# Incidence of Phage Capsid Organization on the Resistance to High Energy Proton Beams
## 3 Results and Discussion
Figure 3: Susceptibility trend of pC89, 12 III1 and P9b to proton beam radiation in terms of survival percentage (**a**) and logarithmic viability reduction (**b**). also found in terms of the dose rate in the range 0.0615-0.615 Gy/s, as specified in Table 1. Interestingly, when evaluated in terms of logarithmic reduction as a function of the proton absorbed dose, the tested phages showed different trends (Figure 3b). In fact, pC89 showed a linear trend of the logarithmic reduction with increasing proton absorbed dose, whereas the two engineered phages exhibited a quadratic trend with a saturation phase of the log reduction at the maximum tested dose for 12 III1 and a lag phase at the lowest dose for P9b. These findings indicate that the action of resistance to the proton radiation was maintained in P9b up to the dose of 375 Gy, beyond which the phage began to lose vitality; on the other hand, 12 III1 showed a loss of vitality at all doses, even higher than that of pC89, revealing an almost absence of resistance already at the first exposure dose of proton radiation. It is known that coat proteins have a critical role both during the infectious process of entry and assembly of the new virion [45]. Consequently, the coat protein structure and its dynamicity during the virus reproduction cycle must be maintained. Our findings show that P9b did not suffer damage (including any lethal mutations) that would interfere with its correct infectious cycle. Based on Equation (3), we deduced the radiation damage (RD) for the proton beam by the logarithmic values of the phage survival. These findings are supported by the RD ranges for each phage clone between the minimum (50 Gy) and high levels (500 Gy), which are reported in Table 3.  
RD was very high in the case of 12 III1, arriving at values of the orders of \(35\times 10^{-3}\)/Gy at 50 dose Gy, and decreasing with increasing exposure doses. These findings agree with those from Figure 3 and confirm the almost total absence of resistance of 12 III1 to proton radiation, as evidenced by higher RD values at lower exposure doses. On the other hand, P9b showed a low and constant RD value, with a slight increase at the highest dose tested (500 Gy), revealing high resistance to all doses tested. Finally, an intermediate trend was observed for pC89, which showed the maximum RD value at the dose of 125 Gy. The RD trend of each phage as a function of the absorbed proton dose and the dose rate is shown in Figure 4.  
Figure 4: Radiation damage (RD) for each phage as a function of absorbed proton dose and the dose rate. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Phage Clone** & **50 Gy** & **125 Gy** & **250 Gy** & **375 Gy** & **500 Gy** \\ \hline pC89 & 3.1 & 13 & 8.0 & 6.5 & 6.95 \\ \hline P9b (QRKLAAKLT) & 2.28 & 2.99 & 2.86 & 2.75 & 4.83 \\ \hline
12 III1 (RWPPHEWHFDD) & 35.4 & 24.0 & 12.8 & 10.1 & 10.7 \\ \hline \end{tabular}
\end{table} 
Table 3: Radiation damage (RD, \(\times 10^{-3}\) Gy\({}^{-1}\)) for each phage at different doses (Gy) of 5 MeV proton irradiation. Results indicated that, while 12 III1 and pC89 reached the maximum RD at 35.4 and 13 dose Gy, respectively, P9b had still not reached the maximum RD value. Moreover, RD values from all phages tended to become closer at the maximum total dose. Since RD represents the logarithmic inactivation of phage titer per absorbed dose in Gy, the more susceptible phages will have high values at low doses, as in the case of 12 III1 and pC89, and a negative correlation as the dose Gy is increased, due to the fact that most of the phage titer has already been lost at the first doses. Along with those described above, these results indicated that the resistance to irradiations was probably dependent on the maintenance of the capsid structural integrity. The different behaviours of the three phages exposed to the proton beam could be correlated with a different degree of interaction between pVIII-pVIII proteins, due to the peptides exposed in their N-terminus.