In Situ Rheological Method to Evaluate Feedstock Physical Properties Throughout Enzymatic Deconstruction

Feedstock physical properties determine not only downstream flow behavior, but also downstream process yields. Enzymatic treatment of pretreated feedstocks is greatly dependent on upstream feedstock physical properties and choice of pre-processing Technologies. Currently available enzyme assays have been developed to study biomass slurries at low concentrations of ≤ 1% w/w. At commercially relevant biomass concentrations of ≥15% w/w, pretreated feedstocks have sludge-like properties, where low free water restricts movement of unattached enzymes. This work is an account of the various steps taken to develop a method that helps identify the time needed for solid-like biomass slurries transition into liquid-like states during enzymatic hydrolysis. A pre-processing technology that enables feedstocks in achieving this transition sooner will greatly benefit enzyme kinetics and thereby overall process economics. Through this in situ rheological properties determining method, we compared a model feedstock, Avicel®PH101 cellulose, with acid pretreated corn stover. Novozymes Cellic®CTec2 (80 mg protein/g glucan) can reduce 25% (w/w) Avicel from solid-like to liquid-like state in 5.5 h, as the phase angles rise beyond 45° at this time. The same slurry needed 5.3 h to achieve liquid-like state with Megazyme endoglucanase (40 mg protein/g glucan). After 10.8 h, CTec2 slurry reached a phase angle of 89° or complete liquid-like state but Megazyme slurry peaked only to 64.7°, possibly due to inhibition by cello-oligomers. Acid pretreated corn stover at 30% (w/w) with a CTec2 protein loading of 80 mg/g glucan exhibited a solid-like to liquid-like transition at 37.8 h, which reflects the combined inhibition of low water activity and presence of lignin. The acid pretreated slurry also never achieved complete liquid-like state due to the presence of biomass residue. This method is applicable in several scenarios comparing varying combinations of pre-processing technologies, feedstock types, pretreatment chemistries, and enzymes. Using this method, we can generate a process chain with optimal flow behavior at commercially-relevant conditions

Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression

BACKGROUND: RNA interference (RNAi) has emerged as a powerful tool for the targeted knockout of genes for functional genomics, system biology studies and drug discovery applications. To meet the requirements for high throughput screening in plants we have developed a new method for the rapid assembly of inverted repeat-containing constructs for the in vivo production of dsRNAs. METHODOLOGY/PRINCIPAL FINDINGS: The procedure that we describe is based on tagging the sense and antisense fragments with unique single-stranded (ss) tails which are then assembled in a single tube Ligase Independent Cloning (LIC) reaction. Since the assembly reaction is based on the annealing of unique complementary single stranded tails which can only assemble in one orientation, greater than ninety percent of the resultant clones contain the desired insert. CONCLUSION/SIGNIFICANCE: Our single-tube reaction provides a highly efficient method for the assembly of inverted repeat constructs for gene suppression applications. The single tube assembly is directional, highly efficient and readily adapted for high throughput applications

Survival Data and Predictors of Functional Outcome an Average of 15 Years after the Fontan Procedure: The Pediatric Heart Network Fontan Cohort

ObjectiveMulticenter longitudinal outcome data for Fontan patients surviving into adulthood are lacking. The aim of this study was to better understand contemporary outcomes in Fontan survivors by collecting follow‐up data in a previously well‐characterized cohort.DesignBaseline data from the Fontan Cross‐Sectional Study (Fontan 1) were previously obtained in 546 Fontan survivors aged 11.9 ± 3.4 years. We assessed current transplant‐free survival status in all subjects 6.8 ± 0.4 years after the Fontan 1 study. Anatomic, clinical, and surgical data were collected along with socioeconomic status and access to health care.ResultsThirty subjects (5%) died or underwent transplantation since Fontan 1. Subjects with both an elevated (>21 pg/mL) brain natriuretic peptide and a low Child Health Questionnaire physical summary score (<44) measured at Fontan 1 were significantly more likely to die or undergo transplant than the remainder, with a hazard ratio of 6.2 (2.9–13.5). Among 516 Fontan survivors, 427 (83%) enrolled in this follow‐up study (Fontan 2) at 18.4 ± 3.4 years of age. Although mean scores on functional health status questionnaires were lower than the general population, individual scores were within the normal range in 78% and 88% of subjects for the Child Health Questionnaire physical and psychosocial summary score, and 97% and 91% for the SF‐36 physical and mental aggregate score, respectively. Since Fontan surgery, 119 (28%) had additional cardiac surgery; 55% of these (n = 66) in the interim between Fontan 1 and Fontan 2. A catheter intervention occurred in 242 (57%); 32% of these (n = 78) after Fontan 1. Arrhythmia requiring treatment developed in 118 (28%) after Fontan surgery; 58% of these (n = 68) since Fontan 1.ConclusionsWe found 95% interim transplant‐free survival for Fontan survivors over an average of 7 years of follow‐up. Continued longitudinal investigation into adulthood is necessary to better understand the determinants of long‐term outcomes and to improve functional health status.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110738/1/chd12193.pd

Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch

Factors were defined for elongation, rooting and acclimatization of micropropagated shoots of Larix x eurolepis Henry initiated from short shoot buds of plagiotropic stecklings serially propagated for 9 years from an 8-year-old tree. Initiation and multiplication were on Schenk and Hildebrandt (SH) medium supplemented with 5 μM 6-benzyladenine (BA) and 1 μM indole-butyric acid (IBA). Stem elongation was obtained in 36% of the shoots on SH medium containing 0.5 μM BA and 63% of the remaining non-elongated shoots initiated stem elongation after transfer on SH medium devoid of growth regulators. Rooting involved 2 steps: root induction on Campbell and Durzan mineral salts and Murashige and Skoog organic elements, both half-strength (CD-MS/2), supplemented with 1 μM of both naphthaleneacetic acid (NAA) and IBA, and root elongation following transfer to CD-MS/2 medium devoid of growth regulators. Repeating this 2-step sequence yielded up to 67% rooted shoots. Acclimatization of plantlets ranged from 83% to 100%. Over 300 plants were transferred to the greenhouse; some showed plagiotropic growth

Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy

Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries

MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.This report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2. BRCA2 is a key homologous recombination mediator in vertebrates. Brandsma et al. show that it directly interacts with a testis-expressed protein, HSF2BP, and that male mice deficient for HSF2BP are infertile due to a meiotic recombination defect. They also find that HSF2BP contributes to DNA repair in mouse embryonic stem cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Effect Of In Vitro Silica Treatment On Lung Fibroblasts

Pulmonary fibrosis is a debilitating lung disease that is caused by an over proliferation of fibroblasts, which scars the lung tissue. Currently, the disease is regularly modeled by an in vivo approach, which does not allow for the molecular level examination that an in vitro model would. Silica is often used as a stimulus for pulmonary fibrosis, with in vivo experiments, so it was used in this in vitro experiment. To test the impact of silica on lung fibroblasts, lung samples were extracted from an Ly5.1 mouse, the type of mouse for basic research, and plated to allow for fibroblast proliferation. The fibroblasts were treated with varying doses of silica and then tested for alpha-smooth muscle actin (-SMA) and collagen I (Col I) protein levels. TGF-beta (TGF-ß) treatment was used as a positive control as it has been shown in previous to induce activation in NIH3T3 fibroblast cells. The TGF-ß treatment resulted in an increase of both alpha-smooth muscle actin and collagen I levels, demonstrating the activation of fibroblasts. The silica treatment resulted in an increase of collagen I levels in the lower dose, but a decrease in the higher doses. Fibroblast death may have caused this decrease in collagen I levels. Silica treatment did not affect the alpha-smooth muscle actin levels. The constant alpha-smooth muscle actin levels and decrease in collagen I levels suggests that the untreated fibroblasts were activated, creating a successful in vitro model of pulmonary fibrosis

The Evaluation of Community Engagement In a Science Musical Performance

In response to Australia's declining interest in science, the - Commonwealth Scientific and Industrial Research Organisation sponsors Ologism, a science-based rock band, to improve science engagement. Using observations, peer-assisted informal interviews, and a case-control study we assessed Ologism's effectiveness in stimulating an interest in science during a live performance. We concluded that Ologism can stimulate scientific interest among attendees but alone cannot stimulate prolonged scientific engagement. However, in coordination with similar efforts, Ologism has the potential to engage Australians in science