*5.2. Traffic Analysis: Historical Traffic under Adverse Weather Conditions at Milano Malpensa and Bergamo Airports*

In order to evaluate the influence of adverse weather conditions on air traffic in the extended TMA around MXP and BGY, located at ca. 77 km distance from each other, FlightRadar24 datasets from three different selected days with severe events—11 May 2019, 7 July 2019, and 6 August 2019—were analyzed.

In the case of MXP, there were two available days—7 July 2019 and 6 August 2019 without airport closure under disturbed weather conditions and one day—11 May 2019 when adverse weather conditions caused temporal closure of the airport. For BGY, there were two days—11 May 2019 and 7 July 2019—in the dataset where adverse weather did not lead to an airport closure and one day—6 August 2019—when the airport was temporarily closed. As the airports are located adjacent to each other, it is of interest to investigate the datasets in pairs in order to analyze the influence of the changing capacity of one airport on the second one. Therefore, the following pairs of data were considered:

The first two datasets recorded on 7 July 2019 contain 322 arrivals at MXP and 144 arrivals at BGY. The travel time of arrivals in the extended TMA and touchdown time are illustrated in Figure 9 in orange for MXP and in blue for BGY. In order to detect peak times while smoothing the influence of outliers, a moving mean value over five neighbor travel times was calculated. These mean values are shown as dark blue and dark orange lines in Figure 9. In the case of MXP, the time until landing in the extended TMA varied between 693 and 2913 s with an average time of 1246 s and five peak periods, the most noticeable of which was between 19:30 UTC and 21:30 UTC (Figure 9). The maximal and the average flown distances in the extended TMA were approximately 302 and 168 km, respectively, whereas the average flown distance at the mentioned peak period was equal to 225 km (i.e., 57 km longer than the average flown distance over the whole day).

**Figure 9.** Visualization of travel time in the extended TMA and touchdown time for historical traffic at MXP (orange color) and at BGY (blue color) on 7 July 2019.

In the same day, there were two outlier arrivals at BGY in terms of travel time in the extended TMA. Taking them into account, the time until landing varied between 817 and 4836 s, with an average travel time of 1186 s. The detectable peak period is between 16:15 UTC and 17:30 UTC (Figure 9). The maximal and the average flown distances in the extended TMA are approximately 718 and 160 km, respectively. The average flown distance at the peak time amounts to 247 km with the outlier arrival and 194 km without it. Even without the outlier, the average flown distance during the peak period is 34 km longer than the corresponding value over the whole day.

Summing up, there are two possible coherent peaks at the considered airports that may have been caused by weather conditions. These peaks are pairwise-marked by the areas bounded by the dotted lines in Figure 9.

The second pair of datasets collected on 6 August 2019 contains 421 arrivals. It includes 99 more arrivals than the traffic scenario on 7 July 2019 for MXP and 144 arrivals for BGY. The travel time in the extended TMA and the touchdown time of arrivals are shown in Figure 10. The time until landing in MXP's extended TMA was between 715 and 2436 s, with an average travel time of 1161 s. The maximal and the average flown distances in the extended TMA were approximately 352 and 161 km, respectively. In spite of the fact that this scenario is larger than the previous one for MXP, it has lower values for maximal and average travel time and, as a consequence, average travel distance.

**Figure 10.** Travel time in the extended TMA vs. touchdown time for historical traffic at MXP (orange color) and at BGY (blue color) on 6 August 2019.

In the same day, the time until landing in the extended TMA around BGY was between 703 and 5679 s, with an average of 1332 s. There are two peaks with respect to travel time in the extended TMA between 19:30 UTC and 23:15 UTC, which are marked by the areas bounded by the dotted lines in Figure 10. The maximal and the average flown distances in the extended TMA were approximately 739 and 185 km, respectively.

The average flown distance during peak time amounts to 310 km and is, through temporal closures of runways, at least 63 km greater than the corresponding average flown distance during the peak time on 7 July 2019, when BGY was not closed. Figure 11 illustrates the accumulated rainfall per ten minutes on 6 August 2019 at BGY, which correlates with the peaks for travel time shown in Figure 10. However, there are no significant peaks between 19:30 UTC and 23:15 UTC at the nearby MXP airport, as illustrated in orange in Figure 10.

**Figure 11.** Histogram of the rainfall (blue bars) and accumulated rain (red line) at the Bergamo airport on 6 August 2019 [46].

The last pair of datasets was recorded on 11 May 2019 and contains 233 arrivals to MXP and 122 arrivals to BGY. On this day, a squall line hit MXP between 14:00 UTC and 16:00 UTC causing intense precipitation and heavy hail formation [46]. These adverse weather conditions (Figure 12) led to the airport's closure for about 40 min and some flight delays. In addition, nine aircraft were diverted to other airports. Figure 13 illustrates travel time at the extended MXP TMA, which was between 719 and 4693 s with an average of 1196 s. This distribution indicates that controllers had to guide many aircraft into holdings. According to Figure 13, the airport was closed at around 16:00 UTC. The maximal and average flown distances in the extended TMA were approximately 676 and 165 km, respectively. The average flown distance during the peak time was equal to 349 km.

**Figure 12.** Apron of a runway at MXP on 11 May 2019 [53].

**Figure 13.** Visualization of travel time in the extended TMA and touchdown time for historical traffic at MXP (orange color) and at BGY (blue color) on 11 May 2019.

Historical traffic at BGY on 11 May 2019 indicated a time until landing in the extended TMA of between 782 and 2985 s, with an average travel time of 1197 s (Figure 13). There was a peak period with respect to travel time between 17:30 UTC and 19:30 UTC, probably as a consequence of the weather conditions at MXP airport described above. The maximal and average flown distances at the BGY extended TMA were approximately 415 and 109 km, respectively, and the average flown distance during the peak period is equal to 215 km. Although the weather conditions at BGY did not cause a runway closure, one can notice the direct influence of the weather conditions at MXP on operations at BGY. Therefore, the historical dataset for MXP on 11 May 2019 was chosen to construct simulation traffic scenarios for investigation of the influence of adverse weather on arrival traffic. Table 1 summarizes the mentioned parameters for the three considered case studies.

**Table 1.** Data summary of available historical traffic.

### *5.3. Traffic Scenario Definition*

The simulations involved three traffic scenarios for the TMA at MXP, which were based on FlightRadar24 datasets of the three days (11 May 2019, 7 July 2019, and 6 August 2019). There were two days when weather conditions did not cause airport closure (7 July 2019 and 6 August 2019) at MXP, which were used as baseline scenarios, and one day with an airport closure for some period of time (11 May 2019), which was used as the disturbed scenario.


