**Palmitate but Not Oleate Exerts a Negative E**ff**ect on Oxygen Utilization in Myoblasts of Patients with the m.3243A**>**G Mutation: A Pilot Study**

#### **Leila Motlagh Scholle 1, \*, Helena Schie**ff**ers 1 , Samiya Al-Robaiy 2 , Annemarie Thaele 1 , Diana Lehmann Urban 3 and Stephan Zierz 1**

Received: 5 August 2020; Accepted: 15 September 2020; Published: 16 September 2020

**Abstract:** It is known that exposure to excess saturated fatty acids, especially palmitate, can trigger cellular stress responses interpreted as lipotoxicity. The effect of excessive free fatty acids on oxidative phosphorylation capacity in myoblasts of patients with the m.3243A>G mutation was evaluated with the mitochondrial (Mito) stress test using a Seahorse XF96 analyzer. ß-oxidation, measured with the Seahorse XF96 analyzer, was similar in patients and controls, and reduced in both patients and controls at 40 ◦C compared to 37 ◦C. Mito stress test in the absence of fatty acids showed lower values in patients compared to controls. The mitochondrial activity and ATP production rates were significantly reduced in presence of palmitate, but not of oleate in patients, showing a negative effect of excessive palmitate on mitochondrial function in patients. Diabetes mellitus is a frequent symptom in patients with m.3243A>G mutation. It can be speculated that the negative effect of palmitate on mitochondrial function might be related to diacylglycerols (DAG) and ceramides (CER) mediated insulin resistance. This might contribute to the elevated risk for diabetes mellitus in m.3243A>G patients.

**Keywords:** fatty acid oxidation; palmitate; oleate; m.3243A>G mutation


**1. Introduction**

[75] Perez-Martin E, Weiss M, Diaz-San Segundo F, Pacheco JM, Arzt J, Grubman MJ, et al. Bovine type III interferon significantly delays and reduces severity of foot-and-

[76] Dias CC, Moraes MP, Weiss M, Diaz-San Segundo F, Perez-Martin E, Salazar AM, et al. Novel antiviral therapeutics to control foot-and-mouth disease. J Interferon Cyto‐

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mouth disease in cattle. J Virol. 2012;86(8) 4477-4487.

212 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

kine Res. 2012;32(10) 462-473.

agents. Antiviral Res. 2012;96(2) 213-220.

The Age of Enlightenment is the period in time when the method of reasoning known as the Scientific Method was developed. This revolution in science began with the description of the sun as the center of our solar system rather than the earth. Natural phenomena previously explained by spiritualists were now described by science. Given our still evolving under‐ standing of influenza, it is perhaps no coincidence that we describe the combined effects of the influenza virus gene segments with the word 'constellation', which has astrological roots describing the position of the stars. Interestingly, the name influenza also has astrological roots: it was borrowed from the Italian word *influenza* in the mid-17th century which, in turn, was derived from the Medieval Latin word *influentia*, a 14th century term that refers to the influence of the stars. The scientific rational to describe the influence of the influenza gene constellation on virus phenotype is currently being resolved. Here we try to shine some light on the subject by providing the reader with background information, recent experimental results and provide a framework for questions that remain unanswered.

Influenza is a common infectious respiratory disease caused by influenza viruses. The host range of these viruses can include birds, humans and other mammals. Influenza viruses cause seasonal epidemics and are almost globally ubiquitous. They cause significant morbidity and mortality each year yet some infected persons remain asymptomatic. Influenza is typically transmitted by aerosols produced by coughing or sneezing. Although virus particles on contaminated surfaces can be easily inactivated, the virus is still able to spread easily and rapidly. Vaccination is the recommended approach to prevent disease because of the possible emergence of drug resistance.

Vaccines are produced each year to counter the currently circulating seasonal strains. The influenza vaccine seed viruses used to produce the immunogenic proteins are reassortant viruses. That is, they contain a mix of gene segments from different viruses. The genomes of

© 2013 Plant and Ye; licensee InTech. This is a paper 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, provided the original work is properly cited.

the influenza A and B viruses are made up of eight negative-strand RNA segments. The haemagglutinin (HA) and neuraminidase (NA) proteins found on the surface of the virus are found on two different segments. Usually these two segments from a seasonal virus are combined with another six gene segments from a high yield strain to make a vaccine seed virus. HA is the major immunogenic protein recognized by the host immune system. Because of influenza's high rate of mutation, and the capacity of the genome to tolerate many mutations, there is a need to update the influenza vaccine seed virus strains each year. The influenza virus is able to avoid control by the host immune system via two major types of mutation. Antigenic drift is the process of gradual genetic mutation, especially in the HA gene, that results in newer viruses not being well recognized by antibodies that recognized the progenitor virus. Anti‐ genic shift is the replacement of one or more segments from one influenza virus with those of another. This unpredictable event can lead to a change in host range, transmission or patho‐ genicity. Likewise, genetic reassortment, the mixing of genomic segments from different strains, can generate undesirable characteristics in the influenza vaccine seed viruses. Here we explore possible reasons for this and describe approaches that might be beneficial to the development of influenza vaccine seed viruses.

that one of each segment is packaged into one virion. However, it also helps alleviate a problem faced by many RNA viruses, the high error rate inherent in RNA synthesis. The error rate for the influenza viruses has be calculated to be 2.0 x 10-6 and 0.6 x 10-6 mutations per site per infectious cycle for influenza A and B respectively [5]. Rates ranging from 3.72 to 6.77 x 10-4 substitutions per site per year have been calculated for the influenza C segments [6]. Influen‐ za viruses can exist as a quasispecies, that is, a group of diverse viruses that collectively contribute to the characteristics of the population (reviewed in [7]). This enables mutations to exist that, by themselves, may not increase the fitness of the virus and could even be detrimen‐ tal. A combination of these mutations, that together increase the fitness of the virus, may result in a virus with some selective advantage. Such a combination could occur by gene reassort‐ ment. The separation of different mutations on different virus segments facilitates this proc‐

PB2 (2348 nt)

C D

A B

PB1 (2319 nt)

PA (2269 nt)

HA (1833 nt)

NP (1806 nt)

M1 BM2 (1149 nt)

**Figure 1.** Genomes of influenza viruses. A) The ends of the negative strand influenza genomic RNA are complexed with the three polymerase proteins and the remaining sequence is encapsidated with nucleoprotein (vRNP (-)). The positive strand cRNP is similarly complexed. The mRNA is transcribed with a 5' cap structure and poly-A tail (see main text for details). Figure used with permission from Resa-Infante et al., 2011. B) Schematic of the influenza A virus ge‐ nome. The bold black lines represent the 3' and 5' untranslated regions. The blue and pink boxes represent the major protein coding regions. C) Schematic of the influenza B virus genome. The green and brown boxes represent the ma‐ jor protein coding regions. D) Schematic of the influenza C virus genome. The red and purple boxes represent the ma‐ jor protein coding regions. The protein coding regions are not to scale. Coding regions in a different reading frame are

shown above or below each other, coding regions in the same frame are show as contiguous blocks.

(1055 nt) NS1

(1513 nt) NB

NS2

NA

PB2 (2365 nt) PB1 (2363 nt) P3 (2183 nt)

(2363

(863 nt) NS1

PB2 (2280 nt)

(2274 nt) PB1 (& PB1-N40)

Gene Constellation of Influenza Vaccine Seed Viruses

http://dx.doi.org/10.5772/55289

215

HA (1701 nt)

2152 nt) PA

NP (1497 nt) NA (1410 nt) (982 nt) M1 M2

(2365 (2280 (1701 (1497 (1410

NP (1802 nt) M1 CM2 (1180 nt)

(935 nt) NS1

NS2

NS2

PB1-F2

PA-X

HEF (2073 nt)


*<IP_ADDRESS>. Electron beam‐induced current (EBIC) [117]*

Another method to obtain the electrical parameters is EBIC from which the calculated carrier diffusion length forCH3 NH3 PbI3−*x* Cl*x* is 1.5–1.9 μm [40]. The carrier diffusion length is compa‐ rable or longer than that of other polycrystalline semiconductors with direct bandgaps used in solar cells [76, 77, 118–120]


*3.6. Rhabdoid Cells*

Rhabdoid tumors have been documented in many different topographies across the body [62], always linked to biological aggressiveness and bad prognosis. Its histogenesis is still unclear. A handful of rhabdoid tumors of the bladder have been published, particularly in children and young adults [63–65]. There are, however, isolated cases reported in adulthood [66–68].

