**1. Introduction**

Leishmaniases are zoonosis caused by heteroxenous flagellate protozoa of genus *Leishmania* (Ross, 1903), order Kinetoplastida and family Trypanosomatidae. The infection is transmitted by the bite of infected female sand flies, dipteran insects of family Psychodidae, and subfamily Phlebotominae, genus *Lutzomyia* (New World) and *Phlebotomus* (Old World) [1]. These diseases are major public health problems, affecting indiscriminately men, women, and children, and are the ninth leading cause of infectious diseases in the world, despite remaining within the framework of neglected diseases [2–4]. Leishmaniases manifest in different clinical forms, mainly due to the variety of parasites that affect the human population [5]. They are endemic in 98 countries, reaching America, Europe, Asia, Africa, and Australia, with about 350 million people living in risk areas [2, 6, 7].

American visceral leishmaniasis (AVL) has become one of the most important tropical diseases, due to its high incidence, high mortality rates in untreated individuals, and malnourished children, and it can also progress to death [3]. In Latin America, AVL has been recorded in 12 countries, with 90% of cases occurring in Brazil. The geographical expansion of AVL in Brazil has enabled its establishment in urban areas of medium and large cities [3]. Autochthonous human cases are recorded in most Brazilian states, except Acre, Amapá, Amazonas, Paraná, Rondônia, and Santa Catarina [3, 8].

The etiologic agent of AVL in the Americas is *Leishmania (Leishmania) infantum chagasi* (Cunha and Chagas, 1937), whose main vector is *Lutzomyia (Lutzomyia) longipalpis* (Lutz and Neiva, 1912), a species with strong evidence of vector competence, and closely linked to the expansion process of the disease, as revealed by its wide geographical distribution in the Americas [9]. In Brazil, it is currently considered the main vector of AVL in all regions; however, its presence was not yet detected in the states of Acre, Amazonas, Rondônia, and Santa Catarina. The ability of *L. (L.) longipalpis* to often feed on domestic and synanthropic animals, as well as its remarkable anthropophily favored its adaptation to changing environments, favoring the maintenance of the transmission cycle in the rural environment and, at the same time, the spread of the disease into urban areas [9–12].

A variant epidemiological situation is observed in the central region of Brazil, in Corumbá and Ladário (Mato Grosso do Sul State), and Jaciara (Mato Grosso State), where *L. (L.) longipalpis* is absent and *Lutzomyia (Lutzomyia) cruzi* (Mangabeira, 1938) has been incriminated as a vector because of its high abundance, anthropophily, and natural infection with *L. (L.) infantum chagasi* [13–15].

Leishmaniases produce major impacts on human health as a consequence of environmental change, mainly through the possible expansion of transmission areas. Continuous environmental change processes, such as deforestation, fires, agriculture, mining, construction of dams and hydroelectric power plants, migration, unplanned urbanization, and lack of urban infrastructure are examples of situations that have led to an increase in people at risk of infection, and fostered the emergence of outbreaks of leishmaniasis in a new ecoepidemiological pattern [10, 16].

Geographic information systems (GIS) have generated valuable contributions to the control and prediction of vector-borne diseases [17–19], and to evaluate the influence of environmental factors on the habitats of vectors and hosts, and the risk of transmission to humans [20]. Such studies aim to characterize and analyze the spatial and temporal dynamics of the diseases and consequently identify epidemiological patterns, generating information that can be valuable tools when planning control actions.

**1. Introduction**

92 The Epidemiology and Ecology of Leishmaniasis

350 million people living in risk areas [2, 6, 7].

the disease into urban areas [9–12].

*infantum chagasi* [13–15].

Amazonas, Paraná, Rondônia, and Santa Catarina [3, 8].

Leishmaniases are zoonosis caused by heteroxenous flagellate protozoa of genus *Leishmania* (Ross, 1903), order Kinetoplastida and family Trypanosomatidae. The infection is transmitted by the bite of infected female sand flies, dipteran insects of family Psychodidae, and subfamily Phlebotominae, genus *Lutzomyia* (New World) and *Phlebotomus* (Old World) [1]. These diseases are major public health problems, affecting indiscriminately men, women, and children, and are the ninth leading cause of infectious diseases in the world, despite remaining within the framework of neglected diseases [2–4]. Leishmaniases manifest in different clinical forms, mainly due to the variety of parasites that affect the human population [5]. They are endemic in 98 countries, reaching America, Europe, Asia, Africa, and Australia, with about

American visceral leishmaniasis (AVL) has become one of the most important tropical diseases, due to its high incidence, high mortality rates in untreated individuals, and malnourished children, and it can also progress to death [3]. In Latin America, AVL has been recorded in 12 countries, with 90% of cases occurring in Brazil. The geographical expansion of AVL in Brazil has enabled its establishment in urban areas of medium and large cities [3]. Autochthonous human cases are recorded in most Brazilian states, except Acre, Amapá,

The etiologic agent of AVL in the Americas is *Leishmania (Leishmania) infantum chagasi* (Cunha and Chagas, 1937), whose main vector is *Lutzomyia (Lutzomyia) longipalpis* (Lutz and Neiva, 1912), a species with strong evidence of vector competence, and closely linked to the expansion process of the disease, as revealed by its wide geographical distribution in the Americas [9]. In Brazil, it is currently considered the main vector of AVL in all regions; however, its presence was not yet detected in the states of Acre, Amazonas, Rondônia, and Santa Catarina. The ability of *L. (L.) longipalpis* to often feed on domestic and synanthropic animals, as well as its remarkable anthropophily favored its adaptation to changing environments, favoring the maintenance of the transmission cycle in the rural environment and, at the same time, the spread of

A variant epidemiological situation is observed in the central region of Brazil, in Corumbá and Ladário (Mato Grosso do Sul State), and Jaciara (Mato Grosso State), where *L. (L.) longipalpis* is absent and *Lutzomyia (Lutzomyia) cruzi* (Mangabeira, 1938) has been incriminated as a vector because of its high abundance, anthropophily, and natural infection with *L. (L.)*

Leishmaniases produce major impacts on human health as a consequence of environmental change, mainly through the possible expansion of transmission areas. Continuous environmental change processes, such as deforestation, fires, agriculture, mining, construction of dams and hydroelectric power plants, migration, unplanned urbanization, and lack of urban infrastructure are examples of situations that have led to an increase in people at risk of infection, and fostered the emergence of outbreaks of leishmaniasis in a new ecoepidemiological pattern [10, 16].

The AVL shows a persistent scenario in Brazil, with most of the factors contributing for its endemicity residing in processes that are external to the health sector. This makes the strengthening of new strategies necessary. Thus, this study is aimed to analyze the spatial and temporal distribution of AVL in Tocantins State, through evaluation of epidemiological and environmental factors that are potentially related to its expansion: disease incidence, presence of the vector *L. (L.) longipalpis*, type of land use, and deforestation. In the current scenario, where environmental changes impact public health, it is essential to intensify research in diseases related to the environment, especially vector-borne diseases such as AVL.


**6. Acknowledgement**

The author is a scholar within Sub-measure 8.2.2 Regional Innovation Strategies, Measure 8.2 Transfer of knowledge, Priority VIII Regional human resources for the economy Human Capital Operational Programme co-financed by European Social Fund and state budget.


