The fundamental, still unanswered question is the origin of HIV (Gallo et al., 1985; Baltimore, 1985; Duesberg, 1987; Temin, 1988, 1989; Varmus, 1989; Sharp et al., 2001; Zhu et al., 1998; Apetrei & Marx, 2005). Is HIV a unique relative of other retroviruses? When looking at retroviruses, the rule applies that practically every species in the animal kingdom has its own retrovirus – mouse, rat, chicken, goat, sheep, cow, horse, monkey, chimpanzee, etc. Some of them are very contiguous, due to their phylogenetic relationship - mouse and rat, monkey and chimpanzee. Similarly, retroviruses of monkeys, chimpanzee and humans are evolutionarily collateral. Yet there is no substantial evidence about transmission of retroviruses between different species in nature. On the basis of these facts and our achieved results, it is possible to conclude - all retroviruses have been an integral part of a particular host organism since the beginning of their existence as species and they are passed on from generation to generation. The hosts – including humans - inherited them from their ancestors.  Gentamicin protection assay. The number of colonies represents the number of intracellular bacteria in normal human lymphocytes. Number of used cells: normal human

To analyze the role of bacteria in immunodeficiency, reduction of their amount in the intestinal tract of 20 AIDS patients was performed by oral administration of probiotic bacteria *Escherichia coli* strain Nissle 1917. After three months of probiotics treatment the viral load decreased or remained on the detection limit (<400 c/ml) in 55.5% of the patients tested. According to these findings, it is assumed that not only one biological component plays the most important role in the AIDS process, but that there are two – HIV and the bacterial host. These bacteria bearing rich extrachromosomal genetic information are able to penetrate from the GIT into the human body and attack strongly the human immune

The fundamental, still unanswered question is the origin of HIV (Gallo et al., 1985; Baltimore, 1985; Duesberg, 1987; Temin, 1988, 1989; Varmus, 1989; Sharp et al., 2001; Zhu et al., 1998; Apetrei & Marx, 2005). Is HIV a unique relative of other retroviruses? When looking at retroviruses, the rule applies that practically every species in the animal kingdom has its own retrovirus – mouse, rat, chicken, goat, sheep, cow, horse, monkey, chimpanzee, etc. Some of them are very contiguous, due to their phylogenetic relationship - mouse and rat, monkey and chimpanzee. Similarly, retroviruses of monkeys, chimpanzee and humans are evolutionarily collateral. Yet there is no substantial evidence about transmission of retroviruses between different species in nature. On the basis of these facts and our achieved results, it is possible to conclude - all retroviruses have been an integral part of a particular host organism since the beginning of their existence as species and they are passed on from generation to generation. The hosts – including humans - inherited them from their ancestors. In accordance with this idea, it is assumed that transfer of the HIV from apes to humans in Africa, as a consequence of their accidental contacts, is not the cause of AIDS

negative controls (healthy subjects) <5

P15/7 P3/3 725/5 M22/5 P1/4 K1/1 M1/6 M12/5

lymphocytes 1,5x107; bacteria 1x108.

**3. Etiology and possible evolution of disease**

system.

pandemics.

But there is a very serious objection – if HIV were in our bacteria from our beginning, why did they emerge only about 25-35 years ago? Were they hidden without any implication all the time, for many thousands of years, in our bodies? To answer this question, we should go back into the ancient history of humankind. In the past, major epidemics frequently occurred when there were new patterns of communication and transportation between separately populated areas and/or new patterns of settlement. The first recorded great plague occurred in Athens during the Peloponnesian war in 430 BC. In the middle of the sixth century AD (542), there was a great outbreak of bubonic plague (Justinianus) in the Mediterranean countries, where 40 to 50% of the population died. The biggest cataclysm of bubonic plague epidemic started in 1346 in Europe and the population was finally decimated by 50 to 60%. Plague subsequently spread to Asia and at least 60 million people died just only in China alone. This plague continued up to the seventeenth and partially to the eighteenth century. In the sixteenth century, after Europeans reached America, new devastating epidemics started among the native peoples resulting in a 90% population loss among American Indians within 120 years.

Who were in particular the victims of these plagues? Mostly people who were malnourished, sick, with a debilitated immune system. As discussed above, bacteria carrying the rich extra-chromosomal genetic information, as plasmids, phages, transposomes, viruses and virus-like particles (HIV included), are mostly pathogenic for the host organism and continually attack the immune system after penetration. This attack depends on the amount of the microbes which penetrate into the blood. Thus the immune system of people with low amounts of these bacteria was stronger and their carriers had a chance to overwhelm any plague. One of the most important results of the set of plagues that occurred in the history of humankind, mainly in Europe, North Africa, North America and partially in Asia, was natural selection, resulting in sanitation of the human population. Hand in hand in this tremendous evolution process was the selection of people with a low level of bacteria carrying pathogenic, mainly extra-chromosomal genetic information, able to penetrate into the human body and attack the immune system. The outcome of this complex evolutional proceeding, which finished towards the end of the nineteenth century, was a great reduction of pathogenic bacteria in the human population and eventually the establishment of a balance between non-pathogenic and pathogenic ("good" and "bad") microbes in the microflora of surviving individuals. The effect of this process was limitation of bacteria bearing HIV sequences and the selection of people carrying CCR-5 receptor deficiency on white blood cells.

A consequence of many plagues in the history of mankind was the establishment of a balance between prokaryotic and eukaryotic kingdoms in our body, which led to a positive selection of people with a low amount of viral and retroviral genetic information in their intestinal bacteria. This tremendous process, whose victims were many millions of humans, was very intensive particularly in Europe. The stability, acquired over many centuries, between eukaryotic and prokaryotic kingdoms was interrupted in the middle of the twentieth century due to the use of antibiotics, narcotic drugs and changes in life style (homo and anal sex).

 It is generally accepted that due to ATB practically all primary bacterial pathogens were liquidated throughout two-three decades. The success of antibiotics in elimination of many diseases induced by microbes was such expressive that in the mid-1950s US Surgeon General Steward effusively proclaimed that the era of bacterial diseases finished. This trend

The Role of Bacteria and Yeasts in AIDS 387

Hypothetically, bacteria are probably hosts not only for HIV but also for other retroviruses. The presence of HIV sequences in commensurate bacteria of the patients may be explained as follows: 1) intestinal bacteria were infected by HIV or virus-like particles (VLP) previously produced by human cells, in particular by macrophages and lymphocytes; 2) intestinal bacteria of AIDS patients are a natural host of HIV sequences in the form of a virus

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will have to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4+ Tcell depletion (Bealshe et al., 1994; Bartlett et al., 2003). While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific

Recent studies suggest that induction of primary immune responses by recombinant bacterial vectors leads to gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses. Genetically modified bacteria have been experimentally used as recombinant probiotics also in gastrointestinal disorders, but clinical usage is currently not the case. Nevertheless, in the future a greater use of this kind of approach is expected. The expression of HIV-1 antigens in specific probiotic strains may be

We would like to thank Danica Stanekova and all the people working at HIV/AIDS Reference Centre, Slovak Medical University, Bratislava, to Milos Mokras from Department of Infectious Diseases and Geographic Medicine, Derer's Hospital, Bratislava and to

This work was supported by the grant VEGA 2/5025/27, 2/0081/08 and the grant APVV-

Apetrei, C., and Marx, P.A. (2005). African lentiviruses related to HIV. *J. Neurovirol*. 11: 33-

Baltimore, D. (1985). Retroviruses and retrotransposos: The role of reverse transcription in

Bartlett, J.G., Carlos, del Rio, DeMaria, A., Sepkowitz K.A. (2003). Smallpox Vaccination and the HIV-Infected Patient: A Roundtable. *AIDS Clinical Care*, 15: 61-63. Belshe, R.B., Bolognesi, D.P., Clements, M.L., Corey, L., Dolin, R., Mestecky, J., Mulligan, M.,

Beachey, E.H. (1981). Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surface. *J. Infect. Dis.*, 143: 325-345. Benjamin, P., Federman, M., and Wanke, Ch.A. (1995). Characterization of an invasive

Stablein, D., Wright, P. (1994). HIV infection in vaccinated volunteers*. JAMA*, 10:

phenotype associated with enteroaggregative *Escherichia coli. Infection and*

Vladimir Krcmery from St. Elizabeth University, Bratislava for patient's samples.

shaping the eukaryotic genome. *Cell* 40: 481-482,

immunogens has proven challenging (Wang et al., 2009).

suitable for clinical use as a potential vaccine.

**5. Acknowledgement**

0404-07.

**6. References**

49.

431.

*Immunity*, 63: 3417-342.

**4. Conclusion**

or VLP.

of ATB glorification was spread very quickly from USA to Europe and microbiology got to the scientific periphery. In 1969, only a few people heard the voice of the English scientist J. A. Reaburn who said that in the future years it would be shown that antibiotics could bring us unforeseen vicious tricks.

In this way resistant, potentially pathogenic microbes, bearing rich extra-chromosomal genetic information as plasmids, phages, transposomes, virus-like particles (VLP) or viruses (including HIV), went from minority to majority and attacked the human immune system. Microbes employed their ability to change their shape – cell wall deficient (CWD) to survive in these hard conditions or were internalized into host cells (Swidsinski et al., 1998, 2002; Martin et al., 2004; Beachey, 1981; Benjamin et al., Cohen & Laux, 1995). Some of these microbes are able to overcome insurmountable barriers existing between microbial DNA and DNA of more highly organized beings. The result of this process are health troubles and one of them, induced by HIV bacteria with strong affinity to T lymphocytes, is immunodeficiency.

