*2.6. Effects of Leachate Sample from the UASB Reactor on BeWo Cells and Their Production of β-hCG*

The BeWo cell line was supplied by ATCC® CCL-98™, and it was cultured using Roswell Park Memorial Institute (RPMI)-1640 medium with 2% fetal bovine serum under a 5% CO2 atmosphere at 37 ◦C and 95% humidity (confluence > 70% and viability > 90%). A negative control was prepared with 100 μL of PBS in 5 mL of medium containing 5 × 105 cells and similar conditions. It was tested with 100 μL of the leachate sample from the UASB reactor. Additionally, 100 μL of the LNG stock solution was assayed. All experiments were carried out in duplicate for 24 h. A colorimetric assay (MTT assay) was carried out for cell viability according to Lomonte et al. [16]. For more details, see the Supplementary Materials (Supplementary Materials SM2). The analysis of the levels of β-hCG hormone in the medium was assayed after 24 h by ElectroChemiLuminescence using the COBAS® 411 Integrated System (HITACHI) analyzer for the immunoassay test. A total of 1 mL of the medium was taken out from each experimental well and added to a conical Eppendorf tube for the analysis of progesterone and β-hCG using the COBAS® 411 device. The UV–vis absorption and photoluminescence were recorded on the COBAS® 411 by the HITACHI provider. This device was supplied by the Hospital Marco Fidel Suarez (Bello, Colombia).


**Conflict of interest**

Poonam Velagapudi received speaking fees from Medtronic, Abiomed, Opsens, and Shockwave and participated in advisory boards for Abiomed and Sanofi. Muhammad Asim Shabbir and Nidhish Tiwari have nothing to disclose.


**Table 4.**

*Bromocriptine and cabergoline specifications.*

#### *Pituitary Adenomas: Classification, Clinical Evaluation and Management DOI: http://dx.doi.org/10.5772/intechopen.103778*

with microadenomas, risk of microadenoma progression is low and prolactin level monitoring is not necessary [15].

Macroadenomas always need management. Bromocriptine should be the drug of choice for patients who need fertility restoration. Pregnant women with macroadenoma without extrasellar extension can be followed similarly as microadenoma patients. However, if the suprasellar extension was detected before pregnancy, tumor debulking is advisable as the risk of macroadenoma growth during pregnancy is up to 35% [15, 16]. In these patients, it is also prudent to have a visual field assessment every 3 months till delivery.

Surgical management of prolactinoma is indicated in patients who are nonresponders to dopaminergic therapy, with intolerable adverse effects from medical therapy (e.g. bromocriptine), CSF fistulas under DA, cystic tumors with intramural hemorrhage, or progressive neurological deficits [11, 17]. Stable visual field defect is not considered an indication for surgery as most patients will have tumor shrinkage on medical therapy with improvement on visual symptoms.

When to consider a prolactinoma as being medication resistant?

Seventy-percent of bromocriptine resistant patients will respond on cabergoline. Around 10–16% of prolactinoma patients will need surgical management [11]. Most patients will have a reduction of PRL levels 2–3 weeks after dopamine agonist initiation, which generally precedes the tumor size reduction. Periodic PRL measurements and pituitary MRI every 6–12 months is advised. After 2 years of continuous therapy, if prolactin levels have been normalized and >50% reduction of tumor size has been achieved, medication dose can be reduced. Typically, a low-dose of dopamine agonist after 2 years of tumor control will usually keep prolactin within normal range and prevent tumor recurrence [18].


**2.1. Introduction**

Alopecia areata (AA) is a clinical condition characterized by well circumscribed, round, or oval patches of hair loss on the scalp or other parts of the body. Sometimes, alopecia totalis (AT), loss of all scalp hair, or alopacia universalis (AU), loss of all body hair, may develop. This disorder affects both sexes equally and occurs at all ages, although children and young adults are affected most often. The etiopathogenesis of AA is still unclear, but there is evidence that autoimmunity and endocrine dysfunction may be involved [24-26]. The autoimmune etiology has been proposed on the basis of its association with various autoimmune diseases, the presence of autoantibodies and various underlying immune abnormalities in the affected sites of these patients [27, 28]. One of the main associations is with thyroid abnormalities. This association was further supported by an increased incidence of abnormal thyroid structure, function tests and/or presence of thyroid autoantibodies found in many studies [29-32].

The aim of this study was to determine the prevalence of thyroid autoimmunity in patients with AA.

### **2.2. Patients and methods**

The study included 70 patients with AA (40 female and 30 male). A detailed history and examination were taken in all study subjects, including patients age, age at onset, duration of disease, associated diseases, history of thyroid disorders and the extent and severity of disease. The diagnosis of AA was made on clinical grounds. Skin biopsy was performed in selected cases. No patient was diagnosed before this study as having any type of thyroid dysfunction. The control group consisted of 70 volunteers (40 female and 30 male) who had skin diseases other then AA or autoimmune disorders. Blood samples were taken and a physical examination and thyroid sonography was performed. All subjects gave their informed consent in accordance with the requirements of the institutional Ethichs Committee. Thyroid autoantibodies (thyroglobulin antibody, anti-Tg, and thyroid peroxidase antibody, anti-TPO) and thyroid hormones (thyroxine (T4), triiodthyronine (T3) and thyroid stimulating hormone (TSH) were measured in all subjects. Total T4 (normal range: 70-180 nmol/L) and total T3 (normal range: 1.3-3.3 nmol/L) were measured by use of radioimmunoassay (RIA); TSH (normal range: 0.3-4.2 mlU/L) was determined by use of immunoradiometric assay (IRMA) (BRAHMS Aktiengesellshaft, Hennigsdorf, Germany). Serum levels of anti-Tg (threshold value: 115 IU/mL) and anti-TPO (borderline value: 34 IU/mL) were measured by use of electrochemiluminiscence immunoassay (ECLIA) according to standard protocols (COBAS, Roche Diagnostics GmbH, Mannheim, Germany).

Baseline clinical characteristics for the two groups were compared with the use of Student's t-test for continuous variables, the chi-square test or Fisher's exact test (two-sided) for categorical variables, as appropriate. Data were considered statistically significant at P <0.05.

Statistical analyses were performed using MedCale for Windows, version <IP_ADDRESS> (MedCale Software, Mariakerke, Belgium).