Aside from genuine rhabdoid tumors, a focal *rhabdoid* phenotype can be observed sometimes in UC [68], where large and ovoid cells with large atypical nuclei and lateralized eosinophilic cytoplasm may appear growing without any specific pattern usually in high-grade neoplasms. A possible rhabdomyoblastic dedifferentiation in the context of a sarcomatoid UC should be ruled out, at least theoretically, in these cases.

By immunohistochemistry, rhabdoid cells are positive for CK7, CK20, vimentin, E-cadherin, and β-catenin, p63, and INI-1 [68].

#### *3.7. Lipid*/*Lipoblast-Like Cells*

These two terms refer to a rare variant of UC composed of lipidic appearing tumor cells intermingled with transitional cells in variable proportions. It was first recognized by Mostofi et al. in 1999 [69]. Since then only single case reports and two short series [70,71] have been published. The longest series so far analyzes 27 cases collected from different international institutions [71]. Lipidic-appearing cells may resemble either adipocytes or adipoblasts (Figure 2e) and usually take part

in a high-grade UC, not otherwise specified. Immunohistochemistry confirms the epithelial nature in all cases, including the co-expression of CK7 and CK20 [70,71].


**Abbreviations**

The following abbreviations are used in this manuscript:

#### **Appendix A. Supplementary Case Data from the Guizhou Grid, China**

Table A1 shows the BESS properties of the 14 microgrids in the Guizhou Grid, including capacity, initial SOC, charge and discharge restriction and charge and discharge efficiency.

**Table A1.** Battery energy storage system properties of 14 microgrids in the Guizhou Grid.

The peak/flat/valley electricity price formulated by Guizhou Grid, China is presented in Table A2, which divides a day into three types of time internals.

**Table A2.** Peak/flat/valley electricity price formulated by the Guizhou Grid.

The learning rate *α*, discount factor *γ* and greedy degree *ε* parameters of the 14 microgrids are given in Table A3.

**Table A3.** Q-Learning Parameters of 14 Microgrids.

The values of hyper parameters that appear in this paper are given in Table A4.

**Table A4.** Hyper parameters settings for the proposed Q-learning algorithm.


**6.1 Janmarg: Ahmedabad**

The city has decided to develop and implement an integrated public transit system including:

The primary objective of the integrated public transit initiative in Ahmedabad is to make the city more accessible – with physical, social and economic accessibility.

Ahmedabad city has developed a Bus Rapid Transit System under the Jawaharlal Nehru National Urban Renewal Mission (JNNURM) with the name "Janmarg" or "the people's way" and the slogan 'Accessible Ahmedabad'.Janmarg boasts of an innovative plan anddesign which includes pedestrian only sections and one-way bus lane etc.

#### **6.2 "Namma" metro: Bengaluru**

Bangalore Metro Rail Corporation Limited (BMRCL), a joint venture of Government of India and Government of Karnataka is a Special Purpose Vehicle entrusted with the responsibility of implementation of the Bengaluru Metro Rail Project. "Namma Metro" is an environment friendly initiative as it aims at reducing carbon emissions in the city. The project has an East–West corridor - 18.10 km long, starting from Baiyappanahalli in the East and terminating at Mysore Road terminal in the West and a 24.20 km North–South corridor commencing at Nagasandra in the North and terminating at Puttenahalli in the South.

In connection with the construction of Bengaluru Mass Rapid Transit System, the Government of Karnataka has introduced a number of innovative measures to create a dedicated resource pool, including special cess to capture land value increments due to transit [19]. The Government has taken up several value capture instruments like development ceases, chess on additional FAR, Transferable Development Right (TDR) etc. to finance Bengaluru mass rapid transit system.

*Smart Growth and Transit Oriented Development: Financing and Execution Challenges in India DOI: http://dx.doi.org/10.5772/intechopen.95034*


*8.1.1 Pelvic floor exercise*

Pelvic floor exercises helps limit the progression of mild prolapse and relief milder prolapse symptoms. However, pelvic floor exercises on its own does not to restore normal support and are not useful if the prolapse extends to or beyond the vaginal introitus. It is reported to salivate 42–53% of mild and moderate prolapses up to grade two descent but it is unlikely to have any improvement in patients with more than grade two descent [25, 27, 28].

Urogynaecology nurse specialist should be able to guide the patient through pelvic floor exercises along with the physiotherapist and assess the response in 3–4 months.

#### *8.1.2 Pessary management*

Vaginal pessaries offer help to women who decline surgery, who are unfit for surgery, or for whom surgery is contraindicated [29].

Long-term pessary use has a high drop rate because of side effects such as vaginal ulceration, discharge and bleeding. It has been reported that only 15–20% of women uses pessaries as long term approach [30, 31].

Fitting the correct pessary size requires a trial and error. A digital examination allows some estimate of vaginal size. In general, it is best to start with smaller pessaries, increasing the size if not staying in place. Change of pessaries usually with in 6 months if no complications such as ulceration and bleeding. If the vagina is atrophic, topical oestrogen cream should be used twice to reduce the risk of ulceration and bleeding. Pessaries can ring pessaries ranging in diameter from 52 to 129 mm and they placed between the back of symphysis pubis and posterior fornix. They usually help with prolapsed when there is uterus as the cervix fits in the middle of the ring. However patients who had hysterectomy they are more likely to benefit off shelf pessaries compared to the ring pessaries as the shape of those provides better support for the vaginal vault where shape and axis could have changed by the hysterectomy [32, 33].

Urogynaecology nurse specialist should be able to fit the pessaries and change them and identify any ulceration and bleeding caused by pessary use. The nurse led pesky clinic in primary and secondary care is a well established model and proved to be cost effective and reduces patient loads on the outpatients clinics with high patient satisfaction rate [34].


*2.4. Technological Characteristics of the Bread*

## 2.4.1. Bread Texture

Bread texture was evaluated using a texturometer TA-XTplus (Stable MicroSystems, Surrey, UK) in penetration mode. Each bread sample was cut into slices with a height of 20 mm and 120 × 100 mm rectangular shape, and slices rested for 15 min before testing. An acrylic cylindrical probe with 10 mm diameter pierced 5 mm of the sample at 1 mm/s of crosshead speed, with a load cell of 5 kg, according to the method earlier described in reference [12]. Comparison of the bread texture with different Yg and Cc contents was performed in terms of firmness.

Bread staling was evaluated measuring firmness during a storage time of 96 h, and the aging kinetics of the bread was described as a function of the dairy product incorporation according to a linear Equation (1)

$$\text{Firmness} = \text{A}^\* \text{ time} + \text{B},\tag{1}$$

where A can be considered the aging kinetics and B the initial firmness.

## 2.4.2. Quality Parameters of Bread

Bread moisture was determined according to the standard method AACC 44–15.02. Water activity (aW) variations were determined at room temperature (Hygrolab, Rotronic).

Bake loss (BL) of breads, which is defined as the amount of water and organic material (sugars fermented and CO2 released) lost during baking [15], was calculated, according to the Equation (2).

$$\text{Bake loss } (\%) = \left[ (\text{W}\_{lb} - \text{W}\_{ab}) / \text{W}\_{lb} \right] \times 100 \tag{2}$$

where

*Wab*: weight of the loaf after baking;

*Wbb*: weight of the loaf before baking.

Bread volume was determined by rapeseed displacement according to the standard method AACC 10-05.01 after 2h of cooling down [8]. Specific bread volume was calculated using Equation (3).

$$\text{Specific volume (cm}^3\text{/g)} = \text{bread volume (cm}^3\text{)} \text{bread weight (g)} \tag{3}$$

Knowing the weight and the volume of the bread, the specific weight (g/cm3) can also be estimated using the Equation (4), i.e., the reciprocal of specific bread volume.

$$\text{Specific weight (g/cm}^3) = \text{bread weight (g)} \text{bread volume (cm}^3) \tag{4}$$

All the experiments were done at least in triplicate.

