Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

Machine learning enhanced evaluation of semiconductor quantum dots.

A key challenge in quantum photonics today is the efficient and on-demand generation of high-quality single photons and entangled photon pairs. In this regard, one of the most promising types of emitters are semiconductor quantum dots, fluorescent nanostructures also described as artificial atoms. The main technological challenge in upscaling to an industrial level is the typically random spatial and spectral distribution in their growth. Furthermore, depending on the intended application, different requirements are imposed on a quantum dot, which are reflected in its spectral properties. Given that an in-depth suitability analysis is lengthy and costly, it is common practice to pre-select promising candidate quantum dots using their emission spectrum. Currently, this is done by hand. Therefore, to automate and expedite this process, in this paper, we propose a data-driven machine-learning-based method of evaluating the applicability of a semiconductor quantum dot as single photon source. For this, first, a minimally redundant, but maximally relevant feature representation for quantum dot emission spectra is derived by combining conventional spectral analysis with an autoencoding convolutional neural network. The obtained feature vector is subsequently used as input to a neural network regression model, which is specifically designed to not only return a rating score, gauging the technical suitability of a quantum dot, but also a measure of confidence for its evaluation. For training and testing, a large dataset of self-assembled InAs/GaAs semiconductor quantum dot emission spectra is used, partially labelled by a team of experts in the field. Overall, highly convincing results are achieved, as quantum dots are reliably evaluated correctly. Note, that the presented methodology can account for different spectral requirements and is applicable regardless of the underlying photonic structure, fabrication method and material composition. We therefore consider it the first step towards a fully integrated evaluation framework for quantum dots, proving the use of machine learning beneficial in the advancement of future quantum technologies.

Loss of ANCO1 Expression Regulates Chromatin Accessibility and Drives Progression of Early-Stage Triple-Negative Breast Cancer.

Mutations in the gene ankyrin repeat domain containing 11 (ANKRD11/ANCO1) play a role in neurodegenerative disorders, and its loss of heterozygosity and low expression are seen in some cancers. Here, we show that low ANCO1 mRNA and protein expression levels are prognostic markers for poor clinical outcomes in breast cancer and that loss of nuclear ANCO1 protein expression predicts lower overall survival of patients with triple-negative breast cancer (TNBC). Knockdown of ANCO1 in early-stage TNBC cells led to aneuploidy, cellular senescence, and enhanced invasion in a 3D matrix. The presence of a subpopulation of ANCO1-depleted cells enabled invasion of the overall cell population in vitro and they converted more rapidly to invasive lesions in a xenograft mouse model. In ANCO1-depleted cells, ChIP-seq analysis showed a global increase in H3K27Ac signals that were enriched for AP-1, TEAD, STAT3, and NFκB motifs. ANCO1-regulated H3K27Ac peaks had a significantly higher overlap with known breast cancer enhancers compared to ANCO1-independent ones. H3K27Ac engagement was associated with transcriptional activation of genes in the PI3K-AKT, epithelial-mesenchymal transition (EMT), and senescence pathways. In conclusion, ANCO1 has hallmarks of a tumor suppressor whose loss of expression activates breast-cancer-specific enhancers and oncogenic pathways that can accelerate the early-stage progression of breast cancer.

A Unipolar Quantum Dot Diode Structure for Advanced Quantum Light Sources.

Triggered, indistinguishable single photons are crucial in various quantum photonic implementations. Here, we realize a novel n+-i-n++ diode structure embedding semiconductor quantum dots: the gated device enables spectral tuning of the transitions and deterministic control of the charged states. Blinking-free single-photon emission and high two-photon indistinguishability are observed. The line width's temporal evolution is investigated across over 6 orders of magnitude time scales, combining photon-correlation Fourier spectroscopy, high-resolution photoluminescence spectroscopy, and two-photon interference (visibility of VTPI,2ns = (85.8 ± 2.2)% and VTPI,9ns = (78.3 ± 3.0)%). Most of the dots show no spectral broadening beyond ∼9 ns time scales, and the photons' line width ((420 ± 30) MHz) deviates from the Fourier-transform limit by a factor of 1.68. The combined techniques verify that most dephasing mechanisms occur at time scales ≤2 ns, despite their modest impact. The presence of n-doping implies higher carrier mobility, enhancing the device's appeal for high-speed tunable, high-performance quantum light sources.

Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment.

Radiation therapy is an effective cancer treatment, although damage to healthy tissues is common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damage in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type-specific reference DNA methylation atlases. We found that cell-type-specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damage was reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from patients with breast cancer undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation, indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type-specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

[Development of circumcision rates in Germany since the approval of ritual circumcision : A population-based analysis from 2013 to 2018]. / Entwicklung der Zirkumzisionszahlen in Deutschland seit Billigung der rituellen Beschneidung : Eine bevölkerungsbezogene Analyse von 2013 bis 2018.

