In addition, the LC50 value is >1,000,000 mg/L water-soluble fraction; therefore, its toxicity is <10% of that of diesel in oil-based drilling fluid and, as the flash point of NSF is about 134°C, the related fire hazard is low. The synthetic-based drilling fluid with NSF has the advantage of causing no swelling damage to the reservoir, and saves the cost of drilling fluid because it doesn't need to deal with the environmental problems (the sulfur and aromatic hydrocarbon contents were <0.5 mg/L) and field problems caused by the reaction between drilling fluid and reservoir, and greatly increases the ROP through reducing the drill pipe sticking and with C12-C22 hydrocarbons in synthetic-based fluid, and safely uses, and has stable drilling fluid performances which are flash point (134°C) and low fire hazard. We have therefore achieved our aim of preparing a high-quality and non-toxic synthetic fluid, of which the aromatic hydrocarbon and sulfur contents meet international standards. Using this synthetic-based drilling fluid therefore facilitates environment protection drilling.In view of the pollution problems related to using oil-based drilling fluids, we prepared a new environmentally friendly synthetic fluid, namely, NSF, for use in synthetic-based drilling fluid instead. We used heavy hydrocarbons from Daqing as raw material in our preparation and employed few-steps refining techniques, such as hydrodesulfurization, hydrodearomatization, and hydrogenation. After the treatment, the sulfur and aromatic hydrocarbon contents in the NSF were <0.5 mg/L, meeting international environmental standards. NSF is composed mainly of C The drilling fluids that commonly used in the oil field can be divided into water-based drilling fluid, oil-based drilling fluid and synthetic-based drilling fluid according to their properties. Water-based drilling fluid is one of the earliest used systems in drilling. And with the increasing complexity of drilling conditions, water-based drilling fluids with high temperature resistance, high density, and strong inhibitor are gradually studied, such as using nanomaterials to replace KCl and produce a new kind of inhibitor which can reduce drilling and disposal cost. Also, some researchers reported a novel kind of water-soluble acrylic acid grafted activated carbon and Cetyltrimethylammonium modified grapheme which can also reduce water invasion and protect the clay from water. However, for drilling long sections in wellbore, it still can cost much and cause environmental problems. Therefore, it is always a problem to find biogradable materials and nanomaterials which are good for environment and reservoir  a distillation range of 80–280°C, (2) condensation point of−20 to−40°C, (3) flash point of 30–50°C, (4) sulfur content of 250 mg/L, (5) aromatic hydrocarbon content of 6–8%, and (6) alkene content of 2–3%.As shown in After fractionation, hydrogenation, desulfurization and aromatization, cooling, and other processes, the aromatic content of the final synthetic fluid was <0.5 mg/L and the sulfur content was also <0.5 mg/L. This means that the quality of this fluid was consistent with the accepted standards. The main processes are as follows.(1) Fractionation# naphtha), which is separated from the fractionator (3). Finally, the agent fluid is heated to the separation temperature (200–260°C) with the heating furnace (2) and transported into the fractionator (3). After the agent fluid is separated from the fractionator (3), the clean gasoline is obtained at the top of the fractionating tower and the 200# naphtha is measured in the tower. The bottom fluid of the fractionating tower is transported to the first hydrogenation reactor (4) at a pressure of 0.5 MPa, with the supercharged pump (11).The heavy hydrocarbons in the stock cushion tank (1) is transported by the stock hydrocarbons pump (9), in turn, with the second hydrogenation-generated fluid of the second-order hydrogenation reactor (7) to exchange the heat in the first heat exchanger (13) and, subsequently, into the second heat exchanger (12) to exchange the heat with the generated fluid (220°C). After mesothermal temperature zinc oxide fine desulfurization (220°C), it is transported into the third heat exchanger (10) to exchange the heat with the solvent oil (200(2) First hydrogenation−1, under the existence of catalyst, the sulfide in oil reacts with hydrogen, and after once of this process, the organic sulfur at the bottom of the tower is converted into H2S, and most of the alkenes are converted into alkanes. The amount of this process is depend on if the sulfur content meets the demand of standard which is 0.5 mg/L, if not, repeat the process again. The first-order hydrogenated synthetic fluid is discharged from the bottom of the first-order hydrogenation reactor (4) into the zinc oxide desulfurization sweetener.The additional hydrogen supplied by the compression device is mixed with the bottom fluid before being pumped by the supercharged pump (11), and the mixture of bottom fluid and hydrogen enters the first-order hydrogenation reactor (4) from the upper part of the tower. In this process, the mixture of bottom fluid and hydrogen is always in the condition of 200–260°C and 0.5 MPa. Next, the hydrogenation reaction is conducted at a temperature of 200–220°C, pressure of 0.5 MPa, and airspeed of 1 h(3) Zinc oxide desulfurization2S from the first-order hydrogenated synthetic fluid is then reduced to <0.5 mg/L in the zinc oxide sweetener through the reaction between H2S and ZnO, the temperature is controlled at 200–220°C and the pressure is 0.5 MPa. After this period, and the generated synthetic fluid after desulfurization is transported into the second hydrogenation reactor (7) under 200–220°C and 0.5 MPa.The H(4) Second hydrogenation−1 in the second-order hydrogenation reactor (7), the arene reacts with hydrogen and is converted into alkane and cycloalkane. Subsequently, after second-order hydrogenation, the generated synthetic fluid and excess hydrogen are discharged from the bottom of the second-order hydrogenation reactor (7) to the first heat exchanger (13).The hydrodearomatization is conducted at a temperature of 120–160°C, pressure of 0.5 Mpa, and airspeed of 1 h(5) Cooling and separationThe second-order hydrotreated synthetic fluid enters the condenser (14) after heat exchange in the first heat exchanger (13) with the stock heavy hydrocarbons and, subsequently, enters the cooling separator (8) after cooling at the condenser (14). The final product is obtained after gas–liquid separation in the cooling separator (8). The final product is transported to the product cushion tank if the synthetic fluid product satisfies the demand of both aromatics and sulfur content being <0.5 mg/L. The fluid in the product tank is the synthetic fluid of the environmentally friendly drilling fluid. If the content of aromatics and sulfur are not below the standard condition (0.5 mg/L), repeat the above process. The hydrogen is compressed by the circulating hydrogen compressor after mixing the circulating hydrogen from the cooling separator (8) and the supplemental hydrogen and, subsequently, it is transported to be mixed with the raw material or to be used as cold hydrogen.In this experiment, the sulfur content in the raw material was sorted into thiol, sulfur ether, and sulfur dioxide, which have high aromatics content, as shown in Thiol and sulfur dioxide are both active sulfides with unpleasant odor, are significantly corrosive to Cu metal, and can reduce the stability of oil products. Sulfur ether is inactive sulfide, but it is also corrosive. The sulfur oxides produced by the combustion of these three sulfurs cause environmental pollution. Furthermore, if the aromatic content is too high, the drilling fluid will be highly toxic and will cause serious pollution. The international standards for sulfur and aromatics contents are shown in We compared the synthetic fluid of the drilling fluid required particularly for the exploitation of shale formations with the above criteria, finding that the conventional raw material has both high sulfur and high aromatic contents. Therefore, to improve the material to be used as synthetic fluid in drilling fluid, it needed to be desulfurized and dearomatized.Catalytic cracking was used in this experiment, as it can convert the organic sulfur compounds in the stock hydrocarbons into hydrogen sulfide, which escapes in the form of gas. Subsequently, the performance of the stock hydrocarbons could be improved by catalytic oxidation, after it meets the above standards Wu, .Catalytic cracking desulfurization is essentially the carbon-sulfur bond in the organic sulfide molecule being broken by the catalyst (acidic condition) to form hydrogen sulfide, changing the original form of the sulfur, after which it is removed  + 3% emulsifier + 3% viscosifier + 2% wetting agent (WETA-2) + 3% acryl resin + lime + barite.250 mL synthetic fluid + 20% brine . Finally, pumping the different drilling fluids from the negative part, and then measuring the positive permeability (K1) with kerosene after damaging. The ratio of K1 to K0 is the permeability recovery values. All of the above process is implemented under ambient temperature for about 120 min. The drilling fluid affected the reservoir in both solid and liquid phases, i.e., the invasion of the solid particles and the invasion of the filtrate  Designed synthetic fluid is obtained from stock hydrocarbons by fractionation, hydrogenation, desulfurization, dearomatization, cooling, and other processes. NSF is composed mainly of C3 and that of the lower was 1.52 g/cm3, minor dynamic filtration which is 0.3 mL in 2 h, and insignificant pollution of the reservoir.