**Acknowledgements**

blood vessels and the establishment of specific microenvironment within the tumor. These

**Figure 6.** Effect of portacaval anastomosis on thiobarbituric acid reactive substances (TBARS) assay supplemented with FeSO4 50 (μM) of liver homogenate, subcellular fractions and serum. Sham, false‐operated rats (white bars); shunt, rats with portacaval anastomosis for 6‐8 weeks (black bars). Data are average ± SEM from at least eight independent

Warburg's effect [99]; hypoxia also induces adaptive responses as the expression of special‐ ized proteins named hypoxia‐inducible factors (HIFs) [100]. HIF‐1 is a transcription factor formed by two subunits (HIF‐1α and HIF‐1β). Although HIF‐1 is constitutively expressed in normoxic conditions, HIF‐α is constantly degraded by prolyl‐hydroxylases; hydroxylated residues serve as docking site for von Hippel‐Lindau tumor suppressor protein that is a con‐ stituent of an E3 ubiquitin ligase complex. The ubiquitinated HIF‐1α suffers proteasomal deg‐ radation [101]. Low oxygen tension and some proinflammatory cytokines stabilize HIF‐1α and allow its nuclear translocation to regulate key genes for the hypoxic response [100]. HIF‐1α mediates the expression of genes that supports tumor growth such as NADH dehydrogenase (ubiquinone) 1α subcomplex, 4‐like 2 (NDUFA4L2), a protein that attenuates the activity of

It was shown that the kinase inhibitor sorafenib, an antineoplasic extensively used in oncol‐ ogy with potent antiangiogenic effects, induces intratumor oxidative stress that favor drug resistance in HCC; the insensibility to the drug requires the action of HIF‐1α regulating the

concentration environment with

conditions [102].

favors the onset of aerobic glycolysis, by the

adaptations make the tumor cells adapt to highly variable O2

Significant statistical difference by *t*‐student test, *p* < 0.05.

the mitochondrial complex I, reducing the ROS production in low O2

successive lapses of hypoxia‐reperfusion [98].

observations. \*

170 Redox - Principles and Advanced Applications

From the metabolic point of view, lack of O2

M.D‐M. research investigation is supported by DGAPA, PAPIIT grant no. IN200815. We thank LN Fernando López‐Barrera for his artistic assistance and Jééssica González‐Norris for critically editing the text.


**7. Application Case: Results**

Four different solutions were analyzed in this paper: the traditional diesel configuration (S0), the PTO solution (S1), the PTO of higher power size, and the fully electric one (S3). Table 9 provides the results of the total costs obtained with a complete LCPA analysis: we mainly focused on BLD, OPEX, and M&R values to compare the solutions. EEDI, Sox, and NOx are approximated values, but they can give an idea of the solution quality. Speaking in terms of BLD, the reference ship layout (S0) is the less expensive, while the fully electric configuration, as expected, is the most expensive; solution S1 and S2 are very similar, S2 is penalized by the third gen-sets installed for a redundancy aspect but not needed in terms of power generation (two gen-sets covered all scenarios). In terms of OPEX, the best solution is S2, while the reference configuration (S0) is the worse. It is important to remind that OPEX value includes fuel consumption, so, this value could also be read as, S0 has a higher fuel consumption than S2 and S3. For each configuration, fuel consumption has been calculated, combining the utilization factors and the complete engines diagrams. Referring to data reported in Tables 5–8, the results obtained are truthful.

The M&R are lower in S2 and higher in S1: as expected, S2 is the configuration that has been designed to ensure engines' better working point and this is underlined in OPEX and M&R values that are the best. S1 that has a worse working point, as shown in Table 8, also has higher M&R costs: a bad engine utilization reduces the MTBM and increases the maintenance times over ship life cycle.

**Table 9.** Results of the layout comparison.

The EEDI value points out that all alternative configurations have better efficiency and lower emissions than the reference one. S1 is the solution with the lower EEDI value, but we should underline that value in S2 is affected by the third gen-set installed as a stand-by unit. The EEDI value is a function of installed power.

It is important to note that despite S1 has worse engines working point, and therefore higher M&R is the best solution. Speaking in terms of NPV, the total value not differs so much from the other ones.

