**4. Evaluation of fitness cost of insecticide resistance**

The main approach to investigate the fitness cost of resistance in field populations is to monitor the levels of resistance along the time in environments distinctly exposed to insecticides. Moreover, if the principal mechanism selected for resistance is known, the genotyping of resistance genes in place and time scales render important assumptions about their fitness cost. It is very difficult to access this kind of data from the field, however, since there are many variables occurring simultaneously.

For example, one population of *A. gambiae* from M'Bé, Côte D'Ivoire, used to be considered susceptible to most insecticides up to 2002, when a civil crisis broke and the monitoring was discontinued. Ten years later, a new study revealed important changes of the resistance mechanisms among *A. gambiae* populations from that locality. The main mechanisms that led them to become highly resistant to OC, PY, and CA were the L1014F *kdr* mutation and elevated activity of MFO and EST. The only well-known contexts that might explain this severe shift from susceptible to highly resistant were the pressure with deltamethrin-based products from rice paddles and the distribution of long-lasting PY impregnated nets (LLINs) since 2006 [56]. The alteration in the resistance profile over the time would suggest a low cost of the resistance alleles. However, little was known regarding the actual levels of insecticide pressure, migra‐ tion from vicinity areas, and about the extent of the influence of surrounding environment. In this case, controlled laboratory assays could help to estimate the fitness costs of the selected resistance mechanisms.

For fitness studies in the laboratory, population cage experiments can evaluate the fluctuation of resistance itself and the selected mechanisms over successive generations, under an environment clearly free of insecticide and without interference of migration. In this matter, the cost of resistance can be measured according to the velocity that the resistance alleles decrease in confined lineages along the time. A laboratory lineage of *A. aegypti* resistant to PY due to the NaVR2 *kdr* mutation4 presented deleterious effects in a series of life-trait parameters. Population cage assays corroborated these negative costs, showing that the *kdr* allele severely decreased from 75% to almost zero along 15 generations [57]. Most of the studies have been making use of an opposite direction: populations from the field are confined and submitted to a selection pressure in the laboratory. In another example, also with *A. aegypti*, populations

4 NaVR2 is the kdr allele mutant in both 1016 (Val to Ile) and 1534 (Phe to Cys) of the voltage gated sodium channel (NaV), found in American populations of *A. aegypti*.

from distinct Mexican localities were pressured with the PY permethrin for at least five generations in the laboratory. All the lineages had an increase in the resistance levels, corre‐ lated with an augment in the frequency of the Val1016Ile *kdr* mutation and with a number of detoxifying genes differentially transcribed, generally distinct at each lineage. Interestingly, the lineages that reached the highest frequencies of the *kdr* mutation presented a lower number of altered detoxifying genes [58]. These results strongly suggested that this *kdr* mutation had a lower fitness cost compared to the metabolic resistance genes occurring at each genetic background.

One has to consider that the evaluation of the overall fitness effects of a given mutation is very challenging, once it is difficult to separate their own effects from those caused by other mechanisms possibly coselected for resistance. In these aforementioned *rdl* examples, the reduced fitness might be related to the A302S mutation itself, and/or to metabolic resistance mechanisms. Similarly, the persistence of the resistance allele in an environment free of dyeldrin might be explained by the rdl cross-resistance with other insecticide that had been continually applied, as well as by the selection of modifiers genes, as previously discussed.

The main approach to investigate the fitness cost of resistance in field populations is to monitor the levels of resistance along the time in environments distinctly exposed to insecticides. Moreover, if the principal mechanism selected for resistance is known, the genotyping of resistance genes in place and time scales render important assumptions about their fitness cost. It is very difficult to access this kind of data from the field, however, since there are many

For example, one population of *A. gambiae* from M'Bé, Côte D'Ivoire, used to be considered susceptible to most insecticides up to 2002, when a civil crisis broke and the monitoring was discontinued. Ten years later, a new study revealed important changes of the resistance mechanisms among *A. gambiae* populations from that locality. The main mechanisms that led them to become highly resistant to OC, PY, and CA were the L1014F *kdr* mutation and elevated activity of MFO and EST. The only well-known contexts that might explain this severe shift from susceptible to highly resistant were the pressure with deltamethrin-based products from rice paddles and the distribution of long-lasting PY impregnated nets (LLINs) since 2006 [56]. The alteration in the resistance profile over the time would suggest a low cost of the resistance alleles. However, little was known regarding the actual levels of insecticide pressure, migra‐ tion from vicinity areas, and about the extent of the influence of surrounding environment. In this case, controlled laboratory assays could help to estimate the fitness costs of the selected

For fitness studies in the laboratory, population cage experiments can evaluate the fluctuation of resistance itself and the selected mechanisms over successive generations, under an environment clearly free of insecticide and without interference of migration. In this matter, the cost of resistance can be measured according to the velocity that the resistance alleles decrease in confined lineages along the time. A laboratory lineage of *A. aegypti* resistant to PY

Population cage assays corroborated these negative costs, showing that the *kdr* allele severely decreased from 75% to almost zero along 15 generations [57]. Most of the studies have been making use of an opposite direction: populations from the field are confined and submitted to a selection pressure in the laboratory. In another example, also with *A. aegypti*, populations

4 NaVR2 is the kdr allele mutant in both 1016 (Val to Ile) and 1534 (Phe to Cys) of the voltage gated sodium channel

presented deleterious effects in a series of life-trait parameters.

**4. Evaluation of fitness cost of insecticide resistance**

variables occurring simultaneously.

250 Insecticides Resistance

resistance mechanisms.

due to the NaVR2 *kdr* mutation4

(NaV), found in American populations of *A. aegypti*.

The knowledge of alterations in physiological and reproductive aspects is generally achieved by comparing life-trait parameters between susceptible and resistant individuals. As the result of pleiotropic effects of an altered gene will depend on the whole genomic structure, it is important that susceptible and resistant groups have the most similar genetic background as possible. The parameters usually evaluated are larval developmental time, adult longevity, ability to avoid predators, fecundity, fertility, mating competitiveness, and reproductive potential. When treating of blood-sucking insects, probing, acceptance of blood meal, and amount of ingested blood can also be tested. Such studies demand well-controlled conditions and are generally highly laborious, so that most of them follow few parameters at a time. In addition, the knowledge of the biology of the species under investigation is a prerequisite for the definition of which aspects would be more informative.

Fitness studies in the laboratory necessarily have to consider a well-representative collection from the field, in order to contemplate most part of the whole amplitude of variable traits from the original population. An F1 offspring of this sampling may then be raised in the laboratory to sufficiently amplify the number of individuals to be tested, as well as to normalize the physiological condition among the different populations. A laboratory lineage control of susceptibility and vigor should also be raised in parallel, as an endogenous control of experi‐ mental conditions, whenever possible.

Selection pressure for insecticide resistance in the laboratory has the advantage of controlled strength of selection and environmental conditions, population size, and absence of migration. On the other hand, if a monogenic key-mechanism for resistance was under lower frequency in the field, it is likely that this gene is not present in the sampling that established the first generation in the laboratory. For this reason laboratory pressures tend to result in polygenic resistance, where several resistance traits of minor effects are selected, but with a larger response when emerged together in the same genetic background [30]. This could also explain the different patterns of selected mechanisms to the same class of insecticide, especially metabolic resistance, in a same species.

Another important issue to be aware of when evaluating fitness costs in the laboratory environment is that most of the studies have investigated the possible life-trait alterations under optimal conditions. The amount and quality of food, the composition of substrate (or water in case of aquatic insects), density of individuals along life cycle phases, and mainly temperature and humidity are usually controlled. By contrast, insects are continually exposed to a wide range of abiotic or biotic stresses in the field. Therefore, the physiological costs of resistance alleles are probably underestimated in laboratory optimal conditions [41, 59]. The

evaluation of the fitness costs in resistant insects under stress conditions (in terms of nutrition, temperature, and larval density, for instance) can bring forth relevant data related to the evolution of resistance in the field. However, such investigations are still scarce [60-62].


*4.10. Urate Transport Assay*

The urate transport assay, with ABCG2-expressing plasma membrane vesicles, was conducted using a rapid filtration technique described in our previous studies [27,36], with some minor modifications. In brief, each plasma membrane vesicle (0.5 mg/mL) was incubated with 20 µM of radiolabeled urate in a reaction mixture (total 20 µL: 10 mM Tris/HCl, 250 mM sucrose, 10 mM MgCl2, 10 mM creatine phosphate, 1 mg/ml creatine phosphokinase, pH 7.4, and 50 mM ATP, or AMP as an ATP substitute) for 10 min at 37 ◦C. After incubation, the reaction mixture was mixed with 980 µL of ice-cold stop buffer (2 mM EDTA, 0.25 M sucrose, 0.1 M NaCl, 10 mM Tris-HCl, at a of pH 7.4); the resulting solution was rapidly filtered through an MF-Millipore Membrane (HAWP02500; 0.45 µm pore size and 25 mm diameter; Millipore). After washing with 5 ml of ice-cold stop buffer five times, the plasma membrane vesicles on the filter were dissolved in Clear-sol II (Nacalai Tesque). Then, the radioactivity in the plasma membrane vesicles was measured using a liquid scintillator (Tri-Carb 3110TR; PerkinElmer, Waltham, MA, USA).

The urate transport activity was calculated as the incorporated clearance (µL/mg protein/min) defined as the incorporated level of urate [disintegrations per minute (DPM)/mg protein/min]/urate level in the incubation mixture [DPM/µL]. ATP-dependent urate transport was calculated by subtracting the urate transport activity in the absence of ATP from that in the presence of ATP; ABCG2-mediated urate transport activity was calculated by subtracting ATP-dependent urate transport activity of control plasma membrane vesicles from that of ABCG2-expressing plasma membrane vesicles.


