage. In addition, since the library yield is higher, a lower amount of input DNA can be used, saving precious clinical material. DDAT does not require DNA shearing or sonication as FFPE treatment in itself causes DNA fragmentation, and only a short heat step is required to denature the dsDNA rendering it accessible for random primer amplification. By using DDAT, samples considered not amplifiable with standard methods can be used to generate sequencing libraries of improved quality, and furthermore the per-sample cost of DDAT is lower than that of commercially available kits. In other words, for the same sequencing throughput, 3- to 4-fold more usable reads are produced. The quality of the DDAT sequencing data is dependent on inclusion of an enzyme digestion step to remove FFPE-induced damaged DNA bases, minimizing FFPE-associated sequencing artefacts. Finally, the quality of the sequencing is significantly improved; therefore, more robust biologically relevant information can be extracted.We have established a new methodology for generating WGS libraries using DDAT, which gives superior library yield and quality of WGS data from FFPE DNA compared to a standard commercially available kit. The improved efficiency is due to the two random priming and extension steps that enable ssDNA and dsDNA capture. As a result, the input DNA does not require an additional DNA fragmentation step (e.g. by sonication) before using DDAT, which further maintains the integrity of the DNA. This is particularly important when the input DNA is extracted from FFPE-treated tissue that is often already highly fragmented and single stranded.During optimization of the protocol, we discovered that the ramp rate used to reach the 37°C incubation step during the first and second strand syntheses was crucial for efficient library preparation, with a faster ramping rate (132°C/min versus 4°C/min) reducing the overall library yield . The reaTo detect the level of DNA degradation in our FFPE DNA, we used multiplex PCR of the GAPDH gene , as thisWe have shown that removing damaged DNA bases in the DDAT method is sufficient to rescue the WGS data from FFPE-induced sequencing artefacts. Removal is the only option as the damaged bases in ssDNA cannot be repaired because there is no complementary strand to use as a template. Removal rather than repair does not seem to negatively impact the resulting WGS data as the yield and quality of data from the DDAT preparation with damaged base removal are generally improved compared to the standard method; furthermore, this type of damaged based removal has been shown to be effective for low DNA input targeted sequencing .https://sequencing.qcfail.com/articles/pbat-libraries-may-generate-chimaeric-read-pairs/). However, based on the alignment statistics this does not appear to be the case when using DDAT as in fact we observe a lower proportion of chimeric reads for our DDAT prepared libraries than for our standard libraries . DDAT increases the yield and quality of FFPE WGS data and we anticipate that this method can be applied to generate high-quality WGS data from low input quantities, particularly from good quality starting material, improving our ability to obtain relevant data from samples previously deemed unsuitable for WGS.Whole genome sequencing data that support the findings of this study have been deposited in Sequence Read Archive, accession number PRJNA531154.lqz017_Supplemental_FileClick here for additional data file.


To investigate the expression of Mitofusin-2 (MFN2) in HCC tissues and its role in the development of HCC.A total of 107 HCC specimens were collected for tissue microarray analysis and immunohistochemistry (IHC) analysis. The relationship between MFN2 expression and clinical features of patients with HCC was analyzed.P = 0.049). Kaplan-Meier survival analysis showed that HCC patients with low expression of MFN2 had a worse prognosis in overall survival than HCC patients with high expression of MFN2 (P = 0.027). Patients with high expression of MFN2 had a better prognosis in disease-free survival compared with HCC patients with low expression of MFN2 (P = 0.047). Vascular invasion and MFN2 expression were shown to be prognostic variables for overall survival in patients with HCC. Multivariate analysis showed that vascular invasion (P < 0.001) and MFN2 expression (P = 0.045) were independent prognostic factors for overall survival. Vascular invasion (P < 0.001) and MFN2 expression (P = 0.042) were independent risk factors associated with disease-free survival.Expression level of MFN2 in HCC tissues was 0.92 ± 0.78, significantly lower than that of matched paracancerous liver tissues (1.25 ± 0.75). Patients with low expression of MFN2 had significantly higher rates of cirrhosis than those with high expression of MFN2 (Our data revealed that MFN2 expression was decreased in HCC samples. High MFN2 expression was correlated with longer survival times in patients with HCC and served as an independent factor for better outcomes. Our study therefore provides a promising biomarker for the prognostic prediction of HCC and a potential therapeutic target for the disease. Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world –3. ApproMitochondrial fusion protein 2 (MFN2) is localized on the mitochondrial outer membrane and is involved in the regulation of fusion of mitochondrial outer membrane , 16. MFNThis study was designed to explore the expression level of MFN2 in the cancer tissues of patients with hepatitis B-related HCC, analyze the relationship between the expression level of MFN2 and clinicopathological features, and investigate its role in the regulation of hepatitis B-related HCC.We collected a total of 107 HCC tissues from August 2000 to Jun 2010. All patients were sero-positive of hepatitis B surface antigen. None of the patients received any chemotherapy or radiotherapy before collection of the tissue. The follow-up period was defined as the time interval between the date of operation and the date of death or the last follow-up. The study was approved by the medical ethics committee of Yantai Yuhuangding Hospital. Since all specimens used were anonymous, the Medical Ethics Committee exempted patients from the need for informed consent.Tissue microarray (TMA) was constructed. Each tissue core (diameter: 0.6 mm) was perforated and re-embedded from the labeled area by using a tissue array. The specimens were fixed with 4% paraformaldehyde. The biotin blocking Kit  was closed. After closure, the tissues were incubated with MFN2 antibodies  in a humid chamber at 4 °C for the night. The tissues were washed with PBS three times and incubated with biotinylated goat anti-rabbit antibodies for 1 hour. Finally, the slices were stained with hematoxylin and observed under a microscope. Semi-quantitative IHC was used to assess MFN2 protein expression levels according to the following criteria: “0” (negative staining), “1” (weak staining), “2” (moderate staining) and “3” (strong staining). The final score was calculated as the percentage of positive expression multiplied by the intensity score. The median IHC score was used as a cut-off value for determining high and low levels of expression.P value less than 0.05 was defined as statistically significant.Statistical analysis was performed using SPSS software . Student’s t test or Chi square test was used to examine the correlation between MFN2 expression and clinical and pathological variables. The Kaplan-Meier method (logarithmic rank test) was used to construct the survival curve. Multivariate Cox proportional hazards regression model was used to assess the independent predictive value of MFN2. P < 0.001, Fig. The expression level of MFN2 in 107 pairs of HCC tissues and matched paracancerous liver tissues were measured by IHC staining. The results showed that the expression level of MFN2 in HCC tissues was 0.92 ± 0.78, significantly lower than that of matched paracancerous liver tissues , as shown in Table According to the median score (0.9) of MFN2 expression in HCC tissues, all enrolled HCCs were divided into MFN2 low expression group (MFN < 0.9) and MFN2 high expression group (MFN ≥ 0.9). Demographic data and clinical features of the patients in the two groups were compared. We found that patients with low expression of MFN2 had significantly higher rates of cirrhosis than those with high expression of MFN2 (P = 0.605).There are 107 paratumor tissues we have collected and enrolled, 85 of them were diagnosed with cirrhosis and 22 were not diagnosed with cirrhosis. The MFN2 IHC scores were 1.27 ± 0.75 in cirrotic tissue and 1.18 ± 0.74 in non-cirrotic tissue (P = 0.027). We also compared the difference in the disease-free survival between the MFN2 high-expression group and the low-expression group. The results showed that patients with high expression of MFN2 had a better prognosis in disease-free survival compared with patients with low expression of MFN2 (P = 0.047), as shown in Fig. To determine the prognostic value of MFN2 in HCC, we performed a Kaplan-Meier survival analysis. The results showed that HCC patients with low expression of MFN2 had a worse prognosis in overall survival than patients with high expression of MFN2 (P < 0.001) and MFN2 expression (P = 0.045) were independent prognostic factors for overall survival  and MFN2 expression (P = 0.042) were independent risk factors associated with disease-free survival.We further explored the risk factors associated with disease-free survival Table . UnivariMitochondria are involved in the regulation of cellular energy metabolism, signaling, and programmed cell death , 22. TheMFN2 is located at the 36.22 locus of human chromosome 1 short arm. Cytogenetic analysis found that human chromosome 1 short arm 3 region 6 is the mutation-prone area of many malignant tumors , 17, 18.Previous studies have shown that MFN2 plays an important anti-proliferative effect in breast cancer cells , 28. MFNThere are some limitations in this study. Firstly, the sample size is relatively small, so the results may be biased. Secondly, the data collected in this study came from a single center and this may lead to some enrollment bias. A multicenter prospective study is needed to further validate the role of MFN2 in HCC and its potential prognosis prediction value.In summary, our study results revealed a role of MFN2 in the development of HCC. Our data showed that MFN2 expression was decreased in HCC samples. High MFN2 expression was also correlated with longer survival times in patients with HCC and served as an independent predictor for better outcomes. Collectively, our data suggest that MFN2 is a promising biomarker for the prognosis of patients with HCC and a potential target in HCC treatment.