In Figure 10, it has been reported the LCC values maintenance over time for the four layouts S0, S1, S2, S3. As just said, solution S2 is the best in terms of operational maintenance; indeed, it has been designed to minimize maintenance costs. An unexpected result is given by solution S0: it is a cheaper solution than S1 even though the main engines are used less efficiently than in solution S1 (characterized by the introduction of the PTO).

From these results, it is also possible to see that the fully electric, besides being the most expensive solution, will not guarantee a significant advantage during the years. From this analysis, we can also say that, at least for our application case, it is not true that the most expensive solution is always the best in terms of maintenance costs. Analyzing the last years of the ship life in the graph (25–30 years), it seems that solution S2 would bring a good saving compared to the original solution.

**Figure 10.** Integration over 30 years of Operational Maintenance costs.

Table 10 provides the results in terms of dimensionless index ready for the LCPA calculations: the coefficient is always defined between zero (worst) and one (best). S1 is the best, and S2 is the second-best solution, even if it is affected by the power surplus installed. Not purchasing the third gen-set, decision subjected to owner agreement, implies less BLD and M&R. The S3 is the worst layout, but it is important to stress that LCPA tool does not take into account noise and vibration problems, new possible weights distribution onboard, engine room locations, and off-limits routes.

The last column in Table 10 is part of the LCPA tool structure: for each KPI, a weight has been assigned. The assignation is arbitrary and given by the designer or the owner or the shipbuilder. In this case, we selected BLD and M&R as most interesting KPIs, followed by OPEX. In this perspective, despite NPV value is one of the most popular economic indicators, it has not been deeply discussed and analyzed during this work focused on maintenance actions and costs: the weight assigned in Table 10 is equal to 0 for this reason. The NPV value does not present significant changes in S0, S1, and S2 because the technology used in the propulsion system is the same (i.e., diesel engines), while the S3 configuration employs large electric motors as main propulsion item.

**Table 10.** Results of the layout comparison.

The same results are shown in Figure 11 below.

**Figure 11.** KPIs coefficients in spider graph.

#### **8. Conclusions**

For a research vessel, all the necessary data have been provided to start developing and assess a maintenance prediction model based on a real maintenance plan and not on a parametric formulation. The methodology described in Section 4 is a result of multiple interactions: it has been developed for the reference vessel only and then readapted to a general application for various vessel types. Figure 3 provides a simplified scheme of the maintenance tool and calculation structure.

During the tool development, a corrective Formula (4) for diesel engines MTBM has been defined to take into account design mistakeS on the maintenance costs. Application cases have been carried out on a research vessel, as exposed in Sections 5 and 6. Different propulsion solutions have been identified, and the tool can characterize them in terms of different performances; but, at the same time, it not so simple to identify the best solution because of the final evaluation that is strictly related to the stakeholder point of view.

Further improvements could derive from the following activity:

**Author Contributions:** For conceptualization, methodology, validation, formal analysis and investigation P.G., G.F., M.M. and G.M. gave their contribution. For software and data curation, G.F., M.M. and G.M. gave their contribution. The writing—original draft preparation was made by G.M. and the writing—review and editing has

been carried out by P.G. and G.F. Resources have been provided by M.M. Supervision has been carried out by P.G., G.F. and M.M. Project administration and funding acquisition was in charge of P.G.

**Funding:** The reported research is still under development in the frame of the HOLISHIP (HOLIstic optimization of SHIP design and operation for life cycle) project (2016–2020), which has been funded by the European Commission in the HORIZON 2020 Transport Program, Grant Agreement nr 689074.

**Acknowledgments:** The Authors would like to express their gratitude to Alberto Dachà (Fincantieri s.p.a) for the valuable contribution he gave in terms of constructive discussion and advise during the development of the research activity.

**Conflicts of Interest:** The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


*Article* **Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom** *Thalassiosira pseudonana* **Using a Multiprong Approach**

**Erik L. Jensen 1,† , Véronique Receveur-Brechot 1, Mohand Hachemane 1, Laura Wils 1 , Pascale Barbier 2, Goetz Parsiegla 1, Brigitte Gontero 1,\* and Hélène Launay 1,\***

**Citation:** Jensen, E.L.;

Receveur-Brechot, V.; Hachemane, M.; Wils, L.; Barbier, P.; Parsiegla, G.; Gontero, B.; Launay, H. Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom *Thalassiosira pseudonana* Using a Multiprong Approach. *Int. J. Mol. Sci.* **2021**, *22*, 8723. https://doi.org/ 10.3390/ijms22168723

