Organic synthesis: march of the machines.

Organic synthesis is changing; in a world where budgets are constrained and the environmental impacts of practice are scrutinized, it is increasingly recognized that the efficient use of human resource is just as important as material use. New technologies and machines have found use as methods for transforming the way we work, addressing these issues encountered in research laboratories by enabling chemists to adopt a more holistic systems approach in their work. Modern developments in this area promote a multi-disciplinary approach and work is more efficient as a result. This Review focuses on the concepts, procedures and methods that have far-reaching implications in the chemistry world. Technologies have been grouped as topics of opportunity and their recent applications in innovative research laboratories are described.The authors gratefully acknowledge support from UK Engineering and Physical Sciences Research Council (SVL and RMM), Woolf Fisher Trust (DEF) and Pfizer Worldwide Research and Development (CB, RJI).This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1002/anie.201410744/abstract

Machine-Assisted Organic Synthesis.

In this Review we describe how the advent of machines is impacting on organic synthesis programs, with particular emphasis on the practical issues associated with the design of chemical reactors. In the rapidly changing, multivariant environment of the research laboratory, equipment needs to be modular to accommodate high and low temperatures and pressures, enzymes, multiphase systems, slurries, gases, and organometallic compounds. Additional technologies have been developed to facilitate more specialized reaction techniques such as electrochemical and photochemical methods. All of these areas create both opportunities and challenges during adoption as enabling technologies

Association of Serum Antibody Levels Following Vaccination with A Modified Live BVDV Vaccine and Protection from Clinical Disease upon Challenge

Two studies were conducted to examine the range of virus neutralizing serum antibody (VNSA) response after vaccination with a modified-live viral vaccine in cattle and the level of protection elicited when subsequently challenged with a highly virulent type 2 BVDV. Study 1 examined responses in colostrum deprived (CD) calves with no passively acquired antibodies whereas Study 2 examined responses in conventionally raised (CR) calves which had varying levels of passive antibodies prior to vaccination. Study 1 used CD calves averaging 120 days of age. For Study 2, calves averaged 130 days of age and were stratified into CR-Low, Mid and High response groups based response to vaccination. Grouping was based on standard deviations from the mean value obtained from ELISA assay and reconfirmed by virus neutralization. Twelve calves, from each Rep in Study 2, were selected to represent low, mid and high response groups (n = 4 calves per group). The VNSA values for the three groups were as follows; CR-Low (titer \u3c 1:4), CR-Mid (titer 1:4 to 1:16) and CR-High (titer \u3e 1:16). Calves were challenged with a high virulence BVDV2 strain. Samples were collected for both studies on days -2, 2, 4, 6, 9, 11 and 13 post challenge to determine levels of circulating white blood cells, virus isolation and levels of BVDV VNSA. While, overt respiratory or enteric disease was not observed in any of the vaccinated calves, clinical symptoms were more likely to be observed in calves with lower BVDV VNSA levels following the BVDV challenge demonstrating significant association between VNSA levels and clinical protection. However, the extent of clinical symptoms including decreases in WBC and pyrexia, were more severe in the non-vaccinated animals which indicates vaccination did provide a level of protection

The rapid synthesis of oxazolines and their heterogeneous oxidation to oxazoles under flow conditions.

A rapid flow synthesis of oxazolines and their oxidation to the corresponding oxazoles is reported. The oxazolines are prepared at room temperature in a stereospecific manner, with inversion of stereochemistry, from β-hydroxy amides using Deoxo-Fluor®. The corresponding oxazoles can then be obtained via a packed reactor containing commercial manganese dioxide.We are grateful to the Swiss National Science Foundation (DNT), the Ralph Raphael fellowship (RJI), the German Academic Exchange Service DAAD (SF), the Royal Society Newton International Fellowship (ZEW), Pfizer Worldwide Research and Development (CB) and the EPSRC (SVL, SF and ZEW, grant nº EP/K0099494/1) for financial support. The Labtrix Start was kindly loaned by Chemtrix BV and the liquid-liquid phase separator by Zaiput Flow Technologies (we are grateful to Dr Andrea Adamo for technical support with this device).This is the final version. It was first published by the Royal Society of Chemistry at http://dx.doi.org/10.1039/C4OB02105

Cyclopropanation using flow-generated diazo compounds.

