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BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5(+) cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

This study aimed to reassess the duration of neoadjuvant therapy (NAT) for patients with borderline resectable pancreatic cancer (BRPC). The medical records of patients with BRPC who received NAT before intended curative resection were retrospectively reviewed. Patient demographics, clinicopathological factors, and prognostic factors for overall survival were analyzed. The serum carbohydrate antigen (CA) 19-9 level was examined monthly during NAT. A total of 118 patients with BRPC were enrolled. The median survival time and 5-year overall survival were 28.0 months and 31%, respectively. Three months after NAT, the CA19-9 levels were normal in 57% of the patients, and 92% underwent resection. Multivariate analysis showed that radiological partial response (hazard ratio (HR), 0.53; 95% confidence interval (CI), 0.26–0.99; p = 0.047); a normal CA19-9 level after NAT (HR, 0.30; 95% CI, 0.22–0.66; p = 0.006); and tumor resection (HR, 0.29; 95% CI, 0.13–0.67; p = 0.005) were independent predictors of better survival. The median CA19-9 level and the rate of normal CA19-9 levels before and after NAT were 256 (interquartile range (IQR), 23–1197) U/mL and 33%, and 27 (IQR, 7–176) U/mL and 57%, respectively. A normal CA19-9 level after NAT was an independent predictor of better survival in patients with BRPC. A longer NAT duration might contribute to improved prognosis of patients with elevated CA19-9 levels.

In a systematic study of light charged particle induced nuclear reactions we investigated the excitation functions of deuteron induced nuclear reactions on natural lutetium targets. Experimental excitation functions up to 50 MeV on high purity natLu were determined using the standard stacked foil activation technique. High resolution off-line gamma-ray spectrometry was applied to assess the activity of each foil. From the measured activity direct and/or cumulative elemental cross-section data for production of 171,172,173,175Hf, 171,172,173,174g,176m,177m,177gLu and 169Yb radioisotopes were determined. The experimental data were compared to results of the TALYS theoretical code taken from the TENDL databases and results of our calculations using the ALICE-IPPE-D and the EMPIRE-D codes. No earlier experimental data were found in the literature. Thick target yields for the investigated radionuclides were calculated from the measured excitation functions.

Background The purpose of this study was to determine whether advanced quantitative magnetic resonance imaging (MRI) can be deployed outside of large, research-oriented academic hospitals and into community care settings to predict eventual pathological complete response (pCR) to neoadjuvant therapy (NAT) in patients with locally advanced breast cancer. Methods Patients with stage II/III breast cancer (N = 28) were enrolled in a multicenter study performed in community radiology settings. Dynamic contrast-enhanced (DCE) and diffusion-weighted (DW)-MRI data were acquired at four time points during the course of NAT. Estimates of the vascular perfusion and permeability, as assessed by the volume transfer rate (Ktrans) using the Patlak model, were generated from the DCE-MRI data while estimates of cell density, as assessed by the apparent diffusion coefficient (ADC), were calculated from DW-MRI data. Tumor volume was calculated using semi-automatic segmentation and combined with Ktrans and ADC to yield bulk tumor blood flow and cellularity, respectively. The percent change in quantitative parameters at each MRI scan was calculated and compared to pathological response at the time of surgery. The predictive accuracy of each MRI parameter at different time points was quantified using receiver operating characteristic curves. Results Tumor size and quantitative MRI parameters were similar at baseline between groups that achieved pCR (n = 8) and those that did not (n = 20). Patients achieving a pCR had a larger decline in volume and cellularity than those who did not achieve pCR after one cycle of NAT (p Ktrans, ADC, cellularity, and bulk tumor flow from baseline (pre-treatment) were all significantly greater (p Conclusions Quantitative analysis of DCE-MRI and DW-MRI can be implemented in the community care setting to accurately predict the response of breast cancer to NAT. Dissemination of quantitative MRI into the community setting allows for the incorporation of these parameters into the standard of care and increases the number of clinical community sites able to participate in novel drug trials that require quantitative MRI.

This is a Correspondence submitted to Nature GeoScience following the undermentioned paper: Gabriel Chiodo, Jessica Oehrlein, Lorenzo M. Polvani, John C. Fyfe & Anne K. Smith, Insignificant influence of 11-year solar cycle on the North Atlantic Oscillation, Nature Geoscience, volume 12, pages 94–99 (2019). It is a non-peer reviewed preprint.

This study aimed to explore the prognostic significance of the resection margin (R) status of pancreatic ductal adenocarcinoma (PDAC) patients receiving neoadjuvant therapy (NAT) or adjuvant chemotherapy (AC). We retrospectively reviewed 427 consecutive patients, and the overall survival (OS) and disease-free survival (DFS) were analyzed based on the R status by a propensity score analysis (PSA). The R0 ratio of the NAT (+) group was significantly higher than that of the NAT (−) group (97.2% vs. 69.6%, P

The feasibility of neoadjuvant therapy (NAT) for elderly patients with rectal cancer has not been evaluated well. Between 2004 and 2014, 506 patients with locally advanced low rectal cancer underwent curative resection. Fifty-four were over 75 years old (elderly group), and 452 were under 75 years old (young group). The patients were divided into sub-groups according to whether they received NAT. Nineteen (35.2%) patients from the elderly group and 348 (77.0%) from the young group received NAT. The proportion of patients who received NAT was significantly lower in the elderly group. In the elderly group, the median age and prevalence of co-morbidities were significantly lower in patients with than in those without NAT. The incidence of severe adverse events was similar in the two groups. On multivariate analysis, age was not related to postoperative complications in patients who received NAT. The 5-year local recurrence rate was significantly lower in the elderly patients who received NAT, and similar to that of the young patients who received NAT. Neoadjuvant therapy was feasible and should be considered as a treatment option for carefully selected elderly patients with locally advanced low rectal cancer.