**1. Introduction**

The Lower Rio Grande Valley (LRGV) region has undergone sudden hydrologic change due to urbanization. This abrupt change has produced a decline in water quality in the primary waterways of the region. The Laguna Madre is an estuarine wetland system along the Gulf of Mexico that receives freshwater from the LRGV [1]. This watershed is known for its recreational activities and is currently threatened by the inflows of main drainage pathways that carry significant levels of contaminants. According to the Texas Commission on Environmental Quality (TCEQ) 2020 Integrated Report [2], two water segments from the Lower Laguna Madre are considered impaired due to high levels of bacteria and low dissolved oxygen. The watershed is comprised of three waterways, Hidalgo/Willacy Main Drain (HWMD), Raymondville Drain (RVD), and International

Ernest, A.; Oubeidillah, A.; Johnstone, J.; Chavez, I.R.S.; Fuller, C. Development of a Cyberinfrastructure for Assessment of the Lower Rio Grande Valley North and Central Watersheds Characteristics. *Sustainability* **2021**, *13*, 11186. https://doi.org/10.3390/ su132011186

**Citation:** Navarro, L.; Mahmoud, A.;

Academic Editors: Julian Scott Yeomans and Mariia Kozlova

Received: 25 July 2021 Accepted: 26 September 2021 Published: 11 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Boundary & Water Commission North Floodway (IBWCNF), that provide freshwater inflows to the Lower Laguna Madre. Prior to this study, these waterways had not been characterized. Watershed characterization can enable proper identification of potential sources of pollution to help reduce water impairments to the Laguna Madre and preserve the ecosystem.

One of the emerging tools for watershed characterization is cyberinfrastructure that can assist in both data collection and decision-making processes within the watershed. Cyberinfrastructure supports the process of accessing data via an extensive network and provides updated water quality data for further research. The introduction of a cyberinfrastructure can provide an efficient data collection to well demonstrate the watershed characteristics. In one study, cyberinfrastructure not only utilized widespread data but also allowed researchers to analyze large amounts of data over time at different locations [3–5]. This platform offers a rapid generation of new relationships between wide inventories of data. Cyberinfrastructure secures data and delivers interpreted information via a sequence of web services and portals in forms that are universally coherent by distinct stakeholders [6]. Further, it serves as the center for a variety of data from distinct sources, such as non-point and point source and watershed delineation characteristics. Cyberinfrastructure and the watershed delineation are crucial for the watershed characterization since together they will help identify sources of pollution data within the drainage area.

An ample watershed delineation is key for a successful watershed characterization. A watershed delineation is developed by using elevation data and computing several elevation-based files that represent the overall drainage area as well as the hydrological characteristics of a watershed [7]. Each watershed can be divided into sub-watersheds to produce a more detailed drainage structure. The Geographical Information systems (GIS) platform has facilitated the development of hydrological analysis, such as drainage areas based on elevation data. In 2010, a watershed GIS-based applications study performed a hydrological analysis which showed positive outcomes regarding GIS-applications for watershed management and water quality by providing a full overview of watershed characteristics, such as land cover [8]. Hydraulic and hydrological modelling as well as water resource management commonly require investigation of landscape and hydrological features, such as terrain slope, drainage networks, drainage divides, and catchment boundaries [9]. Additionally, high resolution in data resources is important to obtain accurate results in watershed drainage areas [10]. When the land slope is very flat and has few contours, it is challenging for the acquisition of topographic maps. Light Detection and Ranging (LIDAR) is a high-resolution digital elevation model (DEM) that is an ideal source for the type of topography characterized in low elevation areas [11]. Although the terrain in the LRGV is flat, the complex hydrologic features make the process difficult and challenging with even high-resolution DEM. Hence, a previous study focused on enhancing streamlines and watershed boundaries derived from a high-resolution DEM for future hydrologic modeling and flood forecasting [12]. To determine accurate stream networks, an effective method of eliminating pits or depressions is the stream burning algorithm. This algorithm often identifies river channels or lakes that are not recorded in the DEM, avoiding serious errors in the streaming [3,4]. A stream-burning algorithm can enhance the replication of streams' positions by using raster representation of a vector stream network to trench known hydrological features into a DEM, resulting in a comprehensive watershed delineation [4,13,14]. In addition, delineation of watersheds will not only serve to determine drainage boundaries but to distinguish existing sources of nonpoint sources (NPS) and point sources (PS) pollution.

Part of watershed characterization is to identify potential sources of pollution within the watershed. Pollutant sources have been divided into two different classifications: NPS and PS; with this distinction, it becomes easier to study, analyze, understand, and propose actions to mitigate the pollutant load. NPS pollutants are difficult to identify because they cannot be tracked and usually come from several land uses. The major contributor of NPS pollution is stormwater runoff originated by rainfall [15] and other forms of water

flow through several different land uses. They ultimately discharge to lakes, canals, and coastal waters. This runoff carries significant levels of pollution caused by fertilizers, oil, grease, sediments, bacteria, and nutrients [16]. NPS pollutants contained a significant amount of nutrients, such as nitrogen and phosphorus [17]. There has been increasing emphasis on tackling NPS pollution from agricultural land for the presence of high nutrient contamination [18]. Currently, urbanization has led to increased water transfers from agriculture to urban uses [1,19]. These changes are altering the nature, location, and scope of wastewater loadings into the river. Urban runoff has caused negative results on water quality due to high bacteria and low dissolved oxygen (DO) levels [15]. Recent reports indicated that more than 40% of all impaired waters were affected solely by NPS pollutants, while only 10% of impairments were caused by PS pollutant discharges alone [20].

Unlike NPS pollutants, PS pollutants can be identified because they come from only one source. However, they still present a problem when addressing the pollution issues in primary waterways. To establish the proper actions to reduce or stop the pollutant load into waterbodies, it is necessary to identify the source of the pollutant. PS pollution identification is a challenging task because of the uncertainties and nonlinearity in the transport process of pollutants [21]. The typical way to identify PS pollution requires obtaining prior information of the pollution source, gaining complex information about pollution such as incidents regarding flow simulation dimensions, tabulating the number of PS pollutants involved, and evaluating the pollutant release process [22]. Determining potential sources is the first step in acting toward reducing the effects of water quality problems. Unlike NPS, PSs can be identified because they come from only one source. However, they still present a problem when addressing the pollution issues in primary waterways. To establish the proper actions to reduce or stop the pollutant load into waterbodies, it is necessary to identify the source of the pollutant. PS identification is a challenging task because of the uncertainties and nonlinearity in the transport process of pollutants [21]. The typical way to identify a PS requires obtaining prior information of the pollution source, gaining complex information about pollution such as incidents regarding flow simulation dimensions, tabulating the number of PS involved, and evaluating the pollutant release process [22]. Determining potential sources is the first step in acting toward reducing the effects of water quality problems.

Almost 70% of all rivers and streams in the United States are unassessed. In the State of Texas, 88% of all rivers and streams are unassessed. In the United States, 53% of the assessed water bodies were considered impaired due to high levels of *E. coli* and fecal coliform [23]. In addition, fecal coliform bacteria and other pathogens present in stormwater discharges threaten public health and have been responsible for numerous beach closings in the region [24]. Some studies have found that both livestock and manure management can potentially be agricultural sources of fecal indicator bacteria in watersheds [25]. Moreover, estuaries have faced eutrophication because of increased inputs of nutrients, such as nitrogen and phosphorus. This phenomenon is now considered to be a worldwide issue [26–28]. Ammonia can enter the aquatic environment via direct means of municipal effluent discharge and excretion of nitrogenous wastes from animals. It may also contaminate certain areas through indirect means such as nitrogen fixation, air deposition, and runoff from agricultural lands [29]. Improper wastewater management practices in this under-served region have caused severe water quality problems, and sections of the river have experienced poor water quality with regard to dissolved oxygen, bacteria, and algae [30].