*Article* **Discrimination of Spatial Distribution of Aquatic Organisms in a Coastal Ecosystem Using eDNA**

### **Hyunbin Jo 1, Dong-Kyun Kim 1, Kiyun Park 1 and Ihn-Sil Kwak 1,2,\***

Received: 4 July 2019; Accepted: 15 August 2019; Published: 21 August 2019


5. Conclusion

We propose an integrated dynamic treatment of emissions from LULUC, caused by domestic agricultural production, and from iLULUC that is linked to international agricultural commodity trade, which may be used in LCA frameworks and other assessment methods that include GHG emissions accountings. iLULUC effects are accounted for which are induced by countries with increasing demand for certain agricultural commodities. LULUC emissions are not only caused by growing national agricultural land use, but also by the growth of builtup areas. Indirect LULUC emissions related to an increase in net agricultural imports represent the balance of (a) (positive) iLULUC emissions from import increases and (b) (negative) dLUC emissions from exported commodities. Our model thus reflects a dynamic rather than a static perspective of agricultural commodity production and trade—it uses the increases of production, exports, and imports in place of their absolute values.

Indirect LULUC factors are derived by converting data on agricultural commodity production and trade to the commodity's corresponding energy content on an LHV basis. A (hypothetical) global iLULUC pool reflects the global interconnectedness of agricultural commodity trade; national iLULUC emissions may be derived from it and represent the LULUC emissions inherent in the traded products.

Our results account for the allocation of emissions to specific product groups consumed in a country in proportion to their corresponding energy content on an LHV basis. This allows for the aggregation of agricultural product group data on different spatial levels, and it provides a more detailed focus compared to generic agricultural land-related emission estimates. With this approach, 3150 new results from 175 countries are provided with the respective indirect (LU)LUC effects. The results vary substantially between nations, with clear differences between producing and exporting countries versus importing countries. A similar differentiation applies to specific product groups within a country.

LUC-related GHG-accounting should rest on a well-documented computational basis as a prerequisite for a fair differentiation of "LULUC-emitting/exporting nations" versus "LULUC-importing nations" on the one hand and between (LU)LUC-driving product groups versus product groups with little or no effects on LULUC emissions on the other. Further work should address the validation and improvement of the model and its input data.


2.6.4. Wettability

The wettability test was carried out according to Stoltenberg and Breitkreutz [29]. A 96-well plate (Biologix Group Limited, Lenexa, KS, USA) was used and a cellulose filter disc was placed in each well, which was then moistened by adding 20 μL of 0.3% aqueous solution of methylene blue. The time from placing the ODMT on moistened paper to complete coloring of the MT matrix was noted.

#### 2.6.5. Differential Scanning Calorimetry

RUP raw material (API), microparticles placebo (MP AQ placebo, MP SUR placebo), microparticles (MP AQ RUP, MP SUR RUP), ODMT placebo (F1, F5, F9, F13), ODMT with pure RUP (F2, F6, F10, F14) and ODMT with RUP enclosed in SUR MP (F3, F7, F11, F15) and AQ MP (F4, F8, F12, F16) were tested using thermal analyzer system (DSC Mettler Toledo, Greifensee, Switzerland). Swatches were accurately weighed (5 mg), inserted in pans made of aluminum, then warmed up to 300 ◦C with 10 ◦C/min rate with 20 mL/min flow of nitrogen.

#### *2.7. Evaluation of Taste Masking E*ff*ectiveness*

#### 2.7.1. In Vivo

The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of Medical University of Białystok approval number R-I-002/438/2016. The efficiency of taste masking level was tested by six probands undergoing a test conducted as follows: five ODMTs were placed in the oral cavity for 30 s (the maximum time to dissolve/disintegrate in accordance to FDA guidelines [30]), spitted and mouth were rinsed with water. Sensory evaluation was marked as follows: 0—no bitterness, 1—slightly bitterness, 2—moderately bitterness, 3—significant bitterness. Before the experiment was carried out, the participants were chosen on the basis of sensory sensitivity test, utilizing four main flavors [31].

#### 2.7.2. RUP Dissolution

RUP dissolution was carried out in apparatus II (paddle) (Erweka Dissolution Tester DT 600HH, Heusenstamm, Germany) with phosphate buffer (pH 6.8, 50 mL) imitating natural spit in following conditions: 75 rpm and 37 ◦C (+/−0.5). The quantity of dissolved RUP was assessed as pointed in 2.5.3.

#### 2.7.3. Electronic Tongue

#### Reagents and Membrane Materials

Analytical reagent grade chemicals and purified water with 0.07 μS/cm conductivity (Elix Advantage System Mili-Q plus Milipore, Spittal an der Drau, Austria) were used. The membrane consisted of poly(vinylchloride) (PVC) (Tarwinyl, Tarnów, Poland); bis(2-ethylhexyl) sebacate (DOS), and o-nitrophenyl octyl ether (o-NPOE) (Fluka, St. Gallen, Switzerland); potassium tetrakis [3,5-bis(trifluoromethyl)phenyl]-borate (KTFPB), tridodecylmethylammonium chloride (TDMAC), 1-dodecylpyridinium chloride (DDPC), (Sigma—Aldrich, St. Luis, MO, USA), and potassium tetrakis(p-chlorophenyl)borate (KTpCPB) (Fluka, St. Gallen, Switzerland); calix[6]arene-hexaacetic acid hexaethylester (amine ionophore I) (Fluka, St. Gallen, Switzerland), and 3-mercapto-5-/2-hydroxynaphthyl-azo-triazole (METRIAN) (Department of Drug Chemistry, Medical University of Lublin, Poland).


