**About the Editor**

**Joachim Wink** (Dr.) completed his Ph.D. in 1985 at Frankfurt University. He then went to the pharmaceutical industry and started his career at the Hoechst AG where he was responsible for strain collection and specialized in the cultivation and taxonomic characterization of Actinobacteria and Myxobacteria. During this period, he was responsible for the strain library within the pharmaceutical research and a number of screening projects with Hoechst Marion Russel, Aventis and Sanofi. In the year 2005, he did his habilitation at the Carolo Wilhelma University of Braunschweig and in 2012, he went to the Helmholtz Centre for Infection Research in Braunschweig where he founded the working group of the strain collection with its focus on Myxobacteria. Here, he is now working on the isolation and taxonomic characterization of Myxobacteria and Actinobacteria as well as the analysis of their secondary metabolites with a focus on the antibiotic active ones. The Compendium of Actinobacteria on the homepage of the German Culture Collection is a permanent actualized working tool for people working with Actinobacteria, which was prepared by him. He has published more than 60 papers on secondary metabolites and the taxonomy of the producing microorganisms in reputed journals, a number of reviews as well as book chapters and more than 35 patents. He is a member of the editorial board of a number of international journals.


4.4.2. Organized Groups

Solo entities are those who work alone with minimal security. They are usually experts in their field, using a single piece of code as equipment, such as viruses, worms, and sniffers to abuse frameworks. They generally gain access to the organization's framework through hardware damage and web loopholes. Their targets are usually small, and attacks are somewhat less critical. In addition, large and complex systems that may contain flaws are more vulnerable to attack. Monetary institutions are also more exposed to attacks because they exchange sensitive information [72].


**6.1 Anesthesia**

There are many options for anesthesia. These are; local, regional and general anesthesia.

## *6.1.1 Local anesthesia*

Usually, local anesthetics and opioids are frequently used. These medicines pass through the placenta and thus, before delivery, the medicine/drugs should be given in small doses to avoid toxicity in the neonate. The common toxicities related to local anaesthia are; central nervous system [CNS] depression and bradycardia [13].

It must be noted that opioids used alone do not provide adequate analgesia and so are most often used with anesthetics.

In the option for anesthesia, most of the local anesthetic methods used include; pudendal blocks and perineal infiltration as well as para-cervical blocks.

a.**Pudendal block:** Pudendal blocks are used rarely. Instead of this, epidural injections are more preferred. In this, a local anesthetic is injected through the vaginal wall so as to bathe the pudendal nerve as it crosses the ischial spine. In this way, the block anesthetizes the lower vagina, perineum and posterior

#### *Vaginal Delivery DOI: http://dx.doi.org/10.5772/intechopen.96097*

vulva. However, the anterior vulva, innervated by lumbar dermatomes, is thus not anesthetized [13]. Usually, pudendal block is considered a safe and simple method for spontaneous vaginal deliveries if the delivery is uncomplicated. It is even more so especially if the mother wishes to bear down and push or when the labor is advanced such that there is no time for epidural injection. It should be noted that pudendal block may have some complications which include intravascular injection of anesthetics, hematoma and infections.


*Article* **Safety Management and Safety Performance Nexus: Role of Safety Consciousness, Safety Climate, and Responsible Leadership**

**Farida Saleem 1,\* and Muhammad Imran Malik 2**

**Abstract:** Drawing from social system theory, social identity theory, and social exchange theory, this study examines how safety management practices are linked with employee safety performance through safety consciousness and safety climate. Furthermore, responsible leadership is introduced as a boundary condition in the safety consciousness—safety performance and safety climate—safety performance relationships. Data were collected from employees belonging to pharmaceutical firms located in different industrial zones of Lahore, Pakistan. The support is found for full mediation of safety consciousness and safety climate for the safety management and safety performance relationships. Responsible leadership moderates the safety consciousness—safety performance and safety climate—safety performance relationships so that when the safety climate is weak or the safety consciousness is low, a high level of responsible leadership enhances safety performance.

**Keywords:** safety performance; safety climate; safety consciousness; responsible leadership; industrial safety


**2. Reviewing livelihood and disaster**

**Livelihood**: the term 'livelihood' as a systematic study developed from basically the rural studies, since then it has been used in various dimensions in academia. The conventional meaning of the term is an economic activity, while in fact, apart from the economic aspect it comprises a wide range of issues such as ecological, political, cultural, environmental and so forth. The formal analysis of the term can be given as "a livelihood comprises the capabilities, assets (both natural & social) and activities required for a means of living; a livelihood is sustainable which can cope with the recovery from and shocks, maintain or enhance its capabilities and assets, both now and in the future, while not undermining the natural resource base" [9]. This analysis primarily consists of three concepts: capability, equity and sustainability; and these are interconnected and interdependent in its proper implication. Scholars emphasize the relationship as "each is also both and means…. linked together, capabilities, equity and sustainability present a framework or paradigm for development thinking which is both normative and practical" [9]. Here, through separate descriptions, the core meaning of the terms can be understood.

**Capability**: in the developmental studies this was firstly used and systematically studied by Amartya Sen as a theoretical foundation to understand human development, he maintains that income, utilities, resources, and wealth act as means towards an end not an end in themselves, further he states that development should be understood as the removal of major barriers to our freedom like poverty, illiteracy, poor economic opportunities and so on [10].

**Equality**: the traditional method of measuring the equity has been concerned with only relative distribution of income, while it keeps a vast meaning like property, opportunity and capability; and avoids discrimination on any ground [9].

**71**

nature [13].

**3. Glimpses of the tragedy**

3 *World Health Organization, 2002*. World Disaster Report.

*Question of Livelihood in the Light of Disaster: With Special Reference to Flood of Bahraich, India*

Hence, the term 'livelihood' does not merely denote the issue of employment or source of income, but the entire construction of a community viz., polity, society, culture, economy, wherein people live for centuries, comes under the

**Disaster**: The disaster is a natural phenomenon or just a deleterious consequence of human's unnecessary intervention in the natural world, which is a controversial debate across the world. Difficult to conclude that this is an 'act of God' or the human's self-made tragedy, but cannot be denied the fact that modern technological advancement has multiplied the intensity and consistent occurrence of the disaster. The term 'disaster' is derived from astrological science and has been understudies for more than one century, but in terms of its specific academic studies, it has expanded since the 1950s [11]. To formulate the term, the World Health Organization on Emergency and Humanitarian Action (EHA) states that a disaster is an occurrence a level of suffering that exceeds the capacity of adjustment

holds the view as "a disaster is a situation or event which overwhelms local capacity, necessitating a request to a national or international level for external assistance, an unforeseen and often sudden event that causes great damages, destruction and human suffering" [11]. Similarly, "disaster is seen as a process leading to an event that involves a combination of a potentially destructive agent from the natural or technological sphere and a population in a socially produced condition of vulnerability" [12]. There is no major dispute on the character of disaster as 'massive human suffering', but has big dissension over it is either 'man-made' or the 'revenge of god'. Of course, nature naturally generates a number of disasters like cyclones, earthquakes, floods, famine and many more, but the role of modern science and technology should not be overlooked that has escalated the pace of disaster [1]. For instance, the flood of Uttarakhand in 2013, and the Kosi flood of Bihar in 2008 are basically the consequences of unnecessary human encroachment in the domain of

The history of flooding in the region goes back in the deep past. In the known history the region has been gravely affected by flooding from 1922 to 1925. 1946, 1954, 1955, 1960, 1961 and 1963 were also the year of flooding, but the flooding of 1969 was much more devastative than previous years. 1978, 1980, 1981, 1982 and 1983 were also the year of tragedy. In the year of 1983, the sudden release of water from Girija Bairaj of Nepal caused severe damage to the region, while this was not the year of heavy rainfall. Hundreds of people died due to the sudden release of water without any prior information. 1986, 87 and 1988 were not untouched by the flood. In the year 1990 flood-affected a few pockets of the region. In the year 1991, there was no flood, but the Ghaghara river induced soil erosion. Again, in the year 1992 floods made a catastrophe. In the year 1993, it broke the previous records. The

. The Centre on the Epidemiology of Disaster, Brussels

**Sustainability**: there is a prosperous series of intellectual criticism on the developmental model in which production, employment, and poverty reduction are a key concern. It claims that this is mainly made for the welfare of the capitalism and market, and lesser responsible towards the people of the rural and marginalized communities. In recent decades, the developmental model moved to the diverse framework of the society and inclined towards the social, cultural and political field

*DOI: http://dx.doi.org/10.5772/intechopen.95287*

of the communities [9].

periphery of 'livelihood'.

of the affected community3

*Question of Livelihood in the Light of Disaster: With Special Reference to Flood of Bahraich, India DOI: http://dx.doi.org/10.5772/intechopen.95287*

**Sustainability**: there is a prosperous series of intellectual criticism on the developmental model in which production, employment, and poverty reduction are a key concern. It claims that this is mainly made for the welfare of the capitalism and market, and lesser responsible towards the people of the rural and marginalized communities. In recent decades, the developmental model moved to the diverse framework of the society and inclined towards the social, cultural and political field of the communities [9].

Hence, the term 'livelihood' does not merely denote the issue of employment or source of income, but the entire construction of a community viz., polity, society, culture, economy, wherein people live for centuries, comes under the periphery of 'livelihood'.

**Disaster**: The disaster is a natural phenomenon or just a deleterious consequence of human's unnecessary intervention in the natural world, which is a controversial debate across the world. Difficult to conclude that this is an 'act of God' or the human's self-made tragedy, but cannot be denied the fact that modern technological advancement has multiplied the intensity and consistent occurrence of the disaster. The term 'disaster' is derived from astrological science and has been understudies for more than one century, but in terms of its specific academic studies, it has expanded since the 1950s [11]. To formulate the term, the World Health Organization on Emergency and Humanitarian Action (EHA) states that a disaster is an occurrence a level of suffering that exceeds the capacity of adjustment of the affected community3 . The Centre on the Epidemiology of Disaster, Brussels holds the view as "a disaster is a situation or event which overwhelms local capacity, necessitating a request to a national or international level for external assistance, an unforeseen and often sudden event that causes great damages, destruction and human suffering" [11]. Similarly, "disaster is seen as a process leading to an event that involves a combination of a potentially destructive agent from the natural or technological sphere and a population in a socially produced condition of vulnerability" [12]. There is no major dispute on the character of disaster as 'massive human suffering', but has big dissension over it is either 'man-made' or the 'revenge of god'. Of course, nature naturally generates a number of disasters like cyclones, earthquakes, floods, famine and many more, but the role of modern science and technology should not be overlooked that has escalated the pace of disaster [1]. For instance, the flood of Uttarakhand in 2013, and the Kosi flood of Bihar in 2008 are basically the consequences of unnecessary human encroachment in the domain of nature [13].


