Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering.

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three-dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10-times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high-throughput screening to 150-mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.

Autonomic Responses to an Acute Bout of High-Intensity Body Weight Resistance Exercise vs. Treadmill Running.

The aim of this study was to compare postexercise autonomic nervous system (ANS) recovery between a high-intensity training protocol (HITP) and high-intensity treadmill running (TM) in 10 physically fit males. For each trial, ANS activity was measured through the heart rate variability markers of log-transformed square root of the successive R-R differences (lnRMSSD) and high frequency power (lnHF). These markers were analyzed in 5-minute segments at 5-10 minutes of the pre-exercise period (PRE) and during the postexercise period at 15-20 minutes (POST15-20min), 20-25 minutes (POST20-25min), 25-30 minutes (POST25-30min), and 1 hour (POST60min). Plasma epinephrine (E) and norepinephrine (NE) were also examined at PRE, immediately post exercise (IPE), 1-hour post (1HP), and 2-hour post (2HP). The results of this study demonstrate a significant overall time-dependent decreases in lnRMSSD and lnHF (p = 0.003 and 0.001, respectively) in both trials. Trial-dependent differences were also observed in postexercise lnRMSSD and lnHF measures, HITP being significantly lower than TM (p = 0.002 and 0.000, respectively). lnRMSSD at POST60min-HITP remained significantly lower compared to PRE (p ≤ 0.05). lnHF returned to baseline in HIPT and TM (p = 0.081 and 0.065, respectively). A time-dependent increase in E and NE was observed in both trials at time point IPE when compared to PRE (p ≤ 0.05). E at 1HP and 2HP returned to near resting levels (p = 0.62, p = 0.26), whereas NE remained slightly elevated in both groups (p = 0.003, p = 0.021). A trial-dependent increase was observed with the HITP eliciting a greater E response (p = 0.025) and NE response (p = 0.03). The HITP causes a greater disruption of the ANS than intensity-matched TM exercise.

PDGF-AB Reduces Myofibroblast Differentiation Without Increasing Proliferation After Myocardial Infarction.

After myocardial infarction (MI), fibroblasts progress from proliferative to myofibroblast states, resulting in fibrosis. Platelet-derived growth factors (PDGFs) are reported to induce fibroblast proliferation, myofibroblast differentiation, and fibrosis. However, we have previously shown that PDGFs improve heart function post-MI without increasing fibrosis. We treated human cardiac fibroblasts with PDGF isoforms then performed RNA sequencing to show that PDGFs reduced cardiac fibroblasts myofibroblast differentiation and downregulated cell cycle pathways. Using mouse/pig MI models, we reveal that PDGF-AB infusion increases cell-cell interactions, reduces myofibroblast differentiation, does not affect proliferation, and accelerates scar formation. RNA sequencing of pig hearts after MI showed that PDGF-AB reduces inflammatory cytokines and alters both transcript variants and long noncoding RNA expression in cell cycle pathways. We propose that PDGF-AB could be used therapeutically to manipulate post-MI scar maturation with subsequent beneficial effects on cardiac function.

The relationship between resting heart rate variability and heart rate recovery.

OBJECTIVE: There is limited research available regarding a possible relationship between resting heart rate variability (HRV) and post-exercise heart rate recovery (HRR). The aim of this study was to examine the relationship between resting HRV and HRR after maximal exercise. METHODS: Sixty-six college age men participated in this study. HRV was measured in a supine position before and for 30 min after a maximal exercise test on a treadmill. HRV was assessed in the time (i.e., SDNN) and frequency (i.e., normalized HF power [HFnu] and normalized LF:HF ratio [LFnu:HFnu]) domains. Heart rate was recorded at maximal exercise (MHR), and at 1- (HR1) and 2- (HR2) min of the cool-down recovery period. HRR was determined from the difference between MHR and HR1 (HRR1) and the difference between MHR and HR2 (HRR2). RESULTS: No significant relationship was found between resting HRV and HRR1 or HRR2. However, SDNN was significantly inversely correlated to MHR (P < 0.05), and HFnu was significantly inversely correlated to MHR (P < 0.01), HR1 (P < 0.01), and HR2 (P < 0.05). Furthermore, MHR accounted for the greatest variation in both SDNN and HFnu (P < 0.05). INTERPRETATION: Therefore, the HRV may not be related to the recovery of HR expressed as a slope (i.e., HRR) within 2 min following a maximal exercise test. This is possibly due to a significant inverse relationship between HRV and MHR, HR1 and HR2 post-maximal exercise.

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.

Iron status of female collegiate athletes involved in different sports.