(2) We evaluated NSF systematically in the laboratory, with the results indicating that the properties of the designed NSF being clearly superior to those of other synthetic fluid. It is the preferred synthetic fluid for preparing environmentally friendly drilling fluid systems because of its high permeability recovery value which is 95.2%, excellent suspension property which with the upper drilling fluid was 1.49 g/cm(3) The prepared NSF is a quality product that does not cause pollution to the environment. This new synthetic-based drilling fluid is not only environmentally friendly but also its performance is superior and economic, because it can improve the rate of penetration without inducing borehole problems. We have therefore achieved our aim of preparing a kind of low-cost, high-quality, non-toxic synthetic fluid, of which the aromatic hydrocarbon and sulfur contents meet international standards. Using this synthetic-based drilling fluid therefore facilitates environment protection drilling.All datasets generated for this study are included in the article/supplementary material.YS: conceptualization, resources, and supervision. XZ and JZ: methodology. XZ and DS: validation and data curation. YS and JZ: formal analysis. XZ: investigation. YS and XZ: writing—original draft preparation and visualization. YS, XZ, and JZ: writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Expanded application of antibody-based therapeutics renewed the interest in radiolabelled antibodies. RadioimmunoPET emerged as a powerful tool for evaluation of pharmacokinetics of and target engagement by biotherapeutics. In addition to monoclonal antibodies, new radiolabelled engineered proteins have recently appeared, offering high-contrast imaging of expression of therapeutic molecular targets in tumours shortly after injection. This creates preconditions for noninvasive determination of a target expression level and stratification of patients for targeted therapies. Radiolabelled proteins hold great promise to play an important role in development and implementation of personalised targeted treatment of malignant tumours. This article provides an overview of biodistribution and tumour-seeking features of major classes of targeting proteins currently utilized for molecular imaging. Such information might be useful for researchers entering the field of the protein-based radionuclide molecular imaging.The use of radiolabelled antibodies was proposed in 1970s for staging of malignant tumours. Intensive research established chemistry for radiolabelling of proteins and understanding of factors determining biodistribution and targeting properties. The use of radioimmunodetection for staging of cancer was not established as common practice due to approval and widespread use of [ One of the pioneers in RID, Prof. Angelica Bischof Delaloye wrote in 2000: “The more and more generalized availability of positron emission tomography (PET) with Fluorine-18 fluorodeoxyglucose (FDG) for diagnosis and staging of malignant diseases will probably definitively seal the fate of radioimmunodiagnosis as it has been conceived up until now.“ (Bischof The first attempt for radionuclide imaging of tumours using radiolabelled antibodies was made in the seventies to avoid shortcomings of existing methods for cancer staging.  Bischof . Current111In (T1/2= 2.8 d)  and 89Zr (T1/2= 3.27 d) are currently commercially available and protocols for their coupling to mAbs are established , such as human epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor type 2 (HER2) have been used for targeted treatment of different types of cancers since 90s. Labelling of these mAbs with radionuclides enabled visualization of their therapeutic targets in clinical trials  results in dozens of hits concerning development of 89Zr-labelled mAbs for support of clinical trials.It would not be an exaggeration to state that immunoPET is being established as a valuable tool in drug development coupled with a steady and rapid growth in development of antibody-based therapeutics. Currently, over 570 antibody therapeutics are at various clinical trial phases, including 62 in late-stage clinical studies  effect typical for large macromolecules  , or medium half-life positron emitters 55Co (T1/2= 17.5 h), 64Cu(T1/2= 12.7 h), 76Br (T1/2= 16.2 h), and 86Y(T1/2= 14.7 h). Methodology for production of medium half-live positron emitters using low-energy cyclotrons is well developed  is established only in a few PET centres, which makes their translation into clinical practice slow.The major issues in the use of intact IgG for radionuclide imaging are associated with their large size. Digestion of antibodies with the enzymes pepsin or papain results in the smaller fragments Fab (ca. 55 kDa) and (Fab’)2 ca. 10 kDa (FDa) Fig. . A good 99mTc-labeled anti-EpCAM murine Fab fragment nofetumomab merpentan  has been approved for staging of breast, lung, gastrointestinal, ovary, bladder, kidney, cervix, and pancreas carcinomas and has been found useful. Imaging using [99mTc]Tc-NR-LU-10 demonstrated reasonable sensitivity (up to 90% for primary tumours) in imaging of lung cancer  derived by proteolytic pepsin cleavage from the parental murine antibody IMMU-4, and approved for diagnostic imaging of colorectal cancers Decrease of apparent binding affinity compared to IgG. Particularly, this is typical for Fab fragments due to the loss of the avidity effect of bivalent binding;2 are still too large to have efficient extravasation;Both Fab and (Fab)2 are still above the EPR border (45 kDa for globular proteins (Wester and Kessler Both Fab and (Fab)Still, there are some issues as:124I-labeled anti CA19-9 diabody . scFvs have a molecular weight of ~25 kDa, i.e. they are smaller than Fab. scFvs have only one binding site, i.e. they are monovalent, but scFv’s with subnanomolar affinities to their targets have been generated   , derived from either phage display human antibody repertoires (designated as VH or dAbs) or from camelid immunoglobulins (designated VHH or nanobodies) 3 to histidine tags of nanobodies is possible  are quite small with a typical molecular weight in the range between 4 and 20 kDa, which meets the requirements for an optimal balance between extravasation and excretion rates . Their affinity is sufficiently high for imaging applications. Therefore, a number of scaffold proteins were evaluated for radionuclide imaging , pH in the range from 3.5 to 11.5 and lipophilic solvents during labelling and purification.The Affibody scaffold consists of three tightly packed alpha-helices, stabilized by a hydrophobic core. The size is only 58 amino acids and the molecular weight of affibody molecules is thus 6-7 kDa making them one of the smaller scaffolds available -labelled anti-HER2 affibody molecules ABY-025 was compared with the properties of [111In]In-CHX-A”-trastuzumab in prostate cancer DU-145 xenografts with low HER2 expression  for site-specific labelling with 99mTc, 188Re or 186Re 3 .111In-label and 17.5 ± 0.3 with 68Ga  and 99mTc were evaluated as labels for ADAPTs. As a result of these studies, an optimized variant of ADAPT6 provided a substantial increase of tumour-to-liver and tumour-to-bone ratios  have been developed using a 46-amino acid 5 kDa minimized scaffold derived from an engineered albumin-binding domain (ABD)  scaffold consists of 4-6 repeating 33-amino acids blocks, each organized as a β-turn and two antiparallel α-helices  is formed by three anti-parallel β-strands linked by loops and three disulphide bonds (Colgrave and Craik 64Cu-labeled FN3(CD20), binding to lymphoma-associated CD20 with affinity of 20 nM was evaluated  Lipovsek . This 9418F]F-BMS-986192 adnectin was evaluated for the imaging of the programmed death protein ligand (PD-L1) for measuring this target expression in tumours for stratification of patients for immunotherapy , it would be trapped inside the cell and be retained for a long time . Therefore, tumour-to-organ ratios can differ several-fold depending on a label. The issue is that the influence of labelling chemistry on biodistribution of ESPs is difficult to predict as it depends both on properties of a label and properties of ESPs.Detailed optimisation of labelling chemistry could facilitate the selection of a conjugate, providing the best sensitivity and specificity for imaging. Unfortunately, affibody molecules and, to less extend, ADAPTs are the only type of scaffold proteins that are studied in sufficient detail. This is important to remember when reading the following section. It is possible that non-optimal variants of some ESPs are taken for comparison, and the potential of some imaging probes may not be completely revealed.Another important issue of such comparison is that only literature data are compared. Very often, different tumour models were used by different groups. We did try to select studies where the same xenografts or xenografts with approximately equal target expression level were used. Still, there might be a substantial variation in e.g. subclones of cell lines. Ideally, the comparisons should be performed in the same laboratory and using the same batch of xenografted mice. Unfortunately, we can only dream about this.89Zr]Zr-DFO*-trastuzumab  is a tyrosine kinase receptor regulating cellular proliferation. Overexpression of HER2 has been detected in a large fractions of breast . However, the activity was cleared from a majority of tissues (except from kidneys) already by 1.5-3 h after injection. At these time points, small imaging probes provided much higher tumour-to-organ ratios compared to [89Zr]Zr-DFO*-trastuzumab even six days after injection  demonstrated tumour-to-blood ratio for [124I]-PIB-ZHER2:342 six hours after injection that was six-fold higher than for [124I]-PIB-trastuzumab 72 h after injection. This resulted in appreciably better imaging contrast  resulted in the tumour uptake exceeding the hepatic uptake  is receptor tyrosine kinase, which is overexpressed in many malignant tumours 2 2 but lower than the uptake of [88Y]Y-DOTA-G250 antibody (Garousi et al. 111In]In-DOTA-HE3-ZCAIX:2 affibody molecule. However, the activity was cleared from the majority of normal tissues providing the superior tumour-to-tissue ratios for the affibody molecule already 4 h after injection. This has been confirmed by small-animal SPECT/CT imaging (Fig. CAIX is a membrane-bound cell-surface enzyme, which is overexpressed in a large fraction of renal cell carcinomas (Krieg et al. Radioimmunodetection evolved from experimental tool for tumour staging to the immunoPET, a powerful method for evaluation of pharmacokinetics and target engagement in development of new biotherapeutics. New formats of protein-based imaging probes, such as sdAb and engineered scaffold proteins, have emerged. Preclinical and early clinical data suggest that these novel formats would be superior to radiolabelled antibodies for stratification of patients for targeted therapies. These promising data are waiting for confirmation in prospective clinical trials.