*Edited by Dorota Kopacz*

The ocular surface refers to the cornea, conjunctiva, lacrimal glands, and lid of the eye. It is composed of the mucosa that lines the globe and palpebrae, the corneoscleral limbus, the corneal epithelium, and the tear film. This book focuses on the tear film, describing its physiology, dynamics, and role in maintaining the health of the ocular surface. It also examines keratoconus, a condition affecting both the preocular tear film and the ocular surface in which the cornea thins and bulges outward, as well as recent possibilities in molecular genetics.

ISBN 978-1-83880-959-1

Ocular Surface Diseases - Some Current Date on Tear Film Problem and Keratoconic Diagnosis

Published in London, UK © 2021 IntechOpen © Ideas\_Studio / iStock


**3. Classification of biomedical waste**

The medical waste generated is a multi-component waste and varies from regular nonhazardous waste. It consists of general items, biological materials to chemicals

#### **Table 3.**

*Classification of medical waste.*

including radioactive substances. The classification of medical waste, as per EPA (USA), EU, the government of UK, and WHO is depicted in **Table 3**.

All these classifications have commonly focused on Infectious waste, hazardous waste, chemical waste, and general waste. WHO's classification is most elaborate and very specific and generally followed irrespective of country and continent.


*2.3. Antimicrobial Activity*

Strains used for determining antimicrobial activity included *Bacillus subtilis* ATCC 6051, *B. alvei* ATCC 6344, *B. cereus* ATCC 21772, *B. megaterium* ATCC 25848, *B. pumilus* ATCC 7061, *Staphylococcus aureus* ATCC 6538, *S. epidermidis* ATCC 14990, *S. saprophyticus* ATCC 15305, *Enterococcus faecalis* ATCC 29212, *Mycobacterium smegmatis* ATCC 19420, *Escherichia coli* ATCC 9637, *Proteus mirabilis* CECT 170, (from Type Culture Spanish Collection), *Pseudomonas aeruginosa* AK 958 (from the University of British Columbia, Department of Microbiology collection), *Salmonella sp*. CECT 456, *Klebsiella oxytoca* LMM2 (clinical isolate, University of La Laguna), and *Candida albicans* CECT 1039. The bacteria cultures were developed in nutrient broth (NB) or brain heart infusion broth (for *E. faecalis* and *M. smegmatis* containing 0.06% Tween 80), and the yeast was cultured in Sabouraud liquid medium at 37 ◦C. All culture media were purchased from Oxoid. The minimum inhibitory concentrations (MICs) were determined for each compound in triplicate by broth microdilution method (range 0.08–40 μg/mL) in 96 well microtitre plates, according to the M07-A9 of the CLSI (Clinical and Laboratory Institute) [32]. Wells with the same proportions of dimethyl sulfoxide (DMSO) were used as controls and never exceeded 1% (*v*/*v*). The starting microorganism concentration was approximately 1–5 × 105 CFU/mL, and growth was monitored by measuring the increase in optical density (OD) at 550 nm (OD550) with a microplate reader (Multiskan Plus II, Tritertek, Huntsville, AL, USA) and viable count in agar plates. The MIC was defined as the lowest concentration of compound that completely inhibits growth of the organisms compared with that of the control after incubation time.

#### **3. Results and Discussion**


*2.2. Tissue Sampling and DNA Extraction*

Plant tissues were sampled from 36 individuals and dried immediately with silica gel at room temperature for DNA extraction. Unique identifiers were provided to the specimens and the tissue samples. Further, the tissue samples were ground to a fine powder using BeadBlaster 24 microcentrifuge homogenizer. Genomic DNA extraction was then performed using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol with minor modifications, where proteinase K was added, followed by the AP1 buffer and RNase A, and the incubation time was increased to 1–3 h. Samples were eluted in Nuclease-Free Water. The isolated DNA was tested for quality by gel electrophoresis (BioRad, Hercules, CA, USA) on a 1% agarose gel and quantity using spectrophotometric analysis (Denovix, Wilmington, DE, USA).


**2. Diagnosis**

The diagnosis is based on clinical and imaging features, histological confirmation, and exclusion of other diseases that can create similar histopathological and clinical findings.

Imaging is mandatory for diagnosis of sarcoidosis and includes as follows:

### **2.1 Chest X-rays and HRCT scan**

A correct staging of pulmonary sarcoidosis based on radiological stage of the disease is necessary to evaluate the sarcoidosis prognosis [1]. Particularly, there are five radiologic stages (forms) of intrathoracic sarcoidosis on chest radiography [2–4] as follows:

**33**

*Early Diagnosis of Sarcoidosis*

*DOI: http://dx.doi.org/10.5772/intechopen.90523*

• Stage IV: pulmonary fibrosis.

older than 50 years old of age;

• pulmonary ground glass opacity;

• consolidation or large nodular opacities;

zones and with subpleural involvement.

**2.2 Cardiopulmonary and laboratory tests**

distribution;

cysts;

and

accepted HRCT scoring system.

infiltrates with parenchymal lesion;

• Stage III: infiltrates alone, parenchymal disease only; and

Main findings of HRCT sarcoidosis are as follows:

• thickening of the peribronchovascular interstitium;

• Stage II: bilateral mediastinal lymphadenopathy (hilar lymphadenopathy) and

HRCT, however, is more accurate in identifying the different manifestations of pulmonary sarcoidosis as well its complications, but we are still searching for an

• hilar and/or mediastinal lymph node enlargement (sometimes with calcifications), classically bilateral hilar and right paratracheal nodal enlargement (Garland triad); left paratracheal and aortopulmonary nodes can be also enlarged. Atypical pattern of nodal enlargement can be observed in patients

• pulmonary interstitial nodules (micro or macronodules) in a perilymphatic

• coarse linear opacities, interlobular septal thickening, honeycombing, and

• architectural distortion, superior hilar retraction, or traction bronchiectasis;

• pulmonary fibrosis (Stage IV) with linear bands of fibrosis from hila to all directions with distortion of normal lung architecture; honeycombing is rare, only in patients with severe fibrosis, mainly in the middle and upper lung

The diagnosis of pulmonary sarcoidosis requires a compatible clinical picture supported by radiologic (X-rays chest and/or HRCT of lungs) and pathologic data (pulmonary functional tests and laboratory tests). But for a certain diagnosis, biopsy is required in most cases, and endobronchial biopsy via bronchoscopy is often performed; its results may be positive even in patients with normal chest radiographs. The central histologic finding is the presence of noncaseating granulo-

Pulmonary function tests are also routinely used in sarcoidosis for early diagnosis and follow-up. They include the evaluation of forced vital capacity (FVC), forced expiratory volume in one second (FEV1), the total lung capacity determination (TLC), vital capacity (VC), residual volume (RV) associated with body

Pleural disease is rare and may be observed when disease is extensive.

mas with special stains negative for fungi and mycobacteria [2].

*Sarcoidosis and Granulomatosis - Diagnosis and Management*

and scleral plaques.

production (rare) [2].

**2. Diagnosis**

clinical findings.

and biopsy yield [2]; and

**2.1 Chest X-rays and HRCT scan**

• Stage 0: normal chest radiographic findings;

therapy) [3].

[2–4] as follows:

patients, but quite uncommon in African-American and Japanese patients.

*Cutaneous involvement* may be present not only with erythema nodosum associated with Löfgren syndrome, but also with lupus pernio, violaceous rash on the cheeks or nose (quite common) and maculopapular plaques (quite uncommon) [2].

*Ocular involvement* is also possible, which may lead to blindness for untreated anterior or (most frequent) posterior granulomatous uveitis, conjunctival lesions

Other uncommon possible manifestations are [2] *nervous system involvement* with lymphocytic meningitis (rare), cranial nerve palsies, hypothalamic/pituitary dysfunction (rare) with diabetes insipidus and myelopathy; *heart failure* from cardiomyopathy (rare) or heart block and sudden death; *osseous involvement* with arthritic syndromes; *blood abnormalities*: anemia, leukopenia, thrombocytopenia and hemolytic anemia with or without splenomegaly (without splenomegaly may reflect bone marrow involvement); *gastrointestinal and genitourinary involvement* (rare): hepatomegaly, cholestasis, portal hypertension, Crohn's disease, pancreatic involvement, nephrocalcinosis, vulva itchiness and male infertility (rare); *exocrine and endocrine manifestations* with hyperprolactinemia, amenorrhea, galactorrhea, or nonpuerperal mastitis in women; hypercalciuria and hypercalcemia likely result from the increased 1,25-dihydroxy vitamin D

The diagnosis is based on clinical and imaging features, histological confirmation, and exclusion of other diseases that can create similar histopathological and

• routine chest computed tomography (CT), usually high-resolution CT (HRCT) scanning of the chest [2] (has a significantly superior detection rate to chest X-ray for mediastinal and pulmonary parenchymal changes and is also useful to identify active alveolitis or fibrosis), and the agreement between findings

• fluoro-deoxy glucose-positron emission tomography (FDG-PET) combined with CT-scan (is fundamental to determinte the extent of organ involvement and the disease activity and to evaluate the response to pharmacological

A correct staging of pulmonary sarcoidosis based on radiological stage of the disease is necessary to evaluate the sarcoidosis prognosis [1]. Particularly, there are five radiologic stages (forms) of intrathoracic sarcoidosis on chest radiography

• Stage I: bilateral mediastinal lymphadenopathy (hilar lymphadenopathy);

Imaging is mandatory for diagnosis of sarcoidosis and includes as follows:

• chest radiography (is fundamental for a first evaluation);

**32**

HRCT, however, is more accurate in identifying the different manifestations of pulmonary sarcoidosis as well its complications, but we are still searching for an accepted HRCT scoring system.

Main findings of HRCT sarcoidosis are as follows:

Pleural disease is rare and may be observed when disease is extensive. The diagnosis of pulmonary sarcoidosis requires a compatible clinical picture supported by radiologic (X-rays chest and/or HRCT of lungs) and pathologic data (pulmonary functional tests and laboratory tests). But for a certain diagnosis, biopsy is required in most cases, and endobronchial biopsy via bronchoscopy is often performed; its results may be positive even in patients with normal chest radiographs. The central histologic finding is the presence of noncaseating granulomas with special stains negative for fungi and mycobacteria [2].


**12. Benzo[4,5]thiazolopyrimido[5,4-d]pyrimidines**

Medhabati *et al*. [90] reported the synthesis of 2-thio-5-arylbenzo[4,5] thiazolopyrimido[5,4-d]pyrimidin-4-one derivatives under aqueous medium. Benzo[4,5]thiazolopyrimido[5,4-d]pyrimidines were synthesized by condensation

**Figure 14.** *Synthesis of 2-thio-5-arylbenzo[4,5]thiazolopyrimido[5,4-d]pyrimidin-4-ones.*

of thiobarbituric acids, 2-aminobenzothiazole and aldehydes using metal-proline as catalyst (**Figure 14**).


**5. Conclusion**

372 Practical Applications in Biomedical Engineering

acquisition adjustments and/or image subtractions.

**Cardiac Index Mouse**

failure).

and human myocardium.

**4. Future perspectives**

**4.1. Molecular imaging**

As an extension of the development and use of such techniques (DENSE, tagging, HARP) has been the tremendous value for regional cardiac functional quantification and direct applicability to transgenic mice and in pathological states (myocardial infarction, heart

Such approaches are, however, associated with a number of limitations and drawbacks, including the necessity for use of complex algorithms and laborious data post-processing (tagging, HARP, and DENSE), the inherently low spatial resolution for strain quantification (tagging vs. DENSE), the T1-tag dependence, the low SNR performance of DENSE (often with a temporal decreasing dependence following ECG-triggering), and the necessity to eliminate the anti-echo and free-induction decay signals in DENSE through proper

**(n=9)**

Myocardial Mass - 153.1±37.2 g rEF 38.0±25.0 % 52.2±15.8 % Wall Thickness 27.9±22.4 % 42.6±21.8 % Wall Motion 2.8±3.0 % 4.8±2.4 % **Table 2.** Summary of mean global and regional cardiac mechanical functional indices (±sd) of murine

In the new era of molecular imaging, MRI faces major challenges in accomplishing detection of molecular probes with increased sensitivity and specificity, comparable to other diagnostic techniques (such as PET/SPECT). Currently, 10-100 μmolar sensitivity is attained by MRI in contrast to the established nano- and picomolar sensitivity of PET and SPECT. While a new generation of contrast agents is anticipated to extend current limits shedding new light into cellular and molecular mechanisms, current efforts focus primarily on stem cell technology,

Advances in the biology of stem cells have evoked great interest in cell replacement therapies for the regeneration of heart tissue after myocardial infarction. Despite the initial

cellular tracking, and construction of hybrid PET/MRI systems, as discussed below.

*Regenerative techniques – Stem cell technology and cellular tracking*

EF 50.7±3.7 % 64.4±2.4 % EDV 45.4±11.0 μl 117±32.7 ml ESV 22.5±6.2 μl 46.5±10.3 ml SV 22.9±5.4 μl 83±15.4 ml CO 1.4±0.3 ml/min 5.0±0.9 l/min

**Human (n=8)**

> In the short-lived period of mouse cardiac MRI of the past 15 years, tremendous strides have been made for image-based phenotyping of the cardiovascular system. Such were realized in terms of the scalability of equipment, ease of handling and maintaining animals under proper physiological homeostatic conditions, adaptation of conventional imaging techniques, and inception of new, fast imaging acquisition schemes that have revolutionized cardiac, image-based phenotyping with efficient, high-throughput imaging protocols.

> In conclusion, despite the usefulness, practicality, and low costs associated with the study of the mouse, important genetic, developmental, morphological, and cardiovascular system differences exist between mouse and man especially as such are unmasked in pathological conditions or under stress. Physiological results indicate that the optimal ISO anesthetic regimen for mouse studies under anesthesia is approximately 1.5% v/v, yielding stable MAP and HR values comparable to those observed in the animal's conscious state, with a minute-tominute variability in MAP and HR of ≤11%. Based on such recordings, the optimal FiO2 appears to be 50%. The additional use of N2O was associated with higher and more stable values of MAP and HR (at a mixture of 25–50% O2 and 75–50% N2O). Arterial pH values are within the physiological range and varied between 7.20 and 7.43. ISO anesthesia at 1.5% v/v is also associated with mild hyperglycemia (+47%) whereas insulin levels remain unaltered. The protocol described for physiological studies of mice under anesthesia has the potential for high reproducibility in diagnostic modalities, including MRI, microCT, ultrasound, and microPET. This regimen can be useful in phenotypic screening and pharmacological studies of cardiac function in mice and can facilitate the transfer of such work to noninvasive imaging platforms, with tremendous potential for both basic science and translational research.

> Basic MRI studies of murine global cardiac structure and function under optimal physiological conditions, in combination with PCA and other image processing techniques,

can identify modal components of shape variability and disseminate components of global mechanical motion. Such atlases can be population-based instead of single-subject based and can serve as a powerful reference tool for morphological and functional inter-strain mouse studies, complementary to current ongoing efforts for image-based phenotyping that target the cardiovascular system. Based on constructed morphometric maps and atlases using principal component analysis in C57BL/6J, it is found that in probabilistic atlases, a gradient of probability exists for both strains in longitudinal locations from base to apex. Based on the statistical atlases, differences in size (49.8%), apical direction (15.6%), basal ventricular blood pool size (13.2%), and papillary muscle shape and position (17.2%) account for the most significant modes of shape variability for the left ventricle of the C57BL/6J mice. Correspondingly, for DBA mice, differences in left ventricular size and direction (67.4%), basal size (15.7%), and position of papillary muscles (16.8%) account for significant variability. These data reason in favor of existing variability in the apical location in both strains, a direct consequence of the heart's effort to re-establish the position of the apex in a consistent manner at end-diastole. Additionally, higher variability exists in DBA mice in the location of the papillary muscles, as well as in the epicardial areas of the left ventricle.

Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging:

I would like to thank Professors G. A. Johnson, G. Truskey, L. Hedlund, and Dr. E. Bucholz, Mr. G. Cofer, and Mr. J. Cook at Duke University, Drs. R. Balaban and A. Koretsky, and Mr. D. Despres and Dr. M. Lizak at the National Institutes of Health, Prof. D. Rueckert at Imperial College, London, Professor BA Janssen at Maastricht Cardiovascular Institute, and my fellows and students Dr. D. Perperidis, Mr. N. Aristocleous, Mr. R. Mean, Mr. S. Gkagkarelis, and Mr. S. Angeli at the University of Cyprus. I am indebted to the help and support from Remcom Inc. I am also grateful for the availability of the Image Registration Toolkit used under License from IXICO Ltd and to Dr. E. Treiber from Bruker Biospin for

I would also like to acknowledge grant support from the Research Promotion Foundation (RPF) under grants RPF/TEXNOLOGY/MHXAN/0609(BE)/05, RPF/STOXOS/0302/02, and

[1] Aletras A, Ding S, Balaban RS, Wen H. DENSE: Displacement encoding with stimulated

[2] Ali A, Dale AM, Badea A, Johnson GA. Automated segmentation of neuroanatomical structures in multispectral MR microscopy of the mouse brain. Neuroimage 2005;

[3] Axel L, Dougherty L. MR Imaging of motion with spatial modulation of magnetization.

[4] Badea C, Fubara B, Hedlund L, Johnson G. 4D micro-CT of the mouse heart. Molecular

[5] Balaban RS, Hampshire VA. Challenges in Small Animal Noninvasive Imaging. ILAR

[6] Barbee RW, Perry BD, Re RN, Murgo TP. Microsphere and dilution techniques for the determination of blood flows and volumes in conscious mice. Am J Physiol Regulatory

[7] Barbee RW, Perry BD, Re RN, Murgo JP, Field LJ. Hemodynamics in Transgenic Mice

[8] Beg MF, Helm PA, McVeigh E, Miller MI, Winslow RL. Computational cardiac anatomy

[9] Bernston GC, Bigger JT Jr, Eckberg DL, Grossman P, Kaufmann PG, Malik M, Nagaraja HN, Parges SW, Paul JP, Stone PH, Van Der Molen MW. Heart rate variability: origins,

[10] Berr SS, Roy RJ, French BA, Yang Z, Gilson W, Kramer CM, Epstein FH. Black blood gradient echo Cine magnetic resonance imaging of the mouse heart. Magn Reson in

with Overexpression of Atrial Natriuretic Factor. Circ. Res. 1994; 74:747-751.

methods, and interpretive caveats. *Psychophysiol*. , 1997; 34:623- 648.

echoes in cardiac functional MRI. J. Magnetic Resonance 1999; 137: 247-252.

**Acknowledgement**

RPF/PROSVASI/0302/01.

**6. References**

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the provision of the cardiac MR images.

Radiology 1989; 171(3):841-5.

Imaging 2005; 4(2):110-116.

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The Current Status, Challenges, and Future Perspectives 375

On the forefront of direct, high-field acquisitions using RF technologies with mouse cardiac MRI, the commercially available birdcage outperforms cylindrical spiral multi-turn surface coils in relative signal-to-ratio (SNR) by a factor of 3–5 times as assessed by experimental measurements, simulations, and experiments in free space, and under phantom and animal loading conditions. Nevertheless, quantitative comparison of the performance of the two spiral coil geometries in anterior, lateral, inferior, and septal regions of the murine heart yield maximum mean percentage rSNR increases to the order of 27–167% post-mortem (cylindrical compared to flat coil), values that are by far higher than previous designs of surface coils and comparable to receiver phased array performance.