Academic Editors: Istvan Simon and Csaba Magyar

Received: 21 July 2021 Accepted: 11 August 2021 Published: 13 August 2021

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

**Copyright:** © 2021 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/).

**Abstract:** Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3 −. The "iota" class (ι-CA) was first found in the marine diatom *Thalassiosira pseudonana* (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium–calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

**Keywords:** analytical ultracentrifugation; CO2 concentrating mechanism; diffusion-ordered NMR spectroscopy; electrospray ionization mass spectrometry; homotetramer; manganese; metalloprotein; photosynthesis; small-angle X-ray scattering


4. Molecular analysis

are considered the strongest parameters discriminating HCC from high-grade dysplastic

If high-grade malignant tumour is found in the liver, the differential diagnosis includes metastatic malignancy versus HCC and cholangiocarcinoma. Any malignant tumour can ultimately spread to the liver via bloodstream, lymphogeneous dissemination or transperitoneal spread. In some biopsy series, metastatic lung, colorectal, pancreatic and breast carcinomas have been the most common secondary liver tumours [3]. However, frequency of different metastatic malignant tumours in liver biopsies depends on many factors, including the biological potential of the tumour and its incidence in the population as well as institutional approach to liver biopsy in different oncological patients. This, in turn, may depend on the patient's general status, presence of contraindications for biopsy or significant oncological treatment and the

In order to distinguish HCC from metastatic tumours, it is advisable to combine at least two hepatocellular markers and at least two antigens that are more frequently seen in adenocarcinomas. Among hepatocellular markers, Arg1 should be combined with either HepPar1 or GPC3. Most of adenocarcinomas express cytokeratin (CK) 19, MOC-31 and CK7 [55]. The spectrum of immunohistochemical panel should be planned in accordance with tissue availability within the biopsy. The suggested minimal panel includes ARG1 and CK19 [55], while maximal investigation might include several HCC markers accounting for different grades of HCC, several adenocarcinoma markers and antigens that are characteristic for certain tissues (neuroendocrine or melanocytic differentiation) or epithelia of specific organs, e.g., breast, large bowel, lung, thyroid, kidney and others. Panels of immunohistochemical markers can disclose the location of primary tumour giving rise to metastasis. Thus, CK20 and CXD2 are typical for metastatic colorectal carcinoma; CDX2 and CK7 for gastric carcinoma; TTF-1 and napsin A for lung adenocarcinoma and oestrogen receptor, mammaglobin, GATA3 or GCDFP-15 for breast cancer [65]. The expression frequencies of different tissue- and organ-specific antigens in metastases and corresponding primary tumours are further outlined in Table 4.

Antigen Tumour Frequency, % References CDX2 Colorectal carcinoma 100 [66] CDX2 Metastatic colorectal carcinoma 96.7–100 [67, 68] SATB2 Primary colorectal carcinoma 96.0 [68] SATB2 Metastatic colorectal carcinoma 92.2 [68] CK20 Metastatic colorectal carcinoma 97.1 [68] TTF-1 Lung adenocarcinoma 83.3 [69] Napsin A Lung adenocarcinoma 86.7 [69] HMB-45 Metastatic melanoma 76–81 [70, 71] MART-1 Melanoma 48.4–83 [72, 73]

nodule [3].

3.5. Hepatocellular carcinoma versus metastasis

34 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

availability of effective treatment.

The molecular classification of hepatocellular carcinoma is still developing. Thus, different approaches have been proposed. Although the present tools of molecular analysis assure the

technical background for in-depth studies, HCC might be more difficult target for the systematisation of molecular findings than other tumours. The problems are associated with heterogeneity of etiological factors and their geographic distribution in different populations

Diagnostic Algorithm of Hepatocellular Carcinoma: Classics and Innovations in Radiology and Pathology

http://dx.doi.org/10.5772/intechopen.76136

37

A trans-ancestry study has been carried out involving 608 cases of HCC. The cohort was created to reflect both etiological and geographic/genetic diversity of HCC. The main identified molecular targets were TP53–Rb pathway, Wnt pathway, modulators of chromatin and transcription,

