Chloride Ion Transport Blockade Enhances Cytocidal Effects of Sterile Water on Bladder Cancer Cells

Background:Bladder cancer is a major health concern worldwide. The conventional intravesical Bacillus Calmette–Guérin therapy has certain shortcomings; thereby, demanding novel alternatives. Although sterile water is a probable agent for such novel intravesical therapies, bladder cancer cell lines differ in their sensitivity to hypotonic shock due to sterile water. Therefore, we aimed to investigate whether Cl- channel blockers enhance the cytocidal effect of hypotonic shock on bladder cancer cells resistant to sterile water.Methods:Bladder cancer cell lines of varying grades (RT112, T24, and J82) were exposed to sterile water, and morphological changes were closely observed using microscopy. Sterile water-induced changes in cell membrane integrity and cell viability were analyzed to determine the effects of hypotonic shock. These effects were further analyzed using a Cl- channel blocker.Results:T24 and J82 cells started swelling immediately upon exposure to sterile water and ruptured within 10 min. RT112 cells demonstrated limited hypotonic swelling with few cell ruptures. After treatment with the Cl- channel blocker, RT112 cells ruptured faster as compared to that in cells treated with sterile water. The percentages of viable dimethylsulfoxide and 5-nitro-2-(3-phenylpropylamino) benzoic acid -treated (50, 100, 200, and 300 µM) RT112 cells after 10 min of exposure to sterile water were 13.6 % ± 3.4 %, 6.3 % ± 1.2 %, 2.0 % ± 1.1 %, 0.7 % ± 0.7 %, and 0 %, respectively.Conclusions:Taken together, the Cl- channel blockers enhanced the cytocidal effects of hypotonic shock in bladder cancer cells. Intravesical therapy with sterile water after treatment with a Cl- channel blocker represents a potential new adjuvant therapy after TURBT with high efficacy.

Wide field of view quantitative imaging of cellular ATP release

Although several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none have been clearly identified. To facilitate progress, we report here a wide field of view (∼20 × 20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2–5 s−1) or swelled by hypotonic shock. The amount of ATP secreted in response to a series of five graded stretch pulses (5–37% linear deformation, 1-s duration at 25°C) changed nonmonotonically with respect to strain amplitude and was inhomogeneous across the cell monolayer. In a typical experiment, extracellular ATP density averaged 250 fmol/mm2, but the area of detectable signal covered only ∼40% of the cells. In some areas, ATP accumulation peaked around 900 fmol/mm2, which corresponded to an estimated concentration of 4.5 µM. The total amount of ATP released from the combined stretch pulses reached 384 ± 224 pmol/million cells ( n = 4). Compared with stretch, hypotonic shock (50%, 30°C) elicited a more homogeneous ATP secretion from the entire cell population but at a lower yield totaling 28 ± 12 pmol/million cells ( n = 4). The quantitative extracellular ATP mapping of several thousand cells at once, with this wide field of view imaging system, will help identify ATP release pathways by providing unique insights on the dynamics and inhomogeneities of the cellular ATP secretion that are otherwise difficult to assess within the smaller field of view of a microscope.

Comparison of Platelet Concentrates Prepared from Platelet Rich Plasma-Platelet Concentrate and Buffy Coat Poor-Platelet Concentrate on Storage Days 1 and 5

Objective: The purpose of this study is to assess the quality of platelet concentrates on storage days 1 and 5 prepared by platelet rich plasma-platelet concentrate (PRP-PC) and buffy coat poor-platelet concentrate (BC-PC) methods comparing to the American Association of Blood Banks (AABB) recommendations.Material and Method: Totally of 120 platelet concentrates (PC) units on storage days 1 and 5 (60 of PRP-PC triple blood bag and 60 of BC-PC quadruple AS-5 blood bag) were separated from whole blood donations at Songklanagarind Hospital. The prepared PC were assessed with 5 parameters such as volume, platelet count, white blood cell count per unit, pH, swirling phenomenon score and hypotonic shock response. The independent t-tests, paired Student’s t-tests and SPSS program were utilized in statistical analysis step.Results: The mean±standard deviation (S.D.) of each parameter were as follow : (1) Volume of PRP-PC and BC-PC met the standard (40-70 ml). (2) All of the platelet concentrates met the standard (≥ 5.5x1010/unit). The mean±S.D.: PRP-PC and BC-PC (day 1) were 6.820±1.480 x1010 and 7.010±1.300 x1010/unit (p-value=0.260), while PRP-PC and BC-PC (day 5) were 6.620±1.160x1010 and 6.720± 1.150x1010/unit (p-value=0.040). (3) The white blood cell in platelet concentrates met the standard (<0.2x1010/unit). The mean±S.D.: PRP-PC and BC-PC (day 1) were 0.030±0.017 x1010and 0.026±0.019x1010/unit (p-value=0.040), while PRP-PC and BC-PC (day 5) were0.033±0.013x1010 and 0.027±0.019x1010/unit (p-value= 0.580). (4) The pH of all units (PRP-PC and BC-PC) met the standard (≥6.2). The mean±S.D.: PRP-PC and BC-PC (day 1) were 7.430±0.330 and 7.750±0.160 (p-value=0.006), while PRP-PC and BC-PC (day 5) were 7.590±0.350 and 7.620±0.280 (p-value=0.710). The swirling phenomenon score and hypotonic shock response were the same as standard AABB and were not statistically difference.Conclusion: The quality of PRP-PC and BC-PC after storing on days 1 and 5 as follow (1) Volume of PRP-PC and BC-PC met the standard. (2) The platelet count per unit of PRP-PC and BC-PC (day 1), PRP-PC and BC-PC (day 5) were not statistically difference. (3) The white blood cell count per unit of PRP-PC and BC-PC (day 1) were statistically difference, while PRP-PC and BC-PC (day 5) were not statistically difference. (4) The pH of PRP-PC and BC-PC (day 1) were statistically difference, while PRP-PC and BC-PC (day 5) were not statistically difference. The swirling phenomenon score and hypotonic shock response of PRP-PC and BC-PC were not statistically difference. Platelet concentrates of both method storing on days 1 and 5 fulfilled the quality guideline of AABB.

Robust Generation of Transgenic Mice by Hypotonic Shock Mediated Transgene Delivery in Testicular Germ Cells

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.