Such hardware improvements, in association with fast radial pulse sequence acquisitions, may also lead to quantification of global and regional functional parameters in various mouse strains. While morphological differences appear only to relate to increased papillary muscle variability in the DBA/2J mice, nevertheless interstrain cardiac hemodynamics, based on dynamic cardiac MRI acquisitions, do not exhibit significant differences for neither the LV nor RV in C57BL/6J or DBA/2J mice. Comparative to previous reports of global functional indices [Bucholz 2010], similar mouse and human results were also observed. The work supports the validity of the hypothesis of functional scaling in mice and humans.


A puzzling finding of our study is that the observed rescuing did not correspond to an improvement in the analysed bioenergetic aspects. Steady-state levels of representative OXPHOS subunits of complex I and IV remained low in the MELAS cybrids overexpressing Cterm, as did the level of assembled complex I and IV. Accordingly, complex IV activity and mitochondrial oxygen consumption rate did not recover in the presence of the Cterm domain. Moreover, Cterm expression did not influence the higher glycolytic flux peculiar of MELAS cells, as shown by lactate measurement. Interestingly, our MELAS cell line displayed a strong accumulation of ATP synthase F1-c subcomplexes, named V\*. It was shown in cultured cells [38] that intermediate subcomplexes V\* increase when mitochondrial translation is inhibited by doxycycline, indicating that, in order for the subcomplex to evolve to the fully assembled holoenzyme, the mitochondrially encoded subunits ATP6 and ATP8 are needed. The strong increment of subcomplex V\* in MELAS cells is in accordance with the observed decreased level of subunits ATP6 and ATP8 (see Figure 2). Moreover, Cterm did not influence the complex V profile either on a quantitative or qualitative basis. The fact that overexpressed Cterm did not improve these bioenergetic defects could be explained by assuming that, although increased, the observed translation efficiency is still below a threshold for respiratory chain improvement. Even a longer timescale of Cterm expression was not able to produce any increment in the OXPHOS subunit level (data not shown). In light of this, the slight increase in mitochondrial protein synthesis observed in

mock cells, which is anyhow lower than that caused by Cterm, has a negligible functional significance, especially considering that no recovery of cell viability occurs upon mock transfection. Finally, Cterm peptide had no impact on mitophagy and mitochondrial mass.

It is important to point out that, unlike what has been here described, some of us reported an increase in oxygen consumption using another MELAS cell line transfected with Cterm [14,16]. This discrepancy suggests that distinct MELAS cybrid lines may have peculiar molecular pathways and compensatory mechanisms, in parallel with the various phenotypes observed in MELAS patients with similar levels of heteroplasmy.

Overall, the reported data tend to exclude that Cterm capacity to restore cell viability to the wild type level derives from a direct involvement of the mitochondrial functions analysed herein, suggesting that other mitochondrial pathways, as well as those extra-mitochondrial, that are possibly triggered by the mitochondrial status are involved. Mitochondrial dysfunction can influence nuclear gene expression in mammalian systems by means of retrograde signalling mediated by ROS, Ca2+, ADP/ATP, NAD/NADH, and non-coding RNAs [39,40]. A study of transcriptional reprogramming conducted in the MELAS cybrids highlighted various metabolic pathways that are differentially triggered by variations in mtDNA 3243 mutation heteroplasmy [41]. These pathways, which represent both adaptive and maladaptive cellular responses, refer to a series of genes related to glycolysis for energy production, antioxidant and redox regulatory systems, autophagy/mitophagy, apoptosis, chromatin remodelling, and compartment-specific unfolded protein stress response. In this context, nuclear mRNAs or non-coding RNAs of either nuclear or mitochondrial origin, such as processed mt-tRNA fragments exported to the cytoplasm or circular RNAs [40,42–44], may represent possible targets of cytosolic RNA-binding proteins for regulation of nuclear gene expression. In addition to mitochondrial retrograde signalling to the cytoplasm, the full recovery of cell viability might be the result of Cterm capacity to bind cytosolic RNA targets. This hypothesis is supported by the observation that: (i) a certain amount of overexpressed Cterm is not imported into mitochondria and remains in the cytosol [13]; and (ii) Cterm interacts also with non-tRNA targets (see Figure 1). This circumstance is in accordance with the hypothesis that the Cterm mode of RNA recognition relies more on structural rather than on specific base– base interactions, as observed in the available 3D structures of whole LeuRS-tRNALeu complexes from bacterial species [45]. This feature is reminiscent of the promiscuous RNA binding mode typical of aaRSs [22,46].

In conclusion, our results on the rescuing ability of Cterm in MELAS cybrids open up to pathways that, in our cellular model, seem to be independent of the mitochondrial bioenergetics. Importantly, the demonstration that Cterm has rescuing activity in MELAS cybrids, in spite of differences in the apparent mechanism of action, strongly supports the therapeutic potential of the Cterm and of molecules derived thereof [17], opening up new perspectives for the treatment of mitochondrial diseases. Further work on additional cellular models, preferably tissue-specific, will be required to fully unveil the molecular basis of Cterm rescuing activity.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/life11070674/s1, Figure S1: Stable expression of Cterm-FLAG transcript, Figure S2: Raw data from RIP experiments, Figure S3: RFLP analysis and quantification of m.3243A>G heteroplasmy in MELAS cybrids, Figure S4: Transient expression of Cterm-FLAG transcript, Figure S5: Gel scans and densitometry data of metabolic [35S]-methionine labelling replicates after 48-h transfection, Figure S6: Gel scans and densitometry data of metabolic [35S]-methionine labelling replicates after 72-h transfection; Figure S7: Relative quantification of [35S]-labelling signals, Figure S8: Effect of pBAD and pQE60 transfection on metabolic [35S]-methionine labelling of mitochondrial translation products, Figure S9: Effect of pBAD and pQE60 transfection on mt-tRNALeu(UUR) and mt-tRNAGlu steady-state levels, Figure S10: Relative quantification of SDS-PAGE and BN-PAGE signals.

**Author Contributions:** Conceptualization, G.d., P.C., M.R., P.L.P. and F.B.; methodology, M.R., P.L.P. and F.B.; formal analysis, F.C., F.R., G.P. and F.B.; investigation, F.C., F.R., G.P. and F.B.; resources, G.d. and P.L.P.; writing—original draft preparation, M.R., P.L.P. and F.B.; writing—review and editing, G.d., V.M., P.C., M.R., P.L.P. and F.B.; supervision, M.R., P.L.P. and F.B.; project administration, M.R., P.L.P. and F.B.; funding acquisition, G.d., M.R., P.L.P. and F.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Progetto competitivo "Sviluppo Catalisi dell'Innovazione nelle Biotecnologie" (CIB); Fondi di Ateneo "Contributo Ordinario di Supporto alla Ricerca" 2015/16 and 2017/18 (University of Bari Aldo Moro); AFM Télethon and Istituto Pasteur Italia-Fondazione Cenci Bolognetti.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Aleksandra Filipovska (University of Western Australia) for supplying MELAS transmitochondrial cybrids and to Claudio Lo Giudice (University of Bari Aldo Moro) for his kind help with statistical analyses.

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

#### **References**

## *Article* **Subcellular Localization of Fad1p in** *Saccharomyces cerevisiae***: A Choice at Post-Transcriptional Level?**

**Francesco Bruni 1, Teresa Anna Giancaspero 1, Mislav Oreb 2, Maria Tolomeo 1, Piero Leone 1, Eckhard Boles 2, Marina Roberti 1, Michele Caselle 3 and Maria Barile 1,\***

**Abstract:** FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In *Saccharomyces cerevisiae*, the gene encoding for FAD synthase is *FAD1*, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that *FAD1* generates two echoforms that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3 region of *FAD1* mRNA was analyzed by 3- RACE experiments, which revealed the existence of (at least) two *FAD1* transcripts with different 3- UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3- UTRs allowed us to predict the existence of a *cis*-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3- UTR context. Here, we propose that the long *FAD1* transcript might be responsible for the generation of mitochondrial Fad1p echoform.

**Keywords:** FAD synthase; *FAD1*; mitochondria localization; *Saccharomyces cerevisiae*; mRNA; mitochondrial localization motif

#### **1. Introduction**

Riboflavin (Rf or vitamin B2) deficiency in humans and experimental animals has been linked to several diseases, such as cancer, cardiovascular diseases, anemia, abnormal fetal development, different neuromuscular and neurological disorders, some of which are treatable with high doses of riboflavin. Among the latter, there are the multiple acyl-CoA dehydrogenase deficiency (MADD) and the Brown–Vialetto–Van Laere syndrome (BVVLS) (see [1] and refs therein). The role of this vitamin in cell metabolism depends on its conversion into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are the redox cofactors of a large number of dehydrogenases, reductases, and oxidases. Most of these flavoenzymes are compartmented in the cellular organelles, where they are involved in energy production and redox homeostasis as well as in different cellular regulatory events including protein folding, apoptosis, and chromatin remodeling [2–4]. The relevance of such processes merits further research aimed to better describe flavin homeostasis and flavoenzyme biogenesis, especially in those organisms that can be simple and suitable models for human pathologies. The conservation of the main biological processes within all eukaryotes, together with the possibility of simple and quick genetic manipulation, make the budding yeast, *Saccharomyces cerevisiae,* a suitable model to understand the molecular mechanisms underlying human diseases [5–8].

**Citation:** Bruni, F.; Giancaspero, T.A.; Oreb, M.; Tolomeo, M.; Leone, P.; Boles, E.; Roberti, M.; Caselle, M.; Barile, M. Subcellular Localization of Fad1p in *Saccharomyces cerevisiae*: A Choice at Post-Transcriptional Level? *Life* **2021**, *11*, 967. https:// doi.org/10.3390/life11090967

Academic Editor: Theodoros Rampias

Received: 29 July 2021 Accepted: 13 September 2021 Published: 14 September 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/).