**8. Acknowledgment**

The research was supported by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation, HA 1400/14-1-4) and the Jacobs Foundation, Zürich (Switzerland). I am very grateful to the families for participating in the studies and to Mrs Joswig-Gröttrup and Mrs Hamilton Kohn, Jugendamt der Stadt Braunschweig, Department of Preschools, as well as to all preschool teachers for their very good cooperation. My warmest thanks also to Dr. Kurt Hahlweg, PhD Frank C. Verhulst and Dr. Nina Heinrichs for their comments on earlier versions of this manuscript.

#### **9. References**

156 Epidemiology Insights

Furthermore, a teacher perspective could add valuable information about problem behaviour at school, which might possibly result in reports of more externalizing problems. Due to principles of data collection, frequent change of teachers, and the transition from kindergarten to primary school it was only possible to obtain the parental report. For international comparison of the results, the age at school entry in Germany,

To sum up, the study contributes to a more complete understanding of externalizing behaviour problems and their continuity from kindergarten to primary school. The results point to the need for early child psychiatric research on child mental health beginning in infancy and the preschool years. The development of problem behaviour in specific clinical or risk groups may differ from the pattern found in the present data. An increased utilization of child mental health services by older children, who already show disturbances in the preschool years, has important implications for early preschool recognition of child mental health problems and indicates the need for the prevention and development of a differentiated delivery of child mental health services. Clinicians working in primary care, day care, or school systems need to be attentive to opportunities for early detection and intervention regarding preschoolers' externalizing behavioural problems, particularly since efficacious prevention and treatment exists for the psychopathology of young children (e.g.

Since the first child psychiatric epidemiological studies in the 1950´s and 1960´s, epidemiological research has provided a wealth of empirical findings that may aid develop strategies for improving the mental health outcomes of children. Descriptive epidemiological data on prevalence rates, historical trends, and outcomes of mental health problems can help planning mental health services for children and provide evidence for setting priorities when resources are limited. Etiologic epidemiological research forms the basis for prevention interventions by unravelling the causative mechanisms in the development of psychopathology. Clinical epidemiological strategies are important for more evidence-based approaches to diagnostic assessment and intervention strategies, and outcome research may help improving the quality of mental health services. Finally, more efforts should be put into improving partnerships between epidemiological researchers and prevention specialists and between epidemiological researchers and policy-makers for improving strategies for preventing and treating mental health

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**7. Conclusion**

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**8. Acknowledgment**

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**9**

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**Epidemiology of Tics**

Blair Ortiz, William Cornejo and Lucía Blazicevich

*Child and Adolescent Diseases Research Group PEDIACIENCIAS*

Tics are the most frequent movement disorders during childhood; their highest prevalence occurs at scholar and adolescence-ages. Most tics are transient but some of them become

Moreover, some tics are related to disorders with significant impact, like attention-deficit hyperactivity disorder (ADHD), obsessive-compulsive (OCD) and affective disorders. Epidemiological studies are the initial approach to diagnose them and properly begin

In this chapter, we explained the clinical bases of tics, review the more representative epidemiological studies that assess the prevalence of tics and make a critical standpoint of

**2.1 Definition of tics and their practical ways to characterize them since clinical view**  As we described elsewhere, tics are defined as "sudden, rapid, recurrent, nonrhythmic, stereotyped motor movements or vocalizations"and have clinical concerns1.Tics are likely to differ in terms of the body location, number, frequency, complexity, intensity or forcefulness, noticeable and resulting social consequences2. Intervals between tics can range from seconds, hours or even days. The forcefulness with which a tic is performed can range from slight and barely noticeable to intense and obvious. Over time, the frequency and intensity of tics may wax and wane and maybe influenced by a variety of internal and external stimuli including private events, contextual variables and social reinforcement

Tics can also vary considerably in their complexity. Tics that involve the contraction of a single muscle group are typically referred as simple tics and those that involve the contraction of multiple muscle groups are typically considered complex. Simple tics are typically of very short duration (*i.e.* 1 second) and include such behaviors as eye blinking; jerking of the face, head, torso, or limbs; coughing; sniffing; throat clearing and making singles syllable sounds. Complex tics are often sustained for longer durations or occur in paroxysms and can include virtually any orchestrated pattern of behavior otherwise

chronic having educational, familial and social negative implications.

these studies about epidemiology applicable to clinical practice.

**1. Introduction**

treatment.