Iron status was assessed in 70 female athletes aged 18-25 yr participating in collegiate cross-country track, tennis, softball, swimming, soccer, basketball, and gymnastics. No significant differences in mean hemoglobin, hematocrit, serum iron, total iron-binding capacity, transferrin saturation, and ferritin were found among teams. The mean concentrations of each parameter for each of the teams were within the normal ranges. However, several athletes from different sports had suboptimal iron status indexes. Of 17 athletes with a serum ferritin concentration < or = 15 microg/L, 8 (4 freshmen, 2 sophomores, 2 unknown) also exhibited low serum iron concentrations (< 60 microg/dL) and low transferrin saturation (< 16%). Thirteen (6 freshmen, 3 sophomores, 2 juniors, 2 seniors) of 51 (25%) athletes failed to consume two-thirds of the Recommended Dietary Allowance for iron and exhibited suboptimal serum concentrations of ferritin, iron, and/or transferrin saturation. Of nine athletes taking iron supplements, one exhibited suboptimal iron status. In summary, nonanemic iron depletion was present among female collegiate athletes involved in many different sports and in all years of participation (freshmen, sophomore, junior, and senior athletes). Female athletes should continue to be individually and routinely evaluated for nutritional deficiencies throughout their collegiate athletic careers.

Adeno-associated virus and lentivirus vectors: a refined toolkit for the central nervous system.

The last two decades have witnessed the increasing instrumentalization of viruses, which have progressively evolved into highly potent gene transfer vehicles for a wide spectrum of applications. In the context of the central nervous system (CNS), their unique gene delivery features and targeting specificities have been exploited not only to improve our understanding of basic neurobiology, but also to investigate diseases or deliver therapeutic candidates. As a result, we have started moving away from the opportunistic use of recombinant vectors that are derived from naturally existing viruses toward the rational engineering of tailored lentivirus (LV) and adeno-associated virus (AAV) vectors for specific use in the CNS.

The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology.

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.

The effect of experience and gender on cardiovascular and metabolic responses with dynamic Tae Kwon Do exercise.

This study, conducted at the Exercise Physiology Laboratory of Auburn University, AL, addressed and compared the acute cardiovascular and metabolic effects elicited by novice and experienced men and women participants during a single bout of dynamic Tae Kwon Do exercise and investigated whether or not dynamic Tae Kwon Do practice is an exercise modality that provides sufficient cardiorespiratory demand for enhancing aerobic fitness and promoting weight and fat loss. Twenty-eight men and women (aged 19-42) were assigned to 1 of the following 4 groups: Tae Kwon Do experienced and trained men (ME), Tae Kwon Do experienced and trained women (FE), novice Tae Kwon Do men (MN), and novice Tae Kwon Do women (FN). The results of this investigation indicate that this form of exercise can be performed for an extended period of 20 minutes. All 4 groups achieved the recommended stimulus for effective initiation of cardiovascular adaptations and conditioning. The mean exercise heart rate responses (88.3-92.2% of maximal heart rate [HR max]) were similar for all groups. The observed exercise intensity ranged from 67.9 to 72.1% VO2max, and no significant difference based on the experience and gender and exercise oxygen uptake could be established. Data in this study indicate a high caloric expenditure for this mode of exercise. Total caloric cost of 20 minutes of dynamic Tae Kwon Do, 194.8 and 201.6 kcal for novice women and experienced women, respectively, was significantly lower in comparison with that of their men counterparts (316.5 and 286.5 kcal, respectively), but no significant relationship between experience and energy cost was found. The conclusion of this study indicates that dynamic Tae Kwon Do is an exercise modality that can be appropriately prescribed for cardiovascular conditioning, weight control, and fat loss.

Copper status of collegiate female athletes involved in different sports.

Copper status was assessed in 70 female collegiate athletes aged 18 to 25 years participating in cross country track, tennis, softball, swimming, soccer, basketball, and gymnastics during the 2000-2001 season. A group of 8 college-aged females, 20 to 23 years of age, who were not collegiate athletes, served as controls. Mean copper intakes including supplements did not differ significantly among the controls and athletic teams. Mean copper intakes including supplements as micrograms/day and percent recommended dietary allowance (RDA) were as follows: controls 1071 +/- 772 microg (119 +/- 86%), cross country track 1468 +/- 851 microg (163 +/- 95%), tennis 1099 +/- 856 microg (122 +/- 95%), softball 654 +/- 420 microg (73 +/- 47%), swimming 1351 +/- 1060 g (150 +/- 118%), soccer 695 +/- 368 microg (77 +/- 41%), and gymnastics 940 +/- 863 microg (104 +/- 96%). Forty-one percent of athletes and 29% of controls failed to consume two thirds of the RDA for copper. Mean serum copper and ceruloplasmin concentrations were within the normal range and did not differ significantly among the controls (117 +/- 22 microg/dl, 445 +/- 122 mg/L) and cross country track (98 +/- 17 microg/dl, 312 +/- 59 mg/L), tennis (140 +/- 84 microg/dl, 424 +/- 244 mg/L), softball (95 +/- 30 microg/dl, 310 +/- 77 mg/L), swimming (98 +/- 25 microg/dl, 312 +/- 40 mg/L), soccer (93 +/- 15 microg/dl, 324 +/- 54 mg/ L), basketball (85 +/- 10 microg/dl, 280 +/- 62 mg/L), and gymnastics (96 +/- 21 microg/dl, 315 +/- 68 mg/L) teams. Copper status of female collegiate athletes appears to be adequate in this cross-sectional assessment.