*3.2. Risk of Bias of the Included Studies*

In random sequence generation, five studies were graded as unclear [36,40,46,48,49]. In allocation concealment, 10 studies were graded as unclear [14,15,36,40,44–46,48–50]. Nine studies were graded as high in blinding of participants because several articles were add-on study designs that cannot blind participants [14,38,41–43,46,48–50]. Furthermore, four studies were graded as unclear. In the blinding of outcome assessment, 11 studies were graded as unclear [14,15,39,43–50]. In incomplete outcome data, two studies were graded as unclear [15,44]. Six studies were graded as high as the DR was more than 10% [36–39,41,45]. One study was graded as high in selective reporting because it did not report the outcome variables previously described in the protocol [36]. Detailed visualization of each study with the risk of bias graph is presented in Supplementary Materials Digital Content 3.

**Table**

**1.**

Characteristics

of

the

included

studies.

**Table**

**1.**

*Cont.*

**Table 2.** Detailed description of the acupuncture treatment.

*Healthcare* **2021**, *9*, 1502

**Table 2.** *Cont.*

treatment group.


*Editorial* **Silkworm and Silk: Traditional and Innovative Applications**

**Silvia Cappellozza 1,\*, Morena Casartelli 2,3, Federica Sandrelli 4, Alessio Saviane 1 and Gianluca Tettamanti 3,5**

The various subjects covered in the present Special Issue "Silkworm and Silk: Traditional and Innovative Applications" demonstrate how sericulture, a practice deeply rooted in human history, can act as a bridge to bring together an exceptionally wide range of scientific and technical expertise in both conventional topics and cutting-edge technologies.

As with many other species, *Bombyx mori* was obtained through human-driven selection from a wild ancestor but, in contrast to other animals, the genetic stocks of silkworm strains have been preserved in public research facilities for centuries. Therefore, in nations with a history of sericulture, there are wide collections of various genotypes that must be maintained through a customary activity. Additionally, the majority of sericultural regions in the world continue to practice agricultural production of cocoons through conventional silkworm rearing. Since the mulberry leaf is the only food source for the larvae of this insect (or an artificial diet based on it), there has always been a need for moriculture, and the same organizations dedicated to maintaining *B. mori* genetic resources typically preserve mulberry germplasm collections as well; a significant amount of research is devoted to understanding the traits of these varieties and how they relate to the silkworm. A multilevel study on the effects of regional mulberry genotypes on the health of the silkworm is reported in this issue; the authors demonstrated that the selection of mulberry varieties characterized by specific qualitative and quantitative features can strongly impact larval development, cocoon production, and raw silk parameters [1]. Scientists also learnt how to use mulberry varieties to feed other animals or as a source of raw materials and active chemicals; further, they adopted new technologies to obtain advanced materials from silk. This issue presents a bibliometric paper by Giora et al. [2], which analyzed recent research trends in the fields of sericulture and moriculture and provides an overview of the past 20 years of research in this sector of rising interest. Two other reviews [3,4] aimed to connect advances in or criticisms of sericulture to societal and economic changes in various regions of the world, highlighting how this activity may be heavily dependent not only on technological improvements, but also on many external factors.

One of the newest trends in sericulture is to promote the development of circular economy supply chains to employ silk by-products for feed and food, a topic that is comprehensively discussed and documented in the article by Tassoni et al. [5]. Nowadays, silk by-products are easily exploitable via melting silk with the right solvents to obtain solutions of silk proteins, opening the door to entirely new applications in industry including in the textile sector, which is still of the utmost importance for an economy dependent on traditional sericultural agroindustry. Furthermore, this issue contains numerous articles that address these novel applications and cover both the general role of silk fibroin in biomedicine [6] and methods for creating hybrid materials, for instance, by adding nanoparticles [7]. The paper by Bassani et al. [8] examined particular devices, such as silk

**Citation:** Cappellozza, S.; Casartelli, M.; Sandrelli, F.; Saviane, A.; Tettamanti, G. Silkworm and Silk: Traditional and Innovative Applications. *Insects* **2022**, *13*, 1016. https://doi.org/10.3390/ insects13111016

Received: 27 October 2022 Accepted: 30 October 2022 Published: 3 November 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

nerve conduits, in a study case illustrating how research findings can be translated into medical products for use in clinical settings.

Advances in methodology and knowledge are not limited to the new biotechnological uses of silk proteins. A significant part of the Special Issue is devoted to papers using the most recent techniques to study silkworm physiology to enhance silk production. This can improve the general knowledge of silkworm biology and hasten the potential use of this organism in various applied fields. Specifically, Yokoi et al. [9] undertook an RNA-seq investigation of several silkworm larvae tissues, including the midgut, fat body, testis, ovary, and the different subregions of the silk gland, generating reference transcriptome data for a *B. mori* strain. Masuoka et al. [10] identified novel silk protein regulation factors, using a proprietary software, to conduct co-expression networks and time-course expression analyses on transcriptomic data from various tissues, including different subregions of the silk glands, in reference silkworm strains. Zhang et al. [11] performed a transcriptomic analysis of fat body derived from silkworms treated with hydrogen sulfide, a molecule that, at specific doses, exerts beneficial effects on the developmental and economical traits of *B. mori*. All these results provide data and methods with which to develop strategies for enhancing silk production in *B. mori*. Moreover, de la Peña et al. [12] compared RNA-seq transcriptional profiles of silk glands belonging to various silkworm strains commonly reared in the Philippines and maintained under different temperature conditions, producing a set of information useful for the genetic improvement of *B. mori* strains and to increase their silk productivity in this geographical area. Finally, the article by Baci et al. [13] reports a comprehensive overview of the most advanced genome-editing techniques performed in *B. mori,* using the CRISPR/Cas9 methodology. In their paper, the authors illustrated strategies used to obtain both knock-out and knock-in *B. mori* strains. They also focused on the possible applications of CRISPR/Cas9 to generate silkworm strains characterized by a higher resistance to viral infections, highlighting the importance of this technology for both basic research and applied purposes in *B. mori*. Since *B. mori* is considered a model species among Lepidoptera, it has been extensively employed in studies of physiology, genetics, molecular biology, and pathology. Zhang et al. [14] made use of this lepidopteron to investigate how hemolymph ecdysteroid titer affects maternal genes during oogenesis, demonstrating that maternal mRNAs begin accumulating in the ovary during the larval period, at the wandering stage. In the article by Chang et al. [15], the role of polyamines in regulating cell cycle progression and DNA replication was explored as well as the expression of genes coding for enzymes involved in the polyamine pathway in different tissues. Since it is known that the administration of polyamines to *B. mori* larvae increases the expression of the gene coding for fibroin heavy chain and improves the quantity and quality of silk, this study opens the door to manipulating the expression of key genes involved in the polyamine pathway in silk glands to improve silk production. The use of silkworms and insects in general as model organisms began in response to growing concerns about animal health and welfare, with the goal of preventing, or at least reducing, the usage of higher animals in all sectors of the life sciences. In fact, the principles of the 3Rs (Replacement, Reduction, and Refinement), developed in the 1950s, offer a foundation for conducting more compassionate animal research. Since then, they have become an integral part of national and international laws and regulations governing the use of animals in research and are progressively leading to the development of suitable alternative models, among invertebrates, for experimental aims. Holometabolous insects represent a promising option due to their lower costs, convenience in handling, lack of ethical restrictions, and features that can successfully reproduce different biological mechanisms occurring in mammals. In this Special Issue, three articles pave the way for the development of the silkworm as an alternative model for screening antimicrobial compounds against nosocomial pathogens, such as *Staphylococcus epidermidis* [16], and bacteria that cause skin diseases and systemic infection, such as *Cutibacterium acnes* [17], as well as for studying the effects of natural compounds with hypoglycemic activity [18]. Although it is still up for debate whether these insect-based models are applicable in clinical settings, the findings of these three