#### *2.5. Image Analysis of the Bread Slices*

The gas cell number of the crumb breads was evaluated 120 min after production using image analysis technology. Images of three slices of breads (control, yoghurt, and curd cheese breads) were scanned full-scale using an Image Scanner (Xerox Corporation, Webster, NY, USA).

A threshold method was used for differentiating gas cells, and the segmentation was performed manually by binarization of grey-scale images into black-and-white images using the Otsu and default ImageJ algorithms, carried out in the ImageJ-based Fiji 1.46 software package [16].

#### *2.6. Nutritional Composition of the Bread*

Total nitrogen content was analysed by the MicroKjeldahl method (ISO 20483:2006). The quantification was performed by molecular absorption spectrophotometric equipment (Skalar-Sanplus System, Auto-Analyser, Giesen, Germany) [17,18].

Fat content was determined according to NP 4168. Ash content was determined by incineration at 550 ◦C in a muffle furnace (AACC Method 08-01.01).

Total mineral contents were determined by inductively coupled plasma atomic emission spectrometer (ICP-AES: Thermo System, ICAP-7000 series). All the experiments were done in triplicate.


2.1.2. Daily Variations in T

The temperature ranges from 15 to 35 ◦C in the afternoons. Because the bare surface will likely be heated above the atmospheric temperature, the range of ground surface temperature will likely be greater than the average atmospheric temperature range.


*Article* **Post-Traumatic Sepsis Is Associated with Increased C5a and Decreased TAFI Levels**

**Jan Tilmann Vollrath 1, Ingo Marzi 1, Anna Herminghaus 2, Thomas Lustenberger 1 and Borna Relja 1,3,\***

Received: 8 March 2020; Accepted: 20 April 2020; Published: 24 April 2020

**Abstract:** Background: Sepsis frequently occurs after major trauma and is closely associated with dysregulations in the inflammatory/complement and coagulation system. Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a dual role as an anti-fibrinolytic and anti-inflammatory factor by downregulating complement anaphylatoxin C5a. The purpose of this study was to investigate the association between TAFI and C5a levels and the development of post-traumatic sepsis. Furthermore, the predictive potential of both TAFI and C5a to indicate sepsis occurrence in polytraumatized patients was assessed. Methods: Upon admission to the emergency department (ED) and daily for the subsequent ten days, circulating levels of TAFI and C5a were determined in 48 severely injured trauma patients (injury severity score (ISS) ≥ 16). Frequency matching according to the ISS in septic vs. non-septic patients was performed. Trauma and physiologic characteristics, as well as outcomes, were assessed. Statistical correlation analyses and cut-off values for predicting sepsis were calculated. Results: Fourteen patients developed sepsis, while 34 patients did not show any signs of sepsis (no sepsis). Overall injury severity, as well as demographic parameters, were comparable between both groups (ISS: 25.78 ± 2.36 no sepsis vs. 23.46 ± 2.79 sepsis). Septic patients had significantly increased C5a levels (21.62 ± 3.14 vs. 13.40 ± 1.29 ng/mL; *p* < 0.05) and reduced TAFI levels upon admission to the ED (40,951 ± 5637 vs. 61,865 ± 4370 ng/mL; *p* < 0.05) compared to the no sepsis group. Negative correlations between TAFI and C5a (*p* = 0.0104) and TAFI and lactate (*p* = 0.0423) and positive correlations between C5a and lactate (*p* = 0.0173), as well as C5a and the respiratory rate (*p* = 0.0266), were found. In addition, correlation analyses of both TAFI and C5a with the sequential (sepsis-related) organ failure assessment (SOFA) score have confirmed their potential as early sepsis biomarkers. Cut-off values for predicting sepsis were 54,857 ng/mL for TAFI with an area under the curve (AUC) of 0.7550 (*p* = 0.032) and 17 ng/mL for C5a with an AUC of 0.7286 (*p* = 0.034). Conclusion: The development of sepsis is associated with early decreased TAFI and increased C5a levels after major trauma. Both elevated C5a and decreased TAFI may serve as promising predictive factors for the development of sepsis after polytrauma.

**Keywords:** polytrauma; sepsis; complement; patients

#### **1. Introduction**

Trauma is responsible for one out of 10 deaths worldwide and is among the most frequent causes of mortality, notably among young people [1]. Traumatized patients who survive the initial post-injury phase are at high risk to die from late-occurring complications such as multiple organ failure (MOF), respiratory complications, and/or sepsis [2–5]. During sepsis, the activation of both coagulation and complement systems, which can be triggered by a pathogen itself or damaged tissues, is essential as a reaction to tissue injury and for an adequate host defense against pathogens [6,7]. Nevertheless, abnormalities in the well-regulated interactions between these systems or their excessive activation can be harmful and lead to thrombosis or disseminated intravascular coagulation [6,7].

Thrombin-activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase 2 or carboxypeptidase U, is a carboxypeptidase B-like proenzyme with a molecular weight of 56 kDa [8]. Upon activation by the thrombin-thrombomodulin complex at the vascular endothelial surface, its activated TAFI form (TAFIa) inhibits fibrinolysis by removing C-terminal lysines from fibrin, which are imperative for the efficient formation of plasmin [8,9]. In a septic rat model with *Pseudomonas aeruginosa*, the inhibition of TAFIa reduced the systemic inflammatory response, thus suggesting that TAFI plays an important role in the deterioration of organ dysfunction in sepsis [10]. In a rat model of a sepsis-complicating burn injury, TAFI was elevated after 24 and 72 h, confirming the close link between inflammation and coagulation [11]. Septic patients, as well as healthy study subjects with low-grade lipopolysaccharide (LPS)-induced endotoxemia, showed decreased levels of TAFI [12,13], and in polytraumatized patients, TAFI levels inversely correlated with the inflammation-associated development of complications [14].

Anaphylatoxin C5a is a strong chemoattractant signal that is involved in the regulation of the innate immune system, playing a key role in host homeostasis, inflammation, and defense against pathogens [15–19]. Upon activation through the cleavage of C5 to C5a and C5b by the C5-convertase, C5a takes part in the recruitment and activation of inflammatory cells like neutrophils, eosinophils, T lymphocytes, and monocytes [19,20]. Recently, in a baboon model of *Escherichia coli* bacteremia, it was shown that complement-mediated bacteriolysis had a detrimental effect by inducing a release of LPS and fulminant inflammation [21]. The inhibition of C5 cleavage blocked sepsis-induced inflammation, decreased the associated consumptive coagulopathy, and protected organ functions, resulting in improved survival [21].

Besides its role in coagulation, TAFI has been shown to have anti-inflammatory properties, thus being able to inactivate activated complement factors C3a and C5a [22,23]. Therefore, in the present study, we included severely injured trauma patients with sepsis to determine whether TAFI might represent a possible link between inflammation/complement and coagulation in sepsis.

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

#### *2.1. Ethics*

This study was performed at the University Hospital of the Goethe University Frankfurt with the institutional ethical approval in accordance with the Declaration of Helsinki and following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines (167/05). Written informed consent was obtained from all enrolled patients in accordance with ethical standards. All patients signed the informed consent forms themselves, or informed consent was obtained from the nominated legally authorized representative consenting on the behalf of participants, as approved by the ethical committee.