INTRODUCTION: The religious and cultural circumcision of male infants in Germany is controversially discussed. After the passing of the religious circumcision bill in 2012, an increase of infant circumcisions without medical indication was feared. The aim of this study was to analyze the development of the circumcision case numbers. MATERIALS AND METHODS: We used the research database of the German Institute for Applied Health Research with a representative anonymous sample of 4.9 million insured persons to estimate the annual circumcision numbers in Germany from 2013-2018. We stratified the data according to age (< 18 vs. ≥ 18 years). The number of male adolescents in the study period was taken from the database of the German Federal Statistical Office. RESULTS: In the study period, 673,819 circumcisions were performed. From 2014, there was a significant decrease in the number of cases across all age groups (p = 0.049). Thereby, circumcisions in minors significantly increased (p = 0.002) and procedures in adults significantly decreased (p = 0.01) during the entire study period. The number of male minors increased by 4% from 6,709,137 (2013) to 6,992,943 (2018). The corresponding population-based number increased from 7.5 circumcisions per 1000 minors in 2013 to 8 in 2018 (p = 0.037). CONCLUSIONS: After the passage of the circumcision bill in 2012, there was a significant increase of circumcisions in the age group of < 18 years in Germany. A major limitation of our study is that presumably many ritual circumcisions might not be provided within the health care system.

Stromal Senescence following Treatment with the CDK4/6 Inhibitor Palbociclib Alters the Lung Metastatic Niche and Increases Metastasis of Drug-Resistant Mammary Cancer Cells.

BACKGROUND: CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. METHODS: We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. RESULTS: Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-ß- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-ß-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. CONCLUSIONS: These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche.

Environmentally Induced Sperm RNAs Transmit Cancer Susceptibility to Offspring in a Mouse Model.

DNA sequence accounts for the majority of disease heritability, including cancer. Yet, not all familial cancer cases can be explained by genetic factors. It is becoming clear that environmentally induced epigenetic inheritance occurs and that the progeny's traits can be shaped by parental environmental experiences. In humans, epidemiological studies have implicated environmental toxicants, such as the pesticide DDT, in intergenerational cancer development, including breast and childhood tumors. Here, we show that the female progeny of males exposed to DDT in the pre-conception period have higher susceptibility to developing aggressive tumors in mouse models of breast cancer. Sperm of DDT-exposed males exhibited distinct patterns of small non-coding RNAs, with an increase in miRNAs and a specific surge in miRNA-10b levels. Remarkably, embryonic injection of the entire sperm RNA load of DDT-exposed males, or synthetic miRNA-10b, recapitulated the tumor phenotypes observed in DDT offspring. Mechanistically, miR-10b injection altered the transcriptional profile in early embryos with enrichment of genes associated with cell differentiation, tissue and immune system development. In adult DDT-derived progeny, transcriptional and protein analysis of mammary tumors revealed alterations in stromal and in immune system compartments. Our findings reveal a causal role for sperm RNAs in environmentally induced inheritance of cancer predisposition and, if confirmed in humans, this could help partially explain some of the "missing heritability" of breast, and other, malignancies.

Pleiotrophin drives a prometastatic immune niche in breast cancer.

Metastatic cancer cells adapt to thrive in secondary organs. To investigate metastatic adaptation, we performed transcriptomic analysis of metastatic and non-metastatic murine breast cancer cells. We found that pleiotrophin (PTN), a neurotrophic cytokine, is a metastasis-associated factor that is expressed highly by aggressive breast cancers. Moreover, elevated PTN in plasma correlated significantly with metastasis and reduced survival of breast cancer patients. Mechanistically, we find that PTN activates NF-κB in cancer cells leading to altered cytokine production, subsequent neutrophil recruitment, and an immune suppressive microenvironment. Consequently, inhibition of PTN, pharmacologically or genetically, reduces the accumulation of tumor-associated neutrophils and reverts local immune suppression, resulting in increased T cell activation and attenuated metastasis. Furthermore, inhibition of PTN significantly enhanced the efficacy of immune checkpoint blockade and chemotherapy in reducing metastatic burden in mice. These findings establish PTN as a previously unrecognized driver of a prometastatic immune niche and thus represents a promising therapeutic target for the treatment of metastatic breast cancer.

[Outpatient before inpatient treatment?-Reality of care and economic analysis for minor urological interventions in Germany between 2013 and 2018]. / Ambulant vor stationär? ­ Versorgungswirklichkeit und ökonomische Analyse von kleinen urologischen Eingriffen in Deutschland von 2013 bis 2018.