*2.3. Key Diagnostic Sugar Residues in the Sea Cucumber Saponins*

Characterization of common key fragments expedited the structure elucidation of new and reported saponins. Tandem mass spectrometry analysis of saponins showed the presence of several diagnostic key fragments corresponding to certain common structural element of saponins as summarized in Table 1. Here we report a new diagnostic key fragment at *m/z* 643 corresponding to the sodiated hydrated sugar residue MeGlc-Glc-Xyl-Xyl.

**Table 1.** Key diagnostic ions in the MS2 of the holothurians saponins. It has been adapted from [12] and modified.

The structures of sugar components of saponins were established by the identification of these diagnostic ions produced by tandem mass spectrometry. Observing these oligosaccharide moieties (*m/z* 493 and/or 507 and/or 511 (493 + H2O) and/or 523 and/or 643 and/or 657) simplified the characterization of the saponin structure.


**6. Lymphatic transport of lipid carriers**

There are two pathways for entry to the lymphatic system: M-cell uptake of particles and absorption of lipidic molecules through lymph vessels. The vascular region throughout the body is surrounded by specialized lymphatic vessels that absorb the molecules giving entry to a complex network of lymphatic systems [69, 70]. The pore size of blood vessels is not enough for the uptake of large molecules of triglycerides and phospholipids. However, the lymphatic capillaries are single-layer, thin-walled and non-fenestrated due to which it is easily able to permit these large molecules [71]. Upon absorption through lymph vessels, the lymph fluid containing absorbed molecules enters the thoracic duct followed by subclavian vein and finally is drained into the circulatory system. This pathway bypasses the liver before entering the blood circulatory system thus avoiding first-pass hepatic metabolism [72]. When drug reaches the circulatory system, it is in form of micelles or mixed micelles which dissociate into monomers because of dilution with the large volume of lymph/blood (the surfactant concentration decreases due to dilution and results in a concentration below its CMC value) [73]. In other cases where the drug is carried in lipid vesicle form, the release of drug becomes prolonged. It has been reported in literature that the presence of triglyceride-rich lipoproteins diverts the absorption of drug exclusively through

lymphatic route. Moreover, it has been also reported that greater lipoprotein synthesis particularly after postprandial administration diverts absorption of drugs dominantly through lymphatic system [67, 74]. This indicates that lipid derived either from diet or formulation determines the transport of drug either through lymphatic system or portal blood circulation [75]. This further relates to the new era for formulation development ruling out the necessity of traditional pathways like increasing solubilization capacity. It also brings new opportunities for various excipients that are emerging as bioactive recently in the last decade [76]. Further, excipients such as Cremophor EL and pluronic polymers have been shown to decrease chylomicron production and reduce the P-gp efflux transport of drug molecules [77, 78]. One of the versatile excipients in this category is tween 80 which has been found as excellent solubilizer, inhibitor of P-gp efflux and increase chylomicron production [76, 79]. Another pathway for lymphatic absorption through M cells has also been explored in the last two decades. These cells are able to recognize pathogens due to the presence of specific ligands on their surface. The particles of formulation have thus been engineered to mimic these ligands that are recognized by M-cells. Such ligands include peptides like Arg-Gly-Asp, glucans, proteins like lectins etc. [80–82]. Another peptide ligand investigated for M-cell uptake is RGD which recognizes a5b1 integrin on M-cells. However, due to its instability in gastrointestinal tract media, its analogue RGD peptidomimetic has been developed. This new moiety has been grafted on the surface of poly(lactic-*co*-glycolic acid) nanoparticles and tested for M-cell uptake. It was found that the efficacy of this new molecule remained the same as compared to its parent moiety (RGD) but the stability in GI media was improved [83, 84].


**8. Oleocanthal concentration in olive oil and factors which may affect its concentration**

Oleocanthal concentration in EVOO is highly variable, ranging from as little as 0.2 mg/kg to as high as 498 mg/kg (Gomez-Rico et al., 2006). Such variation in oleocanthal concentration amongst differing EVOOs may be due to multiple factors that have the capacity to modify the concentration of this compound (Carrasco-Pancorbo et al., 2005). These factors include: method of phenolic extraction and quantification, geographic region of olive growth, olive tree cultivar, agricultural techniques applied to cultivate olives, olive maturity, processing of the olives to oil, storage of oil, and domestic heat application to oil.


**4. Conclusion**

This chapter has analyzed several strategies related to the movement of goods during the Covid-19 pandemic and expressed by international organizations, including ICC, WTO and WCO. Knowing that the pandemic is not likely to end soon, and that automatization may be the path to be followed to pursue a faster and more secure trade exchange, the common idea is to emphasize the usage of new technologies such as blockchain, IoT, machine learning, data analytics, artificial intelligence, and big data.

Although, most countries have already started implementing some or all of them, getting several advantages by their use such as speed control and tracking—as collected in the WCO annual consolidated survey—, there still are several issues to face to have widespread usage. Costs, lack of knowledge, good practices and/or trust, legislation and privacy issues, and lack of governmental strategy and coordination are the one to be faced shortly to ensure an interconnected international customs system.

This chapter focuses only on strategies proposed by international organizations, without acknowledging national responses to the pandemic, and this may represent a limit in the investigation.

Future research directions may be, on the one hand, understanding whether any country would be able to implement the proposed approaches, investing in new technologies for its borders and Customs, and seeing the actual improvements in the relationship among the international trade stakeholders; on the other hand, selecting a specific geographical scope, examining regional and national response to the downshift of international trade and the proposed solutions at a local level.


**B. APF-Application Case**

In this case, the constructed inverter was experimentally used for APF application. That is, the control scheme was able to work as an APF only. The parameters used for the experiment can be seen in Table 3. In addition, the harmonic current term in Equation (2) was be included so as to be compensated by the MLI. The resulting PWM pulses were then sent to the IGBTs via the DS1202 board. The constructed MLI was controlled to only compensate the harmonic contents of the load currents, while the grid supplied the three-phase positive-sequence currents of the nonlinear load.

The values of the nonlinear load parameters were *RL*1 = *RL*2 = 48 Ω, and *XL*1 = *XL*2 = 154 mH. To allow the grid to feed power to the nonlinear load, the duty cycle of the DC-DC converter was kept constant at 0.3 (no MPPT is used). The DC-link controllers were successful in maintaining the DC-link voltages at reference levels, as seen in Figure 23. These voltages were measured via analog-to-digital converters by using voltage sensors and plotted in the control desk of the MicroLabBox.

