Digital PCR provides sensitive and absolute calibration for high throughput sequencing

<p>Abstract</p> <p>Background</p> <p>Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.</p> <p>Results</p> <p>We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.</p> <p>Conclusion</p> <p>The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.</p

Nut production in Bertholletia excelsa across a logged forest mosaic: implications for multiple forest use

Although many examples of multiple-use forest management may be found in tropical smallholder systems, few studies provide empirical support for the integration of selective timber harvesting with non-timber forest product (NTFP) extraction. Brazil nut (Bertholletia excelsa, Lecythidaceae) is one of the world’s most economically-important NTFP species extracted almost entirely from natural forests across the Amazon Basin. An obligate out-crosser, Brazil nut flowers are pollinated by large-bodied bees, a process resulting in a hard round fruit that takes up to 14 months to mature. As many smallholders turn to the financial security provided by timber, Brazil nut fruits are increasingly being harvested in logged forests. We tested the influence of tree and stand-level covariates (distance to nearest cut stump and local logging intensity) on total nut production at the individual tree level in five recently logged Brazil nut concessions covering about 4000 ha of forest in Madre de Dios, Peru. Our field team accompanied Brazil nut harvesters during the traditional harvest period (January-April 2012 and January-April 2013) in order to collect data on fruit production. Three hundred and ninety-nine (approximately 80%) of the 499 trees included in this study were at least 100 m from the nearest cut stump, suggesting that concessionaires avoid logging near adult Brazil nut trees. Yet even for those trees on the edge of logging gaps, distance to nearest cut stump and local logging intensity did not have a statistically significant influence on Brazil nut production at the applied logging intensities (typically 1–2 timber trees removed per ha). In one concession where at least 4 trees ha-1 were removed, however, the logging intensity covariate resulted in a marginally significant (0.09) P value, highlighting a potential risk for a drop in nut production at higher intensities. While we do not suggest that logging activities should be completely avoided in Brazil nut rich forests, when a buffer zone cannot be observed, low logging intensities should be implemented. The sustainability of this integrated management system will ultimately depend on a complex series of socioeconomic and ecological interactions. Yet we submit that our study provides an important initial step in understanding the compatibility of timber harvesting with a high value NTFP, potentially allowing for diversification of forest use strategies in Amazonian Perù

Demonstration of an ultracold micro-optomechanical oscillator in a cryogenic cavity

Preparing and manipulating quantum states of mechanical resonators is a highly interdisciplinary undertaking that now receives enormous interest for its far-reaching potential in fundamental and applied science. Up to now, only nanoscale mechanical devices achieved operation close to the quantum regime. We report a new micro-optomechanical resonator that is laser cooled to a level of 30 thermal quanta. This is equivalent to the best nanomechanical devices, however, with a mass more than four orders of magnitude larger (43 ng versus 1 pg) and at more than two orders of magnitude higher environment temperature (5 K versus 30 mK). Despite the large laser-added cooling factor of 4,000 and the cryogenic environment, our cooling performance is not limited by residual absorption effects. These results pave the way for the preparation of 100-um scale objects in the quantum regime. Possible applications range from quantum-limited optomechanical sensing devices to macroscopic tests of quantum physics.Comment: Published versio

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3′,4′,5′-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3′,4′,5′-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3′,4′,5′-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5–20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug

Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer

Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis

Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease

Developing an agenda for research about policies to improve access to healthy foods in rural communities: a concept mapping study

Background Policies that improve access to healthy, affordable foods may improve population health and reduce health disparities. In the United States most food access policy research focuses on urban communities even though residents of rural communities face disproportionately higher risk for nutrition-related chronic diseases compared to residents of urban communities. The purpose of this study was to (1) identify the factors associated with access to healthy, affordable food in rural communities in the United States; and (2) prioritize a meaningful and feasible rural food policy research agenda. Methods This study was conducted by the Rural Food Access Workgroup (RFAWG), a workgroup facilitated by the Nutrition and Obesity Policy Research and Evaluation Network. A national sample of academic and non-academic researchers, public health and cooperative extension practitioners, and other experts who focus on rural food access and economic development was invited to complete a concept mapping process that included brainstorming the factors that are associated with rural food access, sorting and organizing the factors into similar domains, and rating the importance of policies and research to address these factors. As a last step, RFAWG members convened to interpret the data and establish research recommendations. Results Seventy-five participants in the brainstorming exercise represented the following sectors: non-extension research (n = 27), non-extension program administration (n = 18), “other� (n = 14), policy advocacy (n = 10), and cooperative extension service (n = 6). The brainstorming exercise generated 90 distinct statements about factors associated with rural food access in the United States; these were sorted into 5 clusters. Go Zones were established for the factors that were rated highly as both a priority policy target and a priority for research. The highest ranked policy and research priorities include strategies designed to build economic viability in rural communities, improve access to federal food and nutrition assistance programs, improve food retail systems, and increase the personal food production capacity of rural residents. Respondents also prioritized the development of valid and reliable research methodologies to measure variables associated with rural food access. Conclusions This collaborative, trans-disciplinary, participatory process, created a map to guide and prioritize research about polices to improve healthy, affordable food access in rural communities

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: a report of the international immuno-oncology biomarker working group

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland