Tumor Tissue Explant Culture of Patient-Derived Xenograft as Potential Prioritization Tool for Targeted Therapy

Despite of remarkable progress made in the head and neck cancer (HNC) therapy, the survival rate of this metastatic disease remain low. Tailoring the appropriate therapy to patients is a major challenge and highlights the unmet need to have a good preclinical model that will predict clinical response. Hence, we developed an accurate and time efficient drug screening method of tumor ex vivo analysis (TEVA) system, which can predict patient-specific drug responses. In this study, we generated six patient derived xenografts (PDXs) which were utilized for TEVA. Briefly, PDXs were cut into 2 × 2 × 2 mm3 explants and treated with clinically relevant drugs for 24 h. Tumor cell proliferation and death were evaluated by immunohistochemistry and TEVA score was calculated. Ex vivo and in vivo drug efficacy studies were performed on four PDXs and three drugs side-by-side to explore correlation between TEVA and PDX treatment in vivo. Efficacy of drug combinations was also ventured. Optimization of the culture timings dictated 24 h to be the time frame to detect drug responses and drug penetrates 2 × 2 × 2 mm3 explants as signaling pathways were significantly altered. Tumor responses to drugs in TEVA, significantly corresponds with the drug efficacy in mice. Overall, this low cost, robust, relatively simple and efficient 3D tissue-based method, employing material from one PDX, can bypass the necessity of drug validation in immune-incompetent PDX-bearing mice. Our data provides a potential rationale for utilizing TEVA to predict tumor response to targeted and chemo therapies when multiple targets are proposed

Thermotaxis of Human Sperm Cells in Extraordinarily Shallow Temperature Gradients Over a Wide Range

<div><p>On the basis of the finding that capacitated (ready to fertilize) rabbit and human spermatozoa swim towards warmer temperatures by directing their movement along a temperature gradient, sperm thermotaxis has been proposed to be one of the processes guiding these spermatozoa to the fertilization site. Although the molecular mechanism underlying sperm thermotaxis is gradually being revealed, basic questions related to this process are still open. Here, employing human spermatozoa, we addressed the questions of how wide the temperature range of thermotaxis is, whether this range includes an optimal temperature or whether spermatozoa generally prefer swimming towards warmer temperatures, whether or not they can sense and respond to descending temperature gradients, and what the minimal temperature gradient is to which they can thermotactically respond. We found that human spermatozoa can respond thermotactically within a wide temperature range (at least 29–41°C), that within this range they preferentially accumulate in warmer temperatures rather than at a single specific, preferred temperature, that they can respond to both ascending and descending temperature gradients, and that they can sense and thermotactically respond to temperature gradients as low as <0.014°C/mm. This temperature gradient is astonishingly low because it means that as a spermatozoon swims through its entire body length (46 µm) it can sense and respond to a temperature difference of <0.0006°C. The significance of this surprisingly high temperature sensitivity is discussed.</p> </div

Human sperm distribution in a linear temperature gradient.

<p><b>A:</b> Schematic illustration of the drinking straw, fitted to the dimensions of the thermoseparation device, and used to measure the sperm distribution in a linear temperature gradient. The straw, homogeneously filled with human spermatozoa at the commencement of a measurement, was frozen in liquid nitrogen at the end of the measurement and cut into 8 equal parts at the locations marked by dashed lines. <b>B:</b> Temperature preferences of human spermatozoa. The temperatures shown in the abscissa were measured inside the straw. The number of spermatozoa in the straw’s outermost segment (the warmer side) could not be counted due to loss of volume (since this was the segment cut last). For this reason the highest temperature shown in the figure is 41.7°C rather than 42.3°C. The results with the inhibitor U73122 were corrected for the solvent (ethanol) effect by subtracting the difference between ethanol and untreated cells (control) from the values obtained with U73122 at each temperature tested. Both the results (mean ± SEM) of untreated cells (9 experiments, 20 measurements in total) and the results of cells treated with U73122 (7 experiments, 13 measurements) were normalized according to the results of dead cells (4 experiments, 12 measurements) at 36.8°C. Sperm accumulation of untreated cells at 40.4°C, 41.2°C and 41.7°C was significantly higher than sperm accumulation at 36.8°C, 38.1°C, 38.5°C, 38.9°C and 39.7°C (<i>P</i><0.0001, according to the contrast <i>t</i>-test). The connecting line is a hypothetical sigmoidal-curve fit (R² = 0.91; Origin 6.1 software, OriginLab). The differences between the untreated cells and the negative controls (dead and U73122-treated cells) were statistically significant only at temperature values ≥40°C (Marked with Asterisks; <i>P</i>≤0.04, according to the one-way ANOVA). The straight line was calculated according to the average of the negative controls at each tested temperature.</p

Migration of human spermatozoa in a descending temperature gradient.

