*2.4. Electrochemistry*

A single-line flow system was coupled with potentiostat using a flow cell provided by DropSens, for the SPCE electrodes (DRP110). The Electrochemical Flow Injection Analysis system (FIA-EC) used is composed of a four-channel Peristaltic Pump—MINIPULS® 3 (Gilson, Villiers-le-Bel, France), Injection Valve 77521 Rheodyne (with a 100 μL sample

loop), Flow Cell from DropSens (model DRP-FLWCL), and Potentiostat (Autolab PGSTAT 101). A boxed connector for screen-printed electrodes from Dropsens was used to connect the SPCEs. Each measurement was performed in triplicate.

#### *2.5. Determination of Apparent Rate Constants and Half-Lives*

The kinetics of PON decay and the scavenging effect of myoglobin on peroxynitrite at pH 9 were assessed using the calculation of half-lives and apparent rate constants of peroxynitrite decay. The static method was used for the measurements of concentrations over time, as pH 9 offers the possibility of having slower decays and changes dynamics toward specific chemical decay reactions. The terms "peroxynitrite" and "PON" are widely accepted for a mixture of ONOO− and ONOOH, depending on the pH. At pH 9, ONOO− is assumed to be in excess over the protonated form ONOOH, based on the pKa of PON 6.8. The term "peroxynitrate" refers to a mixture of O2NOO− and O2NOOH, depending on the pH. Trivial names: ONOO−, peroxynitrite; ONOOH, peroxynitrous acid; O2NOO−, peroxynitrate; O2NOOH, peroxynitric acid.

For the determination of the kinetic parameters, we took in consideration the previously proposed model by which the bimolecular decomposition of PON is dominant at pH 9, according to the following reaction [30,31]:

$$\text{HOONO} + \text{ONOO}^- \rightarrow 2\text{NO}\_2^- + \text{O}\_2 + \text{H}^+ + 2\text{e}^- \tag{1}$$

According to this model, the rate law should follow pseudo first-order kinetics at pH 9, due to the excess of ONOO−. Our aim was to validate the FIA-EC method as a tool for the determination of rate order and kinetic parameters by comparing the results with the classical UV-Vis method. In this particular case, the first purpose was to establish by both UV-Vis and FIA methods if the reaction follows indeed pseudo first-order kinetics or second/higher-order kinetics at pH 9.

Two approaches were used for the confirmation of rate order and the calculation of apparent rate constants and half-lives: the first method (namely called from now on "Method A") uses the plotting of all the integrated rate law data, according to the assumed rate order. Briefly, this was done as follows: the linearity (from the value of R2) of the graphs *ln(concentration)* (for (pseudo) first-order) or *1/concentration* (for second-order) vs. *time* and the correlation of the observed half-life (extrapolation from the graph t1/2obs) with the calculated half-life (t1/2calc). The half-lives for (pseudo) first-order and secondorder reactions were calculated with the following formulas, respectively: ln2/k and 1/k·C0PON. C0PON is the initial concentration of PON and k is the (apparent) rate constant. For the linearity, we considered the R2 values. If R2 approaches 1, is significantly higher than the R2 of the other model, *and* there is good similarity between t1/2calc and t1/2obs, the corresponding apparent order of the reaction is attributed to the detriment of the other apparent order. Each decay rate constant determination was plotted for a total of 180 s.

The second method (namely called from now on "Method B") was the "half-life method" described by Ira Levine, in the "Physical Chemistry" book, chapter 16, "Reaction kinetics" [32]. This method can be applied when the rate law has the form r = k[A]n. Based on Equation (1) and considering the excess of ONOO−, this method would follow the equation r = k[ONOOH]n, therefore determining the rate order in ONOOH. According to this method adapted to our particular case, one first plots the *concentration* vs. *time* and then should be able to fit the equation with a single-exponential decay function if n = 1 (based on Equation (2), where parameter k (the apparent pseudo first-order rate constant in our case, kobs) is solved after compilation. Secondly, one performs the extrapolation of t1/2 for various concentrations and then plots the *logt*1/2 vs. *logC*0 [32]. The slope of the logarithmic graph (that should have a linear fit) will establish the order of the reaction in reactant A, *n*, where *n* = 1 − *slope*. The two methods, A and B, were compared at the end.

$$\mathbf{C}\_{\rm{PON}} = \mathbf{C}\_{\rm{OPON}} \times \mathbf{e}^{-\rm{kt}} \tag{2}$$

where CPON is the PON concentration at a specific moment in time.