studies are undoubtedly encouraging. Another article focused on the health status of the silkworm and its pathologies. In particular, the study dealt with nuclear polyhedrosis, a major viral disease in sericulture that causes high larval lethality and great economic losses. By using a genomic approach that considered different BmNPV strains, the authors demonstrated that mutation and rearrangement of the genome can lead to differential pathogenicity of this virus [19]. The holobiont concept, which has recently emerged as a theoretical and experimental context in which to study the interactions between hosts and their associated microbial communities, is worth mentioning while discussing the silkworm as a laboratory model. It is becoming increasingly clear that the development, growth, and health of macro-organisms are influenced by the complex microbial communities they host. The article by Dee Tan and Bautista [20] deals with this topic; the authors studied the gut microbial composition of four *B. mori* strains reared in the Philippines to construct a database of useful information fostering an improved understanding of whether and how the gut microbiota affects silk production and to develop strategies for future improvement of the strains. Another topic that has attracted increasing attention in recent years is the administration of probiotics to provide health benefits, generally by improving or restoring the gut microbial community. Unban et al. [21] isolated from the midgut of *Samia ricini*, another lepidopteran species used for silk production, a xylose-utilizing lactic acid bacterium, *Enterococcus hirae* SX2, which acts as a probiotic in this insect due to its tannin tolerance and antimicrobial activity against insect pathogens. The results presented in this article demonstrate that the oral administration of *E. hirae* SX2 to Eri silkworm not only improves its growth and reduces mortality, therefore positively influencing its economic traits, but also represents a starting point for developing strategies to select novel probiotics that can positively affect *B. mori* larvae and improve sericulture research.

Taken together, the articles in this Special Issue cover the transversality of all the topics encompassed by sericulture, which goes far beyond silkworms and silk. This collection of many articles from different countries in a relatively limited period of time testifies to the great interest the scientific community takes in sericulture, encouraging the guest editors to repropose the subject in the near future.;

**Author Contributions:** Conceptualization, S.C., M.C., F.S., A.S. and G.T.; writing—original draft preparation, S.C., M.C., F.S., A.S. and G.T.; writing—review and editing, S.C., M.C., F.S., A.S. and G.T. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**


*Article* **Measuring Green Marketing: Scale Development and Validation**

**Neringa Vilkaite-Vaitone 1,\*, Ilona Skackauskiene 1 and Gonzalo Díaz-Meneses 2**

**Abstract:** Academics and practitioners are paying increasing attention to green marketing as lesser damage to the environment and future generations become a priority in a current complex business environment. Despite the expanding studies in this field, there is still a lack of psychometrically sound scales to measure green marketing practices. To fill this gap, the research aimed to develop and evaluate a multifaceted green marketing scale. First, we draw on theoretical evidence to define and conceptualize the construct of green marketing. Then, we use a multistudy scale development process to create and validate the Green Marketing Scale (GMaS). Two groups of participants were used for the validation of the scale. Study 1 (*n* = 102), with the help of exploratory factor analysis (EFA), refined and reduced the items, proposed the factor structure. Study 2 (*n* = 155) established the validity of the construct and the reliability of the scale. The authors have tested the six-factor model against the four-factor models using confirmatory factor analysis (CFA) with a sample of marketing managers. The results of the CFA have indicated that the revised version of the four-factor model appears to be the most tenable solution, as it shows the best fit for the data. The resulting 14-item GMaS captures a variety of green marketing manifestations across organizational settings and involves the dimensions of Strategy, Internal Marketing, Product, and Marketing Communication. In general, the research confirms the validity and reliability of the GMaS scale and can be used to measure green marketing in organizational settings in the energy industry.

**Keywords:** green marketing scale; scale development; scale validation; internal marketing; external marketing; strategy; tactics; operations; clean technology


**1. Introduction**

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88 Research on Soil Erosion Soil Erosion

305-342, 0-94757-111-6, IAHS.

Conservation of natural forest ecosystems will require a land ethic as prelude to under‐ standing the functioning of forest ecosystems, ecological and physiological impacts of dis‐ turbances on ecosystems, and the processes involved in recovery of disturbed ecosystems. Many of the harmful effects of pollution, fire, flooding, and soil compaction can be abated by judicious planning measurements to create and perpetuate the critical components of for‐ est stand structure and species composition. Strategies for continuous production of the products and services that can be supplied by forest ecosystems will need to be reinforced by expanded long-term research and close cooperation among various disciplines such as forest biologists, social scientists, economists, and regulatory government agencies [17].

Nowadays, multi-objective planning is necessary in forestry because of increased and varied demand for forest products and services. Management objective such as production of qual‐ ity potable water, carbon stocking, aesthetic, recreation and community health in forest es‐ pecially adjacent to big cities are of great importance. Forests have managed to produce wood products at various diameters and quality classes as the society demanded overtime [24]. Afterwards, the importance of these objectives has gradually diminished and over‐ whelmed by other management objectives such as conservation of water resources, preven‐ tion of soil erosion, carbon stocking, creation of landscape aesthetic, camouflaging military facilities and allocation of land for recreation [2]. The forest values on be grouped as static and dynamic forest values (Figure 1).

© 2012 Misir and Misir; licensee InTech. This is an open access article 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. © 2012 Misir and Misir; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**3. Forest Management and Soil Erosion**

every expert, there is an equal and opposite expert" [6].

sion of changes in soil productivity [12].

All forest management activities affect soils, with effects ranging over a continuum from nearly none where the activity is minimal to large. To foster communication, a threshold should be established above which effects merit attention and below which further consider‐ ation is not justified. The magnitude of that threshold varies with the state of knowledge, about forest dynamics and must include recognition of uncertainty. Failure to identify thresholds inhibits communication to a wider audience and even among ourselves [12].