BACKGROUND: Although outpatient provision of services is economically desirable, many minor urological interventions in Germany are currently carried out on an inpatient basis. The aim of our study is to investigate whether the current health policy framework contributes to more outpatient treatment. MATERIALS AND METHODS: We used a sample of 4.9 million anonymous, insured persons representative according to age and region provided by the Institute for Applied Health Research (InGef GmbH). We report extrapolations for the number of outpatient and inpatient services throughout Germany between 2013 and 2018. In addition, we performed an economic analysis for two selected interventions. RESULTS: During the study period, the total number of prostate biopsies declined from 184,573 to 174,558 cases. The share of outpatient biopsies declined continuously by 0.9% per year from 81% to 76% (p < 0.001). For botulinum toxin injection into the bladder, the total increased from 15,630 to 26,824 cases. The share of outpatient treatments increased by 2.7% per year from 3% to 19% (p = 0.01). For the other examined interventions (insertion of suprapubic urinary catheters, the insertion, removal, and changing of ureteral stents, cystoscopies and urethral dilatation), there were no significant changes in the share of outpatient procedures. CONCLUSIONS: The significant increase of outpatient botulinum toxin injections shows the successful control effect through adapted remuneration options. A shift to the inpatient sector was observed for prostate biopsies. This may be due to higher hygienic standards and technical requirements for MRI fusion.

Traceless enzymatic protein synthesis without ligation sites constraint.

Protein synthesis and semisynthesis offer immense promise for life sciences and have impacted pharmaceutical innovation. The absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets, however. Here we introduce the PALME (protein activation and ligation with multiple enzymes) platform designed for sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization and protein N/C-terminal or internal functionalization. The practical utility of this methodology is demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. The modular PALME platform exhibits unprecedentedly broad accessibility for traceless protein synthesis and functionalization, and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

Impaired CXCL12 signaling contributes to resistance of pancreatic cancer subpopulations to T cell-mediated cytotoxicity.

Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cell-autonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+ /LSL-Trp53R172H/+ /p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro co-culture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack.

Contemporary treatment trends for upper urinary tract stones in a total population analysis in Germany from 2006 to 2019: will shock wave lithotripsy become extinct?

PURPOSE: To describe the change in upper urinary tract stone management in Germany over a 14-year period. METHODS: Using remote data processing we analyzed the nationwide German billing data from 2006 to 2019. To analyze the clinics' case numbers and regional trends, we used the reimbursement.INFO tool based on standardized quality reports of all German hospitals. To also cover shock wave lithotripsy (SWL) as an outpatient procedure, we analyzed the research database of the Institute for Applied Health Research with a representative anonymous sample of 4 million insured persons. RESULTS: The number of inpatient interventional therapies for upper tract urolithiasis in Germany increased from 70,099 cases in 2006 to 94,815 cases in 2019 (trend p < 0.0001). In-hospital SWL declined from 41,687 cases in 2006 to 10,724 cases in 2019 (decline of 74%; trend p < 0.0001). The percentage of SWL as an outpatient procedure increased between 2013 and 2018 from 36 to 46% of all performed SWL, while total SWL case numbers declined. Contrarily, the number of ureteroscopies increased from 32,203 cases in 2006 to 78,125 cases in 2019 (increase of 143%; trend p < 0.0001). The number of percutaneous nephrolithotomy also increased from 1673 cases in 2006 to 8937 in 2019 (increase of 434%; trend p < 0.0001). CONCLUSION: We observed an increase in interventional therapy for upper tract urolithiasis in Germany with a dramatic shift from SWL to endoscopic/percutaneous treatment. These changes may be attributed to enormous technological advances of the endoscopic armamentarium and to reimbursement issues.

Decreasing Number of Urodynamics in Urological and Gynaecological Clinics Reflects Decreased Importance for Surgical Indications: German Population-Based Data from 2013 to 2019.