ARs) have long been an enigmatic receptor subtype of interest due to their specific brain distribution, unusual surface localization and key role in synaptic plasticity, cognition and memory. These receptors are uniquely positioned to sculpt both the developing and mature hippocampal circuitry due to high overall expression and a distinct peak within the critical synapse formation period during the second postnatal week. Unlike the majority of other GABAARs, they exhibit both receptor clustering at extrasynaptic sites via interactions with the radixin scaffold as well as synaptic sites via gephyrin, thus contributing respectively to tonic currents and synaptic GABAergic neurotransmission. α5 GABAAR signaling can be altered in neurodevelopmental disorders including autism and mental retardation and by inflammation in CNS injury and disease. Due to the unique physiology and pharmacology of α5 GABAARs, drugs targeting these receptors are being developed and tested as treatments for neurodevelopmental disorders, depression, schizophrenia, and mild cognitive impairment. This review article focuses on advances in understanding how the α5 subunit contributes to GABAAR neurobiology. In particular, I discuss both recent insights and remaining knowledge gaps for the functional role of these receptors, pathologies associated with α5 GABAAR dysfunction, and the effects and potential therapeutic uses of α5 receptor subtype targeted drugs.α5 subunit containing GABA type A receptors (GABA ARs) are heteropentameric ligand-gated chloride (Cl−) ion channels typically composed of two α (α1–6), two β (β1–3), and one γ (γ1–3) or δ subunit  with slow decay rates and isoform-specific effects of pharmacological inhibitors . Mutation of the α5 subunit H105 residue, a key alpha subunit residue required for forming the benzodiazepine binding site with the γ2 subunit, led to repositioning of α5 H105R subunits into the pharmacologically inactive alpha subunit location  and are contributors to tonic inhibition in dentate gyrus granule cells . Further support for the specific contribution of α5 GABAARs to slowly decaying IPSCs is seen in early neurodevelopment during the switch from α5 to α1 and α3 subunit expression , evoked IPSCs (eIPSCs) and GABAARs show input-specific synaptic localization and function in different brain regions both for pyramidal cells and interneurons. Recent work demonstrates preferential localization of α5 GABAARs to inhibitory synapses on dendrites of somatostatin-expressing interneurons in CA1 that are targeted by vasoactive intestinal peptide and calretinin-positive interneurons  and circuit-specific localization and function are critical areas of current research and future pharmacological development  and the α5H105R point mutation mice. Although originally generated to render α5 receptors insensitive to benzodiazepines, α5H105R mice also have a 25% decrease in hippocampal α5 protein level  exhibited decreased dendritic arborization and reduced expression of the AMPA type glutamate receptor GluA2 subunit .While acute reduction in α5 GABAARs indicated no pronounced changes in surface or total α5 levels, while functional deficiencies ranged from reduced currents and gating defects to altered channel activation and deactivation. A V294L  mutation identified in a patient with severe early-onset epilepsy and developmental delay showed receptors with 10 times greater GABA sensitivity, although maximal GABA currents were reduced by increased receptor desensitization  show cognitive impairment due to excessive GABAergic inhibition. Acute treatment with α5IA reversed deficits in novel object recognition and spatial learning and was able to restore deficits of immediate early genes expression during memory processing  identified improved hippocampal-dependent memory in aged rats with cognitive impairment  (Luscher et al., Another important unmet need where α5 GABAARs, these receptors are being targeted and tested as treatments for neurodevelopmental disorders, mild cognitive impairment, depression and schizophrenia. The recent cryo-EM studies of heteropentameric synaptic GABAARs and binding of GABA, antagonists, and benzodiazepines should further advance α5 subtype specific structure-based drug design. Despite the progress in understanding of α5 GABAAR neurobiology, comparatively little is understood regarding mechanisms that regulate α5 GABAAR trafficking, stability, and both synaptic and extrasynaptic clustering. Furthermore, understanding of α5 GABAAR plasticity occurring from endogenous signaling mechanisms and from drug treatments in the developing, mature and aging brain will be needed to effectively and safely advance therapeutic application of α5 GABAAR preferring drugs.Due to the unique physiology and pharmacology of α5 GABATJ prepared the figure, table and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living cells.How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast  Standard fluorescence in situ hybridization, FISH, is a traditional microscopy-based approach, which is widely used in DNA localization studies. 3D-FISH in combination with confocal microscopy and image reconstruction enables the analysis of the spatial arrangement of chromosomes. However, this technique, in its traditional form at least, requires sample fixation There are various existing tools to study 3D genome configuration, such as probing RNA-chromatin interactions, chromosome conformation capture (3C) techniques and microscopy-based approaches, including the 3C variant Hi-C that extends the capability of the technology by identifying longer range interactions across the whole genome Here we describe a novel approach for achieving 3D spatial resolution at millisecond time scales and single-molecule detection sensitivity directly in single living eukaryotic cells using astigmatism imaging Saccharomyces cerevisiae and its DNA-binding Mig1 protein as a reference for genome mapping. Mig1 is a Zn-finger transcription factor which binds to target DNA sequences and under glucose-rich extracellular conditions represses expression of genes essential for metabolism of non-glucose carbon sources in vivo 2D Slimfield imaging of Mig1-GFP under glucose rich and depleted conditions. Our results indicated that Mig1 operates as 6–9-mer clusters, the main fraction of which, upon glucose repletion, is located in the nucleus and immobile. Glucose deprivation causes an increase of the clusters mobility and cytoplasmic import, however, a small portion of Mig1 was still detectable in the nucleus. We showed that immobile Mig1 molecules with apparent 2D diffusion coefficients lower than ∼0.1 µm2/s were likely to be bound to DNA, and that we could use 3C models combined with bioinformatics analysis to predict the likely Mig1 binding sites in 3D not enable full 3D genomic architectures in yeast to be determined as a function of time, but rather enables snapshots of parts of the 3D genomic architecture to be resolved with exceptionally high time resolution.We utilize the budding yeast 22.1Ti-series epifluorescence microscope. A schematic of the optical design is shown in 2. The image was collected by a 300 mm focal length tube lens onto a Photometrics Evolve 512 Delta EMCCD camera, with a DV2 color splitter to enable separate, simultaneous imaging of GFP and mCherry fluorescent protein components in the sample. A cylindrical lens was placed between the tube lens and the camera for astigmatism imaging. The resulting magnification at the sample is 93 nm per pixel. The sample was held on a Mad City Labs XYZ positioning nanostage. Full details of filters and lenses in We constructed a bespoke astigmatism super-resolution fluorescence microscope, built around the body of a Nikon 2.22.2.1z = 0 position. Images of single immobile GFP molecules were acquired at a 4.7 ms exposure time at axial positions between z = −0.5 µm and z = +0.5 µm in 0.25 µm intervals  to obviate any difficulties transfecting cells with a dye and labelling efficiency. Novel synthetic dyes (such as azetidine-substituted Janelia Fluor® fluorescent dyes) have higher quantum yields and are more photostable than fluorescent proteins but require electroporation to be introduced into the yeast cell To calibrate the fluorescent foci PSF image deformation due to the cylindrical lens, we imaged immobilized GFP on a coverslip at different axial distances, using a surface-immobilization assay adapted from earlier studies, where the presence of single-molecules was verified using single-step photobleaching ntervals A spaced 2.2.2x and σy. The mean σx and σy values were collated for each axial position and are shown in x/σy,r = σ to the axial position, z, was obtained by fitting an optimized 2nd order polynomial  lynomial C:r(z)=2.2.3z, comparable to the approximate axial working range over which we could reliably detect single GFP molecules. 