The Laguna Madre is identified as an impaired waterbody due to the presence of high concentrations of bacteria and low dissolved oxygen [2]. The Lower Laguna Madre receives freshwater inflows from three waterways located in the north and central part of the LRGV. The three waterways are HWMD, RVD and IBWCNF, which are not fully characterized due to insufficient data. The aim of this paper is to provide a comprehensive characterization of the north and central watersheds to analyze pollution sources. A cyberinfrastructure database was developed to facilitate navigating through distinct information

to obtain potential sources of pollution. Watershed delineation was developed using as GIS platform to determine the watersheds' drainage areas. Quantifying this information will support the identification of which of the three watersheds contribute the most to water impairments in the Lower Laguna Madre by assessing each watershed independently. The watershed characterization has been shown to support stakeholders in the region for optimal watershed management and enhance their decision-making process.


**5.1 Auxiliary liver transplantation**

An alternative to traditional orthotopic liver transplantation for patients with ALF is that of auxiliary transplant, based on the well-established regenerative capacity of hepatocytes. Unlike traditional transplantation, where native hepatectomy is performed simultaneously with donor engraftment, in auxiliary transplants, the patient's native liver is left surgically intact, while a partial or smaller sized donor graft is transplanted. This procedure allows assumption of hepatic functions by the donor graft, resolution of multiorgan failure, and clinical stabilization of the patient. In turn, the native liver benefits both from additional time, as well as improved physiological conditions, thus maximizing the opportunity for hepatic regeneration. Younger patients (below 40 years of age) with ALF due to viral hepatitis or acetaminophen toxicity appear to have the best outcome with this strategy. After native hepatic function is demonstrated, the auxiliary graft can be removed, but is most frequently allowed to atrophy by withdrawal of immunosuppression. Complete cessation of immunosuppression can be achieved in many patients (*Boudjema et al., 1995*).

#### **5.2 Artificial and bioartificial hepatic support systems**

Mechanical hepatic support systems serve as a bridge to transplant in patients with ACLF, and as a bridge to transplant or spontaneous recovery in patients with ALF. These systems are designed to reproduce the detoxifying functions of the liver, and mimic the principles upon which renal replacement therapy is based. While conventional renal dialysis removes small toxins and water-soluble toxins, the liver detoxifies larger toxins and protein-bound toxins. Dialysis of these larger and protein-bound toxins through unbound human albumin solutions allows the removal from the patient's circulation.

Several artificial systems have been developed utilizing albumin dialysis. The Molecular Adsorbent Recirculating System (MARS), Single Pass Albumin Dialysis (SPAD), and Prometheus are examples; of these, MARS has been the most widely studied. MARS has been utilized for management of hepatic encephalopathy, cerebral edema, hepatorenal syndrome, treatment of drug overdoses, and as a bridge to transplantation (*Mitzner, 2011*). MARS has been shown to improve hemodynamic parameters and organ perfusion during circulatory collapse, and has been associated with improvement in hepatic synthetic function. For patients with ACLF, MARS can provide temporarily relief of intractable pruritis and fatigue.

Bioartificial systems work similarly to artificial systems to remove toxins by albumin dialysis, but additionally utilize human or porcine hepatocytes to mimic hepatic synthetic function. Advances in the development of bioartificial systems have been limited by the challenges in maintaining hepatocyte viability.


**1.1.2 Robust approaches for general mismatch**

Several other approaches are known to provide the improved robustness against more general types of mismatches, for example, the algorithms that use the diagonal loading of the sample covariance matrix [14][16], the eigenspace-based beamformer [11][30][31], and the covariance matrix taper (CMT) approach [32]-[34]. For the diagonal loading method, a serious drawback is that there is no reliable way to choose the diagonal loading level, F.Vincent and O.Besson propose the method to select the optimal loading level with a view to maximizing the signal-to-noise ratio (SNR) in the presence of steering vector errors and it is shown that the loading is negative, but they can't give the exact solution, instead of the approximate solution, moreover, they can't give the expression of steering vector errors [35]. The eigenspace-based approach is essentially restricted in its performance at low SNR and when the dimension of the signal-plus-interference subspace is high, and the dimension must be known in the latter technique [31]. The CMT approach is known to provide an excellent robustness in scenarios with nonstationary interferers, however, its robustness against mismatches of the desired signal array response may be unsatisfactory, furthermore, it can also be explained as the diagonal loading [33].


*2.3. Free-Fall Drop Test*

A Lansmont Model PDT 227 Precision Drop Tester (Lansmont Corporation, Monterey, CA, USA) was used to perform the free-fall drop events (Figure 2). Impact bruises were produced by dropping each corrugated container five times from a predetermined drop height onto the concrete floor surface of the drop test equipment. In this study, the packages were dropped onto the bottom panel of the package from two drop heights, 30 cm and 50 cm. The testing was performed in duplicate for each package configuration at the two different drop heights.

**Figure 2.** Packaged product on the Lansmont PDT 227.

To record the impact acceleration of the apple from the free-fall drop event, a model HT356B21 triaxial accelerometer (PCB Piezotronics, Inc., Depew, NY, USA) was attached to the apple located in the corner cell of the top tray. The sensor was placed in this position to ensure it would not shear off during the impacts with surrounding apples. The Lansmont Test Partner 3 (Lansmont Corporation, Monterey, CA, USA) processed the signal events and the resultant acceleration from each impact recorded.


**6. Conclusions**

In this paper, every component shown in Figure 1 was modeled and coded in Matlab along with the objective function (COEPS). A wind–hydro grid connected power system was proposed as an adjunct to an existing power grid. This was mathematically modeled and then coded in Matlab. The GA of the Matlab optimization toolbox was used to find the optimal feasible value of the COEPS, which was 0.0955388 \$/kWh. This was 28.7% less than the conventional energy from the power grid. The discounted payback period was 10 years, 3 months, and 7 days. Furthermore, carbon emissions were reduced by 68.66% compared with experimentally estimated data. As a result, the grid energy purchases were also reduced. Specifically, comparing the system described in this study with the

formerly studied on-grid wind-only system showed that the COEPS, ECO2, COERenewables, and grid energy purchases were reduced by 12.26%, 24.69%, 1.52%, and 24.68%, respectively. These are very promising results, especially for oil-importing countries, such as Jordan, where imported energy is a significant financial burden to the economy. The proposed wind power system with hydro storage is recommendable for its clean and economical features, compared with the conventional fossil-fueled grid or wind-only on-grid renewable configurations.

Finally, this paper is a case study to demonstrate the important point of local solutions to the global problem of global warming. The paper is necessarily limited to the specific data and assumptions of the local case study. Future work will include applying the above principle and the methodology of this paper to many other local engineering boundary conditions.