Yeasts, as well as other fungi, plants and bacteria, have the ability either to synthesize Rf de novo or to take it from outside, whereas mammals must obtain Rf from diet. The first eukaryotic gene coding for a cellular Rf transporter was identified in *S. cerevisiae* as the product of the *MCH5* gene [9]. More recently, three human Rf transporters have been cloned and characterized; they belong to a novel family of Rf transporters, namely, RFVT/SLC52, which exhibit no significant similarity to Mch5p [10,11]. Mutations in RFVTs have been recently linked to BVVLS, a neurodegenerative disorder characterized by cranial nerve deficits, bilateral sensorineural deafness, respiratory insufficiencies, and the degeneration of some spinal cord neurons [1,12].

Intracellular conversion of Rf to FAD is a ubiquitous pathway and occurs via the sequential actions of ATP:riboflavin 5'-phosphotransferase or riboflavin kinase (RFK, EC <IP_ADDRESS>), which phosphorylates the vitamin into FMN, and of ATP:FMN adenylyl transferase or FAD synthase (FADS, EC <IP_ADDRESS>) that adenylates FMN to FAD. Eukaryotes generally use two different enzymes for FAD production, whereas most prokaryotes depend on a single bifunctional enzyme [13,14]. The first eukaryotic genes encoding for RFK and FADS were identified in *S. cerevisiae* and named *FMN1* [15] and *FAD1* [16], respectively.

Fmn1p is a 24.5 kDa protein showing a sequence and structure similarity to the RKF-module of prokaryotic FADS and appears largely conserved through evolution. Immunoblotting analysis of subcellular fractions revealed that Fmn1p is localized in microsomes and in mitochondria [15]. Orthologs of *S. cerevisiae FMN1* have been cloned and the corresponding proteins purified from the yeast *Schizosaccharomyces pombe* and from *Homo sapiens*. The crystal structures of both these proteins have been solved, revealing a novel ATP and Rf-binding fold [17–19].

Fad1p, the sole known protein isoform generated by *S. cerevisiae FAD1* gene, is a 35.5-kDa soluble enzyme that is essential for yeast life, whose crystal structure has been solved in a complex with FAD in the active site [20]. Fad1p is a single-domain monofunctional enzyme belonging to the 3- -phosphoadenosine 5- -phosphosulfate (PAPS) reductase family and shows little or no sequence similarity to the prokaryotic FAD-forming enzymes. The human gene for FADS, named *FLAD1* [14,21], generates different alternatively spliced transcripts encoding different isoforms, most of them containing the PAPS reductase domain fused with an N-terminal molybdopterin-binding (MPTb) domain with FAD hydrolyzing activity [22].

Our group previously demonstrated that *S. cerevisiae* mitochondria are able to catalyze FAD hydrolysis via enzymatic activity, which is different from the already characterized NUDIX hydrolases and regulated by the mitochondrial NAD redox status [23].

The relevance of *FLAD1*, postulated in our previous papers [24,25], emerged in 2016 when *FLAD1* was identified as a novel Riboflavin-responsive MADD disease gene [26]. MADD defines a heterogeneous class of lipid storage myopathies associated with impaired fatty acid, amino acid and choline metabolisms [27]. In the frame of these studies, a novel isoform containing the sole PAPS domain, hFADS6, was identified and then characterized in some detail [28]. The homology modeling of the PAPS reductase domain of human FADS was performed using the orthologue from *Candida glabrata* as a template [28].

The simultaneous presence in the same cell of the different *FLAD1* transcripts is in line with a possible different subcellular localization for the different isoforms in mammals. Consistently, the existence of distinct cytosolic, nuclear and mitochondrial FADS was demonstrated in different rat models [29,30]. The human transcript variant 1 encodes a 65.3 kDa protein, hFADS1, which contains a predictable mitochondrial-targeting peptide [21]. Mitochondrial localization of hFADS1 was proven in vitro by mitochondrial import assay and confocal microscopy [29].

Mitochondrial supply of FAD is crucial also in yeast, where it is expected to be delivered to a number of nascent client apo-flavoenzymes [31]. This occurs in some cases, such as for the flavoprotein subunit of succinate dehydrogenase (Sdh1p), via an autocatalytic covalent flavinylation mechanism, which could be assisted by ancillary proteins [32–36].

The first eukaryotic gene encoding for a mitochondrial FAD translocator was identified in *S. cerevisiae* and named *FLX1*, whose human orthologue is *SLC25A32.* Interestingly, mutations in the *SLC25A32* gene have been identified as the cause for RR-MADD [37,38], thus confirming the essential role of flavin cofactor supply for mitochondrial proteome.

The existence of a mitochondrial FADS isoform in yeast is still controversial as well as the physiological role of the mitochondrial FAD translocator, Flx1p. Initially, it was reported that FAD is synthesized by Fad1p exclusively in the cytosol [16] and, consequently, imported into mitochondria via Flx1p [39]. However, results from our laboratory showed that, besides in the cytosol, FAD-forming activities can be specifically revealed in mitochondria, entailing FAD precursors uptake in mitochondria and mitochondrial FAD export to cytosol via Flx1p [40,41]. Moreover, FAD movement across the mitochondrial membrane catalyzed by Flx1p plays an additional regulatory role on apo-Sdh1p biogenesis at the post-transcriptional level [42].

As a matter of fact, the protein responsible for FAD synthesis in *S. cerevisiae* mitochondria remains to be identified and characterized. In this paper, we report the existence of *S. cerevisiae* Fad1p as two distinct echoforms localized to both cytosol and mitochondria, and the presence of two populations of *FAD1* mRNAs, which differ for their 3- UTRs. Whether and how the 3- UTRs play a role in the mechanism that destines Fad1p to either cytosol or mitochondria will be dealt with.

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

#### *2.1. Materials*

All reagents and enzymes were from Sigma-Aldrich (St. Louis, MO, USA), ThermoFisher Scientific (Waltham, MA, USA) and Merck KGaA (Darmstadt, Germany). Zymolyase was from MP Biomedicals (Aurora, OH, USA). Bacto Yeast Extract and Yeast Nitrogen base were from BD Difco (Franklin Lakes, NJ, USA). Monoclonal antibody against STREP-tag (α-STREP) was obtained from IBA Lifesciences (Göttingen, Germany), monoclonal antibody against actin (α-ACTIN) was from Abcam (Cambridge, MA, USA), secondary anti-rabbit or anti-mouse IgG antibodies conjugated with peroxidase were from ThermoFisher Scientific (Waltham, MA, USA). The RNeasy Midi Kit was from Qiagen (Hilden, Germany). The enhanced AMV Reverse transcriptase kit was from Sigma-Aldrich (St. Louis, MO, USA). The Dynabeads mRNA Purification kit was from Invitrogen (Waltham, MA, USA). The Wizard SV Gel and PCR Clean-up system was from Promega (Madison, WI, USA).

#### *2.2. Yeast Strains and Recombinant Multicopy Plasmids*

The wild-type *S. cerevisiae* strain CEN.PK113-13D (also named EBY157, WT, or K26; genotypes *MAT*α, *MAL2*-*8c* , *SUC2* and *ura3*-*52*) was previously described in [40,42]. The CENPK2-1C, derived from the CEN.PK yeast series, was used as a recipient strain to express a recombinant Fad1p carrying a STREP tag at the N- or C-terminal end. To construct the multicopy plasmids p426HXT7-FAD1-STREP, p426-MET25-FAD1-STREP, and p426-MET25- STREP-FAD1, the *FAD1* gene was cloned by PCR with a selected pair of primers designed to amplify the complete *FAD1* open reading frame (ORF), skipping the contiguous 5 and 3- UTR regions. The amplified fragments were cloned into the multicopy plasmid p426- HXT7-STREP or p426-MET25-STREP using the standard gap-repair procedure. In these vectors, the recombinant *FAD1* construct, encoding Fad1p fused to the STREP tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at the N- or C-terminal end, was placed between the *HXT7* or the *MET25* promoter and *CYC1* terminator. The transformation of the CEN.PK113-13D and CENPK2-1C strains with the recombinant or empty vectors was carried out according to the frozen competent cell procedure [43]; the transformed strains were selected for the presence of URA3 genetic marker, which confers the ability to grow in a minimal synthetic liquid medium (SM, 6.7 g/L yeast nitrogen base supplemented with 25 mM Histidine, 0.44 mM Leucin and 0.19 mM Tryptophan) without uracil. All the transformed strains used in this study are summarized in Table 1.

**Table 1.** *S. cerevisiae* strains used in this study.

#### *2.3. Media and Growth Conditions*

The wild-type K26 cells were grown aerobically at 30 ◦C with constant shaking in rich liquid medium (YEP, 10 g/L yeast extract, 20 g/L Bacto Peptone). The K26 transformed strains were grown aerobically at 30 ◦C with constant shaking in SM medium. The CENPK2-1C strain and the novel derived strains (see Table 1) were grown aerobically at 30 ◦C with constant shaking in SM medium. Ethanol, glycerol, galactose or glucose (2% each) were used as carbon sources. The solid medium (YEP or SM) contained 18 g/L agar.

#### *2.4. Preparation of Cellular Extracts*

Cells grown up to the early exponential phase (5 h) were harvested by centrifugation (8000× *g* for 5 min), washed with sterile water, resuspended in 250 μL of lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethanesulfonylfluoride (PMSF), supplemented with one tablet of Roche protease inhibitor cocktail every 10 mL of lysis buffer), and vortexed with glass beads for 10 min at 4 ◦C. The supernatant was removed and centrifuged at 3000× *g* for 5 min to remove cell debris.

#### *2.5. Preparation of Spheroplasts, Cytoplasm, Mitochondria, and Submitochondrial Fractions*

Spheroplasts were prepared using Zymolyase. Mitochondria and cytoplasm were isolated from spheroplasts as described in [23,40]. Mitochondria, ruptured by osmotic shock, were centrifuged at 20,000× *g* for 30 min to separate the mitochondrial soluble fraction and the mitochondrial membrane-enriched fraction.