4D Foci positions  were simulated using Brownian motion with a nominal diffusion coefficient of 1 µm2/s based on sensible experimental estimates from earlier 2D measurements in vitro calibration data  and Nrd1-mCherry (Nrd1 is a protein component of the RNA polymerase as is a clear marker for the position of the nucleus) fusions 4)2SO4, 5 g/l (NH4)2SO4, 0.79 g/l complete amino acid supplement as indicated by the manufacturer) supplemented with 4% glucose until mid-logarithmic growth phase, washed and placed into 3 ml of fresh medium for about 1 h. 5 µl of the culture was applied onto a 1% agarose pad perfused with YNB, formed using a 125 µl volume Gene Frame® (Thermo Scientific) and covered with a plasma-cleaned BK7 22 × 50 mm glass coverslip. Typically 1–4 cells per field of view were imaged using conditions similar to those described previously For live cell imaging, we used the model unicellular eukaryote of budding yeast We employ a non-sparse approach to single-molecule imaging. Unlike PALM and STORM where only single emitters are excited and imaged, we excite everything. By exciting all fluorophores at once and using step-wise photobleaching, we can quantify the stoichiometry and copy number of proteins in live cells dynamically, as in previous studies 2.52/s Images of Mig1-GFP  were tra33.1in vitro calibration data and comparing the measured to the known axial distance. , although if pairwise distances greater than our working 1 µm range are excluded from the predicted distribution, the mean is 368 ± 25 nm which although marginally closer to our experimental measurements is still statistically different (P < 0.0001).We took the 3D centroid positions of immobile Mig1 tracks as the putative positions of Mig1 binding sites in the genome and an indicator of 3D genome architecture . We compThis intriguing observation may result from several possibilities. For example, if only certain areas of the genome are undergoing active transcription, then it is possible that the range of the pairwise differences could be relatively higher than expected but that the peak value could potentially be smaller if the regions of active transcription are themselves relatively clustered. Similarly, the clustered nature of Mig1 implies multivalency of binding to DNA, and this in turn may result in condensation effect of separate DNA strands which are linked by a cluster, thus shifting the mean pairwise distance. It may also be the case that only a subset of spatially clustered Mig1-regulated genes have Mig1 bound to the target promoters at any one time: a subset of Mig1 clusters might also bind transiently (at least over a duration of four consecutive image frames or 20 ms used for the diffusion coefficient estimates), but in a relatively immobile state, to regions of the genome which are not specific promoter regions. Such a phenomenon could indeed be functionally important in intersegmental transfer of clusters between DNA segments Our 3D imaging approach has revealed significant tiers of complexity to the dynamic architecture of the genome, which slower, less precise and less physiologically relevant techniques would not be able to render. It remains to be determined in future studies precisely what are the key explanations for this heterogeneity in the genomic architecture, but our methodology shows promise in being able to enable such future insights. While the 3C model is an ensemble technique which represents an average conformation of a genome, our method reconstructs 3D genome architecture at a given time and thus potentially enables detailed studies of its dynamics in a single living cell.3.3vs. time interval relation) as calculated based on the signal to noise of the fluorophore Here we describe a method which enables us to map out the 3D positions of immobile fluorescent DNA-binding molecules, which thereby act as a valuable proxy to indicate the genome architecture in live yeast cells. We apply it to the Mig1 transcription factor for which we have a predicted model of the 3D position of binding sites within the genome from a knowledge of its binding sequences within the target gene promoters applied to prior 3C data. This allows us to provide more information towards understanding the functional 3D genome architecture in live cells. Recent articles, mainly based on ChIP-Seq technique, suggest that for many DNA binding proteins not only sequence specificity but also DNA shape defines target sites within the genome Lac operon of Escherichia coliRecent methods now allow single-cell 3C to be combined with microscopy European Commission via the Marie Curie Network for Initial Training ISOLATE (grant number 289995); the Biological Physical Sciences Institute (BPSI); a Royal Society Newton International Fellowship (grant number NF160208) and the Wellcome Trust (grant number 204829) through the Centre for Future Health at the University of York; and the BBSRC (grant numbers BB/P000746/1and BB/N006453/1.This work was supported by the None.


High-performance stretchable conductive fibers are desired for the development of stretchable electronic devices. Here we show a simple spinning method to prepare conductive hydrogel fibers with ordered polymer chain alignment that mimics the hierarchically organized structure of spider silk. The as-prepared sodium polyacrylate hydrogel fiber is further coated with a thin layer of polymethyl acrylate to form a core–shell water-resistant MAPAH fiber. Owing to the coexistence and reversible transformation of crystalline and amorphous domains in the fibers, MAPAH fibers exhibit high tensile strength, large stretchability and fast resilience from large strain. MAPAH fiber can serve as a highly stretchable wire with a conductive hydrogel core and an insulating cover. The stretchability and conductivity of the MAPAH fiber are retained at −35 °C, indicating its anti-freezing property. As a prime example of stretchable conductive fibers, MAPAH fibers will shed light on the design of next generation textile-based stretchable electronic devices. High-performance stretchable conductive fibers are desired for the development of stretchable electronic devices but preparation of conductive hydrogel fibers is challenging. Inspired by spider silk the authors demonstrate here a spinning method to prepare stretchable conductive hydrogel fibers with ordered polymer chain alignment. Among various functional hydrogels11, conductive hydrogels are very promising as stretchable conductive materials, which have been extensively studied for applications such as stretchable sensors12 and supercapacitors14. Most of the conductive hydrogels are made into two-dimensional films or three-dimensional monoliths by molding methods16, which result in an amorphous material with polymer chain’s random orientation and disordered alignment. If conductive hydrogels can be made into one-dimensional fibers with ordered alignment of polymer chains, some properties of conductive hydrogel fibers  would be greatly enhanced over conventional conductive hydrogel films and monoliths17. However, the ordered chain alignment of conductive polymers has been achieved in solid-state microfibers prepared by template directed polymerization19 or alignment20, which are not suitable for the preparation of conductive hydrogel fibers at macroscopic scale. On the other hand, spinning methods are widely used to prepare polymer fibers with ordered chain alignment22. But conductive hydrogels are rarely made into long fibers by spinning, due to the poor spinnability of current conductive hydrogels or their precursor solutions. In addition, conductive hydrogel films and monoliths are also limited by their slow resilience from large deformation due to the moderate reversibility of polymer chain alignment11, and the loss of functions at subzero temperature23. Previously reported polyelectrolyte fibers based on polycation–polyanion interfacial complexation are rigid and insulating materials, which are mainly used for biomedical applications, such as tissue engineering and drug delivery25. Previously reported stretchable conductive fibers based on carbon materials26 or metal nanomaterials28 coating on elastomeric fibers are also limited by their sophisticated fabrication process and moderate stretchability29. Therefore, high-performance conductive hydrogel fibers with ordered and reversible chain alignment are highly desired for the development of stretchable electronics, especially for the textile-based stretchable electronic devices30, but remain as a challenge.Stretchable conductive materials are essential for the emerging stretchable electronic devices31. The hierarchically organized structure of spider silk32 and its unique spinning process33 are the key factors to achieve its superb properties34. For example, spider dragline silk is a semi-crystalline protein polymer, where alanine-rich crystalline regions are connected by soft glycine-rich amorphous regions as linkers32. Inspired by the organized structure and the unique spinning process of spider silk, we propose to develop a simple spinning method to prepare conductive hydrogel fibers with ordered and reversible chain alignment from aqueous solution of polyelectrolytes at ambient conditions. Aiming at the production at low cost, a mass-produced synthetic polyelectrolyte: sodium polyacrylate (PAAS), was chosen as the starting material, which has been widely utilized in daily supplies and in industry. In water solutions, the anionic carboxylate groups on a PAAS chain repel each other via double layer forces, which causes the polymer chain to adopt an expanded, rigid-rod-like conformation. Adjusting pH, addition of salts or poor solvents tune the electrostatic interactions and alter the conformation of PAAS chains in solution, leading to the change of bulk properties (such as viscosity and sol–gel transition)35. Based on the knowledge learned from spider silk33, we expect that the stimuli-responsive properties of PAAS in solution would enable PAAS solution a good spinnability, where the spinning and drawing process would enable an ordered alignment of PAAS chains in the as-spun filaments.In