**Figure 23.** The experimental DC-link voltages.

On the other hand, Figure 24 shows the experimental load currents measured by using Hall-effect current sensors. As seen in Figure 24, the load currents are not sine waves since they are highly distorted due to the nonlinear loads. As explained in the control scheme, the harmonic currents of the load were extracted from the load current so as to be compensated by the inverter. The experimental extracted harmonic load currents (the experimental currents of APF) are shown in Figure 25.

**Figure 24.** The experimental nonlinear load currents.

**Figure 25.** The experimental APF currents.

These harmonic currents were then used as a reference for the inverter currents so that the inverter compensated only these load current harmonics, and the grid supplied the nonharmonic currents. The experimental three-phase grid currents are shown in Figure 26.

**Figure 26.** The experimental three-phase grid currents.

The line currents and the grid voltages were out of phase, as revealed in Figure 27, so that the power factor was kept unified. The out-of-phase grid currents were due to the current sensors measuring the current from the grid to the load. On the other hand, the THD of the line currents was 4.8%, which had acceptable harmonic content of less than 5%, as defined by the IEEE-519 standard. The harmonic spectrum of the grid current, *ia*, is shown in Figure 28.

**Figure 27.** The experimental grid current and grid voltage.

**Figure 28.** Harmonic spectrum of the grid current *ia*.


*2.2. β-Hairpin Structure Characterization by NMR*

The peptides were analyzed by 1H–1H homonuclear two-dimensional solution NMR spectroscopy, including double-quantum filtered-correlated spectroscopy (DQF-COSY) [33], total correlation spectroscopy (TOCSY) [34], and rotating-frame nuclear Overhauser effect

*β*

β

α

β

spectroscopy (ROESY) [35] at 298 K. Sequence-specific assignment of all peptides was performed based on the TOCSY and ROESY spectra (Tables S1–S36) [36]. For a given Xaa4- Zbb9 pair, the chemical shift dispersion of the peptides followed the trend HPTFXaaZbb > HPTXaaZbb > HPTUXaaZbb (Tables S1–S36). Since the higher the fraction folded population, the higher the chemical shift dispersion [37], this trend is consistent with the intended designs of the peptides. −

The β structure of the experimental and fully folded reference peptides was confirmed by the chemical shift deviations of the Hα signals, the 3 JHNα spin–spin coupling constants, and the NOE cross-peaks. The Hα chemical shift deviation (∆δHα) is the difference between the Hα signal for the residue of interest and the corresponding random coil Hα signal [38]. In this study, the fully unfolded reference peptides were considered to be random coil [11,22,23,26]. A positive ∆δHα value suggests an extended β-sheet conformation [38,39]. The ∆δHα values of the residues Thr2 through Val5 and Orn8 through Leu11 for the experimental HPTXaaZbb peptides and the fully folded reference HPTFXaaZbb peptides were positive (Figure 2, Figures S1 and S2), suggesting an extended β-strand conformation for these residues. This is consistent with the intended design. In general, the ∆δHα values for the residues in the strand regions (residues 2–5 and residues 8–11) of the fully folded reference peptides were more positive compared to those for the corresponding experimental peptides (Figure 2, Figures S1 and S2), suggesting that the fully folded reference peptides were more well folded than the corresponding experimental peptides. The ∆δHα values of the terminal residues Arg1 and Gln12 for the experimental peptides were near zero (Figure 2, Figures S1 and S2), most likely due to end fraying effects [21]. The ∆δHα values for Gly7 were negative or mostly close to zero (Figure 2, Figures S1 and S2), consistent with turn formation [12]. α α α Δδ α α Δδ α β Δδ α Δδ α Δδ α Δδ α

Δδ α **Figure 2.** The chemical shift deviation (∆δHα) for the residues in peptides HPTFLysGlu (**a**) and HPTLysGlu (**b**).

α α β α α The DQF-COSY spectra were used to determine the 3 JHNα spin–spin coupling constants for each residue in the peptides (Tables S37–S45) [33,40]. The 3 JHNα coupling constants of the residues in the fully folded reference HPTFXaaZbb peptides showed values higher than 7 Hz (Tables S43–S45), consistent with a β-hairpin structure [36,41]. The experimental HPTXaaZbb peptides also exhibited 3 JHNα coupling constants higher than 7 Hz, but slightly lower 3 JHNα values compared to those for the fully folded reference HPTFXaaZbb peptides (Tables S37–S39 and S43–S45). This suggested that the experimental HPTXaaZbb peptides may not be as well folded as the fully folded reference HPTFXaaZbb peptides. For the unfolded reference HPTUXaaZbb peptides, some residues exhibited 3 JHNα values near or less than 7 Hz (Tables S40–S42), suggesting that these peptides may not be as well folded as the experimental HPTXaaZbb peptides or the fully folded reference HPTFXaaZbb peptides.

The NOE cross-peaks in the ROESY spectra included sequential, intra-residues, medium-range, and long-range NOEs with a number of cross strand Hα–Hα, Hα–HN,

HN–HN correlations (Figures S3–S50). All sequential Hα–HN NOE correlations in every strand for all peptides were observed (Figures S39–S50), consistent with β-strand formation [42,43]. In addition, the lack of dαN(*i*, *i* + n) (*n* = 2,3,4) and dNN(*i*, *i* + n) (*n* = 1, 2) patterns rules out the formation of other secondary structures (Figures S39–S50) [42,43]. A network of cross strand side-chain-side-chain NOEs between residues on the two βstrands was observed for the experimental peptides HPTXaaZbb and fully folded reference peptides HPTFXaaZbb (Figures S3−S38), consistent with β-hairpin formation for these peptides. Long-range NOE cross-peaks between Thr2 and Xaa9 were observed for most of the experimental HPTXaaZbb peptides and fully folded reference HPTFXaaZbb peptides (Figures S3−S38), consistent with a right-handed twist [11,27,28]. The number of cross-peaks in the ROESY spectra followed the general trend HPTFXaaZbb > HPTXaaZbb > HPTUXaaZbb (Figures S3−S38), consistent with the intended fraction folded population for our designs [3,22,23].