Modeling of Soil Erosion and Its Implication to Forest Management

http://dx.doi.org/10.5772/ 53741

91

There are two kinds of effects of forest management on soils. The first, direct effect is an al‐ teration of soil properties such as an increase in bulk density following passage of heavy equipment. Soil scientists generally agree on those direct effects; recognition of those altera‐ tions is literally axiomatic. The second effect of management on soils is indirect; a change in site productivity due to alteration of soil properties. Some of those secondary effects are ob‐ vious enough that can be considered corollaries. Specific studies and personal and vicarious experience have led to this worldview. Conversely, some of the indirect effects of manage‐ ment on soils are not as clear, and can be considered postulates. The distinction between axi‐ oms, corollaries, and postulates is often in the eye of the beholder, and depends on interpretation of both published reports and personal observations. Papers that support a position are evaluated differently than those in opposition. I offer no excuses for bias; "For

Erosion is a natural process, but one whose rate and extent is exacerbated by forest manage‐ ment [36]. Most emphasis on erosion has been directed towards its effects on water quali‐ ty and fish habitat, but because it involves displacement of soil, the growing medium, erosion also can affect site productivity [21]. However, forest management activities are necessary parts of forestry, and there may be minimal control over the circumstances under which they are carried out. Alterations of soil physical properties are extensive, immediate, and their effects in reducing productivity are well-documented. Chemical and biological prop‐ erties of soils are also changed by management activities, but the effects on productivity are less well-documented and of longer term; their influence is not clear. Historical evidence shows that forest ecosystems are dynamic and resilient. Assessment of the consequences of changes in properties must recognize that shifts in preferred species should not be equa‐ ted with changes in productivity, and that short-term effects, measured by the length of most experiments or observations, may not be indicative of long-term effects [12]. Accu‐ rate assessment of the effects of its change, however, is likely to continue to be obscured by the influence of the many other elements that also affect forest productivity [40]. At our current state of ignorance, a reasonable approach may be a simple sensitivity analysis that uses spatially based techniques (geographic information systems) and reasonable esti‐ mates of effects of the many factors that affect forest productivity to develop an impres‐

Use of more sophisticated simulation models implies greater knowledge than we currently possess. Both ethical and economic considerations demand good stewardship with profes‐ sional accountability for our natural resources. Extensive forest management, if carried out

**Figure 1.** Classification of Forest values [23]

### **2. Soil Erosion**

Erosion, the detachment of soil particles, occurs by the action of water, wind, or glacial ice. Such 'background' soil erosion has been occurring for some 450 million years, since the first land plants formed the first soil. Only erosion caused by water will be considered here. Wa‐ ter related erosion occurs when raindrops, spring runoff, or floodwaters wear away and transport soil particles. Erosion is a complex natural process that has often been accelerated by human activities such as land clearance, agriculture, construction, surface mining, and urbanization.

Soil erosion by water and wind affects both agriculture and the natural environment, and is one of the most important of today's environmental problems. It isn't easy to find compre‐ hensive information about erosion, as the subobject is multidisciplinary involving geomor‐ phologists, agricultural engineers, soil scientists, hydrologists and others; and is of interest to policy-makers, farmers, environmentalists and many other groups.

Schumm and Harvey [32] believe some of the terms used to describe erosion are misleading. Normal erosion and geologic erosion are often meant to imply pre-agricultural conditions of low erosion rates, whereas accelerated erosion and historic erosion imply greatly increased erosion rates caused by man. Because of the great variability in natural erosion both spatial‐ ly and temporally at present and throughout geologic time, neither concept is correct. They prefer the term natural erosion for normal and geologic erosion, and the term man-induced erosion for accelerated and historic erosion.


2.8.7. Structural Classification of the Selected VDA Proteins

*Rv0554* is an integral part of the menaquinone regulatory operon in Mycobacterium. Menaquinone is a crucial factor in the mycobacterial electron transport chain [45]. *Rv1477* is a mycobacterial resuscitation promoting factor interacting protein (*RipA*) and participates in the cleavage of peptide cross linkages between peptidoglycans similar to other cleavage enzymes, such as *OwlT* and *Spr* in *B. subtilis* and *E. coli*, respectively [46]. *Rv1495* is designated as *mazEF*, which is a subcomponent of the type II toxin–antitoxin system in mycobacteria [47]. *Rv1566c* (*RipD*) is a predicted peptidoglycan specific peptidase of the NlpC/p60 family. The unusual peptide linkages of *Mycobacterium* i.e., L-D and D-D and other isopeptide linkages makes the peptide resilient to cleavage by mycobacterial peptidases [48]. *Rv1636* is a universal stress protein (USP) in *M. tuberculosis*. In *M. tuberculosis*, it is classified into class I USP, based on the presence of only a single conserved domain of USP, which is of similar size to *UspA*. CAMP is bound to *Rv1636*, which regulates the signaling associated with the cAMP molecule [49]. *Rv2010* of *M. tuberculosis* codes for *VapC*-15 toxin. These proteins belong to the PIN domain family proteins and contain ribonuclease activity [50]. *Rv2549c* is designated as *VapC20* of *M. tuberculosis* and along *VapB*, it forms *VapBC* toxin antitoxin complex [51]. *Rv2623* is a universal stress protein of *M. tuberculosis*, that helps mycobacteria in different stress conditions and is also important for mycobacterial growth and persistence [52]. *Rv2757c* of *M. tuberculosis* codes for *VapC*-21 toxin. These proteins also belong to the PIN domain family proteins and contain ribonuclease activity [53]. *Rv2801c* encodes for the *MazF*-mt1 protein of *M. tuberculosis*. *MazF* is a known component of the *MazEF* toxin–antitoxin system in many prokaryotic cells [54].


**2. Evaluation of risk factors by specific organ system disease**

#### **2.1. Recurrence of glomerular disease of the native kidneys**

Recurrence of glomerular disease is the third most common cause of graft loss after chronic allograft injury and death with a functioning graft. Currently available data on the incidence of recurrent disease and resultant graft loss are heterogeneous due to different study design, follow-up durations, patient samples, and the variable use of surveillance biopsies among centers. The reported incidence of recurrent renal disease after renal transplantation and the risk of graft loss from disease recurrence are shown in table 3. The clinical course and impact on graft survival vary between different types of glomerulonephritis (Colgert et al., 2008; Kasiske et al., 2009). Nonetheless, with the exception of primary focal segmental glomerulo‐ sclerosis (FSGS), recurrent glomerular disease is usually a late complication after transplan‐ tation. FSGS secondary to reflux nephropathy or obesity does not recur after transplantation. In patients with hypertensive renal disease or other causes of chronic kidney disease, focal segmental sclerosis may be found on histologic evaluation and must be differentiated from the primary disorder. Suggested risk factors for recurrence of primary FSGS include history of recurrence in a previous transplant, younger age at diagnosis, rapid progression to end stage renal disease from the time of initial diagnosis ( < 3 years), presence of mesangial proliferation in the native kidneys, older donor kidneys, Caucasian ethnicity, and the collapsing variant. Living donor kidneys (versus deceased donor) have not consistently been demonstrated to be associated with an increased risk of recurrence. Familial and sporadic forms of FSGS with podocin mutation, slow progression to end stage kidney disease (ESKD), and non-nephrotic range proteinuria in the native kidney disease are associated with low risk of recurrence (Ponticelli et al., 2010).

transplant centers as an essential component of the transplant evaluation process. A detailed cardiovascular history not only predicts the operative risk but also helps in postoperative cardiac management to improve short- and long-term cardiac outcomes. Over the years there has been much controversy over the best strategy for pre-transplant assessment and manage‐ ment of coronary artery disease (CAD) to prevent adverse peri-operative cardiac events. Recently, the American Heart Association / American College of Cardiology (AHA/ACC) have developed the 2012 AHA/ACC guidelines for "Cardiac Disease Evaluation and Management Among Kidney and Liver Transplantation Candidates" based on a comprehensive review of the literature pertinent to perioperative cardiac evaluation of potential kidney or liver transplant recipients (Lentine et al., 2012). These guidelines are endorsed by the American Society of Transplant Surgeons, American Society of Transplantation, and the National Kidney Foundation (discussed below). The AHA/ACC classifications of evidence to perform a test or