To analyze the risk and reason of false-negative HBV DNA results of NAT reagents among blood donations of China and discuss the necessity of two amplification targets for HBV DNA tests among donations. In this study, samples that showed discordant results on two commercially available assay platforms were further detected by established in-house methods based on conserved regions of the HBV genome. The HBV concentration of these samples was determined using two commercially available reagents. The samples with high titers of HBV were detected by an in-house method. The samples showing high Ct differences between two regions in the in-house method were further sequenced and aligned with primers and probes. The results showed that the established method has a good detection performance. The mismatch between reverse primers and sample sequences led to decreased detection capacity of S and C regions by the in-house method, but it could be compensated by another region. Among the false-negative samples detected by commercial reagents, most were because of low titers; however, there were 7 samples with HBV DNA concentrations much higher than the LOD of the commercial reagents, which may be due to mismatch of the primer or probe. This study highlights the potential risk of HBV false-negative detection by commercial NAT reagents. The dual-target assay may be helpful for HBV screening and reduce the risk of false-negative detection.

Background Hepatitis B virus (HBV) infection is a major concern for blood safety in high-prevalence HBV countries such as China. In Shenzhen, dual hepatitis B surface antigen (HBsAg) enzyme-linked immunosorbent assays (ELISAs) have been adopted in parallel with nucleic acid testing (NAT) for donors for over a decade. A small proportion of blood donors test reactive (R) for HBsAg but negative through routine NAT, which can lead to HBV infection with an extremely low viral load. Objectives We aimed to investigate and analyze the molecular characteristics of HBV among blood donors that tested HBsAg R in a single ELISA test. Methods Blood donations were evaluated in this study if confirmed HBsAg R through one of two ELISA kits. Samples with non-reactive (NR) results by NAT were collected and tested for HBsAg by chemiluminescent microparticle immunoassay (CLIA) with a neutralization test. The level of HBsAg was further assessed by electrochemiluminescence immunoassay (ECLIA). The viral basic core promoter (BCP) and pre-core (PC) and S regions were amplified by nested PCR. Quantitative real-time PCR (qPCR) for viral load determination and individual donation (ID)-NAT were adopted simultaneously. HBsAg was confirmed with CLIA, ECLIA, nested PCR, qPCR, and ID-NAT. Results Of the 100,252 donations, 38 and 41 were identified as HBsAg R with Wantai and DiaSorin ELISA kits, respectively. Seventy-nine (0.077%, 79/100,252) blood samples with ELISA R-NR and NAT NR results were enrolled in the study. Of these, 17 (21.5%,17/79) were confirmed as HBsAg-positive. Of the 14 genotyped cases, 78.6% (11/14) were genotype B, and C and D were observed in two and one sample, respectively. Mutations were found in the S gene, including Y100C, Y103I, G145R, and L175S, which can affect the detection of HBsAg. A high-frequency mutation, T1719G (93.3%), was detected in the BCP/PC region, which reduced the viral replication. Conclusion A small number of blood samples with HBsAg ELISA R-NR and NAT NR results were confirmed as HBV infection, viral nucleic acids were found in most of the samples through routine NAT methods. It is necessary to employ more sensitive and specific assays for the detection of HBV infection among blood donors.

Background Malaria can be transmitted by blood transfusion through donations collected from asymptomatic donors. Transfusion-transmitted malaria (TTM) poses a great risk to blood services worldwide. A good screening tool for Plasmodium spp. detection in blood banks must have a high sensitivity for prevention of TTM. However, in Brazilian blood banks, screening for malaria still relies on microscopy. Methods In Brazil, screening for human immunodeficiency virus type 1 (HIV), RNA/DNA for hepatitis C (HCV) and hepatitis B (HBV) viruses is mandatory for every blood donation and uses nucleic acid amplification testing (NAT). The aim of this study was to evaluate the inclusion of an assay for malaria to identify Plasmodium sp. from total nucleic acid (TNA; DNA/RNA) by targeting the 18S rRNA gene of the parasite. Results Considering the limitations of microscopy and the wide availability of the Brazilian NAT platform in the screening of blood units for HIV, HCV, and HBV, a molecular diagnostic tool was validated for detection of Plasmodium sp. in blood banks; a pilot study showed that using this novel NAT assay could reduce the risk of TTM. Conclusion The prototype HIV/HCV/HBV/malaria NAT assay was effective in detecting infected candidate donors and has good prospects to be applied in routine screening for preventing TTM.