#### *2.6. Western Blotting*

Protein from subcellular fractions or from cellular extracts were separated by SDS-PAGE and transferred onto a PVDF membrane as in [42]. The immobilized proteins were incubated with either a monoclonal antibody against the STREP-tag raised in mouse (α-STREP) to detect the recombinant Fad1p or a polyclonal α-FADS antiserum (2000-fold dilution) raised in rabbit to detect Fad1p [29]. Immuno-reactive materials were visualized with the aid of a secondary anti-rabbit or anti-mouse IgG antibody conjugated with peroxidase. PVDF membranes were also probed with monoclonal α-ACTIN antibody (10,000-fold dilution, raised in mouse), to reveal yeast actin (Act1p).

#### *2.7. RNA Isolation*

The RNeasy Midi Kit was used to extract total RNA from WT cells grown up to early exponential phase. Total RNA concentration was quantified by monitoring absorbance to 260 nm (OD260 nm), RNA integrity was verified by formaldehyde agarose gel electrophoresis and its purity was evaluated by measuring the OD260 nm/OD280 nm ratio.

#### *2.8. 3*- *RACE Analysis*

3- RACE analysis was performed on poly(A)+ RNA obtained from 75 μg of total RNA using the Dynabeads mRNA Purification kit, according to the manufacturer's instructions. The purified poly(A)+ RNA was reverse-transcribed using the Enhanced AMV Reverse Transcriptase Kit and the oligo(dT)-anchor primer [oligo(dT)-AP], according to the manufacturer's instructions. The cDNA pool was diluted 10-fold and these were used

as a template (2.5 μL) for PCR reactions using a gene-specific primer and the AP primer. The specificity of the first round PCR-amplified fragment was proved by performing a semi-nested PCR. The sequence of primers, used in this study, are reported in Table 2. The 3- RACE products were separated by 2% agarose gels. The fragments obtained with the first round PCR were purified from preparative agarose gel by using the Wizard SV Gel and PCR Clean-up system and then sequenced.

**Table 2.** Primers used in this study.

#### *2.9. Semiquantitative RT-PCR Assay*

Semiquantitative RT-PCR assay was performed on 200 ng of total RNA that was reverse-transcribed using the Enhanced AMV Reverse Transcriptase Kit. The cDNA pool was diluted 10-fold and 1 μL was used as a template in PCR-amplification using genespecific primers (Table 2). Amplification conditions were as follows: 94 ◦C for 2 min, then 25 cycles of 94 ◦C for 15 s, 60 ◦C for 30 s, and 68 ◦C for 1 min. For each sample, *ACT1* mRNA was used as an internal control. *SDH1* mRNA was used as a control of inducible expression under nonfermentable carbon source growth conditions.

#### *2.10. Other Assays*

Fumarase (FUM), succinate dehydrogenase (SDH) and phosphoglucoisomerase (PGI) activities were spectrophotometrically measured as in [40,42,44]. Protein concentration was assayed according to Bradford [45], using bovine serum albumin as the standard.

#### **3. Results**

#### *3.1. Mitochondrial Localization of Recombinant Fad1p*

Our previous results based on functional approaches showed that, besides in the cytosol, FAD synthesizing activity can be specifically revealed in mitochondria from *S. cerevisiae* [40,46]. This result was somewhat surprising since a single gene, named *FAD1*, was reported as coding for a sole cytosolic FADS [16]. *FAD1* gene, as normally occurs in yeasts, has no introns; thus, different transcripts generated by alternative splicing are not expected. Consistently, a single transcript is reported in the Ensemble database (http://www.ensembl.org/index.html, accessed on 13 September 2021) from which a single translation product, Fad1p, is predictable with NCBI tool ORF finder (www.ncbi.nlm. nih.gov/orffinder, accessed on 13 September 2021). No mitochondrial-targeting peptide could be found in this product using several prediction programs, such as iPSORT, MitoProt, TargetP and PSORT II. Concerning these aspects, the yeast *FAD1* differs from the human *FLAD1* gene, which generates alternatively spliced isoforms with different subcellular localizations [29,30]. Nevertheless, one of these isoforms, hFADS6, strongly resembles the yeast protein Fad1p, showing a high level of conservation for the PAPS reductase domain (Figure 1).

**Figure 1.** Alignment of FAD synthase sequences from yeast and humans. The alignment of yeast Fad1p with human FAD synthase isoform 6 (hFADS6) was performed by the *Pairwise Sequence Alignment* tool available at the EMBL-EBI website. The PAPS reductase domain (InterPro accession: IPR002500), present in both *S. cerevisiae* Fad1p (amino acids 55-231) and hFADS6 (amino acids 132-286), is underlined and colored in red. Asterisks depict identical residues in the same positions, whereas colons and periods indicate strongly and weakly similar residues, respectively.

To gain some insight into the cellular destination of Fad1p, we investigated the subcellular localization of the recombinant form of Fad1p carrying the STREP-tag at the C-terminus and overexpressed in the CENPK2-1C strain (Table 1). Therefore, subcellular fractions (spheroplasts, mitochondria and cytoplasm) were prepared from CENPK2- 1CFAD1-STREP cells grown in ethanol up to early exponential growth phase and equal amounts of each fraction were analyzed by immunoblotting using the α-STREP antibody (Figure 2a). Even though the recombinant Fad1p–STREP was enriched in the cytoplasmic fraction, an evident immuno-reactive band was observed in the mitochondrial fraction, suggesting a mitochondrial localization for the recombinant Fad1p–STREP. Interestingly, the recombinant Fad1p–STREP was immunodecorated at the same size both in mitochondria and in cytoplasm. When the STREP-tag was moved from the C- to the N-terminal end, the ability of the recombinant STREP–Fad1p to enter mitochondria was completely lost, accompanied by a significant enrichment in the cytoplasmic fraction (Figure S1).

To further prove the ability of the recombinant Fad1p to enter mitochondria, we placed Fad1p–STREP expression under the control of HXT7 promoter in the multicopy plasmid p426-HXT7-FAD1-STREP (Table 1) and used a different genetic strain, namely, K26. Subcellular fractions were prepared from K26FAD1-STREP cells grown in ethanol at the early exponential phase and analyzed by immunoblotting carried out with the α-STREP (Figure 2b). The recombinant protein Fad1p–STREP was immunodecorated at the same size in both cytoplasm and mitochondria, where it was significantly enriched. When the K26FAD1-STREP cells were in the presence of the uncoupler carbonyl cyanide-ptrifluoromethoxy-phenylhydrazone (FCCP), the Fad1p–STREP import into mitochondria was prevented (Figure 2b), as expected for a process depending on mitochondrial membrane potential [47]. This finding clearly indicates that the recombinant Fad1p is imported and localized to mitochondria.

**Figure 2.** Mitochondrial localization of recombinant Fad1p. Spheroplasts (Sphero), mitochondria (Mito) and cytoplasm (Cyt) were prepared from CENPK2-1C (**a**) and K26 (**b**) cells transformed with the multicopy recombinant plasmid (Table 1). Both strains were grown up to the early exponential phase on ethanol. K26FAD1-STREP cells were incubated in the absence or presence of FCCP (25 μM). Proteins (0.1 mg) were separated by SDS-PAGE, transferred onto PVDF membrane and detected with either α-STREP or α-ACTIN antibodies.

#### *3.2. Natural Fad1p Exists in Mitochondria*

To ascertain whether the activity of FADS that we detected inside mitochondria is actually accountable to Fad1p, we analyzed the subcellular localization of natural Fad1p in cells grown either on nonfermentable (glycerol and ethanol) or fermentable (glucose and galactose) carbon sources up to the early exponential phase (5 h) (Figure 3a). A Fad1p band was detected in mitochondria prepared from glycerol-grown cells by using the α-FADS antiserum. It should be noted that even though actin (Act1p) was used as a cytosolic marker [48], a minimal amount of Act1p was also found to be associated to the mitochondrial fraction in agreement with [49]. In ethanol, an even more intense band was detected in the mitochondrial fraction, whereas in glucose-grown cells, the mitochondrial Fad1p level was very low. Thus, the Fad1p content in mitochondria seems to depend on the carbon source. As a control, the dependence of the mitochondrial SDH specific activity on different carbon sources was analyzed (Figure 3b), thus verifying the expected glucose repression due to both transcriptional and post-transcriptional events [50,51]. It is noteworthy that in the mitochondria prepared from galactose-grown cells, no band was detected (Figure 3a), even though we observed a high (induced) level of SDH activity (Figure 3b), in agreement with the presence of mitochondria in cells cultured in galactose as the carbon source [52]. Differently from what was observed for the mitochondrial Fad1p, the cytosolic echoform did not appear to depend on the carbon source (Figure 3a).

**Figure 3.** Subcellular distribution of Fad1p in different carbon sources. (**a**) Spheroplasts (Sphero), mitochondria (Mito) and cytoplasm (Cyt) were prepared from WT cells grown up to the early exponential phase at 30 ◦C in YEP liquid medium supplemented with the indicated carbon sources (2% each). Proteins (0.1 mg) were separated by SDS-PAGE, transferred onto PVDF membrane and detected with either α-FADS or α-ACTIN antibodies. (**b**) The carbon source dependence of the mitochondrial SDH-specific activity is reported as the means (±SD) of two independent measurements.

Cellular sublocalization of Fad1p was also evaluated in K26 cells grown in glycerol up to the stationary phase (24 h), as in [40]. Cytoplasmic and mitochondrial fractions were prepared from spheroplasts and equal amounts of each fraction were used to reveal Fad1p by immunoblotting analysis (Figure S2). A faint α-FADS immuno-reactive band was found in the cytoplasmic fraction. A much more evident band migrating at the same Mr was observed in total mitochondrial fraction, consistently enriched with respect to the spheroplast specific amount. A similar enrichment was observed for the fumarase (FUM) and succinate dehydrogenase (SDH), two mitochondrial markers (Figure S2, histogram). The absence of cytosolic contamination in mitochondria was demonstrated by the absence of phosphoglucoisomerase (PGI) activity, a marker of cytosolic fraction (Figure S2, histogram). Isolated mitochondria were further subfractionated in a membrane-enriched fraction (Mfr) and in a soluble fraction (Sfr). The Fad1p band was clearly detectable in both submitochondrial fractions, whose purity was confirmed by measuring the inner membrane marker SDH and the matrix marker FUM enzymatic activities (Figure S2, histogram).