**Table 3.** Rates of recurrent renal disease after transplantation and risk of graft loss from disease recurrence1

*FSGS*: focal segmental glomerulosclerosis; *MPGN*: membranoproliferative glomerulonephritis; *GN*: glomerulonephrop‐

2Diarrhea (+) HUS usually does not recur; Diarrhea (-) or familial may recur in 21-28%; Factor H or I mutation may recur in 80% to 100%; Patients with mutation membrane cofactor protein does not have recurrence (reference Kasiske et

FSGS 20-50 50 Ig A nephropathy 20-60 10-30 MPGN I 20-50 30-35 MPGN II 80-100 10-20 Membranous GN 3-30 30 HUS2 10-40 10-40 Anti-GBM disease 15-50 < 5 ANCA-associated 7-25 < 5 Vasculitis SLE 3-10 < 5

athy; *HUS*: hemolytic uremic syndrome; *SLE*: systemic lupus erythematosus.

**Recurrence rates (%) Graft loss from disease recurrence (%)**

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**a.** Determining whether the transplant candidate has an active cardiac condition

The primary goal of pre-operative evaluation is to determine whether potential transplant candidates have any active cardiac condition both during the initial evaluation and immedi‐ ately before an anticipated transplantation procedure. "Active" cardiac conditions are defined as unstable coronary syndromes (eg, unstable angina, severe angina, or recent myocardial infarction (MI), decompensated heart failure, significant arrhythmias, and severe valvular disease). The presence of one or more of these conditions is associated with high rates of perioperative cardiovascular morbidity and mortality, hence delay or cancellation of the

therapy is shown in table 4.

1Only selected renal disease are listed.

al., 2009)

Despite the propensity for certain kidney disease to recur, the risk generally does not preclude transplantation and recurrence rarely results in early graft loss. However, systemic primary amyloidosis (AL amyloidosis) and light chain deposition disease are associated with high rates of disease recurrence and increased morbidity and mortality after transplantation and are considered contraindication to transplantation by most centers. In rare selected patients with sustained complete remission of the hematological disorder kidney transplantation can be performed at the discretion of the transplant nephrologist and hematologist/oncologist (Bridoux et al., 2011; Canaud et al., 2012).

#### **2.2. Cardiovascular disease and peripheral vascular disease**

Cardiovascular disease (CVD) is the leading cause of death after renal transplantation. Deaths with a functioning graft occurring within 30 days after transplantation are due to ischemic heart disease in nearly half of the cases. Cardiovascular screening is considered by most

*FSGS*: focal segmental glomerulosclerosis; *MPGN*: membranoproliferative glomerulonephritis; *GN*: glomerulonephrop‐ athy; *HUS*: hemolytic uremic syndrome; *SLE*: systemic lupus erythematosus.

1Only selected renal disease are listed.

The following section describes specific medical and urological issues that should be addressed

Recurrence of glomerular disease is the third most common cause of graft loss after chronic allograft injury and death with a functioning graft. Currently available data on the incidence of recurrent disease and resultant graft loss are heterogeneous due to different study design, follow-up durations, patient samples, and the variable use of surveillance biopsies among centers. The reported incidence of recurrent renal disease after renal transplantation and the risk of graft loss from disease recurrence are shown in table 3. The clinical course and impact on graft survival vary between different types of glomerulonephritis (Colgert et al., 2008; Kasiske et al., 2009). Nonetheless, with the exception of primary focal segmental glomerulo‐ sclerosis (FSGS), recurrent glomerular disease is usually a late complication after transplan‐ tation. FSGS secondary to reflux nephropathy or obesity does not recur after transplantation. In patients with hypertensive renal disease or other causes of chronic kidney disease, focal segmental sclerosis may be found on histologic evaluation and must be differentiated from the primary disorder. Suggested risk factors for recurrence of primary FSGS include history of recurrence in a previous transplant, younger age at diagnosis, rapid progression to end stage renal disease from the time of initial diagnosis ( < 3 years), presence of mesangial proliferation in the native kidneys, older donor kidneys, Caucasian ethnicity, and the collapsing variant. Living donor kidneys (versus deceased donor) have not consistently been demonstrated to be associated with an increased risk of recurrence. Familial and sporadic forms of FSGS with podocin mutation, slow progression to end stage kidney disease (ESKD), and non-nephrotic range proteinuria in the native kidney disease are associated with low risk of recurrence

Despite the propensity for certain kidney disease to recur, the risk generally does not preclude transplantation and recurrence rarely results in early graft loss. However, systemic primary amyloidosis (AL amyloidosis) and light chain deposition disease are associated with high rates of disease recurrence and increased morbidity and mortality after transplantation and are considered contraindication to transplantation by most centers. In rare selected patients with sustained complete remission of the hematological disorder kidney transplantation can be performed at the discretion of the transplant nephrologist and hematologist/oncologist

Cardiovascular disease (CVD) is the leading cause of death after renal transplantation. Deaths with a functioning graft occurring within 30 days after transplantation are due to ischemic heart disease in nearly half of the cases. Cardiovascular screening is considered by most

**2. Evaluation of risk factors by specific organ system disease**

**2.1. Recurrence of glomerular disease of the native kidneys**

during the transplant evaluation process.

6 Current Issues and Future Direction in Kidney Transplantation

(Ponticelli et al., 2010).

(Bridoux et al., 2011; Canaud et al., 2012).

**2.2. Cardiovascular disease and peripheral vascular disease**

2Diarrhea (+) HUS usually does not recur; Diarrhea (-) or familial may recur in 21-28%; Factor H or I mutation may recur in 80% to 100%; Patients with mutation membrane cofactor protein does not have recurrence (reference Kasiske et al., 2009)

**Table 3.** Rates of recurrent renal disease after transplantation and risk of graft loss from disease recurrence1

transplant centers as an essential component of the transplant evaluation process. A detailed cardiovascular history not only predicts the operative risk but also helps in postoperative cardiac management to improve short- and long-term cardiac outcomes. Over the years there has been much controversy over the best strategy for pre-transplant assessment and manage‐ ment of coronary artery disease (CAD) to prevent adverse peri-operative cardiac events. Recently, the American Heart Association / American College of Cardiology (AHA/ACC) have developed the 2012 AHA/ACC guidelines for "Cardiac Disease Evaluation and Management Among Kidney and Liver Transplantation Candidates" based on a comprehensive review of the literature pertinent to perioperative cardiac evaluation of potential kidney or liver transplant recipients (Lentine et al., 2012). These guidelines are endorsed by the American Society of Transplant Surgeons, American Society of Transplantation, and the National Kidney Foundation (discussed below). The AHA/ACC classifications of evidence to perform a test or therapy is shown in table 4.