Background Breast cancer clinical management requires the assessment of hormone receptors (estrogen (ER) and progesterone receptor (PR)), human epidermal growth factor receptor 2 (HER2) and cellular proliferation index Ki67, by immunohistochemistry (IHC), in order to choose and guide therapy according to tumor biology. Many studies have reported contradictory results regarding changes in the biomarker profile after neoadjuvant therapy (NAT). Given its clinical implications for the disease management, we aimed to analyze changes in ER, PR, HER2, and Ki67 expression in paired core-needle biopsies and surgical samples in breast cancer patients that had either been treated or not with NAT. Methods We included 139 patients with confirmed diagnosis of invasive ductal breast carcinoma from the Colombian National Cancer Institute. Variation in biomarker profile were assessed according to NAT administration (NAT and no-NAT treated cases) and NAT scheme (hormonal, cytotoxic, cytotoxic + trastuzumab, combined). Chi-squared and Wilcoxon signed-rank test were used to identify changes in biomarker status and percentage expression, respectively, in the corresponding groups. Results We did not find any significant variations in biomarker status or expression values in the no-NAT group. In cases previously treated with NAT, we did find a statistically significant decrease in Ki67 (p and PR (p = 0.02605) expression. When changes were evaluated according to NAT scheme, we found a significant decrease in both Ki67 status (p = 0.02977) and its expression values (p ) in cases that received the cytotoxic treatment. Conclusions Our results suggest that PR and Ki67 expression can be altered by NAT administration, whereas cases not previously treated with NAT do not present IHC biomarker profile variations. The re-evaluation of these two biomarkers after NAT could provide valuable information regarding treatment response and prognosis for breast cancer patients.

Many surgeons advocate the use of neoadjuvant treatment for resectable pancreatic cancer, however little is known about variation in the utilization of neoadjuvant therapy (NAT) at the hospital level.The National Cancer Data Base was used to identify patients undergoing resection for pancreatic cancer between 2006 and 2014 at high-volume centers. Hospitals were grouped by NAT utilization using standard deviations (SD) from the mean as follows: high neoadjuvant utilizers ( 2 SDs above the mean, 40% of patients receiving NAT); medium-high (1-2 SDs, 27-40%), medium (0-1 SD, 14-26%); or low (- 1.1 to 0 SDs, 14%). Overall survival (OS) was compared across NAT utilization groups.Among 107 high-volume centers, 20,119 patients underwent resection. The proportion of patients receiving NAT varied widely among hospitals, ranging from 0 to 74%, with only five centers using NAT in 40% of patients. These five hospitals had the longest median OS at 28.9 months, compared with 21.1 months for low neoadjuvant utilizers (p 0.001). On multivariable analysis, high and medium-high NAT utilization predicted improved OS, with a hazard ratio (HR) of 0.68 (95% confidence interval [CI] 0.56-0.83, p 0.001) and 0.80 (95% CI 0.68-0.95, p = 0.010), respectively, compared with low utilizers. After excluding patients who underwent NAT, there remained an association of improved OS with high NAT utilization (HR 0.74, 95% CI 0.60-0.93, p = 0.009).High-volume hospitals that more commonly utilize NAT demonstrated longer survival for all patients treated at those centers. In addition to altering patient selection for surgery, high neoadjuvant utilization may be a marker of institutional factors that contribute to improved outcomes.

This protocol is an extension to: Nat. Protoc. 10, 1643-1669 (2015); doi:10.1038/nprot.2015.103; published online 01 October 2015RNAs play key roles in many cellular processes. The underlying structure of RNA is an important determinant of how transcripts function, are processed, and interact with RNA-binding proteins and ligands. RNA structure analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) takes advantage of the reactivity of small electrophilic chemical probes that react with the 2'-hydroxyl group to assess RNA structure at nucleotide resolution. When coupled with mutational profiling (MaP), in which modified nucleotides are detected as internal miscodings during reverse transcription and then read out by massively parallel sequencing, SHAPE yields quantitative per-nucleotide measurements of RNA structure. Here, we provide an extension to our previous in vitro SHAPE-MaP protocol with detailed guidance for undertaking and analyzing SHAPE-MaP probing experiments in live cells. The MaP strategy works for both abundant-transcriptome experiments and for cellular RNAs of low to moderate abundance, which are not well examined by whole-transcriptome methods. In-cell SHAPE-MaP, performed in roughly 3 d, can be applied in cell types ranging from bacteria to cultured mammalian cells and is compatible with a variety of structure-probing reagents. We detail several strategies by which in-cell SHAPE-MaP can inform new biological hypotheses and emphasize downstream analyses that reveal sequence or structure motifs important for RNA interactions in cells.

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.

The natAg(n, xn)106m,g,104m,g,105gAg reaction cross-sections within the neutron energies of 14–37 MeV were determined by using an activation and off-line γ-ray spectrometric technique. The quasi-mono energetic neutron beams were produced from the 9Be(p, xn)9B reaction by using the proton beams of 25, 35, and 45 MeV at the Korean Institute of Radiological and Medical Sciences, Korea. Monte Carlo simulation code MCNPX was used to simulate the neutron energy spectra, whereas the theoretical cross-sections were calculated using the TALYS 1.8 code. The experimental and theoretical reaction cross-sections show similar trends from lower to higher neutron energy. The isomeric cross-section ratios of 104m,gAg and 106m,gAg for the natAg(n, xn) reactions were also obtained from the experimental values of present work and compared with the literature data.

The 7th International Workshop on N-Acetyltransferases (NAT), held from 18 to 20 June 2016, was hosted by Brunhilde Blomeke and her team at the Trier University (Germany). The workshop addressed important aspects and latest advancements in the fields of NAT enzymes, endogenous functions of NATs, NAT gene nomenclature, genetic polymorphisms, and their associations with diseases as well as their use in diagnosis. Representatives from the leading teams performing research on NATs presented their excellent work, discussed the latest results, and created new ideas in the field of N-acetyltransferase research.