This proceeding started between Afro-American drug users because they had not been going through plague selection and sanitation, as had other American inhabitants. Disease spread mainly in big American cities as Los Angeles, San Fransisco, New York, Chicago, and others. Microbes which might be harmless in a healthy body became deadly in conjunction with drugs. This hypothesis was put forward in an editorial of the New England Journal of Medicine by David Durack: Some new factors may have distorted the host-parasite relation. So-called "recreational" drugs are one possibility. They were widely used in the large cities where the most of these cases have occurred, and the only patients in the series reported with this issue who were not homosexual were drug users. Perhaps one of these recreational drugs is an immunosuppressive agent (Durack and Phil, 1981). The leading candidates were nitrites, which were commonly inhaled in the years 1960 to 1977 to intensify orgasm. Butyl nitrite very probably plays an important role in induction of Kaposi's sarcoma in AIDS (Mirwish & Hawerkos, 1987). Mice exposed to isobutyl nitrite showed dose-related immune system damage and became highly susceptible to disease and death caused by mycobacterial infection (Neefe et al., 1983).

The "classical" drugs have a similar but more intensive effect on the immune system and micro-flora of drug users. The elimination of "good" intestinal bacteria by continually used drugs opened the space for amplification of microbes, which had existed before in the minority. The result of this process is that the immune system is not able to react to the massive attack and many diseases are induced, as: pneumonia, tuberculosis, toxoplasmosis, Cryptococcus, hepatitis B, herpes simplex, cytomegalovirus, bacterial, yeast and fungal infections, lymph gland infections and immunodeficiency. Amplified pathogenic microbes are equipped with antibiotic resistance and some of them are bearing viral and retroviral genetic information. Many of these infections are chronic or recurring, often unrecognized at first, and difficult to treat. Consequent antibiotic treatment forwards the spiral process of natural micro-flora destruction. And finally, ingestion of antiretroviral drugs as AZT completed this distortion. The result of such continual, step by step attack of intestinal microbes is their unification, reduction of their heterogeneity and loss of their basic function for the host. In the intestinal tract of two American AIDS patients in the final phase we found only one type of bacteria bearing HIV-1 sequences – multi-resistant *Escherichia coli* with plasmids of about 50 kbp.

### **4. Conclusion**

386 Microbes, Viruses and Parasites in AIDS Process

of ATB glorification was spread very quickly from USA to Europe and microbiology got to the scientific periphery. In 1969, only a few people heard the voice of the English scientist J. A. Reaburn who said that in the future years it would be shown that antibiotics could

In this way resistant, potentially pathogenic microbes, bearing rich extra-chromosomal genetic information as plasmids, phages, transposomes, virus-like particles (VLP) or viruses (including HIV), went from minority to majority and attacked the human immune system. Microbes employed their ability to change their shape – cell wall deficient (CWD) to survive in these hard conditions or were internalized into host cells (Swidsinski et al., 1998, 2002; Martin et al., 2004; Beachey, 1981; Benjamin et al., Cohen & Laux, 1995). Some of these microbes are able to overcome insurmountable barriers existing between microbial DNA and DNA of more highly organized beings. The result of this process are health troubles and one of them, induced by HIV bacteria with strong affinity to T lymphocytes, is

This proceeding started between Afro-American drug users because they had not been going through plague selection and sanitation, as had other American inhabitants. Disease spread mainly in big American cities as Los Angeles, San Fransisco, New York, Chicago, and others. Microbes which might be harmless in a healthy body became deadly in conjunction with drugs. This hypothesis was put forward in an editorial of the New England Journal of Medicine by David Durack: Some new factors may have distorted the host-parasite relation. So-called "recreational" drugs are one possibility. They were widely used in the large cities where the most of these cases have occurred, and the only patients in the series reported with this issue who were not homosexual were drug users. Perhaps one of these recreational drugs is an immunosuppressive agent (Durack and Phil, 1981). The leading candidates were nitrites, which were commonly inhaled in the years 1960 to 1977 to intensify orgasm. Butyl nitrite very probably plays an important role in induction of Kaposi's sarcoma in AIDS (Mirwish & Hawerkos, 1987). Mice exposed to isobutyl nitrite showed dose-related immune system damage and became highly susceptible to disease and

The "classical" drugs have a similar but more intensive effect on the immune system and micro-flora of drug users. The elimination of "good" intestinal bacteria by continually used drugs opened the space for amplification of microbes, which had existed before in the minority. The result of this process is that the immune system is not able to react to the massive attack and many diseases are induced, as: pneumonia, tuberculosis, toxoplasmosis, Cryptococcus, hepatitis B, herpes simplex, cytomegalovirus, bacterial, yeast and fungal infections, lymph gland infections and immunodeficiency. Amplified pathogenic microbes are equipped with antibiotic resistance and some of them are bearing viral and retroviral genetic information. Many of these infections are chronic or recurring, often unrecognized at first, and difficult to treat. Consequent antibiotic treatment forwards the spiral process of natural micro-flora destruction. And finally, ingestion of antiretroviral drugs as AZT completed this distortion. The result of such continual, step by step attack of intestinal microbes is their unification, reduction of their heterogeneity and loss of their basic function for the host. In the intestinal tract of two American AIDS patients in the final phase we found only one type of bacteria bearing HIV-1 sequences – multi-resistant *Escherichia coli*

death caused by mycobacterial infection (Neefe et al., 1983).

with plasmids of about 50 kbp.

bring us unforeseen vicious tricks.

immunodeficiency.

Hypothetically, bacteria are probably hosts not only for HIV but also for other retroviruses. The presence of HIV sequences in commensurate bacteria of the patients may be explained as follows: 1) intestinal bacteria were infected by HIV or virus-like particles (VLP) previously produced by human cells, in particular by macrophages and lymphocytes; 2) intestinal bacteria of AIDS patients are a natural host of HIV sequences in the form of a virus or VLP.

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will have to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4+ Tcell depletion (Bealshe et al., 1994; Bartlett et al., 2003). While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging (Wang et al., 2009).

Recent studies suggest that induction of primary immune responses by recombinant bacterial vectors leads to gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses. Genetically modified bacteria have been experimentally used as recombinant probiotics also in gastrointestinal disorders, but clinical usage is currently not the case. Nevertheless, in the future a greater use of this kind of approach is expected. The expression of HIV-1 antigens in specific probiotic strains may be suitable for clinical use as a potential vaccine.

#### **5. Acknowledgement**

We would like to thank Danica Stanekova and all the people working at HIV/AIDS Reference Centre, Slovak Medical University, Bratislava, to Milos Mokras from Department of Infectious Diseases and Geographic Medicine, Derer's Hospital, Bratislava and to Vladimir Krcmery from St. Elizabeth University, Bratislava for patient's samples.

This work was supported by the grant VEGA 2/5025/27, 2/0081/08 and the grant APVV-0404-07.

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### *Edited by Vladimír Zajac*

The main goal in compiling this book was to highlight the situation in Africa in terms of AIDS and opportunistic diseases. Several chapters reveal great poverty, an apocalyptic situation in many parts of Africa. Global migration of people resulted in their exposure to pathogens from all over the world. This fact has to be acknowledged and accepted as African reality. New, unconventional hypotheses, not determined by established dogmas, have been incorporated into the book, although they have not yet been sufficiently validated experimentally. It still applies that any dogma in any area of science, and medicine in particular, has and always will hinder progress. According to some biologists, in the future, AIDS is very likely to occur in a number of variations, as a direct result of the ongoing processes in the global human society. Thus, we urgently need a comprehensive solution for AIDS, in order to be ready to fight other, much more dangerous intruders.

Microbes, Viruses and Parasites in AIDS Process

Microbes, Viruses and

Parasites in AIDS Process

*Edited by Vladimír Zajac*

Photo by xrender / iStock


**Author details**

160mg once daily on a 3 weeks on/1 week off schedule. The primary endpoint was overall response rate. Preliminary efficacy data of the 33 patients evaluable for response show a 27%

Currently, we have the fortunate problem of having several agents demonstrating effica‐ cy in the first- and second-line setting, with a number of other small molecule inhibitors that target VEGFR tyrosine kinase being evaluated in mRCC consistently showing activi‐ ty. With similar mechanisms of action, clinical responses have been observed, including in patients that have previously received TKI therapy. Part of the challenge in moving forward is the lack of understanding of the biologic underpinnings of resistance to the currently approved agents and uniform clinical definitions of what truly constitutes treat‐

Studies combining targeted therapies are being performed with the known caveat that combinations are associated with high financial cost and risk of increased toxicity due to additive and overlapping side effect profiles. Rational combinations of active agents continue to be evaluated. Currently, combinations of targeted therapy remain experimental and they

Targeted agents are also being studied in the adjuvant setting for patients with resected highrisk RCC. The Adjuvant Sorafenib or Sunitinib for Unfavorable Renal Carcinoma (ASSURE) intergroup trial randomizes high-risk nephrectomized patients to 1 year of sor‐ afenib, sunitinib or placebo (estimated enrolment: 1332, primary endpoint: disease-free survival (DFS)) (NCT00326898). Other trials such as the phase III sunitinib versus placebo study for the treatment of patients at high risk of recurrent RCC (S-TRAC: estimated en‐ rolment 236, primary endpoint: DFS) (NCT00375674) and the sorafenib versus placebo tri‐ al in patients with resected intermediate or high-risk RCC (SORCE: estimated enrolment 1656, primary endpoint: DFS) (NCT00492258) will further help elucidate the effect of

One of the next directions in the therapy of advanced RCC involves the combination of several targeted agents to better inhibit a single pathway at several different levels or inhibition at the same level of several pathways mediating different effects. As combinations of targeted agents undergo investigation, it will be critical for these combinations to demonstrate clinical benefit above and beyond those of sequential monotherapy with the same agents, in order to justify the added toxicity and risk. Thus, prospective data in this regard are critical, and some data

partial response (PR) and a 42% stable disease (SD) rate [27].

should only be employed in the context of a clinical trial.

these agents in the adjuvant setting.