#### **a.** Determining whether the transplant candidate has an active cardiac condition

The primary goal of pre-operative evaluation is to determine whether potential transplant candidates have any active cardiac condition both during the initial evaluation and immedi‐ ately before an anticipated transplantation procedure. "Active" cardiac conditions are defined as unstable coronary syndromes (eg, unstable angina, severe angina, or recent myocardial infarction (MI), decompensated heart failure, significant arrhythmias, and severe valvular disease). The presence of one or more of these conditions is associated with high rates of perioperative cardiovascular morbidity and mortality, hence delay or cancellation of the

it should also be noted that in ESKD patients both myocardial perfusion study (MPS) and DSE have reduced sensitivity and specificity compared with that of the general population. In the general population, abnormalities on myocardial perfusion study has been suggested to correlate well with the presence of coronary artery disease (CAD) with mean weighted sensitivity of 88% and specificity of 74% (Klocke et al., 2003). In patients with stage 5 CKD (GFR < 15 ml/min or dialysis dependent) DSE and MPS have been reported to have sensitivities ranging from 44% to 89%, and 29% to 92%, respectively, and specificities ranging from 71% to 94% and 67% to 89%, respectively, for identifying ≥ 1 coronary stenosis > 70% (Lentine et al. 2009, Lentine et al, 2012). Furthermore, abnormal MPS and DSE test results have not been consistently shown to be associated with prognostic value for cardiac events and mortality in ESKD patients. In a meta-analysis of 12 studies involving either thallium-201 scintigraphy or DSE, Rabbat *et al*. demonstrated that ESKD patients with inducible ischemia had 6 times higher risk of MI and 4 times higher risk of cardiac death than patients without inducible ischemia (Rabbat et al., 2003). In contrast, in a small prospective study of 106 kidney transplant candi‐ dates clinically classified as moderate (age ≥50 years) or high (diabetes mellitus, extra-cardiac vascular disease, or known CAD) coronary risk who underwent MPS, DSE, and coronary angiography, De Lima *et al.* found that clinical risk stratification and coronary angiographic findings of CAD (defined as ≥70% stenosis in ≥1 epicardial arteries by visual estimation by 2 observers) predicted major adverse cardiac events (MACEs) [defined as sudden death, myocardial infarction, arrhythmia, heart failure, unstable angina, or revascularization] after a median follow-up of 46 months but results of MPS and DSE did not predict MACEs (De Lima

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Given the wide ranges of sensitivities and specificities of the MPS and DSE and the inconsistent associations of angiographically defined CAD with subsequent survival in ESKD patients, the AHA/ACC Writing Committee acknowledges that there are currently no definitive data to sup‐ port or refute screening for myocardial ischemia among potential kidney transplant candidates without active cardiac conditions. However, it is recommended that until further data are avail‐ able, it may be useful to use aggregate CAD risk factors to target screening of patients with the highest pretest probability of having significant CAD. Suggested algorithm for pretransplant

In general, high cardiac risk candidates should undergo a formal evaluation by cardiology. If necessary, percutaneous coronary intervention or coronary bypass surgery and cardiac rehabilitation should be performed prior to transplantation. If coronary intervention is indicated, caution should be made especially if stenting is planned. The 2012 AHA/ACC guidelines do not recommend transplant surgery within 3 months of bare metal stent (BMS) and within 12 months of DES placement, particularly if the anticipated time of poststent dual antiplatelet therapy will be shortened (Class III; level of Evidence B). Transplant surgery is also not advisable in patients within 4 weeks of coronary revascularization with balloon

In patients with established CVD or in those at risk for CV events, aggressive risk factor modification and treatment *per* ACC/AHA guidelines (Pearson et al., 2002) are recommended. The cardioprotective effects of statins, aspirin, ACE inhibitors, and/or β blockers have been

cardiac evaluation based on the 2012 AHA/ACC guidelines is shown in figure 1.

angioplasty (Clas III; Level of Evidence B) (Lentine et al., 2012).

et al., 2003).

**Table 4.** Evidence Grading

surgical procedure may be required. The 2012 AHA/ACC guidelines recommend that a thorough history and physical examination be performed in all patients preoperatively to identify any active cardiac conditions (Class I; Level of Evidence C). In prospective transplant candidates with chronic cardiac conditions, re-assessment of their cardiac status before surgery may be necessary. The former is defined as chronic limiting angina, an MI that is < 30 days old but without symptoms of unstable angina, prior history of coronary artery bypass graft (CABG) or percutaneous coronary intervention (PCI), decompensated heart failure, moderate valvular disease or prior valve surgey, or stable arrhythmias.

**b.** Noninvasive stress testing in kidney transplant candidates without active cardiac conditions

The AHA/ACC recommend noninvasive stress testing in kidney transplant candidates with no active cardiac conditions based on the presence of multiple CAD risk factors regardless of functional status. Eight relevant risk factors among transplant candidates –as defined in the Lisbon Conference report include: diabetes, prior cardiovascular disease, dialysis duration of greater than 12 months, left ventricular hypertrophy, age > 60 years, smoking, hypertension, and dyslipidemia (Abbud-Filho et al., 2007). Although the exact number of risk factors required to initiate noninvasive stress testing has not been well defined, the AHA/ACC Committee suggests that the presence of 3 or more risk factors should prompt further evaluation with noninvasive stress testing (Class IIb; Level of Evidence C) (Lentine et al., 2012)

Noninvasive stress testing for CAD may be performed with exercise or with a pharmacological agent, and gauged by electrocardiography (EKG) changes (exercise stress test), myocardial perfusion distribution (myocardial perfusion imaging), or left ventricular wall motion (stress echocardiogram). Myocardial perfusion studies (MPS) and dobutamine stress echocardiogram (DSE) are more commonly used due to the frequent abnormalities detected on baseline EKGs in patients with ESKD. In addition, dialysis patients may not be able to achieve an adequate level of exercise during an exercise stress test because of their sedentary lifestyles. However, it should also be noted that in ESKD patients both myocardial perfusion study (MPS) and DSE have reduced sensitivity and specificity compared with that of the general population. In the general population, abnormalities on myocardial perfusion study has been suggested to correlate well with the presence of coronary artery disease (CAD) with mean weighted sensitivity of 88% and specificity of 74% (Klocke et al., 2003). In patients with stage 5 CKD (GFR < 15 ml/min or dialysis dependent) DSE and MPS have been reported to have sensitivities ranging from 44% to 89%, and 29% to 92%, respectively, and specificities ranging from 71% to 94% and 67% to 89%, respectively, for identifying ≥ 1 coronary stenosis > 70% (Lentine et al. 2009, Lentine et al, 2012). Furthermore, abnormal MPS and DSE test results have not been consistently shown to be associated with prognostic value for cardiac events and mortality in ESKD patients. In a meta-analysis of 12 studies involving either thallium-201 scintigraphy or DSE, Rabbat *et al*. demonstrated that ESKD patients with inducible ischemia had 6 times higher risk of MI and 4 times higher risk of cardiac death than patients without inducible ischemia (Rabbat et al., 2003). In contrast, in a small prospective study of 106 kidney transplant candi‐ dates clinically classified as moderate (age ≥50 years) or high (diabetes mellitus, extra-cardiac vascular disease, or known CAD) coronary risk who underwent MPS, DSE, and coronary angiography, De Lima *et al.* found that clinical risk stratification and coronary angiographic findings of CAD (defined as ≥70% stenosis in ≥1 epicardial arteries by visual estimation by 2 observers) predicted major adverse cardiac events (MACEs) [defined as sudden death, myocardial infarction, arrhythmia, heart failure, unstable angina, or revascularization] after a median follow-up of 46 months but results of MPS and DSE did not predict MACEs (De Lima et al., 2003).