*2.5. Sample Demographics*

The sample consisted of 111 participants from across the Greater Western Sydney (see Table 1). The majority of participants were female (51.4%), with a nearly equal number of males (47.7%) and one participant identifying as transgender. Participants' ages at the time of participation ranged between 16 and 60, with a mean age of 22.90 (*SD* = 5.25). Most participants were single (*n* = 82.9%) and had no children (94.6%). Seventy-six participants arrived in Australia between 2000 and 2009 (68.4%) with their close kin (mother 83.8%, father 71.2%, sibling 46.8%). The majority migrated from Sub-Saharan Africa (25%), closely followed by South-East Asia (24%), with the others migrated from East Asia (13%), the Middle East (11%), Eastern Europe (9%), the Pacific (6%), the Americas (6%), Western Europe (4%), and North Africa (2). The mean age at the time of migration was 11 years old (Mean (M) = 11.90, Standard Deviation (SD) = 4.67). The majority spoke English as a primary language (66.7%). Twenty-four languages were noted by those whose primary language was not English. The majority indicated a religious affiliation (87.4%), with 55% of those being Christian/Catholic. Ninety-five participants were heterosexual (85.5%), eight were bisexual (7.2%), five were homosexual (4.5%), one identified as lesbian (0.9%) and one identified as other (0.9%), and prefer not to say (0.9%), respectively.

**Table 1.** Demographic information for the study sample.

#### **3. Results**

The present study sought to examine the role an individual's culture has in the construction of their sexual and reproductive health. Table 2 presents the degree to which a participant's cultural identity was determined by their cultural connectedness to their Country of Origin, Australian Culture, Community, or Family.

**Table 2.** Cultural identity in relation to participants' perceived cultural connectedness (%).

The results indicate that stronger identification with one's family positively correlates with seeking help from an intimate partner, a doctor, community leaders, and seeking no help. Table 3 depicts correlations between the measures of cultural connectedness and sources of help. Table 4 depicts correlations between the sources of help. The results indicate significant positive correlations between a strong identification with one's country of origin and seeking help from an intimate partner, parents, a sexual health clinic, the Internet, and a doctor.

**Table 3.** Bivariate correlations between measures of cultural connectedness and sources of sexual and reproductive health (SRH) help-seeking.

*Note*. Correlations marked with an asterisk (\*) and double asterisk (\*\*) were significant at *p* < 0.05 and *p* < 0.01, respectively.

*Note*. Correlations marked with an asterisk (\*) and double asterisk (\*\*) were significant at *p* < 0.05 and *p* < 0.01, respectively.

Analyses indicated significant correlations between the identification with one's country of origin, Australian culture, one's community, and one's family and various sources of help, whereby stronger connections related to stronger inclinations toward seeking help from specific sources. Interestingly, seeking help from an intimate partner or doctor/general practitioner (GP) was significant across all measures of cultural connectedness. Additionally, seeking help from various sources often related to seeking help from other

sources. However, stronger inclinations to seek help from a relative or sexual health clinic were significantly related to lower inclinations to seek no help.

To identify group differences between participant's religious identifications (No Religion, Catholic/Christian, Greek Orthodox, Islamic, Buddhist, Other) among the various sources of help (Intimate Partner, Friend, Parent, Relative, Sexual Health Clinic, Internet, Doctor/GP, Community Leaders, No Help), Kruskall-Wallis nonparametric tests were conducted to accommodate the uneven group sizes. A statistically significant difference was identified for receiving help from parents (*X* 2 [5, *N* = 111] = 11.30, *p* < 0.05, *η* 2 = 1.16).

These results suggest significant differences between religious groups in regard to seeking help from parents. No significant differences, however, were found between the six religious categories—most likely due to small group sample sizes. However, the results show a significant difference only between religious affiliation and seeking help from a parent. Table 5 depicts the degree to which individuals of various religious identities seek help from their parent(s).

**Table 5.** Participants' perceived likelihood of SRH help-seeking from parent among religious identities.

The present study also sought to determine which sources individuals felt most comfortable seeking help from. Table 6 indicates participants' perceived likelihood (in percentage) to seek help from various sources. Doctors/GP (92.7%), sexual health clinics (88.1%), the Internet (84.1%), and intimate partners (81.1%) were among the most likely sources of help, while community leaders (72.5%), relative(s) (60%), and no help (56.8%) were among the most unlikely sources of help.

**Table 6.** Perceived likelihood (%) of SRH help-seeking depending on source.

The present study also sought to ascertain the most dominant barriers and facilitators to individual's help-seeking attitudes. Among the barriers hindering individuals' helpseeking, a lack of knowledge was identified as the most dominant barrier (45.9%). This was followed by concerns regarding concealment from one's family and community (36.0%). These results are complimented by the facilitator of help-seeking, whereby an increase in knowledge was identified as the most dominant facilitator of help-seeking (63.1%). Similarly, assurance of concealment was identified as the second most dominant facilitator of help-seeking (45.9%). Tables 7 and 8 depict the barriers and facilitators of help-seeking.

**Table 7.** Participants perceptions of potential barriers to SRH help-seeking.

**Table 8.** Participants perceptions of potential facilitators of SRH help-seeking.

To contextualise the key findings, participants' sexual and reproductive health histories were recorded. It was identified that 60.40% (*n* = 67) of the participants were currently sexually active. Of the 111 participants, 49.50% (*n* = 55) used contraceptives, 11.70% (*n* = 13) did not use contraceptives, and 38.70% (*n* = 43) preferred not to answer. Table 9 depicts the types of contraceptives participants have previously used.

**Table 9.** Types of contraceptives used by 1.5 generation migrants in Australia.

With regard to prior sexual health concerns, 2.7% (*n* = 3) of participants had previously been diagnosed with an STI. Among those, 66.7% (*n* = 2) were diagnosed with gonorrhoea, while 33.30% (*n* = 1) were diagnosed with herpes. Additionally, 66.7% (*n* = 2) took antibacterial medications, while 33.3% (*n* = 1) sought help from a doctor. When queried about the duration leading to their help-seeking behaviours, it was revealed that 66.70% (*n* = 2) sought help within 1—3 days of having sex while 33.3% (*n* = 1) sought help within 4—7 days. Participants justified this by saying that they were not aware that they were infected with an STI (*n* = 2, 66.7%) and that they were hoping that the STI would go away without intervention (*n* = 1, 33.3%).

In terms of pregnancy, 9.0% (*n* = 10) had previously experienced an unplanned pregnancy. Among these participants, 40% (*n* = 4) kept the child, 40% (*n* = 4) terminated the pregnancy, 10% (*n* = 1) organised an adoption, and 10% (*n* = 1) preferred not to answer on the outcome of the pregnancy.