To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM.n = 32) and without circumferential AIT (n = 32). Cultures were exposed to 0.1, 1, and 10 μM netarsudil (N = 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared with their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time series).Sixty-four porcine anterior segment cultures were randomly assigned to groups with (p = 0.01) and in eyes without TM . At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 μM netarsudil elevated IOP in eyes with TM  as well as in eyes without TM . Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 μM elevated IOP both in eyes with TM  and in eyes without TM . At this concentration, outflow tract vessels constricted by 27%.Netarsudil at 1 μM reduced IOP both in eyes with TM (− 0.60 ± 0.24 mmHg, In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal. Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, with 42% of patients eventually losing vision in one eye . The annPresent ocular hypotensive medications either reduce aqueous humor production  or increase the uveoscleral outflow as the main mechanism  . Older mIn this study, we examined netarsudil, a Rho-kinase inhibitor (RKI) and an inhibitor of the norepinephrine transporter . It can Porcine eyes were obtained from a local abattoir  and cultured within 2 h of sacrifice. No Institutional Animal Care and Use Committee approval was required because animals were not being sacrificed for the purpose of doing research. Extraocular tissues, including the conjunctiva, were carefully removed. Eyes were decontaminated by submersion in 5% povidone-iodine ophthalmic solution  for 2 min and hemisected in a biosafety cabinet. After removal of the posterior segment, lens, and ciliary body, anterior segments were mounted on custom perfusion dishes. A total of 72 eyes were cultured of which 64 eyes completed the study.We studied the outflow facility responses of anterior segment organ cultures to 0.1, 1, and 10 μM netarsudil. To establish the contribution by the TM to these responses, treatment groups were created for each concentration with and without circumferential ab interno trabeculectomy (AIT) using a trabectome as described before . Eight eWe determined the structural response of outflow tract vessels distal to the TM at the same concentrations of 1 of 0.1, 1, and 10 μM netarsudil using two eyes per treatment group. Time-series volumetric scans of the perilimbal region were captured via wide-spectrum spectral-domain optical coherence tomography (SD-OCT) pre- and post-treatment. Outflow tract cross-sectional areas were compared with respective baselines.Ocular perfusion and outflow measurements were done with a system established for the anterior chamber perfusion model , 18–21. SD-OCT imaging and analysis were performed with a system established for ex vivo , 22, 23 The TM was removed by a glaucoma surgeon experienced in ab interno trabeculectomy. Anterior segments were placed under an ophthalmic surgery microscope  and positioned with the cornea facing down in an aseptic holder. TM removal was then performed over the entire circumference via trabectome  as described before [t test in Python 3.6 [t test.IOP measurements were down-sampled into 2-h blocks and normalized to respective controls. Pretreatment was compared with post-treatment with a one-sample thon 3.6 . SD-OCT thon 3.6   reduction. We observed again a significant IOP elevation of 1.91 ± 0.19 mmHg (p < 0.001) at a higher concentration of 10 μM.Eyes with an intact TM that were exposed to 0.1 μM netarsudil experienced an IOP elevation by 1.59 ± 0.36 mmHg , as seen in nonablated eyes. At 10 μM, the highest concentration tested, netarsudil resulted again in an IOP elevation by 3.65 ± 0.86 mmHg (p < 0.001), as seen in eyes with an intact TM.Eyes that had undergone a circumferential removal of TM by AIT showed an IOP elevation by 0.23 ± 0.32 mmHg at 0.1 μM netarsudil  and a higher concentration (10 μM) of netarsudil had the opposite effect, resulting in IOP elevation and outflow vessel constriction. This effect did not require the TM but appeared to be mediated by distal outflow tract vessels.2+ [The biochemistry and pharmacokinetics of netarsudil have been examined in animals , 33 and 2+ . Specifi2+  and by d2+ .Our findings in porcine eyes are similar to Li et al.’s results in mouse  and KielThis ex vivo study had several limitations. The observed changes were statistically highly significant but small due to a perfusion rate that was only slightly higher than normal. We chose this rate out of concern to trigger NO release by excessive endothelial shear force –48. In vIn conclusion, we found that netarsudil was able not only to decrease but also increase IOP in the porcine anterior segment culture depending on the concentration tested. The IOP changes matched the diameter changes of distal outflow tract vessels. The hyper- and hypotensive property of netarsudil persisted after TM removal.


M-values Gaussian-MIxture Normalization), to correct the probe designs based on M-values of DNA methylation. Our strategy includes fitting Gaussian mixture distributions to type I and type II probes separately, a transformation of M-values into quantiles and finally a dilation transformation based on M-values of DNA methylation to maintain the continuity of the data. Our method is validated on several public datasets on reducing probe design bias, reducing the technical variation and improving the ability to find biologically differential methylation signals. The results show that MGMIN achieves competitive performances compared to BMIQ which is a well-known normalization method on β-values of DNA methylation.The Illumina Infinium HumanMethylation450 Beadchips have been widely utilized in epigenome-wide association studies (EWAS). However, the existing two types of probes (type I and type II), with the distribution of measurements of probes and dynamic range different, may bias downstream analyses. Here, we propose a method, MGMIN ( However, the methylation values  derived from the two types of designs exhibit different distributions. Particularly, the type I probes possess a larger range of measurement than the type II probes  method normalizes type II probes to render them comparable with type I probes . The method includes three steps: (i) fit Gaussian-mixture distributions to type I and type II probes separately, (ii) utilize a transformation of M-values into quantiles, (iii) perform a dilation transformation based on M-values to maintain the continuity of the data. We evaluate MGMIN using several independent datasets in terms of reducing the replicate technical variance and correcting the type II bias. By comparison with BMIQ, the results show that MGMIN improves the overall performance of normalization.In this work, we propose a method to correct the probe design bias based on the Gaussian Mixture Model (GMM) of the The β-value of DNA methylation for each probe is defined by the ratio of the methylated intensity (M) and the overall intensity (sum of methylated intensity and unmethylated intensity: M + U):M-value is calculated by the log2 ratio of the methylated intensity (M) vs. the unmethylated intensity (U):M-value close to zero indicates that the measured CpG site is about hemimethylated. A positive M-value suggests that more copies of the measured CpG site are methylated than unmethylated and a negative M-value means more copies of the CpG site are unmethylated. The M-value has been widely used in two-color expression microarray analysis :M-value is more statistically valid in an analysis by modeling the distribution of M-values because of it's homoscedastic. So we choose to adjust the M-values of type II probes into the distribution property of type I probes to correct the probe design bias.Due to more than 95% CpG sites have intensities more than 1,000 in Illumina methylation data, the α in β-value and u et al. , the M-vM-values of a single 450K microarray can be viewed as a finite Gaussian mixture model of several methylation states . The probability density function of the M-value for a single CpG site (Mi) is defined as:Gaussian Mixture Model (GMM) has been widely applied as a clustering method in analyzing gene-expression microarray data  represents the model density for Mi with unknown parameter vector θ, K is the number of different methylation states (components), kth Gaussian component, and πk is the mixing proportions which satisfy the constraint that k ≤ 1. The parameter vector θ consists of the mixing proportions πk, the mean value μk and the standard deviation σk, which can be estimated by the EM algorithm.where M-values of type I and type II probes are modeled by GMM separately. Let S ∈  and T ∈ . KI and KII are the numbers of components for type I and type II probes, which are both set as 3 by default.Next, we describe MGMIN in detail. First, UT), hemimethylated (HT), or hypermethylated (FT) states by using the maximum probability criterion. Let UT probes with M-values smaller (larger) than FT probes with M-values smaller (larger) than T ∈ . Then, we calculate the probabilities of M-value) by using the parameters P−1 returns the value of the inverse cumulative density function given the probability p and q is the normalized M-values for Second, each probe is assigned to hypomethylated  transformation is performed. Some parameters are identified as minG = min{MG}, maxG = max{MG} and G is M-values of set G, M-values for type II probes consist of q for q for M-values are transformed to β-values.Then, we merge the M-value of DNA methylation, (ii) KI will be changed to 4 automatically when M-values of hypermethylated type II probes, (iii) if KI = 4, the FI will be the set of probes belonging to the component with the largest μ, while the UI contains the probes belonging to the component with the smallest μ and the other two components are assigned to HI, (iv) no thresholds need to be set by default or estimated by manual to distinguish the three different states of DNA methylation.There are some important points to notice: (i) the initial values for μ and σ in EM algorithm are set as  and  and small perturbations to the initial μ and σ will not affect the final model because MGMIN captures the natural property of the We selected several public 450K datasets as following:Dataset 1: GSE29290 downloaded from GEO considered in Dedeurwaerder et al. . We usedDataset 2: GSE38268 downloaded from GEO considered in Lechner et al.  which coDataset 3: GSE38266 downloaded from GEO considered in Lechner et al.  which coDataset 4: GSE95036 downloaded from GEO considered in Degli Esposti et al.  which coM-values instead of beta-mixture models for β-values. MGMIN also uses quantile information to correct the M-values of the type II probes into a distribution which is comparable with that of type I probes. MGMIN complies the inherent Gaussian mixture distributions for M-values of type I and type II probes to avoid setting any parameters manually, which is different from the default breakpoints in BMIQ. Thus, MGMIN needs less manual intervention than BMIQ. However, MGMIN is slightly inferior to BMIQ on some dataset  for each probe across the three replicates was computed using no normalization (RAW), BMIQ, and MGMIN separately. As can be seen in M-values of the type II probes such that the distribution curves for the M-values of the type I and type II probes show similar dynamic ranges and peaks  under the least restrictive condition. Let τp represent the p-value threshold for bad probes and τr represent the threshold for the ratio of bad probes in a sample. The maximum value of τr is set to 0.3 here in our opinion because a sample with more than 30% bad probes is vulnerable. We can get the same test dataset from GSE38266 with the one described in Teschendorff et al.  