surgical procedure may be required. The 2012 AHA/ACC guidelines recommend that a thorough history and physical examination be performed in all patients preoperatively to identify any active cardiac conditions (Class I; Level of Evidence C). In prospective transplant candidates with chronic cardiac conditions, re-assessment of their cardiac status before surgery may be necessary. The former is defined as chronic limiting angina, an MI that is < 30 days old but without symptoms of unstable angina, prior history of coronary artery bypass graft (CABG) or percutaneous coronary intervention (PCI), decompensated heart failure, moderate

I Conditions for which there is evidence for and/or general agreement that the procedure/therapy is useful

II Conditions for which there is conflicting evidence and/or a divergence of opinion about the usefulness/

III Conditions for which there is evidence and/or general agreement that the procedure/therapy is not useful/

B Consistent retrospective cohort, exploratory cohort, ecological, outcome research, or case-control studies, or

**b.** Noninvasive stress testing in kidney transplant candidates without active cardiac

The AHA/ACC recommend noninvasive stress testing in kidney transplant candidates with no active cardiac conditions based on the presence of multiple CAD risk factors regardless of functional status. Eight relevant risk factors among transplant candidates –as defined in the Lisbon Conference report include: diabetes, prior cardiovascular disease, dialysis duration of greater than 12 months, left ventricular hypertrophy, age > 60 years, smoking, hypertension, and dyslipidemia (Abbud-Filho et al., 2007). Although the exact number of risk factors required to initiate noninvasive stress testing has not been well defined, the AHA/ACC Committee suggests that the presence of 3 or more risk factors should prompt further evaluation with

Noninvasive stress testing for CAD may be performed with exercise or with a pharmacological agent, and gauged by electrocardiography (EKG) changes (exercise stress test), myocardial perfusion distribution (myocardial perfusion imaging), or left ventricular wall motion (stress echocardiogram). Myocardial perfusion studies (MPS) and dobutamine stress echocardiogram (DSE) are more commonly used due to the frequent abnormalities detected on baseline EKGs in patients with ESKD. In addition, dialysis patients may not be able to achieve an adequate level of exercise during an exercise stress test because of their sedentary lifestyles. However,

noninvasive stress testing (Class IIb; Level of Evidence C) (Lentine et al., 2012)

valvular disease or prior valve surgey, or stable arrhythmias.

**Evidence Class: Magnitude of procedure/treatment effect**

8 Current Issues and Future Direction in Kidney Transplantation

efficacy of performing the procedure/therapy

effective and in some cases may be harmful

extrapolation from level A studies

IIa Weight of evidence/opinion is in favor of usefulness/efficacy IIb Usefulness/efficacy is less well established by evidence/opinion

C Case-series studies or extrapolations from level B studies

**Evidence Level: Estimate of certainty (precision) of procedure/treatment effect**

A Consistent direction and magnitude of effect from multiple randomized controlled trials

and effective

**Table 4.** Evidence Grading

conditions

Given the wide ranges of sensitivities and specificities of the MPS and DSE and the inconsistent associations of angiographically defined CAD with subsequent survival in ESKD patients, the AHA/ACC Writing Committee acknowledges that there are currently no definitive data to sup‐ port or refute screening for myocardial ischemia among potential kidney transplant candidates without active cardiac conditions. However, it is recommended that until further data are avail‐ able, it may be useful to use aggregate CAD risk factors to target screening of patients with the highest pretest probability of having significant CAD. Suggested algorithm for pretransplant cardiac evaluation based on the 2012 AHA/ACC guidelines is shown in figure 1.

In general, high cardiac risk candidates should undergo a formal evaluation by cardiology. If necessary, percutaneous coronary intervention or coronary bypass surgery and cardiac rehabilitation should be performed prior to transplantation. If coronary intervention is indicated, caution should be made especially if stenting is planned. The 2012 AHA/ACC guidelines do not recommend transplant surgery within 3 months of bare metal stent (BMS) and within 12 months of DES placement, particularly if the anticipated time of poststent dual antiplatelet therapy will be shortened (Class III; level of Evidence B). Transplant surgery is also not advisable in patients within 4 weeks of coronary revascularization with balloon angioplasty (Clas III; Level of Evidence B) (Lentine et al., 2012).

In patients with established CVD or in those at risk for CV events, aggressive risk factor modification and treatment *per* ACC/AHA guidelines (Pearson et al., 2002) are recommended. The cardioprotective effects of statins, aspirin, ACE inhibitors, and/or β blockers have been

Figure 1. Suggested algorithm for pretransplant cardiac evaluation

well-described. Omega-3 fatty acid consumption from fish or fish oil has also been suggested to confer a cardioprotective effect. If feasible, β1 cardioselective agents should be given several weeks prior to a planned living donor renal transplant. This allows time to maximize the efficacy of beta blockers and time to slowly titrate beta blockers, avoiding bradycardia and hypotension. Avoidance of these adverse effects may decrease the risk of stroke and all-cause mortality, leading to a positive net clinical benefit (Deveraeaux et al., 2008, Harte et al. 2008).

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In recent years, cardiac troponin T (cTnT) has been suggested to provide prognostic informa‐ tion in the cardiac evaluation of patients with ESKD. Independent investigators have demon‐ strated an association between increased levels of cardiac toponin T isoforms and all-cause and cardiac death risk in asymptomatic patients with ESKD (Lentine et al., 2009, Khan et al., 2005). In a study consisting of 644 wait-listed renal transplant candidates, Hickson *et al.* demonstrated that increasing cTnT levels were associated with progressively reduced survival independent of low serum albumin and history of stroke. The survival of patients with cTnT levels between 0.01 and 0.03 ng/mL did not differ from that of patients with levels < 0.01 ng/mL. In contrast, cTnT levels between 0.03 and 0.09 ng/mL were associated with significantly increased mortality (hazard ratio, HR=3.01, p=0.040). Notably, mortality was further increased in patients with cTnT levels >0.1 ng/mL (HR=4.085, p=0.009) whereas in patients with normal cTnT, excellent survival was achieved independent of other risk factors (Hickson et al., 2008). The 2012 AHA/ACA guidelines support the use of cTnT level at the time of evaluation for kidney transplantation as an additional prognostic marker (Class IIb; Level of Evidence B) (Lentine et al., 2012). However, the routine use of cTnT as adjunctive tools in cardiac risk

