A communication process for global requirements engineering

Globally distributed software development teams face problems with software development life cycle phases, as the distributed nature of each of these phases make it even more challenging to communicate between the stakeholders. Global distance can give rise to incomplete requirements handovers which make the situation more difficult. It is important to address this issue as the end product is likely to deliver less business value when such problems arise. In this research, we propose a process to facilitate non-verbal communication among globally distributed requirements engineering teams. The focus of this research is the situation that occurs after requirements are handed to another site. Our proposed process endeavors to ensure that incomplete and conflicting requirements are identified and mitigated

<i>STN1</i> KD exacerbates DNA damage induced by H₂O₂.

(A) Box-and-whisker plots showing distributions of neutral comet tail lengths for each experimental condition. Cells were treated with 500 μM H2O2 in DMEM for 2 hours, followed by 5 hours incubation in DMEM, then were subjected to a neutral comet assay (see Materials and methods for details). Numbers of analyzed comets (n) are shown above each lane. p-values for Anderson-Darling tests are shown above respective paired lanes. (B) Box-and-whisker plots showing distributions of alkaline comet tail lengths for each experimental condition. Prior to the assay, cells were treated with 500 μM H2O2 in DMEM for 10 minutes or 5 μM HU in DMEM for 3 hours. The apparent lack of DNA strand breaks in the HU-treated cells does not necessarily contradict the flow cytometry data shown in Fig 2: histone H2AX can be phosphorylated by ATR, which is activated by exposed ssDNA within stalled replication forks [45]. Tail lengths were measured by the “OpenComet” plugin in ImageJ software [35]. For each condition, numbers of analyzed comets (n) are shown above each lane. p-values for Mann–Whitney U tests are shown above respective paired lanes.</p

Classification of tail lengths in comet assays.

(A, B) The same data set shown in Fig 3A or 3B was divided into two classes. Fractions of each class per condition are represented by 100% stacked bar charts. Values in boxes represent the percentage fraction for each class. In each condition, numbers of analyzed comets (n) are shown above each bar. (A) Classification of the neutral comet assay data. Class 1, tail length shorter than 80 μm; Class 2, tail length longer than 80 μm. Chi-square test (with one degree of freedom) rejected the null hypothesis that the class distributions are the same between KD control and shSTN1 cells (p = 2.4E-9). (B) Classification of the alkaline comet assay data. Class 1, tail length shorter than 155 μm; Class 2, tail length longer than 155 μm. Chi-square test (with one degree of freedom) rejected the null hypothesis that the class distributions are the same between KD control and shSTN1 cells (p 2O2-treated HeLa cells. The horizontal axis shows comet tail length (μm) and the vertical axis indicates counts for each binned value. Blue-colored regions indicate “Class 2” fractions. (PDF)</p

H₂O₂ -treated sh<i>STN1</i> cells fail to resume DNA synthesis in S phase.

(A) Experimental design. Asynchronous cell populations were pulse-labeled with BrdU prior to H2O2 treatment (500 μM, 2 hours. The mock-treated cells were allowed to grow for 2 hours.). Immediately after washing out the H2O2, the cells were allowed to incorporate EdU for one hour. (B) Representative microscopic images for KD control cells. DAPI staining was used to identify nuclei. The merged column shows superpositions of BrdU (detected by Cy5-conjugated antibody) and EdU (detected by conjugated Alexa Fluor 488) signals. Top row: Mock-treated cells. The majority of the BrdU-positive nuclei are also EdU-positive, indicating unperturbed progression of chromosomal replication. Bottom row: H2O2-treated cells. The majority of the BrdU-positive nuclei are EdU-negative, indicating suppression of DNA replication upon H2O2 treatment. An example of a BrdU/EdU-double-positive nucleus is indicated by a white arrowhead, and its enlarged image is shown in the rightmost column. Scale bars, 20 μm. (C) Percentage of BrdU or EdU positive cells in each condition. Data from three biological replicates are shown with mean values (boxes) and SEM. (D) Percentages of EdU-positive cells among BrdU-positive cells. Data from three biological replicates are shown with mean values (boxes) and SEM. For (C) and (D), at least 200 cells per condition were randomly selected in each assay. p-values for unpaired t-tests are shown above respective paired bars.</p

Validation of RAD51 inhibition by B02.

(A) The experimental timeline. HeLa cells were treated with RAD51 inhibitor B02 starting one hour before the beginning of Etposide treatment (25 μM, 1 hour). (B) Representative images of RAD51 foci and γ-H2AX signals in HeLa cell nuclei. The enlarged area shown in the rightmost column is indicated by white squares in the RAD51 column. RAD51 foci formation induced by etoposide treatment was suppressed by B02 treatment. Scale bars, 10 μm. (C) Distributions of the number of RAD51 foci per nucleus in HeLa cells. p-values for Mann–Whitney U tests are shown above the indicated lanes. (D) Distributions of the number of RAD51 foci per nucleus in U2OS cells. p-values for Mann–Whitney U tests are shown above the indicated lanes. (PDF)</p

Validation of mimosine-induced cell cycle arrest and synchronized release into S phase.