**13. Combination therapy**

have recently emerged.

**12. Sequence of targeted therapy**

ment resistance.

152 Renal Tumor

V. Michalaki, M. Balafouta, D. Voros and C. Gennatas

Oncology Unit Areteion Hospital Univesity of Athens, Greece

## **References**

[1] Campbell, S. C, & Lane, B. R. (2012). Malignant renal tumors. In A. Wein, L. Kavoussi, A. Novick, A. Partin, & C. Peters (Eds.), Campbell-Walsh urology (10th ed., Philadel‐ phia: Elsevier Saunders., 1413-1474.

[14] Motzer, R. J, Hutson, T. E, Tomczak, P, Michaelson, M. D, Bukowski, R. M, Rixe, O, et al. (2007b). Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J

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[15] Motzer, R. J, Michaelson, M. D, Rosenberg, J, Bukowski, R. M, Curti, B. D, George, D. J, et al. (2007a). Sunitinib efficacy against advanced renal cell carcinoma. J Urol , 178,

[16] Witteles, R. M, Telli, M. L, Fisher, G. A, & Srinivas, S. (2008). Cardiotoxicity associated

[17] Heng, D. Y, Chi, K. N, Murray, N, Jin, T, Garcia, J. A, Bukowski, R. M, et al. (2009). A population-based study evaluating the impact of sunitinib on overall survival in the

[18] Ahmad, T, & Eisen, T. (2004). Kinase inhibition with BAY in renal cell carcinoma. Clin

[19] Escudier, B, Eisen, T, Stadler, W. M, Szczylik, C, Oudard, S, Siebels, M, et al. (2007a). Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med , 356, 125-134.

[20] Escudier, B, Szczylik, C, Hutson, T. E, Demkow, T, Staehler, M, Rolland, F, et al. (2009a). Randomized phase II trial of first-line treatment with sorafenib versus interferon Alfa-2a in patients with metastatic renal cell carcinoma. J Clin Oncol , 27, 1280-1289.

[21] Atkins, M. B, Hidalgo, M, Stadler, W. M, Logan, T. F, Dutcher, J. P, Hudes, G. R, et al. (2004). Randomized phase II study of multiple dose levels of CCI-779, a novel mam‐ malian target of rapamycin kinase inhibitor, in patients with advanced refractory renal

[22] Hudes, G, Carducci, M, Tomczak, P, Dutcher, J, Figlin, R, Kapoor, A, et al. (2007). Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med ,

[23] Kay, A, Motzer, R, Figlin, R, Escudier, B, Oudard, S, Porta, C, et al. (2009). Updated data from a phase III randomized trial of everolimus (RAD001) versus PBO in meta‐ static renal cell carcinoma (mRCC). Genitourinary Cancers Symposium, February

[24] Rini, B. I, Wilding, G, Hudes, G, Stadler, W. M, Kim, S, Tarazi, J. C, et al. (2007). Axitinib (AG-013736; AG) in patients (pts) with metastatic renal cell cancer (RCC) refractory to

[25] Rixe, O, Bukowski, R. M, Michaelson, M. D, Wilding, G, Hudes, G. R, Bolte, O, et al. (2007). Axitinib treatment in patients with cytokine-refractory metastatic renal-cell

[26] Sternberg, C. N, Szczylik, C, Lee, E, Salman, P. V, Mardiak, J, Davis, I. D, et al. (2009). A randomized, double-blind phase III study of pazopanib in treatment-naive and

treatment of patients with metastatic renal cell cancer. Cancer , 115, 776-783.

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[1] Campbell, S. C, & Lane, B. R. (2012). Malignant renal tumors. In A. Wein, L. Kavoussi, A. Novick, A. Partin, & C. Peters (Eds.), Campbell-Walsh urology (10th ed., Philadel‐

[2] Barrisford, G. W, Singer, E. A, Rosner, I. L, Linehan, W. M, & Bratslavsky, G. Familial renal cancer: molecular genetics and surgical management.Int J Surg Oncol. (2011).

[3] Rini, B. I, & Small, E. J. (2005). Biology and clinical development of vascular endothelial growth factortargeted therapy in renal cell carcinoma. J Clin Oncol , 23, 1028-1043.

[4] Altomare, D. A, & Testa, J. R. (2005). Perturbations of the AKT signaling pathway in

[5] Hudson, C. C, Liu, M, Chiang, G. G, Otterness, D. M, Loomis, D. C, Kaper, F, et al. (2002). Regulation of hypoxia-inducible factor 1alpha expression and function by the

[6] Shen, C. Kaelin WG Jr. The VHL/HIF axis in clear cell renal carcinoma. Semin Cancer

[7] Medici, D, & Olsen, B. R. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-Dependent Expression of VEGF. PLoS One. (2012). e42913.., 1.

[8] Pal, S. K, Williams, S, Josephson, D. Y, Carmichael, C, Vogelzang, N. J, & Quinn, D. I. Novel therapies for metastatic renal cell carcinoma: efforts to expand beyond the VEGF/

[9] Hilmi, C, Guyot, M, & Pagès, G. VEGF spliced variants: possible role of anti-angiogen‐

[10] Bukowski, R. M, Kabbinavar, F. F, Figlin, R. A, Flaherty, K, Srinivas, S, Vaishampayan, U, et al. (2007). Randomized phase II study of erlotinib combined with bevacizumab compared with bevacizumab alone in metastatic renal cell cancer. J Clin Oncol , 25,

[11] Choueiri, T. K, Plantade, A, Elson, P, Negrier, S, Ravaud, A, Oudard, S, et al. (2008). Efficacy of sunitinib and sorafenib in metastatic papillary and chromophobe renal cell

[12] Christensen, J. G. (2007). A preclinical review of sunitinib, a multitargeted receptor tyrosine kinase inhibitor with anti-angiogenic and antitumour activities. Ann Oncol

[13] Mendel, D. B, Laird, A. D, Xin, X, Louie, S. G, Christensen, J. G, Li, G, et al. (2003). In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res , 9, 327-337.

mTOR signaling paradigm. Mol Cancer Ther. (2012). Mar;, 11(3), 526-37.

mammalian target of rapamycin. Mol Cell Biol , 22, 7004-7014.

cytokine-pretreated patients with advanced renal cell carcinoma (RCC). J Clin Oncol 27: 5021.

**Chapter 10**

**New Systemic Treatment Approaches for Metastatic**

Kidney cancer comprises 2-3% of all cancers according to Cancer Research UK statistics. (http:// info.cancerresearchuk.org). Renal Cell Carcinomas (RCC) is the most common subtype (around 90%) with clear-cell variant constituting up to 75% of all RCCs. Non clear-cell variant are less common and consist of papillary (Type I and II; 10-15%), chromophobe (4%) and collecting duct (including the rare medullary variant; <1%). [1, 2] At initial diagnosis, one third of patients have evidence of distant metastases, and amongst patients who undergo curative nephrectomy, a third will have a recurrence within 5 years. Historically, treatment options have been limited in metastatic RCC, as cytotoxic chemotherapy is not effective in this disease and immunotherapy is of modest benefit. [1] The treatment outlook for metastatic RCC has changed in the past decade, with the introduction of new therapeutic agents which target

The discovery of von Hippel–Lindau (VHL) / hypoxia-inducible factor (HIF) oxygen-sensing pathway and its role in the pathogenesis of RCC (clear-cell as well as some of the non-clearcell variant), has led to a new approach in the systemic therapy for RCC. [1, 3] Tumour suppressor gene VHL encodes the VHL protein (pVHL), which interacts with hypoxiainducible factor (HIF) to regulate cellular response to oxygen deprivation. HIF is a gene

> © 2013 Tan et al.; 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,

© 2013 Tan et al.; 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.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Renal Cell Carcinoma**

Michail Charakidis

**1. Introduction**

http://dx.doi.org/10.5772/55280

Thean Hsiang Tan, Sina Vatandoust and

Additional information is available at the end of the chapter

molecular pathways involved in tumour angiogenesis.

**2. Molecular pathogenesis**

**2.1. Clear- cell variants renal cell carcinoma**

[27] Eisen, T, Joensuu, H, Nathan, P, Harper, P, Wojtukiewicz, M, Nicholson, S, et al. (2009). Phase II study of BAY a multikinase inhibitor, in previously untreated patients with metastatic or unresectable renal cell cancer. J Clin Oncol 27: 5033., 73-4506.


**Impact of the Regional Network for AMI in the Management of STEMI on Care Processes, Outcomes and Health Inequities in the Veneto Region, Italy**

**Mario Saia 1, Domenico Mantoan 2, Marco Fonzo 3, Chiara Bertoncello 4,\*, Marta Soattin 3, Milena Sperotto 4, Tatjana Baldovin 4, Patrizia Furlan 4, Maria Luisa Scapellato 5, Guido Viel 6, Vincenzo Baldo 4, Silvia Cocchio 4 and Alessandra Buja 4**

Received: 13 August 2018; Accepted: 9 September 2018; Published: 11 September 2018

**Abstract:** Cardiovascular diseases are a leading cause of death in Europe. Outcomes in terms of mortality and health equity in the management of patients with ST-Elevation Myocardial Infarction (STEMI) are influenced by health care service organization. The main aim of the present study was to examine the impact of the new organizational model of the Veneto Region's network for Acute Myocardial Infarction (AMI) to facilitate primary percutaneous coronary intervention (PCI) on STEMI, and its efficacy in reducing health inequities. A retrospective cohort study was conducted on HDRs in the Veneto Region for the period 2007–2016, analyzing 65,261 hospitalizations for AMI. The proportion of patients with STEMI treated with PCI within 24 h increased significantly for men and women, and was statistically much higher for patients over 75 years of age (APC, 75–84: 9.8; >85: 12.5) than for younger patients (APC, <45: 3.3; 45–64: 4.9), with no difference relating to citizenship. The reduction in in-hospital, STEMI-related mortality was only statistically significant for patients aged 75–84 (APC: −3.0 [−4.5;−1.6]), and for Italians (APC: −1.9 [−3.2;−0.6]). Multivariate analyses confirmed a reduction in the disparities between socio-demographic categories. Although the new network improved the care process and reduced health care disparities in all subgroups, these efforts did not result in the expected survival benefit in all patient subgroups.