French research team has recently proposed molecular classification into six subtypes, designated as G1–G6. The first three subtypes are characterised by TP53 mutations and are highgrade tumours. G1–G2 share AXIN1 and ATM mutations, while G1 also possesses RPS6KA3 mutations. G3 is characterised by mutations in TSC1/TSC2 and FGF19. G3 is also associated with haemochromatosis, macrovascular invasion, macrotrabecular and compact histological pattern as well as presence of multinucleated and pleomorphic cells. Sarcomatoid changes are more frequent in G1–G2, but clear cells—in G1. G4–G6 lack TP53 mutations and are low-grade tumours. G5–G6 exhibit mutations in CTNNB1 gene, while G4 lacks both mutations in TP53 and CTNNB1. G4 tumours are more frequently characterised by small size, steatohepatitic morphology and inflammatory infiltrates as well as absence of satellite nodules and vascular invasion. G5–G6 carcinomas display microtrabecular pattern, cholestasis and lack inflammatory infiltrates. By immunohistochemistry, these HCCs are characterised by nuclear expression

Nowadays, the classic diagnostic algorithm of HCC (see Figure 1) includes the evaluation of risk factors in a given patient to assess the need for surveillance. Cirrhotic patients are referred to ultrasound examination once per 6 months. Suspicious nodules are further evaluated by CT and MRI. Characteristic findings by CT and MRI including arterial hypervascularisation represent the basis of non-invasive diagnostics. In controversial and non-cirrhotic cases, biopsy is indicated that might need supplementation by immunohistochemistry according to the morphological features. Innovations are expected in the field of miRNA-based liquid biopsy to support radiological diagnosis, addition of SIR assessment and miRNA profile to select the optimal treatment, e.g. possibly broadening Milan criteria (see also chapter "Innovative Blood Tests for Hepatocellular Carcinoma: Liquid Biopsy and Evaluation of Systemic Inflammatory Reaction"), and novel immunohistochemical markers for cases that still remain ambiguous.

HCC is a frequent and aggressive malignant tumour, estimated to range sixth by incidence and second by mortality in the global cancer statistics. The high ratio of mortality to incidence (0.95)

mTOR–PIK3CA pathway and mutations in genes regulating telomere maintenance [82].

of beta catenin and strong positivity for glutamine synthetase [52].

5. Diagnostic algorithm of hepatocellular carcinoma

with diverse genetic background [81].

6. Conclusions

Figure 1. Diagnostic algorithm of hepatocellular carcinoma. 1—Recommended by the American Association for the Study of the Liver diseases (AASLD). 2—Recommended by the European Association for the Study of the Liver (EASL). Abbreviations: RFs, risk factors; vs, versus; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; US, ultrasonography; CT, computed tomography; MRI, magnetic resonance imaging; HGDN, high-grade dysplastic nodule; FNH, focal nodular hyperplasia; IHC, immunohistochemistry; SIR, systemic inflammatory response; mi, micro; RNA; ribonucleic acid.

technical background for in-depth studies, HCC might be more difficult target for the systematisation of molecular findings than other tumours. The problems are associated with heterogeneity of etiological factors and their geographic distribution in different populations with diverse genetic background [81].

A trans-ancestry study has been carried out involving 608 cases of HCC. The cohort was created to reflect both etiological and geographic/genetic diversity of HCC. The main identified molecular targets were TP53–Rb pathway, Wnt pathway, modulators of chromatin and transcription, mTOR–PIK3CA pathway and mutations in genes regulating telomere maintenance [82].

French research team has recently proposed molecular classification into six subtypes, designated as G1–G6. The first three subtypes are characterised by TP53 mutations and are highgrade tumours. G1–G2 share AXIN1 and ATM mutations, while G1 also possesses RPS6KA3 mutations. G3 is characterised by mutations in TSC1/TSC2 and FGF19. G3 is also associated with haemochromatosis, macrovascular invasion, macrotrabecular and compact histological pattern as well as presence of multinucleated and pleomorphic cells. Sarcomatoid changes are more frequent in G1–G2, but clear cells—in G1. G4–G6 lack TP53 mutations and are low-grade tumours. G5–G6 exhibit mutations in CTNNB1 gene, while G4 lacks both mutations in TP53 and CTNNB1. G4 tumours are more frequently characterised by small size, steatohepatitic morphology and inflammatory infiltrates as well as absence of satellite nodules and vascular invasion. G5–G6 carcinomas display microtrabecular pattern, cholestasis and lack inflammatory infiltrates. By immunohistochemistry, these HCCs are characterised by nuclear expression of beta catenin and strong positivity for glutamine synthetase [52].