**Acknowledgements**

This work was supported by the São Paulo State Research Support Foundation (FAPESP) [grants: 2017/13876-2; 2019/24237-6], by the Ministerio Español de Ciencia, Innovación y Universidades with Grant RTI2018-097810-B-I00 and the São Paulo State Institute for Technological Research, in the development of materials, whom the authors thank.


Section 2

Pleistocene Environment Change and Human Evolution in Europe and South Asia

**13**

**Chapter 2**

**Abstract**

Neanderthals

**1. Introduction**

Central Europe

Pleistocene Climate Change in

Loess is terrestrial, clastic sediment formed by the accumulation of wind-blown

paleotemperature changes during the Upper Pleistocene (130 ky - 20 ky) in Baranja region. Average growing season (AGS) temperature varied 13.2 °C or 9.5 °C during that time period, depending on which formula is applied for calculations. Magnetic susceptibility (MS) measurements show strong peaks in the paleosol horizons pointing to more humid climate. The overall climate was much cooler then present. Stable carbon isotope values point to dominance of C3 vegetation type during the Late Pleistocene in southern part of Central Europe. Climate change in the Late Pleistocene is very likely a significant but not the only factor that influenced the extinction of Neanderthal population which paved the way for the dominance of

**Keywords:** Pleistocene, climate change, loess, stable isotopes, Central Europe,

silt-sized particles and formed by the accumulation of wind-blown dust. It covers up to 10% of the world's surface area and is usually inter-bedded with paleosol horizons forming loess-paleosol sequences, or LPS [1]. Such successions provide very detailed insight into Pleistocene climatic fluctuations [2–4]. Due to this characteristic, LPS's provide very good records of palaeoclimate and environmental changes in the Pannonian Basin for at least 1 Ma [5]. The region of Baranja situated in the north-eastern part of Croatia is located in the southern edge of Pannonian Basin which is a part of Central Europe (**Figure 1**). It was selected for this research because some of the thickest loess successions in Croatia are exposed along the Danube River in Baranja. The total thickness of these loess deposits (above and under the surface) probably exceeds 50 m [6]. The Zmajevac locality was selected because it is accessible, contains four paleosols in loess and has a total thickness of almost 20 m.

Definition of loess: It is s a terrestrial, clastic sediment, composed dominantly of

dust. It is usually inter–bedded with paleosol horizons, forming loess-paleosol successions (LPS). Due to their characteristics LPS's represent valuable records of climate changes during Pleistocene. The thickest LPS sections in Croatia are in the Baranja region. Stable oxygen (δ18O) and carbon (δ13C) isotope analysis were made on loess malacofauna in order to quantify paleo-temperature changes and describe paleo-vegetation in this part of Central Europe. δ18O values show significant

*Adriano Banak, Oleg Mandic, Davor Pavelić,*

anatomically modern humans (AMH) in Central Europe.

*Marijan Kovačić and Fabrizio Lirer*


**3. Why post harvest technology?**

The food processing industry in India encompasses wide variety of products including fruits and vegetables, milk and poultry, meat and its products, alcoholic beverages, fisheries, grain processing and confectionaries like chocolates, cocoa

#### *Role of Post-Harvest Physiology in Evolution of Transgenic Crops DOI: http://dx.doi.org/10.5772/intechopen.94694*

products, soya based products, high protein products and many more. The agricultural and food products when gets detached from plant or being harvested are prone to various physiological, biochemical and biological events that results in its degradation of loss of commodity. Thus, it is necessary to adopt and practice some measures in order to reduce the loss of food commodities. Several measures can be taken in order to minimize the loss which includes reduction in moisture content, control of microorganisms, denaturation of endogenous enzymes and ensuring proper packaging of food materials. Postharvest technology plays a vital role in flourishing the food industry by minimizing losses and spoilage and value addition of commodities. It ensures the use of optimum harvest factors, utilizes modern machinery to ensure reduced losses due to handling, packaging, storage and transportation as well. It mainly aims to meet the food requirement for growing population by minimizing losses and enhancing nutritive value of food products. Postharvest technology is well established in large as well as small scale industries. The storage of food products can be prolonged by adapting several technologies like use of thermal processing, drying, low temperature, chemical and biological reactions couples with several preservation technologies etc. Postharvest technology opens new opportunities for marketing of food products as processed food commodities are gaining popularity among consumers and it also generate new employment opportunities among individuals. The fundamental objective underlying in postharvest technology sector is to maintain quality of produce after harvest in terms of texture, appearance, flavor etc, to ensure proper safety of food and to reduce loss of food and agricultural commodities from harvest to consumption.


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[81] Sun H. The interaction between pathogens and the host coagulation system. Physiology

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[101] Funk JL, Feingold KR, Moser AH, Grunfeld C. Lipopolysaccharide stimulation of RAW 264.7 macrophages induces lipid accumulation and foam cell formation. Atherosclerosis.

[102] Coppens I, Sinai AP, Joiner KA. *Toxoplasma gondii* exploits host low density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J Cell Biol. 2000;**149**:167–180.

[103] Palacpac NMQ, Hiramine Y, Mi-Ichi F, et al. Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in *Plasmodium falciparum*-

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[105] Moll GN, Vial HJ, Ancelin L, et al. Phospholipid uptake by *Plasmodium knowlesi* infected

[106] Krishnegowda G, Gowda DC. Intraerythrocytic *Plasmodium falciparum* incorporates extraneous fatty acids to its lipids without any structural modification. Mol Biochem

[107] Grellier P, Rigomier D, Clavey V, Fruchart JC, Schrevel J. Lipid traffic between high density lipoproteins and *Plasmodium falciparum*-infected red blood cells. J Cell Biol.

[108] Vial HJ, Thuet MJ, Broussal JL, Philippot JR. Phospholipid biosynthesis by *Plasmodium knowlesi*-infected erythrocytes: the incorporation of phospohlipid precursors and the identification of previously undetected metabolic pathways. J Parasitol. 1982;**68**:379–391.

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[111] Agnello V, Abel G, Elfahal M, Knight GB, Zhang QX. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA.

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**Lipoproteins in Specific Diseases**

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**Provisional chapter**

## **Lipoproteins and Diseases of the Brain Lipoproteins and Diseases of the Brain**

Kwang-Min Kim and G. Tayhas R. Palmore Kwang-Min Kim and G. Tayhas R. Palmore Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67053

#### **Abstract**

Apolipoprotein E4 (apoE4) and outer surface protein A (ospA) are pathogenic lipoproteins involved in the progression of Alzheimer's disease and Lyme neuroborreliosis, respectively. Results from previous studies indicate that apoE4 exhibits neurotoxicity by activating amyloid beta pathways, and ospA causes damage to the brain by stimulating immune activity of microglia and astrocytes. These results, however, lack information about the specific interactions that develop between neurons and these two lipoproteins. It is essential to investigate the effect of these lipoproteins on neuronal morphology and function to better understand the mechanism of damage and disease of the brain. This chapter summarizes previous studies on the role of apoE4 and ospA in diseases of the brain and discusses experimental results from our own work that suggests new roles for apoE4 and ospA in neuronal outgrowth and synaptic loss.

**Keywords:** apolipoprotein E4, bacterial outer surface protein A, neurodegeneration, neuroinflammation, nerve regeneration, synaptic loss

## **1. Introduction**

Lipoproteins in the brain are involved in the onset and progression of neurodegenerative diseases (e.g., Alzheimer's disease) [1, 2] and neuroinflammatory disorders (e.g., neuroborreliosis) [3, 4]. These lipoproteins are either endogenously expressed by astrocytes [5] and microglia [6, 7] or exogenously produced by bacterial pathogens (e.g., *Borrelia burgdorferi, Streptococcus pneumoniae*) [8].

The most abundant endogenous lipoproteins in the brain include apolipoprotein E (apoE) and apoJ [2]. These endogenous lipoproteins mediate transport of lipids between various cells in the brain to maintain and regulate the brain structure and function [9, 10]. The apoE isoform, apoE4, has been investigated intensively because previous studies showed that lipidation of

© 2016 The Author(s). Licensee InTech. 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, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. 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, provided the original work is properly cited.

apoE4 (i.e., apoE4 carrying cholesterol and phospholipids) is the major risk factor indicative of the onset of Alzheimer's disease (AD) [11].

 *B. burgdorferi* causes Lyme disease, which is the most common tick-borne infection in Europe and in North America [14]. A recent study using rats infected with *B. burgdorferi* demonstrated that *B. burgdorferi* was observed across the blood-brain barrier (BBB) and that the expression level of ospA was augmented significantly in the brain [4].

Thus, apoE and ospA have been of interest to both scientists and clinicians who seek to develop new strategies for treatment of brain injuries and brain disorders induced by these pathogenic lipoproteins. It still remains unclear however, if apoE and ospA interact directly with neurons to disrupt the structure and function of the brain, whereas it is documented extensively that these lipoproteins induce pathological states via amyloid beta (Aβ) aggregation [15, 16] and immune activation of microglia and astrocytes [17, 18]. To address the absence of direct evidence of interaction between lipoproteins and neurons, we have studied the effect of apoE4 and ospA on neurons in terms of axonal outgrowth and synaptic loss. This chapter discusses these findings and the potential new roles of apoE4 and ospA in the context of previous studies on these lipoproteins in neurodegeneration and neuroinflammation.

## **2. ApoE4 and neuronal outgrowth**

## **2.1. Lipidation of apoE isoforms**

ApoE transports and clears lipids from one cell to another to maintain lipid homeostasis of the brain [9, 10]. To carry lipids (e.g., cholesterol, phospholipids, and lipoproteins), apoE is lipidated (i.e., lipid-bound apoE) by adenosine triphosphate (ATP)-binding cassette A1 (ABCA1) transporters on astrocytes [19] (**Figure 1a**). The lipidation status of apoE depends on its three isoforms (i.e., apoE2, apoE3, and apoE4) coded by three alleles (ε2, ε3, and ε4 of *APOE* gene) on chromosome 19 [20]. The three isoforms of apoE differ from one another by amino acid interchanges at two residue sites (**Table 1**).

These minor variations cause a change in the structure and function of apoE, which eventually leads to distinct disease mechanisms in AD [21]. ApoE4 has an arginine at residue 112 that connects the N terminus (Arg 61) to the C terminus (Glu 255) to form a folded structure of apoE called *domain interaction* [22]. ApoE2 and apoE3 have a cysteine at residue 112, which is less likely to create the folded structure of domain interaction. The presence of domain interaction results in distinct lipid-bound forms among apoE isoforms. ApoE4 binds preferentially to larger lipid particles due to its folded structure, which interferes with internalization of lipids into neurons [11]. In contrast, apoE2 and E3 bind to various sizes of lipids in more ways that are efficient and thus facilitate lipid transport between cells in the brain. The lipidated apoE can be internalized into cells in the brain (i.e., astrocytes, microglia, and neurons) through the family of low-density lipoprotein receptors (LDLR), low-density lipoprotein receptor-related protein 1 (LRP1), or heparan sulfate proteoglycans (HSPGs) [11, 16].

**Figure 1.** The interaction between apoE and neurons illustrated. (a) ApoE transports lipids to neurons by forming lipopeptide particles (i.e., lipidation of apoE). ApoE is lipidated by ATP-binding cassette A1 (ABCA1) transporters of astrocytes. The lipid-binding affinity of apoE4 is different from that of apoE2 and apoE3 because of structural differences in its *domain interaction*. Both lipid and apoE can bind to Aβ monomers and oligomers. The Aβ-lipidated apoE2/3 complex can be internalized by LDLR, LRP1, or HSPG, which clears Aβ. The efficiency of internalizing large lipidbound apoE4 into cells is low, which increases the probability of Aβ plaque formation because of poor Aβ clearance. (b) ApoE alone can bind to Aβ monomers and oligomers regardless of its isoforms. The Aβ-nonlipidated apoE complex increases the probability of forming Aβ plaques because nonlipidated apoE cannot be internalized via LDLR or LRP1. (c) Nonlipidated apoE4 enhances neuronal adhesion, axon outgrowth, and neurite branching. The receptor in neurons that regulates growth-enhancing effects of nonlipidated apoE4 remains unknown. Abbreviations: ABCA1, ATP-binding cassette A1 transporter; LDLR, low-density lipoprotein receptor; LRP1, low-density lipoprotein receptor-related protein 1; HSPG, heparan sulfate proteoglycans.

apoE4 (i.e., apoE4 carrying cholesterol and phospholipids) is the major risk factor indicative

The exogenous lipoprotein most studied in the brain is the bacterial outer surface protein A (ospA), which is produced by *B. burgdorferi* [12, 13]. *B. burgdorferi* causes Lyme disease, which is the most common tick-borne infection in Europe and in North America [14]. A recent study using rats infected with *B. burgdorferi* demonstrated that *B. burgdorferi* was observed across the blood-brain barrier (BBB) and that the expression level of ospA was augmented significantly

Thus, apoE and ospA have been of interest to both scientists and clinicians who seek to develop new strategies for treatment of brain injuries and brain disorders induced by these pathogenic lipoproteins. It still remains unclear however, if apoE and ospA interact directly with neurons to disrupt the structure and function of the brain, whereas it is documented extensively that these lipoproteins induce pathological states via amyloid beta (Aβ) aggregation [15, 16] and immune activation of microglia and astrocytes [17, 18]. To address the absence of direct evidence of interaction between lipoproteins and neurons, we have studied the effect of apoE4 and ospA on neurons in terms of axonal outgrowth and synaptic loss. This chapter discusses these findings and the potential new roles of apoE4 and ospA in the context of previous studies on these lipoproteins in neurodegeneration and neuroinflammation.

ApoE transports and clears lipids from one cell to another to maintain lipid homeostasis of the brain [9, 10]. To carry lipids (e.g., cholesterol, phospholipids, and lipoproteins), apoE is lipidated (i.e., lipid-bound apoE) by adenosine triphosphate (ATP)-binding cassette A1 (ABCA1) transporters on astrocytes [19] (**Figure 1a**). The lipidation status of apoE depends on its three isoforms (i.e., apoE2, apoE3, and apoE4) coded by three alleles (ε2, ε3, and ε4 of *APOE* gene) on chromosome 19 [20]. The three isoforms of apoE differ from one another by amino acid

These minor variations cause a change in the structure and function of apoE, which eventually leads to distinct disease mechanisms in AD [21]. ApoE4 has an arginine at residue 112 that connects the N terminus (Arg 61) to the C terminus (Glu 255) to form a folded structure of apoE called *domain interaction* [22]. ApoE2 and apoE3 have a cysteine at residue 112, which is less likely to create the folded structure of domain interaction. The presence of domain interaction results in distinct lipid-bound forms among apoE isoforms. ApoE4 binds preferentially to larger lipid particles due to its folded structure, which interferes with internalization of lipids into neurons [11]. In contrast, apoE2 and E3 bind to various sizes of lipids in more ways that are efficient and thus facilitate lipid transport between cells in the brain. The lipidated apoE can be internalized into cells in the brain (i.e., astrocytes, microglia, and neurons) through the family of low-density lipoprotein receptors (LDLR), low-density lipoprotein receptor-related

protein 1 (LRP1), or heparan sulfate proteoglycans (HSPGs) [11, 16].

of the onset of Alzheimer's disease (AD) [11].

**2. ApoE4 and neuronal outgrowth**

interchanges at two residue sites (**Table 1**).

**2.1. Lipidation of apoE isoforms**

in the brain [4].

96 Advances in Lipoprotein Research

When the lipidated apoE is internalized into cells, Aβ monomers and oligomers are also cleared because they bind to both lipids and apoE at residues 12–28 [23]. Thus, Aβ clearance is dependent on the structural difference of apoE isoforms, and this mechanism helps to prevent the Aβ aggregation that is associated with the progression of AD. The Aβ-bound apoE, however, forms aggregates regardless of the isoform of apoE when they are not lipidated and thus, are not internalized [16, 23] (**Figure 1b**).

#### **2.2. Nonlipidated apo E4 and neuronal outgrowth**

When lipidated, apoE4 is known to be toxic to neurons through various pathogenic pathways such as Aβ aggregation and apoE fragment formation [21]. The effect of apoE4 on neurons when it is not lipidated, however, remains unclear. To address this knowledge gap, the effect of apoE4 on neuronal outgrowth was studied *in vitro* without lipids in the medium [24]. This study compared neuronal responses to various culture substrates including glass, laminincoated glass, and apolipoprotein E4-coated glass by quantifying key neuronal outgrowth parameters in terms of cell adhesion, axon length, number of neurites, and number of branches on axons. The results of this study demonstrated that apoE4 not only enhances neuronal adhesion but also significantly increases axon outgrowth and branching when compared to laminin, a protein that is recognized as one of the best extracellular matrix (ECM) proteins for enhancing neuronal growth [25]. As such, results from this study contradict the prevailing view that apoE4 has only a degenerative effect on cells in the brain. Although apoE4 when lipidated predominantly exhibits neurotoxicity when studied *in vivo* and in clinical models, it should be considered that both lipidated and nonlipidated apoE in these models constantly interact with neurons to mediate brain activity. Thus, the results from this study provide a complementary mechanism of action of apoE. In addition, the neuron-growing potential of apoE4 can be applied to transplantable therapeutic systems using stem cells or microstructure devices prior to interaction of lipids *in vivo*.

The mechanism by which nonlipidated apoE mediates axon outgrowth and branching remains elusive, whereas lipidated apoE is known to interact with cells via LRP1, LDLR, or HSPG [16]. It has been reported that apoE does not bind to LDLR or LRP1 without lipidation [26, 27]. Integrin and HSPGs also were tested for their involvement in apoE4-induced axon outgrowth by inhibiting these receptors. Neither of these receptors was found to be responsible for apoE4-induced neuronal outgrowth (**Figure 1c**). The mechanism of interaction between neurons and nonlipidated apoE4 is the subject of ongoing studies.

## **3. Bacterial lipoprotein and synaptic loss**

#### **3.1. Bacterial lipoproteins and neuroinflammation**

Bacterial surface components including lipoproteins and lipopolysaccharide (LPS) have been reported to be elevated in the cerebrospinal fluid (CSF) of patients suffering from a bacterial infection such as bacterial meningitis [28]. These components can cause neuropsychiatric manifestations such as lymphocytic meningitis, cranial and peripheral neuropathy, and cerebral infarcts [29, 30]. When compared to LPS, bacterial lipoproteins activate inflammatory pathways more vigorously [31], leading to more severe damage to tissue [32]. Bacterial lipoproteins still remain in the tissue even after the degradation of bacteria by antibiotic therapies [33, 34]. As a result, many studies suggest that minimizing the production of bacterial proteins or inhibiting bacterial protein synthesis is more effective at preventing neural injury from bacterial infections in animal models or patients [35, 36] than simply using antibiotics to kill bacteria. Bacterial lipoproteins in the brain trigger microglia activation via the toll-like receptors (TLRs) to produce inflammatory mediators (e.g., cytokines and reactive oxygen species) [37–39] and induce migration of immune cells across the BBB [40, 41]. The result is damaged brain tissue including cell death of astrocytes, oligodendrocytes, and neurons [42, 43].

The outer surface protein (osp) is the most studied bacterial lipoprotein that includes ospA, ospB, and ospC from *B. burgdorferi* [12, 44, 45]. Three palmitoyl groups (i.e., the lipid portion) at the N-terminus of the peptide is responsible for immune activation and tissue injury [46, 47], whereas the peptide portion of ospA is not effective at activating immune pathways [32]. Thus, tripalmitoyl-S-glyceryl-cysteine (Pam3-Cys), a synthetic lipopeptide mimicking the N-termini of osp, is often used for studying bacterial infection in a wide range of research fields involving immunology and neuroscience [48, 49]. Although all of ospA, ospB, and ospC share common immune pathways (e.g., NF-κB activation) via TLR2, ospA shows higher toxicity to tissues when compared with ospB and ospC [32]. The reason for distinct toxic effects among these different lipoproteins continues to remain elusive.

## **3.2. OspA and presynaptic loss**

the Aβ aggregation that is associated with the progression of AD. The Aβ-bound apoE, however, forms aggregates regardless of the isoform of apoE when they are not lipidated and

When lipidated, apoE4 is known to be toxic to neurons through various pathogenic pathways such as Aβ aggregation and apoE fragment formation [21]. The effect of apoE4 on neurons when it is not lipidated, however, remains unclear. To address this knowledge gap, the effect of apoE4 on neuronal outgrowth was studied *in vitro* without lipids in the medium [24]. This study compared neuronal responses to various culture substrates including glass, laminincoated glass, and apolipoprotein E4-coated glass by quantifying key neuronal outgrowth parameters in terms of cell adhesion, axon length, number of neurites, and number of branches on axons. The results of this study demonstrated that apoE4 not only enhances neuronal adhesion but also significantly increases axon outgrowth and branching when compared to laminin, a protein that is recognized as one of the best extracellular matrix (ECM) proteins for enhancing neuronal growth [25]. As such, results from this study contradict the prevailing view that apoE4 has only a degenerative effect on cells in the brain. Although apoE4 when lipidated predominantly exhibits neurotoxicity when studied *in vivo* and in clinical models, it should be considered that both lipidated and nonlipidated apoE in these models constantly interact with neurons to mediate brain activity. Thus, the results from this study provide a complementary mechanism of action of apoE. In addition, the neuron-growing potential of apoE4 can be applied to transplantable therapeutic systems using stem cells or microstructure

The mechanism by which nonlipidated apoE mediates axon outgrowth and branching remains elusive, whereas lipidated apoE is known to interact with cells via LRP1, LDLR, or HSPG [16]. It has been reported that apoE does not bind to LDLR or LRP1 without lipidation [26, 27]. Integrin and HSPGs also were tested for their involvement in apoE4-induced axon outgrowth by inhibiting these receptors. Neither of these receptors was found to be responsible for apoE4-induced neuronal outgrowth (**Figure 1c**). The mechanism of interaction

Bacterial surface components including lipoproteins and lipopolysaccharide (LPS) have been reported to be elevated in the cerebrospinal fluid (CSF) of patients suffering from a bacterial infection such as bacterial meningitis [28]. These components can cause neuropsychiatric manifestations such as lymphocytic meningitis, cranial and peripheral neuropathy, and cerebral infarcts [29, 30]. When compared to LPS, bacterial lipoproteins activate inflammatory pathways more vigorously [31], leading to more severe damage to tissue [32]. Bacterial lipoproteins still remain in the tissue even after the degradation of bacteria by antibiotic

between neurons and nonlipidated apoE4 is the subject of ongoing studies.

thus, are not internalized [16, 23] (**Figure 1b**).

98 Advances in Lipoprotein Research

devices prior to interaction of lipids *in vivo*.

**3. Bacterial lipoprotein and synaptic loss**

**3.1. Bacterial lipoproteins and neuroinflammation**

**2.2. Nonlipidated apo E4 and neuronal outgrowth**

OspA from *B. burgdorferi* is able to cross the BBB by binding to CD40 of brain-microvascular endothelial cells [4]. OspA in the brain activates TLR2 on microglia and astrocytes, which initiates immune activity and causes damage to brain tissue [14, 50]. However, information regarding the interaction between ospA and neurons is lacking because the expression level of TLR2 in neurons is extremely low when compared with that of microglia or astrocytes. Thus, the interaction between ospA and neurons has been overlooked [51, 52]. To address this question, the effect of ospA on neurons has been investigated with a specific focus on synaptic loss. The density and transmission of synapses are considered to be the key parameters in determining the functional state of brain tissue (e.g., information processing and storage) because neurons transmit electrical and biochemical signals to adjacent neurons through the synapse. The signal-sending synapse (i.e., presynapse) is located on the axon and the signal-receiving synapse (i.e., postsynapse) is located on the dendrite of a neuron. If neurons lose one of these synapses or have misaligned synapses, the brain cannot function properly even when neurons survive from brain injuries or diseases. Thus, the change in pre- and postsynaptic density was quantified following treatment of cultures of rat E18 hippocampal neurons with ospA (2 μM) for 24 h (**Figure 2**). The quantification of synaptic density was determined by counting the number of synaptic sites (i.e., synapsin or postsynaptic density protein 95 (PSD-95)) in a randomly selected secondary dendrite. OspA expressed from *Escherichia coli* (prepared by the Biomaterials and Advanced Drug Delivery Laboratory at Stanford University) showed that ospA significantly decreased the number of presynaptic sites (i.e., synapsin) (*p* = 0.04), whereas it did not affect the number of postsynaptic sites (i.e., PSD-95) (p > 0.05) (**Figure 2**). This result suggests that ospA directly disrupts neuronal function by damaging presynapses exclusively.

**Figure 2.** OspA and synaptic density. (a) Fluorescent images showing rat primary hippocampal neurons treated with vehicle (endotoxin-free water). (b) Fluorescent images showing rat primary hippocampal neurons treated with ospA (2 μM). (a) and (b) Neurons stained with anti-PSD-95 (postsynaptic protein), anti-synapsin (presynaptic protein), merge of PSD-95 and synapsin, merge of PSD-95, synapsin, and MAP2 (dendrite) from left to right are shown. (c) The postsynaptic density was measured by the number of postsynaptic sites (puncta) per length of selected dendrite. The postsynaptic density was not affected by ospA (*P* > 0.05). (d) The presynaptic density was measured by the number of presynaptic sites (puncta) per length of selected dendrite. The presynaptic density decreased significantly by ospA (*P* = 0.041).

#### **3.3. Mechanism of synaptic dysfunction induced by bacterial lipoproteins**

A recent study demonstrated that viral infection leads to cognitive dysfunction by microglial engulfment of presynapses via the complement C3 pathway [53]. Another recent study showed that viral infection impairs synaptic function via glycogen synthase kinase 3 (GSK-3) activation and intracellular accumulation of Aβ [54]. Thus, an increasing number of studies are being reported that elucidate the mechanism underlying synaptic dysfunction induced by viral infection. Although there is evidence that bacterial lipoprotein ospA also damages presynapses (**Figure 2**), information as to how bacterial infection impairs synaptic function is lacking. Three possible mechanisms may account for synaptic dysfunction during bacterial infection. First, bacterial lipoproteins damage synapses via activation of inflammatory pathways (e.g., TLR2 and TLR4) as discussed in Section 3.1. Second, bacterial lipoproteins damage synapses through neurotransmitter-mediated excitotoxicity. It has been demonstrated that the level of quinolinic acid, the N-methyl-D-aspartate (NMDA) receptor agonist, was elevated significantly in the CSF of Lyme neuroborreliosis patients [55]. The NMDA receptor mediates synaptic transmission, plasticity, and excitotoxicity in the central nervous system (CNS) and it exhibits excitotoxic effects when an excessive flux of calcium occurs by the increase of a neurotransmitter such as glutamate [56]. However, it is yet to be determined whether the presence of bacterial lipoproteins directly mediates the elevation of quinolinic acid. Third, bacterial lipoproteins damage synapses through physical interaction with synapses independent of biochemical pathways (i.e., inflammation and receptor activation). It has been suggested that the physical properties of proteins (e.g., aggregate pattern and size) is a crucial determinant in mediating pathogenic toxicity [57, 58]. This toxicity occurs independent of their sequences or lengths [59] in a manner that is similar to the aggregation of Aβ in Alzheimer's disease [60] or α-synuclein in Parkinson's disease [61]. Previous studies showed that Pam3-Cys, the synthetic N-terminus of ospA, self-assembled and showed aggregating potential *in vitro* assays [58, 62], which can be related to brain tissue damage including the disruption of synaptic function.

## **4. Conclusions**

This chapter describes the new roles of apoE4 and ospA as major pathogenic endogenous and exogenous lipoproteins, respectively, in neuronal outgrowth and function by discussing recent experimental data in the context of previous reports. Recent studies show that apoE4 enhances neuronal adhesion and axonal outgrowth *in vitro* when it acts alone without lipids. New studies also demonstrate the possibility that ospA can induce synaptic dysfunction by damaging exclusively presynaptic sites. These results contribute to a new understanding of how lipoproteins are involved in developing neuropathology by interacting with neurons. Future studies should focus on the specific mechanism of interaction between apoE4 and neurons and the effect of ospA on synaptic function using *in vivo* models. Along with many pathogenic pathways governed by various cell types in the brain (e.g., microglia, astrocytes, and oligodendrocytes), the effect of pathogenic factors on neuronal activity provides a deeper understanding of structural and functional abnormality in neurodegeneration and neuroinflammation [63]. Understanding the interaction between lipoproteins and neurons in the brain should yield new approaches to the treatment of brain injuries and brain disorders.

## **Author details**

**3.3. Mechanism of synaptic dysfunction induced by bacterial lipoproteins**

(*P* = 0.041).

100 Advances in Lipoprotein Research

A recent study demonstrated that viral infection leads to cognitive dysfunction by microglial engulfment of presynapses via the complement C3 pathway [53]. Another recent study showed that viral infection impairs synaptic function via glycogen synthase kinase 3 (GSK-3) activation and intracellular accumulation of Aβ [54]. Thus, an increasing number of studies are being reported that elucidate the mechanism underlying synaptic dysfunction induced by viral infection. Although there is evidence that bacterial lipoprotein ospA also damages presynapses (**Figure 2**), information as to how bacterial infection impairs synaptic function is lacking. Three possible mechanisms may account for synaptic dysfunction during bacterial infection. First, bacterial lipoproteins damage synapses via activation of inflammatory pathways (e.g., TLR2 and TLR4) as discussed in Section 3.1. Second, bacterial lipoproteins damage synapses through neurotransmitter-mediated excitotoxicity. It has been demonstrated that the level of quinolinic acid, the N-methyl-D-aspartate (NMDA) receptor agonist, was elevated significantly in the CSF of Lyme neuroborreliosis patients [55]. The

**Figure 2.** OspA and synaptic density. (a) Fluorescent images showing rat primary hippocampal neurons treated with vehicle (endotoxin-free water). (b) Fluorescent images showing rat primary hippocampal neurons treated with ospA (2 μM). (a) and (b) Neurons stained with anti-PSD-95 (postsynaptic protein), anti-synapsin (presynaptic protein), merge of PSD-95 and synapsin, merge of PSD-95, synapsin, and MAP2 (dendrite) from left to right are shown. (c) The postsynaptic density was measured by the number of postsynaptic sites (puncta) per length of selected dendrite. The postsynaptic density was not affected by ospA (*P* > 0.05). (d) The presynaptic density was measured by the number of presynaptic sites (puncta) per length of selected dendrite. The presynaptic density decreased significantly by ospA

> Kwang-Min Kim<sup>1</sup> and G. Tayhas R. Palmore<sup>2</sup> \*

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## *Edited by Turgay Isbir*

Lipoproteins have key roles in human growth and development, along with promoting, preventing, and/or participating in the pathogenesis or in the treatment of various diseases. This book presents a systematic and comprehensive review about the structure and metabolism of lipoproteins, particularly highlighting the crucial role of those molecules in the body and considering the interest of some lipids in healthy and diseased conditions. This book aims to provide integrative approach to understand the lipoprotein metabolism. Distinguished international experts contributed six chapters about the genetic variations, plasma lipoprotein components, and molecular relationship of lipoproteins with cognition and obesity.

Photo by CreVis2 / iStock

Advances in Lipoprotein Research

Advances in

Lipoprotein Research

*Edited by Turgay Isbir*


**3. The shelf life of processed organic waste and relevance**

The efficiency of organic waste largely depend on its safety at acquiring security. This is required particularly when composing organic waste with the feed [16]. This prolonging the shell life of processed organic waste [17, 18]. The treatment of organic waste can be obtained by cooking, extrusion, pellatizing, dehydration, ensiling and probiotics [17, 18]. The shelf life of the organic waste are better prolonged with extrusion, pellatizing, and dehydration than cooking and ensiling [19]. The dehydration treatment is helpful when considering the treatment of resultant food waste in swine [19, 20]. The moisture content of organic waste varies between 50% and 85%. This may threatened the use of organic waste in animal feed but the application of adequate treatment reduced the moisture content to produce a safe organic waste [19, 20]. Adequate heat could prevent organic waste susceptibility to oxygen. This helps to prolong shelf life of waste during storage and as well prevent lipid peroxidation [20, 21]. Solar energy was identified to be economical and environmental viable at transforming organic waste into dried waste. Solar energy was used at pasteurizing and drying of organic waste. This process produce low carbon emissions and energy efficient [20, 21]. Likewise in a findings, it was also revealed that 92.74% dry matter was obtained from the use of solar energy. The crude proteins, EE and crude fiber content were 25.68%, 21.57% and 0.75% respectively [21, 22].

Furthermore, the quality and quantity of dietary proteins are of importance in poultry and swine feed [21, 22]. 15% - 23% of crude proteins was found in organic waste on a dry basis [22, 23]. The C.P obtained in organic waste in an experiment on a dry basis was estimated to be 18.9%. This was significantly higher than the conventional feed [22, 23]. The amino acid content of organic waste was higher compared to that of conventional feed [22, 23]. It was also revealed that the lysine, methionine, threonine and tryptophan content of organic waste was higher compare to the conventional feed while a similar result was obtained comparing the two diets [24]. Phosphorus and calcium are important element relevant for cell division and tissue formation. The availability of phosphorus and calcium was studied in an investigation [24]. It was found that organic waste contain 0.004% to 0.46%

**69**

*Composting and Organic Waste Recycling a Better Option for Food Safety and Food Security*

calcium on a dry basis [24, 25]. This has a similarity with result obtained with soyabean meal 0.27% - 0.47% with a C P of 47.2%. This was higher than result obtained from corn (0.01–0.03%) [24, 25]. The breeding of broiler was experimented using different concentration of organic waste. Result revealed that the dried leftover food produced 20.02% C P and 9.99% EE [25]. The concentration of DHA in meat from broiler group fed dried leftover was not significantly higher than the control group. It was also recorded that the arachidonic acid content in broiler meat was not different among the treatment. However, the leftover treatment produced a better result than the control [25]. The salt content in organic waste compared with conventional feed made from corn in an experiment. It was reported that salt content of organic waste was higher compare to the conventional feed [25, 26]. The salt content of organic waste was recorded to be between 2.0% to 2.5% and approximately 3.28% on a dry basis. The present of salt in animal diet was very helpful. It enhances palatability [26–28]. It prevent the growth mold. It regulates growth and performance of the animals [28, 29]. Adequate salt level provide a soft texture of pork. It was recorded in a study that the salt content of organic waste was higher compare to convention feed composed from corn and soyabeans [28, 29]. It was also found that the performance of poultry and pig was strictly due to high salt content. Organic waste can be used as a substitute for salt if properly incorporated into diet because high salt level may result into poisoning [29]. It may also be subjected to rancidity [29]. Animal feed contribute to alleviation of food security through utilization of organic waste. Some bioactive compound and nutrients are found helpful to animal feed [29]. The use of organic waste in poultry and pig was found to be promising.

Moreover, waste generated by animal compounds is a more serious problem than waste voided by humans. Human waste is treated before it is released into waterways, but animal waste is not or poorly processed [30]. It has been reported that disease organisms may be transmitted from animal to animal through the use of organic manure in feed processing. The transfer of these diseases may constitute a problem to animal and public health [31]. Chicken waste is well known for Salmonella and Campylobacter spp. [32]. Cattle are known to host a gram of cattle manure with up to 10 million Salmonella organisms [31, 32]. Several methods have been used by researchers to inactivate pathogens before they are introduced into animal feed. The use of the stack method was found to be effective by some researchers [31, 32]. Some Salmonella species require an approximate temperature of 63°C (145°F), whereas *E. coli* requires heat of approximately 68°C (153°F) [31, 32]. Fermentation was also used to breakdown the bacteria in manure [33, 34]. This was an ensilage process. It involves the ensiling of waste with various grasses stored in airtight silo. In this situation, higher temperatures is ideal for killing infectious organisms [34]. Under these conditions, poultry litter buffers acids that destroy bacteria [35, 36]. In other words, some bacteria and coliforms were not completely controlled by ensilage. In a different finding, it was also recorded that stacking and fermentation is not as effective as the use of heating and drying method [35–37]. This involves subjecting manure or litter to the hot air blast from the oven. The temperature here is approximately 150°C (300°F) or higher, depending on the method [36, 37]. Campylobacter jejuni, *E. coli* Samonella, germisia and Listeria

were found to survive prolonged anaerobic digestion [36, 37].

The use of organic manure in feed processing allows the transfer of diseasecausing bacteria to spread resilience strains that may not be associated with organic waste. Most of these diseases inactivate at different temperatures [35–37].

*DOI: http://dx.doi.org/10.5772/intechopen.97683*

**4. Food waste and pathogens**

*Composting and Organic Waste Recycling a Better Option for Food Safety and Food Security DOI: http://dx.doi.org/10.5772/intechopen.97683*

calcium on a dry basis [24, 25]. This has a similarity with result obtained with soyabean meal 0.27% - 0.47% with a C P of 47.2%. This was higher than result obtained from corn (0.01–0.03%) [24, 25]. The breeding of broiler was experimented using different concentration of organic waste. Result revealed that the dried leftover food produced 20.02% C P and 9.99% EE [25]. The concentration of DHA in meat from broiler group fed dried leftover was not significantly higher than the control group. It was also recorded that the arachidonic acid content in broiler meat was not different among the treatment. However, the leftover treatment produced a better result than the control [25]. The salt content in organic waste compared with conventional feed made from corn in an experiment. It was reported that salt content of organic waste was higher compare to the conventional feed [25, 26]. The salt content of organic waste was recorded to be between 2.0% to 2.5% and approximately 3.28% on a dry basis. The present of salt in animal diet was very helpful. It enhances palatability [26–28]. It prevent the growth mold. It regulates growth and performance of the animals [28, 29]. Adequate salt level provide a soft texture of pork. It was recorded in a study that the salt content of organic waste was higher compare to convention feed composed from corn and soyabeans [28, 29]. It was also found that the performance of poultry and pig was strictly due to high salt content. Organic waste can be used as a substitute for salt if properly incorporated into diet because high salt level may result into poisoning [29]. It may also be subjected to rancidity [29]. Animal feed contribute to alleviation of food security through utilization of organic waste. Some bioactive compound and nutrients are found helpful to animal feed [29]. The use of organic waste in poultry and pig was found to be promising.

#### **4. Food waste and pathogens**

*Humic Substances*

to its efficiency [14, 15].

1.3 billion tons of food are produced globally and are wasted during production, postharvest, and processing [12, 13]. A huge portion of organic waste include edible and inedible food. Organic waste generated at the latter part of food supply is comparatively higher than at the early stage of the chain. It was also projected that increasing demand for animal product may result in high demand for feed particularly the coarse grains such as maize and protein meal by 2025 [12–14]. The conventional poultry and swine diet depend on maize and soyabean as energy and proteins sources [12–14]. As well, these same, staple food is largely consumed in many part of the world particularly in the developing countries [13, 14]. As a result, a notable increase in the cost of staple food for animal feed drives the cost of production. However, the likely solution for this could be organic waste from cereal grains and plant proteins sources used in animal nutrition. However, production cost is reduced with introduction of food waste due to its low cost compared to conventional feeds [13–15]. Food organic waste from poultry, cattle, etc. is mixed with livestock feed. This reduces the amount of waste disposal in the community [13–15]. In other words, waste retains nutritive value, which may reduce the cost of feeding. However, the waste, the type and quantity of other foodstuffs, the way the excrement is treated and the species to which the treated excrement is fed is related

**3. The shelf life of processed organic waste and relevance**

The efficiency of organic waste largely depend on its safety at acquiring security.

Furthermore, the quality and quantity of dietary proteins are of importance in poultry and swine feed [21, 22]. 15% - 23% of crude proteins was found in organic waste on a dry basis [22, 23]. The C.P obtained in organic waste in an experiment on a dry basis was estimated to be 18.9%. This was significantly higher than the conventional feed [22, 23]. The amino acid content of organic waste was higher compared to that of conventional feed [22, 23]. It was also revealed that the lysine, methionine, threonine and tryptophan content of organic waste was higher compare to the conventional feed while a similar result was obtained comparing the two diets [24]. Phosphorus and calcium are important element relevant for cell division and tissue formation. The availability of phosphorus and calcium was studied in an investigation [24]. It was found that organic waste contain 0.004% to 0.46%

This is required particularly when composing organic waste with the feed [16]. This prolonging the shell life of processed organic waste [17, 18]. The treatment of organic waste can be obtained by cooking, extrusion, pellatizing, dehydration, ensiling and probiotics [17, 18]. The shelf life of the organic waste are better prolonged with extrusion, pellatizing, and dehydration than cooking and ensiling [19]. The dehydration treatment is helpful when considering the treatment of resultant food waste in swine [19, 20]. The moisture content of organic waste varies between 50% and 85%. This may threatened the use of organic waste in animal feed but the application of adequate treatment reduced the moisture content to produce a safe organic waste [19, 20]. Adequate heat could prevent organic waste susceptibility to oxygen. This helps to prolong shelf life of waste during storage and as well prevent lipid peroxidation [20, 21]. Solar energy was identified to be economical and environmental viable at transforming organic waste into dried waste. Solar energy was used at pasteurizing and drying of organic waste. This process produce low carbon emissions and energy efficient [20, 21]. Likewise in a findings, it was also revealed that 92.74% dry matter was obtained from the use of solar energy. The crude proteins, EE and crude fiber content were 25.68%, 21.57% and 0.75% respec-

**68**

tively [21, 22].

Moreover, waste generated by animal compounds is a more serious problem than waste voided by humans. Human waste is treated before it is released into waterways, but animal waste is not or poorly processed [30]. It has been reported that disease organisms may be transmitted from animal to animal through the use of organic manure in feed processing. The transfer of these diseases may constitute a problem to animal and public health [31]. Chicken waste is well known for Salmonella and Campylobacter spp. [32]. Cattle are known to host a gram of cattle manure with up to 10 million Salmonella organisms [31, 32]. Several methods have been used by researchers to inactivate pathogens before they are introduced into animal feed. The use of the stack method was found to be effective by some researchers [31, 32]. Some Salmonella species require an approximate temperature of 63°C (145°F), whereas *E. coli* requires heat of approximately 68°C (153°F) [31, 32]. Fermentation was also used to breakdown the bacteria in manure [33, 34]. This was an ensilage process. It involves the ensiling of waste with various grasses stored in airtight silo. In this situation, higher temperatures is ideal for killing infectious organisms [34]. Under these conditions, poultry litter buffers acids that destroy bacteria [35, 36]. In other words, some bacteria and coliforms were not completely controlled by ensilage. In a different finding, it was also recorded that stacking and fermentation is not as effective as the use of heating and drying method [35–37]. This involves subjecting manure or litter to the hot air blast from the oven. The temperature here is approximately 150°C (300°F) or higher, depending on the method [36, 37]. Campylobacter jejuni, *E. coli* Samonella, germisia and Listeria were found to survive prolonged anaerobic digestion [36, 37].

The use of organic manure in feed processing allows the transfer of diseasecausing bacteria to spread resilience strains that may not be associated with organic waste. Most of these diseases inactivate at different temperatures [35–37].

#### **Table 2.**

*Lethal- time conditions for some common pathogen sourced: [2].*

In addition, European safety rules for food should be followed by a high level of protection of human health, and the production of edible terrestrial invertebrates as food should be safe and wholesome and reared and marketed as food [37–42]. The lethal time conditions for some common pathogens are shown in the **Table 2**.