**Keywords:** health care research; quality assurance; hospital management; health inequities


Abstract

In this chapter, we study the properties of specific one dimensional photonic quasicrystal (PQCs), in order to design an output multichannel filter. We calculate the transmittance spectrum which exhibits a photonic band gap (PBG), based on the Transfer Matrix Method (TMM) and the two-fluid model. We show that the generalized Thue-Morse (GTM) and generalized Fibonacci GF(m, n) distributions provide a stacking of similar output multichannel with zero transmission when the input was a sharp resonance of peaks at given n ¼ 2pm where p, is a positive integer. Also, we consider GTM configuration and we apply a deformation y ¼ xhþ1 along the PQC filter, which enhanced the band width of each channel with respect to the number of peaks inside the main transmittance. Here, the coefficient h represents the deformation degree, x and y are thicknesses of the layers before and after the deformation, respectively. This improves the characteristics of PBG.

Keywords: hybrid quasiperiodic PC, superconducting materials, GTM sequence, GF sequence, multichannel optical filters, deformed 1D photonic quasicrystals


*Article* **CHIR99021 Maintenance of the Cell Stemness by Regulating Cellular Iron Metabolism**

**Yingying Han 1, Yong He 2, Xiaofang Jin 1, Jiayi Xie 3, Peng Yu 1, Guofen Gao 1, Shiyang Chang 2,\*, Jianhua Zhang 1,\* and Yan-Zhong Chang 1,\***

**Abstract:** CHIR99021 is an aminopyrimidine derivative, which can efficiently inhibit the activity of glycogen synthesis kinase 3α (GSK-3α) and GSK-3β. As an essential component of stem cell culture medium, it plays an important role in maintaining cell stemness. However, the mechanism of its role is not fully understood. In the present study, we first found that removal of CHIR99021 from embryonic stem cell culture medium reduced iron storage in mouse embryonic stem cells (mESCs). CHIR99021 treated Neuro-2a cells led to an upregulation of ferritin expression and an increase in intracellular iron levels, along with GSK3β inhibition and Wnt/GSK-3β/β-catenin pathway activation. In addition, iron treatment activated the classical Wnt pathway by affecting the expression of β-catenin in the Neuro-2a cells. Our data link the role of iron in the maintenance of cell stemness via the Wnt/GSK-3β/β-catenin signaling pathway, and identify intermediate molecules, including Steap1, Bola2, and Kdm6bos, which may mediate the upregulation of ferritin expression by CHIR99021. These findings reveal novel mechanisms of the maintenance of cell stemness and differentiation and provide a theoretical basis for the development of new strategies in stem cell treatment in disease.

**Keywords:** CHIR99021; glycogen synthesis kinase 3; classical Wnt signaling pathway; ferritin; Neuro-2a; cell stemness

## **1. Introduction**

Embryonic stem cells (ESCs), derived from early embryos or primary gonads, have the abilities of self-renewal and pluripotency [1,2]. Oct4, Sox2, and Nanog jointly regulate the pluripotency of ESCs [3]. The Wnt signaling pathway is a very important pathway to affect the stem cell behavior, which contains a complex protein interaction network [4]. When the Wnt signal is absent, the scaffold proteins, Axin, adenomatous polyposis coli (APC), GSK-3β, and casein kinase 1α (CK1α), form a protein destruction complex, which induces β-catenin phosphorylation and then degradation by the proteasome. Conversely, the Wnt ligands bind to the Frizzled receptor and recruit the Dishevelled (DVL) protein, resulting in the dissociation of the destruction complex. Subsequently, β-catenin binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate the target genes [5]. The non-canonical Wnt pathway does not require β-catenin. This alternate signaling cascade activates multiple intracellular targets. The most extensively studied are the Wnt/planar cell polarity (PCP) and Wnt/calcium (Ca2+) pathways [6–8].

The canonical Wnt/β-catenin signaling pathway plays a key role in embryonic development and stem cell stemness. The activation of this pathway has been shown to increase the expression of pluripotent genes in mESCs. Besides, the activation of the

**Citation:** Han, Y.; He, Y.; Jin, X.; Xie, J.; Yu, P.; Gao, G.; Chang, S.; Zhang, J.; Chang, Y.-Z. CHIR99021 Maintenance of the Cell Stemness by Regulating Cellular Iron Metabolism. *Antioxidants* **2023**, *12*, 377. https:// doi.org/10.3390/antiox12020377

Academic Editor: Dimitrios Galaris

Received: 8 December 2022 Revised: 19 January 2023 Accepted: 1 February 2023 Published: 4 February 2023

**Copyright:** © 2023 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/).

pathway can significantly up-regulate the expression of prostate tumor stem cell markers B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), Aldehyde dehydrogenase 1A1 (ALDH1A1), Cluster of differentiation-44 (CD44), aldehyde dehydrogenase 1 family, member A3 (ALDH1A3), and SOX2 mRNA [9]. Additionally, studies in colorectal cancer have found that transmembrane 4L6 family member 1 (TM4SF1) can maintain cancer cell stemness and epithelial-mesenchymal transformation (EMT) through the Wnt/β-catenin/c-Myc/SOX2 pathway [10].

Iron is an element essential to normal physiological functions and metabolic activities [11]. Most of the iron is liganded within functional proteins, including hemoproteins, iron-sulfur (Fe/S) proteins, and enzymes which contain non-heme, non-Fe/S iron. In addition, there is a fraction of uncommitted iron, called the labile iron pool (LIP). These different forms of iron can participate in numerous of important biological processes, such as oxygen transport, electron transport, gene expression, cell proliferation, and cell differentiation [12]. Both iron deficiency and excess can have harmful effects. Therefore, it is essential to maintain iron homeostasis to support the normal physiological activities, while protecting against the toxicity of the catalytic metal. Iron levels are tightly controlled by some proteins that regulate the absorption, storage, circulation, and utilization of iron [12,13]. Cellular iron uptake is mainly mediated by the plasma iron transport protein, transferrin (Tf), and its receptor, transferrin receptor 1 (TfR1). Tf-bound iron enters to the LIP, with some of this iron binding to cytosolic poly (rC) binding proteins PCBP1 and PCBP2, which can transport the metal to the ubiquitous iron storage protein, ferritin, or other non-heme iron-proteins. Excess iron can be transported out of cells by ferroportin 1 (FPN1) [14]. Cellular iron homeostasis is regulated by a post-transcriptional regulation mechanism mediated by iron regulatory proteins (IRPs). These two RNA-binding proteins specifically bind to the conserved motif, iron response element (IRE), in the untranslated regions (UTRs) of mRNAs encoding iron metabolism-related proteins [15].

The fate of stem cells is regulated by many factors [16], among which the endogenous factors mainly include the expression of genes, while the exogenous factors mainly include differentiation and inhibition between cells and the effects of external substances. As an exogenous factor, iron also affect the stemness and differentiation of stem cells. Metal ions, such as Mn, Co, Al and Fe, can regulate cell attachment and affect neuronal differentiation [17,18]. Besides, it was found that the neural differentiation of mESCs decreased upon addition of different concentrations of iron oxide nanoparticles to retinoic acid-induced mESCs [19]. In human dental pulp stem cells, the iron chelator, deferoxamine (DFO), can promote the expression of proteinaceous factors related to the stem cell characteristics [20–22]. Besides, at both cellular level and in clinical treatment, iron also play vital roles in the normal function of mesenchymal stem cells [23].

Both iron and the Wnt/GSK-3β/β-catenin pathway play a role in the maintenance of cell stemness. In fact, the iron and GSK-3β pathways may intersect. It has been found that Deferasirox (DFX) can trigger the proteolysis of cyclin D1 in mantle cell lymphoma (MCL), which requires the participation of GSK-3β [24]. In a human neuroblastoma cell line (SH-SY5Y), FeSO4 can activate GSK-3β, and down-regulate the phosphorylation of GSK-3β [25]. In addition, in APP/PS1 transgenic mice, intranasal administration of DFO can abrogate the activity of GSK-3β, thus inhibiting tau protein phosphorylation [26]. In Parkinson's disease dementia (PDD), the neurotoxicity resulted by iron can promote the activation of GSK-3β-related pathways, thus playing an important role in the pathological synergism of α-syn, tau and Aβ [27]. These studies suggest that there may be a specific relationship between iron and GSK-3β, however it is unclear how GSK3 may affect iron homeostasis. In this study, we investigated the effects of CHIR99021 on cellular iron homeostasis. Our data demonstrate that iron has an effect on cell stemness and that this effect is associated with the Wnt/GSK-3β/β-catenin pathway. Our findings provide insight into hitherto unknown regulatory mechanisms controlling stem cell stemness maintenance and differentiation.