Overall, on a molecular basis, these results support our previous functional data, proving that Fad1p exists in yeast mitochondria. Since no difference in size was observed between cytoplasmic and mitochondrial Fad1p, we postulated that the *FAD1* gene might generate two identical echoforms, destined to two distinct subcellular localizations.

#### *3.3. Detection of Two FAD1 mRNAs with Different 3*- *UTR and Their Carbon Source Dependence Profile*

In order to unravel the mechanism responsible for the subcellular destination of Fad1p, we considered a series of indications derived from the literature, which proposes a role for *cis*-acting 3- UTR elements in targeting transcripts to mitochondria [53,54], and from a genome-wide analysis, which suggests a high probability for *FAD1* mRNA to be located on mitochondria-bound polysomes [54]; hence, the protein would be imported while being translated in a co-translational process [55–57]. Therefore, we hypothesized that important information for Fad1p localization could reside in the 3- UTR of *FAD1* mRNA. Since no information was available, a combination of bioinformatic analyses and experimental procedures were carried out to gain knowledge about this untranslated region.

Firstly, we searched for putative polyadenylation sites (PASs), despite their complexity [58,59], and found two canonical signals (ATTAAA) in the 1 kbp region downstream of *FAD1* ORF using DNAFSMiner [60]. The proximal one was placed 50 nucleotides after the stop codon and the distal one was placed 339 nucleotides after the stop codon. The bioinformatic tool also predicted other different PASs with similar scores, namely, AATAAA and TATCAA (Figure S3). These predictions suggested that *FAD1* mRNA may be present in different forms, whose length depends on the alternative use of distinct PASs in the transcript 3'UTR.

To test this hypothesis, 3- RACE experiments were performed on a template cDNA obtained from purified poly(A)+ RNA prepared from WT cells grown at the early exponential phase on glucose as a carbon source and the gene-specific primers (Table 2; Figure 4a). When the primers FAD1A.for and AP were used, a single amplified product of about 400 bp was obtained (Figure 4b, lane 1). The specificity of the 400 bp product was verified by a semi-nested PCR using the gene-specific primer FAD1C.for located 91 nucleotides downstream of FAD1A.for. The amplified product was obtained at the expected size, which is about 300 bp (Figure 4b, lane 2). The 400 bp fragment was sequenced, revealing the existence of *FAD1* transcript with a 3- UTR of 128 nucleotides (Figure S3). To search for the existence of a longer 3- UTR region of *FAD1* mRNA, a PCR was performed using the primers FAD1C.for and FAD1B.rev, the last being located downstream of the end of the 3- UTR previously identified (Figure 4b); in this case, a product of about 450 nucleotides was obtained (Figure 4b, lane 3), indicating the existence of a longer *FAD1* mRNA. To define the entire length of the 3- UTR of this transcript, a PCR reaction was performed using the primers FAD1B.for and AP, obtaining a product of 550 bp (Figure 4b, lanes 4 and 5). Its specificity was proved by a semi-nested PCR with the primers FAD1B.for and FAD1B.rev, which gave rise to a fragment of about 100 bp, as expected (Figure 4b, lane 6). The 550 bp product was sequenced, showing the existence of an additional *FAD1* mRNA with a longer 3- UTR region of 759 bp (Figure S2). Similar results were obtained when the analysis was performed using cDNA as a template, obtained starting from purified poly(A)+ RNA prepared from WT cells grown on glycerol (Figure S4). Thus, *FAD1* generates (at least) two *FAD1* mRNAs with different 3- UTR lengths. Based on the different length of the 3- UTR, we named them 'short *FAD1* mRNA' and 'long *FAD1* mRNA', respectively.

To understand whether or not the length of 3- UTR is responsible for the fate of Fad1p echoforms, the carbon source dependence of the *FAD1* transcript levels was investigated. To this purpose, total RNA was extracted from WT cells grown at the early exponential phase on different carbon sources, and the amount of *FAD1* transcripts was evaluated by semiquantitative RT-PCR, with *ACT1* mRNA used as an internal control, essentially as in [42]. The long *FAD1* mRNA was amplified using the primer pair FAD1A.for and FAD1B.rev, whereas the total *FAD1* mRNA (short + long) was amplified using the primer pair FAD1A.for and FAD1A.rev (Figure S5). The RT-PCR product (540 bp) relative to the long *FAD1* mRNA complemented the 3- RACE data, confirming the presence of a longer transcript. The relative amount of total *FAD1* mRNA did not depend on the carbon source, whereas a carbon source dependence was observed for the long *FAD1* mRNA: its amount was higher in ethanol grown cells, reduced in cells grown on glucose and almost absent in galactose-grown cells. As a control, the expression pattern of *SDH1* mRNA was analyzed; it

was repressed by glucose, but not by galactose (Figure S5), in agreement with SDH activity reported here (Figure 3, histogram) and in literature [50].

**Figure 4.** Characterization of *FAD1* mRNA 3- UTR by 3- RACE analysis. (**a**) *FAD1* coding region and 1 kbp downstream region are schematized. Arrows designate the position and direction of gene-specific primers used for 3- RACE. (**b**) The experiment was carried out on a poly(A)+ RNA prepared from WT cells grown up to the early exponential phase at 30 ◦C on glucose. PCR products were separated by a 2% agarose gel. Fragments in lanes 1 and 4 were gel-extracted, purified and sequenced (Figure S3). On the left side of each gel, band sizes (bp) of the marker [M, *MassRuler Low Range DNA Ladder*, ThermoFisher Scientific (Waltham, MA, USA)] are indicated.

#### *3.4. A Mitochondrial Localization Motif in FAD1 3*- *UTRs*

To further confirm that Fad1p localization mechanism might be influenced at the mRNA level, we searched for *cis*-acting elements that could be responsible for targeting *FAD1* mRNA to the outer mitochondrial membrane. Sequence inspection revealed the existence of a putative *cis*-acting motif TGTATATACA containing the consensus mitochondrial localization motif M1 (TGTA(a/c/t/)ATA), as defined in [61], and resembling the motif 6 (WTATWTACADG) reported as a mitochondrial downstream motif [62]. This element was located about 90 nucleotides downstream the *FAD1* ORF stop codon; therefore, it was present in both *FAD1* transcripts. The same motif (or similar sequences) was not found in either the upstream *FAD1* ORF region or the *FAD1* ORF.

The 1 kbp genomic downstream region *FAD1* ORF contains, on the opposite strand the *MRP10* gene, encoding a mitochondrial ribosomal protein of 10.7 kDa [63,64]. Mrp10p lacks a mitochondrial-targeting sequence (as revealed by bioinformatic analysis); however, the identified M1 mitochondrial localization motif is present in its 3- UTR, as revealed by the Shalgi's database [61]. This motif is placed 56 nucleotides downstream of the *MRP10* ORF stop codon. It should be noted that the M1 motif in 3- UTR of *FAD1* and *MRP10* is a palindromic sequence that could be read on both DNA strands (Figure 5). This property implies a rather nontrivial extension of the M1 motif and allows the same region to play the role of mitochondrial localization motif for both the *MRP10* and the *FAD1* transcripts.

**Figure 5.** The *cis*-acting motif M1 in the 3- UTR of *FAD1* transcripts. Schematic representation of the partial sequence of the *S. cerevisiae* genomic region comprised between nucleotides 372248 and 373617 (chromosome IV). The white boxes indicate *FAD1* ORF on the Crick strand and *MRP10* ORF on the *Watson* strand; the orange box indicates the mitochondrial localization motif M1, placed about 87 and 55 nucleotides downstream of the stop codon (TAA, highlighted in red) of *FAD1* and *MRP10* ORFs, respectively. The dotted arrows highlight the bidirectionality of the M1 palindromic sequence.

#### **4. Discussion**

Experiments reported in this paper point out the issue of the origin of mitochondrial FAD in *S. cerevisiae* mitochondria, strictly related with the problem of the identity and of the subcellular localization of the FAD forming enzyme, FADS. Immunoblotting experiments reported here reveal the presence of an α-FADS immuno-reactive band in mitochondria, showing the same Mr of that found in the spheroplasts and cytoplasm. This is in line with the absence of a putative cleavable presequence in Fad1p, the sole peptide generated by *FAD1* gene. Thus, these results prove, on a molecular basis, Fad1p localization to yeast mitochondria and support the absence of a cleavable mitochondrial signal peptide in Fad1p.

It is well-documented that in eukaryotic cells, a protein can be located to two different subcellular compartments; this dual localization results in different echoforms—that is, proteins with identical or nearly identical amino acid sequences distinctly placed in the cell [65–67]. This is an important and frequent phenomenon; in fact, more than one third of the yeast mitochondrial proteome seems to be dual localized [68,69]. It has also become evident that dual localization can be regulated, induced and rebalanced in response to either cellular signaling or changing extracellular conditions. Different dual-targeting mechanisms of mitochondrial proteins have been described or suggested, and, in some case, they seem to be tightly regulated in time, location and function [65]. Our results demonstrate that *FAD1* generates two echoforms.

Analyzing the dependence of the Fad1p levels on fermentable and nonfermentable carbon sources (Figure 3), we verified that the amount of Fad1p in mitochondria, but not in the cytoplasm, depends on the carbon source being higher in ethanol and glycerol than in the fermentable glucose. Interestingly, mitochondrial Fad1p almost disappeared in galactose-grown cells. This finding may settle the discrepancy between results from our (see Introduction) and other laboratories [32,39] about the existence of mitochondrial FADS and, consequently, about the direction of FAD transport via the inner mitochondrial membrane translocator Flx1p. The absence of mitochondrial Fad1p, observed in galactose-grown cells, implies that in this condition FAD must be transported from cytosol to mitochondria via Flx1p (Figure 3 [32,39]). Vice versa, in a nonfermentable carbon source and in glucose, in the presence of mitochondrial Fad1p, Flx1p is expected to mediate FAD efflux from mitochondria to cytosol acting as a regulator of apo-flavoprotein biogenesis [40,42].