nature, spiders spin silk fibers from aqueous protein solutions at ambient conditions36 and good mechanical properties. To achieve a good water resistance property, PAH fibers are coated with a thin layer of polymethyl acrylate (PMA) to form core–shell PMA–PAAS hydrogel (MAPAH) fibers. Remarkably, the water-resistant MAPAH fibers exhibit a unique combination of high tensile strength (5.6 MPa) and stretchability (elongation at a break of 1200%), fast resilience (<30 s) from large stretching strain, high electrical conductivity (2 S m−1), and great anti-freezing property. The simultaneous achievements of these prime properties by MAPAH fibers are attributed to the coexistence and reversible transformation of crystalline and amorphous domains in hydrogel fibers, which are enabled by the spinning and gelation process. The multifunctional MAPAH fiber as a high-performance and low-cost stretchable conductive fiber will shed light on the design of next generation textile-based stretchable electronic devices.After a systematic exploration, we have found that long and uniform filaments can be readily drawn from a gel-like PAAS solution in a mixture of water (as a good solvent) and dimethyl sulfoxide . Water evaporation from the PAAS filaments in air enriches the poor solvent DMSO in the filaments, which triggers a quick phase transition to form PAAS hydrogel (PAH) fibers with adjustable diameters and arbitrary length as wish. Just like spider silk, PAH fiber shows a unique beads-on-a-string structureMw ~ 3 × 107 Da) and low cost (~$20 per kilogram) was chosen as the starting material. The preparation procedure of PAH and MAPAH fibers is summarized in Fig. 37. The addition of DMSO into water causes significant desolvation of PAAS chains upon the counterion Na+ binding carboxylate groups, increasing the polymer–polymer interactions and triggering the phase transition37. The optimal solution for PAH fiber preparation has DMSO% = 20 wt%, which is very close to the critical DMSO concentration for the phase transition. Therefore, when a PAAS filament was drawn out from the optimal PAAS solution at room temperature, the DMSO% in the filament increased due to water evaporation and quickly exceeded the critical concentration. Therefore, PAAS in the filament rapidly aggregated to form a fine PAH fiber, while the excess aqueous solution can automatically form liquid droplets due to surface tension . As-prepared PAH fibers were immersed in a 5 wt% PMA solution in ethyl acetate for several seconds and then taken out. After the quick evaporation of ethyl acetate, a thin and uniform layer of PMA was firmly coated on the PAH fiber to form the core–shell MAPAH fiber. As judged by scanning electron microscopy . PMA was synthesized through conventional radical polymerization (G’) are dominant over loss moduli (G”) across the whole range of frequencies studied, which indicates the viscoelastic behavior of PAAS solutions. The optimal PAAS solution (4 wt%) appeared as a physical gel at room temperature , indicating that PAAS chains are more extended in pure water than in water/DMSO mixture. However, filaments drawn from this 4 wt% PAAS solution in pure water were thinner and unstable, which could not form PAH fibers , the solution viscosity was greatly reduced and no PAH fibers can be obtained , where the majority of carboxylate groups are negatively charged. In an acidic solution, PAAS chain is protonated and the uncharged linear polymer chain prefers a random coil conformation in solution38, indicating the viscoelastic nature of both fibers. Remarkably, due to the good balance of strength (4.4 ± 0.5 MPa) and stretchability (elongation at break of 740 ± 100%), PAH fibers achieved a high tensile toughness of 11.1 ± 0.5 MJ m−3, which is the total energy required to break the fiber. More surprisingly, greatly enhanced tensile strength (5.6 ± 0.6 MPa) and stretchability (elongation at break of 1180 ± 100%) were simultaneously achieved by MAPAH fibers, resulting in a tensile toughness of 26.8 ± 3.1 MJ m−3 that is among the best of current tough hydrogels41. Since the PMA film has a much higher stretchability (elongation-at-break ~1900%) than the PAH fiber, the very thin PMA coating layer (thickness 2–3 μm) may reinforce the PAH fiber (diameters of 200 ± 20 μm) by reducing the generation of crack on the fiber surface. Therefore, the mechanical properties of the MAPAH fiber is significantly better than the PAH fiber. Due to the great water-resistance and enhanced mechanical properties of MAPAH fibers, we have focused onto MAPAH fibers for further exploration.The mechanical properties of both PAH and MAPAH fibers were studied based on fibers drawn from the same optimal PAAS solution, whose diameters were controlled as 200 ± 20 μm by adjusting the drawn speed in a proper range. The environmental temperature and relative humidity were maintained at 25 °C and 42 ± 2%, respectively. The water content of PAH fibers and MAPAH fibers measured by thermogravimetric analysis (TGA) were similar, both of which were in the range of 25 ± 3%  and X-ray diffraction (XRD), respectively. As shown in Supplementary Figs.26. Since MAPAH fibers contain a large amount of water that serve as solvent and plasticizer, the amorphous domains could be forced to form ordered alignment upon stretching, providing a high stretchability and high strength. After the stretched fiber being released, the temporarily aligned PAAS chains would relax and adopt entropically favorable random conformation and orientation, enabling the fast recovery with the assistance of water in fibers. Therefore, the high stretchability and fast resilience of MAPAH fibers can be attributed to the stress-driven alignment and entropy-driven molecular relaxation of PAAS chains mainly in the amorphous domains, while the crystalline domains serve as physical crosslinking points and enable a high tensile strength21.Based on the structural analysis, a mechanism was proposed Fig.  to expla−1 at room temperature, which is in the conductivity range of concentrated PAAS solutions in water (1–4 S m−1)29. As shown in Supplementary Fig. R/R0 = (R−R0)/R0; R0 and R correspond to the resistance without and with stretching, respectively) as a function of the strain was studied , which indicates water is the only solvent in the MAPAH fiber. The absence of anti-freezing organic solvents in MAPAH fiber implies a different anti-freezing mechanism from organohydrogels. Indeed, the outstanding anti-freezing property of MAPAH fibers is likely due to the high concentration of sodium ions in the PAAS hydrogel matrix, since concentrated salt solution is anti-freezing. The conductivity of MAPAH fibers is comparable to other conductive hydrogels at room temperature16 and much higher than that of organohydrogels at low temperatures23, which indicates the advantage of MAPAH fibers as stretchable and anti-freezing conductive hydrogel fibers.Conventional conductive hydrogels would freeze and lose stretchability and conductivity at subzero temperatures. Liu has reported organohydrogels based on water-ethylene glycol binary solution that can sustain subzero temperature due to the anti-freezing property of water-ethylene glycol mixture °C Fig. . Indeed, °C Fig. , which i °C Fig. . The molIn summary, we report ultra-stretchable and anti-freezing conductive hydrogel fibers that are facilely prepared by spinning of PAAS solutions at ambient conditions. Crystalline and amorphous domains are coexistent in the as-prepared hydrogel fibers, which may reversibly convert between each other upon mechanical stress and release. The dynamic alignment and relaxation of PAAS chains in the amorphous domains are likely responsible for the large stretchability and fast resilience, while the crystalline domains could serve as physical crosslinking points and enable the high tensile strength. MAPAH fiber can serve as a highly stretchable and anti-freezing wire with good electrical conductivity. Owing to the ordered and reversible chain alignment enabled by the spinning and gelation process, MAPAH fiber represents a unique example of high-performance and low-cost stretchable conductive fibers, which will shed light on the design of next generation textile-based stretchable electronic devices.Mw ~ 3 × 107 Da) was purchased from Sinopharm Chemical Reagent Co. Ltd. Methyl acrylate (MA) was passed through a basic alumina column to remove the inhibitor before polymerization. Tetrahydrofuran (THF) was dried over molecular sieves. 2,2’-Azobisisobutyronitrile (AIBN) was recrystallized twice from ethanol. All other chemicals were of analytical grade, purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received.Sodium polyacrylate  was stirred and heated at 80 °C for 1 h to get a uniform, transparent and viscous solution. As shown in Supplementary Movie The optimal PAAS solution was prepared as follows: 200 mg PAAS solid powder in 4.8 g solvent (3.84 g Hλ = 1.54178 Å). One MAPAH fiber was placed parallel or perpendicular to the X-ray incidence direction. For the XRD of stretched MAPAH fibers, one MAPAH fiber with certain elongation was glued on a silicon wafer and placed parallel to the X-ray incidence direction. Thermogravimetric analysis (TGA) was done by TGA Q5000IR, the temperature was from 20 °C to 800 °C with 10 °C/min. Differential scanning calorimetric (DSC) was performed by using TA Q2000 at identical heating and cooling rate of 2 °C min−1 between −50 °C and 90 °C. The contact angle of water droplet on PMA film and MAPAH fiber was measured by using a contact angle meter SL2008, Solon Tech. Rheological measurements of different PAAS solutions were conducted on a TA AR-G2 rheometer. Mechanical properties of PAH fibers and MAPAH fibers were tested on an Instron 3340 universal testing instrument at 25 °C and relative humidity ~42 ± 2%. The average diameter of each fiber sample was obtained from three measurements along the fiber length using an optical microscope. The electrical resistance of MAPAH fibers was measured by using a multimeter. The electric conductivity was calculated based on the equation: σ = L/(S × R), where L, S, σ, and R are the length, cross section area, conductivity, and electrical resistance of the MAPAH fiber.Field emission scanning electron microscope  was used to image the freeze-dried hydrogel fibers. For all the other characterization, freshly prepared hydrogel fibers were used. X-ray diffraction (XRD) was carried out on a Rigaku D X-ray diffractometer with Cu Kα radiation (Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