INTRODUCTION: The routine use of urodynamic studies (UDS) has been questioned. Additionally, the material and personnel costs are poorly remunerated. We aimed to analyse the UDS utilization in Germany. METHODS: We analysed UDS performed by hospitals based on quality reports from 2013 to 2019. A representative sample of 4 million insured persons was used to estimate outpatient UDS utilization from 2013 to 2018. RESULTS: There was an overall decrease of 14% in UDS in Germany from 2013 to 2018 (60,980 to 52,319; p = 0.003). In the outpatient sector, there was a slight non-continuous drop of 11% from 34,551 to 30,652 from 2013 to 2018 (p = 0.06). UDS utilization in hospitals decreased by 26% from 26,429 in 2013 to 19,453 in 2019 (p = 0.004). University hospitals showed a smaller decrease (3,007 to 2,685; p = 0.02). In urology, the number of UDS (11,758 to 6,409; p < 0.001) and the number of performing departments (328 to 263 clinics; p < 0.001) decreased. Gynaecological departments also showed a decrease in UDS (1,861 to 866; p < 0.001) and performing departments (159 to 68; p < 0.001). However, in paediatrics, there was an increase in UDS (1,564 to 2,192; p = 0.02). By age, the number of children remained constant (1,371 to 1,252; p = 0.2), but there was a strong decrease seen in 60- to 79-year-olds (9,792 to 5,564; p < 0.001). CONCLUSION: UDS appear to be less important in the indication for surgery. Despite high resource expenditure and low remuneration, the decrease in urodynamics in the outpatient sector is less pronounced, indicating a trend to perform UDS in an outpatient setting.

Low Dose Chronic Angiotensin II Induces Selective Senescence of Kidney Endothelial Cells.

Angiotensin II can cause oxidative stress and increased blood pressure that result in long term cardiovascular pathologies. Here we evaluated the contribution of cellular senescence to the effect of chronic exposure to low dose angiotensin II in a model that mimics long term tissue damage. We utilized the INK-ATTAC (p16Ink4a-Apoptosis Through Targeted Activation of Caspase 8) transgenic mouse model that allows for conditional elimination of p16Ink4a -dependent senescent cells by administration of AP20187. Angiotensin II treatment for 3 weeks induced ATTAC transgene expression in kidneys but not in lung, spleen and brain tissues. In the kidneys increased expression of ATM, p15 and p21 matched with angiotensin II induction of senescence-associated secretory phenotype genes MMP3, FGF2, IGFBP2, and tPA. Senescent cells in the kidneys were identified as endothelial cells by detection of GFP expressed from the ATTAC transgene and increased expression of angiopoietin 2 and von Willebrand Factor, indicative of endothelial cell damage. Furthermore, angiotensin II induced expression of the inflammation-related glycoprotein versican and immune cell recruitment to the kidneys. AP20187-mediated elimination of p16-dependent senescent cells prevented physiologic, cellular and molecular responses to angiotensin II and provides mechanistic evidence of cellular senescence as a driver of angiotensin II effects.

Charge Tunable GaAs Quantum Dots in a Photonic n-i-p Diode.

In this submission, we discuss the growth of charge-controllable GaAs quantum dots embedded in an n-i-p diode structure, from the perspective of a molecular beam epitaxy grower. The QDs show no blinking and narrow linewidths. We show that the parameters used led to a bimodal growth mode of QDs resulting from low arsenic surface coverage. We identify one of the modes as that showing good properties found in previous work. As the morphology of the fabricated QDs does not hint at outstanding properties, we attribute the good performance of this sample to the low impurity levels in the matrix material and the ability of n- and p-doped contact regions to stabilize the charge state. We present the challenges met in characterizing the sample with ensemble photoluminescence spectroscopy caused by the photonic structure used. We show two straightforward methods to overcome this hurdle and gain insight into QD emission properties.

An AIB1 Isoform Alters Enhancer Access and Enables Progression of Early-Stage Triple-Negative Breast Cancer.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an "enabler" phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. SIGNIFICANCE: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

The Relationship Between the Perceived Movement Quality and the Kinematic Pattern of Complex Skills in Gymnastics.

The study aimed to investigate the relationship between the perceived movement quality of a gymnastics skill and its kinematic pattern, as well as the influence of expertise. Thirty participants with different levels of gymnastics expertise (n = 10 visual experts, n = 10 motor experts and n = 10 novices) were recruited for the study. They were instructed to compare the movement quality of eleven video sequences, showing different handstand - back handspring performances. To extract the kinematics, the performances were digitized. By means of an ongoing cluster analysis, the kinematic pattern as well as the pattern of the perceived movement quality of the skills were determined for each experimental group. The results of the cluster analysis of the different experimental groups were analyzed and compared. Expertise differences were found regarding the pattern of the perceived movement quality. There was a significant correlation between the dendrograms of the visual experts and the motor experts (p = .021), as well as between the dendrograms of the visual experts and the novices (p = .011). There was no significant correlation between the dendrograms of the motor experts and the novices (p = .173). The pattern of the perceived movement quality was not correlated with the holistic kinematic pattern of judged skills (p > .143). These results suggest perceptual and cognitive differences of the participants due to their different previous visual and motor experience.

From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase.

Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1' pocket and F189W of the S2' pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis.

Cardiomyocyte-specific circulating cell-free methylated DNA in esophageal cancer patients treated with chemoradiation.

Thoracic high dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4-6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of >35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.