τp = 1e − 2 and τr = 0.1 or 0.15. Overall, MGMIN identified more true positive features than BMIQ.The goal of a bias correction approach is to reduce the technical variation and identify the biological informative signals at the same time. We used a strategy similar to Teschendorff et al.  to compad Smyth,  and an Fd Smyth, . Both Dad Smyth,  with no d Smyth, . The resd Smyth,  which isf et al.  which coIn this paper, we have proposed a method called MGMIN for correcting the probe design bias of type II probes in Illumina Infinium 450K BeadChips, which can reduce the technical variation and improve the ability to find biologically differential methylation signals. We have shown that MGMIN outperforms BMIQ on multiple evaluation datasets in correcting the type II design bias and improving the data quality.M-values of type II probes while leaving the M-values of type I probes unchanged. The three-state beta-mixture distribution model in BMIQ sets two default breakpoints  to divide the β-values into three classes: hypomethylated, hemimethylated, and hypermethylated, which works well for most cases. However, the result curves of BMIQ show obviously inconsistent in some samples with high heterogeneity. We set 3 or 4 classes for probes depending on the result of Similar to BMIQ, MGMIN uses quantile information to correct the Based on the results of Dataset 3, we think the high quality of dataset is the base of normalization, in other words, there is no meaning to correct the samples with low quality. It should be pointed out that the parameter estimation of MGMIN is slower than that of BMIQ (about 1.5 times), which can be relieved by reducing the number of iterations.MGMIN can be used in the 450K methylation data preprocessing with other methods to normalize the values of the two type probes and improve the data quality.The datasets for this study can be found in GEO: GSE29290, GSE38268, GSE38266, and GSE95036.ZW performed the experiments and wrote the manuscript. All authors read and revised the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


After FC, CI significantly increased in 24 (58%) cases; a total of 19 (46%) FCs resulted in an increase in PPV. Both CI and PPV increased in 13 responders and neither in 11; the coefficient of agreement was only 0.21. We found no correlation between absolute changes in CI and PPV after fluid challenge. Conclusions: The results of this heterogenous population of critically ill patients suggest incoherence in fluid responsiveness between systemic and microvascular hemodynamics; larger cohort prospective studies with adequate a priori sample size calculations are needed to confirm these findings.Background: The aim of the study was to assess the coherence between systemic hemodynamic and microcirculatory response to a fluid challenge (FC) in critically ill patients. Methods: We prospectively collected data in patients requiring a FC whilst cardiac index (CI) and microcirculation were monitored. The sublingual microcirculation was assessed using the incident dark field (IDF) CytoCam device . The proportion of small perfused vessels (PPV) was calculated. Fluid responders were defined by at least a 10% increase in CI during FC. Responders according to changes in microcirculation were defined by at least 10% increase in PPV at the end of FC. Cohen’s kappa coefficient was measured to assess the agreement to categorize patients as “responders” to FC according to CI and PPV. Results: A total of 41 FC were performed in 38 patients, after a median time of 1 (0–1) days after ICU admission. Most of the fluid challenges  were performed using crystalloids and the median total amount of fluid was 500 (500–500) mL. The main reasons for fluid challenge were oliguria ( Fluid therapy is the first line treatment in patients with acute circulatory failure, as fluids can increase cardiac output and improve tissue perfusion. Although fluid administration remains an early intervention in this setting, the optimal timing, the amount of fluids as well as the effectiveness of such intervention should always be carefully evaluated to reduce the risk of fluid overload . TherefoWith the introduction of new microcirculatory imaging techniques, such as video-microscopy, a direct assessment of the microcirculation at bedside in order to directly evaluate tissue perfusion is now possible. Different studies have shown that fluid administration improved microvascular perfusion in patients with sepsis or trauma ,5 togethThe main purposes of this study were therefore to compare the coherence of fluid responsiveness of the macro and microcirculation and to assess the relationship between changes in microcirculatory and macrohemodynamic variables.This was a retrospective analysis including patients admitted to a 35-bed mixed medical-surgical intensive care unit (ICU). The study was approved by the hospital ethics committee (P2017/225), which waived the need for written informed consent because of the observational and retrospective nature of the study. All adult patients admitted between February and June 2016 to the Department of Intensive Care at Erasme Hospital, Brussels (Belgium) were eligible if: (a) underwent a fluid challenge, according to the decision of the attending physician; (b) had any form of invasive hemodynamic monitoring system that allowed the measurement of beat-to-beat CI; (c) underwent microcirculatory evaluation as routine monitoring of tissue perfusion [2) associated with dynamic fluid responsiveness predicting an increase in cardiac output after fluid therapy. Patients enrolled could be in spontaneous breathing or ventilated in assisted or controlled mode. Additionally, inotropic/vasopressors or vasodilators agents could be administered. Both rhythmic and arrhythmic patients were included.The principal reason for inclusion was the clinical need for fluid therapy according to one or more predefined signs of impaired organ perfusion: mean arterial pressure (MAP) <60 mmHg or >40 mmHg below normal values for at least 15 min; tachycardia >100 bpm; oliguria <0.5 mL/kg per hour for at least 3 h; metabolic acidosis  with hyperlactatemia (>2 mmol/L); reduced cardiac index (CI < 2.2 L/min·m2) value; veno–arterial gap of carbon dioxide (vaCO2 gap).We collected demographic and anthropometric data. Treatments with mechanical ventilation, inotropes and vasoactive drugs, sedative-hypnotics and analgesics drugs were recorded. For a correct macrohemodynamic monitoring, presence and correct functioning of the catheter for invasive arterial pressure detection, of the central venous access and of the advanced monitoring device  were evaluated; some of these patients also had pulmonary artery catheter, as decision of the treating physician, which was not used to assess CI for this study. The following hemodynamic parameters were recorded at the baseline and after the fluid challenge: heart rate; mean arterial pressure; central venous pressure; CI; lactate blood level; central oxygen venous saturation  was used. After an appropriate preparation of the oropharyngeal mucosa at the base of the tongue , and avoiding pressure artefacts, images were obtained from five different locations within the sublingual region. The following parameters were calculated, using the De Backer score : total sAll the macro and micro hemodynamic parameters were collected at two different times: at baseline (T0), i.e., immediately before starting the fluid-challenge test, and at the end of the infusion (T1). No change in infusion speed of inotropes, sedation and ventilation mode was performed between the two detection times, as routine practice to assess the effectiveness of the fluid challenge.Fluid challenge was defined as a fluid bolus  infused within a short time, maximum 30 min. Fluid responders were defined as patients whose variations in CI (CI-responders) or PPV (PPV-responders) after fluid challenge were 10% higher than the baseline value. This threshold was decided according to the inter-observer maximum variability in PPV assessment [Data were expressed as median and interquartile range (25th to 75th percentiles) as the Kolmogorov–Smirnov test showed a non-normal of distribution of continuous variables. The Wilcoxon test was used to compare repeated measurements. Regression analysis was used to test the relationship between microcirculatory and hemodynamic variables. The changes of the variables after fluid challenge were computed as absolute changes from baseline. Cohen’s kappa coefficient was measured to assess agreement to categorize patients as “responders” to fluid challenge using the CI-responder and PPV-responder criteria. Differences between responders and non-responders to fluid challenge were analysed using Mann Whitney U-test. The ability of the PPV to follow variations or trends of CI after a fluid challenge was assessed by analysing the correlation between the changes of the two