We observe a strong defect mediated coupling between the long-range oscillating electric field induced by the longitudinal optical phonon in the dipolar medium and the exciton. This so-called Fröhlich exciton phonon interaction is suppressed by doping. The suppression correlates with a distinct increase of the degree of valley polarization up to 20% even at elevated temperatures of 220 K. Our result demonstrates a promising strategy to increase the degree of valley polarization towards room temperature valleytronic applications.Charge carriers in semiconducting transition metal dichalcogenides possess a valley degree of freedom that allows for optoelectronic applications based on the momentum of excitons. At elevated temperatures, scattering by phonons limits valley polarization, making a detailed knowledge about strength and nature of the interaction of excitons with phonons essential. In this work, we directly access exciton-phonon coupling in charge tunable single layer MoS 2.Owing to the presence of a valley degree of freedom, atomically thin transition metal dichalcogenides show promise for room temperature valleytronic applications. Here, the authors use polarization-resolved Raman spectroscopy to gain insight to the exciton-phonon coupling in charge tunable single layer MoS Valley selective optical excitation allows for optoelectronic applications based on the momentum of excitons9. For both, optical, as well as electronic phenomena, strength and nature of the electron-phonon interaction are crucial. Scattering with optical phonons dominates the mobility in single layer MoS2 at room-temperature11. Moreover, electron-phonon coupling plays a fundamental role in the dynamics of photo excited electron hole pairs and related excitons that are bound by strong Coulomb interaction. The electron-phonon or exciton-phonon interaction is of great importance regarding fast cooling of photo-excited carriers14, the homogeneous linewidth of excitonic luminescence17, optical absorption spectra18 and coherence19. In addition to lattice imperfections and disorder20, scattering by phonons is a significant mechanism for valley depolarization and decoherence of excitons, resulting in a breakdown of valley polarization at temperatures above ~100 K21, thus preventing high-temperature valley polarization required for realistic applications. Exciton-phonon interaction can be directly accessed by resonant Raman spectroscopy, where excitons play an important role as real intermediate states22.A direct band gap2. We use field effect structures with electrolyte gates that enable a tuning of the free electron density ne by two orders of magnitude to demonstrate electronic control over the Fröhlich exciton-LO phonon scattering rate and its correlation to the degree of circularly polarization of the PL of the A exciton.Here, we combine polarization resolved photoluminescence (PL) with resonant and non-resonant Raman spectroscopy to identify Fröhlich exciton-LO phonon interaction as a significant contribution to valley depolarization via direct exchange interaction in single layer MoST = 220 K for an excitation energy of Ei = 1.96 eV. The PL experiments are carried out on a 1L-MoS2 field effect device utilizing an ionic liquid top gate. Figure 13 cm−2 when increasing Vgate by 2 V −I(σ−))/(I(σ+)+I(σ−)) for a series of Vgate. In Fig. 0) and the charged (A−) excitons  are summarized in Fig.  ne Fig. . Thus, t2 visible in backscattering configuration are an out-of-plane oscillation A’1 and the in-plane mode E’ that is represented by one longitudinal optical (LO) and one transverse optical (TO) phonon branch  phonons induce a macroscopic electric field which can strongly couple to electrons or excitons via the Fröhlich interaction (FI)25 as sketched in Fig. êi and ês are the electric field vectors of the incident and the scattered light and A’1 and the E’ phonons, the DP Raman tensors are26:A’1 mode maintains the polarization of the scattered light, whereas light scattered by the E’ mode is unpolarized. In the case of circularly polarized incident light, the A’1 mode maintains the polarization, whereas the E’ mode turns circularly right-handed (σ+) to circularly left-handed (σ−) polarized light27. The polarization dependences of the DP tensors are confirmed in non-resonant (Ei = 2.54 eV) Raman measurements depicted in Fig. A’1 mode is co-polarized and the E’ mode is cross-polarized under circularly polarized excitation. We refer to the configurations  =  and  as co-polarized and cross-polarized configurations, respectively. The polar plot representation of the normalized mode intensities in Fig. A’1 and the E’ modes under circular excitation.The Raman active optical phonon modes in 1L-MoSnch Fig. . Phonons28, hence, the scattering is expected to be co-polarized.In contrast to the DP Raman tensor, the tensor for scattering due to Fröhlich interaction is diagonalEi = 1.96 eV, which is close to the outgoing resonance with the A exciton of MoS2, we observe a very strong contribution of the E’ mode in the co-polarized configuration (E’CO) in addition to a rather weak DP related cross-polarized contribution (E’CROSS)  Fig. . Hence, E’ mode strongly suggests that Fröhlich exciton-LO phonon interaction dominates the Raman scattering over the DP contribution under resonant excitation. Surprisingly, we find a strong suppression of this forbidden Raman scattering for heavily electron doped MoS2. Figure Ei = 1.96 eV) for an electron density n++ that is increased by about two orders of magnitude compared to n0. We estimate the electron density ne from the energy of the A’1 mode29.  for a whole rotation of êi(s) in the plane of the MoS2 crystal . From the comparison of experiment and calculations, we conclude that the observed forbidden Raman scattering is consistent to an impurity assisted second-order Fröhlich exciton-LO phonon scattering process that activates scattering with large q phonons. We would like to stress, that for an increase of the exciton radius a0 by e.g., a factor of 10 with increasing electron density23 and the subsequent increase of qa0 = 0.2, the probability of the intrinsic process is only minor increased  potential fluctuation, and hence to a reduction of the electron-impurity scattering cross section. As the change of the Fermi energy in our experiments is limited to ~13 meV, we expect shallow potential fluctuations induced by local strain or dielectric modifications due to interfacial imperfection or by the interaction with (charged) impurities in the substrate to be responsible for the impurity assisted Fröhlich scattering process. Additionally to the screening of impurities, the suppression of the FI scattering with increasing ne might also result from dielectric screening of the FI because the strength of the FI is inverse proportional to the dielectric constant25. We can exclude that a shift and broadening of the excitonic resonance and the well-studied bleaching of the absorption at the exciton resonance, resulting mainly from Coulomb screening23, to be responsible for the complete suppression of the FI scattering intensity with increasing ne. The extent to which these effects influence the scattering probability can be estimated from the comparison between the DP and the FI contributions  electronic device applications.2 flakes . We use a PDMS stamp to transfer the flakes onto silicon substrates with a 300 nm thick SiO2 layer as dielectric (Siltronic AG). Contacts to the MoS2 flake and for the electrolyte top gate are fabricated by standard optical lithography and e-beam evaporation of 5 nm Ti and 30 nm Au. As an electrolyte top gate, we use either a solid polymer electrolyte consisting of poly-(ethylene oxide) and CsClO4 (ratio 1:0.12) or the ionic liquid Diethyl-methyl-(2-methoxyethyl)-ammonium-bis-(trifluormethylsulfonyl)-imid (Sigma Aldrich).Data shown in the manuscript is taken on micromechanically exfoliated monolayer MoSRaman scattering and photoluminescence measurements are performed in a free beam optical setup using a He/Ne ion laser or a Kr/Ar ion laser (Melles Griot) for resonant and non-resonant excitation, respectively. The excitation power is 50 µW for all measurements. The light is focused onto the sample with a ×50, NA = 0.42 objective (Mitutoyo) on a spot size of <2 µm. Polarization control is realized by a set of linear polarizers and quarter-wave and half-wave plates (Thorlabs). For details refer to Supplementary Note −1 to minimize hysteresis effects. Leakage currents are monitored during all optical measurements to ensure electronic stability of the gate.Electronic control over the gate potentials during the optical measurements is realized by a two-channel source measurement unit (Keysight Technologies) for top and back gate. The top gate voltage is ramped at a rate of 1 mVsSupplementary InformationPeer Review File