The objective of present study was to increase solubility and dissolution performance of a poorly water soluble antidiabetic drug, Nateglinide (NAT), through formation of inclusion complexes with hydroxypropyl-beta-cyclodextrin (HP–β–CD). The effect of L-arginine (ARG), an amino acid, on the complexation efficiency and solubility enhancing power of HP–β–CD was investigated by preparing ternary inclusion complexes. The binary and ternary inclusion complexes were prepared by physical mixing, kneading, co-evaporation, and spray drying methods containing NAT, HP–β–CD, and ARG. The complexes were characterized by FTIR, DSC, PXRD, and 1H–NMR. Molecular modeling study revealed that introduction of ternary agent ARG have improved the interactions of NAT and HP–β–CD. The complex prepared by spray drying method showed the highest increase in solubility and dissolution rate compared to other methods. Molecular docking study revealed that ARG interactions plays an essential role in increasing the stability and solubility of the complex. The present study demonstrated increase in solubility and dissolution of NAT. Hence, ternary complexes of NAT can be used as an efficient tool for the delivery of insoluble drug, NAT.

Background Pooled testing, in which biological specimens from multiple subjects are combined into a testing pool and tested via a single test, is a common testing method for both surveillance and screening activities. The sensitivity of pooled testing for various pool sizes is an essential input for surveillance and screening optimization, including testing pool design. However, clinical data on test sensitivity values for different pool sizes are limited, and do not provide a functional relationship between test sensitivity and pool size. We develop a novel methodology to accurately compute the sensitivity of pooled testing, while accounting for viral load progression and pooling dilution. We demonstrate our methodology on the nucleic acid amplification testing (NAT) technology for the human immunodeficiency virus (HIV). Methods Our methodology integrates mathematical models of viral load progression and pooling dilution to derive test sensitivity values for various pool sizes. This methodology derives the conditional test sensitivity, conditioned on the number of infected specimens in a pool, and uses the law of total probability, along with higher dimensional integrals, to derive pooled test sensitivity values. We also develop a highly accurate and easy-to-compute approximation function for pooled test sensitivity of the HIV ULTRIO Plus NAT Assay. We calibrate model parameters using published efficacy data for the HIV ULTRIO Plus NAT Assay, and clinical data on viral RNA load progression in HIV-infected patients, and use this methodology to derive and validate the sensitivity of the HIV ULTRIO Plus Assay for various pool sizes. Results We demonstrate the value of this methodology through optimal testing pool design for HIV prevalence estimation in Sub-Saharan Africa. This case study indicates that the optimal testing pool design is highly efficient, and outperforms a benchmark pool design. Conclusions The proposed methodology accounts for both viral load progression and pooling dilution, and is computationally tractable. We calibrate this model for the HIV ULTRIO Plus NAT Assay, show that it provides highly accurate sensitivity estimates for various pool sizes, and, thus, yields efficient testing pool design for HIV prevalence estimation. Our model is generic, and can be calibrated for other infections. Published version

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates.

Neutron total cross sections of natFe, 197Au and natW have been measured at the n ELBE neutron time-of-flight facility in the energy range 0.15-8MeV with an uncertainty due to counting statistics of up to 2% and a total uncertainty due to systematic effects of 1%. The neutrons are produced with the superconducting electron accelerator ELBE using a liquid lead circuit as photo-neutron target. By periodical sample-in-sample-out measurements the transmission of the sample materials has been determined using a low-threshold plastic scintillation detector. The resulting effective total cross sections show good agreement with previously measured data that cover only part of the energy range available at n ELBE. The results have also been compared to evaluated library files and recent calculations based on a dispersive coupled channel optical model potential.

In this article, we describe the desired attributes to build a system aimed to content distribution in enterprise networks. As a result of this characterisation, we propose several additions to BitTorrent, which is currently used for personal use, so it could efficiently work with large-scale enterprise networks. The modifications are necessary to address NAT, double NAT, and firewalls that are common in enterprise environments. To deal with these problems, the protocol is augmented and also a description of the mechanisms to handle these variations is given. Simulation experiments show that results for this proposed work are much more efficient than BitTorrent since we took advantage of distinctive characteristics in these networks. By efficient, we mean: 1) decrease time to download content, 2) provide support to external hosts (other LANs), while 3) WAN bandwidth is used in the best possible manner.

Background Recent years have seen standardization of the anatomic definitions of pancreatic adenocarcinoma, and increasing utilization of neoadjuvant therapy (NAT). The aim of the current review was to summarize the evidence for NAT in pancreatic adenocarcinoma since 2009, when consensus criteria for resectable (R), borderline resectable (BR), and locally advanced (LA) disease were endorsed. Methods PubMed search was undertaken along with extensive backward search of the references of published articles to identify studies utilizing NAT for pancreatic adenocarcinoma. Abstracts from ASCO-GI 2014 and 2015 were also searched. Results A total of 96 studies including 5520 patients were included in the final quantitative synthesis. Pooled estimates revealed 36% grade ≥ 3 toxicities, 5% biliary complications, 21% hospitalization rate and low mortality (0%, range 0–16%) during NAT. The majority of patients (59%) had stable disease. On an intention-to-treat basis, R0-resection rates varied from 63% among R patients to 23% among LA patients. R0 rates were > 80% among all patients who were resected after NAT. Among R and BR patients who underwent resection after NAT, median OS was 30 and 27.4 months, respectively. Conclusions The current study summarizes the recent literature for NAT in pancreatic adenocarcinoma and demonstrates improving outcomes after NAT compared to those historically associated with a surgery-first approach for pancreatic adenocarcinoma. Electronic supplementary material The online version of this article (10.1186/s12957-017-1240-2) contains supplementary material, which is available to authorized users.