variables, which were calculated by subtracting the first from the second measurement (T1–T0). After excluding all the pairs of measurements where at least one value was zero, we analysed the direction of change of CI and PPV to assess the percentage of concordance between the two variables ,12.p values < 0.05 at the univariate analysis would have been considered in the multivariable logistic regression.For the prediction of an increase of PPV > 10%, as the number of events was small, only variables measured at baseline with a ® SPSS® Statistics software, version 22.0  for Macintosh. For all statistical tests, a p < 0.05 was taken to indicate significance.Statistical analysis was performed using PRISM version 8.0 , and IBMn = 26; median age of 64 (51–69) years) were included over the study period. Demographic characteristics, comorbidities and reasons of ICU admission are reported in A total of 38 patients , hypotension (n = 10), low cardiac output (n = 3) and suspected hypovolemia (n = 2).Overall, 41 fluid challenges were performed, as three patients received a second fluid challenges on another day. Median time from ICU admission to fluid challenge was 1 (0–1) days. Most of the fluid challenges (39/41 95%) were performed using crystalloids , while two patients received 4% albumin. The median total amount of administered fluid and the time of the infusion were 500 (500–500) mL and 20 (15–20) minutes, respectively. The main reasons for fluid challenge were oliguria  patients were on norepinephrine (median dose 0.21 (0.11–0.49) µg/kg/min), with 11 of these 24 patients receiving also dobutamine. In total, 24 (59%) patients were on continuous intravenous sedative (midazolam in 13 and propofol in 6) or analgesic (morphine in 6 and remifentanil in 18) therapy. Among the 16 patients on controlled mechanical ventilation, median pulse pressure variation was 14% (ranges: 5–21%), with 10/16 (62%) having criteria for fluid responsiveness prediction. After fluid challenge, heart rate and venous arterial COA total of 24 (58%) patients were considered as CI-responders after fluid challenge, while 19 (46%) patients were considered as PPV-responders. In total, 13 patients (31%) were “responders” and 11 (26%) were “non-responders” for both CI and PPV changes . As suchWe found no correlation between absolute changes in CI and PPV after fluid challenge ; similar2 0.260, p < 0.001), as well as absolute change in MFI after fluid challenge and the baseline values of MFI   . Similar< 0.001) . To eval< 0.001) . Conside< 0.001) . In the < 0.001) . Thus, n2 gap, microvascular density and flow. Importantly, in half of fluid challenges, we observed an incoherence between CI-responsiveness and PPV-responsiveness. There was no correlation between the absolute variations in CI and PPV after fluid challenge. A significant but weak correlation was found between absolute variations of PPV at the baseline and after fluid challenge, as previously reported [In the present study, following a fluid challenge test, we observed a significant improvement in both macro and micro-hemodynamic parameters, including mean arterial blood pressure, central venous oxygen saturation, veno-arterial COreported ,13.Concordance between variations in macro and microhemodynamic parameters was weak; only 57% of the patients examined showed a significant improvement in CI (“macrocirculatory responsiveness”) and in terms of PPV (“microcirculatory responsiveness”) or absence of fluid responsiveness for both macro and micro-circulation. Coefficient of agreement was 0.21, indicating poor correlation between CI and PPV in the definition of “responder” or “non-responder” as a result of a fluid challenge. This fluid-responsiveness incoherence between macro and microcirculation was frequent  and provides a significant challenge for physicians. In clinical practice, CI-responsiveness is used to consider a “positive” response to fluid challenge, as increase in CI will eventually result in improved tissue perfusion. However, our study shows that increase in CI will not automatically translate in improved sublingual capillary density or flow in all patients; more importantly, some patients, who are considered as “non-responders” according to changes in CI, might still have significant improvement in microcirculatory flow. The concept of “fluid responsiveness” based on changed in CI might not be appropriate in several patients and changes in CI should not be the only tool to encourage or discourage volume loading, as changes in the microcirculation, which is the main determinant of tissue perfusion, are unpredictable. Interestingly, the physician may erroneously suspend the administration of fluids and prevent the patient to receive an effective intervention to restore microvascular perfusion when only CI-responsiveness is considered. As such, we considered that the concept of “hemodynamic incoherence”, i.e., a disorder in which resuscitation procedures aimed at the correction of systemic hemodynamic variables are ineffective in correcting microcirculatory perfusion , should In total, four types of loss of hemodynamic coherence have been proposed. In type 1, the main finding is the heterogeneous perfusion of the microvessels, which is typical in septic patients who have non-perfused vessels close to perfused capillaries. Type 2 is typically of hemodilution, which is responsible for the loss of RBC-filled capillaries and increased diffusion distance between RBCs and the tissue cells. In type 3, stasis of microcirculatory blood flow, due to increased systemic vascular resistance or central venous pressures, is observed. Finally, type 4 are observed in severe tissue edema, which is responsible for an increased distance between the capillaries and the cells and reduced oxygen delivery to tissues . We coulIn the literature, a number of studies have described such types of loss hemodynamic coherence ,15,16,17Markers of organ perfusion such as lactate, peripheral temperature and capillary refill time can be used to identify loss of hemodynamic concordance between macro and microcirculation. However, such parameters have some limitations: lactates, for example, are a terminal metabolic product whose origin does not always reflect tissue hypoperfusion . PeripheOur study presents several limitations. First, it is a monocentric and retrospective study with a small sample tested; moreover, no sample size calculation was performed and the heterogeneity of patients and reasons for fluid administration can significantly influence the power of the study and its conclusions. Second, we analyzed a heterogeneous population  and the relationship between macro- and micro-hemodynamic is influenced by the underlying pathogenetic mechanism. Future larger cohort studies should specifically evaluate patients suffering from shock and multiple organ failure, in order to understand whether the severity of the disease might potentially affect the dynamic incoherence between macro and microcirculation. However, previous studies that only considered specific subgroups of patients, predominantly with sepsis ,22, wereIn this heterogeneous patients’ population of critically ill patients, an incoherence between CI-responsiveness and PPV-responsiveness in almost half of fluid challenges was observed. Larger cohort prospective studies with adequate a priori sample size calculations are needed to confirm these studies. As such, the definition of fluid responsiveness may be challenged and include, in some patients, microcirculatory assessment to evaluate the effects of fluid administration on tissue perfusion. Whether this approach is feasible and might be used to titrate fluid administration in critically ill patients, has to be demonstrated in larger studies.


Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy.HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls.p = 0.01) and controls (p = 0.007).VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy (VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study. We identified that these effects were, in part, mediated by microparticles (MPs) containing virions that transmitted VZV to neighboring cells.Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries, causing VZV stroke.2THA- or green fluorescent protein (GFP)-tagged ORF 23-expressing VZV was grown in MeWo (human melanoma cell line) cells, mitotically inactivated by mitomycin C, and titered by immunohistochemical staining with anti-VZV antibodies. Infected HBVAFs (either VZV or mock) were inoculated to resting HBVAFs at a ratio of 1:3. Infection was confirmed with detection of cytopathic effect and expression of the following viral proteins: envelope glycoprotein H (gH) expressed at 12 to 24 hours after VZV infection and VZV immediate early protein (IE62 protein) expressed at 4 to 12 hours after VZV infection.HBVAF transdifferentiation and proliferation were assessed with α-smooth muscle actin–APC  and Click-iTTM Plus EdU Alexa Fluor 647 Kit . HBVAF migration was assessed using a scratch assay with a 200-μL pipette tip in cells incubated with 10 μg/mL mitomycin C to inhibit mitosis.CD54-PE  expression on human umbilical vein endothelial cells  was analyzed by flow cytometry. Intracellular reactive oxygen species (ROS) production was determined as oxidation-dependent fluorescence of dichloro-dihydro-fluorescein diacetate .Cytokines were measured with Meso Scale Discovery electrochemilluminescence.g for 60 minutes and stained with phycoerythrin-labeled annexin V (BD Biosciences) or mouse anti-gH VZV monoclonal antibody .MPs were sedimented from 200 μL platelet-poor plasma or culture supernatants after centrifugation at 17,000g for 1 hour. Samples were loaded onto a copper carbon/formvar-coated grid, negatively stained with 2% aqueous uranyl acetate, and imaged with a JEOL JEM-1010 electron microscope.Negative-stain transmission electron microscopy was performed on MPs obtained by sequential centrifugation at 131,000For proteomics, solid-phase extraction was performed before liquid chromatography/mass spectroscopy analysis. Samples were analyzed on nanoelectrospray IMS QToF (Synapt 2GSi). Proteins with a Protein Lynx Global Server score >95% in the Uniprot Human Proteome database were considered.3 Cerebral/cervical vasculopathy/arteriopathy was defined as focal or segmental stenosis or occlusion with abnormalities