#### *2.2. Patients*

Patients were included according to the following criteria: history of penetrating or blunt trauma with an injury severity score (ISS) ≥ 16 and between 18 and 80 years of age. Patients with pre-existing immunological disorders, concomitant acute myocardial infarction, immunosuppressive or anticoagulant medication, burns, thromboembolic events, and/or lethal injury were excluded. All patients were treated according to the Advanced Trauma Life Support (ATLS®) standards and the polytrauma guidelines. While haemodynamically instable patients received immediate surgery, haemodynamically stable patient underwent whole-body computed tomography. Upon arrival to the emergency room, the following demographic and clinical data were collected: age; gender; blood

pressure; respiratory rate; heart rate; temperature; mechanism of injury; abbreviated injury scale for each body region (head, chest, abdomen, and extremity); and the general injury severity (ISS), as described before [24]. Routine blood gas analysis (including pH and lactate) was performed upon admission to the hospital. The numbers of fresh frozen plasma (FFP) and packed red blood cell (PRBC) units transfused within the first 24 h and during further clinical course were recorded. The diagnose of sepsis was assessed by both the 2005 criteria outlined by the International Sepsis Forum [25], as well as by the revised definition criteria according to the Sepsis-3 criteria [26,27]. Systemic inflammatory response syndrome (SIRS) was defined by fulfilling at least two of the following criteria: heart rate > 90 beats per minute, respiratory rate > 20 per minute or arterial carbon dioxide tension (PaCO2) < 32 mm Hg, body temperature >38 ◦C or <36 ◦C, and white blood cell count >12,000 cells/mm3 or <4000 cells/mm3 or with >10% immature forms. According to the old definition, sepsis was diagnosed when the patients fulfilled criteria for SIRS and had evidence for an infection. However, limitations of that definition, including an excessive focus on inflammation and the inadequate specificity and sensitivity of the SIRS criteria, led to the Sepsis-3 definition, in which sepsis was defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. The authors have summarized that, for clinical operationalization, organ dysfunction can be represented by an increase in the sequential (sepsis-related) organ failure assessment (SOFA) score of 2 points or more, which is associated with an enhanced in-hospital mortality risk [26,27]. Medical records were analyzed regarding length of in-hospital stay, length of ICU stay, and in-hospital mortality.

#### *2.3. Blood Processing and Analysis*

Blood samples were obtained from 48 severely traumatized patients as early as possible after admission to the emergency department (ED) and until day ten daily in pre-chilled ethylene-diaminetetraacetic acid (EDTA) or heparin vacuum tubes (BD vacutainer; Becton Dickinson Diagnostics, Aalst, Belgium) for routine diagnostics or for laboratory investigations and were kept on ice until centrifugation at 2000× *g* for 15 min at 4 ◦C. Afterwards, supernatants were stored at −80 ◦C until analysis. The mean time between injury and acquisition of the blood sample was 81 ± 8 min.

TAFI levels were measured by using a commercially available ELISA (IMUCLONE™ TAFI ELISA; American Diagnostica Inc., Stamford, CT, USA) according to the manufacturer's instructions. The TAFI antigen level is expressed as a percentage of normal pooled plasma. C5a levels were determined using a commercially available Human C5a ELISA kit II (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. Blood counts were measured using the Sysmex XE-2100 automated blood cell counter (Sysmex Europe GmbH, Norderstedt, Germany). pH, lactate, and PaCO2 were determined using a standard clinical blood gas analysis device (ABL800 Flex, Radiometer, Krefeld, Germany).

#### *2.4. Statistics*

All data were tested for normal distribution by a Kolmogorov-Smirnov-Lillieford's test. Continuous variables were compared using the Mann-Whitney U test, and categorical variables were analyzed with the two-sided Fisher´s exact test. Data are presented as the mean ± standard error of the mean (SEM) unless otherwise stated. Receiver-operator curves (ROC) were calculated to analyze the optimal cut-off values. The sample size was performed based on the mean TAFI data obtained from the pilot study in Relja et al. [14]. Based on the mean values, a Cohen's d of 1.060 and an effect size of *r* = 0.468 were calculated. Applying a power of 0.8, a group size of 15 per group is necessary to reach significant results in an unpaired *t*-test (α = 0.05). Results were considered as statistically significant when the *p*-value was <0.05. Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA).

#### **3. Results**

#### *Main Findings*

A cohort of 48 patients (33 male) met the inclusion criteria and were included in the study. Mean patient age was 52.23 ± 2.74 years, and all patients were substantially injured with a mean ISS of 24.95 ± 1.75. Of these, 14 patients with an ISS of 23.36 ± 2.79 developed sepsis during the clinical course, while 34 patients with an ISS of 25.78 ± 2.36 did not develop sepsis or septic complications. Table 1 summarizes the general patient and injury characteristics of the study population. There were no significant differences between the sepsis and no sepsis group with regard to the general patient and injury characteristics. The mean time between the onset of trauma and arrival to the ED was 66.17 ± 4.85 min. The mean time until admission to the ED between the cohort of septic patients vs. not septic patients did not statistically differ (65.40 ± 11.55 vs. 66.50 ± 5.17 min).

**Table 1.** Demographic and trauma characteristics of the study population. Data are given as mean ± standard error of the mean; *p* < 0.05. Abbreviations: a, no sepsis group; AIS, abbreviated injury scale; b, sepsis group; ISS, injury severity score; and ns, no significance.

To investigate the influence of blood transfusions concerning the development of sepsis after polytrauma, the number of fresh frozen plasma (FFP) and packed red blood cells (PRBC) during the first 24 h after admittance to the emergency department and during the whole clinical course were determined. Patients belonging to the sepsis group received significantly more units of packed red blood cells during the whole clinical treatment compared to the no sepsis group (10.50 ± 2.50 vs. 5.46 ± 21.74; *p* < 0.05). As depicted in Table 2, there were no significant differences between these two groups concerning the units of packed red blood cells during the first 24 h, the systolic blood pressure <90 mm Hg at admittance to the ED, the number of transfused units of fresh frozen plasma within 24 h or during the whole clinical course, or the body temperature at admittance to the ED. Furthermore, no significant differences among the groups in the international normalized ratio (INR), activated partial thromboplastin time (PTT), or platelet counts (PLT) were assessed.

A significantly prolonged stay in the intensive care unit was observed in septic patients (22.14 ± 4.12 days vs. 7.00 ± 1.16; *p* < 0.0001), and in-hospital stays lasted significantly longer for this group (41.07 ± 6.43 vs. 19 ± 2.90; *p* < 0.001) compared to the group without septic complications. A total of six patients (12.5%) died, with a significantly higher number of patients in the sepsis group compared with the no sepsis group (four vs. two; *p* < 0.05; Table 3).

**Table 3.** Outcome of the study population. Data are given as mean ± standard error of the mean; *p* < 0.05. Abbreviations: a, no sepsis group; b, sepsis group; ICU, intensive care unit.

In order to further investigate the differences of clinical features at admission to the ED between patients developing sepsis during the clinical course and patients who did not develop sepsis, we compared lactate concentrations and pH values in the blood, heart rate, and respiratory rate in our study cohort. As shown in Figure 1, patients who developed sepsis had significantly higher amounts of lactate in their blood and a more acidic constellation of their acid bases balance, as depicted by significantly lower pH values (*p* < 0.05; Figure 1A,B). Furthermore, patients who developed sepsis during the clinical course had significant changes of their vital parameters, as they had higher respiratory and heart rates at their admission to the ED (*p* < 0.05; Figure 1C,D).

In order to investigate changes in coagulation and inflammation, we measured TAFI and C5a levels at admission to the ED. Patients belonging to the sepsis group had significantly lower TAFI levels and significantly higher C5a levels compared to the no sepsis group, as shown in Figure 2A,B, respectively (*p* < 0.05). Moreover, patients from the sepsis group showed significantly increased leukocytes counts compared with the no sepsis group (*p* < 0.05; Figure 2C).

**Figure 1.** Upon admission of severely injured trauma patients (*n* = 48) to the emergency department, lactate levels (**A**), pH values (**B**), heart rate (**C**), and respiratory rate (**D**) were measured. Patients were stratified to those who developed sepsis in their clinical course (sepsis, *n* = 14) vs. those who did not develop infectious complications (no sepsis, *n* = 34). Data are shown as mean ± SEM. \* *p* < 0.05 sepsis vs. no sepsis.

**Figure 2.** Upon admission of severely injured trauma patients (*n* = 48) to the emergency department, plasma thrombin-activatable fibrinolysis inhibitor (TAFI) levels (**A**), plasma C5a levels (**B**), and leukocyte counts (**C**) were determined. Patients were stratified to those who developed sepsis in their clinical course (sepsis, *n* = 14) vs. those who did not develop infectious complications (no sepsis, *n* = 34). Data are shown as mean ± SEM. \* *p* < 0.05 sepsis vs. no sepsis.