To investigate the effects of berberine (BBR) and cinnamic acid (CA), the main active components in Jiaotai Pill (, JTP), on palmitic acid (PA)-induced intracellular triglyceride (TG) accumulation in NIT-1 pancreatic β cells.Cells were incubated in culture medium containing PA (0.25 mmol/L) for 24 h. Then treatments with BBR (10 μmol/L), CA (100 μmol/L) and the combination of BBR and CA (BBR+CA) were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase (AMPK) protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), phosphorylation acetyl-coA carboxylase (pACC), carnitine acyl transferase 1 (CPT-1) and sterol regulating element binding protein 1c (SREBP-1c) were determined by Western blot or real time polymerase chain reaction.PA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-1. Meanwhile, AMPK downstream lipogenic genes including SREBP-1c mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR+CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic β cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study.It can be concluded that in vitro, BBR and BBR+CA could inhibit PA-induced lipid accumulation by decreasing lipogenesis and increasing lipid oxidation in NIT-1 pancreatic β cells.

NMR experiments on proteins in simultaneous equilibria with multiple binding partners can provide a tool to understand complex biological interaction networks. Competition among proteins for binding to signaling hubs is often at the basis of the information transmission across signaling networks in every organism. Changes in affinity towards one or more partners, as well as changes of the relative concentration of the competing partners, can determine pathways alterations that lead to pathological consequences. Overall, the knowledge of the interaction hierarchy of the multiple partners to a single signaling hub can lead to new therapeutic strategies. Smith and Ikura (Nat Chem Biol 10:223–230, 2014) have recently proposed pairwise competition NMR experiments to determine the binding hierarchy in network interactions. We have taken the moves from their approach to show how from pairwise competition NMR experiments the ratios between the equilibrium constants for multiple binding partners can be determined, and thus, given their concentration in solution, the concentrations of all the possible complexes can be obtained.

In Fig. 1b of this Article, the authors mistakenly republished a blot previously published as Fig. 1d in their article in Nature Structural & Molecular Biology (Nat. Struct. Mol. Biol. 12, 589-593; 2005).

Nat. Methods 14, 695–698 (2017); published online 15 May 2017; corrected after print 28 February 2018 We were alerted by readers that the reported Nm consensus sequence in mRNA matches the 3′-adaptor sequence used in sequencing library preparations, and this could be caused by mispriming1. In our approach, the majority of RNA fragments without Nm at the 3′ end are blocked from ligating to the 3′ adaptor because of the presence of 3′ phosphate from the last oxidation elimination step (OE) (Fig. 1a of the original paper), while Nm sites accumulate at the 3′ ends (Supplementary Figs. 1 and 2; Supplementary Notes 1 and 2). However, because of the low Nm abundance in messenger RNA (mRNA), only very limited amounts of mRNA fragments carry 3′ Nm and thus can be successfully ligated to the 3′ adaptor. Mispriming could occur if the 3′ end of the reverse transcription (RT) primer hybridizes to a few bases of the 5′-ligated RNA (Fig. 1a). Although our method effectively identifies Nm sites in abundant ribosomal RNA (rRNA, Supplementary Fig. 1), its application to less abundant mRNA can be contaminated by mispriming, leading to false-positive Nm sites and the erroneous AGAUC motif on mRNA (original Fig. 2d), which corresponds to the 5′-end sequence of the 3′ adaptor.

Blood transfusion is a life-saving approach, although it carries the risk of transfusion-transmitted viruses (TTVs). In Saudi Arabia (SA), nucleic acid amplification test (NAT) has been obligatory for HCV, HBV, and HIV by 2008. Our aim is to evaluate the effectiveness of NAT for detection of TTVs in the blood donated at Saudi German Hospitals (SGH) in SA. A total of 12,437 donor samples from SGH between January 2009 and June 2011 were subjected to routine serologic tests for HBV, HCV, and HIV followed by minipool NAT using Cobas Amplicor analyzer (Roche Diagnostics) for the seronegative samples. Out of 12,032 seronegative plasmas, five (0.042 %) were positive for HCV and two (0.017 %) for HIV-1 by NAT. None of our seronegative donors were NAT positive for HBV, highlighting the value of hepatitis B core antibody detection in donor testing. Our results provide evidence to support the effectiveness of NAT screening to detect TTVs in blood donations.