Using yeast strains overexpressing a recombinant form of Fad1p, the dual location of natural Fad1p was confirmed. Our data demonstrate that Fad1p is, per se, able to enter mitochondria, despite the absence of a canonical mitochondrial-targeting sequence. A "mitochondrial destination" message should be localized at the N-terminal end, since Fad1p loses its ability when the STREP-tag is moved from the C- to the N-terminal end (Figure S1). Presumably, the α1-helix in the first twenty amino acids at the N-terminal end, as well as the contiguous α2-helix, indeed have amphipathic characteristics, with the basic residues exposed to the solvent from the same side of the central axis, as revealed by the crystal structure solved at 1.9 Å resolution (PDB entry: 2wsi) [20]. This would be in agreement with the involvement of this protein moiety in the import process. Alternatively, another putative noncanonical/weak mitochondrial-targeting sequence [70] could be implicated in the mitochondrial destination of Fad1p.

We also observed that in galactose-grown cells, the recombinant Fad1p-STREP entered mitochondria per se [71], behaving differently from the natural Fad1p that was unable to enter mitochondria. All together, these data suggest the existence of a fine mechanism regulating the localization of the natural Fad1p, presumably at the transcript level and dependent on the carbon source. It is well-established that intracellular localization of mRNA has a significant impact on the efficiency of its translation and, presumably, determines its response to changing metabolic conditions or cellular stress [72].

Our attention was focused on the 3- UTR region of *FAD1* mRNA. Since no information was available on this region, we carried out a 3- RACE analysis and found the existence of (at least) two *FAD1* mRNAs with different 3- UTR lengths (Figure 4). This finding could be relevant for Fad1p cell biology, since it is well-known that 3- UTR regulates multiple aspects of mRNA metabolism, including subcellular localization, translation efficiency and stability, in cooperation with different RNA binding proteins [73]. A mechanism that could have a role in generating different transcripts with specific subcellular localizations and/or functions might be alternative polyadenylation [74,75]. We predicted different canonical and noncanonical putative PASs localized in the first 1 kbp downstream of *FAD1* ORF. Whether or not these PASs are actually active and responsible for the generation of the two *FAD1* transcripts are matter of ongoing investigation in our laboratory.

Searching for possible *cis*-acting motifs responsible for the peripheral mitochondrial localization of *FAD1* transcripts, we revealed the existence of the mitochondrial localization motif M1, placed 87 nucleotides downstream the *FAD1* ORF stop codon (Figure 5). The same motif has been identified within a set of genes encoding mitochondrial proteins, whose mRNAs are translated by polyribosomes attached to the cytosolic side of the outer mitochondrial membrane [76]. This motif is present in both *FAD1* transcripts, which might localize close to the mitochondrial outer membrane. Intriguingly, this finding is in agreement with a value of mitochondrial localization of mRNA (MLR) for *FAD1* equal to 87 [54]. This value is similar to the MLR values measured for other mRNAs, whose products have been demonstrated to be internalized into mitochondria (i.e., 87.8 for *SDH1* transcript), thus indicating that Fad1p translation has a high probability to occur on the mitochondrial outer membrane. Recently, it has been reported that mRNAs that initiate translation away from mitochondria experience a significant drop in mobility toward the outer mitochondrial membrane and tend to remain there [77]. The M1 motif is a candidate binding site of the RNA binding protein Puf3p [78], localized on the cytosolic side of the outer mitochondrial membrane, where it interacts with some components of the mitochondrial protein import machinery [57,79]. Besides its role in mRNA targeting to mitochondria, Puf3p controls mRNA stability and translation efficiency according to growth conditions and in response to oxidative stress [80–82]. The identified palindromic M1 motif is shared by the 3- UTR of *FAD1* transcripts and the 3 downstream region of *MRP10* gene, which is transcribed in the opposite direction with respect to *FAD1* gene. Since an interaction between the M1 of *MRP10* mRNA and Puf3p has been proven [83,84], an involvement of Puf3p in mitochondrial localization of Fad1p could be expected. By analyzing the carbon source dependence profile of *FAD1* transcripts (Figure S3), we found that the long transcript is expressed in ethanol and in glycerol at a higher level than in glucose; it is almost absent in galactose. The comparison between this profile and mitochondrial Fad1p levels (Figure 3) shows that the carbon source dependence of the long

*FAD1* mRNA level strictly parallels the carbon source dependence of the mitochondrial Fad1p level. This observation suggests that the long *FAD1* mRNA might be responsible for the generation of Fad1p mitochondrial echoform. Therefore, although the M1 motif is present in both the short and long *FAD1* transcripts, we propose that the different length of the 3- UTRs could be responsible for the distinct subcellular destinations of Fad1p. This diverse destination could be explained by a differential Puf3p–M1 interaction caused by the exposure of M1 motif in different RNA contexts. In this scenario, the secondary structure of the two 3- UTRs may be causative of the selective interaction with Puf3p. Moreover, the recruitment of additional RNA binding proteins, presumably involved in mRNA decay or Puf3p interaction with mitochondrial import machinery [85], could also be affected by 3- UTR length and structure. Therefore, Puf3p might regulate the dynamic balance between translation efficiency, protein folding and import rate, thus establishing whether Fad1p may be released in the cytosol or imported into mitochondria [81]. Additionally, it has been shown that cytosolic ribosomes have the ability to 'sense' features relative to nascent polypeptide chains; between those features, the sequence and structure of 3- UTR mRNAs can influence the polypeptide synthesis rate, modulating translation accordingly [76,86]. It is noteworthy that the M1 motif was not included in the constructs used for the expression of recombinant Fad1p. Nonetheless, the protein was capable, per se, of localizing to mitochondria in the presence of the mitochondrial membrane potential (Figure 2; [71]). Therefore, the M1 sequence would serve to fine-tune the cellular localization of Fad1p echoforms in various metabolic conditions, such as different carbon sources.

Our hypothesis is summarized in Figure 6. We speculate that, when Puf3p is bound to the M1 motif present in the long 3- UTR context (panel a), Fad1p synthesis efficiency and/or folding rate are slowed down, allowing the co-translational import to proceed and generate the Fad1p mitochondrial echoform. Vice versa, when Puf3p interacts with the M1 in the short 3- UTR (panel b), a higher translation rate might cause a premature folding, thus favoring the cytosolic destination of Fad1p.

**Figure 6.** *Cont*.

(**b**)

**Figure 6.** Model depicting the dual location of Fad1p. The cartoon shows a possible role of Puf3p in the different destination of Fad1p echoforms. (**a**) Puf3p binds to the long *FAD1* mRNA and drives the nascent polypeptide in the mitochondrial matrix (MMX) via TOM–TIM complexes before the fully translated protein starts to fold. (**b**) Puf3p binds to the short *FAD1* mRNA. As translation is presumably not sufficiently slowed down, premature and rapid folding of the nascent polypeptide in the cytosol (CYT) prevents Fad1p import across mitochondrial membranes (OMM—outer mitochondrial membrane; IMM—inner mitochondrial membrane).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/life11090967/s1. Figure S1: Sublocalization of recombinant STREP-Fad1p. Figure S2: Endogenous Fad1p mitochondrial localization in WT cells grown up to the stationary phase (24 h) on glycerol. Figure S3: *FAD1* ORF and its 1 kbp downstream region. Figure S4: 3- RACE analysis carried out on cells grown on glycerol (early exponential phase, 30 ◦C). Figure S5: Carbon source dependence of *FAD1* transcripts.

**Author Contributions:** Conceptualization, M.B., M.R., M.C., and E.B.; methodology, E.B. and M.R.; formal analysis, M.C., P.L., and F.B.; investigation, F.B., M.O., T.A.G., M.T., and P.L.; resources, E.B., M.R., and M.B.; writing—original draft preparation, M.B. and T.A.G.; writing—review and editing, M.R., F.B., and M.T.; supervision, M.C. and M.R.; project administration, M.B., M.O., M.R., and M.C.; funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from MIUR PON-ricerca e competitività 2007–2013 (project PON01\_00937: "Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la salute dell'uomo") and from Università degli Studi di Bari "Aldo Moro" (Progetti Competitivi "Effetto di mutazioni di FLAD1 e di alterazioni dell'omeostasi delle flavine sullo stato redox e sulla biogenesi mitocondriale: uno studio integrato su fibroblasti umani") to M.B.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The helpful collaboration of Emilia Dipalo, who participated as a student in the early stages of this work, is gratefully acknowledged.

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

#### **References**

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**2. Materials and Methods**

Quantitative samples were taken in June 2018, in the intertidal zone at Shinyang, close to the eastern corner of Jeju Island (Figure 1). Sediments were fixed in 5% neutralized formalin solution and brought back to the laboratory. Meiofauna were extracted using the Ludox method [11], and postfixed with 70% ethanol dyed with Rose bengal. Nematodes were counted and specimens were packed into a Syracuse glass filled with mixture of glycerin, 95% ethanol, and distilled water (1:29:70). The glass was placed in a drying oven set to 40 ◦C for 1–2 days until completely dehydrated, as in the glycerin-ethanol method [12]. Specimens were mounted in a drop of anhydrous glycerin within a wax-paraffin ring. Specimens were identified, measured, photographed, and drawn under a Leica DM5000 light microscope equipped with Leica Application Suite Version 3.8.0 software and a Leica DFC 425 C digital camera. All the measured sizes are given in μm. For scanning electron microscopy, specimens were dehydrated in a series: 40% ethanol, 70% ethanol, 95% ethanol, 95% ethanol + acetone (50:50), acetone I, and acetone II, and then critical point dried. Once dried, specimens were mounted on a stub to be coated with platinum–palladium alloy and examined with Cam Scan S-2.

**Figure 1.** Map of sampling locality. This map is made with QGIS software v.2.18.14, a free and open-source geographic information system (https://qgis.org).

Type specimens were deposited in National Institute of Biological Resources (South Korea).