In addition, we analyzed correlations between TAFI and/or C5a and clinical (heart and respiratory rate) or laboratory parameters (pH value, leukocyte count, lactate value, INR, thromboplastin time (TPT), PTT, and PLT) and the SOFA score. TAFI and C5a, as well as TAFI and lactate, have shown a

significant negative correlation (TAFI and C5a: Pearson *r* = −0.4933, *p* < 0.0104 and TAFI and lactate: Pearson *r* = −0.4268, *p* < 0.0423; Table 4 and Figure 3A,B). Furthermore, C5a and lactate, as well as C5a and the respiratory rate, had a significant correlation (C5a and lactate: Pearson *r* = 0.4542, *p* < 0.0173 and C5a and respiratory rate: Pearson *r* = 0.4185, *p* < 0.0266; Table 4 and Figure 3C,D). In addition, correlation analysis of TAFI and C5a with the SOFA score have shown a significant negative correlation of TAFI with the SOFA score (Pearson *r* = −0.3865, *p* = 0.0422) and a significant positive correlation of C5a with the SOFA score (Pearson *r* = 0.3795, *p* = 0.0386) (Table 4).

**Table 4.** Correlation analyses between thrombin-activatable fibrinolysis inhibitor (TAFI), C5a, leukocyte counts, lactate levels, pH values, heart and respiratory rates, sequential (sepsis-related) organ failure assessment (SOFA) score, international normalized ratio (INR), thromboplastin time (TPT), partial thromboplastin time (PTT), and platelet (PLT). *p* < 0.05: statistically significant.

To investigate which of the examined parameters in this study (TAFI, C5a, leukocytes count, lactate, heart rate, and respiratory rate) could provide a prognostic marker for the development of sepsis after polytrauma, the predictive power of each parameter has been calculated using ROC analyses. While the ROC analysis for lactate did not show a statistically significant area under the ROC curve (*p* = 0.052), TAFI showed an optimal cut-off for predicting sepsis on admission to the ED at 54,857 ng/mL, with a sensitivity of 64% and specificity of 87.50%. The AUC was 0.7550 (95% CI: 0.585 to 0.925; *p* < 0.032; Table 5 and Figure 4A). C5a showed an optimal cut-off at 17 ng/mL on admission to the ED for predicting sepsis in the clinical course, with a sensitivity of 75.00%, specificity of 70.00%, and an AUC of 0.7286 (95% CI: 0.547 to 0.910; *p* < 0.034; Table 5 and Figure 4B). Leukocytes showed an optimal cut-off at 14.50 U/nl, with a sensitivity of 65.63%, specificity of 69.23%, and an AUC of 0.7043 (95% CI: 0.527 to 0.882; *p* < 0.033; Table 5 and Figure 4C). The heart rate showed an optimal cut-off

at 102.5/min on admission to the ED for predicting sepsis in the clinical course, with a sensitivity of 86.96%, specificity of 64.29%, and an AUC of 0.6957 (95% CI: 0.490 to 0.902; *p* < 0.049; Table 5 and Figure 4E). The respiratory rate showed an optimal cut-off at 15/min, with a sensitivity of 63.64% and a specificity of 64.29%. The AUC was 0.7029 (95% CI: 0.511 to 0.895; *p* < 0.043; Table 5 and Figure 4F).

**Figure 3.** Correlation analysis between TAFI and C5a (**A**), TAFI and lactate (**B**), C5a and respiratory rate (**C**), and C5a and lactate (**D**) are shown. *r*: Pearson coefficient.

**Table 5.** Receiver-operator curve (ROC) analyses on TAFI, C5a, leukocyte count, lactate level, heart rate, and respiratory rate at admission to the emergency department to predict the development of sepsis after polytrauma. Sensitivity and specificity are given as percentage values. Abbreviations: AUC, area under the curve and CI, confidence interval.

The results in Figure 5 reflect the dynamic course of both parameters after trauma and sepsis. The distribution of the TAFI and C5a values in the samples obtained in the ED and subsequent daily measurements for ten consecutive days show that the TAFI was significantly decreased on admission, day 2, and on day 9 in the sepsis group compared to the control group without sepsis (*p* < 0.05; Figure 5A). On the contrary, the C5a values were significantly increased on admission, days 2–6, and on day 9 in the sepsis group compared to the control group without sepsis (*p* < 0.05; Figure 5B).

**Figure 4.** Receiver operating curve analyses on TAFI (**A**), C5a (**B**), leukocyte counts (**C**), lactate levels (**D**), heart rate (**E**), and respiratory rate (**F**) at admission to the emergency department to predict the development of sepsis after polytrauma are demonstrated. *p* < 0.05: statistically significant, AUC: area under the curve, and CI: confidence interval.

**Figure 5.** Upon admission of severely injured trauma patients (*n* = 48) to the emergency department (ED), plasma TAFI levels (**A**) and plasma C5a levels (**B**) were determined daily for ten days (D1–D10). Patients were stratified to those who developed sepsis in their clinical course (sepsis, *n* = 14) vs. those who did not develop infectious complications (no sepsis, *n* = 34). Data are shown as mean ± SEM. \* *p* < 0.05 sepsis vs. no sepsis at the indicated time.

#### **4. Discussion**

Polytraumatized patients who survive the initial post-injury phase are at a serious risk to die from septic complications during their further clinical treatment [18]. Both the inflammatory/complement and the coagulation systems contribute to these harmful events. Regarding this, it is important to note that the cross-talk between the inflammation/complement and the coagulation occurs in both directions, as coagulation can affect the inflammatory activity as well as inflammation is able to activate coagulation [14,28,29]. TAFI is a carboxypeptidase B-like proenzyme, which, on the one hand, upon its activation to TAFIa by the thrombin-thrombomodulin complex inhibits fibrinolysis by removing C-terminal lysines from fibrin [8,9]. On the other hand, activated complement factors C3a and C5a can be inactivated by TAFIa [22,30]. The data suggests that TAFI may be a potential link between the inflammation/complement and the coagulation systems.

In the underlying study, we observed significantly reduced TAFI levels in patients developing sepsis during their clinical course after polytrauma compared to polytraumatized patients without septic complications. This difference occurred early upon admission of the patients to the ED. In contrast, C5a levels were significantly increased in septic patients after polytrauma. A significant negative

correlation between TAFI and C5a levels was found. In addition, the correlation analyses of TAFI and C5a with the SOFA score have demonstrated a significant negative correlation of TAFI and a significant positive correlation of C5a with the SOFA score. These findings enhance markedly the impact of both TAFI and C5a as biomarkers, which may indicate later-occurring development of the post-traumatic sepsis. However, whether reduced TAFI levels in septic patients after polytrauma derive from, e.g., consumption in the course of coagulopathy or whether initially reduced TAFI levels potentially put patients at risk for septic complications still remains elusive. It can be speculated that maybe upon reduced TAFI levels, missing inactivation of C3a and C5a may lead to an immunological imbalance and an excessive immunological reaction. As nicely reviewed and originally published by the expert group around Huber-Lang, anaphylatoxin C5a appears in the circulation of humans within 20 min post-polytrauma, and its levels have been related to the mortality rate [31,32]. That data indicated an almost synchronical rapid activation and dysfunction of the complement, suggesting a trauma-induced complementopathy early after injury [32]. Furthermore, the authors suggest that these events may participate in the impairment of the post-traumatic innate immune response. Within the time latency between 20 min of the complement activation, as shown by Huber-Lang, and 66 min until admission of patients to the ED, as shown in our study, certainly, a trauma-induced complementopathy may have occurred. Thus, levels of both TAFI and C5a may have been different at the onset of trauma, and we cannot exclude any alterations which may have emerged until the blood sampling in the ED was performed. Thus, the latency between the timepoint of trauma and sampling in the ED must be taken into account when interpreting the results. Relja et al. reported elevated TAFI levels in polytraumatized patients with and without infectious complications but with a significantly higher increase in both TAFI and TAFIa in patients without infectious complications [14]. This supports the hypothesis that reduced TAFI levels may increase the risk for the development of septic complications. In line with these findings, Zeerleder et al. as well reported significantly decreased TAFI levels in septic patients compared to healthy controls [33]. Interestingly, in our pilot study, we found that TAFI levels at admission to the ED and at day 4 after the trauma were significantly lower in the smaller cohort of patients with complications compared to the control group [14]. In that study, we were able to demonstrate that TAFI levels inversely correlated with the inflammation-associated development of not-further-defined complications after major trauma. Furthermore, coagulopathic patients, however, experienced significantly lower levels of activated TAFI on admission and on days six to eight [34]. Based on those results, the finding that TAFI and C5a levels immediately at admission to the ED can stratify patients who are going to develop sepsis in their clinical course is certainly a promising result in terms of a search for early sepsis biomarkers. Yet, it remains to be noted that, in our study, we did not prospectively evaluate the levels of both potential biomarkers and that this remains to be elucidated in further studies.