**Author Contributions:** H.M.K.A.-M. wrote the paper, brought the measured data from formal institutions in Jordan, performed the simulation and optimization using Matlab optimization toolbox, mathematically modeled the system and coded these models in Matlab; M.E. helped obtain more meaningful results and reviewed the article before the final submission; A.A.-Q. reviewed the paper and performed the pdf distribution to convert the monthly average wind speed measured data into hourly data; A.A. reviewed the paper and wrote the literature review part.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to acknowledge Yarmouk University, Irbid, Jordan, for their support in this research.

**Conflicts of Interest:** The authors declare no conflict of interest.


*4.4. Chengdao* 䃐䚃 *(The Dao of Sincerity)*

Finally, *cheng*2 developed from meaning the completion of human-spirit communication to meaning the unity of heaven's Dao with humans' Dao. As Mencius said, "Sincerity is the Dao of heaven, and the pursuit of sincerity is the Dao of humans" 䃐㘵, ཙѻ䚃ҏ; ᙍ䃐㘵, Ӫѻ䚃ҏ (*Mencius*, 4A12). The *Doctrine of the Mean* similarly states, "Sincerity is the Dao of heaven, and the attainment of sincerity is the Dao of humans" 䃐㘵, ཙѻ䚃ҏ; 䃐ѻ㘵, Ӫѻ䚃ҏ (Zheng and Kong 2000, 53.1689b). Because Cheng Yi 〻乔 and Zhu Xi ᵡ⟩ explicated *cheng*2 as being the "principle of reality" (Zhu 1992, p. 31), many scholars have assumed that classical Confucians regarded *cheng* as the ontological foundation of all existence. Cheng's and Zhu's explication, however, represented only the neo-Confucian reconception of *cheng*2. In fact, during the Warring States period it was a common concept that *cheng*2 represented the movement and virtue of heaven. For example, the *Heguanzi* 咆ߐᆀ (Master of the Pheasant Cap) states such:

Heaven makes sincerity the virtue of the sun: the sun sincerely emerges and sincerely enters. . . . Heaven makes faithfulness the law of the moon: the moon faithfully dies and faithfully grows. ཙ㘵䃐ަᰕᗧҏ, ᰕ䃐ࠪ䃐ޕ.... ཙ㘵ؑަᴸࡁҏ, ᴸؑ↫ؑ⭏. (Huang 2004, pp. 168–69)

The regular, constant circulation of the sun and the moon represents the movement of heaven's course (*dao*) and its virtue of sincerity and faithfulness. A similar statement is also seen in the silk manuscript *Jingfa* ㏃⌅ (The Canon of Law) excavated from Mawangdui (Yates 1997, pp. 80–81). The *Xunzi* reads as follows:

Heaven does not speak, but people infer its height. Earth does not speak, but people infer its thickness. The four seasons do not speak, but people anticipate them. These things have such constancy because they have utmost sincerity. . . . Heaven and Earth are most vast, but without sincerity they could not transform the myriad things. ཙн䀰㘼Ӫ᧘儈✹, ൠн䀰㘼Ӫ᧘ ✹, ഋᱲн䀰㘼Ⲯဃᵏ✹. ཛ↔ᴹᑨ, ԕ㠣ަ䃐㘵ҏ.... ཙൠ⛪བྷ⸓, н䃐н 㜭ॆ㩜⢙. (Wang 1988, 2.46)6

Heaven, earth, and the four seasons never speak, but they demonstrate their sincerity through regular, constant movement, which generates and transforms the myriad things. The *Annals of Sire Lü* presents similar ideas:

If heaven's movements were not faithful, the year could not complete. If earth's movements were not faithful, grass and trees could not grow. . . . Great as heaven and earth are, and as transforming as the four seasons are, they still could not complete things by being unfaithful, let alone human affairs. ཙ㹼нؑ, н㜭ᡀ↢; ൠ㹼нؑ, 㥹ᵘнབྷ.... ཙൠѻབྷ, ഋᱲѻॆ, 㘼 ⥦н㜭ԕнؑᡀ⢙, ৸⋱ѾӪһ. (Chen 2001, 19.1311)7

This also describes the regular movements of heaven, earth, and the four seasons as representing their virtue of sincerity and faithfulness while setting a model for human affairs. Reading all these statements together with the two passages from the *Mencius* and the *Records of Ritual* cited above, we can see clearly that this was in fact a common natural, cosmological view of the Warring States thinkers, without involving the dimension of an ontological "principle of reality."

Prior to defining sincerity as heaven's Dao, both passages from the *Mencius* and the *Records of Ritual* discuss the significance of sincerity in cultivating one's own good virtues and serving one's parents, friends, superiors, and the people. This shows that the authors' concern still lies in the dimension of ethical and political virtues. With its sincere movements and virtues, heaven generates and transforms the myriad things, including human beings. If humans attain *cheng* (sincerity), *chengren* ᡀӪ (completion of consummate humanity; *Analects*, 14.12), and *chengwu* ᡀ⢙ (completion of things; Zheng and Kong 2000, 53.1694a) through imitating heaven and cultivating themselves, they are assisting heaven in its work of generation and transformation. Thus, the mysterious veil hanging over *cheng* has finally been lifted.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.


**6. Neurological monitoring**

There are currently no consensus guidelines for neuromonitoring on ECMO, with variations in practice at different institutions. Neuromonitoring may include assessment of brain structure or morphology via imaging, assessment of brain function via EEG or SSEPs, assessment of cerebral perfusion via cerebral oximetry or transcranial doppler, and assessment for neurological injury via biomarkers. Bembea and colleagues performed a systematic review of the literature; 39 observational and case-control studies met inclusion criteria, with most of the literature coming from neonatal studies [52]. There was very little data in pediatric and adult cohorts, and the study found limited data on the use and effectiveness of monitoring technologies. A recent review by Lin et al. discusses neuromonitoring in the neonatal ECMO patient [53].

#### **6.1 Exam**

Neuromonitoring of the ECMO patient should begin with daily neurologic assessments that are documented in the patients chart. These are limited by reliability when performed by multiple providers from different disciplines, however are useful for obtaining a daily baseline that can be suggestive of injury when a change is noted. This would also require daily sedation holidays for accurate assessments as well as using the least amount of sedation to keep the patient safe and comfortable. Use of neuromuscular blockade should be reserved for extremely ill patients and those whose movement limits ECMO flows. A change in neurologic exam is often the trigger for seeking additional information such as through neuroimaging.

#### **6.2 Neuroimaging**

Cranial or head ultrasound (HUS) is a mode of imaging limited to neonates and infants with open fontanelles. Ultrasound uses high frequency sound waves transmitted via a probe that are reflected back based on the tissue's composition as well as distance from the probe. Changes in tissue density from hemorrhage or ischemia will reflect back sound waves differently from surrounding tissue. Cranial ultrasounds are portable, easy to use, relatively inexpensive, and do not carry radiation risks. Most neonatal ECMO programs will obtain a HUS prior to ECMO cannulation as well as daily HUS for the 1st few days on ECMO. While it is best for detecting hemorrhages, ischemic changes are harder to interpret on HUS [54]. HUS can also give information on changes in ventricular size that would be seen in hydrocephalus. It is not as sensitive as other imaging techniques and a study showed that MRI was significantly more sensitive for detection of CNS lesions than HUS alone [55, 56]. The quality of images depends on the skill level of the ultrasound technician and interpretation of acquired images can be subjective and variable. HUS findings have not consistently correlated with neurodevelopmental outcomes and should not be used for predicting outcomes in neonatal ECMO survivors [37, 56].

**209**

*Neurologic Complications and Neuromonitoring on ECMO*

Computed tomography (CT) is a diagnostic imaging modality that utilizes X-Rays directed at the patient that are picked up by a detector and sent to a computer to create thin 2D image slices, at different tissue depths. Multiple images can then be stacked to create a 3D picture. It is the most frequently used imaging modality for diagnosis of acute intracranial injury for patients on ECMO. A CT scan can be quickly obtained and has better sensitivity and specificity for detecting intracranial hemorrhage that might lead to clinical changes in management [53]. A disadvantage is exposure to radiation and its associated risks. Transporting a patient on ECMO to a CT scanner in the radiology department can be challenging in the absence of a portable scanner that can be brought to bedside. ELSO currently recommends a CT scan prior to hospital discharge for patients less than 4 years of age and if there is an abnormal neurologic exam for patients older than 4 years of age as part of post-ECMO follow up [57]. Magnetic Resonance Imaging (MRI) is a non-invasive technology that creates 3D anatomic images without exposing the patient to radiation. A strong magnetic field is used to force protons in the body into alignment. Then a brief radiofrequency pulse stimulates protons causing a change in alignment. The scanner can detect electromagnetic energy transmitted as the protons realign. It is reserved for patients after decannulation from ECMO, due to MRI incompatible materials in the cannulae and circuits. MRI is the most sensitive and specific imaging technique available. However it takes much longer time to obtain the study compared to a CT and is more expensive. While diffusion-restriction can be seen up to 10 days after acute ischemic injury, the optimal timing for obtaining an MRI after ECMO remains unclear [53].

While neuroimaging provides information on the structure of the brain, EEG provides real-time information on the electrical activity of the brain. Information is obtained via electrodes placed on the scalp, connected to a monitor, with very little burden to the patient that would include scalp abrasions. Continuous EEG (cEEG) monitoring requires technicians to set up the electrodes as well as neurologists to read the EEGs, which can be time consuming. Amplitude-integrated EEG (aEEG) compresses the raw EEG data from 1 to 2 leads, is easier to set up and interpret, but due to lower sensitivity, can be used as a screening tool or in resource limited settings [53]. Ischemic and hemorrhagic injuries can predispose a patient to seizures that require prompt treatment. Continuous EEG monitoring is important for early identification and treatment of subclinical seizures or electrical status epilepticus that may not be otherwise detected, although studies are needed to show its benefit in improving long term outcomes. EEG monitoring is especially useful in paralyzed patients in whom a neurological exam cannot be elicited. EEG can be used to detect early cerebral ischemia through loss of fast alpha and beta frequencies to slowing and even suppression of all electrical activity as might be seen in an infarct. In 2011, the American Clinical Neurophysiology Society deemed ECMO as a high risk clinical scenario in neonates that would warrant long term EEG monitoring due to cardiac or pulmonary risks for acute brain injury and clinical encephalopathy [58]. This recommendation is supported by ELSO in their guidelines for management of neonatal respiratory failure [59]. In their 2015 consensus statement on continuous EEG in critically ill adults and children, the American Clinical Neurophysiology Society recommended continuous EEG monitoring for patients treated with pharmacologic paralysis, including patients on ECMO [60].

This is a non-invasive, portable test that is based on the Doppler effect. A Doppler probe is used to emit high frequency sound waves through the cranium that are reflected

*DOI: http://dx.doi.org/10.5772/intechopen.85103*

**6.3 Electroencephalography (EEG)**

**6.4 Transcranial doppler ultrasound (TCD)**

*Advances in Extracorporeal Membrane Oxygenation - Volume 3*

**6. Neurological monitoring**

neonatal ECMO patient [53].

**6.1 Exam**

**6.2 Neuroimaging**

gas is decreased to 0.5 –1 L/minute and oxygen increased to 100% FiO2 through the circuit, without any changes to extracorporeal blood flow [50, 51]. In-line gas monitoring on the ECMO circuit can be used to trend venous paCO2, but serial arterial blood gas analysis should be used to confirm the lack of ventilation secondary to central apnea. For patients on VA ECMO, hemodynamics should be maintained through circuit flows and use of vasoactive medications as needed. Patients found to

There are currently no consensus guidelines for neuromonitoring on ECMO, with variations in practice at different institutions. Neuromonitoring may include assessment of brain structure or morphology via imaging, assessment of brain function via EEG or SSEPs, assessment of cerebral perfusion via cerebral oximetry or transcranial doppler, and assessment for neurological injury via biomarkers. Bembea and colleagues performed a systematic review of the literature; 39 observational and case-control studies met inclusion criteria, with most of the literature coming from neonatal studies [52]. There was very little data in pediatric and adult cohorts, and the study found limited data on the use and effectiveness of monitoring technologies. A recent review by Lin et al. discusses neuromonitoring in the

Neuromonitoring of the ECMO patient should begin with daily neurologic assessments that are documented in the patients chart. These are limited by reliability when performed by multiple providers from different disciplines, however are useful for obtaining a daily baseline that can be suggestive of injury when a change is noted. This would also require daily sedation holidays for accurate assessments as well as using the least amount of sedation to keep the patient safe and comfortable. Use of neuromuscular blockade should be reserved for extremely ill patients and those whose movement limits ECMO flows. A change in neurologic exam is often the trigger for seeking additional information such as through neuroimaging.

Cranial or head ultrasound (HUS) is a mode of imaging limited to neonates and infants with open fontanelles. Ultrasound uses high frequency sound waves transmitted via a probe that are reflected back based on the tissue's composition as well as distance from the probe. Changes in tissue density from hemorrhage or ischemia will reflect back sound waves differently from surrounding tissue. Cranial ultrasounds are portable, easy to use, relatively inexpensive, and do not carry radiation risks. Most neonatal ECMO programs will obtain a HUS prior to ECMO cannulation as well as daily HUS for the 1st few days on ECMO. While it is best for detecting hemorrhages, ischemic changes are harder to interpret on HUS [54]. HUS can also give information on changes in ventricular size that would be seen in hydrocephalus. It is not as sensitive as other imaging techniques and a study showed that MRI was significantly more sensitive for detection of CNS lesions than HUS alone [55, 56]. The quality of images depends on the skill level of the ultrasound technician and interpretation of acquired images can be subjective and variable. HUS findings have not consistently correlated with neurodevelopmental outcomes and should not

be used for predicting outcomes in neonatal ECMO survivors [37, 56].

be brain dead on ECMO can be considered as candidates for organ donation.

**208**

Computed tomography (CT) is a diagnostic imaging modality that utilizes X-Rays directed at the patient that are picked up by a detector and sent to a computer to create thin 2D image slices, at different tissue depths. Multiple images can then be stacked to create a 3D picture. It is the most frequently used imaging modality for diagnosis of acute intracranial injury for patients on ECMO. A CT scan can be quickly obtained and has better sensitivity and specificity for detecting intracranial hemorrhage that might lead to clinical changes in management [53]. A disadvantage is exposure to radiation and its associated risks. Transporting a patient on ECMO to a CT scanner in the radiology department can be challenging in the absence of a portable scanner that can be brought to bedside. ELSO currently recommends a CT scan prior to hospital discharge for patients less than 4 years of age and if there is an abnormal neurologic exam for patients older than 4 years of age as part of post-ECMO follow up [57].

