Heating of the handrail surface to human-skin temperature with moderate humidity significantly impairs the survival of other pathogenic bacteria (<i>S</i>. <i>aureus</i>, <i>P</i>. <i>aeruginosa</i>, <i>A</i>. <i>baumannii</i>) as well as <i>E</i>. <i>coli</i> DH5α, and an inhibitor (2-AP) of NhaA, which regulates dry resistance, enhances the warming effect against some bacteria.

The experiment was performed in a thermo-hygrostat incubator. Temperature (T) (15°C) and humidity (M) (55%) were adjusted by the controller installed in the incubator. See the Materials and Methods. A. Survival rates of human pathogens (S. aureus, P. aeruginosa, A. baumannii) and E. coli DH5α on the handrail device warmed to human-skin temperature. Warming was performed under conditions of 15°C and 55% humidity (T 15°C/M 55%). Values show the ratio of CFUs between immediately after drying “0 h” and “18 h” after drying. Bars show the average ± SD. *, ppB. 3D structures of annotated protein sequences of E. coli NhaA (MMDB ID: 33841 PDB ID: 1ZCD) acquired from the NCBI Structure library (https://www.ncbi.nlm.nih.gov/), and the chemical structure of the specific inhibitor (2-aminoperimidine, 2-AP: PubChem ID: 112416) obtained from the PubChem Compound library at the same site. Yellow marks indicate binding sites for 2-AP. Among the pathogens used in the experiment, only S. aureus replaced “N64” with “A64.” Red ladder, extracellular membrane. Blue ladder, inner membrane. C. 2-AP enhances the warming effect on some bacteria (S. aureus and A. baumannii). See above. Bars show the average ± SD. $, pp<0.05, with a statistically significant difference when compared with those without 2-AP at “18 h after drying”.</p

Data sets obtained from experimental matrix analysis (temperature, humidity, and the survival rate of <i>E</i>. <i>coli</i>).

“Survival rate” indicates the value (number of live E. coli) for “T18” vs “T0.” (PDF)</p

Comparison of the number of live bacteria at each sampling site among the four groups classified by environmental factors.

Group 1 was low temperature and high humidity [total 108: n = 12 (each bar)], group 2 was high temperature and high humidity [total 135: n = 15 (each bar)], group 3 was low temperature and low humidity [total 144: n = 16 (each bar)], and group 4 was high temperature and low humidity [total 21: n = 21 (each bar)]. See the detailed sampling sites in S1 Table. Bars show the average ± SD. *, p (PDF)</p

Low temperature and humidity in hospitals increases the survival rate of bacteria on high-touch dry surfaces.

A. PCA scatter plot showing four groups (Groups 1–4) with distinct environmental conditions [temperature (T: T1–3), humidity (M: M1–3), and number of people (N: N1–3)] in three hospitals [designated H, K, and M]. PC1-factor loading shows 0.248(T1)/ 0.275(T2)/ 0.297(T3), ˗0.419(M1)/ ˗0.398(M2)/ ˗0.424(M3), and ˗0.306(N1)/ ˗0.265(N2)/ ˗0.312(N3) (N: number of people at that time). PC2-factor loading shows ˗0.425(T1)/ ˗0.415(T2)/ ˗0.383(T3) and ˗0.404(N1)/ ˗0.392(N2)/ ˗0.423(N3). As specified in the text, the data (66 sets) published in our previous manuscript were reused [19]. “O” ward, Obstetrics. “S” ward, Surgery. “I” ward, Internal medicine. “%,” cumulative contribution rates for PC1 and PC2. See S1 Table. B. Comparison of the number of live bacteria (CFU) on dry surfaces between Groups 1–4. Bars [Group 1 (n = 108), Group 2 (n = 135), Group 3 (n = 144), and Group 4 (n = 189)] show the average ± SD. *, pC. Comparison of the amount of ATP (as an index for the frequency of “human contact”) on dry surfaces between Groups 1–4. Bars [Group 1 (n = 117), Group 2 (n = 135), Group 3 (n = 157), and Group 4 (n = 189)] show the average ± SD.</p

Data sets (temperature, humidity, number of people, and the number of bacteria (CFU) / the frequency of human contact (ATP) in three city hospitals in Sapporo) reused from our previously published manuscript [19].

“CFU” indicates the number of live bacteria on dry surfaces in hospitals, “ATP” shows the human-contact frequency for these surfaces. (PDF)</p

Intermittent warming to human-skin temperature significantly reduced the number of spore-forming <i>B</i>. <i>subtilis</i>.

The experiment was performed in a thermo-hygrostat incubator. Cycle of temperature (22°C and 37°C) and humidity (RH: 45%-90%) were program-controlled by the controller installed in the incubator. See the Materials and Methods. A. Graphs show the schedule of intermittent warming and the temperature (left) and humidity (right) [blue: -B- (repeating 22°C with 45% humidity and 37°C with 90% humidity with an interval of 2 h)]. Fixed schedules were included as controls [red: -A- (22°C and 45%), green: -C- (37°C and 90%)]. B. Temperature and humidity monitoring with a data logger (AD-6324SET, AD Discover Precision, Tokyo, Japan). Red, temperature. Blue, humidity. Arrows indicate the timing of removal of the plate from the incubator. C. Effect of intermittent warming at human-skin temperature on decreasing the number of B. subtilis compared with E. coli. **, p (PDF)</p

Warming to human-skin temperature with moderate humidity significantly reduces the survival rate of human pathogenic bacteria on dry surfaces of a 96-well plastic plate.