**2. Content**

contingencies3.

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**1. Introduction**

Globally, malaria control progress is plateauing, and, in some instances, case numbers are rising [1]. Although the reasons for this are multifaceted, an increasing and intense resistance to pyrethroids in *Anopheles* vectors is almost certainly a contributing factor. Insecticide-treated nets (ITNs) have significantly contributed to the control of malaria over the past two decades [2]. However, currently, all WHO-prequalified ITNs contain pyrethroids [3], and pyrethroid resistance is widespread in all major malaria vectors [4,5].

In response to growing concerns over the sustained effectiveness of solely pyrethroidbased control tools, new products are being developed and evaluated. Examples of these are dual-active ingredient (AI) ITNs containing an additional insecticide, or synergist ITNs which contain an insecticide synergist, in combination with a pyrethroid. These net types are often termed 'next-generation' insecticide-treated nets. The second AIs have a different mode of action (MoA) from their partner pyrethroid, to improve the control of resistant vector populations.

The current methods for measuring ITN durability [6] were developed for pyrethroidonly nets, which cause rapid knockdown and death in susceptible mosquitoes. Consequently, the different MoAs of the new insecticides necessitate the need for new protocols to reliably measure net durability. In nets with the synergist piperonyl butoxide (PBO), the PBO works by improving the efficacy of the pyrethroid it is paired with, in populations with pyrethroid resistance due to increases in oxidase activity, and is itself generally noninsecticidal. Without suitable mosquito strains or net controls, it is difficult to determine if the synergist component of the net is long-lasting using the currently recommended methods. For other AIs, such as chlorfeniapyr, which targets the insect mitochondria, or pyriproxyfen, which is a juvenile hormone analogue, 'non-standard' endpoints such as delayed mortality and insect fertility and fecundity need to be measured to assess biological durability (bioefficacy, measured through direct impact on mosquitoes).

Several of these new types of ITN are being evaluated in large-scale randomized control trials (RCTs) and pilot deployment schemes. These trials are expected to demonstrate the biological durability, attrition, and fabric integrity of these new net types when under long-term household use. Measuring the biological durability of the ITNs involves assessing the insecticidal activity of a sub-sample of randomly selected nets withdrawn from the field. There is an urgent need for methods to reliably measure the bioefficacy of these nets, to collect baseline data, and to subsequently measure the durability of biological efficacy of nets collected from the field after fixed periods of use. This has resulted in methods for measuring net bioefficacy and biological durability being developed and utilized by multiple programme teams, which makes comparing the results of these studies complex. A better approach would be for programme teams to adopt a single, standardized method validated using a multi-site approach.

In this publication, we demonstrate the process used to collate and interrogate several different methods to produce a singular 'consensus standard operating procedure (SOP)', for evaluating the biological efficacy of new net types, suitable for durability monitoring. Our objective was to create procedures that build on the experience from studies already underway. We also considered the feasibility of conducting these methods in as many sites as possible, accounting for factors such as throughput of mosquito colonies and space, which can preclude the use of certain methods and inform choices about sample sizes and replicate numbers.

This project forms part of a package of work to improve entomological methods in vector control and is supported by Innovation to Impact (I2I) at the Liverpool School of Tropical Medicine (LSTM). Three new types [7] of ITN are used as case studies: pyrethroid + piperonyl butoxide (PBO), pyrethroid + pyriproxyfen (PPF), and pyrethroid + chlorfenapyr (CFP). The final consensus SOPs for measuring the biological durability of these net types are included in Additional Files 2–4 (Supplementary materials).

#### **2. Materials and Methods**

For each net type, a collaborative process of method development and iterative drafting was conducted to produce a consensus SOP (Figure 1). Initially, a group of stakeholders was formed. Inclusion in these groups was based on having (1) a research interest in the development or deployment of new net types, (2) experience in the development or testing of new net types, or (3) an involvement in ongoing trials or deployment schemes of new net types. Available methods for measuring the biological durability of each net type were then identified through consultations with stakeholder groups and literature searches. This was not a systematic process, and for each net type, several historical procedures exist which were not considered here. Rather, the focus was to identify SOPs currently being developed or utilized which evaluated the biological durability of new net types and to use them to align the methods on points of difference. For each net type, the experimental parameters of the method were established (i.e., exposure method, controls used, population, replicates, endpoints). Values for each parameter were extracted from all accessible methods and compared before a 'consensus value' was suggested for each experimental element. Other methodological questions were identified for discussion. At this stage, the method development document was shared with the stakeholder group for comment, and further discussed on a group call. The feedback on the method development was then used to prepare a draft consensus SOP. The draft was distributed with the group for a second round of comments and discussion. Following the incorporation of this feedback, a final consensus SOP was produced and submitted to the group for approval.

**Figure 1.** Infographic showing the process of method development used for producing consensus SOPs for biological durability monitoring of new net types.

