Purity and Recovery of crystallized mAb04c.

<p>*Protein redissolved in 100 mM sodium acetate pH 4.0.</p

SDS-PAGE of HCP spiking experiment.

<p>Silver-staining. Lanes: (1) mAb04c, standard; (2) mother liquor with mAb04c and lysozyme; (3) washed mAb04c crystals from mother liquor with lysozyme; (4) the supernatant from the crystallization with lysozyme; (5) mother liquor with mAb04c and BSA; (6) washed mAb04c crystals from mother liquor with BSA; (7) the supernatant from the crystallization with BSA.</p

Micrograph of crystalline mAb04c crystallized from clarified culture supernatant.

<p>Crystallization condition: sitting drop. Reservoir: 0.1 M imidazol, 0.2 M calcium acetate, 9% w/v PEG 8000. 20 µL clarified culture supernatant (8.3 mg/ml mAb04c) plus 20 µl reservoir, RT. Scale bar 100 µm.</p

Phase diagram of mAb04c with PEG 8000 as precipitant.

<p>Buffer: 0.1 M imidazol, 0.2 M calcium acetate, pH 7.0, RT.</p

Micrograph of crystals of mAb04c under polarized light.

<p>3 µl microbatch. Conditions: 1.5 µl 20 mg/ml mAb04c in 20 mM Tris, 50 mM Histidine, pH 7 plus 1.5 µl 12% (w/v) PEG 8000, 0.4 M calcium acetate in 0.2 M Imidazol, pH 7, RT. The broken crystal (intentionally) indicates that the dimension of crystal is about 100∼150 µm×10∼20 µm×10∼20 µm.</p

Coomassie blue stained none-reducing SDS-PAGE of mAb04c before and after crystallization or protein A purification.

<p>7 µg of IgG was loaded per lane. Lanes: (1) clarified mAb04c culture supernatant; (2) washed mAb04c crystals, redissolved in 100 mM sodium acetate pH 4.0; (3) mAb04c, purified via protein A chromatography.</p

SE-Chromatogram of the sample before and after crystallization.

<p>Elution buffer: 0.05 M Tris/0.15 M NaCl, pH 7; flow rate: 1 ml/min; wavelength: 225 nm. (A) Clarified mAb04c culture supernatant. Peak 3: mAb04c, 8.42 min; Peak 4: Contaminating Protein, 9.98 min; Peak 5: Histidine in buffer, 12.19 min. (B) washed mAb04c crystals, redissolved in 100 mM sodium acetate pH 4.0. Peak 2: mAb04c, 8.43 min.</p

Hyperthermia-driven aberrations of secreted microRNAs in breast cancer <i>in vitro</i>

<p><b>Purpose:</b> Expression profile alterations of nine breast cancer (BC)-associated secreted microRNAs (miRs) were determined under microenvironmental alterations occurring in tumour progression, metastasis or specific oncological treatment modalities. Thereto, the potential influence of the exogenic stimuli hypoxia, acidosis and hyperthermia was investigated <i>in vitro</i>.</p> <p><b>Material and methods:</b> Four established BC cell lines were applied as <i>in vitro</i> BC model systems. Quantitative analyses of secreted microRNA specimens were performed by RNA isolation from cell culture supernatant and subsequent real-time PCR in cells under physiological versus hypoxic, acidic or hyperthermia conditions.</p> <p><b>Results:</b> The <i>in vitro</i> application of exogenic stimuli hypoxia, extracellular acidosis and hyperthermia caused heterogeneous expression alterations for the investigated secreted miRNA phenotypes. The majority of relevant exogenic stimuli-dependent microRNA expression alterations were restricted to single events displaying distinct cell type and stimulus dependent correlations only. Most remarkably, hyperthermia triggered a uniform significant down-regulatory effect on the expression levels of the three secreted microRNAs miR-10b, miR-15b and miR-139, respectively. The marked decrease in miR-10b and miR-15b levels was detectable in all four, while miR-139 was found significantly reduced in three out of four BC cell lines.</p> <p><b>Conclusion:</b> Hyperthermia-dependent down-regulatory influence on three distinct BC-related microRNAs <i>in vitro</i> generates translational aspects for clinical BC treatment, since the identified microRNAs miR-10b, miR-15b and miR-139 are known to have oncogenic as well as tumour suppressor functions in BC. However, an evaluation regarding the potential impact of microRNA-related hyperthermia-dependent alterations for innovative BC treatment approaches demands further analysis including <i>in vivo</i> data.</p

Effects of increased temperature and atmospheric drought on δ¹³C. δ¹³C is shown for water soluble organic matter (WSOM) in current year's needles (N10), older needles (N09, N08) and fine roots (FR), and in bulk material of bark and coarse roots (B, CR) from Douglas-fir seedlings of the provenances Pend Oreille and Monte Creek in control C (20°C, 0.35 kPa, white circles) and temperature/atmospheric drought treatment T (30°C, 1.91 kPa, grey circles).

<p>Asterisks indicate significant differences between treatments C and T (Tukey test, *** for p<0.001, ** p<0.01, * p<0.05). Data shown are mean values ± SD (N = 3 (64 DAT) – 10 (76DAT)). Numbers 1, 41, 64, 76 in the x-axis caption indicate the time point of harvest after the onset of the experiment.</p

Effects of increased temperature and atmospheric drought on the metabolite profile of two Douglas-fir provenances (Monte Creek and Pend Oreille) and over all provenances (All) in current year's (N10) and last year's needles (N09).

<p>Treatment effects are shown as the log10 ratios of temperature/atmospheric drought treatment (T) <i>vs.</i> controls (C) obtained from the mean values of the relatively quantified metabolite peaks of the two treatments (N = 4–6 for for each treatment N10 and N = 3–5 for N09). Significance levels are given for group differences (T vs. C) and were obtained by a post-hoc Tukey test (*** for p<0.001, ** p<0.01, * p<0.05,. p<0.1). n. det.: the compound was not detected in at least 50% of the replicates of each provenance and needle age class.</p