SO) is measured to decrease (increase) by approximately 200 meV (240 meV) with the incorporation of 1% Bi, corresponding to a greater than fourfold increase in ΔSO in going from GaP to GaP0.99Bi0.01. The evolution of SO with x is characterised by strong, composition-dependent bowing. We demonstrate that a simple valence band-anticrossing model, parametrised directly from atomistic supercell calculations, quantitatively describes the measured evolution of SO with x. In contrast to the well-studied GaAsx1−Bix alloy, in GaPx1−Bix substitutional Bi creates localised impurity states lying energetically within the GaP host matrix band gap. This leads to the emergence of an optically active band of Bi-hybridised states, accounting for the overall large bowing of SO and in particular for the giant bowing observed for x ≲ 1%. Our analysis provides insight into the action of Bi as an isovalent impurity, and constitutes the first detailed experimental and theoretical analysis of the GaPx1−Bix alloy band structure.Using spectroscopic ellipsometry measurements on GaP Research on dilute bismide alloys has primarily focused to date on GaAsx1−Bix, where incorporation of Bi brings about a strong reduction of the direct Γ-point band gap  and chemical properties (electronegativity) between As and Bi: Bi, being significantly larger and more electropositive than As, acts as an isovalent impurity which primarily impacts and strongly perturbs the valence band (VB) structure34. This is in contrast to dilute nitride alloys, in which small electronegative nitrogen (N) atoms strongly perturb the conduction band (CB) structure in GaNxAsx1− and related alloys38. Additionally Bi, being the largest stable group-V element, has strong relativistic (spin-orbit coupling) effects39. As such, the reduction of x1−Bix is accompanied by a strong increase in the VB spin-orbit splitting energy (ΔSO)40.Highly-mismatched III-V semiconductor alloys containing dilute concentrations of bismuth (Bi) have attracted significant attention in recent yearsx1−Bix alloys, via molecular beam epitaxy42 and metal-organic vapour phase epitaxy43 (MOVPE), has only recently been attempted. Here, we present the first detailed analysis of the GaPx1−Bix electronic band structure. Early experiments on impurities in GaP can be traced back to the advent of semiconductors, with the initial experiments of Trumbore et al.44 revealing that Bi dopants generate bound localised impurity states in GaP, i.e. Bi-related localised impurity states lying energetically within the GaP host matrix band gap. However, there is little further data available regarding the GaPx1−Bix band structure. Here, we explicitly verify that the evolution of the main features of the GaPx1−Bix VB structure with Bi composition x can be understood straightforwardly in terms of an x-dependent valence band-anticrossing (VBAC) interaction between the extended states of the GaP VB edge and localised bound impurity states associated with substitutional Bi impurities. The VBAC interaction between these extended and localised states produces a set of Bi-hybridised bands, with the GaPx1−Bix alloy VB edge then being a primarily Bi-derived band possessing an admixture of GaP VB edge Bloch character30, enabling optical coupling to the comparatively unperturbed Γc6 CB states. Comparison between theory and experiment highlights the emergence of this Bi-derived impurity band lying energetically within the GaP band gap, close in energy to the unperturbed GaP VB edge. Our analysis reveals the giant bowing of SO due to this VBAC interaction: SO) decreases (increases) by ≈200 meV (240 meV) when 1% Bi is incorporated substitutionally in GaP.Epitaxial growth of GaPx1−Bix band structure.We begin here by describing our measurement procedure and the general features of our experimental results. Next, we describe our theoretical model, which is then applied to analyse the trends revealed by the experimental measurements. Finally, we use the results of this combined theoretical and experimental analysis to describe general features of the GaPx1−Bix epitaxial layers containing up to 3.7% Bi. The samples were grown on (001)-oriented GaP by MOVPE, with x-ray diffraction measurements confirming that the layers are in a state of compressive pseudomorphic strain. Full details of the sample growth and characterisation–including data from high-resolution x-ray diffraction, atomic force microscopy, secondary ion mass spectrometry, and scanning transmission electronic microscopy measurements–can be found in ref.43. The SE measurements were performed at room temperature using a J. A. Woollam Co. variable angle spectroscopic ellipsometer. Three incident beam angles were used to generate sufficient data to provide confidence in modelling fits to the measured spectra. Angles of 73.5°, 74.0° and 74.5° were chosen since they are close to the pseudo-Brewster angles of the samples under investigation, thereby ensuring that the phase change on reflection Δ  remained close to 90°. When Δ is close to 0 or 180°, the level of noise in the measured SE spectra is increased, and the sensitivity of the ellipsometic parameters to small changes in the optical properties is decreased. Keeping Δ close to 90° increases both the precision and accuracy of the measurements45. A carefully defined modelling and fitting procedure was used to extract the energies corresponding to critical points in the band structure from the measured SE data, allowing the energies corresponding to the 46. Full details of this fitting procedure are presented as Supplementary Material.Spectroscopic ellipsometry (SE) was used to study bulk-like GaPtan(Ψ) is related to the change in the ratio of the amplitudes of the p- and s-polarisations upon reflection45–in the GaP, GaP0.987Bi0.013 and GaP0.963Bi0.037 samples. Solid (dashed) black lines show the corresponding fits to Δ (Ψ). The GaP sample consists of an epitaxial GaP buffer layer grown on a GaP substrate, and was analysed first in order to obtain accurate input parameters for the SE fits. These parameters were then used to describe the substrate and buffer layer in the subsequent models of the Bi-containing samples. Following this procedure it was possible to achieve good fits to the key features observed in the measured GaPx1−Bix SE spectra  and bowing of SO).Turning to Fig. x1−Bix. This approach does not rely on post hoc fitting to alloy band structure data, thereby providing a predictive capability commonly lacking in models of this type32. In ref.30 we employed an atomistic tight-binding (TB) model to analyse the electronic structure of ordered and disordered GaPx1−Bix alloys. By directly constructing the T2-symmetric localised states |ψBi〉 associated with an isolated, substitutional Bi impurity we predicted the presence of a VBAC interaction having a composition dependence 44. Analysis of the electronic structure of ordered Gax1−Bix alloys eludicates the differences in the impact of Bi incorporation on the band structure: the natural VB offsets between GaP, GaAs and GaBi lead to the 6p valence orbitals of Bi lying below the 4p valence orbitals of As in energy, but higher in energy than the 3p valence orbitals of P. As such, a substitutional Bi impurity forms a resonant localised state lying energetically below the VB edge in GaAs, but a bound localised state lying above the VB edge in energy in GaP30.To understand this unusual behaviour we have used alloy supercell electronic structure calculations to analyse the contributions to the Bi-induced changes in the band edge energies, and to parametrise a suitable VBAC model for GaPx1−Bix we have derived an extended basis set 12-band (VBAC) k ⋅ p Hamiltonian to describe the dilute bismide band structure32. Using the TB model of ref.30 we have directly evaluated the Bi-related parameters of this model, including the distinct VBAC, virtual crystal (VC) and strain-related contributions to the Bi-induced shifts in the band edge energies52. To analyse the SE measurements we focus on the band edge energies at the zone centre: at Γ the 12-band Hamiltonian diagonalises into decoupled blocks describing the CB, heavy-hole (HH), light-hole (LH) and spin-split-off (SO) band edges52. As in GaAsx1−Bix, the energy of the GaPx1−Bix Γ-point CB state Γc6 is well described as α describes the VC shift of the CB edge energy, and x1−Bix/GaP epitaxial layer52.Building on our initial analysis of GaP5252. The energies of the LH- and SO-like VB states are given as the eigenvalues of a 3 × 3 matrix which can be found, along with full details of the model, in ref.52.The energies of the HH-like alloy VB states are given in the 12-band VBAC model as the eigenvalues of the 2 × 2 matrixx1−Bix are summarised in Table x1−Bix are provided. Comparing these two sets of VBAC parameters we firstly note that while a substitutional Bi impurity in GaAs leads to the formation of a resonant localised state lying 183 meV below the VB edge (ΔEBi < 0), in GaP it leads to the formation of a bound localised state lying 122 meV above the VB edge (ΔEBi > 0). In both cases the alloy VB edge consists of an admixture of