#### **3. Case Study 1: ITNs Containing Pyrethroid plus Piperonyl Butoxide (Pyrethroid + PBO Nets)**

Currently, six pyrethroid + PBO nets are prequalified by the WHO (DuraNet Plus, VEERALIN, PermaNet 3.0, Tsara Boost, Tsara Plus, Olyset Plus) [3]. These vary in several

specifications (Additional File 1: Table S1) such as pyrethroid AI, PBO concentration, and location of PBO on the net (roof only or on all panels). A conventional cone test, followed by a tunnel test for those nets which fail to reach cone bioassay thresholds [8], is suitable for exposing mosquitoes to pyrethroid + PBO nets and monitoring mortality. Certain methodological parameters of the WHO cone test, such as replicate number and control nets, vary depending on if the assay is being used for WHOPES (the precursor to WHO prequalification) phase I, II, or III testing. The WHO guidance states "candidate LNs (nets) treated with insecticides with effects on mosquitoes that differ from those of pyrethroids may require proof of principle and new assays" [8]; however, guidance or thresholds on how to interpret PBO-synergism for biological durability monitoring is not available.

Nine methodologies that measure pyrethroid + PBO net biological durability were identified through searching the literature and contacting key stakeholders (Table 1). Of these, methods were accessible for six of them (published or provided on request). Of the remaining three, one study had not yet finalized its methods (ID = 7), one confirmed it was not conducting biological durability monitoring (ID = 8), and one did not have biological durability monitoring listed as an intervention endpoint on its clinical trial registry; the authors were contacted to confirm this, but they did not respond (ID = 9). Values for each methodological parameter were extracted from the accessible SOPs and a 'consensus' value suggested for each parameter (Table 2). It was established that one method (ID = 2) was an updated version of another (ID = 1), so study #2 was later excluded.

**Table 1.** List of identified methods/trials measuring pyrethroid + PBO net biological durability.

Abbreviations: DM = Biological durability monitoring; LSTM = Liverpool School of Tropical Medicine; NIMR = Nigerian Institute of Medical Research; NNP = New Nets Project; PMI = President's Malaria Initiative; PSI = Population Services International; SOP = Standard operating procedure.

*3.1. Other Methodological Considerations Identified*

**Table 2.** Methodological parameters extracted from pyrethroid + PBO net biological durability monitoring methods. Methods were compared

*3.2. Changes Made to the Proposed Pyrethroid + PBO Methods following Stakeholder Discussions*

Following feedback from stakeholders, a final consensus SOP was produced and approved by the group (Additional File 2: I2I-SOP-001: Methods for monitoring the biological durability of insecticide-treated nets containing a pyrethroid plus piperonyl butoxide (PBO)).

#### **4. Case Study 2: ITNs Containing Pyrethroid plus Pyriproxyfen (Pyrethroid + PPF Nets)**

Royal Guard, developed by Disease Control Technologies, is currently the only WHO prequalification listed pyrethroid + PPF net (Additional File 1: Table S2). The WHO cone test is a suitable method for exposing mosquitoes to pyrethroid + pyriproxyfen (PPF) nets for measuring the nets' biological durability, but different endpoints are needed for each active ingredient. Knockdown and mortality can be used to assess the bio-efficacy of the pyrethroid but the most suitable endpoints for PPF, a juvenile hormone analogue that affects fertility and fecundity in mosquitoes, need to be defined.

Seven documents detailing methods for evaluating pyrethroid + PPF nets were provided by stakeholders (Table 3). One of these (ID = 1) did not measure fertility endpoints. Of the remaining documents, four detailed methods for oviposition observations, and two detailed methods for ovary dissection.

**Table 3.** List of identified methods/trials measuring pyrethroid + PPF net biological durability.

Abbreviations: CNRFP = Centre National de Recherche et de Formation sur le Paludisme; CREC = Centre de Recherche Entomologique de Cotonou; DC = Diagnostic concentration; IRD = Institute of Research for Development; KCMUCo = Kilimanjaro Christian Medical University College; LSHTM = London School of Hygiene and Tropical Medicine; LSTM = Liverpool School of Tropical Medicine; PPF = Pyriproxyfen; SOP = Standard operating procedure.

To reach a consensus SOP for both methods, methodological parameter values were extracted from available SOPs and a 'consensus' value was proposed for each one (Oviposition: Table 4; Dissections: Table 5). Methods for both oviposition and dissection are included, as discussions showed differences in preference between labs for one or the other method (Figure 2).

**Figure 2.** Infographic showing the methodological process for measuring sterility via scoring oviposition using chambering, following exposure to pyrethroid + PPF nets.

*Insects* **2022**, *13*, 7

**Table 4.** Methodological parameters extracted from pyrethroid + PPF net biological durability monitoring methods, which scored mosquito

nets and seems to be a suitable method of exposure.

This is the standard exposure time used in WHO cone bioassays [6]. Preliminary validation testing will be conducted to look at effect of exposure time.

Exposure time Not included in SOP 3 min 3 min

*Changes Made to the Proposed Methods following Stakeholder Discussions*

Following feedback from stakeholders, a final consensus SOP was prepared and approved by the group (Additional File 3: I2I-SOP-002: Methods for monitoring the biological durability of insecticide-treated nets containing a pyrethroid plus pyriproxyfen (PPF)).