Patients with CKD frequently suffer from nonischemic cardiac abnormalities including left ventricular hypertrophy (LVH), left ventricular dilatation, left ventricular systolic and/or diastolic dysfunction. Renal transplantation has variably been shown to improve left ventric‐ ular dysfunction and ameliorate LVH (Zolty et al., 2008). Hence, the presence of such abnor‐ malities does not necessarily preclude transplantation. Nonetheless, patients with an ejection fraction of 40% are considered moderate to high risk candidates and warrant a formal Cardiology consultation. An ejection fraction below 40% generally precludes transplantation. It is our practice to refer these patients to Cardiomyopathy Center for further diagnostic and therapeutic interventions. The presence of advanced irreversible cardiomyopathy is a contra‐ indication to solitary kidney transplantation and patients should be referred for possible

Patients with a history of transient ischemic attacks or cerebrovascular accidents should under‐ go carotid Doppler studies. Duplex ultrasonography may be considered in asymptomatic pa‐ tients with symptomatic peripheral arterial disease (PAD), CAD, or atherosclerotic aortic

*2.2.1. Biomarkers for cardiac risk assessment*

**2.3. Nonischemic cardiomyopathy**

combined kidney-heart transplantation.

**2.4. Peripheral vascular disease**

assessment in renal transplant candidates remains to be studied.

1Unstable coronary syndromes: unstable angina, severe angina, recent myocardial infarction 1Unstable coronary syndromes: unstable angina, severe angina, recent myocardial infarction 2Myocardial perfusion study or dobutamine stress echocardiogram (center specific).

2Myocardial perfusion study or dobutamine stress echocardiogram (center specific).

**Figure 1.** Suggested algorithm for pretransplant cardiac evaluation

well-described. Omega-3 fatty acid consumption from fish or fish oil has also been suggested to confer a cardioprotective effect. If feasible, β1 cardioselective agents should be given several weeks prior to a planned living donor renal transplant. This allows time to maximize the efficacy of beta blockers and time to slowly titrate beta blockers, avoiding bradycardia and hypotension. Avoidance of these adverse effects may decrease the risk of stroke and all-cause mortality, leading to a positive net clinical benefit (Deveraeaux et al., 2008, Harte et al. 2008).


*Article* **Holy Communion in Greek Orthodoxy in the Time of Coronavirus: Ideological Perspectives in Conflict**

**Efstathios Kessareas**

Department of Religious Studies, Faculty of Philosophy, University of Erfurt, 99089 Erfurt, Germany<EMAIL_ADDRESS>
**Abstract:** This article examines the controversy over the mode of distribution of Holy Communion that surfaced during the COVID-19 pandemic, with a focus on debates that took place in the Greek Orthodox community. After describing and evaluating the role of secular and religious experts in the context of the pandemic, the paper analyzes three main perspectives on the issue of the Eucharist: (1) the secularist-rationalist viewpoint; (2) the religious–traditionalist outlook; and (3) the "Third Way" perspective. The paper argues that the Church's Holy Communion controversy is indicative of a deeper struggle between religious and secular thinkers and among various voices in the Greek Orthodox Church concerning the latter's place in, and influence over, the modern secular socio-political order.

**Keywords:** Greek Orthodoxy; Holy Communion; COVID-19; ideology; tradition; modernity; secularization


*3.2. Chondroitin Sulfate Blends*

While methacrylated CS forms mechanically robust hydrogels that retain the inherent functionality of CS and have been shown to support chondrogenesis, osteogenesis and neurogenesis in vitro and in vivo, they require photopolymerization, meaning, in clinical

applications, these constructs would require specialized tools for delivery and ultra-violet light for polymerization, which could potentially injure surrounding healthy tissue [74]. Tang, et al. approached this issue by developing a hydrogel scaffold comprised of CS functionalized with graphene oxide. Johnson–Claisen rearrangement chemistry allowed graphene oxide (GO) to be functionalization with primary amines. A solution composed of CS and modified GO gelled in situ within 10 min and the incorporation of graphene improved stiffness and toughness drastically (320 and 70%, respectively) over gels made of just CS. They also proved to be highly porous, resistant to degradation, and enabled MSCs to proliferate and deposit collagen matrix [75]. Of note, however, the entrapment of cells and potential chemical remnants from the EDC/NHS chemistry did initially slow cell metabolism [75]. Potentially of value is the conductive nature of GO, which may confer the scaffolds with the ability to induce natural conductive currents to improve tissue regeneration.

New CS crosslinking methods provide new ways to study the interactions of CS with cells in the absence of other extracellular matrix components and, in that regard, are indispensable. However, CS is not the sole constituent of the extracellular matrix in any tissue in the human body, but rather is interlaced with other GAGs and natural polymers. Therefore, combining CS with different polymers presents an opportunity to further control a gel's rheological properties, present additional biological signals, and better mimic native tissue. For example, collagen scaffolds functionalized with CS have been shown to recapitulate the chondrogenic niche, modulate inflammation, and mimic the mechanical properties of native collagen [55]. Corradetti, et al. demonstrated that such constructs support chondrogenic differentiation in rat bone marrow-derived stem cells in vitro and suppressed inflammation in vivo. MSCs grown on CS-collagen constructs aligned with scaffold pores, whereas cells grown on scaffolds containing only collagen showed clustering behavior, demonstrating that the presence of CS in the CS-collagen scaffolds is essential to influence cell-scaffold adhesion and [55], therefore, cytoskeletal organization and differentiation [76,77]. These cells also developed more intracellular vesicles, which have been associated with enhanced intercellular communication [78]. The constructs innately induce chondrogenic differentiation, and even though they didn't support osteogenesis innately, they displayed a synergistic effect with osteogenic media, showing increased expression of osteogenic factors Alp, Spp1, and Bgla compared to controls [55].

CS has also been combined with collagen using genipin as a crosslinker for tissue engineered scaffolds for regeneration of different types of cartilage, such as the nucleus pulposus. Forming a lightly crosslinked, gelatin-like scaffold, type II collagen and CS crosslinked with genipin are biocompatible and support differentiation of adipose-derived stem cells in vitro. When used as an injectable carrier of adipose derived stem cells in a rat model of NP degeneration CS-collagen gels showed increased disc height, water content, proteoglycan and type II collagen synthesis, and partial recovery of NP structure [57].


5.1.1. Stable Expression

The abovementioned approaches of vector delivery into plant cells, such as biolistic and *Agrobacterium*-mediated transformation, are used to deliver the CRISPR/Cas DNA, and by screening the mutants, the DNA is inserted into the target location in the plant genome and carry out the editing phenomena. Currently, there are numerous applications of this system in plant GE. In 2016, Gao and colleagues incorporated a fluorescent gene in the expression cassette of CRISPR/Cas9 [146]. Recently, an interesting strategy was developed in which *BARNASE* and *CMS* were used as suicide genes to kill embryos and pollens containing the transgene of T0 plant progeny [147].