Naito et al. investigated the link between C5a and TAFI with regard to acute lung injury and demonstrated that, in TAFI-deficient mice, LPS treatment induced a more severe acute lung injury (ALI) compared to wild-type mice [35]. Furthermore, the authors observed significantly higher concentrations of IL-1beta, TNF-beta, and IL-6, along with increased lung infiltration of neutrophils, in TAFI-deficient mice [35]. Naito et al. concluded that TAFI plays a critical role in the pathogenesis of ALI via its ability to regulate the activity of complement factor C5a [35]. Nishimura et al. reported worse alveolitis in TAFI knock-out mice than in wild-type (WT) mice with C5a instillation directly into the lungs [36]. In line with these results, we observed decreased TAFI levels and elevated C5a levels in patients who developed septic complications after polytrauma. More importantly, TAFI correlated negatively with C5a. Fujiwara et al. investigated the role of TAFI in allergic bronchial asthma and showed significantly worse airway responsiveness and increased pulmonary concentrations of IL-5 and osteopontin in a TAFI-deficient mouse model [37]. Pretreatment with a C5a antibody significantly attenuated these effects, and thus, the authors concluded that TAFI plays a protective role in the pathogenesis of allergic inflammation, probably by inhibiting the complement system [37]. In a model of inflammatory arthritis (anticollagen antibody–induced arthritis (CAIA)), Song et al. investigated the

role of TAFI and showed that TAFI deficiency exacerbated inflammatory arthritis and, furthermore, that the cleavage of C5a by TAFI suppressed the ability of C5a to recruit immune cells to the affected joint [38]. Interestingly, the authors showed that the expression of both TAFI and C5a in synovial fluid was higher in patients with autoimmune arthritis than in those with osteoarthritis and concluded that TAFI plays an important role in attenuating local C5a-mediated inflammation and, thus, represents a molecular link between inflammation and coagulation in autoimmune arthritis [38].

While the above-mentioned studies all unanimously reported that decreased/lower levels of TAFI led to aggravated disease severity, Mook-Kanamori et al. showed that higher protein levels of TAFI in cerebrospinal fluid were significantly associated with systemic complications in patients with bacterial meningitis [39]. The authors observed survival benefits in TAFI-deficient mice in a pneumococcal meningitis mouse model as compared to wild-type mice and concluded that, during the initial phase of infection, a low level or an absence of TAFI results in initially increased complement levels, which may improve survival (in the animal model) and reduce systemic complications in humans suffering from bacterial meningitis [39]. Taken together, it still remains elusive whether TAFI acts as an acute reactant marker that links homeostasis and inflammation or whether a lack of TAFI plays an active role in the development of septic complications after polytrauma.

Most clinical studies examining anticoagulant effects on thrombo-inflammation have been performed in the context of sepsis, although some specific examples relevant to ischemia/reperfusion injuries are also reported. Unfortunately, the data in septic patients in which inhibitors of thrombin have been applied, as well as their efficiency, are limited [40]. As an example, the overall benefit of low molecular weight heparin in patients with sepsis remains, despite considerable investigations, uncertain [40]. Although initial trials with the IV infusion to restore plasma ATIII levels were promising, larger studies did not provide any benefit for overall mortality, and consequently, Rhodes et al. have made specific recommendations against the use of ATIII in the updated International Guidelines for the Management of Sepsis and Septic Shock [40,41]. The use of other anticoagulant agents, e.g., recombinant human activated protein C, have failed to consistently demonstrate a reduction in 28-day all-cause mortality in clinical trials [42]. Currently, soluble recombinant human thrombomodulin (rhTM) is undergoing clinical evaluation for the treatment of severe sepsis [40]. ART-123, an rhTM, appears to be a safe intervention in critically ill sepsis patients in phase 2b trials [40,43].

Although this study has several limitations—of those, the most important, the limited sample size—it must be considered that there is no significant difference between demographic and trauma characteristics between evaluated groups, which certainly ensures better compatibility of assessed data. Nevertheless, it has to be taken into account that patients in the sepsis group received significantly more PRBC during the whole clinical course, while there was no significant difference within the first 24 h (Table 2). Sadjadi et al. reported that, in trauma patients, infection was associated with the transfusion of PRBC and that transfused patients had eight times the increased risk of infection independently of their injury severity [44]. Likewise, Claridge et al. reported a dose-dependent correlation between transfusions of PRBC and the development of infection in traumatized patients [45]. Based on the data of our study, we cannot fully exclude an effect of transfused PRBC either on TAFI or on C5a, and this remains to be elaborated in future works. Although several studies have reported that a PRBC transfusion can negatively affect clinical outcomes [46,47], it still remains unclear if TAFI is an acute phase reactant that links hemostasis and inflammation or whether it plays an active role in the development of post-traumatic sepsis. With regard to the effects of PRBC transfusions on, e.g., the complement, certain clinical syndromes such as transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO) that occur within 6 h of blood transfusions indicate an association [48,49]. The major drivers of those complications and their pathogenesis, however, are complex and incompletely understood. The potential connection between the complement system and neutrophils is given, as well as the parallels between these syndromes and other acute pulmonary disorders, which are known for their involvement of the complement. Yet, similar to TAFI, we cannot exclude a direct impact of the PRBS transfusion on C5a levels or its activity; however, since the

transfusion rates within the first 24 h after the trauma did not differ between the septic and nonseptic group, we believe that the data as demonstrated are plausible. Moreover, C5a levels remain increased in septic patients nearly throughout the complete observational period, and thus, we believe that this effect is associated with inflammatory changes caused by sepsis rather than with PRBC transfusions, which, anyway, were initially not significantly enhanced. However, the increased amount of totally transfused PRBC in our study must therefore be considered as a disturbing variable by interpreting the data. Further limitations of the study include the study setting, since this study was conducted in a single center. In addition, the heterogeneity of the traumatized patients, as well as the variable timing of measurements, must be carefully considered in data interpretation. Although there were sufficient measurements made later after the trauma to determine the roles of TAFI and C5a as indicators of sepsis, a long-term study also including septic patients from other pathologies is necessary, but this was not the aim of the study.

Early recognition and effective management of patients at risk for infectious complications are essential for improved outcomes. However, early recognition specifically of post-traumatic sepsis is impeded by the lack of clinically proven and utilized biomarkers. The current work in clinical settings has not yielded the requisite gold standards for the diagnosis and monitoring of post-traumatic sepsis. Yet, identifying patients at risk for the development of sepsis during their clinical course is of great importance for further clinical treatment. TAFI and C5a may be helpful, specifically in, e.g., decision-making about the timing for post-traumatic surgeries or indicate patients that should early receive antibiotic therapy or undergo further specific clinical tests of sepsis markers. With regard to complement factors or TAFI as potential biomarkers in general, clinical studies with large subject numbers are needed to obtain the complete temporal profiles of, e.g., activation of the complement factors or factor dynamics in the course of sepsis development. Presumably, this should provide a multipanel set of biomarkers, which can be coupled with routine lab tests and blood cultures for the diagnosis and monitoring of sepsis.

Taken together, in the underlying study, we were able to show that the development of sepsis after major trauma is associated with decreased TAFI levels and increased C5a levels. The significant negative correlation between TAFI and C5a supports the concept that reduced TAFI levels may lead to an exacerbation of septic complications and that TAFI might play a protective role by downregulating inflammation. Furthermore, irrespective of their possible pathomechanistical roles in the onset of sepsis, both TAFI and C5a might serve as very early diagnostic markers to detect patients at risk for the development of sepsis or septic complications.

