Time of Fusarium inoculation and post-anthesis temperature stress affect FHB severity and DON concentration in winter wheat

Two pot experiments were conducted using elite lines and near isogenic lines (NILs) of winter wheat in a controlled environment to evaluate the effect of time of inoculation and subsequent increase in temperature during grain-filling on Fusarium head blight (FHB) and deoxynivalenol (DON) on the infected wheat grains. The experiments were a complete factorial combination with randomised replicates. Fusarium graminearum was used to spray inoculate wheat spikes at glume loose (GL). GL was established when the lower glume could be opened with a thumbnail. Fusarium inoculation was done at glume loose (GL+0), 4 and 8 days after (GL+4 and GL+8), respectively. Pots transferred to controlled environment cabinets set at 23/15oC or 28/20oC. Results reveal that FHB severity showed a significant (P<0.05) cultivar and temperature interaction in both elite and near isogenic lines. High temperature increased FHB severity by 35% in the NILs. DON concentration showed cultivar sensitive in both sets of cultivars. Inoculation at GL+4 which corresponded with the mid-anthesis gave the highest FHB severity and DON concentration in NILs, while the elite cultivars showed cultivar sensitive to DON accumulation regardless of the time of Fusarium inoculation.publishersversionPeer reviewe

Increasing diversity of production cell lines through miniaturization, automation, and high-throughput analytics

The development of a successful biologic therapeutic manufacturing process begins with the creation of a stable clonal cell line. Since attributes of the production cell line will significantly impact upstream and downstream processes, researchers must find ways to generate several candidate lines with diverse properties. However, a wide diversity is difficult to achieve since cultures are commonly selected, maintained, and screened as populations. In these populations, robust sub-populations can overtake the overall culture and reduce diversity. To combat this, sub-populations must be physically separated by splitting or subcloning, and maintained in individual vessels requiring intensive labor and infrastructure. As a result, researchers must balance between either increasing diversity vs. increasing resources need to maintain and screen hundreds of cultures. In order to shift this balance towards greater diversity, we have developed systems that combines miniaturization of culture vessels, targeted use of automation, and single cell analysis to allow for hundreds of cell lines to be isolated, maintained, and analyzed. We demonstrate cell lines can be easily maintained in simple low volume formats with no impact on cells. We show that we can significantly improve and maintain diversity through separation and isolation of hundreds of cultures. Additionally, higher throughputs allows to assess cell line phenotypes of multiple candidate lines early in development. Benefits achieved through this approach did not increase resources or timelines. Moving towards miniaturization combined with single cell analysis will also enable future possibilities for more precise cell engineering and gene editing

Paraoxonase 2 overexpression inhibits tumor development in a mouse model of ovarian cancer.

Ovarian cancer (OC) is most lethal malignancy among all gynecological cancer. Large bodies of evidences suggest that mitochondrial-derived ROS play a critical role in the development and progression of OC. Paraoxonase 2 (PON2) is a membrane-associated lactonase with anti-oxidant properties. PON2 deficiency aggravates mitochondrial ROS formation, systemic inflammation, and atherosclerosis. The role of PON2 in cancer development remains unknown. In this report, in human, we identified that PON2 expression is higher in early stages (but not in late stages) of OC when compared to normal tissue. Using a mouse xenograft model of OC, we demonstrate that overexpression of PON2 prevents tumor formation. Mechanistically, PON2 decreases OC cell proliferation by inhibiting insulin like growth factor-1 (IGF-1) expression and signaling. Intriguingly, PON2 reduces c-Jun-mediated transcriptional activation of IGF-1 gene by decreasing mitochondrial superoxide generation. In addition, PON2 impairs insulin like growth factor-1 receptor (IGF-1R) signaling in OC cells by altering cholesterol homeostasis, which resulted in reduced caveolin-1/IGF-1R interaction and IGF-1R phosphorylation. Taken together, we report for the first time that PON2 acts as a tumor suppressor in the early stage of OC by reducing IGF-1 production and its signaling, indicating PON2 activation might be a fruitful strategy to inhibit early stage ovarian tumor

Lipid Transfer Inhibitor Protein (Apolipoprotein F) Concentration in Normolipidemic and Hyperlipidemic Subjects

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 mg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) ≤ 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined

Interrogating cell culture populations for the selection of production cell lines using microfluidic culturing, single cell analysis, and predictive modelling

Cell line development for manufacturing is a lengthy, multi-step, resource intensive, critical path activity. Attempts to perform in silico modelling and prediction of cell culture has been difficult due to complexities around heterogeneous cell culture populations that rapidly shift over generations under changing selective conditions. For example, early populations will often change as response to media and culturing conditions from a static colony culturing in microtiter plates, to small scale suspension culturing, and finally in a controlled bioreactor processes. As a result, it is challenging to make the final cell line selection early, while predicting future bioprocess performance, and ultimately estimate the protein product quality. We address this challenge by drastically increasing the amount of early cell culture population data obtained through use of emerging single cell technologies. Data obtained is combined with modelling approaches to select the best cell lines upfront to reduce timelines and processing steps. To achieve this, we have implemented a platform from Berkeley Lights that effectively digitalizes most aspects of cell culture. Thousands of individual cell lines can be manipulated, cultured and interrogated on a perfusion nanofluidic chip resulting in extensive data on cell behavior on an individual cell level as well as the populations. Through multivariate predictive modeling of this data, we can predict the performance of candidate clonal cell lines in larger scale production runs. Incorporation of additional single cell analysis such as digital droplet RT-PCR and next generation sequencing further predicts product quality, such as heterogeneity of bispecifics and sequence variant detection. Similar approaches can further be used to then study the stability and integrity of a final CHO cell banks. When combined, single cell interrogation of early culture populations allow for the dematerialization of the CLD process, make better predictions of bioprocess performance, and reduce select the final production clone earlier

No sex differences in adult telomere length across vertebrates:a meta-analysis

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