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

#### *2.1. Cell Culture*

The Neuro-2a cell line, a mouse neuroblastoma cells, was cultured in DMEM (GIBCO, Grand Island, New York, USA 8121382) with 10% fetal bovine serum (Biological Industries, Cromwell, CT, USA) and 1× Pen/Strep (GIBCO, 10378016). Cells were maintained at 37 ◦C in a humidified incubator containing 5% CO2 (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.2. Preparation of MEF Feeder Cells and Culture of mESCs*

Healthy ICR male and female mice aged 2–3 months were selected to mate, and primary mouse embryonic fibroblast (MEF) feeder cells were obtained from pregnant mice at day 12.5 or 13.5 postfertilization. The embryonic day 12.5 (E12.5) or E13.5 fetal mice were isolated, and the head, limbs, tail, and internal organs were removed, leaving only the torso. The tissue fragments were then cut to <1 mm3 pieces and digested into individual cells with 0.25% trypsin. Next, the cells were cultured in MEF feeder culture medium, which contained DMEM supplemented with 10% FBS, 1× nonessential amino acids (GIBCO, 11140), 1× L-glutamine (Specialty media, TMS-002-C) and 1× Pen/Strep. MEF feeder cells of generation 2–3 were incubated with MEF feeder culture medium with mitomycin C (10 μg/mL) for 2–3 h to inhibit proliferation. Afterwards, the cells were cryopreserved until subsequent use in embryonic stem cell culture experiments.

The mESCs were generated and banked in our laboratory. A 0.1% gelatin was added to cover the wells of 24-well plates and incubated in a tissue culture incubator for 2–3 h in advance of use. The MEF feeder cells were then resuscitated and seeded onto the gelatin-coated plates until the cells covered the bottom. Additionally, mESCs were then resuscitated and maintained in KnockOutTM DMEM (GIBCO, 10829018) supplemented with 15% KnockOutTM serum replacement (GIBCO, A3181502), 1× nucleosides for ES cells (Merck Millipore, ES-008-D), 1× nonessential amino acids, 1× L-glutamine, 1× 2 mercaptoethanol (GIBCO, 21985023), 1× Pen/Strep, 1000 U/mL LIF (GIBCO, A35933), 3 μM CHIR99021 (Selleck, Houston, TX, USA S2924), and 1 μM PD0325901 (Selleck, S1036).

#### *2.3. Drug Treatment*

Cells were treated with CHIR99021, deferoxamine (DFO, Sigma, D9533) or ferric ammonium citrate (FAC, Sigma, St. Louis, MO, USA F5879). The concentrations and treatment times were as indicated in the results and figures.

#### *2.4. Western Blot Analysis*

Cells of the control group and drug-treated group were lysed in RIPA lysis buffer containing 95% RIPA buffer (Solarbio, Beijing, China R0010), 2% protease inhibitors (Roche,<PHONE_NUMBER>1), 2% phosphatase inhibitors (Roche, Basel, Switzerland,<PHONE_NUMBER>1), and 1% PMSF (Solarbio, P0100) for 30 min, shaken every 5 min. The protein concentration was assessed by a BCA kit (Yeasen, Shanghai, China, 20201ES76). Protein samples were resolved by SDS-PAGE (10% or 12% acrylamide) at 25 μg per lane and then transferred onto nitrocellulose (NC) membranes, which were cut appropriately, and blocked in 5% skimmed milk for 1.5 h and then incubated with different primary antibodies overnight at 4 ◦C. The next day, after washing, the membranes were incubated with the corresponding secondary antibodies at room temperature for 1.5 h. Finally, we used an ECL kit (CWBIO, CW0049M) and a chemiluminescence imager (Bio-Rad, Hercules, CA, USA) for visualization. Quantitative analysis of the protein bands was normalized to β-actin, as an internal reference.

 Total RNA Extraction and qRT-PCR*

Total RNA was extracted from neuro-2a cells or mESCs using TRIzol reagent (Invitrogen, 15596018). RNA was then reverse-transcribed to cDNA. Next, we mixed 10 μL SYBR Green PCR Master Mix (CWBIO, CW0957), 1μg cDNA, 0.4 μL forward primer, 0.4 μL reverse primer and 5.2 μL RNase-free water for the subsequent PCR reaction. qRT-PCR amplification was performed using a Bio-RAD CFX96 Connect Real-Time System. The primer sequences used are listed in Table 1.

**Table 1.** Primer sequences used for qRT-PCR.

#### *2.6. RNA-Sequencing (RNA-Seq) and Data Analysis*

The Neuro-2a control group cells and CHIR99021-treatment group samples were collected in TRIzol (Invitrogen, 15596018) and sent to Majorbio (Shanghai, China). The original data and results of RNA-sequencing were analyzed by Majorbio.

#### *2.7. Inductively Coupled Plasma Mass Spectrometry (ICP-MS)*

Total cellular iron content was evaluated by ICP-MS. Cell samples were centrifuged and collected after 0.25% trypsin digestion. Then, the samples were digested in 500 μL 65% nitric acid at room temperature overnight. The nitric acid was evaporated by heating for 20 min in a metal bath at 90 ◦C. Afterwards, 500 μL 30% H2O2 was added and allowed to react at 70 ◦C for 15 min, and then continued to vaporize for at least 6 h at 100 ◦C until the liquid in the tube was almost fully volatilized. The digested samples were re-suspended and mixed in 1 mL ultra-pure water for subsequent ICP-MS.

#### *2.8. Alkaline Phosphatase Staining*

For mESCs, the BCIP/NBT Alkaline Phosphatase Color Development kit (Beyotime, Jiangsu, China, C3206) was used according to the manufacturer's instructions. Cells were carefully washed 3–5 times with PBS and fixed with an appropriate amount of 4% formaldehyde for 20 min. Next, cells were incubated with the BCIP/NBT working solution for 15 min in the dark. An appropriate amount of double-distilled water was added to the plates to wash the cells 1–2 times to stop the color reaction. The results were examined and imaged under a light microscope (OLYMPUS, FV300).

#### *2.9. Electric Cell-Substrate Impedance Sensing (ECIS) Detection of Cell Proliferation*

The proliferation of cells was detected using an ECIS instrument (Applied BioPhysics, Troy, NY, USA). The experimental materials and reagents prepared include: 0.22 μm filter, syringe, sterile water, and 100 mM cysteine. An 8W10E+ electrode plate was pre-treated with 400 μL cysteine solution at a final concentration of 10 mM in each well, and incubated at 37 ◦C overnight. The electrode plate was washed 3 times with sterile water the next day. The cells were then seeded onto the electrode plate with about 1.5 × 104 cells per well, and left for 5 min. The real-time detection was performed after cells placed in the 37 ◦C incubator and connected to the ECIS instrument.

#### *2.10. Profiles of Mitochondrial Respiration and Glycolysis*

An Oxygraph-2k (O2k, TissueGnostics Asia Pacific Limited, Beijing, China) was used to assess the degree of oxidative phosphorylation and glycolysis of cell samples. The cells were digested, centrifuged, resuspended and counted, and then added into the chambers of the instrument. For the levels of oxidative phosphorylation, oxygen consumption rate (OCR) was recorded after injections of pyruvate (P), malic acid (M), ADP (D), oligomycin (Omy), the mitochondrial uncoupling agent, FCCP (U+), and antimycin A (Ama). For the level of glycolysis, the extracellular acidification rate (ECAR) was recorded, after injections of glucose (Glu), oligomycin (Omy), and 2-deoxy-glucose (2-DG), according to the manufacturer's instructions. The data were exported and analyzed by DatLab (New York, NY, USA, <IP_ADDRESS>) software.

#### *2.11. Statistical Analysis*

All data are presented as the mean ± standard error (SEM). The statistical graphs were generated using GraphPad Prism 6 software. Differences between two groups were compared by non-paired *t*-tests. Differences were considered statistically significant when *p* < 0.05.

#### **3. Results**

#### *3.1. The Effects of CHIR99021 and Iron on Cell Stemness in mESCs*

The small molecule compound, CHIR99021, is an aminopyrimidine derivative which can efficiently inhibit GSK-3α and GSK-3β. It is also commonly used to culture ESCs and induced pluripotent stem cells (iPSCs). To explore the effects of CHIR99021 on iron in mESCs, we used qRT-PCR to examine the transcriptional levels of H-ferritin (FtH) and L-ferritin (FtL), which make up the ubiquitous intracellular iron storage protein. The expression levels of *FtH* mRNA were significantly decreased after the removal of CHIR99021 alone or with simultaneous removal of LIF, PD0325901, and CHIR99021 (Figure 1A). Likewise, the expression levels of *FtL* mRNA were also significantly diminished after removal of CHIR99021 alone (Figure 1B). There was no significant difference in the expression levels of *Oct4* mRNA after removing CHIR99021 alone or removing LIF, PD0325901 and CHIR99021 (Figure S1A), while the levels of *Sox2* mRNA were significantly decreased (Figure S1B). These results demonstrated that removal of CHIR99021 reduced iron storage in mESCs.

To further examine the relationship between CHIR99021, iron, and cell stemness in mESCs, we supplemented normal embryonic stem cell medium and embryonic stem cell differentiation medium, in which LIF, PD0325901, and CHIR99021 were simultaneously removed, with 25 μM ferric ammonium citrate (FAC) and examined the mRNA levels of the pluripotency genes, *Oct4* and *Sox2*. Supplementing the embryonic stem cell culture medium with iron did not affect the expression of these mRNAs. However, the levels of *Sox2* mRNA were significantly increased after supplementing the differentiation medium with 25 μM FAC (Figure 1C,D). By alkaline phosphatase staining, we also found that the stemness was weakened in mESCs cultured with differentiation medium, with the cells exhibiting a differentiated, spreading morphology. After supplementing the differentiation medium with 25 μM, 50 μM, or 100 μM FAC, although the mESCs also appeared to differentiate, the color of the cells was darker than that of the DV- group (Figure 1E,F). Together, these results suggested that removal of CHIR99021 decreases iron levels and the stemness of mESCs; supplementation with iron may be beneficial to the maintenance of stemness.