extended (Bloch) and localised (Bi-related) character. However, in GaAsx1−Bix the alloy VB edge states are primarily GaAs-derived, retaining significant Bloch character, while in GaPx1−Bix the alloy VB edge states are primarily Bi-derived, having low overall Bloch character30. As we will describe in further detail below, this key difference describes the fundamentally distinct nature of the perturbed VB structure in GaAsx1−Bix and GaPx1−Bix alloys, and has significant consequences for the electronic and optical properties. We note that this behaviour is qualitatively similar to the stark differences in the CB structure of the dilute nitride alloys GaNxAsx1− and GaNxPx1−: a substitutional N impurity in GaAs creates a N-related localised state which is resonant with the CB, while in GaP it creates a bound impurity state lying energetically within the band gap54.The Bi-related band structure parameters computed for GaPβ, we note that a larger value is calculated for GaPx1−Bix. This describes that the VBAC interaction is more pronounced in GaPx1−Bix than in GaAsx1−Bix–i.e. that a substitutional Bi impurity more strongly perturbs the VB structure in GaP than in GaAs–reflecting the larger differences in size and electronegativity between P and Bi than between As and Bi. We note that β in GaPx1−Bix is comparable to that calculated previously for the GaNxPx1− CB (β = 1.74 eV)54. Turning our attention to the VC contributions to the band offsets–described via the parameters α, κ and γ for the CB, VB and SO offsets respectively –we again note that the calculated differences in these parameters for GaPx1−Bix and GaAsx1−Bix alloys reflect the associated trends in the lattice mismatch and natural band offsets between GaP, GaAs and the fictitious semimetallic zinc blende compound GaBi55. The calculated values α = 4.39 and 2.82 eV for GaPx1−Bix and GaAsx1−Bix respectively describe increases of approximately 44 and 28 meV per % Bi of the type-I zone-centre CB offset in free-standing GaPx1−Bix/GaP and GaAsx1−Bix/GaAs, reflecting the larger difference in energy between the Γc6 CB edge states in GaP and GaBi compared to that between GaAs and GaBi. For the VB offset, the larger calculated value of κ for GaPx1−Bix describes the larger VB offset between GaP and GaBi than between GaAs and GaBi. Similarly, the smaller calculated value of γ for GaPx1−Bix describes the smaller SO band offset between GaP and GaBi than between GaAs and GaBi, and results from a combination of (i) a natural VB offset between GaP and GaAs which exceeds the difference in ΔSO between the two compounds56, and (ii) the extremely large predicted VB spin-orbit splitting in GaBi39.Considering the VBAC coupling parameter 56, we note that the VB structure of the AlAsx1−Bix alloy–growth of which has also recently been established57–is expected to be qualitatively the same as in GaPx1−Bix. That is, based on the known chemical trends for III-V compounds, it can be expected based on the analysis presented here that (i) a substitutional Bi impurity in AlAs leads to the formation of a bound Bi-related localised impurity state, having the same general character as that described here in GaP, and (ii) this leads in turn to a large decrease (increase) and composition-dependent bowing of SO) at dilute Bi compositions x58.Finally, since the III-V compound AlAs has approximately the same natural VB offset relative to GaAs as GaPx in pseudomorphically strained GaPx1−Bix/GaP, for the CB , HH  states, respectively. The hydrostatic component of the pseudomorphic strain pushes the CB  edge(s) upwards (downwards) in energy, while the axial component lifts the degeneracy of VB edge in the usual manner60. Since the GaPx1−Bix/GaP epitaxial layers under investigation are in a state of compressive pseudomorphic strain, this splitting of the HH- and LH-like states leads in general to HH-like VB edge eigenstates52. We calculate that ECB reduces linearly with increasing x, by 18 meV per % Bi. The VBAC interaction produces two Bi-hybridised HH-like bands, the energies x, displaying strong composition-dependent bowing. Beginning from x = 0, we calculate that x = 0 and 1%. Similarly, the VBAC interaction produces a set of LH- and SO-like Bi-hybridised bands52, the energies of which are again strongly dependent on x and characterised by strong composition-dependent bowing. As ESO with increasing x the coupling between the LH- and SO-like states–which is brought about by the axial component of the pseudomorphic strain52–leads to an anticrossing which is manifested in an abrupt increase in the rate at which ESO decreases for Figure c6 CB edge states and the Bi-related localised states61, quantitative assessment of the evolution of SO with Bi composition x depends critically on the character of the hybridised GaPx1−Bix/GaP alloy VB edge eigenstates. As described above, since the GaPx1−Bix/GaP epitaxial layers under investigation are in a state of compressive pseudomorphic strain, the highest energy alloy VB states are expected to be HH-like  of that between Γv8 and Γc6 in GaP. Thus, our analysis indicates the emergence of a hybridised alloy VB edge in the form of an impurity band: this band has (i) energy c6 CB edge states which increases with increasing Bi composition x  and Bi  character we have calculated the four distinct energy gaps ECB − ESO. No clear features were distinguishable in the measured SE spectra close to the calculated transition energy ECB − ESO transition energy associated with the SO band. On the basis of the SE measurements it was not possible to determine whether there exist two distinct transitions in this energy range, although the presence of such transitions may explain the large spectral linewidths required to describe the measured SE spectra in this energy range .The SE measurements are sensitive to optically allowed inter-band transitions, and are hence capable in principle of detecting transitions between (i) the CB edge and the HH-like VBs, having transition energies SO) results from the emergence of an optically active band of primarily Bi-derived impurity states lying energetically within the GaP band gap. That this impurity band lies within the GaP band gap accounts quantitatively for the observed trends: the contribution of the strong, composition-dependent bowing of SO) is combined with the binding energy ΔEBi of the Bi-related localised states. This behaviour is qualitatively distinct from that in GaAsx1−Bix, where substitutional Bi atoms generate localised states which are resonant with the GaAs VB63, but similar to that in dilute nitride GaNxPx1−, where substitutional N atoms produce a band of primarily N-derived states lying deep within the GaP band gap67.The quantitative agreement between the calculated and measured data in Fig. x = 1.3%  of the reduction in SO, from 80 meV in GaP to approximately 360 meV at x = 1.3%. This is again in excellent agreement with the calculated value ΔSO = 355 meV, with the majority (92%) of the increase in ΔSO associated with the emergence of the From the SE measurements we extract x from 1.3 to 3.7% we note that the change in SO per % Bi is significantly reduced. The measured  value x = 3.7%  value ΔSO = 1 eV (0.590 eV) at x = 3.7% Bi represents a further increase of 250 meV (235 meV) from that at x = 1.3%. For x = 0 and 1.3%, despite occuring over a 2.4% increase in x. The measured and calculated changes of ΔSO between x = 1.3 and 3.7% are approximately equal to those between x = 0 and 1.3%, again representing a significantly reduced change per % Bi. These trends highlight the strong dependence of the bowing of SO on x. Our measured and calculated variation of SO with x differs from that predicted using first principles electronic structure calculations48, but is close to that calculated via a VBAC model using parameter estimates based on available data for related alloys49.Increasing x1−Bix electronic structure. Firstly, GaP has an indirect band gap due to the Xc6 CB states lying ≈0.5 eV below Γc6, while in semimetallic GaBi the Xc6 states lie ≈2 eV above Γc655. Applying the VC approximation in conjunction with the TB model we estimate that the Xc6 states shift downwards in energy by ≈12 meV per % Bi in free-standing GaPx1−Bix. This is less than the 44 meV per % Bi reduction of the Γc6 state energy described by the VC parameter α  we estimate that a direct band gap exists for high Bi compositions x1−Bix and GaNxPx1− band structures, since substitutional N in GaP generates localised states lying below the host matrix Xc6 states in energy, bringing about a quasi-direct band gap even at ultra-dilute N compositions66.We now turn our attention to two key qualitative features of the GaP30 demonstrated that the VBAC description of the GaPx1−Bix VB structure breaks down with increasing x in the presence of short-range alloy disorder. Bi clustering creates a distribution of Bi-related localised states with which the GaP VB edge states strongly hybridise. This leads to a distribution of GaP VB edge Γ character over a multiplicity of impurity levels, suggesting that there is no single band possessing sufficient Bloch character to allow for appreciable absorption or emission of light30. While our results above demonstrate that the VBAC model provides a useful approach to analyse the main features of the band structure, the details of the electronic structure are in practice determined primarly by the impact of short-range alloy disorder. The difficulty in obtaining photoluminescence from the samples