#### **4. Discussion**

This study was designed to investigate the role of culture and religion on sexual and reproductive health indicators and help-seeking attitudes amongst 1.5 generation migrants

using a quantitative survey. Overall, the results suggest that 1.5 generation migrants were most likely to seek help from doctors/general practitioners (92.7%), sexual health clinics (88.1%), the Internet (84.1%), and intimate partners (81.1%) regarding clinical SRH issues. For support on non-clinical SRH matters, the results suggest that 1.5 generation migrants feel the least comfortable seeking SRH support from community leaders (72.5%) and relative(s) (60%). These findings can be further contexualised when culture and religiosity are considered.

With regards to the role of cultural connectedness on 1.5 generation migrants SRH helpseeking, the results indicate significant positive correlations between a strong identification with one's country of origin and seeking help from an intimate partner, parents, a sexual health clinic, the Internet, and a doctor. Stronger identification with one's family positively correlates with seeking help from an intimate partner, a doctor, community leaders, and seeking no help. This is in line with research indicating that some youths of minority and migrant backgrounds often struggle to engage with their parents when they experience an SRH concern for fear of the consequences of transgressing ethnocultural or religious protocols held in high esteem by their parents [12,13]. However, this was not the case for all of the 1.5 generation migrants in this study. This may be because these migrants feel more connected to their parents in line with their collectivist ethnocultural values [14]. For those who sought help from parents, it could also be that both the youth and their parents have acculturated more than popular discourses give them credit for [14].

In this study, strong identification with Australian (secular, individualist, capitalist and Eurocentric) culture positively correlates with seeking help from an intimate partner, relatives, a sexual health clinic, a doctor, and community leaders, while stronger identification with one's community positively correlates with seeking help from an intimate partner, relatives, a doctor, community leaders, and seeking no help. Other studies highlighted that culture as a significant factor in SRH help-seeking [6]; however, the findings of this study suggest that 1.5 generation migrants are not influenced by culture to the same extent as their older counterparts [14]. These findings suggest that the colloquially perceived ethnocultural values between more recent migrants and those with a longer history in Australia are not so incongruent [14]. These findings can inform contemporary discourses about young migrants and their SRH help-seeking needs.

The study inquired about whether religious affiliation influenced 1.5 generation migrants' SRH help-seeking. The analyses identified a significant difference only between religious affiliation and seeking help from a parent. This may be because increased religiosity has been linked to difficulties in seeking help for SRH issues from close family members due to fear of social sanctioning, as contemporary Australians youths' sexual behaviour is often at odds with religious doctrine [2]. Notably, those with no religious affiliation were slightly more likely to seek help from parents, yet there were no statistically significant differences between the six religious affiliations. The findings therefore suggest that more inquiry is needed into the role of religiosity and SRH help-seeking amongst young migrants and culturally and linguistically diverse youth.

To support access to SRH supports, the reduction in barriers and increase in facilitators is required. In this study, the top three barriers as perceived by 1.5 generation migrants were; not knowing where to access SRH services (45.90%), ensuring that their family and community did not find out (36.00%), and not having enough money to pay for SRH services (28.80%). Likewise, being made aware of where the services are (63.10%), being confident that no one would find out (45.90%), and access to services which are free/low cost (36.90%) were identified as the most dominant facilitators of help-seeking. These findings are aligned with Australian and international research with minority youth, aged 16 to 24, indicating that increased awareness of services that provide inconspicuous access to free SRH services improve youth SRH outcomes [15–18]. For instance, SRH support provided at university campuses can offer confidentiality from family and the community and often include billing options for local and international students that require minimal to no payment upfront [17,19,20]. However, such services are only accessible to those whose social determinants allow them the privilege of attending university. Considering that religion was an important influence in help-seeking, religious organisations may be well placed liaisons between youths, their families and communities, and SRH services.

#### **5. Limitations**

The study findings reiterate the role of cultural connectedness and religiosity in SRH help-seeking for migrant youths. The study has also highlighted key areas which require further consideration and investigation. The purposeful nature of the sampling strategy helped to achieve a varied sample with the aim of capturing perspectives from various ethnic, religious, and migration backgrounds. However, the country of origin of the sample was not proportional, as most participants were from sub-Saharan Africa. In addition, the majority of participants were Catholic or Christian, which may not reflect many 1.5 generation migrants who do not prescribe to Christianity. This cultural similarity may mean the full breadth of cross-cultural SRH help-seeking perspectives and behaviours have yet to be explored. Additionally, although participants' mean age of migration was 11 years old, those who arrived much younger may not experience as much pressure or culture clash, as they may have been too young to remember or for their families to feel that they had to adhere to the rules of their ethnic origins. The age of participants is also relevant in relation to when they migrated to Australia. For instance, as the participants aged, they may be less likely to recall or recount their experiences as children. Further, their perspectives of SRH help-seeking were asked in relation to the present versus helpseeking in the past, which would have included fewer SRH services and engagement from community services and networks. Irrespective of age at participation, it seemed that for the migrants in this study, religion appeared to hold more weight in determining their SRH help-seeking attitudes. More exploration is needed to determine the interaction between age of migration and SRH help-seeking and outcomes. Finally, the analysis was restricted, as one-way ANOVAs could not be conducted on the studies due to the small and uneven sample sizes; as such, Kruskall-Wallis tests were used instead. Ultimately, generalisations cannot be made about the different perspectives among such groups, and further study is recommended to assess the effect of diverse religious backgrounds on SRH help-seeking amongst migrants in Australia.

Although participants of this study were recruited from a number of Western Sydney suburbs, this was done in relation to seven Western Sydney University campuses and surrounding off-campus venues (e.g., major shopping malls). As a result, the participants are likely to have been university students or staff and therefore well-educated. In such a case, the participants would potentially have a heightened capacity to both understand and critically analyse the statements before sorting them. As such, the sample may not be representative of the many 1.5 generation migrants who may not have high levels of education. With lower levels of education come lower levels of health literacy [21]. Consequently, participants' perspectives on health care services and the engagement of these migrants with those services may be influenced by their increased ability to scrutinise, navigate, and mediate their experiences within the Australian health care system compared to other groups of migrants. Expansion of this study to include a broader variety of 1.5 generation migrants is therefore required.