#### **5. Case Study 3: ITNs Containing Pyrethroid plus Chlorfenapyr (Pyrethroid + CFP Nets)**

Interceptor G2 (IG2), developed by BASF, is currently the only WHO prequalification listed pyrethroid + CFP net (Additional File 1: Table S3). The cone test has been shown to be ineffective in reliably measuring the bioefficacy of the chlorfenapyr component of IG2 nets [16], and so an alternative bioassay is needed. There is a growing consensus around the WHO tunnel test as being the best method to assess IG2 bioefficacy. This should be run in parallel with a standard WHO cone test [6], which assesses the biological durability of the alpha-cypermethrin component of the net. The SOP discussed and included (Additional File 4) here is related to assessing the biological durability of the CFP component.

Eight documents, detailing methods used for evaluating pyrethroid + CFP nets, were provided by stakeholders (Table 6). Of these, three were generic SOPs for conducting the 'net in tube' cylinder assay (ID = 6) or tunnel test (ID = 7, 8), and did not contain specific experimental parameters for testing CFP nets, and, therefore, information was not extracted from them for comparison. Methodological parameters were extracted from the available SOPs, compared, and used to propose a 'consensus' value for each (Table 7).

**Table 6.** List of identified methods/trials measuring pyrethroid + CFP net biological durability.

Abbreviations: CFP = Chlorfenapyr; cRCT = Cluster Randomized Control Trial; CREC = Centre de Recherche Entomologique de Cotonou; IHI = Ifakara Health Institute; KCMUCo = Kilimanjaro Christian Medical University College; LSHTM = London School of Hygiene and Tropical Medicine; LSTM = Liverpool School of Tropical Medicine; NNP = New Nets Project; PAMCERC = Pan-African Malaria Vector Research Consortium; PMI = Presidents Malaria Initiative; SOP = Standard operating procedure.

**Table 7.** Methodological parameters extracted from pyrethroid + chlorfenapyr net biological durability-monitoring methods. Methods

*Changes Made to the Proposed Pyrethroid + CFP Methods following Stakeholder Discussions*

	- a. Data comparing the use of 50 vs. 100 mosquitoes in tunnels with pyrethroid nets are available (Moore, Personal communication), and these data were considered to confirm the number of mosquitoes tested.
	- b. Further to this, preliminary work to compare 50 vs. 100 mosquitoes in tunnels using Interceptor net and Interceptor G2 nets was conducted, and found no significant difference in these two numbers (Kamande, Personal communication).

Following feedback from stakeholders, a final consensus SOP was produced and approved by the group (Additional File 4: I2I-SOP-003: Methods for monitoring the biological durability of insecticide-treated nets containing chlorfenapyr).


*2.3. Preparation of BaTiO3*/*Polymer*/*Al (BPA) Films*

To determine the possibility of the as-obtained BaTiO3 nanocrystals to form a uniform film for capacitor energy-storage application, we chose sample S8 (in Table 1) as an example to prepare BaTiO3/polymer/Al films (BPA films, Figure 2) using the similar method reported in our previous work [25]. Typically, the BaTiO3 nanocrystals (S8) were mixed with a silicon-containing heat-resistant resin (CYN-01), and then some silane coupling agent (KH550) was added into the above mixture. Dimethylacetamide (DMAc, Guangzhou Jinhuada Chemical Reagent Co., Ltd., Guangzhou, China)) was used as the solvent. The mass ratio of *M*BaTiO3:*M*DMAc:*M*Polymer:*M*KH550 was kept at 100:45:25:4. The as-prepared mixture was ultrasonically treated for 30 min for a uniform slurry. The above slurry was coated on an Al foil by a bar coater (T-300CA) and a coating rod (D10-OSP010-L0400) from Shijiazhuang Ospchina Machinery Technology Co., Ltd (Shijiazhuang, China)). The as-formed films were then dried in an oven at 220 ◦C for 10 min and finally used for the test of dielectric properties.