Malassezia commensal yeasts along with multitude of antigens have been found to be associated with various skin disorders including Pityriasis versicolor (PV). Amongst them Mala s1, a 37 kDa protein has been proved to be a major allergen reacting with a large panel of sera. However, there exists no therapeutic alternative to combat such problems in form of plant based natural compounds. The purpose of this study is in the first place, to determine the anti-Malassezia activity of Nyctanthes arbor-tristis L. (NAT) ethanolic leaf extract through turbidimetric growth curves, disruption of plasma membrane and secondly, it aims to present in silico validation of its active constituents over Mala s1a novel allergen. The antifungal susceptibility 50 % ethanolic extract of NAT was determined by broth microdilution method according to CLSI guidelines. Further MICs and IC50 were determined spectrophotometrically using the software SoftMax® Pro-5 (Molecular Devices, USA). Active constituents mediated disruption of plasma membrane was studied through flowcytometry by permeabilization of fluorescent dye Propidium Iodide (PI). Antioxidant activity of the extract was determined using the DPPH stable radical. Molecular validation of fungal DNA from the extract was observed using PCR amplification. In silico analysis of its active constituents over Mala s1 was performed using HEX software and visualized through Pymol. The anti-Malassezia potential of NAT leaf extracts reflected moderate MIC 1.05 μg/μl against M. globosa, while least effective against M. restricta with MIC 1.47 μg/μl. A linear correlation coefficient R 2 = 0.866 was obtained in case of M. globosa while minimum was observed in M. restricta with R 2 = 0.732. The flow cytometric data reveal ~ 75 % cell death when treated with active constituents β-Sitosterol and Calceolarioside A. The docking confirmations and the interaction energies between Mala s1 and the active constituents (β-Sitosterol and Calceolarioside A) from extracts showed an effective binding which suggests Mala s1 as efficient allergen for site specific targeting. This study revealed that Nyctanthes arbor-tristis L. (NAT) extracts possess high anti-Malassezia potential which is driven mainly by disruption of plasma membrane. Also in silico validation and molecular modeling studies establishes Mala s1 as a novel allergen that could be a potential target in disease treatment. Our results would also provide a foundation for the development of new therapeutic approach using NAT extract as lead compound with high antioxidant property as an added trait for skin care.

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPRdn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.

End hopping is one of the good methods to defend against network attack, but has problems with network address translation (NAT) because packets sent from an unknown endpoint would be dropped by NAT. To avoid the dropping of packets, we propose a punching scheme: a client sends a punching packet to create mapping rules in NAT, so that the packets from the server would be able to pass through effectively with such rules. In this paper, some preliminaries and definitions are provided for building the model of end hopping. Then we discuss the main reason of such packet dropping and specify all the failure situations based on the model. What’s more, we analyze how the punching scheme helps end hopping cross NAT. Finally, we validate the feasibility of this scheme with empirical results: if the client is behind a NAT and with punching scheme, the service rate increases to 100%. Therefore, our proposed scheme can greatly improve the performance of crossing NAT in end hopping with little security and computational overhead.

Nat. Methods 14, 547–548 (2017); published online 30 May 2017; updated online 14 June 2017; corrected online 25 July 2017; retracted 30 March 2018 This paper is being retracted because the genomic variants observed by the authors in two CRISPR-treated mice cannot be conclusively attributed to CRISPR–Cas9.

Since serological donor-screening tests for HIV were introduced in 1985, the safety of donated blood components has improved dramatically. However, these tests do not completely prevent the risk of transfusion-associated HIV infection related to the use of blood donated during the pre-seroconversion window period. Testing based on nucleic acid amplification is being implemented to screen for HIV-infected blood donated during this period, which has reduced the probability of transmitting HIV through transfusion by shortening the window period. This article describes a case of acute HIV-1 infection, detected using a nucleic acid amplification test (NAT) in a repeat blood donor who donated during the pre-seroconversion window period and whose antigen and anti-HIV antibody expression was observed after molecular marker detection. In addition, the possible route of infection is discussed based on the patient's history, and finally, the need for NAT technology for blood donor screening is emphasized.

Whole-cell, patch-clamp recordings were carried out in acutely dissociated neurons from entorhinal cortex (EC) layer II to study the effects of Zn2+ on Na+ current kinetics and voltage dependence. In the presence of 200 μM extracellular Cd2+ to abolish voltage-dependent Ca2+ currents, and 100 mM extracellular Na+, 1 mM Zn2+ inhibited the transient Na+ current, I NaT, only to a modest degree (~17% on average). A more pronounced inhibition (~36%) was induced by Zn2+ when extracellular Na+ was lowered to 40 mM. Zn2+ also proved to modify I NaT voltage-dependent and kinetic properties in multiple ways. Zn2+ (1 mM) shifted the voltage dependence of I NaT activation and that of I NaT onset speed in the positive direction by ~5 mV. The voltage dependence of I NaT steady-state inactivation and that of I NaT inactivation kinetics were markedly less affected by Zn2+. By contrast, I NaT deactivation speed was prominently accelerated, and its voltage dependence was shifted by a significantly greater amount (~8 mV on average) than that of I NaT activation. In addition, the kinetics of I NaT recovery from inactivation were significantly slowed by Zn2+. Zn2+ inhibition of I NaT showed no signs of voltage dependence over the explored membrane-voltage window, indicating that the above effects cannot be explained by voltage dependence of Zn2+-induced channel-pore block. These findings suggest that the multiple, voltage-dependent state transitions that the Na+ channel undergoes through its activation path are differentially sensitive to the gating-modifying effects of Zn2+, thus resulting in differential modifications of the macroscopic current’s activation, inactivation, and deactivation. Computer modeling provided support to this hypothesis.