#### **6. Conclusions**

The influence of a cross-cultural upbringing is often noted in the extant literature as a potentially challenging factor in migrant youths' sexual and reproductive health helpseeking. Amongst the 1.5 generation migrants in this study, there were no significant differences between ethnocultural groups or levels of cultural connectedness in relation to sexual and reproductive health help-seeking. While cultural norms of migrants' country of origin can remain strong, it is religion that seems to have more of an impact on how 1.5 generation migrants construct, experience, understand, and engage with various aspects of SRH. The present study's results suggest differences between religious groups in regard

to seeking help specifically from youths' parents. Notably, participants who reported having 'no religion' were more likely to seek help with sexual and reproductive health matters from their parents. Given that religion can play such an important role in youths' sexual and reproductive health religious organisations may be well-placed to encourage youth help-seeking. This may be a means of addressing the barriers that youths perceive to accessing support in ways that ensure equitable and easy access to confidential and low to no cost sexual and reproductive health services.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1660-4601/ 18/3/1341/s1, Text S1: Participant Information Sheet, Demographic Questions and Quantitative Survey.

**Author Contributions:** Conceptualization, T.D.; Data curation, T.D. and D.A.; Formal analysis, T.D., J.T. and Z.M.; Funding acquisition, T.D.; Investigation, T.D.; Methodology, T.D.; Project administration, T.D. and D.A.; Software, T.D.; Supervision, T.D.; Validation, J.T.; Writing—original draft, T.D. and J.T.; Writing—review and editing, T.D., D.A., J.T., Z.M. and V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Western Sydney University as well as the Translational Health Research Institute and the School of Health Sciences (both organisations within Western Sydney University) provided funding for this research through Early Career Research Grants in 2015. However, these units did not have a role in the design, collection, analysis and interpretation of data.

**Institutional Review Board Statement:** This study is part of a larger research project examining the SRH of 1.5 generation migrants in Australia and ethical approval was received from the Human Research Ethics Committee of Western Sydney University. In addition, informed consent to participate in this study was obtained from all participants (approval date and code: 19 June 2015, H11168).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical restrictions on public access.

**Acknowledgments:** We would like to thank Janette Perz whose mentorship and sponsorship of this research made it possible. We would also like to thank the participants who provided their time and voices to this project, which would not have been possible without their candour during data collection and trust in the aims and goals of the research.

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


*Review* **Towards a Better Understanding of the Relationships between Galectin-7, p53 and MMP-9 during Cancer Progression**

**Yves St-Pierre**

**Citation:** St-Pierre, Y. Towards a Better Understanding of the Relationships between Galectin-7, p53 and MMP-9 during Cancer Progression. *Biomolecules* **2021**, *11*, 879. https://doi.org/10.3390/ biom11060879

Academic Editor: Alexander V. Timoshenko

Received: 18 May 2021 Accepted: 11 June 2021 Published: 14 June 2021

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

**Copyright:** © 2021 by the author. 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/).

INRS-Centre Armand-Frappier Santé Biotechnologie, Laval, QC H7V 1B7, Canada<EMAIL_ADDRESS>
**Abstract:** It has been almost 25 years since the discovery of galectin-7. This member of the galectin family has attracted interest from many working in the cancer field given its highly restricted expression profile in epithelial cells and the fact that cancers of epithelial origin (carcinoma) are among the most frequent and deadly cancer subtypes. Initially described as a p53-induced gene and associated with apoptosis, galectin-7 is now recognized as having a protumorigenic role in many cancer types. Several studies have indeed shown that galectin-7 is associated with aggressive behavior of cancer cells and induces expression of MMP-9, a member of the matrix metalloproteinases (MMP) family known to confer invasive behavior to cancer cells. It is therefore not surprising that many studies have examined its relationships with p53 and MMP-9. However, the relationships between galectin-7 and p53 and MMP-9 are not always clear. This is largely because p53 is often mutated in cancer cells and such mutations drastically change its functions and, consequently, its association with galectin-7. In this review, we discuss the functional relationships between galectin-7, p53 and MMP-9 and reconcile some apparently contradictory observations. A better understanding of these relationships will help to develop a working hypothesis and model that will provide the basis for further research in the hope of establishing a new paradigm for tackling the role of galectin-7 in cancer.

**Keywords:** galectin-7; p53; MMP-9; cancer; gain-of-function

#### **1. Introduction: The Discovery of Galectin-7**

The first reports on galectin-7 were published more than 25 years ago by two independent groups conducting systematic searches for differentially expressed proteins in keratinocytes. The first report was published by Prof. Julio Celis. Using two-dimensional gel electrophoresis, his group had been conducting systematic searches for differentially expressed proteins between normal and SV40-transformed human keratinocytes for several years [1]. The researchers noticed a protein, named IEF17, that was constitutively expressed in normal keratinocytes but not in transformed cells. They subsequently cloned the gene and showed that its sequence had strong homology to consensus sequences found in the carbohydrate-binding site of galectins. They further showed that the protein bound asialofetuin via a lactose-dependent interaction. Upon consultation with established galectin researchers who had just published a consensus on the criteria necessary for a protein to be recognized as a galectin the year before [2], they named this new protein galectin-7. During approximately the same period of time, a French group from L'Oréal was conducting a systematic search for epidermal-specific and stage-specific biomarkers using epidermal cDNA libraries [3]. Their attention was focused on the clone 1A12, whose sequence was identical to the sequence encoding the IEF7 protein identified by Madsen and colleagues that had just been submitted to the GenBank database. Magnaldo and colleagues constructed a GST-fusion protein with their cDNA clone, expressed it in an *E. coli* expression system and confirmed its affinity for lactose. Building on their initial discoveries, both the Danish and French groups actively pursued their research on galectin-7 and subsequently concluded that galectin-7 is a keratinocyte-specific marker found in all layers

of the epidermis and other stratified epithelia of tissues, most notably in the tongue, cornea, esophagus, stomach, anus and Hassal's corpuscles of the thymus [4–8]. Subsequent studies showed, however, that galectin-7 is also present in other types of epithelia, including the myoepithelial cells of the mammary gland epithelium [9,10]. In most cases, galectin-7 was found to be expressed in both the cytosolic and nuclear compartments and released into the extracellular space by a nonclassical secretion mechanism, a pattern typical of members of the galectin family [11]. Although most of the literature has focused on the expression of galectin-7 in cells of epithelial origin, it is important to keep an open mind with regards to its expression in other cell types, as we now know that it is also expressed, albeit at a rarer frequency, in cells of other lineages. We and others have reported, for example, that *LGALS7* expression is found in specific subtypes of lymphoid/myeloid cells, including transformed human B and T lymphocytes [12,13] and normal CD8-positive dendritic cells [14,15]. Too often these data are lost in the sea of transcriptomic datasets and we still largely ignore the function of galectin-7 in these normal myeloid cells.