(A) Experimental timeline. HeLa cells were treated with 500 μM mimosine for 23 hours to arrest them at the G1/S boundary [55]. At t = 0 hr, the arrested cells were synchronously released into S phase by replacing the medium with drug-free fresh medium. To monitor fractions of S-phase cells in the released cell population, pulse-labeling by EdU (incubation with 10 μM EdU for 10 min before fixation) was carried out at t = 0, 1, 2 and 4 hr, followed by conjugation with Alexa Flour 488 by Click chemistry (Click-iT EdU Cell Proliferation Kit for Imaging). EdU incorporation into nuclei was assessed by fluorescence microscopy. (B) Jitter plots showing the signal intensity of EdU (i.e., fluorescence intensity of Alexa Fluor 488 in a.u. [arbitrary units]). For each condition, the signal intensities of 500 randomly selected nuclei are shown. A nucleus with fluorescence intensity > 3,500 a.u. was defined to be EdU-positive. The EdU-positive rate per condition is shown above each lane. EdU (-), asynchronous cells without EdU labeling; EdU (+), asynchronous cells with EdU labeling. At t = 0 hr, all the analyzed nuclei were EdU-negative, demonstrating highly efficient cell cycle arrest at G1/S. As time elapsed, the fraction of EdU-positive nuclei gradually increased, indicating cell cycle progression into S phase with a modest synchronization rate. (C) Flow cytometric analysis of DNA content (PI fluorescence) in asynchronous (white) and mimosine-treated (colored) samples. After the release, cells were collected at t = 0 (red), 1 (green), 2 (blue), and 4 (purple) hours, respectively. The horizontal axis shows PI intensity (DNA content), and the vertical axis indicates cell counts for each intensity value. (PDF)</p

Flow cytometric analyses of γ-H2AX levels in HU- or H₂O₂-treated HeLa cells.

(A) Histograms of Alexa488 intensity in undamaged, HU- or H2O2-treated HeLa cells. The horizontal axis shows Alexa488 fluorescence (γ-H2AX level), and the vertical axis indicates cell counts for each intensity value. Blue-colored regions indicate γ-H2AX positive fractions. (B) Another data set for the experiment described in Fig 2A. (C) The minimum processing time of H2O2 treatment required for γ-H2AX signals to appear was 10 minutes. HeLa parental cells were treated with 500 μM H2O2 for indicated periods before fixation. Broken lines show nuclei. Scale bars, 20 μm. (PDF)</p

Flow cytometric analyses of γ-H2AX levels in HU- or H₂O₂-treated HeLa cells.

(A) HeLa cells were treated with either HU (5 μM, 3 hours) or H2O2 (500 μM, 10 min (See S4C Fig for details)) and immediately fixed with 1% PFA. The fixed cells were double-stained with PI (for DNA content) and Alexa488-conjugated anti-γ-H2AX antibody, and subjected to flow cytometry. The horizontal and vertical axes of density scatter plots represent PI fluorescence and Alexa488 fluorescence, respectively. Two lines that divide the density scatter plots into four orthants (1st orthant, high DNA content, high γ-H2AX level; 2nd orthant, low DNA content, high γ-H2AX level; 3rd orthant, low DNA content, low γ-H2AX level; 4th orthant, high DNA content, low γ-H2AX level) were manually determined on the plot for Damage (-) KD control, and the same lines were applied to the other plots. Percentages of each sub-population are shown at the corners of the density plots. A 1D-histogram for PI fluorescence distribution is shown above each scatter plot. The vertical axis indicates the normalized (modal normalization) cell count. PI fluorescence signal values in each panel were adjusted so that the signal intensities for G1 peaks are the same among experiments. (B) γ-H2AX positive rates (percentage) for low-DNA content populations (f2/(f2 + f3)) and high-DNA content populations (f1/(f1 + f4)) were computed, where fi is a population fraction for the i-th orthant. Exp. #1 and #2 refer to sets of experiments shown in Fig 2A and S4 Fig, respectively.</p

<i>STN1</i> knockdown decreased the number of RAD51 foci in H₂O₂-treated nuclei.

The compiled data shown in Fig 6C are decomposed into the original two independent experiments, (A) and (B). (PDF)</p

CST is an upstream factor of RAD51.

(A) The experimental timeline. HeLa cells treated with H2O2 (500 μM, 2 hours) were then incubated for 2 days with DMEM containing RAD51 inhibitor B02 (20 μM final concentration) or the same volume of DMSO as a control. (B) Viabilities of H2O2-treated HeLa cells at t = 4 days measured by trypan blue staining. p-values for unpaired t-tests are shown above respective paired bars. Data from three or four biological replicates are shown with mean values (boxes) and SEM.</p