We have devised a room temperature process for the cyclopropanation of electron-poor olefins using unstabilised diazo compounds, generated under continuous flow conditions. This protocol was applied to a wide range of different diazo species to generate functionalised cyclopropanes which are valuable 3D building blocks.We are grateful to Pfizer Worldwide Research and Development (CB, RJI and JMH), the Swiss National Science Foundation (DNT), CAPES (RL, no 9865/13-6) and the EPSRC (SVL, grant no EP/K0099494/1 and no EP/K039520/1) for financial support.This is the final published article, originally published in Organic & Biomolecular Chemistry, 2015,13, 2550-2554 DOI: 10.1039/C5OB00019

Visualizing tumor evolution with the fishplot package for R

BACKGROUND: Massively-parallel sequencing at depth is now enabling tumor heterogeneity and evolution to be characterized in unprecedented detail. Tracking these changes in clonal architecture often provides insight into therapeutic response and resistance. In complex cases involving multiple timepoints, standard visualizations, such as scatterplots, can be difficult to interpret. Current data visualization methods are also typically manual and laborious, and often only approximate subclonal fractions. RESULTS: We have developed an R package that accurately and intuitively displays changes in clonal structure over time. It requires simple input data and produces illustrative and easy-to-interpret graphs suitable for diagnosis, presentation, and publication. CONCLUSIONS: The simplicity, power, and flexibility of this tool make it valuable for visualizing tumor evolution, and it has potential utility in both research and clinical settings. The fishplot package is available at https://github.com/chrisamiller/fishplot. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-3195-z) contains supplementary material, which is available to authorized users

Genetic heterogeneity of induced pluripotent stem cells: results from 24 clones derived from a single C57BL/6 mouse.

This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.0120585Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs "captures" the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically "fit" for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0-45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40-60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.This work was supported by grants from the NIH (CA101937 and CA162086, to TJL, and HL116605, to JMK), the Barnes Jewish Hospital Foundation (00335-0505-02, to TJL), and the Burroughs Wellcome Fund (to JMK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test’s single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn’t differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn’t differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1–0.4]; Method 3: 0.3U/gHb [IQR: 0.1–0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2–0.6]; Method 3: 0.3U/gHb [IQR: 0.1–0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn’t improve repeatability. Delays of up to 5 hours didn’t result in a clinically relevant difference in measured G6PD activity. The manufacturer’s recommended cut-off for intermediate deficiency is conservative

Pirfenidone in idiopathic pulmonary fibrosis:expert panel discussion on the management of drug-related adverse events

Pirfenidone is currently the only approved therapy for idiopathic pulmonary fibrosis, following studies demonstrating that treatment reduces the decline in lung function and improves progression-free survival. Although generally well tolerated, a minority of patients discontinue therapy due to gastrointestinal and skin-related adverse events (AEs). This review summarizes recommendations based on existing guidelines, research evidence, and consensus opinions of expert authors, with the aim of providing practicing physicians with the specific clinical information needed to educate the patient and better manage pirfenidone-related AEs with continued pirfenidone treatment. The main recommendations to help prevent and/or mitigate gastrointestinal and skin-related AEs include taking pirfenidone during (or after) a meal, avoiding sun exposure, wearing protective clothing, and applying a broad-spectrum sunscreen with high ultraviolet (UV) A and UVB protection. These measures can help optimize AE management, which is key to maintaining patients on an optimal treatment dose.Correction in: Advances in Therapy, Volume 31, Issue 5, pp 575-576 , doi: 10.1007/s12325-014-0118-8</p

BreakTrans: Uncovering the genomic architecture of gene fusions

Producing gene fusions through genomic structural rearrangements is a major mechanism for tumor evolution. Therefore, accurately detecting gene fusions and the originating rearrangements is of great importance for personalized cancer diagnosis and targeted therapy. We present a tool, BreakTrans, that systematically maps predicted gene fusions to structural rearrangements. Thus, BreakTrans not only validates both types of predictions, but also provides mechanistic interpretations. BreakTrans effectively validates known fusions and discovers novel events in a breast cancer cell line. Applying BreakTrans to 43 breast cancer samples in The Cancer Genome Atlas identifies 90 genomically validated gene fusions. BreakTrans is available at http://bioinformatics.mdanderson.org/main/BreakTran