**Author Contributions:** Conceptualization, B.R. and T.L.; methodology, J.T.V. and B.R.; formal analysis, A.H. and B.R.; investigation, J.T.V.; data curation, B.R.; writing—original draft preparation, J.T.V.; writing—review and editing, I.M., A.H., T.L., and B.R.; visualization, A.H. and B.R.; supervision, B.R.; and project administration, B.R. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** We thank Kerstin Wilhelm, Minhong Wang, and Alexander Schaible for their outstanding technical assistance.

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).


**A Case Study on Distributed Energy Resources and Energy-Storage Systems in a Virtual Power Plant Concept: Technical Aspects**

**Tomasz Sikorski 1 , Michal Jasi ´nski 1,\* , Edyta Ropuszy ´nska-Surma 2 , Magdalena W˛eglarz 2,\* , Dominika Kaczorowska 1 , Paweł Kostyla 1 , Zbigniew Leonowicz 1 , Robert Lis 1 , Jacek Rezmer 1 , Wilhelm Rojewski 1, Marian Sobierajski 1, Jarosław Szyma ´nda 1 , Daniel Bejmert 3 , Przemysław Janik 3 and Beata Solak 1**

Received: 6 May 2020; Accepted: 8 June 2020; Published: 15 June 2020

**Abstract:** The article presents calculations and power flow of a real virtual power plant (VPP), containing a fragment of low and medium voltage distribution network. The VPP contains a hydropower plant (HPP), a photovoltaic system (PV) and energy storage system (ESS). The purpose of this article is to summarize the requirements for connection of generating units to the grid. Paper discusses the impact of the requirements on the maximum installed capacity of distributed energy resource (DER) systems and on the parameters of the energy storage unit. Firstly, a comprehensive review of VPP definitions, aims, as well as the characteristics of the investigated case study of the VPP project is presented. Then, requirements related to the regulation, protection and integration of DER and ESS with power systems are discussed. Finally, investigations related to influence of DER and ESS on power network condition are presented. One of the outcomes of the paper is the method of identifying the maximum power capacity of DER and ESS in accordance with technical network requirements. The applied method uses analytic calculations, as well as simulations using Matlab environment, combined with real measurement data. The obtained results allow the influence of the operating conditions of particular DER and ESS on power flow and voltage condition to be identified, the maximum power capacity of ESS intended for the planed VPP to be determined, as well as the influence of power control strategies implemented in a PV power plant on resources available for the planning and control of a VPP to be specified. Technical limitations of the DER and ESS are used as input conditions for the economic simulations presented in the accompanying paper, which is focused on investigations of economic efficiency.

**Keywords:** virtual power plant; distributed energy resources; energy storage systems; grid codes; power systems; smart grids; prosumer; business model; economic efficiency; sensitivity analysis

#### **1. Introduction**

A Virtual Power Plant (VPP) is still an actual approach and there is not a standardized definition for the framework of a VPP in the literature. The origin of the terminology of "Virtual Power Plant" may be traced back to 1997, when S. Awerbuch, in the book "The Virtual Utility—Accounting, Technology and Competitive Aspects of the Emerging Industry" defined Virtual Utility as flexible cooperation of independent, market-driven actors that assures an efficient energy service expected by the consumers without the need for appreciating assets [1]. A VPP manages distributed energy resources (DER) named also distributed generation (DG) units [2]. For example, wind, solar and hydroelectric power generation units are interconnected. Managing them together enables them to be more effective [3–5].

A Virtual Power Plant, as an autonomous, intelligent unit equipped with effective and safe power flow control systems, consists of generators, loads and energy storage that is connected to the distribution network [6]. These devices are equipped with controllers, which usually power electronic converters that allow for power flow control [6]. Coordinating the work of the entire VPP is a difficult and demanding task. The system's architecture must not only enable power flow control but also ensure VPP protection—not only related to power system security but also cybersecurity. In Reference [7], VPP architecture based on a common information model (CIM) and IEC standard 61850 is shown.

There have recently been many attempts to integrate intelligent solutions in power systems. An interesting discussion related to microgrids and the VPP is presented in Reference [8]. Microgrids allow increasing the efficiency of the use of distributed energy sources and energy storage systems. It also allows for regulating the load. Microgrids can be connected to the power system or operate as a standalone system. VPP management is based on computer software that enables the integration of distributed sources. In systems connected to the distribution network, the value of the power generated by the generation sources, the operation of the energy storage and the response of the demand side are optimized. Several propositions proclaiming the idea of transforming microgrids to a virtual power plant have recently been discussed, among others in References [2,6]. Additionally, it is also worth mentioning a new topic called virtual microgrids, which can be recognized as software solutions and algorithms supporting the planning, design and operation of microgrids. As an example of the virtual microgrids, it is worth noting a prosumer cluster connection into virtual microgrids to obtain cost reduction [9] or energy peer-to-peer trading in virtual microgrids [10].

Due to the random nature of the generated power, a large number of independent renewable energy sources can lead to system stability problems and therefore the connecting of distant generation sources, loads and energy storage units into a VPP has many benefits [11]. Work [12] shows the possibility of using charging points for electric vehicles, as well as wind generation, in the VPP concept. It also presents power flow optimization while taking into consideration price, wind generation and electric vehicles. Paper [13] concerns VPP control power consumption for heating. The operation algorithm is based on the application of thermal mass to the building to defer power consumption from electric space heating.

There are many different aspects to VPP power flow control. The main goals correspond to economic aspects related to electrical energy trading. The VPP control algorithms predict energy storage charging at low energy prices, as well as discharging energy storage and the sale of energy at peak demand at high prices. Paper [14] presents a stochastic bi-level optimization model to maximize day-ahead profit and to minimize predicted real-time production and the consumption of imbalance charges. In Reference [15], the bidding strategy of a VPP is determined using mathematical models. Ref. [16] presents decentralized coordination of VPP units, considering both active and reactive power using the novel Lagrangian relaxation-based mechanism. The method takes into account the effect of flexible demand and prevents the creation of new demand peaks and troughs. Another aspect concerns optimizing the use of locally generated energy and using the right strategy for storing energy in energy storage [17,18]. Power flow in a VPP, due to technical aspects, can also be optimized. In Reference [19], the binary backtracking search algorithm (BBSA) is used to optimize power flow in a VPP in order to achieve a reduction of generation cost and power losses, as well as, to increase reliability. To achieve

the same goal, risk-constrained stochastic programming is used in Reference [20] and the Imperialist competitive algorithm is used in Reference [21]. A big problem in the modern generation can also be seen to be carbon dioxide emissions. In Reference [22], binary particle swarm optimization (BPSO) is used to solve the indicated issues.

However, technical issues cannot be overlooked when planning the different strategies for a VPP. For example, voltage levels at all points in the distribution network should be within the range allowed by the standards. The same applies to the values of currents in the transformer lines and windings. Cooperation between units included in the VPP, meeting these expectations, is presented in Reference [23]. Moreover, issues regarding the operation of the storage itself are also important. Studies on the impact of energy storage parameters on VPP strategy and performance are presented in References [24,25]. The crucial technical aim of the VPP is concentrated on the aggregation control of the number of distributed generation units, which are grid-connected close to consumers (end-users, households). The aggregation verification may be a centralized or not system supported by a logic control algorithm, as well as, a communication infrastructure [26]. The control strategies must concern reliability, uncertainty, stability, demand response, power quality, active and reactive power management, protection and balancing and reliability in various load circumstances [27–29].

Additionally, when technical aspects are indicated it is worth noticing the management entities—virtual power players issues. Virtual power players' aim is the generation and services remuneration or charging energy consumption. The diversity of players to expedite participation in the electricity markets is described in the literature [11,30,31]. Reference [32] proposes the methodology to DER management. The article includes resource scheduling, aggregation and remuneration. The aggregation process is realized by k-means algorithm. Clustering is realized for different approaches, that concerns tariffs definitions for the period of a week. Customer remuneration is realized in accordance to Portuguese time-of-use tariffs. The research corners twenty thousand consumers and five hundred distributed generation units. The paper [33] deal with the same issues. However, it is realized for 2592 operation scenarios. Those cases consider over 5 hundred DGs, over 20 thousand consumers and ten suppliers. The article [34] is another example of using clustering to prosumers aggregation. The article [35] presents the discussion of demand response in point economic pros and effectiveness. This article presents a sensitivity analysis of demand response prices for the virtual power player operation costs. Additionally, the analysis comparison of cost of distributed resources and demand-side response during facing supply unavailability. This calculation is performed in a real smart grid on buses with associated micro-production. This allows the creation of sub-groups of data according to their correlations. The clustering process is evaluated so that the number of data sub-groups that brings the most added value for the decision-making process is found, according to players' characteristics. In addition to the technical aspects, selected issues concerning the roles of VPP partners are discussed in the accompanying paper [36]. Physical and financial streams between them are highlighted in point of the decision model which is concentrated on profits maximization. The results show that the number of distributed energy resources and the available storage capacity of battery energy storage has an impact on the economic efficiency of the VPP.

This article aims to study the technical aspects of integration of the above-mentioned units with an electrical power system (EPS) with regards to their prospective application in the VPP. The mentioned limitations are related to the regulation and protection procedures applied in the control of generation units and storage systems and also their possible influence on power system parameters. This article presents calculations and power flow of a real virtual power plant (VPP), containing a fragment of low and medium voltage distribution network. The model contains the hydropower plant (HPP), a photovoltaic system (PV), energy storage system (ESS). The problem is based on the identification of the limitations, which are dictated by the technical requirements of cooperation of power generation units and energy storage systems with the power system; the application of the simplified calculation, which is supported by precise simulation techniques so that the maximum power capacity of the planned energy storage system in the planed VPP can be indicated and the investigation of real

measurements of a photovoltaic power plant in order to reveal the impact of power control strategy on the potential of the resources integrated with the VPP. The presented review of VPP definitions and aims, as well as a summary of VPP projects, are the motivation for this paper. Additionally, to obtain the economic issues and impact on electricity marked firstly the technical requirements must be assured. The contribution of this paper covers the current gap of knowledge related to the VPP project which exhibits in the real case limitations of utilization the DER and ESS. For example, this paper provides the real case example of calculation and simulation focused on the determination of maximum power capacity of the ESS planned In terms of VPP efficiency and sensitivity, it is important to identify the maximum level of ESS power capacity that can be connected in the planned node to be installed in the selected network node. Results of presented investigations formulate margin condition for the VPP resources. Another example of the contribution of the paper to the current knowledge gap is the attempt to determine the influence of the power regulation strategies applied in PV plants on the real power range available for the VPP control strategies. Using the real measurement investigations it was shown that reactive power consumption implemented in the PV inverters reduces the energy volume potentially available for VPP form the PV installations. Mentioned examples constitute the limitation for the VPP project and can be adapted to other VPP investments.

The aim of this paper is to identify possible limitations in the development of the VPP which might be related to the regulation, protection and integration of power generation units with power systems. The mentioned problem can be seen to be a crucial issue, especially on the preliminary stage of the VPP concept and when different approaches for the economic strategies for VPP are created. After the introduction section, which highlights the main motivations of this paper, Section 2 presents a literature review of the technical aspects of VPP. This includes recent investigations regarding network integration, as well as a review of a selected real case of VPP realization. The aim of Section 2 is to identify current problems and solutions related to VPP and to provide the range of functionality of current VPP projects. Section 3 highlights the problems regarding the investigations presented in this paper. It has to be emphasized that technical aspects of DER and ESS, associated with the VPP, can be considered on several levels. Thus, Section 3 consists of the identification of codes and technical standards that define the requirements and permissible limits of electrical power system parameters and power quality, protection schemes and active and reactive power control issues applied for distributed energy resources. An additional element described in Section 3 is the energy storage control and limitations coming from the charging and discharging characteristics of energy storage systems. The revealed aspects can be treated as boundary conditions for the identification of their impact on VPP planning and operation strategies. The main investigations are presented in Section 4, which starts with a description of the topology of a medium voltage network in the area of the investigated VPP. The investigations are based on a real VPP project and present results considering the investigation of the cooperation of a 1 MW hydropower plant with 0.5 MW battery energy storage connected to the same node of medium voltage distribution network and impact of their operating condition on power flow and voltage level in observed network belonging to the VPP, (b) identification of maximum power capacity of battery energy storage which can be connected to considered node of the VPP as well as identification of general grid capacity of the investigated fragment of the distribution network to connect possible DERs/ESSs, (c) the identification of the impact of power control strategy applied in a PV power plant on resources available for the VPP. The maximum power capacity of the ESS is understood as the rated power of the ESS determined by requirements for power quality parameters of the grid and requirements for the integration of the generation units with power systems. In presented investigations, the storage capacity of the considered ESS is fixed and is used as a margin condition for the simulations. The storage capacity determines the ESS operability usually reflected by available time for charging and discharging. The selection of storage capacity of the ESS is usually based on specific aim functions considering selected intentions of using the ESS like economic aspects or islanding mode. The maximum power capacity of the ESS is restricted by the standards and regulations addressed to cooperation with the grids. The presented analysis is based on simulations conducted in Matlab

combined with real measurements. The initial condition for the calculation was based on the real measurements of load and generated power that represent a day of summer load peak demand. As a result of the investigation, the maximum power capacity of the considered ESS is identified with regards to the requirements for the permissible level of rapid voltage changes. Additionally, the impact of the power control strategy applied in a PV power plant on resources available for the VPP was also identified. In Section 5 a discussion about the influence of requirements for grid connection applicable to power generation units and its impact on limitation of the maximum power capacity of distributed energy resources and energy storage systems considered for planning operation of the VPP is provided in the broadest context. Section 6 presents the conclusions.

The identified limitations for the VPP, resulting from the technical aspects presented in this paper, are used as the input conditions for the economic investigations presented in the accompanying paper [36]. The paper presents the results of economic efficiency, including sensitivity analysis on price factors and DER production volumes, as well as the capacity of ESS.


*Edited by Sayed Hemeda and Mehmet Barış Can Ülker*

This book discusses contemporary issues related to soil mechanics and foundation engineering in earthworks, which are critical components in construction projects and often require detailed management techniques and unique solutions to address failures and implement remedial measures. The geotechnical engineering community continues to improve the classical testing techniques for measuring critical properties of soils and rocks, including stress wave-based non-destructive testing methods as well as methods used to improve shallow and deep foundation design. To minimize failure during construction, contemporary issues and related data may reveal useful lessons to improve project management and minimize economic losses. This book focuses on these aspects using appropriate methods in a rather simple manner. It also touches upon many interesting topics in soil mechanics and modern geotechnical engineering practice such as geotechnical earthquake engineering, principals in foundation design, slope stability analysis, modeling in geomechanics, offshore geotechnics, and geotechnical engineering perspective in the preservation of historical buildings and archeological sites. A total of seven chapters are included in the book.

Published in London, UK © 2020 IntechOpen © ballykdy / iStock

Geotechnical Engineering - Advances in Soil Mechanics and Foundation Engineering

Geotechnical Engineering

Advances in Soil Mechanics

and Foundation Engineering

*Edited by Sayed Hemeda*

*and Mehmet Barış Can Ülker*


*4.1. Identification and Annotation of B3 Genes in Grapevine*

To identify a complete list of B3 genes in the grapevine genome, annotated grapevine proteins were downloaded from three public databases: Grape Genome Database (http://www.genoscope.cns.fr), the National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/), and the Grapevine Genome CRIBI Biotech website (http://genomes.cribi.unipd.it/). The Hidden Markov Model (HMM) profile of the B3 domain (PFAM 02362) downloaded from the Pfam database (http: //pfam.xfam.org/) was used to survey all grapevine proteins in the 12X coverage assembly of the *V. vinifera* PN40024 genome to identify B3 genes [58]. The sequence integrity of the B3 domain (PFAM 02362) was assessed using SMART (http://smart.embl-heidelberg.de) with default parameters and genes with incomplete domains or with non-significant E-value were omitted from consideration [66]. All non redundant protein sequences with a conserved B3 domain were identified as grapevine B3 family members. Additionally, manual annotation was performed to resolve any discrepancy between incorrectly predicted genes and the actual chromosomal locations of involved genes in question.