The experiment was performed in a thermo-hygrostat incubator. Various combinations of temperature (T) (25°C -37°C) and humidity (M) (45%-90%) were adjusted by the controller installed in the incubator. See the Materials and Methods. A. PCA scatter plot showing the impact on the survival rate of E. coil of temperature (25°C–37°C) and humidity (45%–90%). PC1-factor loading shows ˗0.646 (T: temperature), ˗0.288 (M: humidity), and 0.707 (S: the survival rate of E. coli). PC2-factor loading shows ˗0.408 (T) and 0.913 (M). Six runs were performed for each combination of matrices. B. Comparison of the survival rate of E. coli on dry surfaces (18 h/0 h) with 45%–90% humidity at 25°C. Bars (n = 6) show the average ± SD. *, pC. Comparison of the survival rate of E. coli on dry surfaces (18 h/0 h) with 45%–90% humidity at 37°C. Bars (n = 6) show the average ± SD. *, p (PDF)</p

Differences in NF-κB activation and intracellular AA content between human leukemic and CB-CD34⁺ cells in the presence of high AA.

<p>A) Western blotting analysis of p-IκB in HL60 cells. Cells were treated with the vehicle or with high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There was a significant difference in the expression levels (*<i>P</i><0.001). Values represent the mean ± SD of triplicate samples. B) Immunocytochemical (left) and Western blotting (right) analyses of NF-κB in CB-CD34⁺ and HL60 cells. Cells were treated with vehicle or high AA for 1 h, then washed, cultured, and analyzed after 24 h. Note that translocation of NF-κB into the nucleus was markedly decreased in high AA-treated HL60 cells. Green and blue signals represent NF-κB and DAPI, respectively. Bars indicate 20 μm. There were significant differences in the expression levels (*<i>P</i><0.001, **<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples. C) Intracellular AA content of human leukemic cells and 2 different isolates of CB-CD34⁺ cells. Cells were treated with high AA for 1 h, washed in PBS, and analyzed immediately. There were significant differences in the content between leukemic and CB-CD34⁺ cells. *<i>P</i><0.001, as compared with CB-CD34⁺ cells (1) or (2). The values are mean ± SD values of triplicate samples.</p

Validity of the results that heating of handrails to human-skin temperature kills <i>E</i>. <i>coli</i> DH5α, evidenced by live/dead staining.

The experiment was performed in a thermo-hygrostat incubator with temperature 15°C and humidity 55%. Images show representative staining patterns (× 1,000). Area (red or green) was measured as the number of pixels by ImageJ software. Survival rate is shown as a ratio (T24/T0). Experiments were performed at least three times. Bars show the average ± SD. *, p (PDF)</p

Relationship between antileukemic effects of high AA and HIF-1α expression.

<p>A) Quantitative real-time PCR analysis of <i>HIF-1α</i> mRNA expression in K562 and K562-HIF1α cells. Cells were treated with the vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. After high AA exposure, <i>HIF-1α</i> mRNA expression significantly reduced in K562 (*<i>P</i><0.01), but not in K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. High AA exposure significantly reduced the HIF-1α protein level in both types of cells. However, the HIF-1α protein level in K562-HIF1α cells was significantly higher than that in K562 cells after vehicle or high AA exposure. *<i>P</i><0.01, **<i>P</i><0.0001, ***<i>P</i><0.00001. The values represent the mean ± SD values of triplicate samples. C) Flow cytometric measurement of apoptosis of K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 18 h. There was a significant difference in the number of apoptotic (annexin V⁺ propidium iodide (PI)⁺) cells between high AA-treated K562 and K562-HIF1α cells (*<i>P</i><0. 001). The values represent the mean ± SD values of triplicate samples. D) Flow cytometric measurement of cleaved caspase-3 expressed by K562 and K562-HIF1α cells. Cells were treated with vehicle (gray lines) or high AA (black lines) for 1 h, washed, cultured, and analyzed after 24 h. Activation of caspase-3 by high AA was lower in K562-HIF1α than in K562 cells. E) Western blotting analysis of Mcl-1, Bcl-x_L, and Bcl-2 in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.05) and between the vehicle-treated and high AA-treated K562 cells (**<i>P</i><0.0001). There was no significant difference between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. F) Western blotting analysis of Sp1, Sp3, Sp4, and VEGF. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels of these molecules between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.01, ** <i>P</i><0.0001). There were significant differences in the expression levels of Sp1, Sp3, and Sp4 between the vehicle-treated and high AA-treated K562 or K562-HIF1α cells (^†<i>P</i><0.01, ^††<i>P</i><0.001, ^†††<i>P</i><0.0001). There was a significant difference in the expression level of VEGF between the vehicle-treated and high AA-treated K562 (^†††<i>P</i><0.0001), but not between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05).</p