**Figure 1.** Effects of removal CHIR99021 and iron on the cell stemness in mESCs. (**A**,**B**) qRT-PCR analysis of *FtH* mRNA levels (**A**) and *FtL* mRNA levels (**B**) after incubation for 72 h in full derivation medium (DV) or without LIF, PD0325901, CHIR99021 or all three combined (DV-). (**C**,**D**) qRT-PCR analysis of pluripotency genes *Oct4* (**C**) and *Sox2* (**D**) expression after incubation for 72 h in full derivation medium or without LIF, PD0325901 and CHIR99021 (DV-), without or with the addition of 25 μM FAC. The mRNA levels are normalized to GAPDH mRNA levels. (**E**) Alkaline phosphatase (ALP) staining of mESCs treated for 72 h in full derivation medium (DV) or without LIF, PD0325901 and CHIR99021 (DV-), without or with the indicated amounts of FAC. Scale bar = 100 μm. (**F**) Quantification of area of ALP-positive cells (%). The data are expressed as the mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, ns, not significant.

#### *3.2. CHIR99021 Increasing the Expression of Ferritins, Cellular Iron Levels and Inhibiting the Expression of FPN1 in Neuro-2a Cells*

In order to further explore the effects of CHIR99021 on iron metabolism, we treated Neuro-2a cells with CHIR99021 and assessed the expression of iron metabolism related proteins, including FtH, FtL, TfR1, FPN1, and DMT1 (+/-IRE) (divalent metal transporter 1). The protein levels of FtH and FtL increased significantly in CHIR99021-treated Neuro-2a cells (Figure 2A,B). The levels of *FtH* and *FtL* mRNA were consistent with the increased protein levels (Figure 2C). As ferritin is the main intracellular iron storage protein, we next investigated whether total intracellular iron levels were changed by ICP-MS; total intracellular iron levels increased significantly after CHIR99021 treatment (Figure 2D). We also evaluated the levels of TfR1 protein and mRNA and found no significant differences (Figure 2E–G). However, the protein and mRNA levels of FPN1 [28] decreased significantly (Figure 2E,F,H). Additionally, the protein level of DMT1 decreased significantly (Figure S2). These results indicated that iron metabolism has been affected, and iron export decreased, which is expected to increase the amount of uncommitted iron in cells and, in turn, stimulate an increase in ferritin expression.

**Figure 2.** Changes in iron metabolism in Neuro-2a cells treated with CHIR99021. (**A**,**E**) Western blot analysis of FtH and FtL (**A**), TfR1 and FPN1 (**E**) protein after the cells were treated with or without 3 μM CHIR99021 [29,30] for 72 h. (**B**,**F**) Quantification of Western blot data of FtH and FtL (**B**), TfR1 and FPN1 (**F**). (**C**,**G**,**H**) qRT-PCR analysis of *FtH* and *FtL* (**C**), *TfR1* (**G**), and *FPN1* (**H**) mRNA levels. (**D**) ICP-MS detection of total cellular iron levels in Neuro-2a cells treated with or without CHIR99021. The relative expression levels are normalized to β-actin and then expressed as the fold of control group. The data are presented as the mean ± SEM. \* *p* < 0.05, \*\*\* *p* < 0.001, ns, not significant.

#### *3.3. DFO Abrogates the Increase of Ferritin Induced by CHIR99021*

In order to further confirm that CHIR99021 increases cellular iron levels, we co-treated cells with CHIR99021 and the iron chelator, DFO. We initially performed a concentrationdependence experiment, using 0–40 μM DFO. We found that the protein levels of FtH and FtL were decreased in the presence of 5 μM DFO (Figure 3A–D). Therefore, we proceeded to treat cells with 5 μM DFO and 3 μM CHIR99021. As previously shown, CHIR99021 treatment significantly increased the levels of FtH and FtL, while the expression of these proteins significantly decreased when DFO was included in the incubation (Figure 3E–H). These results suggested that the sequestration of iron could alleviate the increase of ferritin expression induced by CHIR99021.

**Figure 3.** DFO prevents the increase of ferritin expression induced by CHIR99021. (**A**–**D**) Western blot analysis of FtH and FtL protein after treatment with the indicated concentrations of DFO. (**E**,**F**) Western blot analysis of FtH levels after treatment with or without CHIR99021 and/or DFO. (**G**,**H**) Western blot analysis of FtL levels after treatment with or without CHIR99021 and/or DFO. The relative expression levels are normalized to β-actin. The data are expressed as the mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, ## *p* < 0.01, ns, not significant.

#### *3.4. CHIR99021 Stimulates the Expression of Steap1, Bola2 and Kdm6bos Increasing in Neuro-2a Cells*

In order to further explore the effect of CHIR99021 treatment on iron homeostasis and the mechanism of increased ferritin expression, we performed RNA-seq analysis in cells treated with or without CHIR99021. As shown in Figure 4A, we identified 1364 differentially expressed genes between the two groups, of which 580 genes were up-regulated and 784 genes were down-regulated. Gene Ontology (GO) enrichment analysis categorizes groupings of genes into three general areas: biological process, cellular component, and molecular function. From the results of this type of analysis, we found most differentially expressed genes to be concentrated in cellular processes: biological regulations and metabolic processes of biological processes; the composition of cells and organelles in cellular components; and molecular binding parts in molecular functions (Figure 4B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database divides biological metabolic pathways into six areas, including metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, and human diseases. We found that the differentially expressed genes were more heavily distributed in the process of translation and signal transduction (Figure 4C). These results helped us to hone in on the genes targeted by CHIR99021.

**Figure 4.** Comparison of RNA-seq results and verification of the differentially expressed genes between control and CHIR99021-treated cells. (**A**) Volcano plot of mRNA expression differences. (**B**) GO enrichment analysis between control and CHIR99021-treated Neuro-2a cells. (**C**) Statistical classification analysis of KEGG pathways between control and CHIR99021-treated Neuro-2a cells. (**D**–**F**) qRT-PCR analysis of *Steap1* (**D**), *Bola2* (**E**) and *Kdm6bos* (**F**) mRNA levels with or without CHIR99021 treatment. The mRNA levels are normalized to β-actin mRNA levels and then expressed as the fold of control group. The data are expressed as the mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01.

Next, based on the results of sequencing and alignment, we determined the genes which both |log2FC| ≥ 1 and were related to iron or metal ion binding from 1364 differentially expressed genes. Six-segment transmembrane epithelial antigen of prostate 1 (STEAP1) has oxidoreductase activity and metal ion binding function in the molecular functional grouping enriched by GO, and also plays a key role in iron homeostasis [31]. BolA-like protein (Bola2) participates in the assembly of iron-sulfur clusters on biological process grouping [32,33]. KDM6B is a KDM1 lysine-specific demethylase 6B, which has methyltransferase activity and metal ion binding function in the molecular function grouping. By qRT-PCR, we confirmed that the mRNA levels of *Steap1*, *Bola2* and *Kdm6bos* increased significantly upon CHIR99021 treatment (Figure 4D–F).

#### *3.5. Both CHIR99021 and Iron Promote the Expression of β-Catenin*

The small molecular compound, CHIR99021, is a highly effective inhibitor of GSK-3 α/β, while β-catenin is a downstream target gene of GSK-3β in the classical Wnt pathway [34]. To verify the effect of CHIR99021 on the Wnt/GSK-3β/β-catenin pathway, we examined the related proteins. We found that CHIR99021 significantly downregulated the expression of GSK-3α/β, concomitantly upregulating the levels of p-GSK3β and β-catenin (Figure 5A,B,D,E). Thus, the ratio of p-GSK3β/GSK-3β increased significantly with the treatment of CHIR99021 (Figure 5C). In addition, we found that 25 μM and 50 μM FAC significantly increased the expression of β-catenin (Figure 5F–K). In consideration of these and the previous results, we hypothesized that there may be a relationship between β-catenin and iron level, particularly on the context of CHIR99021 exposure.

**Figure 5.** Both CHIR99021 and iron stimulate the expression of β-catenin in Neuro-2a cells. (**A**,**D**) Western blot analysis of GSK-3 α/β, p-GSK3β and β-catenin protein in Neuro-2a cells treated with or without CHIR99021. (**B**,**C**,**E**) Quantification of the Western blot analysis of GSK-3 α/β (**B**), p-GSK3β/GSK-3β (**C**), and β-catenin (**E**). (**F**,**H**,**J**) Western blot analysis of FtH (**F**), FtL (**H**) and βcatenin (**J**) protein after treatment with the indicated concentrations of FAC. (**G**,**I**,**K**) Quantification of the Western blot analysis of FtH (**G**), FtL (**I**) and β-catenin (**K**). The relative expression levels are normalized to β-actin and then expressed as the fold of control group. The data are expressed as the mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, ns, not significant.

#### *3.6. CHIR99021 Affects the Growth of Neuro-2a Cells*

Our above results demonstrate that CHIR99021 treatment altered intracellular iron homeostasis, including changes in iron metabolism-related molecules, as well as significant increases in intracellular iron levels. Among its many roles in enzymes and other proteins, iron participates in the synthesis and replication of DNA, thus playing important roles

in the process of cell growth [35]. We therefore proceeded to examine the expression of cyclin D1 and Ki67 proteins. Treatment of cells with CHIR99021 led to significant decreases in both of these proteins (Figure 6A–C). In addition, we found by electric cell-substrate impedance sensing that CHIR99021 inhibits cell proliferation (Figure 6D).

**Figure 6.** CHIR99021 inhibits the growth of Neuro-2a cells. (**A**–**C**) Western blot analysis and quantification of the expression of cyclin D1 and Ki67 protein. The relative expression levels are normalized to β-actin and then expressed as the fold of control group. (**D**) Detection of cell proliferation by electric cell-substrate impedance sensing. The data are expressed as the mean ± SEM. \* *p* < 0.05.