Natalizumab (NAT) is an effective therapy for relapsing-remitting multiple sclerosis (MS), but is associated with an increased risk of progressive multifocal leucoencephalopathy after 2 years therapy. Thus, NAT treated patients often decide to stop NAT therapy after 2 years. Reports on recurrence of disease activity after NAT discontinuation are controversial. We studied disease activity in 13 MS patients who stopped NAT therapy and either remained without disease modifying therapy (no DMT, n = 6), or switched to glatiramer acetate (GLAT, n = 7). Annual relapse rate (ARR), expanded disability status scale (EDSS), and number of patients with contrast-enhancing-lesions (Gd+) on MRI before, during and within 1 year after NAT were determined. We observed recurrence of disease activity in both groups (5/7 GLAT treated patients and 6/6 patients without DMT) within 12 months after cessation of NAT (mean time to first relapse was 5.5 months for all patients). One of the GLAT treated patients and three patients without DMT had severe relapses with sustained EDSS worsening. No differences in ARR, EDSS and MRI parameters were seen between both groups. Patients with relapses after NAT therapy, however, tended to show higher disease activity (EDSS, ARR) before initiation of NAT therapy compared to patients without relapses. Duration of NAT treatment was not associated with higher disease activity after NAT discontinuation. In this observation the majority of patients showed reappearance of disease activity after discontinuation of NAT regardless of whether they switched to GLAT or remained without DMT. Further treatment strategies are warranted for patients who discontinue NAT therapy.

In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0017-6, 2018) and a Reply by the authors of the Protocol (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0018-5, 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced.

Nat. Methods 15, 207–212 (2018); published online 5 February 2018; corrected after print 20 April 2018 In the version of this Article originally published, an amino acid substitution introduced into the B. subtilis biotin ligase sequence was incorrectly written as R142G. The correct mutation is R124G.

Nat. Methods 14, 547–548 (2017); published online 30 May 2017; updated online 14 June 2017; corrected online 25 July 2017; retracted 30 March 2018 This paper is being retracted because the genomic variants observed by the authors in two CRISPR-treated mice cannot be conclusively attributed to CRISPR–Cas9.

Nat. Methods 14, 607–614 (2017); published online 1 May 2017; corrected after print 20 April 2018 In the version of this Article originally published, the wrong Protocol Exchange DOI and link (10.1038/protex.2017.047) were included in ref. 40. The URL in the reference should have been http://dx.doi.org/10.

There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury, either from cancer therapy or accidental exposure1,2. In addition to their role in promoting sexual dimorphisms, increasing evidence suggests that sex hormones regulate HSC self-renewal, differentiation, and proliferation3–5. We and others previously reported that sex steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice5–7. Here we show that a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant), currently used widely in the clinic for sex steroid inhibition, promoted hematopoietic recovery and mouse survival when administered 24 h after an otherwise lethal dose of total body irradiation (L-TBI). Unexpectedly, this protective effect was independent of sex steroids, but instead relied on suppression of luteinizing hormone (LH) levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the luteinizing hormone/choriogonadotropin receptor (LHCGR) and expand ex vitro when stimulated with LH. In contrast, suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting quiescence of HSCs and protecting them from exhaustion. These findings reveal a role for LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after injury.

Nat. Med. 22, 771–779 (2016); published online 6 June 2016; corrected after print 7 March 2018 In the version of this article initially published, the colors of the lines were switched in the graph that shows the glucose infusion rates for wild-type mice and Adipoq-Tst transgenic mice in Figure 3b. The top line should be purple, and the bottom line should be black.

Nat. Methods 15, 17 (2018); published online 3 Jan 2018; corrected after print 7 February 2018 In the version of this article initially published, the affiliation of Leo James was incorrectly stated as Cambridge University; the correct affiliation is MRC Laboratory of Molecular Biology, Cambridge. Furthermore, the antibody receptor described was incorrectly called TRIM2; the correct name is TRIM21.

Nat. Methods 13, 515–520 (2016); published online 25 April 2016; corrected after print 7 February 2018 In the version of this article initially published, an important funding source, the Agence National de Recherche (ANR-10-BINF-0003 BIP:BIP to M.N.), was omitted. The error has been corrected in the HTML and PDF versions of the article.

Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products.

Serologic testing for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV core antigen (anti-HBc) has historically been the foundation of blood screening, while HBV nucleic acid testing (NAT) was recently developed to detect HBsAg-negative, anti-HBc-negative blood units donated during early acute infection. Comparison data on seroconversion panels using HBsAg assays of varying sensitivities and pooled- or single-sample NAT, along with viral load estimates corresponding to HBsAg assay detection limits, have provided information on the theoretical benefits of NAT relative to HBsAg. Model-derived estimates have generally been predictive of the yields of DNA-positive, HBsAg-negative window period blood units detected in a number of studies from Europe, Japan, and the US. Studies indicate that the added benefit of pooled-sample NAT is relatively small in areas of low endemicity, with greater yields in areas highly endemic for HBV. Single-sample NAT would offer more significant early window period closure and could prevent a moderate number of residual HBV transmissions not detected by HBsAg assays; however, no fully automated single-sample HBV NAT systems are currently available. Even single-sample HBV NAT may not substitute for anti-HBc screening, as indicated by studies of donors with isolated anti-HBc who have extremely low DNA levels undetectable by standard single-sample NAT and who have been associated with transfusion-transmitted HBV. Moreover, HBsAg testing may still be needed even in the setting of combined anti-HBc and NAT screening. HBsAg-positive units from donors in the chronic stage of infection may contain very low or intermittently detectable DNA levels that single-sample NAT would miss. Although such donors are usually anti-HBc reactive and would be interdicted by anti-HBc screening, some lack anti-HBc. Extensive parallel testing will be needed to determine whether single-sample NAT in combination with anti-HBc might be sufficient to detect all the infectious donors currently interdicted by HBsAg testing. In countries that do not screen for anti-HBc, HBsAg testing would be the only means of detecting donations from chronically infected individuals with low/intermittently detectable DNA, since even single-donor NAT would not identify these potentially infectious blood units. In the future, the current fully automated HBsAg assays may incorporate significant sensitivity improvements, and automated single-sample HBV NAT may become a reality. Each country will need to develop its blood screening strategy based on HBV endemicity, yields of infectious units detected by different serologic/NAT screening methods, and cost effectiveness of test methods in ensuring blood safety.