Magnetic Resonance Imaging (MRI) is a non-invasive technology that creates 3D anatomic images without exposing the patient to radiation. A strong magnetic field is used to force protons in the body into alignment. Then a brief radiofrequency pulse stimulates protons causing a change in alignment. The scanner can detect electromagnetic energy transmitted as the protons realign. It is reserved for patients after decannulation from ECMO, due to MRI incompatible materials in the cannulae and circuits. MRI is the most sensitive and specific imaging technique available. However it takes much longer time to obtain the study compared to a CT and is more expensive. While diffusion-restriction can be seen up to 10 days after acute ischemic injury, the optimal timing for obtaining an MRI after ECMO remains unclear [53].

### **6.3 Electroencephalography (EEG)**

While neuroimaging provides information on the structure of the brain, EEG provides real-time information on the electrical activity of the brain. Information is obtained via electrodes placed on the scalp, connected to a monitor, with very little burden to the patient that would include scalp abrasions. Continuous EEG (cEEG) monitoring requires technicians to set up the electrodes as well as neurologists to read the EEGs, which can be time consuming. Amplitude-integrated EEG (aEEG) compresses the raw EEG data from 1 to 2 leads, is easier to set up and interpret, but due to lower sensitivity, can be used as a screening tool or in resource limited settings [53]. Ischemic and hemorrhagic injuries can predispose a patient to seizures that require prompt treatment. Continuous EEG monitoring is important for early identification and treatment of subclinical seizures or electrical status epilepticus that may not be otherwise detected, although studies are needed to show its benefit in improving long term outcomes. EEG monitoring is especially useful in paralyzed patients in whom a neurological exam cannot be elicited. EEG can be used to detect early cerebral ischemia through loss of fast alpha and beta frequencies to slowing and even suppression of all electrical activity as might be seen in an infarct. In 2011, the American Clinical Neurophysiology Society deemed ECMO as a high risk clinical scenario in neonates that would warrant long term EEG monitoring due to cardiac or pulmonary risks for acute brain injury and clinical encephalopathy [58]. This recommendation is supported by ELSO in their guidelines for management of neonatal respiratory failure [59]. In their 2015 consensus statement on continuous EEG in critically ill adults and children, the American Clinical Neurophysiology Society recommended continuous EEG monitoring for patients treated with pharmacologic paralysis, including patients on ECMO [60].

### **6.4 Transcranial doppler ultrasound (TCD)**

This is a non-invasive, portable test that is based on the Doppler effect. A Doppler probe is used to emit high frequency sound waves through the cranium that are reflected back by moving red blood cells in the blood vessels. The difference in frequencies of emitted and reflected waves is proportional to the cerebral blood flow. Studies have found that TCD velocities (TCDV) are much lower for pediatric patients on ECMO when compared to normative values for healthy and critically-ill children [15, 61]. While there was no significant association between global TCDV (systolic flow velocity, diastolic flow velocity, mean flow velocity) and neurologic injury, increased pulsatility index and regional increases in velocities or asymmetries might be predictive of neurologic injury.

#### **6.5 Cerebral near infra-red spectroscopy (NIRS)**

NIRS monitoring is a non-invasive technology that uses near-infrared wavelength of light that penetrates brain tissue via a scalp electrode. It provides a continuous measurement of regional tissue oxygen saturation (rSO2), which is a marker of the balance between oxygen delivery and demand in the tissues. When the probe is placed on the forehead, it measures cerebral oximetry. An analysis of adult patients on VA ECMO showed that cerebral desaturation was common and mortality higher for patients with cerebral desaturation compared to those without [21]. A sudden decrease in cerebral saturation can be associated with an acute neurological event, prompting further investigation. It can also serve as an early predictor of inadequate oxygenation and cardiac output especially peri-cannulation [62]. It can influence management by prompting a need for increased flows in VA ECMO or alternate cannulation strategies if there is differential hypoxia. A very high rSO2 could also be suggestive of very poor oxygen extraction and poor neurologic outcomes.

#### **6.6 Biomarkers**

Several plasma proteins have been evaluated as potential markers for brain injury [63]. These biomarkers include substances associated with glial injury (glial fibrillary acidic protein and s-100b), neuronal injury (neuron-specific enolase and brain-derived neurotrophic factor) and neuro-inflammation (intercellular adhesion molecue-5). Unfavorable neurologic outcomes have been associated with higher biomarker concentrations [64], with combinations of biomarkers providing higher sensitivities and specificities for detection of neurologic injury. These tests are more expensive and require laboratory equipment and processing availability. While not currently a routine component of neuromonitoring on ECMO in most institutions, there is potential for further research and applicability if these results can be obtained in real time to influence management.

#### **6.7 Somato-sensory evoked potentials (SSEPs)**

SSEPs measure electrical signals in the somatosensory cortex after a peripheral stimulus, assessing the pathway of neuronal conduction from the peripheral nerve to the cortex. They are assessed as normal, abnormal (increased latency) or absent. ECMO cannulation is not thought to alter the ability to assess SSEPs from the hemispheres [65]. Small studies have shown an association between abnormal SSEPs and poor neurologic outcome after ECMO [66], but the predictive value of evoked potentials remains to be determined. In one study, absence of bilateral SSEPs was associated with progression to brain death for patients treated with ECPR [67].

#### **6.8 Optic nerve sheath diameter (ONSD)**

It is a simple bedside test used to detect elevated intracranial pressure. A cut-off of 5.2 mm is sensitive and specific for intracranial hypertension [68]. Its use in

**211**

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
*Neurologic Complications and Neuromonitoring on ECMO*

ECMO management is still in its infancy, although a study showed that higher ONSD was associated with poor neurological outcome after ECPR [69].

Therapeutic hypothermia has been shown to be neuroprotective for term neonates at risk of hypoxic ischemic encephalopathy secondary to perinatal asphyxia. However a randomized controlled study out of the United Kingdom did not show an improvement in outcomes for neonates on ECMO treated with mild hypothermia [70]. On the other hand, therapeutic hypothermia has been associated as a risk factor for intracranial hemorrhage and should be avoided [30]. In 2015, the American Heart Association recommended targeted temperature management of 32–36°C for comatose patients with return of spontaneous circulation after cardiac arrest [71]. This was also applied to patients who suffered in- hospital cardiac arrest leading to ECPR. A more recent large, multicenter, randomized control trial failed to show a benefit in survival with favorable neurological outcome for children with in-hospital cardiac arrest. There is no data to support routine therapeutic hypothermia for children undergoing ECPR although maintaining normothermia is still encouraged.

Neurologic complications contribute to significant morbidity and mortality for patients on ECMO, who constitute a high risk population. There are many modalities currently available for neuromonitoring, and as we gain more experience and information through more frequent use, we will be able to develop consensus guidelines and protocols to provide better care. A multimodal approach to active surveillance, early recognition and prompt management of neurologic injuries as

they arise, may improve outcomes for patients on ECMO.

The author has no "conflict of interest" to disclose.

*DOI: http://dx.doi.org/10.5772/intechopen.85103*

**7. Therapeutic hypothermia**

**8. Conclusion**

**Conflict of interest**

**Author details**

Venessa Lynn Pinto

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Texas Children's Hospital and Baylor College of Medicine, Houston, Texas, USA

ECMO management is still in its infancy, although a study showed that higher ONSD was associated with poor neurological outcome after ECPR [69].