of the arterial wall and categorized according to current consensus definitions.3 For the 10 patients with non-VZV–related cerebral vasculopathy, the following tests were negative or normal: intrathecal VZV immunoglobulin (Ig) G production, CSF PCR for VZV, and blood test investigations (serum IgM and VZV PCR) for acute VZV infection. There was no history of chickenpox in any of these 10 children. Three patients were classified as having VZV vasculopathy on the basis of radiologic evidence of cerebral vasculopathy/arteriopathy and history of VZV infection within 12 months from the acute AIS event and/or positive CSF  findings.4 Age-matched healthy controls were children with no AIS, no acute or recent history of VZV infection, or any other vascular pathology (n = 10).We recruited patients with cerebral vasculopathy   referredThis study was approved by the Bloomsbury Ethics Committee, London (07/Q0508/58), and all participants provided consent or age-appropriate assent.p < 0.05 by unpaired t test or Mann-Whitney U test were considered significant.Results are expressed as mean and SEM or median and range. Values of All data are presented within this article and its supplemental material.First, we confirmed that VZV could infect HBVAFs. Cells were infected/mock infected with either wild-type VZV or GFP-ORF23 VZV. We detected positive expression of GFP-ORF23 and of VZV-related viral proteins gH and VZV IE62 in infected HBVAFs, with no detectable VZV protein or GFP-ORF23 expression in mock-infected cells from day 2 onward.p = 0.04 and p = 0.03, respectively). VZV infection also enhanced proliferation of HBVAFs, as indicated by increased expression of the marker EdU in VZV-infected cells, compared to mock-infected cells at days 2 and 6 . Significantly more HBVAFs migrated into the marked scratch area of an in vitro scratch assay at 17 hours in the VZV-infected cultures compared to mock-infected cultures (p = 0.01).We observed increased myofibroblast transdifferentiation of VZV-infected HBVAFs compared to mock-infected cells, as indicated by increased expression of α-smooth muscle actin (a myofibroblast surface marker) in VZV-infected HBVAFs at 2 and 6 days after infection  and resulted in enhanced ROS production as assessed through oxidation-dependent fluorescence of dichloro-dihydro-fluorescein (p = 0.0005). No such changes were observed with media from mock-infected cells.Adventitial fibroblasts produce biologically active proteins that affect endothelial function and vasculopathy progression.p = 0.02); tumor necrosis factor-α (p = 0.02); interleukin-8 (p = 0.018), and monocyte chemoattractant protein-1 (p = 0.02).5To further characterize what could be driving this endothelial activation, we profiled cytokine production in the culture supernatant from VZV-infected HBVAF compared to mock-infected cells. These cells released significantly higher levels of cytokines/chemokines that activate endothelium, promote leucocyte adhesion, and enhance fibroblast migration: interleukin-6 (3 We identified a significantly higher number of annexin V+ GFP+ ORF23+ MPs in supernatants from cultures of VZV-infected cells compared to mock-infected cells (p = 0.02). These MPs also expressed the VZV protein gH.We next investigated signaling between HBVAFs and endothelial cells mediated by cellular MPs. MPs (0.1–1 μm) are released from apoptotic or activated cells and have high surface phosphatidylserine content, which binds annexin V.Transmission electron microscopy uranyl acetate-negative staining of MPs confirmed the presence of VZV associated with MPs in pellets from VZV-infected cells; these virus particles were not detected in MP pellets from mock-infected cells . MPs werdoi.org/10.5061/dryad.466fk4h) and did not express RAB (Ras superfamily of monomeric G) proteins commonly found in endosomes.6Proteomic analysis of MPs derived from VZV-infected HBVAFs revealed proteins consistent with their fibroblastic origin. MPs were highly enriched for VZV proteins that were not detected in the MPs derived from mock-infected samples (p = 0.001) and higher ROS production (p = 0.002) in HUVECs compared to MPs from mock-infected HBVAF  but did not differ from total annexin V+ MP levels detected in patients with non-VZV–related cerebral vasculopathy (n = 10). When costained for the detection of VZV gH protein, however, a significantly higher number of annexin V+ VZVgH+ MPs were identified in children with VZV-related vasculopathy compared to children with non-VZV vasculopathies (p = 0.01) and controls  compared to healthy controls  may also play a role. Moreover, whether MP-mediated viral propagation is specific to VZV or also applies to other viruses causing AIS requires further investigation. It would also be interesting to establish whether VZV-infected cells release phenotypically and quantitatively distinct MPs in resting, activated, and apoptotic states.Our findings support a novel model of VZV vasculopathy pathogenesis, with adventitial fibroblast transformation to myofibroblasts contributing to arterial remodeling by proliferation and migration and further adventitial cell-to-cell viral dissemination and endothelial activation mediated by VZV-MPs. MPs are important intercellular signaling mediators,The small size of our clinical study with a few cases with VZV vasculopathy precludes any definitive conclusions about our proposed mechanism in patients but does at least provide some preliminary face validity regarding our model in a clinical context for this rare disease. Although applied to a pediatric cohort here, the detection of MP-VZV complexes might also be an approach that is diagnostically useful for adult patients, in whom establishing a diagnosis of VZV vasculopathy can be equally challenging. Thus, while the diagnostic or prognostic relevance of circulating VZV+ MPs is currently unknown, study of this as a potential novel biomarker could be fruitful. This would require a prospective study of large numbers of patients, with or without vasculopathy, and further characterization of the cellular origin of these circulating MP-VZV complexes. It would also be interesting to use sensitive assays such as fluorescent antibody against membrane antigen and VZV-IgG time-resolved fluorescence immunoassay to quantify varicella antibodies in patients with VZV vasculopathy and correlate them with levels of VZV MPs.While challenging in a rare disease setting in which patient numbers are limited, further insights might be obtained by exploring our model in more complex 3-D in vitro tissue culture systems. This might unveil novel druggable targets for VZV vasculopathy.


Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.In the model organism  In Escherichia coli, the UvrAB damage sensor recognizes helix-distorting lesions by itself or via Mfd bound to stalled RNA polymerase. Here authors use single-molecule fluorescence imaging to quantify the kinetic signatures of interactions of UvrA with Mfd and UvrB in live cells. At sites of putative DNA damage, these damage recognition factors load specific DNA damage verification factors  that unwind the DNA and verify the location of the damage with nucleotide resolution . Subsequently, specialized endonucleases  are recruited to the site of the DNA, resulting in cleavage of a single-stranded DNA (ssDNA) patch containing the lesion (reviewed in refs. 3).Across the various domains of life, the recognition and repair of bulky, helix-distorting lesions in chromosomal DNA is coordinated by nucleotide excision repair (NER) factors. Damage detection occurs in two stages during GGR: a dedicated set of damage surveillance factorsion Fig.  (reviewe5), strand-specific removal of DNA damage also occurs following stalling of RNA polymerase (RNAP) at sites of lesions (reviewed in ref. 6). In this case, a ternary elongation complex (TEC) of RNAP that is unable to catalyse nascent RNA synthesis manifests as an ultra-stable protein-DNA roadblock. Dedicated transcription-repair coupling factors including the prokaryotic Mfd (and eukaryotic homologs Rad26/CSB) remodel these TECs and terminate transcription11. In prokaryotes, Mfd subsequently recruits the UvrA2B complex to the stall site . Consequently, the rate of removal of UV-induced lesions from the transcribed strand during TCR is enhanced compared to that from non-transcribed DNA in GGR19. This observation has sparked several studies targeted at understanding the mechanistic basis of rate enhancement21.In several studied organisms . UvrA mediates damage recognition by itself and by interacting with Mfd1 by surveying the genome constantly for the presence of DNA damage or Mfd-bound TECs. Following detection of a putative damage site, damage verification is then orchestrated by the damage verification enzyme UvrB26  in MG1655 cells using λ Red recombination  at 30 °C. Exponentially growing cells were deposited on a modified glass coverslip at the bottom of a flow cell and visualized by illumination with 514-nm laser light under continuous flow of growth medium  expressing UvrA-YPet under its native promoter. UvrA-YPet was expressed to the extent of 120 ± 28 copies per cell in ΔuvrA/pUvrA-YPet cells   between consecutive frames of duration (τint) extends the observation time window. Here, the time between consecutive frames is denoted as time-lapse time (τtl). By acquiring the same number of frames in each video collected with a different dark interval, the photobleaching rate (kb) is maintained constant (see Tables keff)32. The product (keffτtl) is a linear function of true off rate (koffτtl) and the normalized photobleaching rate (kbτint)32. For purposes of illustration, the data can be represented as keffτtl vs. τtl plots where the slope reveals the off rate koff and the Y-intercept reveals kb. This interval imaging strategy enables accurate quantification of binding lifetimes over three orders of magnitude from 0.1 s to several minutes34.Continuous imaging of UvrA-YPet could not be used to measure DNA-binding lifetimes, since the loss of signal in a focus represents dissociation of bound UvrA-YPet from the site or photobleaching of the YPet in bound UvrA-YPet molecules. Consequently, measurement of interactions that last longer than the photobleaching lifetime is impossible. Instead we imaged UvrA-YPet using a strategy of performing time-lapse imaging with dark periods of