#### *3.7. CHIR99021 Treatment Decreases Mitochondrial Oxidative Phosphorylation and Enhances Glycolytic Capacity in Neuro-2a Cells*

Oxidative phosphorylation (OXPHOS) and glycolysis are two major pathways of energy metabolism in mammalian cells. Normally, these two pathways are regulated to adapt to changes of external environment. As described above, we found that the GSK3 inhibitor, CHIR99021, can not only alter cellular iron levels and iron metabolism-related proteins, but also activate the Wnt/GSK-3β/β-catenin pathway. Both iron and this Wnt pathway could affect the maintenance of cell stemness [9,23,36]. When stem cells are in an undifferentiated state, mitochondria are immature and perinuclearly distributed, with lower OXPHOS capacity and higher glycolytic activity. Importantly, cellular energy metabolism patterns can also regulate cell fate through epigenetic mechanisms [37,38]. Therefore, we used the cellular energy metabolism analyzer, O2k, to evaluate cellular oxygen consumption and glycolytic capacity. The overall mitochondrial metabolic capacity decreased in cells treated with CHIR99021 (Figure 7A,B); basal respiration, ATP production-related oxygen consumption, and maximal respiration were all significantly decreased. Spare respiratory capacity, which represents the potential responsiveness of cells to energy demands, showed a downward trend (Figure 7C). In contrast, the overall glycolytic capacity of the cells was increased after CHIR99021 treatment (Figure 7D), while the maximum glycolytic capacity and glycolysis reserve capacity were all significantly increased (Figure 7E). These results suggest that CHIR99021 treatment diminishes cellular OXPHOS levels and enhances cellular glycolytic capacity, which is consistent with the characteristic that cells in a state of stemness mainly rely on glycolysis rather than OXPHOS.

**Figure 7.** CHIR99021 treatment decreases mitochondrial oxidative phosphorylation and enhances glycolytic capacity in Neuro-2a cells. (**A**) Overall oxygen consumption capacity of cells, as measured by O2k. Top panel, control group; bottom panel, CHIR99021-treated group. (**B**) Oxygen flux of cells after adding the indicated reagents. (**C**) Basal respiration, ATP-linked respiration, maximal respiration and spare respiratory capacity, as indicated by oxygen consumption rate (OCR) in Neuro-2a cells treated as shown. (**D**) Proton flux of cells after adding the indicated reagents. (**E**) Basal glycolysis, maximal glycolytic capacity and glycolysis reserve capacity in cells treated as shown. The data are expressed as the mean ± SEM. \* *p* < 0.05, \*\*\* *p* < 0.001, ns, not significant.

#### **4. Discussion**

A multifunctional protein, GSK3, is involved in a variety of cellular processes, including proliferation, metabolism and embryonic development [39]. It contains GSK-3α and GSK-3β. The latter is one of the main members of the canonical Wnt pathway. Recent studies have revealed that this pathway plays an important role in the maintenance of stemness [9,10,40]. As an essential element, iron also has many biological functions. Among these, iron has been identified as a key factor in the maintenance of cancer stem cell (CSC)

stemness. The iron chelation can inhibit the expression of the stem cell marker, Nanog, thus revealing a new treatment strategy for cancer [41]. However, it is not clear whether there is a relationship between GSK3 and iron, and, if so, what that mechanism may entail.

CHIR99021, a small molecule compound, can efficiently inhibit both GSK3. The compound is also commonly used in the culture of stem cells. In this study, we found that the expression of stemness-related genes and the transcription level of FtH and FtL in mESCs were significantly decreased upon removal of CHIR99021 from mouse embryonic stem cell culture medium. On the other hand, iron supplementation of embryonic stem cell differentiation medium was beneficial to the maintenance of mESCs stemness. In fact, we found that CHIR99021 can affect cell stemness by affecting intracellular iron levels. To further explore the relationship between GSK3 and cellular iron homeostasis, we treated Neuro-2a cells with CHIR99021, which led to increased expression of FtH and FtL at both the mRNA and protein levels, as well as increased intracellular iron content. Cellular iron homeostasis is regulated at the post transcriptional level by the IRE/IRP system. When intracellular iron levels increase, IRPs are unable to bind to the IREs in the 5- -UTR of the mRNAs encoding ferritin and FPN1, thus permitting their translation, while binding to the 3- -UTR in the mRNA encoding TfR1 is required to stabilize the message, so TfR1 levels decrease under elevated cellular iron conditions. Regulation occurs in the opposite direction upon depletion of cellular iron [42]. Our finding of increased ferritin, with concomitant decreases in FPN1 and unchanged TfR1 after treatment with CHIR99021, indicates that the changes in iron homeostasis induced by CHIR99021 may not be regulated by the system. However, treatment of cells with iron chelation and CHIR99021 alleviated the increase of ferritin expression induced by CHIR99021. Thus, we conclude that CHIR99021 indeed affects cellular iron homeostasis.

To further explore the mechanism of ferritin up-regulation by CHIR99021, we used RNA-seq and narrowed the differentially regulated genes based on iron or metal iron binding, finally identifying three genes of interest: *Steap1*, *Bola2* and *Kdm6bos*. The mRNA expression of all three of these genes increased significantly after CHIR99021 treatment. Interestingly, STEAP1 is highly expressed in prostate cancer [43]. In 2016, Kim et al. reported the presence of a single heme b prosthetic group in STEAP1, which can reduce metal ion complexes and oxygen. Diferric transferrin (Fe2Tf) binds to TfR1, which mediates iron uptake into cells. Then, Fe3+ is released from Tf and must be reduced prior to export from the endosome by the Fe2+ transporter, DMT1. This reduction can be mediated by STEAP1. After export from the endosome, iron can be incorporated into functional proteins or stored in ferritin [31]. Therefore, the increase of ferritin expression induced by CHIR99021 treatment may be a consequence of increased levels of STEAP1. Bola2 has been shown to bind to the multi-functional binding protein PCBP1 to form an intermediate iron chaperone complex and participate in the assembly of [2Fe-2S] clusters, while PCBP1 may bind iron in the LIP to transfer iron to ferritin to form a PCBP1-Fe-GSH-BolA2 complex [32,33]. Therefore, there may be a connection between ferritin and BolA2. KDM6B is a KDM1 lysine-specific demethylase with methyltransferase activity and metal ion binding function. KDM6B requires Fe2+ as a cofactor [44]. Together, our data provide support for a link between GSK3 activity and ferritin synthesis, possibly via the proteins encoded by the above genes, however, the specific mechanisms of these processes require further study.

As one of the main members of Wnt/β-catenin pathway, GSK-3β can phosphorylate β-catenin and cause it to be degraded by proteasome. When the phosphorylation of GSK-3β at Ser9 is increased, the activity of GSK-3β decreases [34]. Our results show that, with CHIR99021, the levels of GSK-3α/β were significantly decreased, while the p-GSK3β/GSK-3β ratio was significantly increased, ultimately leading to promotion of the expression of the downstream target molecule β-catenin. Thus, the Wnt/GSK-3β/β-catenin pathway was significantly activated. We therefore asked, since iron levels increased upon CHIR99021 treatment, could iron also affect the expression of β-catenin? To this end, we treated cells with FAC, which promoted the expression of β-catenin. The finding is consistent with a report demonstrating a positive correlation between FtL and β-catenin in glioma cells [45].

Iron can involve in the regulation of cell growth. Both iron deficiency and excess can affect the cell cycle, which may then affect a series of critical organismal processes [46,47]. Our data that cell proliferation was inhibited with the treatment of CHIR99021 is consistent with these properties of iron in mammals.

Iron has also been found to play a biological role in maintaining the stemness of mesenchymal stem cells and cancer stem cells [23,41]. The activation of the classical Wnt pathway has been reported to inhibit the differentiation of stem cells [9,10,36]. Additionally, in stem cells, the mode of energy metabolism mainly depends on glycolysis rather than oxidative phosphorylation. On the other hand, the mode of cell energy metabolism can also determine the fate of stem cells, with respect to stemness or differentiation, through epigenetic regulation [37,38]. We found that oxidative phosphorylation decreased, along with increased glycolysis, upon treatment with CHIR99021.

#### **5. Conclusions**

In summary, our study uncovers a relationship between GSK3 and ferritin, along with alterations in cellular iron metabolism that affect cells stemness and proliferation. These findings not only open new avenues for further exploration of stemness maintenance and differentiation, but also provide an insight for the treatment of stem cells in disease.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12020377/s1, Figure S1: Effects of removal CHIR99021 on the expression of Oct4 and Sox2 genes. Figure S2: Expression of DMT1 in Neuro-2a cells treated with CHIR99021.

**Author Contributions:** Formal analysis, Y.-Z.C., S.C., J.Z., P.Y. and G.G.; writing—review and editing, Y.-Z.C., S.C. and J.Z.; supervision, Y.-Z.C., S.C. and J.Z.; writing—original draft preparation, Y.H. (Yingying Han); methodology, Y.H. (Yong He), X.J. and J.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant numbers<PHONE_NUMBER>8, 32070962).

**Institutional Review Board Statement:** This study does not involve humans or animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All of the data is contained within the article.

**Acknowledgments:** The work was supported by the National Natural Science Foundation of China (grant numbers<PHONE_NUMBER>8, 32070962).

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

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.


*1.3. Research Questions*

Given the importance of ET in water management generally, and the potential uncertainty of EOS ET estimation for large seemingly homogeneous cropping areas with temporal variance, we explored several research questions to help parameterize the spatiotemporal uncertainty of EOS ET estimation using sUAS RSE. Answers to these research questions can provide practitioners valuable qualifiers of uncertainty as EOS ET estimates are now more routinely used in determining irrigation volumes and scheduling. Through cross-platform comparisons, we asked:


*2.2. Smart Phone Application*

The SP application (SP app) used in this study was the Smart Protractor application (Smart Tools Co., Dae-gu, South Korea). Measurement with the SP app is obtained by positioning a virtual protractor, visible on the SP screen, on photography obtained using the SP camera. The assistant takes a photo of the Schanz pins, saves it, measures the angles, and observes the value. The picture should be taken with the camera positioned along the imaginary line between two pins. After the photo has been taken and saved, two red lines appear on the screen. The lines can be dragged across the screen to place the virtual goniometer on the axis of the Schanz pins, finely adjusting them until the goniometer is positioned correctly. Pictures judged subjectively wrong by the surgeon because of a perspective error can be deleted. The angle-SP measurement was repeated three times, and the average value was determined.

