*<IP_ADDRESS>.6 Mitochondrial cytochrome oxidase*

Mitochondrial cytochrome oxidase is the last enzyme in the respiratory chain and detoxifies superoxide (O2 − ) [16]. Maeda et al. [17] have suggested that mitochondrial cytochrome oxidase is a useful marker enzyme for demonstrating sensory receptors in the periodontal ligament.


**1. Introduction**

Ankle sprains are among the most common of sports injuries, and lateral ankle sprains comprise more than 80% of these [1]. Even though most of them heal well with conservative treatment, a 56–74% recurrence rate has been reported [2]. Up to 30% of patients with lateral ankle sprains end up having chronic ankle instability (CAI) [3]. The predictors of patients with single or repeated ankle sprains who may develop CAI include a grade II–III sprain [4], postural instability [5], lower limb muscle weakness or imbalance [6], and decreased ankle dorsiflexion [7].

#### **1.1 Pathophysiology**

The pathophysiology of CAI includes anatomical and/or functional deficiencies. Anatomical factors consist of pathological laxity of ankle ligaments and problematic bony structures such as hindfoot varus [8, 9]. Functional factors include neuromuscular and proprioception impairments [6, 10].

The traditional spectrum of CAI is divided to mechanical instability, which means a structurally unstable ankle and functional instability, which means a perceptionally unstable ankle. Mechanical instability of the ankle can be demonstrated manually or subjectively. Functional instability is much more difficult to evaluate; a comprehensive questionnaire is usually needed for better communication and understanding [11].


**7. Biological magnification of radioactive material**

Radioactive material may become concentrated or "biologically magnified" during food chain transfer. Numerous biology and ecology text books include information on how living organisms take up nutrients pesticides and radioactive material and concentrate them. Because this concept is well known, we direct the reader to several early studies involving the concentration of radioactive material (See the work of Foster and Rostenbach, 1954; Hanson and Kornberg 1956; Davis and Foster 1958). Ophel (1963) reported a concentration of strontium 90 in perch flesh as 5x that of lake water while that in perch bone was 3000x! Additional information on radioecological concentration can be found in Auberg and Crossley (1958), Auberg and Hungate (1967) and Polikarpov (1966).


*3.3. In Vivo siRNA Delivery Performance of the Double-Tail Self-Assembling Dendron AD*

The final steps to assess the therapeutic potential of siRNA delivery based on the double-tail self-assembled amphiphilic dendron **AD** were constituted by in vivo experiments using prostate cancer (PC-3) xenograft mouse models and Hsp27 as the target gene for knockdown. Figure 12a,b reveal that, following a 5-week animal treatment by intraperitoneal injections (2/week) of siRNA/**AD** complexes (3 mg/kg Hsp27 siRNA, **AD** at N/P = 5), there was a remarkable downregulation of Hsp27 at both mRNA (62%) and protein (73%) levels. This was paralleled by an effective inhibition of tumor growth, as shown in Figure 12c. Further, as for the case of the single-tail amphiphilic analogue **4**, no major organ injury or histopathological changes were evidenced in the animal subjected to **AD**/siRNA treatments (Figures 12d and A2).

**Figure 12.** In vivo effective gene silencing of Hsp27 at the mRNA (**a**) and protein (**b**) levels observed in prostate cancer (PC-3) xenograft mice after treatment with Hsp27 siRNA delivered by **AD** (3 mg/kg siRNA and **AD** at N/P = 5, injection twice a week for 5 weeks). Hsp27 mRNA and protein levels were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Non-treated mice were used for the control. **AD** alone, siRNA alone and a scrambled (non-silencing) siRNA sequence delivered by **AD** were used as negative controls. (**c**) Tumor volume during treatment of PC-3 mice xenografts with siRNA/**AD** complexes (red symbols), compared with non-treated animals (green), and animals treated by AD alone (light blue), siRNA alone (dark blue), and a scrambled (non-silencing) siRNA sequence delivered by **AD** (gray symbol) used as negative controls. (**d**) Hepatic enzyme levels (alanine transferase ALT (light green bars), gamma-glutamine transferase γ-GLT (teal bars), (kidney functions (creatinine CRE (mmol/L, sky blue bars), blood urea nitrogen BUN (μmol/L, dark blue bars), and cholesterol level in the blood (gray bars, values on the right axis) measured in male C57BL/6 mice treated with intravenous administration of siRNA/AD (3 mg/kg siRNA and **AD** at N/P = 5). Untreated mice were used for control. Adapted from [6] with permission of John Wiley and Sons, 2014.

In summary, the nanosized micelles generated upon self-assembly of the double-tail amphiphilic PAMAM-based dendron **AD** were not able to deliver siRNA to a different panel of cancer cell lines with efficiency in vitro and in vivo comparable to those formed by its single-tail precursor **4**. However, they were also capable of eliciting gene silencing effects in the highly challenging and treatment-refectory human primary cells and stem cells. Accordingly, these versatile and safe delivery properties of **AD**, coupled with easy formulation, render AD a promising nanocarrier for highly efficient, cell-type-independent RNAi therapeutics.


*3.2. Prevalence of PA before and during the COVID-19 Pandemic*

Tables 1 and 2 demonstrate the number and proportion of individuals exposed to the coronavirus disease and engaged in PA at a sufficient level (≥150 min/week, according to theWHO recommendations) before and during the national coronavirus quarantine. As shown in Table 2, most students reported being inactive both before and during the COVID-19 quarantine. During the COVID-19 outbreak, 43% of university students were engaged in PA at the desired level. In comparison, 46% of students reported being active before lockdown. A Pearson's χ2 test of independence indicated that the differences between active and inactive participants significantly changed over time; χ2 (1) = 254.93, *p* < 0.001, φ = 0.41. The same status of PA before and during the coronavirus pandemic was reported by 457 active individuals (30.22%) and 613 (40.54%) inactive individuals. People active before the quarantine but becoming inactive during the COVID-19 pandemic numbered 246 (16.27%). Conversely, those inactive before the pandemic outbreak but becoming active during the coronavirus lockdown totaled 196 (12.96%). It suggests that students with a reduced level of PA during the COVID-19 pandemic outnumbered those becoming more active.