**Figure 2.** A schematic diagram of the BaTiO3/polymer/Al film for capacitor cells.


**5. Future directions**

There are 4306 predicted *E. coli* K12 protein sequences present in the UniProt proteome database (http://www.uniprot.org/proteomes/) [129]. An initial analysis of their compartmentalization within the cell using the prediction software TOPCONS2 (http://topcons.cbr.su.se/ pred/) [130] allowed us to putatively assign each of these proteins to one of three subcellular compartments: cytoplasmic, transmembrane in the inner membrane (referred to as transmembrane hereafter), or secreted. To hone in on proteins exhibiting possible oxidoreductase activity, the CXXC motif was used as a signature to identify 406 proteins, which showed that approximately 10% of all predicted *E. coli* proteins contain this motif, thereby demonstrating its relative ubiquity. Of these 406 proteins, ~75% are cytoplasmic, ~18% are transmembrane, and ~7% are secreted (see **Table 2**). The pool of non-CXXC-containing proteins comprises the remaining 3900 proteins, of which ~63% are cytoplasmic, ~23% are transmembrane, and ~14% are secreted (omitted from **Table 2**). The transmembrane and secreted compartments have a lower fraction of CXXC-containing proteins in keeping with the exclusion of cysteine residues from these compartments in aerobes [77]. A comparison of the non-CXXC sequence pool with the CXXC sequence pool shows a slight enrichment of CXXC proteins in the cytoplasm (~75%) versus non-CXXC cytoplasmic proteins (~63%). The distribution of CXXC and non-CXXC proteins in the transmembrane is similar (18 and 23%, respectively); however, about twice as many non-CXXC proteins are secreted (14%) compared to CXXC proteins. Approximately 22% (90 of 406) of CXXC proteins are annotated in the UniProt data as binding metal ions or as iron-sulfur cluster-containing proteins. While 46% of all CXXC proteins have been functionally characterized, the remaining majority (54%) should be characterized to develop a better understanding of the reactions they catalyze, how those identified to be oxidoreductases may contribute to the redox biology of bacteria, and to identify novel targets for therapeutics.

Secreted refers to proteins in the periplasm and secreted outside of the cell. Compartment location was predicted using topological and signal sequence input data on the TOPCONS server. Gene ontology (GO) codes EXP and IDA were used to identify proteins with experimentally verified function from the UniProt database; those lacking these codes were defined as having unknown function. GO codes were also used to identify CXXC proteins annotated to bind metals [129].

**Table 2.** The *E. coli* proteome separated by compartment, the presence of CXXC motifs, and known function.


**3.3 Measurement of the Vesicular Monoamine Transporter (VMAT) 2**

In neurodegenerative diseases there are typically characteristic losses of particular types of neurons in the human brain. As outlined above, progressive losses of dopaminergic neurons is the hallmark of Parkinson's disease. Reflecting this, a number of strategies have been developed for *in vivo* imaging of such neuronal losses. One such approach involves targeting the vesicular monoamine transporter type 2 (VMAT2) using radioligands such as (+)-α-[11C]dihydrotetrabenazine ([11C]DTBZ: (2*R*,3*R*,11b*R*)-(1,3,4,6,7,11b-hexahydro-9- [11C]methoxy-10-methoxy)-3-(2-methylpropyl)-2-hydroxy-2H-benzo[a]quinolizine) (K. A. Frey, et al., 2001). The VMAT2 is not specific for any monoamine, but is a common protein capable of transporting dopamine, norepinephrine, serotonin and histamine (Eiden and Weihe, 2011). Despite this non-specificity, the utility of VMAT2 imaging in neurodegenerative disease is still possible due to the compartmentalization of neuronal types in the human brain (K. A. Frey, et al., 2001). For example, dopaminergic nerve terminals predominate in the basal ganglia, and so enable specificity for examining losses of such terminals in PD patients (Figure 3). The VMAT2 is found in presynaptic vesicles, and transports monoamines from the cell cytosol into the storage vesicle, from where they can be released into the synapse (Wimalasena, 2011).

Lee and colleagues conducted a comparison between [11C]DTBZ, [18F]DOPA and [11C]methylphenidate (a radiopharmaceutical targeting the dopamine transporter (DAT)) (C. S. Lee, et al., 2000). Reflecting the upregulation of aromatic amino acid decarboxylase, and concomitant down regulation of the DAT, that occurs to increase dopamine turnover and reduce its reuptake in Parkinson's disease patients, this study found that [18F]DOPA Ki was reduced less than the [11C]DTBZ binding potential in the PD striatum, and [11C]DTBZ binding was reduced when compared to [11C]methylphenidate binding. The authors suggest that [11C]DTBZ PET is the most reliable method for quantifying dopaminergic terminal density although, per Pavese and Brooks (Pavese and Brooks, 2009), this needs to be validated and the effect of dopaminergic drugs upon [11C]DTBZ uptake determined.

Reflecting the drive to convert short lived carbon-11 labeled radiopharmaceuticals (t1/2 = 20 min) into longer lived fluorine-18 labeled analogs (t1/2 = 110 min) to facilitate distribution to satellite PET centers that do not own a cyclotron, [18F]FP-TBZ ([18F]AV-133) has also been developed to image the VMAT2, and is licensed to Avid Radiopharmaceuticals (H. F. Kung, et al., 2008). In studies by Frey and colleagues, AV-133 PET of normal and PD patients were compared (K. A. Frey, et al., 2008). Findings were similar to [11C]DTBZ, and AV-133 PET provided excellent images of the VMAT2. All PD patients had severe reduction of AV-133 accumulation in the striatum, most severe in the PP contralateral to worst PD symptoms. Similar findings were confirmed in further studies by Okamura and co-workers in 2010 (N. Okamura, et al., 2010), and very clear images showing the differences in AV-133 PET between PD patients and healthy controls were obtained (Figure 4).