#### **2. The First Stage of the Relationship between Galectin-7 and p53**

In 1997, the Bert Vogelstein group published a seminal article in Nature on p53 [16]. Similar to many teams working on p53, the Vogelstein group was studying the molecular mechanisms involved in p53-induced cell death and p53-mediated regulation of the cell cycle. They were particularly interested in identifying transcripts that were under the control of p53. For this, they used an adenoviral vector to express wild-type p53 in the p53-defective human colon cancer cell line DLD-1. They identified 14 transcripts including a transcript encoding the *p21* gene and 13 other transcripts that they called p53-induced genes (PIGs) 1–13. Many of these transcripts were functionally linked to the generation of reactive oxygen species (ROS), allowing establishment of a clear and strong connection between p53 and oxidative stress. Other transcripts encoded multiple genes, including *LGALS7* (identified as PIG1). The authors paid very little attention to galectin-7 other than a comment that this gene was part of the galectin family and that some members (such as galectin-3) were capable of inducing superoxide production in neutrophils. In fact, there were at that time no more than a handful of papers that had been published on galectin-7 (cited above). Following this article by Polyak and colleagues, several groups confirmed that upregulation of galectin-7 was associated with apoptosis in a p53-dependent manner. For example, Bernerd et al. [17] showed that galectin-7 was upregulated in skin keratinocytes upon exposure to UVB irradiation, a known inducer of the *p53* gene and apoptosis in keratinocytes. They also found that induction of galectin-7 paralleled that of wild-type (wt) p53, further confirming that induction of galectin-7, at least in UVB-exposed skin keratinocytes, is dependent on wt p53. Consistent with this hypothesis, they found no detectable expression of galectin-7 in the SCC13 keratinocyte cell line, which does not express wt p53 but rather p53 mutated at position 179 [18]. This mutation at position 179 is known to destabilize the p53 protein by altering its ability to interact with a zinc molecule, causing loss of DNA-binding specificity [19]. Our group also reported that treatment of human MCF-7 breast cancer cells (which harbor wt alleles of p53) with doxorubicin, a well-known inducer of p53, induced the expression of galectin-7 at both the mRNA and protein levels and that this induction was abrogated following knockdown of wt p53 [20]. However, after all these years, the function of galectin-7 in this physiological context related to wt p53 is still unclear. Could GAL-7 be induced by p53 to promote DNA repair? This is certainly a real possibility considering that (1) DNA repair is a pathway activated by p53, (2) galectin-7 is expressed at high levels in the nucleus, (3) galectins have been shown to have nuclear functions and (4) other members of the galectin family, such as galectin-1 and galectin-3, are known to regulate DNA damage repair [21–23].

#### **3. A Twisted Relationship**

While initial studies in colon cancer cells indicated that p53-induced galectin-7 expression was associated with cell death, a number of reports published at that time brought the

universality of this phenotype into question. These studies indicated that galectin-7 might instead be associated with cancer progression (Figure 1). For example, data obtained from a study using an experimental breast carcinoma model [24] and a study evaluating clinical specimens collected from patients with thyroid cancer [25] revealed that high galectin-7 expression levels were associated with cancer progression and aggressiveness. Conducting a comparative transcriptomic analysis between a low-tumorigenic lymphoma cell line (164T2) and its aggressive variants (S11 and S19) generated by serial in vivo passaging, our group further showed that the most prominent change that occurred in highly metastatic variants was the strong upregulation (160-fold) of galectin-7 [12]. This change was unexpected, as, at that time, galectin-7 was considered a keratinocyte marker expressed in all subtypes of stratified epidermis. Analysis of gene expression profiles stored in public datasets in the Gene Expression Omnibus (GEO) repository showed that such strong upregulation of galectin-7 in lymphoid cancer cells is not unusual, as it was also observed following in vivo passaging of the parental FL5.12 cell line, a murine prolymphocytic cell line [26]. Using our lymphoma model, we further provided evidence that such upregulation of galectin-7 in lymphoma cells is not an epiphenomenon. Transfection of an expression vector containing the galectin-7 gene into low-metastatic lymphoma cells increased their metastatic behavior [27]. Conversely, inhibition of galectin-7 in aggressive lymphoma variants decreased their invasive behavior in vivo [13]. These studies established for the first time that galectin-7 can promote metastasis, a conclusion that contradicted previous observations, indicating that galectin-7 leads to cell death. However, this should have come as no surprise given previous findings showing that other members of the galectin family are well known to have a dual role in controlling cell survival depending on the cellular context, differentiation state and/or cellular localization [28–30]. These reports showing that galectin-7 can promote cancer progression, however, challenged the existence of a universal p53–galectin-7 axis and questioned the relevance of continuing to define galectin-7 solely as a p53-induced gene, as originally described by Polyak et al. [16]. This conclusion is supported by data showing that many cancer cells with no detectable p53 transcriptional activity, such as breast cancer cells and HaCaT cells, which carry mutations in both alleles of p53 (R282W and H179Y) that render the protein transcriptionally inactive, express high levels of galectin-7 [17,20].

**Figure 1.** The dual role of galectin-7 in cancer. In most cancer types, overexpression of galectin-7 is associated with and/or promotes cancer progression. For some types of cancer, such as colon cancer and prostate cancer (in green), expression of galectin-7 is rather associated with an anti-tumor role.