**6.4 Immunophenotype of LSCs**

Several immunophenotypes of LSCs have been identified, such as CD34, CD38, CD123, CD117, CD71, CD44, HLA-DR, TIM3, CLL-1, CD96, CD47, CD32 and CD25. Although these surface molecules are not expressed in all LSCs, their high expression may lead to a significant deterioration of the disease prognosis. It is also because of the difference in markers and functions between LSCs and HSCs that targeted therapy for leukemia stem cells is possible. CD33 is the first AML targeted therapeutic antigen approved by the US FDA, which is highly expressed in AML but not in normal HSCs. The monoclonal anti-tumor drug Gemtuzumab ozogamicin, consisting of the CD33 antibody, hP67.6, and the cytotoxic drug, is

**21**

*Advances in Acute Myeloid Leukemia Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.84263*

effect in the AML mouse model [22].

immunophenotype of LSCs.

**7. Summary**

in future.

**Acknowledgements**

QNRC2016833).

against CD33+

a good candidate for selective killing of CD33+

years, tumor-specific chimeric antigen receptor T-cell immunotherapy (CART)

that the anti-CD123 monoclonal antibody CSL362 has a good tumor cell killing

achieved initial clinical success, it is undeniable that LSCs are diverse among different patients, and even in the same individual, the phenotypic differences are quite different. This brings new challenges to clinical treatment. Moreover, studies have shown that in patients with newly diagnosed AML, the distribution of LSCs is uniform and the number is small, but once the patient relapses after chemotherapy, the number of LSCs can be significantly increased, and some new phenotypes appear [97]. The phenotypic changes in LSCs at different stages of the same patient's disease also lead to difficulties in clinical application of this targeted LSC immunophenotypic treatment strategy. Therefore, the current targeted therapy based on this strategy is still in the exploration stage, and the development of related drugs is significantly limited due to the plasticity of the

Although the current monoclonal antibodies against the LSCs phenotype have

LSCs play an important role in the origin, recurrence, and drug resistance of leukemia. Although the current research on LSCs has made some progress, the biological characteristics of LSCs and its mechanism in the pathogenesis of leukemia remain unclear, and the treatment strategy for targeted clearance of LSCs is still in its infancy. Therefore, clarifying its biological characteristics and developing drugs for targeted therapy of LSCs is an important direction for leukemia research

The authors acknowledge financial support from the National Natural Science Foundation of China (Grant no. 81502663), Social Development Foundation of Zhenjiang City (Grant no. SH2018063), Six talent peals project of Jiangsu Province (Grant no.WSW-039), Six for one project of Jiangsu Province (Grant no. LGY2018093), Young medical talents of Jiangsu (Grant no.

cells has become increasingly popular [96]. Busfield et al. detected

LSCs [95]. In addition, in recent

*Advances in Acute Myeloid Leukemia Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.84263*

a good candidate for selective killing of CD33+ LSCs [95]. In addition, in recent years, tumor-specific chimeric antigen receptor T-cell immunotherapy (CART) against CD33+ cells has become increasingly popular [96]. Busfield et al. detected that the anti-CD123 monoclonal antibody CSL362 has a good tumor cell killing effect in the AML mouse model [22].

Although the current monoclonal antibodies against the LSCs phenotype have achieved initial clinical success, it is undeniable that LSCs are diverse among different patients, and even in the same individual, the phenotypic differences are quite different. This brings new challenges to clinical treatment. Moreover, studies have shown that in patients with newly diagnosed AML, the distribution of LSCs is uniform and the number is small, but once the patient relapses after chemotherapy, the number of LSCs can be significantly increased, and some new phenotypes appear [97]. The phenotypic changes in LSCs at different stages of the same patient's disease also lead to difficulties in clinical application of this targeted LSC immunophenotypic treatment strategy. Therefore, the current targeted therapy based on this strategy is still in the exploration stage, and the development of related drugs is significantly limited due to the plasticity of the immunophenotype of LSCs.


*2.3.2 Recovery*

This phase of the disaster cycle is the period taken from the end of the disaster response phase, as defined by stabilizing of the situation, to the return of normal function. From a health systems standpoint, restoration of all services, provision of normal care including elective care, financial and administrative normalization are all goals of the recover period.


*2.6. Evaluating Reutilization Capabilities of Nb2O5 Catalyst Aiming for Sustainable Implementation and Comparison with Other Catalysts' Performance*

Sustainable processes must consider the continuous reutilization of photocatalytic materials within catalytic converters [44]. Reutilization capabilities and stabilities of novel materials should be assessed in order to demonstrate material stability. Therefore, Nb2O5 was submitted to continuous treatment operation. Suspended Nb2O5 photocatalyst was recovered and tested over consecutive decolorization cycles of MO solutions. As depicted in Figure 10, the niobium metal oxide semiconductor presented excellent stability, and retained its catalytic properties over 10 cycles. Please note that consecutive cycles demonstrated reproducible MO removal performance. It is important to remark that previous reports in the literature reported higher stability of niobium oxides than for conventional TiO2 or ZnO catalysts [9,27,28]. In this context, the results reported here support these previous studies, indicating Nb2O5 as an emerging photocatalyst for environmental remediation. However, more studies related to catalyst aging and fouling during continuous operation are required to ensure the lifetime of these emerging catalysts.

**Figure 10.** Percentage of color removal achieved after 40 min of solar photoelectrocatalytic treatment of 5 mg L−1 of MO with 1.0 g L−1 of catalyst Nb2O5 in slurry at pH 5.0 with 0.20 M of H2O2 after consecutive reuse of Nb2O5.

A quick comparison with other photocatalytic materials reported in the literature demonstrates the promising performance of novel niobium oxide semiconductors for water treatment applications. As summarized in Table 2, the results reported in this work are highly competitive, since Nb2O5 makes it possible to reduce operational times by more than half when compared with doped TiO2 and other complex mixed oxides.

**Table 2.** Comparative performance of visible photocatalytic decolorization of Methyl Orange azo dye with different catalysts to attain over 95% color removal.

### **3. Materials and Methods**

#### *3.1. Chemicals*

MO azo dye of 85.0% purity and the H2O2 of 33% (*w*/*w*) were supplied by Sigma-Aldrich. The ammonium salt of the niobium oxalate complex (NH4[NbO(C2O4)2(H2O)]. 3H2O) of 99.0% purity used in the photocatalyst synthesis was purchased from CBMM. The pH was adjusted prior to experiments using H2SO4 or NaOH of analytical grade, supplied by Sigma-Aldrich. All solutions were prepared with high-purity water obtained from a Millipore Milli-Q system with resistivity >18 MΩ cm at 25 ◦C.


*2.6. Thin Layer Chromatography of Methanolic Extract of Tomato Leaves*

Leaves of tomato plant were harvested 24 h after infestation (80 DAS); shade dried at room temperature and grinded to fine powder. Powdered leaves (20 g) was extracted in 400 mL methanol for 72 h.The methanolic extract was dried using rotary evaporator (RFE), the residue (3 mg) was dissolved in 500 μL methanol and collected in eppendorf tube and stored at −4 ◦C.

Extract was spotted on a TLC plate (20 × 20 cm) coated with silica gel HF (250-350 nm). The mobile phase used was chloroform: methanol (95:5 *v*/*v*). The bands, representing various compounds were visualized under UV (254 nm and 380 nm) [39]. The Rf value of each band was calculated and identification of the compound from each band at specific Rf was made from the literature documented.