<p>The temperatures shown in the abscissa were measured by the thermocouples at both ends of the tube (externally to the tube). The results are the mean ± SEM of 6–19 determinations (4 experiments for each temperature gradient tested). Asterisks above the columns indicate a statistically significant difference from the respective no-gradient control (<i>P</i>≤0.01, according to Student’s <i>t</i>-test).</p

Dependence of sperm accumulation on the magnitude of the temperature difference.

<p>The temperature of the sperm-containing compartment was 36.5°C in all runs. The temperature differences shown in the abscissa were measured by the thermocouples at both ends of the tube (externally to the tube). Net accumulation was calculated by subtracting the no-gradient control accumulation from the sperm accumulation in a temperature gradient. The results are the mean ± SEM of 7–9 determinations (3–5 experiments). The difference between capacitated and non-capacitated sperm is very significant (<i>P</i><0.0001, according to one-way ANOVA with Tukey-Kramer Multiple Comparisons post-test).</p

Kinetic parameters of human sperm motility under the conditions of the accumulation assay^a.

a<p>The sperm samples contained 3.5% PVP. The results are the mean ± SEM of 3 experiments (each being the average of duplicate determinations carried out for 80 s). The significance of the difference between temperatures was tested for each parameter by one-way ANOVA with Tukey-Kramer Multiple Comparisons post-test and found insignificant.</p

Migration of human spermatozoa in an ascending temperature gradient.

<p><b>A:</b> Schematic illustration of the Lucite tube composed of two compartments for the separation process <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041915#pone.0041915-Bahat3" target="_blank">[5]</a>. Thermocouples at both ends of the tube holder measured the temperatures at these locations. The two compartments were separated by a thin disc (316 stainless steel) having pores, 40 µm in diameter. <b>B:</b> Migration at various temperatures. The results are the mean ± SEM of 6–19 determinations (3–4 experiments for each temperature gradient tested). The temperatures shown in the abscissa were those measured by the thermocouples at both ends of the tube (externally to the tube). Asterisks above the columns indicate a statistically significant difference from the respective no-gradient control (<i>P</i>≤0.02, according to Student’s <i>t</i>-test).</p

Lowering mutant huntingtin by small molecules relieves Huntington’s disease symptoms and progression

Abstract Huntington’s disease (HD) is an incurable inherited disorder caused by a repeated expansion of glutamines in the huntingtin gene (Htt). The mutant protein causes neuronal degeneration leading to severe motor and psychological symptoms. Selective downregulation of the mutant Htt gene expression is considered the most promising therapeutic approach for HD. We report the identification of small molecule inhibitors of Spt5-Pol II, SPI-24 and SPI-77, which selectively lower mutant Htt mRNA and protein levels in HD cells. In the BACHD mouse model, their direct delivery to the striatum diminished mutant Htt levels, ameliorated mitochondrial dysfunction, restored BDNF expression, and improved motor and anxiety-like phenotypes. Pharmacokinetic studies revealed that these SPIs pass the blood-brain-barrier. Prolonged subcutaneous injection or oral administration to early-stage mice significantly delayed disease deterioration. SPI-24 long-term treatment had no side effects or global changes in gene expression. Thus, lowering mutant Htt levels by small molecules can be an effective therapeutic strategy for HD

Human sperm chemotaxis: Both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants

Background: Human sperm chemotaxis to pre-ovulatory follicular fluid is well established in vitro. However, it is not known whether the female's oocyte-cumulus complex secretes sperm chemoattractants subsequent to ovulation (for enabling sperm chemotaxis within the Fallopian tube) and, if so, which of these cell types - the oocyte or the cumulus oophorus - is the physiological origin of the secreted chemoattractant. Methods: By employing a directionality-based chemotaxis assay, we examined whether media conditioned with either individual, mature (metaphase II) human oocytes or the surrounding cumulus cells attract human sperm by chemotaxis. Results: We observed sperm chemotaxis to each of these media, suggesting that both the oocyte and the cumulus cells secrete sperm chemoattractants. Conclusions: These observations suggest that sperm chemoattractants are secreted not only prior to ovulation within the follicle, as earlier studies have demonstrated, but also after oocyte maturation outside the follicle, and that there are two chemoattractant origins: the mature oocyte and the surrounding cumulus cells.Fil: Sun, Fei. Weizmann Institute of Science. Department of Biological Chemistry; IsraelFil: Bahat, Anat. Weizmann Institute of Science. Department of Biological Chemistry; IsraelFil: Gakamsky, Anna. Weizmann Institute of Science. Department of Biological Chemistry; IsraelFil: Girsh, Eliezer. Ben Gurion University of the Negev; IsraelFil: Katz, Nathan. Ben Gurion University of the Negev; IsraelFil: Giojalas, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Centro de Biología Celular y Molecular; ArgentinaFil: Tur-Kaspa, Ilan. Ben Gurion University of the Negev; IsraelFil: Eisenbach, Michael. Weizmann Institute of Science. Department of Biological Chemistry; Israe