studied here, despite their high crystalline quality43, supports this interpretation: the GaPx1−Bix optical properties are intrinsically limited not solely by growth-related defects commonly associated with Bi incorporation, but by a combination of the indirect band gap and breakdown in VB edge Bloch character.Secondly, our analysis in ref.x1−Bix alloys is unlikely to lead to efficient light emitters: the optical properties are expected to be intrinsically limited by the nature of the material band structure. However, just as quaternary GaNxAsyPx−y1− alloys have found applications in III-V semiconductor lasers monolithically integrated on Si68, it is possible that similar progress could be made using As-rich quaternary GaPx−y1−AsyBix alloys for, e.g., applications in multi-junction solar cells, due to the fact that these alloys can be grown lattice-matched to either GaAs or germanium (Ge) while having band gaps close to 1 eV71.Despite having lattice constants commensurate with growth on Si, our analysis suggests that refinement of the epitaxial growth of GaPx1−Bix band structure. Measurements performed on GaPx1−Bix/GaP epitaxial layers reveal giant bowing of SO, whereby SO) decreases (increases) by approximately 200 meV (240 meV) between x = 0 and 1%. These changes are characterised by strong, composition-dependent bowing. Electronic structure calculations confirm that substitutional Bi in GaP generates localised impurity states lying energetically within the GaP band gap, and that the main features of the GaPx1−Bix band structure can be understood in terms of a VBAC interaction between the extended states of the GaP VB edge, and highly localised Bi-related impurity states. A VBAC model was derived and parametrised directly from atomistic supercell calculations, allowing quantitative prediction of the evolution of the main features of the band structure with x. Our analysis suggests that the highest energy VB in GaPx1−Bix is a hybridised impurity band: admixture of the GaP VB edge Γ character into this primarily Bi-derived band allows optical coupling to the comparatively unperturbed CB states. Aspects of the GaPx1−Bix band structure are broadly comparable to GaNxPx1−, but key qualitative differences highlight the distinction between Bi and N as isovalent impurities in conventional III-V semiconductors.In conclusion, we have presented a combined experimental and theoretical investigation of the GaPSupplementary material


To the editor,Chronic idiopathic neutropenia (CIN) of adults is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils below the lower limit of the normal range in a given ethnic population, for a prolonged period (more than 3 months) . The diaThe etiology of CIN is not entirely known. However, there is evidence suggesting that the pathophysiology of CIN is related to an inflammatory BM microenvironment consisting of proinflammatory cytokines such as tumor necrosis factor-α, interferon-γ and interleukin-1β, proapoptotic mediators such as Fas-ligand, and activated oligoclonal or monoclonal T lymphocytes with myelosuppressive properties that collectively induce the accelerated apoptotic death of the granulocytic progenitor cells . Increasbright/CD16−) monocytes which represent the majority of PB monocytes displaying mainly phagocytic and tissue repair capacity, the intermediate (CD14bright/CD16+) monocytes producing inflammatory cytokines in response to inflammatory stimuli, and the non-classical (CD14dim/CD16+) monocytes displaying proinflammatory properties in association with patrolling and antimicrobial functions [+  monocytes have been reported in infectious and inflammatory conditions such as cardiovascular, chronic kidney, and autoimmune diseases [The possible involvement of the monocytic lineage in the pathophysiology of CIN has not been studied so far. Normally, circulating monocytes consist of phenotypically and functionally heterogeneous subpopulations, namely the classical (CD14unctions . Increasdiseases .n = 70) compared to age- and sex-matched healthy individuals (n = 22). All patients fulfilled the above-described diagnostic criteria of CIN and had mean neutrophil counts 1127 ± 529/μL (range 100–1700/μL) for a mean period of 144 ± 78 months (range 24–312 months). For the definition of neutropenia, we used the threshold of 1800/μL for the absolute neutrophil count according to the World Health Organization [bright/CD16−, intermediate CD14bright/CD16+, and non-classical CD14dim/CD16+ cells, was evaluated on the basis of their relative CD14 and CD16 surface expression using fluorescein isothiocyanate (FITC)-conjugated mouse antihuman CD16 (clone 3G8) and phycoerythrin (PE)-conjugated anti-CD14 (clone RMO52) monoclonal antibodies or the isotypic controls  following red blood lysis . Analysis was performed in a Cytomics FC500 flow cytometer  in the gate of the CD14+ cells representing the pure monocytic population . Grouped data are expressed as mean ± 1 standard deviation.In the present study, we sought to evaluate by flow cytometry the quantitative characteristics of PB monocyte subsets in CIN patients  corroborating further the hypothesis for a BM defect in CIN [Consistent with our previous observations, CIN patients displayed lower PB absolute monocyte counts (419 ± 215/μL) compared to healthy individuals  compared to the controls . On the contrary, a significant increase was observed in the proportion of the intermediate CD14bright/CD16+ and the non-classical CD14dim/CD16+ monocyte subsets in CIN patients  compared to the controls   compared to the controls  that was associated with a significant increase in the absolute number of the intermediate CD14bright/CD16+ and the non-classical CD14dim/CD16+ monocytes in the patients  compared to the controls . An inverse correlation was found between the absolute neutrophil and the absolute classical monocyte counts in CIN patients  indicating possibly a compensatory effort of the BM in response to neutropenia   Fig. . We thennia Fig. . Given t38) Fig. . No corrIn conclusion, we show here for the first time that the monocyte cell subsets are altered in CIN patients and consist of decreased number of classical monocytes and increased intermediate and non-classical ones. The intermediate and non-classical monocytes display proinflammatory properties in terms of higher expression of proinflammatory cytokines and higher potential for antigen presentation . We may Supplemental Table 1(DOC 121 kb)


Cornus mas L.) fruits are known for their significant amounts of anthocyanins which can be used as natural food colorants. The storage stability of anthocyanins from these fruit extracts, at different temperatures , pH 3.02, in the presence of two of the most widely employed food preserving agents (sodium benzoate and potassium sorbate) was investigated. The highest stability was exhibited by the anthocyanin extract stored at 2 °C without any added preservative, with half-life and constant rate values of 1443.8 h and 0.48 × 10−3 h−1, respectively. The highest value of the degradation rate constant (82.76 × 10−3/h) was obtained in the case of anthocyanin extract stored at 75 °C without any added preservative. Experimental results indicate that the storage degradation of anthocyanins followed first-order reaction kinetics under each of the investigated conditions. In aqueous solution, the food preservatives used were found to have a slight influence on the anthocyanins’ stability.Cornelian cherry ( CornusmasL.) is a species of dogwood native to Southern Europe and Southwest Asia. The fruit is an oblong, red drupe, 2–3 cm long, containing a single seed, edible, but astringent when unripe. Fresh cornelian cherry fruits contain twice as much ascorbic acid (vitamin C) as oranges, being also rich in sugar, organic acids and tannins  = initial total anthocyanin content (mg/L); k = reaction rate constant (h−1); t = reaction time (h); t1/2 = half−life (h).The kinetics for the degradation reaction of the investigated anthocyanins can be expressed by the equations:−1); K0 = frequency factor (h−1); Ea = activation energy (kJ/mol); R = universal gas constant (8.314 J/mol∙K); T = absolute temperature (K).The effect of temperature on the kinetics of the degradation process was determined by fitting the rate constants to an Arrhenius type equation [Equation (5)]:10 temperature coefficient was calculated according to Equation (6):10 = the temperature coefficient (K−1); k1,2 = rate constant (h−1) at temperature T1,2 (K).The Qp < 0.05 were considered statistically significant.Data are reported as mean values of at least four experiments. Results were analyzed using one-way variance analysis (ANOVA). Analysis of variance was performed using XLSTAT Release 10 . Differences at The total anthocyanin content of Cornelian cherries and the storage stability of these compounds indicated that these fruits can be used as an important source of natural red pigment for the food industry. The results of the present study have provided detailed information on the degradation kinetic parameters of anthocyanins during storage and heating. Increasing the temperature resulted in higher degradation rate constants: the degradation rate of anthocyanins from Cornelian cherries extract at 22 °C was 1.8 times faster than at 2 °C, while at 75 °C this process was 172 times faster than at 2 °C. Comparison of the rate constants and half-life values showed that the anthocyanin stability was slightly influenced by the kind of the added organic food preservative.