**Figure 7.** (**A**) A 15-year-old girl with a tibia shaft fracture; (**B**) minimally invasive plate osteosynthesis was carried out; (**C**) postoperative CT scan showing a moderate difference (over 16 degrees) in rotation compared with the noninjured side.

**Figure 8.** (**A**,**B**) Parallel Schanz pins placed in the proximal and distal tibia; (**C**) screws were removed at the proximal segment; (**D**) external rotation of the distal segment and the smartphone application measured about 14◦ correction; (**E**) screws were fixed at the proximal segment; (**F**) postoperative radiographs showing the improved alignment after revision procedure.


Air Depleted and Solvent Impregnated Cork Powder as a New Natural and Sustainable Wine… DOI: http://dx.doi.org/10.5772/intechopen.85691

 anthocyanin

and ethanol

 and phenolic acid composition

impregnation

 (CKNI and CKFI) and cork powders with a particle size below 75 μm at two application

 of spiked red wines (TF) and after treatment with natural cork and

dichloromethane

 doses (250 and 500 g/hL)

 and ethanol extractive free cork after air removal

(CKFI75250

 and

CKFI75500).

Samples

238

T CKNI CKFI CKFI75250

CKFI75500

Data are presented as

corresponding

p < 0.05.

Table 4. Chromatic

(CKNI and CKFI) and cork powders with a particle size below 75 μm at two application

characteristics

 of red wines before (TF) and after treatment with natural cork and

dichloromethane

 doses (250 and 500 g/hL).

 and ethanol extractive free cork after air removal and ethanol

impregnation

 to ΔE\* were obtained taking as a reference the untreated wine (T) and wines treated with corks (CKNI, CKFI, CKFI75250

 14.84

X

 s; data within a column followed by the same letter are not significantly

 0.20c

45.32

 0.34b

36.17

 0.27b

57.98

 0.44b

 different (Tukey, p 0.05). L\*, lightness; a\*, redness; b\*, yellowness; ΔE\*, colour difference. The values

0.67

 0.00c

6.35

 0.41b

 and CKFI75500).

 A.U., absorbance units.

8.34

 0.01b

0.76

 0.01c

 10.85

 0.72a

40.72

 1.14a

34.08

 0.99ab

53.10

 1.51ab

0.70

 0.00b

1.99

 0.48a

9.40

 0.07ab

0.74

 0.00c

13.87

 0.08c

44.37

 0.01b

36.22

 0.29b

57.28

 0.19b

0.68

 0.00c

4.88

 0.01b

8.97

 0.17b

0.71

 0.01b

13.17

 0.18b

43.32

 0.23b

35.47

 0.00ab

55.99

 0.18b

0.69

 0.00c

3.54

 0.29ab

9.09

 0.07ab

0.72

 0.00b

10.84

 0.49a

40.55

 0.68a

35.62

 0.31ab

53.97

 0.72ab

0.72

 0.00a

—

10.04

 1.04a

L\*

a\*

b\*

C\*

h°

ΔE

Colour intensity (A.U.)

 Hue

Advances in Grape and Wine Biotechnology

0.66

 0.00a

Table 6.

Mean scores of each attribute after sensory analysis of volatile phenol-free (T0) and volatile phenol-spiked (TF) red wine after treatment with natural cork and dichloromethane and ethanol extractive free cork after air removal and ethanol impregnation (CKNI and CKFI) and cork powders with a particle size below 75 μm at two application doses (250 and 500 g/hL) (CKFI75250 and CKFI75500).

(CKFI75500) presenting a significantly lower score than CKFI75250 and CKFI. This decrease in colour intensity in the CKFI75500 is also followed by a decrease in the sensory hue, being in accordance with the significant change in h° and L\* for this sample. Neither natural nor extractive free cork powders changed significantly the

…

Air Depleted and Solvent Impregnated Cork Powder as a New Natural and Sustainable Wine

For VP-spiked wine, the application of all cork powders in two application doses (250 and 500 g/hL) of CKFI75 decreased the negative phenolic attribute significantly compared to the spiked wine (TF); however, the scores obtained were also significantly higher than those observed for the initial unspiked wine (T0). For the fruity aroma attribute, the application of all cork powder allowed recovering significantly the fruity aroma attribute in relation to the VP-spiked wine (TF); nevertheless, the scores were also significantly lower than that observed for the original unspiked wine (T0). The fruity aroma attribute was significantly higher for the CKFI75250 than for all other cork powder samples even higher than CKFI75500. This could be due to the higher decrease in headspace aroma abundance responsible

For the floral attribute, only CKFI and CKFI75250 allowed increasing significantly this sensory attribute in relation to the TF, and again the scores obtained for the cork-treated wines were significantly lower than that obtained for T0. As observed for the fruity attribute, also for the floral attribute the increase in application dose of CKFI75 decreased the floral attribute of the wine (Table 6). The TF wine presented an increased vegetable attribute that did not decreased with the application of cork powder samples, nevertheless, the scores observed was very low (Table 6). No significant differences were observed for the oxidised aroma attri-

The application of cork powder did not change the acidity and body of the wine samples significantly; however, significant differences were obtained for bitterness, astringency, balance and persistence (Table 6). The spiking wine resulted in a significant increase in the bitterness attribute in relation to the T0. Except for CKFI75500, the other cork powders did not decrease bitterness to the levels observed for T0. For astringency, spiking of wine with VPs increase this sensory attribute, and no cork-powder sample decreased the astringency to the initial levels (T0), nevertheless CKFI and CKFI75250 were able to decrease significantly the astringency in relation to TF. For CKNI, a significant increase of astringency in relation to TF was observed, and this can be explained probably by a migration of phenolic compounds from this cork-powder [58, 59]. For balance, TF significantly decreased this sensory attribute, and the application of all cork powders did not lead to scores significantly different from the TF. For persistence, the application of cork powders to TF significantly increased the persistence of wine; however, the scores

for the fruity notes for this application dose as discussed previously.

bute in all samples (T0, TF and cork powder treated wines).

obtained were significantly lower than the persistence of T0 (Table 6).

application doses

241

3.4 Impact of wine chemical composition on sensory profile of red wine treated with extractive free and ethanol impregnated cork powder and

tactile/textural descriptors (Figure 7) and the chemical composition of wines, concerning the abundance of headspace aroma compounds and phenolic compounds, respectively, were subjected to multiple factor analysis. From the variable map, it can be concluded that for the first and second factors, both groups of variables contribute almost equally (53 and 46%, and 36 and 64% for the sensory and

chemical data for the first and second factors, respectively) (Figure 6b).

The sensory scores provided by the expert panel for aroma (Figure 6), taste and

limpidity and oxidised visual sensory attributes.

DOI: http://dx.doi.org/10.5772/intechopen.85691

### Air Depleted and Solvent Impregnated Cork Powder as a New Natural and Sustainable Wine… DOI: http://dx.doi.org/10.5772/intechopen.85691

(CKFI75500) presenting a significantly lower score than CKFI75250 and CKFI. This decrease in colour intensity in the CKFI75500 is also followed by a decrease in the sensory hue, being in accordance with the significant change in h° and L\* for this sample. Neither natural nor extractive free cork powders changed significantly the limpidity and oxidised visual sensory attributes.

For VP-spiked wine, the application of all cork powders in two application doses (250 and 500 g/hL) of CKFI75 decreased the negative phenolic attribute significantly compared to the spiked wine (TF); however, the scores obtained were also significantly higher than those observed for the initial unspiked wine (T0). For the fruity aroma attribute, the application of all cork powder allowed recovering significantly the fruity aroma attribute in relation to the VP-spiked wine (TF); nevertheless, the scores were also significantly lower than that observed for the original unspiked wine (T0). The fruity aroma attribute was significantly higher for the CKFI75250 than for all other cork powder samples even higher than CKFI75500. This could be due to the higher decrease in headspace aroma abundance responsible for the fruity notes for this application dose as discussed previously.

For the floral attribute, only CKFI and CKFI75250 allowed increasing significantly this sensory attribute in relation to the TF, and again the scores obtained for the cork-treated wines were significantly lower than that obtained for T0. As observed for the fruity attribute, also for the floral attribute the increase in application dose of CKFI75 decreased the floral attribute of the wine (Table 6). The TF wine presented an increased vegetable attribute that did not decreased with the application of cork powder samples, nevertheless, the scores observed was very low (Table 6). No significant differences were observed for the oxidised aroma attribute in all samples (T0, TF and cork powder treated wines).

The application of cork powder did not change the acidity and body of the wine samples significantly; however, significant differences were obtained for bitterness, astringency, balance and persistence (Table 6). The spiking wine resulted in a significant increase in the bitterness attribute in relation to the T0. Except for CKFI75500, the other cork powders did not decrease bitterness to the levels observed for T0. For astringency, spiking of wine with VPs increase this sensory attribute, and no cork-powder sample decreased the astringency to the initial levels (T0), nevertheless CKFI and CKFI75250 were able to decrease significantly the astringency in relation to TF. For CKNI, a significant increase of astringency in relation to TF was observed, and this can be explained probably by a migration of phenolic compounds from this cork-powder [58, 59]. For balance, TF significantly decreased this sensory attribute, and the application of all cork powders did not lead to scores significantly different from the TF. For persistence, the application of cork powders to TF significantly increased the persistence of wine; however, the scores obtained were significantly lower than the persistence of T0 (Table 6).