Both A and B kinetic methods were used for the UV-Vis method and for our proposed FIA-EC method. The molar extinction coefficient of 1670 M−1cm−1 was used to calculate the concentration of ONOO− at 302 nm [33,34].


*1.1. The Origins of Negligence*

There are twenty (tropical) diseases that are officially classified as 'neglected tropical diseases' by the World Health Organization (WHO) [2]. Neglected tropical diseases persist under conditions of poverty and are concentrated almost exclusively in impoverished populations in the developing world. They are: Buruli ulcer, Chagas disease, Dengue and Chikungunya (only WHO, not CDC), Dracunculiasis, Echinococcosis, Yaws, Fascioliasis, African trypanosomiasis, Leishmaniasis, Leprosy, Lymphatic filariasis, Onchocerciasis, Rabies, Schistosomiasis, Soiltransmitted helminthiasis, Cysticercosis, Trachoma, Scabies and other ectoparasites, Snakebite envenoming, Mycetoma and deep mycoses. These diseases are common in 149 countries, affecting more than 1.4 billion people (including more than 500 million children) and costing developing economies billions of dollars every year.

The importance of neglected tropical diseases has been underestimated since many are asymptomatic and have long incubation periods. The connection between a death and a neglected tropical disease that has been latent for a long period of time is not often realized. Additionally, neglected tropical diseases are often associated with some kind of social stigma, making their treatment more complex.

From the toxinology perspective, one can also consider that there are 'neglected' venomous and poisonous animals by employing very similar criteria to justify such negligence: Human accidents occur with individuals who are often amongst the poorest populations, living in remote, rural areas, urban slums or conflict zones; the accident causes no rapid death of the victim and/or such animals are stigmatized (cause bad luck, carry evil spells or are cursed).

Depending on the nature and origin of the venom or toxin, one can clearly perceive that there are 'preferred' subjects and matters in the field of toxinology (Table 1). Probably due to historical and/or epidemiological factors, some animals and venoms—normally the ones that elicit acute, severe lesions due to some pronounced biological activity—were selected (or elected) as 'more relevant' to the field and have been thoroughly studied throughout the years. Endemic animals, such as spiders and scorpions that have adapted to urban environments, have also 'deserved' more attention than other species. All the consulted databases indicated that there is more literature on snakes, spiders and scorpions (the triad) than the others. Interestingly, Scopus and Web of Science present the same publications ratio for triad:neglected (7.8), whereas Google and PubMed display lower ratios (5.5 and 1.8 respectively), probably due to the differences in indexed publications queried.

**Table 1.** Total results retrieved according to the searched terms in different academic databases.

Search performed in 11 September 2021. 1 Limited to skin, in order to exclude ecological studies; 2 Limited to toxin, in order to exclude developmental/reproductive models; 3 Limited to toxins and excluding dinoflagellates.

> The aim of this review is, therefore, to shed a light upon such amazing animals and their venoms and secretions, presenting a non-anthropocentric view of their venom composition and the (few, but consistent) biomedical 'cases' derived from the study of such species, and review the literature and the biotechnological developments derived from venoms and secretions from toxic animals that have not received proper attention from the scientific community over the past years and cast a light on their unique features and interesting molecules. Afterall, just like the neglected tropical diseases, it was never about the 'importance' of these animals, only their 'relevance', i.e., their economic impact, geopolitical localization, affected population, endangerment status and profit potential, in addition to formerly listed reasons.


**4. Stability of fixed points in delayed PWM switched converters**

In previous section, we show that the procedure based on variational equation can be used to compute Floquet exponents for any number of delays (*τ*). In this section, we compare this approach with other methods which determine stability in switched converters. One of them is the computation of characteristic multipliers based on the jacobian matrix. Another one is the computation of Lyapunov exponents using a numeric routine. Each method gives equivalent information. However Floquet approach is the most appropriated when delays appear since this method does not require the evaluation of the jacobian matrix (its dimension increases when the number of delays is higher). The other two methods have this disadvantage [13].