#### *2.7. FTIR Spectroscopy*

All spectra were obtained with the help of an OMNI-sampler attenuated total reflectance (ATR) accessory on a Nicolet FTIR spectrophotometer followed by the method of Lu et al. [40] and Liu et al. [41] with some modifications. Small amount of TLC eluent corresponding to the Rf value of major bands were placed directly on the germanium piece of the infrared spectrometer with constant pressure applied and data of infrared absorbance, collected over the wave number ranged from 4000 cm–1 to 675 cm–1 and computerized for analyses by using the Omnic software [42].

#### *2.8. Statistical Analysis of Data*

The data was subjected to analysis of variance using Statistix 8.1 software. The differences among various treatment means were compared using the least significant differences test (LSD) at *p* ≤ 0.05 probability level (Table S1).

#### **3. Results**

#### *3.1. Plant Growth Attributes*

The plant spread, which is a measurement of plant width, was significantly (31%) higher in PGPR treated plants under unstressed condition over control (Figure 1). Insect infestation decreased the plant spread by 41%, the decrease was ameliorated by the PGPRs and the value was even greater than the control. The plant height was significantly increased in PGPR inoculated plants (Figure 1). The insect infestation significantly reduced (*p* ≤*0.05*) the height of the plant by 40%, and root area by 50% of the control (Figure 1). The PGPR inoculated plants alleviated the inhibitory effects of insect infestation on plant height and root area such that the root area was significantly higher than the control. Both the shoot and root fresh weights were significantly (44% and 34%) increased in PGPR inoculated plants (Figures 2 and 3). Infestation with the insect decreased the fresh weights of both the root and shoot, the shoot fresh weight was more adversely affected. The PGPR inoculation had ameliorated the insectinduced decrease in the root and shoot fresh weight.

**Figure 1.** Mean plant height and plant spread (cm) of tomato under control and infested conditions. Data are means of four replicates along with standard error bars. Different letters on the bar represent significant differences (*p* < 0.05) among treatments.

**Figure 2.** Root area (cm2) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 3.** Fresh weight of shoot and root (g) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

The dry weight of root and shoot was also higher (*p* ≤*0.05*) in PGPR inoculated plants (Figure 4). The root was more responsive and the % increase in root weight was greater. The leaves were almost eaten by the insect and the shoot weight was significantly decreased to 81% whereas root weight was decreased by 38% over the control.

**Figure 4.** Dry weight of leaf, shoot and root (g) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

#### *3.2. Physiological Parameters*

The proline production was lower (*p* ≤ *0.05*) in the untreated control plants (Figure 5). Under unstressed conditions the PGPR treatments stimulated proline content of leaves by 18% over control. Similar percent of increase was recorded in plants infested with *S. litura*. Both the PGPR inoculated plants infested with *S. litura* exhibited marked increase in proline content of leaves over infested plants. The maximum (59%) increase was recorded in the *Bacillus endophyticus* inoculated plants infested with *S. litura.* Chlorophyll a, b and carotenoids followed the similar pattern of response to PGPR and *S. litura* infestation (Figure 6). The response of PGPR was higher (*p* ≤ *0.05*) particularly for carotenoids content. Both the protein and the sugar contents were higher (*p* ≤ *0.05*) in PGPR inoculated plants (Figure 7) under unstressed conditions. *Pseudomonas aeruginosa* showed maximum (1.4 fold) increase in sugar content over infested plants. The infestation with *S. litura* had increased sugar and protein contents significantly higher than the control. The inoculated plants receiving insect infestation exhibited up to 2.25 fold increase in sugar content as compared to that of infested plants.

**Figure 5.** Proline content (μg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 6.** Chlorophylls and carotenoids content (mg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 7.** Sugar and protein content (mg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

The weight of tomato fruit was about 35% greater in plants inoculated with *Bacillus endophyticus* while *Pseudomonas aeruginosa* inoculated plants exhibited 44% increase over control. There was 26% decrease in the weight of tomato fruit in infested plants (Figure 8). The PGPR inoculated plants ameliorated the inhibitory effect of the insect and showed up to 78% increase in the fruit weight over infested plants.

**Figure 8.** Weight of tomato fruits/plant (g) infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

The infestation with insects enhanced the SOD activity. The SOD activity was three fold higher in leaves of plants inoculated with *Bacillus endophyticus* (T1). Plants inoculated with *Pseudomonas aeruginosa* (T2) on infestation further augmented SOD (Figure 9).

**Figure 9.** Superoxide dismutase (SOD) activity in tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

#### *3.3. Phytohormones Contents of Leaves*

The data in Figure 10 revealed that uninoculated uninfested control leaves of tomato had traceable amounts of Salicylic acid. Insect infestation produced very little amounts of SA. Both the PGPR produced significantly higher amounts of SA in plants, *Pseudomonas* sp. being more efficient. The SA was 1.8 folds greater than infested plant leaves. In *Pseudomonas* inoculated plants, this was further augmented and significantly higher (3.6 fold) SA was recorded in infested plant leaves pretreated with *Pseudomonas aeruginosa*. IAA was not detected in the control and insect infested plants but both the PGPR produced significant amount of IAA in the leaves of inoculated plant which was further augmented and up to 449 μg IAA/g leaves was detected in the leaves of plants infested with *S. litura* and pretreated with *Pseudomonas aeruginosa* (Figure 10). Insect infestation increased the GA content of leaves significantly over control. Several fold increases in GA production were recorded in both the PGPR inoculated plants: *Pseudomonas aeruginosa* being most efficient. Both the PGPR inoculated plants overcame the insect infestation induced decrease in GA content (Figure 10). The ABA content was significantly lower in the infested plant leaves as compared to control. *Bacillus endophyticus* inoculation showed significantly higher ABA production under controlled conditions and the value was several times greater than control in the inoculated plant infested with *S. litura*.

**Figure 10.** Phytohormone content in the leaves of tomato plants infested with *S. litura* and under control condition. (**A**): Salicylic acid; (**B**): Indole acetic acid; (**C**): Gibberellic acid; (**D**): Abscisic acid.

#### *3.4. Detection of Secondary Metabolites from Extract of Tomato Leaves*

Thin layer chromatography of tomato leaf extract showed 29 bands of different colors under UV light (Table 1). Calculated Rf values of leaf extract were compared with Rf values of standard compounds ferulic acid (0.72), salicylic acid (0.60), o-coumeric acid (0.74), trans-cinnamic acid (0.74), caffeic acid (0.85), p-coumaric acid (0.77).

The un-inoculated non infested control plant leaves extract contained caffeic acid (Rf 0.85) and quercetin (Rf 0.88). This was in contrast to *Bacillus endophyticus* inoculated plant leaves which exhibited some unidentified compounds at Rf 0.50 in addition to myricitin (Rf 0.73) o-coumaric (Rf 0.74) whereas, *Pseudomonas aeruginosa* inoculated plants showed the presence of flavonoids, ferulic acid, o-coumaric, kaempferol-7-neoheps-eridiside-glycosides in addition to some unidentified compounds of low polarity. Infestation with *S. litura* resulted in the production of caffeic acid and o-coumaric acid in addition to low and high polarity unidentified compounds. PGPR inoculated plants on infestation produced salicylic acid, rutin and kaempferol in addition to p-Coumaric acid and some unidentified compounds.

**Table 1.** Putative secondary metabolites identified on the basis of the Rf values in the extract of tomato leaves of different treatments.