varying intervals25) Fig.  that eleuvrA ΔuvrB Δmfd cells) with pUvrA-YPet. In these cells, we expected that interactions of UvrA-YPet with DNA would reflect two of its key activities: binding to non-damaged DNA and binding to endogenous DNA damage produced as a by-product of cellular metabolism  of 1.6 ± 0.1 s (72 ± 2%) and a slow dissociating population with a lifetime  of 24 ± 1 s (28 ± 2%) . The measured lifetimes of UvrA-YPet in this strain were found to be identical within error  and τuvrA-YPet ΔuvrB Δmfd, fastUvrA| = 1.4 ± 0.4 s (73 ± 4%)) to those in the ΔuvrA ΔuvrB Δmfd/pUvrA-YPet strain -YPet, would be a faithful reporter of binding of kinetics of UvrA alone in uvrB+mfd+ cells -YPet expressed from a low-copy plasmid (pUvrA(Δ131–250)-YPet) in ΔuvrA cells produced a single population with a binding lifetime (τUvrA(Δ131−250)) of 29.7 ± 0.8 s  of 1.5 ± 0.1 s (amplitude: 78 ± 2%) and a long lifetime  of 8.7 ± 0.4 s (22 ± 2%) . This mutant, UvrB(ΔβHG), is inefficiently loaded on DNA in vitro42. The lifetime (τuvrB(ΔβHG) ΔmfdUvrA|) of UvrA-YPet in cells expressing UvrB(ΔβHG) from the chromosome was 148 ± 36 s (100%); over 15-fold longer than that of UvrA-YPet in cells lacking Mfd  and interact with Mfd engaged with failed TECs. Interval imaging of UvrA-YPet in cells lacking UvrB (uvrA-YPet ΔuvrB) revealed a single long-lived UvrA species with a lifetime (τuvrBUvrA|Δ) of 97 ± 18 s  and Mfd (22 per cell)23, and the reported picomolar affinity of UvrA for Mfd12, a large fraction of entire population of UvrA can be efficiently sequestered in complex with Mfd, consistent with the observation of only a single, slowly dissociating species in cells lacking uvrB. Our attempts at co-localization of UvrA and Mfd using the spectrally separated probes, YPet and PAmCherry were limited by the poor expression of Mfd-PAmCherry and UvrA-PAmCherry in cells under our standard growth conditions  of 1.9 ± 0.2 s (79 ± 0.2%) and a long-lived species of UvrA with a lifetime  of 12.0 ± 0.8 s (21 ± 2%)  treatment. Interval imaging of UvrA-YPet in rif-treated cells revealed a short-lived species with a lifetime  of 1.5 ± 0.3 s (63 ± 3%) and a long-lived species of UvrA-YPet possessing a lifetime  of 9.6 ± 0.6 s (37 ± 3%)  Fig. , Table 13%) Fig. , Table 13%) Fig. . The dec2-UvrB complex but not the UvrA2UvrB2 complex in cells, measurement of the lifetime of UvrA in the Mfd-UvrA2-UvrB complex is difficult. Nevertheless, it is conceivable that the lifetime of the Mfd-UvrA2-UvrB complex could be influenced by the availability of downstream factors. We tested this hypothesis by measuring the lifetime of UvrA-YPet in wild-type cells expressing UvrA-YPet from the plasmid (ΔuvrA/pUvrA-YPet). Since UvrA can engage UvrB in solution, we reasoned that at higher expression levels, UvrA would bind UvrB to form the UvrA2B2 surveillance complex, leaving little free UvrB for engagement with Mfd-UvrA2 complexes. Consistent with this expectation, the data revealed a short-lived species with a lifetime  of 2.0 ± 0.1 s (74 ± 2%) and a previously unencountered population of long-lived UvrA possessing a lifetime  of 19 ± 1 s (26 ± 2%)  Fig. , Table 12B2  of 11.5 ± 0.6 s (25 ± 2%) and a short lifetime  of 1.7 ± 0.1 s (75 ± 2%)  Fig. , Table 1uvrA/pUvrA-YPet cells yielded a lifetime (11.5 s) that is longer than that measured for rif-treated uvrA-YPet cells (9.6 s), and cells lacking mfd (8.7 s). The simplest explanation consistent with these observations is that under conditions of high relative UvrA/UvrB abundance the population is composed of UvrA2B(2) complexes .Time-lapse experiments on UV-irradiated ght Fig. . This water Fig. 47,48  by performing interval imaging in four rounds, each lasting 25 min. Analysis of the complete data set revealed binding kinetics of UvrA-YPet corresponding to a short-lived species with a lifetime  of 1.6 ± 0.1 s (77 ± 3%) and a long-lived species of UvrA corresponding to a lifetime of  13.1 ± 0.6 s (23 ± 3%)  Fig. .We wondered if the longer lifetime of UvrA detected in these experiments represented temporally averaged measurements. Since each set of interval measurements lasted 25 min, we proceeded to disaggregate each data set into the four constituent 25-min intervals after UV exposure. Analysis of the resulting data from each time window revealed that the measured lifetime of UvrA changes as a function of the experimental timeline after the UV pulse . In this case, UvrA-YPet exhibited two kinetic populations after UV exposure, a short-lived population with a lifetime of 1.5 ± 0.1 s (74 ± 2%) and a second, longer-lived population with a lifetime of 9.9 ± 0.4 s (26 ± 2%) , and subsequently dropped rapidly to 13 ± 2% at 100 min after UV. In comparison, uvrA-YPet Δmfd cells exhibited a non-linear trajectory that nevertheless exhibited a similar reduction in the fraction of UvrA-YPet possessing a slow off rate. The most striking difference between the two strains was apparent at the 50 min time point—cells carrying mfd exhibited a 1.5-fold greater fraction of UvrA molecules dissociating with the slow lifetime. These results are broadly consistent with the expectation that as repair progresses, fewer unrepaired lesions are available for UvrA binding.Next, we plotted the amplitude of the long-lived species in each time window after UV irradiation Fig. . In uvrA23. Interval imaging of Mfd-YPet in mfd-YPet cells exposed to UV light revealed that the lifetime of Mfd-YPet dropped during the course of the SOS response, leading to an average lifetime (τMfd|UV) of 12 s  and loads UvrB in GGR and second, it stimulates TCR by interacting with Mfd. In this work, we quantified the binding behaviour of fluorescently tagged UvrA in living cells over the experimentally accessible, and biologically relevant timescale of 0.1–1000 s.UvrA is the central player in NER since it performs critical functions in both GGR and TCR: first, it recognizes DNA damage as UvrA44. Using TCR-deficient strains and rif treatment, we assigned the lifetime of DNA-bound UvrA-UvrB interactions to be 8.7 s. This lifetime likely corresponds to loading of UvrB by UvrA on DNA since the loading-deficient UvrB mutant arrests UvrA for 148 s  may explain the faster turnover of UvrA during the SOS response.Curiously, the DNA-binding lifetimes of UvrA in Mfd-UvrAlls Fig. . A predi46. The differences observed in our experiments may fundamentally be attributable to the efficiencies with which UvrA recognizes cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts. CPD lesions51, being less helix distorting than 6–4 photoproducts53, are inefficiently recognized by damage recognition factors in NER across organisms55. CPD recognition by RNAP and subsequent TCR at such sites is a major mechanism for CPD removal after UV treatment46. Compared with untreated cells that remove greater than 80% of CPD lesions within 40 min following UV treatment, rif-treated cells removed less than 50% of CPD lesions at the same time point46. In contrast, 6–4 photoproduct removal was uninfluenced by rif treatment46. Further, this report also demonstrated that the copy number of UvrAB determines whether CPDs are efficiently removed following UV treatment. Considering the low expression level of UvrA-YPet in our strain , the initial time window in which UvrA is turned over rapidly in Δmfd cells may reflect repair of 6–4 photoproducts. The later time windows in which UvrA exhibits a lifetime of 15 s may reflect repair of CPD lesions that are poorly detected by UvrA. Extending this argument, the prediction from the Crowley and Hanawalt study is that the rapid turnover of UvrA in wild-type cells in the 25–100 min time window reflects the repair of CPDs via TCR. Indeed, we found that the binding lifetimes of Mfd mirror those of UvrA in this time period. Further, this finding is consistent with previously established biphasic repair of UV lesions—rapid removal of 6–4 photoproducts is followed by a slower repair of CPDs46.The lifetime of UvrA in TCR-deficient UV-treated cells increases to 15 s, but remains low in UV-treated wild-type cells. An explanation for this striking difference may be found in work by Crowley and Hanawalt12. In this model, stalled RNAP is recognized by Mfd, leading to the exposure of Mfd’s UvrB-homology module that in turn acts as a flag for UvrAB. Damage recognition by UvrAB would then follow initial recruitment to the site of the lesion. In contrast, target search during global genomic repair would comprise of repeated cycles of three-dimensional diffusion of UvrA(B) to sites of undamaged DNA followed by subsequent turnover of UvrA by UvrB until the damage surveillance complex stochastically encounters damaged DNA.Finally, our data suggest that the enhancement of the rate of repair in TCR vs. GGR measured in bulk is best explained by enhanced target search in TCR compared with GGR. This model has been previously proposed in the literature based on evidence from in vitro studiesEscherichia coli MG1655 uvrA-YPet was constructed using the Datsenko–Wanner protocol28 for λ Red recombination using the linker sequence (SAGSAAGSGEF)23. Briefly, the uvrA-YPet strain was constructed as follows: first, the YPet gene from pYpet  were created by replacing the indicated gene with a kanamycin cassette flanked by FRT sites23. Briefly, ΔuvrA cells were created by applying the Datsenko–Wanner protocol using the UvrA_del_507_fw primer (CGGTA GCACC ATGCC ACCGG GCAAA AAAGC GTTTA ATCCG GGAAA GGTGA ccctttcgtcttcaagaattc) and UvrA_del_507_rev (GCTGG TGCAA CTCTG AAAGG AAAAG GCCGC TCAGA AAGCG GCCTT AACGA ggccacgatgcgtccggcgta) that prime the pEAW507 plasmid (see Supplementary Table uvrA gene. ΔuvrB strains were similarly constructed using the UvrB_del_507_fw (GACGA GTAAA ATTAC ATACC TGCCC GCCCA ACTCC TTCAG GTAGC GACTC ccctttcgtcttcaagaattc) and UvrB_del_507_rev (TAGCG CATCA GGCTG TTTTC CGTTT GTCAT CAGTC TTCTT CGCTA TCCTG ggccacgatgcgtccggcgta) primers. In each case, wild-type electrocompetent MG1655 cells  were transformed with 50 ng of gel purified PCR amplicons  and plated on Luria Bertani (LB) agar plates containing kanamycin (40 μg/mL) incubated at 37 °C overnight. Colonies obtained in the kanamycin plates were then transferred to two plates each containing either ampicillin (100 μg/mL) or kanamycin (40 μg/mL). Colonies that grew on kanamycin plates but not on ampicillin plates were then checked for the presence of YPet (or loss of the uvrA or uvrB gene) using sequencing primers (see Supplementary Table uvrA-YPet in the indicated deletion backgrounds.Wild-type MG1655 was a generous gift from the Cox lab (UW Madison). uvrA-YPet expressing mutant UvrB(ΔβHG) from the chromosome were constructed using scar-less CRISPR-Cas9 assisted λ Red recombination as previously described57. Briefly, uvrA-YPet electrocompetent cells were transformed with pKD4628 and pCas956 and selected on LB plates containing ampicillin (50 μg per mL) and chloramphenicol (25 μg per mL) following incubation at 30 °C overnight. Since pCas9 is prone to recombination events56, the resulting colonies were screened with colony PCR to confirm the presence of the full-length Cas9 gene, using primers  and chloramphenicol (25 μg per mL). In these cells, Cas9 was constitutively expressed. The expression of λ Red recombination proteins was induced with 0.2% L-arabinose (w/v) when cells reached a density around 0.4 (OD600). When the optical density reached 0.8, cells were harvested at 4 °C and washed twice with ice-cold water, with an additional wash using 10% glycerol to obtain electrocompetent cells. Finally, aliquots containing 40 μL of cells in microcentrifuge tubes were snap-frozen in liquid nitrogen and stored at −80 °C.Next, E. coli chromosome with the help of guide RNAs expressed from pCRISPR-UvrB-Y96A (Supplementary Table 56. The point mutations were introduced by recombining the foreign ssDNA (Supplementary Table E. coli chromosome on either sides of the desired mutation. Base changes were selected to be within five bases from the PAM sequence.Cas9 endonuclease activity was targeted to the vicinity of the desired point mutations on the uvrA-YPet uvrB(ΔβHG) Δmfd. Additional information on the cell lines can be found in Supplementary Table Aliquots of cells expressing Cas9 and λ Red recombination proteins were transformed with 30 ng of pCRISRP-UvrB-Y96A plasmid and 500 ng of ssDNA. Positive colonies were selected on LB plates containing 50 μg per mL of kanamycin and 25 μg per mL of chloramphenicol at 37 °C, and were screened by colony PCR, and the promoter and gene sequences were sequence verified. pCRISPR-UvrB-Y96A was cured by propagating cells on LB plates for a week at 42 °C. This is critical for subsequent rounds of P1 transduction to create uvrA gene from MG1655 using UvrA_FWc primer (TGG GTA CCG GGC CCG CGA TTG TAC CAT TAC CAA TAG) and UvrAYPET_revc (AGC CAG CCG Aag cga tcg cCA GCA TCG GCT TAA GGA) using Phusion High-Fidelity DNA polymerase (New England Biolabs) and an annealing temperature of 63 °C. The insert thus generated was PCR purified  and digested using ApaI/XbaI. The pHH001 backbone23 (carrying a spectinomycin marker) digested using ApaI/XbaI and additionally with XhoI to digest the released Mfd fragment first at 25 °C for 2 h, followed by an additional 2 h at 37 °C. Following this, standard molecular biology techniques were employed to ligate the digested insert into the vector, followed by selection on spectinomycin plates. Colonies were then screened for the presence of the insert, followed by plasmid isolation . Isolated plasmids were sequence verified to obtain pUvrA-YPet. The UvrA(Δ131–250) sequence was synthesized by IDT (United States) and received as an insert in the standard backbone IDT vector (ampR). This insert was PCR amplified using UvrA_FWc and the reverse primer RecASZ2_rev (GTGGCGGCCGCTCTA) using Phusion polymerase  and visualized in a 1% agarose gel to obtain a single band of the expected size. Following PCR cleanup , the insert was then digested with ApaI/AsisI (4 h at 25 °C followed by 4 h at 37 °C) followed by further purification . In parallel, pHH002 (low-copy vector that expresses Mfd(L499R) under its native promoter cloned between ApaI/AsisI23) was digested to release the fragment containing the mfd(L499R) gene. The PCR amplified mutant uvrA sequence was thus sub-cloned into pHH002 using standard molecular biology protocols. Successful transformants following ligation were screened for the presence of the insert, followed by sequencing to obtain pUvrA(Δ131–250)-YPet. Plasmids were sequenced on both strands prior to use.pUvrA-Ypet was constructed by first amplifying the 23. Briefly, flow cells were assembled using a clean quartz piece  and a bottom cleaned and (3-Aminopropyl)triethoxysilane -treated coverslip  using double-sided sticky tape , and sealed with 5-min epoxy (Parfix). Quartz top pieces were designed to be able to insert inlet and outlet tubing . Prior to imaging, cells were revived from a −80 °C DMSO stock in 500 μL of EZ-rich defined media , supplemented with 0.2% (v/v) glucose in 2-mL microcentrifuge tubes at 30 °C. Cultures were set to shake in an Eppendorf Thermomixer C  at 1000 rpm. On the following day, cultures were reset by inoculating fresh growth medium 1:200 fold, and continued to shake for ~3 h at 30 °C prior to imaging. For experiments involving plasmid-expressed UvrA-YPet or UvrA(Δ131–250)-YPet, spectinomycin (50 μg per mL) was added to the growth media. Cells in early exponential phase were loaded in flow cells at 30 °C, followed by a constant supply of aerated EZ-rich defined media at a rate of 30 µL per min, using a syringe pump .Cells were imaged in quartz-top flow cells as described previously23. Briefly, the microscope comprised a Nikon Eclipse Ti body, a 1.49 NA 100× objective, a 514-nm Sapphire LP laser (Coherent) operating at a power density of 71 W cm−2, an ET535/30m emission filter (Chroma) and a 512 × 512 pixel2 EM-CCD camera (either Photometrics Evolve or Andor iXon 897). The microscope was operated in near-TIRF illumination58 and was controlled using NIS-Elements (Nikon). PAmCherry-tagged proteins were imaged as described previously23.Single-molecule fluorescence imaging was carried out with a custom-built microscope as previously describedτd).Fluorescence images were acquired in time-series format with 100 ms frames. Each video acquisition contained two phases. The first phase (50 frames) aimed to lower background signal by continuous illuminating, causing most of the fluorophores to photo-bleach or to assume a dark state. The second phase (single-molecule phase lasting for 100 frames) is when single molecules can be reliably tracked on a low background signal. In the second phase, consecutive frames were acquired continuously or with a delay time , and MATLAB. First, raw data were converted to TIF format, following by background correction and image flattening as previously described23. Next, foci were detected in the reactivation phase by applying a discoidal average filter , then selecting pixels above the intensity threshold. Foci detected within 3-pixel radius (318 nm) in consecutive frames were considered to belong to the same binding event.Image analysis was performed in Fiji25. Briefly, the photobleaching phase contained 50 continuous 0.1-s frames. In phase II, 100 discontinuous 0.1-s frames (τint = 0.1 s) were collected with time-lapse time (τtl) ranging from τtl = . In each experiment, videos with varying τd were acquired. Foci were detected using a relative intensity threshold of 7 or 8 above the background as appropriate. Depending on the construct being imaged, between 3 and 15 repeats of each experiment were collected for each strain. Cumulative residence time distribution of binding events detected in all data sets were generated for each τtl. Cumulative residence time distributions were global fit to single- or double-exponential models to obtain binding lifetimes and amplitudes of kinetic sub-populations (custom-written MATLAB codes25). Here, the choice of fitting model was decided based on the shape of keffτtl vs. τtl plot (ref. 32 and guidelines from ref. 25), in which the effective decay rate keff represents the sum of the normalized photobleaching rate kb and the off rate koff (keff = kbτintτtl−1 + koff). The linearity of keffτtl vs. τtl plot represents a single kinetic population, whereas deviations from linear fits indicate multiple kinetic sub-populations. The corresponding keffτtl vs. τtl plot was obtained as described previously23, with the shaded error bar representing standard deviations of ten bootstrapped samples deriving from 80% of the compiled binding events (custom-written MATLAB codes). Given that this method is based on fitting mixtures of exponential models to the data, this approach reliably resolves off rates that are at least threefold apart. In experiments involving rif treatment, cells were incubated in growth media containing rif (50 μg per mL) for 30 min in the flow cell prior to imaging.Interval imaging was performed as described previouslyuvrA-YPet and ΔuvrA cells . Following UV irradiation, 5 μL of the cell suspension were taken and serially diluted tenfold, five times. Finally, 50 μL of the 105 dilution were plated in triplicate on LB plates. Plates were then incubated overnight at 37 °C, and colony forming units were counted the next day. Surviving fraction was calculated as the ratio of colonies observed in the irradiated condition to those observed in the un-irradiated condition. Experiments were repeated with two biological repeats, and three technical repeats for each strain and each condition. Plate reader-based UV-survival assays were performed as described previously in refs. 60. Briefly, cells were grown in LB medium to an OD600 between 0.6 and 1 and resuspended in 100 mM MgSO4 following two washes in MgSO4. A 20 μL drop was placed on a quartz plate. UV irradiation  was provided as before using a short-wave UV lamp . Five microliters of the cell suspension were recovered and diluted tenfold, five times in LB. For each condition, sample was plated in triplicate in a flat-bottom 96-well plate (Costar). In addition six wells contained only LB medium to serve as blanks. Data presented reflect mean and standard error of the mean from two biological repeats, each performed in triplicate. UV irradiation in microscopy experiments was delivered in situ as described previously60. The UV flux was measured prior to UV irradiation in all experiments, and the exposure time was adjusted to provide a dose of 20 Jm−2. For experiments involving interval imaging following UV exposure, τtl =  values were used to minimize the time taken to complete one round of interval imaging.UV survival of wild-type, Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary