**4. Discussion**

This project was initiated in an effort to identify the olfactory proteins of parasitoid wasps. In recent years, many aspects about the perception of pheromones and other odorants have been elucidated [36]. Olfaction is used by insects to recognize volatile cues that allow the detection of food, predators, and mates [37]. We identified 43 OBPs, 69 CSPs, 60 GRs, 689 ORs, 26 IRs, and 14 SNMPs in *D. longicaudata* (Table 2, Table S2) and some of them were differentially expressed when they were maintained with fruit flies fed with different fruits (Figure 4C, Table S4). This is the first time the chemosensory genes in *D. longicaudata* have been investigated and our findings provide a basis for elucidating the molecular mechanisms of the olfactory-related behaviors of parasitoid wasps.

OBPs are a group of proteins that specialize in the transport of lipids. In this study, we identified 43 transcripts encoding OBPs and this number is similar to the number of genes encoding OBPs in the *Drosophila* genome [38]. Addittionally, classic OBPs have been reported to contain six Cys in their sequences [38,39]. Because all the identified OBPs in this study were general OBPs (GOBPs) (Table S2), we found conserved Cys in their sequences (Figure S1), which increased the confidence of using the transcriptome to identify the OBPs in *D. longicaudata*. In detail, the *D. longicaudata* OBPs were grouped into four subfamilies: OBP-56, -69, -72, and -83 (Table S2). However, no studies have been demonstrated to distinguish their functions. In general, according to their names, they are called GOBPs because they bind general odorants that are likely to be represented by the same volatiles for most of the species [40]. We also showed the differential expression of nine transcripts encoding OBPs in parasitoid wasps in response to the fruit flies with different scents (Figure 4, Table S4). This might indicate that some specific OBPs are expressed to discriminate different scents.

Similar to OBPs, CSPs are another group of proteins that mediate the olfactory recognition in insects [41]. They are thought to be expressed during nearly the whole life circle of insects [42,43]. The number of CSP genes varies in species. For example, only 4~8 CSP genes in ants, flies, bees, wasps, and anopheline mosquitoes [44]; 19~20 in butterfly, moth, and beetle [45–47]; and 27 to 83 in *Culex* mosquito species [48]. We identified 69 CSP transcripts produced by 58 genes (Table S2), the number of which is similar to *Culex* mosquito species. CSPs function not only in the chemical communication between insects and the environment but also in some other cellular processes, such as lipid transport, general immunity, insecticide resistance, and xenobiotic degradation [45,49]. In ants, CSPs have been proposed to mediate the recognition of chemical signatures composed of cuticular lipids [50]. The differential expression of CSP transcripts identified in this study (Table S4) may support the ability to recognize different scents from their hosts.

In the present study, we also identified some other olfactory gene families, such as OR, IR, GR, and SNMP, which were differentially expressed in response to the fruit fly with different scents (Table S2, Table S4). OR is the name for all molecules that are expressed in the cell membranes of olfactory receptor neurons and are responsible for the detection of odorants. The ORs form a multigene family consisting of around 800 genes in humans and 1400 genes in mice [51]. We identified 689 OR transcripts derived from 637 genes (Table 2, Table S2). The diversity of ORs might help insects to discriminate as many different odors as possible. GRs are found be expressed exclusively in gustatory receptor neurons [52]. However, many GRs are not related to taste receptors but function in the detection of sugars, bitter compounds, and non-volatile pheromones [53]. Interestingly, GR28B represents a new class of thermosensor and is required for thermotaxis [54]. We found GR28B differentially expressed in C and M (Figure 4C, Table S2). This might be evidence of its new role in the detection of different scents. IR genes are expressed in coeloconic sensilla of the antenna and respond, among others, to water and amines [55]. IRs are not related to insect ORs but rather have evolved from ionotropic glutamate receptors (iGluRs), a conserved family of synaptic ligand-gated ion channels [56]. In this study, we identified 26 IR transcripts, and 18 of them encode IR25A (Table 2, Table S2). It is conceivable that the IR25A ancestor initially evolved as a sensory detector for external glutamate, analogous to the synaptic function of iGluRs, and that it only later acquired a co-receptor function after duplication and diversification of the IR repertoire [56]. We also identified 14 SNMP transcripts in the parasitoid wasps, including 11 encoding SNMP1 and 3 encoding SNMP2 (Table 2, Table S2). The SNMP1 has been shown to be antenna specific and play an important role in pheromone detection [57]. While SNMP2, which acts as a second lepidoperan and also associates with pheromone-sensitive sensilla, has been shown to be expressed in sensilla support cells rather than neurons [58,59]. The identification and differential expression of olfactory-related transcripts revealed the complex chemosensory system of *D. longicaudata* and supported a diverse function of olfactory genes in discriminating different chemical cues.

The limitations of this project may include the use of the tissue mixture of insects.  For example, MsepOBP5 exhibited female-biased expression in 0- and 5-day-old adults; MsepOBP22 displayed female-biased expression in 0- and 5-day-old adults but was male-biased in 3-day-old adults [60]. Due to the difficulty of the sample preparation, it is hard to get enough material for sequencing with the same sex. Additionally, it is difficult to determine the tissue-specific olfactory genes. However, our findings provide a basis of future studies about the olfactory system in *D. longicaudata*.


**1. Introduction**

With the rapid development of the social economy under globalization, various kinds of public emergencies in traditional and nontraditional fields have become increasingly prominent, which has become an important hidden threat to national security and people's property and life security [1]. Frequent food security incidents have resulted in the repeated questioning of the credibility of the government [2]. Researchers have found that an effective emergency system can reduce accident losses to less than 6% compared to a situation without an emergency system [3]. In view of this, in the process of dealing with crisis events, the appropriateness of the emergency response system has become the focus of researchers.

In recent years, major food security incidents have occurred frequently in Mainland China, the number and degree of harm of which are both increasing. Food security has attracted more attention from all walks of life than ever before, resulting in a crisis of public confidence in food security [4]. In terms of recent food security incidents, poisonous milk powder incidents broke out in March 2008, but due to the preparation for the Olympic Games, the local government of Hebei did not deal with these in time. The poisonous milk powder incident continued until the New Zealand government reported the incident to China in September. This incident a ffected China's foreign trade and seriously damaged China's international image [5]. This incident caused social unrest because the melamine mixed in the infant milk powder caused kidney lesions in infants [5]. In view of the importance of food security issues, the Food Security Law of Mainland China regulates scientific and e ffective supervision measures with a clear and systematic division of responsibilities for food security supervision, which provides a legal guarantee to resolve the current prominent food security issues in a proper and orderly manner. However, due to the unique and complex market environment of Mainland China, food security supervision will still face multiple dilemmas with respect to the system and technology [6].

Taiwan's food security crisis has also been spreading [7]. From the "plasticizer" to the "Datong Tainted Oil" incidents, food security issues have also been emerging in Taiwan one after the other. The emergence of the poisonous starch incident in 2013 in Taiwan set o ff a panic in Taiwanese society. The o fficial poisonous compound in the starch was maleic anhydride. It was originally used as a food packaging material and adhesive, but some practitioners mixed it into edible starch to increase the viscosity and elasticity of the starch. This substance was illegally added and may cause kidney damage. In April 2013, seven county and municipal health bureaus, such as New Taipei City, began to investigate the manufacturers using the poisonous starch closely. In May, the New Taipei City Health Bureau found that Mingji tofu contained the toxic starch elements, and its raw material was procured from Sanjin Powder Trading Company, located in Tainan; therefore, the Health Bureau of Tainan City continued to investigate the incident. In order to eliminate the circulation of the toxic starch in the market and require the raw material manufacturers of the starch to start providing security certificates from the downstream manufacturers, pitchmen, and food practitioners starting in June, the Health Department launched the "0527 Food Security Project". The "0527 Food Security Project" was a project for food safety control initiated by the resolution of the Conference on Research and Strategies organized by the Ministry of Health and Welfare, which invited experts from food management, science and technology, and public health and medical fields in order to solve the food safety problem caused by the malicious addition of maleic anhydride to starch. It was launched by the Health Department to check all starch manufacturers in Taiwan. Because the poisonous starch incident was a significant issue, it brought food security issues to the public's attention, as well as governmental actions on food security issues [4].

Mainland China and Taiwan are facing a major impact from food security issues. In the face of many major food security incidents in Mainland China and Taiwan over the years and in view of the fact that food security a ffects people's lives and health, this article performs a comparative study of food security in the two regions, which should help governments and nongovernmental organizations to learn from each other's progress at the levels of food supervision and the legal system and to establish an e ffective food supervision mechanism and rigorous legal system to protect consumers' rights and interests. Taking advantage of the promotion of the "Food Security Law" in Mainland China, this study explores the legal system of food security supervision, which should have theoretical and practical significance to solve food security problems e ffectively [8]. A comparative analysis of the current situation of food security in Taiwan could be helpful in implementing a cross-strait food security agreement, enhancing food security communication and mutual trust in Mainland China and Taiwan, thereby ensuring the security and health of local citizens [9].


**6. Conclusion**

The present chapter discusses the modeling of the laser-irradiated biological tissue to understand its thermal behavior, which may help improve the efficacy of the laserbased photo-thermal therapy to destroy the cancerous cells with minimal damage to the surrounding healthy tissue. The light propagation through the biological tissue was mathematically modeled using the RTE. The RTE was solved using the DOM to determine the intensity distribution inside the biological tissue subjected to shortpulse laser irradiation. Once the intensity distribution was obtained, the divergence of radiative heat flux was calculated, which acts as the source term in the Fourier/non-Fourier model-based bio-heat transfer equation to determine the temperature distribution inside the laser-irradiated biological tissue. However, it is a multi-time scale problem because of the two different time scales used to solve the RTE and bio-heat transfer equation. So, the algorithm for solving this multi-time scale was presented. The non-Fourier model-based bio-heat transfer equation was numerically solved using the FVM to determine the temperature distribution. The numerical results were compared with the analytical results obtained using the FIT technique and found that the numerical solution predicted relatively lower temperature values than the analytical solution. A comparative study between the Fourier and non-Fourier (C-V and DPL) models was conducted and found that the temperature predicted using the DPL model lies between the C-V and Fourier models.


*4.9. Growth Studies and Analysis of Ampicillin*−*Resistant Gene Mutants*

Cell growth was monitored by measuring the cell optical density (OD) values using a Bioscreen C high−throughput growth measurement instrument (Bioscreen C MBR, Helsinki, Finland). The single colony was inoculated in 1 mL of RMG5 at 30 ◦C overnight as the seed culture. Then, the seed culture of *Z. mobilis* was transferred to RMG5 until reaching the exponential phase. Subsequently, the cultures were inoculated to the well of the Bioscreen C plate containing 300 µL bacterium suspension with an initial OD600 nm value of 0.05. Three technical replicates were used for each condition.

#### *4.10. Cell Morphology Observation*

*Z. mobilis* strains were cultured in RMG5 or RMG5 with 150 µg/mL ampicillin overnight at 30 ◦C. Bacterial pellets were collected at the exponential phase, washed twice with 1× phosphate buffer saline (1× PBS), resuspended in the same buffer, and observed under a light microscope (Leica Dmi8, Buffalo Grove, IL, USA) at 400 magnifications. Each image was taken using the image−based autofocus system LAS X software of the Leica Dmi8 system, and the cell size was measured using ImageJ software [41].

#### **5. Conclusions**

In summary, genes associated with antibiotic resistance in *Z. mobilis* were systematically investigated, and six ampicillin−resistant genes in *Z. mobilis* were identified. Five of them were knocked out individually, and four ampicillin−resistant gene deletion mutant ZM4∆Ars was also constructed to verify their functions. The strains of ZM4∆0103 and ZM4∆Ars were sensitive to ampicillin at a lower concentration of 150 µg/mL with a long and filament shape. The putative membrane protein ZMO0103 is probably the essential β−lactamase, and other ampicillin−resistant genes may have a synergetic effect with it. This study not only identified the ampicillin−resistant genes in *Z. mobilis* ZM4, but also verified their functions on cell growth, morphology, and transformation efficiency. In addition, ampicillin−sensitive mutants can serve as the parental strains for metabolic engineering practices in *Z. mobilis* enabling the usage of antibiotics, such as ampicillin, that are routinely used for genetic engineering.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics11111476/s1, Figure S1: Secondary structure prediction of ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967 in NCBI database; Figure S2: Multiple sequence alignment of the AmpC superfamily and β−lactamase class A protein in Z. mobilis; Figure S3: Loss of editing plasmids in ampicillin−resistant (AR) gene knockout strains; Figure S4: Cell growth of single and multiple ampicillin−resistant gene knockout strains cultured under 0 (A), 150 (B), and 300 (C) µg/mL of ampicillin; Figure S5: Electroporation efficiency of ZM4∆0103, ZM4∆Ars, and ZM4 by pEZ15A; Figure S6: Electroporation efficiency of ZM4∆0103, ZM4∆Ars, and ZM4 by pEZ15A and pE39−MVA; Figure S7: Distance tree of ZMO0103 after multiple sequence alignment with other proteins collected in NCBI using BALSTP; Figure S8: The detailed result of multiple sequence alignment with other proteins collected in NCBI using BALSTP; Table S1: List of the putative efflux pump−type antibiotic−resistant genes by BLASTP with CARD and MEGARes databases; Table S2: List of the putative β−lactamase genes by BLASTP in the UniProt database; Table S3: Strains used in this study; Table S4: Plasmids used in this study; Table S5: Primers used in this study.

**Author Contributions:** Conceptualization, S.Y.; methodology, B.G., X.H. and Y.W.; validation, X.H.; formal analysis, B.G. and X.H.; investigation, B.G., X.H., Y.W., Q.H. and S.Y.; data curation, B.G. and X.H.; writing—original draft preparation, B.G. and X.H.; writing— review and editing, Q.H. and S.Y.; visualization, Y.W.; supervision and project administration, S.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** Supported by National Key Research and Development Program of China (2018YFC1801200), the National Natural Science Foundation of China (21978071 and U1932141), Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang Province (2018R01014), and the Innovation Base for Introducing Talents of Discipline of Hubei Province (2019BJH021).

**Institutional Review Board Statement:** Ethical review and approval were waived for this study as the study did not involve animal interventions.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing does not apply to this article.

**Acknowledgments:** We acknowledge the support from the State Key Laboratory of Biocatalysis and Enzyme Engineering.

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

#### **References**


The Effect of Dietary Sodium Restriction on Vascular Stiffness in Hypertension

*Henrique Cotchi Simbo Muela, Mujimbi José Viana, António Gerson Bastos Francisco, Isaura da Conceição Almeida Lopes and Valeria Aparecida Costa-Hong*


**3. Intercalary structural bone grafts**

Observations regarding 32 cases of structural bone grafts used for large osteoperiosteal gaps are summarized in **Table 4**. The largest group was 28 cases of giant cell tumors, 40% of these obtained uneventful success (**Figure 5**), 38% needed supplementary operation in the form of

**Table 4.** Large osteoperiosteal gaps (32) and structural grafts (1979–1999).

further autologous bone grafts for areas of non-union at host graft junctions or for pseudarthrosis in the intermediate part of the graft 20%, for control of infection, or for a combination of these factors (18%).

Six patients were considered a failure because the reconstruction failed. Two had recurrence of tumor, one had uncontrolled infection, and these ended up in amputations. In three patients despite two attempts at supplementary grafting, the areas of pseudarthrosis did not heal; these patients accepted an orthosis till further decision. One case of malignant fibrous histiocytoma failed because of recurrence of tumor within 4 months of limb salvage attempt.

Of the 11 patients of posterior or posterolateral spinal fusion, 10 were considered to have obtained satisfactory osseous fusion based upon clinical assessment and stress X-rays done 12–24 months after the operation (**Figure 6**). One young nurse who had posterior fusion along with Steffi's fixation at L3–L4 for spondylolisthesis was considered a failure because of the implant breakage observed 2 years after operations.

#### **3.1. Cytological and histological observation**

infection (three cases) and massive recurrence of tumor (three cases). All patients of solitary osseous lesions healed successfully. Polyostotic fibrous dysplasia (4), multiple fibrous defects (6), enchondromas (3), chondromyxoid fibroma (2), and enchondromatosis (2) by their biological nature may need reoperation for an increase in the size of the lesions which were insignificant at the time of the first surgery. One patient of fibrous dysplasia had to undergo a second operation for a new lesion, and one patient of enchondromatosis is awaiting surgery

**Figure 4.** A case of polyostotic fibrous dysplasia. The cyst in the trochanteric area remained healed after curettage and decal-bone grafting. After about 14 years of gap, another cyst in the supra-acetabular region needed treatment.

**Figure 3.** A recurred giant cell tumor of distal femur with pathological fracture. The healed status achieved by

Observations regarding 32 cases of structural bone grafts used for large osteoperiosteal gaps are summarized in **Table 4**. The largest group was 28 cases of giant cell tumors, 40% of these obtained uneventful success (**Figure 5**), 38% needed supplementary operation in the form of

for the additional area (**Figure 4**).

**3. Intercalary structural bone grafts**

intralesional curettage and decal-bone grafting following 12 years.

48 Bone Grafting - Recent Advances with Special References to Cranio-Maxillofacial Surgery

Fine-needle aspiration cytology (FNAC) was done from the perigraft region (in 20 patients) between the 10th and 40th day after grafting. The FNAC showed high cellularity composed of

**Figure 5.** Intercalary reconstruction after en bloc resection of giant cell tumor of distal femur. Note gradual incorporation and remodeling as observed in the 12-year follow-up.

agent. Its viability however depends upon the influence of processes of allograft preparation

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**Bone graft incorporation:** The biological process of incorporation of bone grafts is practically similar to that of a fracture healing. Under favorable environment, the following major steps occur in a cascadal fashion from the time of placement of the bone graft in the recipient bed to its incorporation and remodeling according to Wolff's law: (i) hematoma formation and its organization by invasion of neocapillaries surrounded by perivascular pluripotent mesenchymal cells; (ii) osteoclastic and phagocytic resorption of nonviable mineral (calcium hydroxyapatite), cellular debris and marrow fat, and tunneling of the graft-making channels for ingrowth and propagation of neocapillaries and osteoprogenitor cells; (iii) conversion (tissue engineering) of osteoprogenitor cells to osteoblastic cells under the influence of local osteoinductive agents (bone morphogenetic protein, other inductive agents, and growth factors present in the organic matrix of the bone graft) and the platelets. Laying down of the new bone (neo-osteogenesis) on the surface of matrix framework and along the vascular spaces/channels; and (iv) remodeling of the newly formed bone to conform to the trabecular pattern along the lines of functional loading and stress (according to Wolff's law).  The most challenging clinical condition of structural (intercalary) bone grafting for large osteoperiosteal gaps in the lower limb may take 2–4 years for adequate incorporation permitting unprotected loading [11, 12]. The mechanical strength of the reconstruct is weak for 1 and 1/2 to 2 years, after which the strength increases by more neo-osteogenesis. The least time is taken in a cavitary pathology which offers a very large osteogenic bed and copious surface for intimate contact with the graft [13–15]. As incorporation takes place from periphery to the center, the time taken for large cavities and large grafts is correspondingly higher. In un-demineralized cortical (e.g., fibula) graft, 20–30% (deepest sector) of the grafted bone may never get incorporated; it may stay

By HCL decalcification we aimed at the removal of nearly 50% of mineral, thus providing adequate structural integrity. The said treatment removed all cell debris and fat providing opened-up channels for ready penetration of neocapillaries and perivascular mesenchymal cells. Acid demineralization also removed the mineral from the surface of the bone, along the vascular channels, and on the lacunar spaces, thus exposing the matrix for intimate contact with the invading perivascular tissues, and facilitated the interaction between the graft matrix (most active osteoinductive principle) and the pluripotent mesenchymal cells from the host. The technique used by us does not destroy the biological osteoinductive property of the bone

Immunogenicity of allogenic bone is now better understood. Fresh unmatched and untreated allogenic bone inevitably evokes an immune response in the host. The immune response in general is delayed and mild and develops slowly; however, it results in "unexplained" graft resorption and delay or failure in its incorporation. In clinical practice deep-freezing, freezing, freeze-drying, and irradiation are currently employed to reduce immunogenicity. Pure BMP from an allogenic source or even a xenogenic source is considered to have negligible immunogenicity. We feel that a simple treatment of allogenic bone by HCL decalcification and ethanol preservation practically eliminates the antigenic material (cells and debris) to permit unhindered incorporation in clinical practice as observed in our cases. Overall analysis

and preservation [5–10].

incarcerated surrounded by newly formed bone.

matrix.

**Figure 6.** A child having tuberculosis of the spine involving multiple vertebrae: posterior spinal fusion was performed using allogenic decal-bone. Note the incorporation of the graft with remodeling during a 3-year follow-up.

polymorphs, lymphocytes, and macrophages between 10 and 20 days. The cellularity gradually reduced with relative increase in the number of lymphocytes. By the 40th postimplantation day, the macrophages were practically absent, and one could see appreciable osteogenic activity by the presence of osteoblasts and osteoclasts. No cellular immune reaction was discernable.

Periodic core biopsy in early stages and biopsy of the graft in patients who required a second operation showed histological and tetracycline fluorescence evidence of neo-osteogenesis between 6 and 12 weeks. The fluorescence in the implanted allogenic bone was quantitatively the same as the bone of a patient's iliac crest in specimens available 12 months after the grafting.

### **3.2. Discussion**

**Contents of bone grafts and their roles:** In general calcium hydroxyapatite, the predominant mineral in bones provides an inert framework providing mechanical stability and offering a lattice work for penetration of neocapillaries, reparative tissues, and osteoconduction (**Table 5**). Only the most superficial bone-forming cells in fresh autografts which survive getting nutrition by tissue perfusion provide direct osteogenic activity. In allografts no viable cells are expected; however, the debris of dead cells act as the most potent immunogenic agent. Organic matrix provides the most potent bone morphogenetic (bone induction principles)

**Table 5.** Contents of bone graft and their role.

agent. Its viability however depends upon the influence of processes of allograft preparation and preservation [5–10].

**Bone graft incorporation:** The biological process of incorporation of bone grafts is practically similar to that of a fracture healing. Under favorable environment, the following major steps occur in a cascadal fashion from the time of placement of the bone graft in the recipient bed to its incorporation and remodeling according to Wolff's law: (i) hematoma formation and its organization by invasion of neocapillaries surrounded by perivascular pluripotent mesenchymal cells; (ii) osteoclastic and phagocytic resorption of nonviable mineral (calcium hydroxyapatite), cellular debris and marrow fat, and tunneling of the graft-making channels for ingrowth and propagation of neocapillaries and osteoprogenitor cells; (iii) conversion (tissue engineering) of osteoprogenitor cells to osteoblastic cells under the influence of local osteoinductive agents (bone morphogenetic protein, other inductive agents, and growth factors present in the organic matrix of the bone graft) and the platelets. Laying down of the new bone (neo-osteogenesis) on the surface of matrix framework and along the vascular spaces/channels; and (iv) remodeling of the newly formed bone to conform to the trabecular pattern along the lines of functional loading and stress (according to Wolff's law).  The most challenging clinical condition of structural (intercalary) bone grafting for large osteoperiosteal gaps in the lower limb may take 2–4 years for adequate incorporation permitting unprotected loading [11, 12]. The mechanical strength of the reconstruct is weak for 1 and 1/2 to 2 years, after which the strength increases by more neo-osteogenesis. The least time is taken in a cavitary pathology which offers a very large osteogenic bed and copious surface for intimate contact with the graft [13–15]. As incorporation takes place from periphery to the center, the time taken for large cavities and large grafts is correspondingly higher. In un-demineralized cortical (e.g., fibula) graft, 20–30% (deepest sector) of the grafted bone may never get incorporated; it may stay incarcerated surrounded by newly formed bone.

polymorphs, lymphocytes, and macrophages between 10 and 20 days. The cellularity gradually reduced with relative increase in the number of lymphocytes. By the 40th postimplantation day, the macrophages were practically absent, and one could see appreciable osteogenic activity by the presence of osteoblasts and osteoclasts. No cellular immune reaction was discernable. Periodic core biopsy in early stages and biopsy of the graft in patients who required a second operation showed histological and tetracycline fluorescence evidence of neo-osteogenesis between 6 and 12 weeks. The fluorescence in the implanted allogenic bone was quantitatively the same as the bone of a patient's iliac crest in specimens available 12 months after the grafting.

**Figure 6.** A child having tuberculosis of the spine involving multiple vertebrae: posterior spinal fusion was performed

using allogenic decal-bone. Note the incorporation of the graft with remodeling during a 3-year follow-up.

50 Bone Grafting - Recent Advances with Special References to Cranio-Maxillofacial Surgery

**Contents of bone grafts and their roles:** In general calcium hydroxyapatite, the predominant mineral in bones provides an inert framework providing mechanical stability and offering a lattice work for penetration of neocapillaries, reparative tissues, and osteoconduction (**Table 5**). Only the most superficial bone-forming cells in fresh autografts which survive getting nutrition by tissue perfusion provide direct osteogenic activity. In allografts no viable cells are expected; however, the debris of dead cells act as the most potent immunogenic agent. Organic matrix provides the most potent bone morphogenetic (bone induction principles)

**3.2. Discussion**

Ca hydroxyapatite Mechanical stability Autogenous surviving cells Osteogenesis Allogenic non-surviving cells Immunogenesis

**Table 5.** Contents of bone graft and their role.

Matrix Bone morphogenetic agents(weak mechanical strength)

By HCL decalcification we aimed at the removal of nearly 50% of mineral, thus providing adequate structural integrity. The said treatment removed all cell debris and fat providing opened-up channels for ready penetration of neocapillaries and perivascular mesenchymal cells. Acid demineralization also removed the mineral from the surface of the bone, along the vascular channels, and on the lacunar spaces, thus exposing the matrix for intimate contact with the invading perivascular tissues, and facilitated the interaction between the graft matrix (most active osteoinductive principle) and the pluripotent mesenchymal cells from the host. The technique used by us does not destroy the biological osteoinductive property of the bone matrix.

Immunogenicity of allogenic bone is now better understood. Fresh unmatched and untreated allogenic bone inevitably evokes an immune response in the host. The immune response in general is delayed and mild and develops slowly; however, it results in "unexplained" graft resorption and delay or failure in its incorporation. In clinical practice deep-freezing, freezing, freeze-drying, and irradiation are currently employed to reduce immunogenicity. Pure BMP from an allogenic source or even a xenogenic source is considered to have negligible immunogenicity. We feel that a simple treatment of allogenic bone by HCL decalcification and ethanol preservation practically eliminates the antigenic material (cells and debris) to permit unhindered incorporation in clinical practice as observed in our cases. Overall analysis in our clinical material has been approximately 80–90% successful for benign cavitary lesions; for impaction grafts in revision joint surgeries, 50–70% success for structural reconstruction in circumferential osteoperiosteal gaps; and 70–90% clinical success in extensive spinal fusion. Supplementary procedures for obtaining success in difficult cases, especially for osteoperiosteal gaps, are an accepted norm in 20–30% of cases. The success rate in our clinical cases is compatible with the observations of the outcome where allogenic bone was used from more sophisticated bone banks. Allogenic bone graft is a rational option when the recipient's patient owns bones that are inherently defective (e.g., fibrous dysplasia, enchondromatosis).

[3] Tuli SM, Singh AD. The osteoinductive property of decalcified bone matrix: An experi-

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osteoperiosteal defects. Acta Orthopaedica Scandinavica. 1982;**53**:857-865


2.5.1. Lymphocyte Apoptosis

The Annexin V-FITC apoptosis detection kit (BioVision, Milpitas, CA, USA) was applied to detect the lymphocyte apoptosis. After being gently mixed, 200 μL of the whole blood sample was transferred to a conical polypropylene test tube, and 4 mL of lysing buffer (Thermo Fisher Scientific, Waltham, MA, USA) was added. The solution was incubated at room temperature for 7 min before being centrifuged at 1500× *g* for 5 min at 20 ◦C, and the supernatant was then discarded. Subsequently, 3 mL of phosphate buffered saline (PBS) (Corning®, Corning, NY, USA) was added to wash the cells, the solution was centrifuged for 5 min, and the supernatant was then discarded. Lymphocytes were suspended with 300 μL of binding buffer. Thereafter, 3 μL of Annexin V-FETC and propidium iodide were added, and the solution was incubated in the dark at room temperature for 5 min. Finally, samples were gently drawn and then the subject to analysis by the BD FACSCaliburTM flow cytometry (Becton Dickson, San Jose, CA, USA).


*Diet*

## *Support measures*

#### *A professional view on symptom management and relief*

*"Periods of rest and "pacing" are important components of all management strategies for ME/CFS patients. Physicians should advise people with ME/CFS on the role of adequate rest, how to introduce breaks into their daily routine, and their frequency and length which may be appropriate for each patient. Excessive rest may be counterproductive, except in the initial stages of disease, in the very severe cases, or in cases of acute exacerbation; so it is important to introduce 'low level' physical and cognitive activities within the patient's capacity, according to the severity of symptoms.*

*Sleep management is tailored to the individual, the role and effect of disordered sleep is explained, common changes in sleep dysfunction that may exacerbate fatigue symptoms are identified; common manifestations include insomnia, hypersomnia, sleep reversal, altered sleep-awake cycle and non-refreshing sleep. The professional provides general advice on good sleep hygiene and encourages gradual changes in sleep pattern, though of course there is no implication that poor sleep hygiene is the cause of non-refreshing sleep. Relaxation techniques appropriate for ME/CFS should be offered for the management of pain, sleep problems and comorbid stress or anxiety. Examples include guided visualisation and breathing techniques, which can be incorporated into daily routines and rest periods", while mindfulness ma be of value as a sympathetic nervous system modulator. Although exclusion diets are not generally recommended for managing ME/CFS, many people find them helpful for some symptoms, including bowel symptoms. The patient may attempt an exclusion diet or dietary manipulation under professional guidance and supervision, e.g., from a dietitian. For those with nausea, advice includes eating small portions and snacking on dry starchy food and sipping fluids. The use of anti-emetic drugs should be considered if the nausea is severe."* Dr. L. Lorusso (personal communication)

#### *4.7. Symptoms Relief and Management Using Available Pharmacological Drugs*

Treatment of pain and sleep dysfunction are key, as they may have an indirect impact on other symptoms. Options for the pharmacological treatment of fatigue, including mental fatigue are more restricted. A balance between benefits and side effects, and significant individual variability in treatment response, call for individualised treatment. Costs are also a consideration, especially in settings where patients pay for medications out of pocket or where there are restrictions in prescribing medications.

Evidence of the effects of various drugs or supplements is scarce and often based on their use for related conditions or on reported use in ME/CFS and clinicians experience. It is important to observe legislations in different countries and to ensure that prescription of any drugs not specifically approved or licensed for ME/CFS is discussed with the patient and an informed choice is made. In some settings, it may be appropriate to obtain formal signed consent from the patient before introduction of a drug that has not been approved for use in ME/CFS. Regulations on supplements and over the counter medications are usually much less strict, but again, use by patients should be based on informed decision. Finally, it is important to note that many patients have already been taking a range of medications and supplements before reaching the ME/CFS specialist; again, in these cases, it is important to discuss continuation or otherwise with the patient; evidence of benefit on the individual patient, costs, potential side effects, or interactions with other medicines are important considerations. Some examples of pharmacological drugs that could be considered, where appropriate, are listed in Box 12. The use of medications that may address multiple symptoms may be considered.

#### *4.8. Following the Consultation and Clinical Monitoring*

Regular follow-ups are opportunities for education, including on self-management, assessment of usefulness of medications and other treatments and side-effects. Follow-up should include monitoring of symptoms, using similar instruments to those used at or before the initial consultation. Examples of instruments that may be used in monitoring patients include hand grip strength measurement, standing test, serum CK, severity assessment using specific instruments or scales (such as analogue scales for pain, fatigue, sleep, and other symptoms), and specific questionnaires for assessing symptom severity.

#### *4.9. Needs of Patients with Different Severities*

People who have severe ME/CFS may be unable to carry out activities of daily living and may spend a significant proportion, or all, of the day in bed. The symptoms experienced by patients with severe ME/CFS are diverse and debilitating, and these may fluctuate and change, both in type and in severity. It is therefore important that the management and care plan is flexible and reviewed regularly. People may have severe ME/CFS for years, and recovery is uncertain. Health services need to be prepared to attend to the specific needs of the severely affected, including home visits or virtual health consultations.

#### **5. Concluding Remarks and Recommendations for Developing and Organising ME/CFS Services**

The following are general recommendations for fully implemented services, but we appreciate that they are not achievable in the short term in many places, especially where knowledge and training in the field are limited or other resources are scarce. We encourage countries and regions to plan for their services, training, and educational needs according to the specific needs and characteristics of their population and patients and their organizational structures and resources. A national champion for each country or regions within countries would be highly desirable, especially in places with no or very scarce provision of services for ME/CFS.

**Box 12.** Examples of pharmacological approaches for relieving/managing ME/CFS symptoms\*.

*Pain*


*Edited by Mahmood S Choudhery*

Regenerative medicine is a promising interdisciplinary field that applies basic principles of engineering and life sciences to repair, replace, or regenerate damaged or lost tissues and organs. Unlike conventional medicine, regenerative medicine uses human cells and other substances to regrow tissues or restore their functions. Regenerative medicine combines approaches such as the use of cell-based, cell-free soluble molecules, stem cells from different sources, gene therapy, tissue engineering, reprogramming of cells, and, more recently, cell-free regenerative therapies. Regenerative Medicine provides details of the recent advancements in regenerative therapies for regenerative medicine applications.

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

Regenerative Medicine

Regenerative Medicine

*Edited by Mahmood S Choudhery*


**5. Cadmium-containing quantum dots as a platform for nanoparticle drug delivery vehicle design**

and traceable drug delivery. However, high-quality QDs are mainly made with heavy metals, like cadmium, whose long-term toxicity is currently largely unknown. Despite this limitation, QDs have been applied to cells and small animals as drug carriers, serving as an outstanding discovery tool for drug screening and validation, and as prototype materials for drug carrier engineering [75]. One primary challenge of drug delivery is maintaining a useful concentration

Pharmacokinetic Properties and Safety of Cadmium-Containing Quantum Dots as Drug Delivery Systems

http://dx.doi.org/10.5772/58553

479

Nanoparticle-based drug delivery is on its way to overcoming the fundamental limitations of simple free drug formulations, providing means to change their pharmacological properties and also understand their biological fate in great detail. Among many contrast agents for studying nanoparticle-based drug delivery vehicles, QDs are particularly suitable. Their unique amalgamation of useful features, such as small size, versatile surface chemistry, and exquisite optical properties make them an ideal platform for the comprehensive characteriza‐ tion of nanoparticle-based drug delivery vehicle behavior across single-cell to whole organism levels. In this new field, QDs have already made substantial contributions, enabling dynamic monitoring of nanocarrier cell uptake, intracellular distribution, circulation half-times, and biodistribution. Early on, the design of QDs drug delivery vehicles was governed by the intrinsically poor pharmacokinetic (PK) properties of conventional drugs. Low drug solubility, rapid metabolism and clearance and, most importantly, a lack of selectivity, regularly lead to therapeutic failure by causing severe systemic toxicity in healthy tissues, thus prohibiting the dose escalation necessary to eliminate tumor cells. Incorporating these drugs into nanocarriers offers an exciting opportunity to redefine the PK properties, improving therapeutic efficacy

When we utilize a nanopharmaceutical, it is important to realize that, in contrast to delivering a drug that is an organic molecule, we are delivering something of a discrete entity in a nanoparticle-comprised of atomic scale parts. Due to the quantum effects and electronic interactions that predominate at the nanoscale, we need to alter the way in which we think about pharmacological parameters in order to adapt to nanoscience. Nanopharmacology is further complicated by the need to establish the behavior of nanoparticles such as QDs within the traditional pharmacological parameters of absorption, distribution, metabolism and excretion (ADME). Nanoconstructs, in many cases, have limited metabolism and excretion and persist in biological systems; this becomes particularly important when toxic atoms such as cadmium are involved. The pharmacological parameters of the behavior of cadmiumcontaining quantum dots in biological systems is currently still under investigation. Dosing parameters, absorption, distribution, metabolism and excretion require considerable further

The first factor we need to establish before we study the pharmacokinetics parameter is the dose. Adequate estimates of QDs exposure during treatments such as cancer therapy must be in place so that both pharmacological and toxicological studies can be conducted within a

study, since we have little information on these parameters at this point.

of the drug in the targeted tissue while preventing toxicity.

and reducing side effects.

**6. Pharmacokinetics of cadmium-containing quantum dots**

Recently, there has been an explosion in the development of nanoparticle-based drug delivery vehicles composed of lipids, polymers, carbon materials, and even hybrid combinations of those materials tailored not only for the dramatic improvement of the pharmacological properties of existing drugs, but also for enabling the delivery of new classes of potent anticancer drugs for gene therapy and immunotherapy [67, 68]. With QDs, a combination of unique physical, chemical, and optical properties facilitates in-depth study of nanocarrier interactions with biological systems through real-time monitoring of QDs biodistribution, intracellular uptake, drug release, and long-term nanocarrier fate. At the same time, compact size and compatibility with a variety of surface modification strategies enables substitution of virtually any QD core with a QD within single-nanoparticle drug delivery vehicles, or incorporation of QD tags within larger multicomponent vehicles. The combination of superior brightness and resistance to photo-degradation represents another set of QD properties that highly useful for long-term nanocarrier tracking.

The great interest in engineering NP-based drug delivery vehicles is driven by the powerful capability of nanocarriers to completely redefine the pharmacokinetic properties of virtually any drug, ranging from small-molecule therapeutics to large proteins and DNA plasmids. Encapsulation of the drug within the nanoparticles keeps it shielded from the biological environment until the moment of carrier degradation and drug release, thus minimizing nonspecific and potentially adverse interactions en route to the target [69].

A few reports have appeared recently regarding this ambitious goal. It has previously conjugated captopril, an antihypertensive drug, to the QD surface and studied its pharmaco‐ dynamics and pharmacokinetics in stroke-prone spontaneously hypertensive rats [70]. The results show that the administered QD-captopril conjugates are capable of decreasing rat blood pressure to the same extent as the captopril alone in the first 30 min, but the therapeutic effect of QD-captopril disappears after 60 min. It is unclear whether the therapeutic effect results from the QD-captopril conjugates or captopril molecules detached from the QD surface. Another piece of interesting work was previously reported, wherein a targeting functionality was added to QDs by linking them with RNA aptamers (A10) that specifically bind to prostate specific membrane antigen (PSMA) [71]. Doxorubicin, a DNA-interacting drug widely used in chemotherapy, was immobilized onto QDs by intercalation within the A10 RNA aptamer [72]. Another study reported the ability of nanoconjugates of CdSe/CdS/ZnS and doxorubicin (Dox) to target alveolar macrophages, cells that play a critical role in the pathogenesis of inflammatory lung injuries. The results demonstrated that nanoparticle platforms can provide targeted macrophage-selective therapy for the treatment of pulmonary disease [73]. This was previously reported regarding siRNA delivery using QDs as delivery vehicles [74].

QDs already play an important role in fundamental biology and *in vitro* disease diagnostics and prognostics. Their unique structural and surface properties, such as their tunable and uniform size, flexible drug-linking and doping mechanisms, large surface-to-volume ratio and wide spectrum of surface reactive groups have enabled a new avenue of research: targeted and traceable drug delivery. However, high-quality QDs are mainly made with heavy metals, like cadmium, whose long-term toxicity is currently largely unknown. Despite this limitation, QDs have been applied to cells and small animals as drug carriers, serving as an outstanding discovery tool for drug screening and validation, and as prototype materials for drug carrier engineering [75]. One primary challenge of drug delivery is maintaining a useful concentration of the drug in the targeted tissue while preventing toxicity.


*3.3.2 Effects of lead on human health*

Drinking water is one of the major sources of human exposure to lead [115]. Lead particularly targets the nervous system, blood, and kidney [116]. Many studies found associations between low level environmental Pb exposure and chronic kidney disease, a general term for heterogeneous disorders affecting the structure and function of the kidney (CKD) [117, 118]. Long-term lead exposure may generate irreversible functional and morphological renal changes [119], distal motor neuropathy and possibly seizures and coma [120]. Infants and small children are more sensitive to the effects of lead, which moreover is transported through the placenta to the foetus [121]. Lead accumulation in fetuses and small children might cause developmental disruption in terms of neurological impairment characterized by a decrease of cognitive faculties, which can be reversible or not, evaluated by psychomotor tests such as the verbal IQ (Intellectual Quotient) test [27, 109]. The period when IQ is most affected is from birth to about 4 years of age [122].

Scientific literature on lead water pollution reports "Lead remains a problem in drinking water in many parts of the world, with millions of properties served by distribution systems containing lead components. Strong links have been established between human exposure to lead and health effects in both adults and children. As a result, the allowable levels of lead indrinking water have generally become lower. Implementation of these regulations is difficult with the controls available. Future recommendations for aspiring to zero lead in drinking water are: (i) improving sampling, monitoring and modeling; (ii) Wider application of shortterm pointof- use devices; (iii) replacement of all lead pipes and plumbing through applicable regulations and increased awareness public" [123–126].

*Chemical Pollution of Drinking Water in Haiti: An Important Threat to Public Health DOI: http://dx.doi.org/10.5772/intechopen.97766*

#### *3.3.3 Effects of nickel on human health*

Nickel is insoluble in water. However, when it is in the form of exceptionally fine particles, it ionizes as Ni (II) in water and in body fluids such as blood. During oral exposure, the major effects observed are the death of a child after ingestion of 570 mg of nickel/kg [127] and intestinal disorders such as nausea, abdominal cramps, and diarrhea [128]. Immunological, hematological, hepatic, renal, genotoxic effects on embryonic development and reproduction have been reported depending on the route of entry into the body [129].