Nucleic acid testing (NAT) has reduced the risk of transmitting infectious disease through blood transfusion. Currently NAT for HIV-1 and HCV are FDA licensed and performed by nearly all blood collection facilities, but HBV NAT is performed under an investigational study protocol. Residual risk estimates indicate that NAT could potentially reduce disease transmission through transplanted tissue. However, tissue donor samples obtained post-mortem have the potential to produce an invalid NAT result due to inhibition of amplification reactions by hemolysis and other factors. The studies reported here summarize the development of protocols to allow NAT of deceased donor samples with reduced rates of invalid results. Using these protocols, inventories from two tissue centers were tested with greater than 99% of samples producing a valid test result.

The impact of neoadjuvant therapy (NAT) for rectal cancer on lymph node yield is not well known. This study evaluates the impact of NAT on tumor regression and lymph node harvest. The subjects were 40 patients with rectal cancer; 20 receiving high-dose, long-course neoadjuvant therapy, and 20 age- and sex-matched controls who did not receive neoadjuvant therapy. Tumor regression (TRG) was graded from 1 to 5 as: TRG1, no residual tumor cells; TRG2, occasional residual tumor cells with marked fibrosis; TRG3, marked fibrosis with scattered tumor cells or groups; TRG4, abundant cancer cells with little fibrosis; TRG5, no tumor regression. We also evaluated the number of lymph nodes retrieved from excised specimens, the size of the largest node, and the extent of lymph node involvement by the tumor. Tumor regression was seen in all patients; as TRG1 in 6 (30%), TRG2 in 2 (10%), TRG3 in 3 (15%), and TRG4 in 9 (45%). The median nodal harvest was 4 (range (0–12) in the NAT group vs 9 (range 1–19) in the control (P = 0.001). The median size of the largest lymph node was 5 mm (range 2–12 mm) in the NAT group vs 9 mm (range 4–15 mm) in the control group (P = 0.004). Tumor-positive nodes were identified in 4 of 17 of the NAT group patients and in 9 of the 20 controls (P = 0.308). Although NAT down-stages rectal cancer, it results in a significantly low yield of lymph nodes, which are also significantly smaller than those in nonirradiated controls. Therefore, surgeons and histopathologists must ensure adequate sampling and accurate staging is done for patients with irradiated rectal cancer.

High-performance liquid chromatography with mass spectrometric detection was used for the structure elucidation of eighteen primary and secondary amines and ammonia derivatised with naphthylisothiocyanate (NIT). A fragmentation scheme was established using reference compounds and the scheme was applied to real air samples from a tyre repair shop and from the air above a bacterial culture. The sampling was performed using a solid sorbent, XAD-2, impregnated with NIT, and the derivatives were extracted with acetonitrile and analysed with LC-MS/MS. A three-step process was developed for screening and identifying of volatile amines. The first step, selected reaction monitoring; SRM was applied in order to screen the samples for NIT derivatives. In the second step, a precursor ion scan gave the [M+H](+) ion, and in the third step a product ion scan gave the fragments needed for identification. The detection limits varied between 0.12 and 0.25 ng microL(-1) when screening for unknown derivatised amines. It was possible to separate and identify all the amines with the structural information obtained and the method proved to be general, sensitive and well suited for sampling and analysis of complex environmental samples.

An in vitro domainal shuffling strategy for protein evolution was proposed in (J. Kolkman and W. Stemmer, Nat. Biotech. 19 (423) 2001). Due to backhybridization, however this method appears unlikely to be an efficient means of iteratively generating massive libraries of combinatorially shuffled genes. Recombination at the domain level (30–300 residues) also appears too coarse to support the evolution of proteins with substantially new folds. In this work, the module (10–25 residues long) and pseudomodule are adopted as the fundamental units of protein structure. Each protein is modelled as an N to C-terminal tour of a digraph composed of pseudomodules. An in vitro method based on PNA-mediated Whiplash PCR (PWPCR), RNA-protein fusion, and restriction-based recombination, XWPCR is then presented for evolving proteins with a high affinity for a given motif, subject to the constraint that each corresponds to a walk on the pseudomodule digraph of interest. Simulations predict that PWPCR is an efficient method of producing massive, shuffled gene libraries encoding for proteins as long as roughly 600 residues.

NAT— from bugs to brains An overview of the 2nd International Workshop on the arylamine N-acetyltransferases

Nat. Biotechnol. 32, 364–372 (2014); published online 16 March 2014; corrected after print 8 November 2017 In the version of this article initially published, the cells labeled Me290 were Me275 cells. Both Me275 and Me290 are human metastatic HLA-A201+ melanoma cell lines, and both of them were obtained from the Ludwig Cancer Institute.