κ

#### **4. De Novo Expression of Galectin-7 by Mutated p53**

Although it is clear that the presence of wt p53 is not essential for a cell to express galectin-7, it is important to remember that p53 has protumorigenic functions when mutated [31,32]. Could it be that galectin-7 is induced by mutated forms of p53? Considering that p53 is mutated in approximately half of cancer cases and that galectin-7 is readily expressed in many cancer types, this is a real possibility. Our group decided to further examine this relationship between galectin-7 and p53 mutants. We studied the most frequent cancer-associated "hot spots" p53 mutations, including missense mutations within the region encoding the DNA-binding domain that involve amino acid residues in direct contact with DNA or amino acid residues that locally or globally affect the conformation of the p53 protein structure [33]. In all cases, these mutations not only eliminate the protective function normally mediated by wt p53 against stressors but also often confer cancer cells with new protumorigenic properties through a gain-of-function (GOF) mechanism. The GOF mechanism was first proposed by Arnold Levine [34]. Using a series of vectors encoding either mouse or human mutant p53 transfected into p53null cells, new or additional phenotypes were conferred on these cells, suggesting that cancer cells with mutant p53 are likely to be more aggressive and have a poorer prognosis than cancer cells with no p53. This suggested that mutant p53 not only acts through a dominant-negative effect on the remaining wt p53 allele but can also trigger the selection of clonal cells with new properties. This could occur through at least two nonexclusive transcriptional and non-transcriptional mechanisms, thereby altering specific signaling pathways [35]. This is particularly well documented in the case of the NF-κB pathway, which is activated by mutant p53, causing chronic inflammatory conditions that favor the onset of transformation [36]. Using expression vectors encoding specific p53 mutants, we found that R175H induces de novo expression of galectin-7 in p53null human breast (MDA-MB-453) and ovarian (SKOV-3) cancer cell lines [20,37]. This does not, however, mean that p53 mutants universally induce galectin-7 in every cancer cell line. The cellular context is likely to play a role. For example, another mutant, R273H, induces galectin-7 in breast cancer cells but not in ovarian cancer cells [20,37]. We also found that treatment of MDA-MB-231 (R280K) and MCF-7 (harboring wt p53) breast cancer cells with doxorubicin, a widely used chemotherapeutic agent, induced galectin-7 in both cell lines. In fact, we found that both can bind to the *LGALS7* promoter in breast cancer cells [20]. It is thus logical to suggest that both wild-type and mutant p53 can induce galectin-7 in breast cancer cells, but given the intrinsic resistance of these cells to cell death, the balance is shifted towards the protumorigenic role of galectin-7 (Figure 2). Interestingly, we found that R273H-induced expression of galectin-7 was inhibited by caffeic acid phenethyl ester (CAPE) and quercetin, two inhibitors of NF-κB [20,38,39]. It is important to note, however, that constitutive NF-κB activity is not sufficient to induce galectin-7. For example, p53-null MDA-MB-453 cells exhibit high levels of NF-κB activity but do not express detectable levels of galectin-7.

Given the multiple roles of galectins in controlling the tumor microenvironment, it is reasonable to hypothesize that the production and release of galectin-7 by cancer cells is one of multiple mechanisms used by GOF mutants of p53 to facilitate a protumorigenic microenvironment [40]. Such a role for galectin-7 in the tumor microenvironment would include its ability to induce local immunosuppression, as we know now that galectin-7, like many galectins, induces apoptosis in activated immune cells [37,41,42]. This role would also include the regulation of the expression of protumorigenic genes in cancer cells. In this context, a number of studies have established a new functional link between galectin-7 and MMP-9, a member of the zinc metalloproteinases family involved in the degradation of the extracellular matrix and several steps of the metastatic process [43]. Such an association between galectin-7 and MMP-9 has been observed in many cancer types. It was first reported in an experimental lymphoma mouse model, in which the expression of galectin-7 by lymphoma cells was shown to increase the aggressive behavior of lymphoma cells by inducing MMP-9 [27].  In some cases, this association between galectin-7 and MMP-9 was found to be specific, as no such association was found between MMP-9 and galectin-1 or galectin-3 [44,45]. Today, when studying the role of galectin-7 in tumor progression, most notably during metastasis, it is difficult to ignore the relationship of galectin-7 with MMP-9. κ κ κ

**Figure 2.** Reconciling the relationship between galectin-7 with wt and mutant p53. In normal cells harboring wt p53 alleles, galectin-7 is induced by de novo expression induced by wt p53 following stress signals. This pathway is also active in some cancer types, such as colon cancer [16]. Expression of nuclear galectin-7 may then regulate the cell cycle and/or DNA repair. In cancer cells with intrinsic resistance to cell death, galectin-7 can be induced by a gain-of-function (GOF) mechanism via mutant p53, shifting the balance towards the pro-tumorigenic role of galectin-7.5. The galectin-7–MMP-9 relationship in cancer.

#### **5. How Does Galectin-7 Induce MMP-9?**

Increased expression of MMP-9 induced by galectin-7, which occurs at both the mRNA and protein levels, has in many cases been documented by adding recombinant human galectin-7 to cancer cells. The use of this approach is largely due to the fact that galectin-7 is relatively easy to produce and purify by standard lactosyl-Sepharose affinity chromatography [41,50]. We can propose a model that integrates the multiple findings on the mechanisms by which galectin-7 can induce MMP-9 (Figure 3). Binding of recombinant galectin-7 to specific cell surface receptors can trigger a number of signaling pathways, including the p38 MAPK, ERK1/2 and JNK pathways [41,47]. The use of pharmacological inhibitors, such as SB203580, an inhibitor of the p38 pathway, and inhibitors of the ERK1/2 and JNK pathways, has shown that both pathways are likely important for galectin-7-

mediated induction of MMP-9 [44,47]. This is not very surprising, as these pathways are commonly used by growth factors to induce MMP-9 expression in cancer cells [43]. The ability of galectin-7 to augment MMP-9 expression in cancer cells has also been established using cancer cells transfected with eukaryotic expression vectors containing the gene encoding galectin-7 (i.e., *LGALS7*). Given the propensity of galectins to be released into the extracellular microenvironment, the ability of galectin-7 to induce MMP-9 expression is again likely to occur via extracellular binding of galectin-7 to cell-surface receptors. This hypothesis is supported by data showing that the increased MMP-9 expression induced by galectin-7 transfectants is inhibited by the addition of lactose to the culture medium [27]. However, another possible pathway that may contribute to the increased expression of MMP-9 induced by galectin-7 involves galectin-7 re-entry by endocytosis via clathrincoated pits in a manner similar to that reported for other galectins [51]. This intracellular pool of galectin-7 is targeted by a relatively large diversity of binding partners depending on the cell type [41]. This has been well documented in the case of galectin-7/Bcl-2 interactions [52]. Galectin-7 is also targeted by Tidf1, which represses the oncogenic role of galectin-7 by promoting its degradation [49]. Whether de novo expression of Tidf1 represses galectin-7-induced MMP-9 expression is an interesting possibility that has not, to our knowledge, been studied.

**Figure 3.** A working model for the control of MMP-9 gene expression in cancer cells via galectin-7. De novo expression of galectin-7 in cancer cells by the GOF pathway leads to accumulation of intracellular galectin-7 (1). Intracellular galectin-7 translocates directly to the nucleus to trigger MMP-9 gene expression (5) or be released by cancer cells in the extracellular tumor microenvironment (2). Once outside cancer cells, extracellular galectin-7 can bind and activate glycoreceptor-mediated signaling pathways that lead to MMP-9 gene expression (3). Such a mechanism can be activated in adjacent cancer cells or in the same cell following an autocrine mechanism (4).

The main question that remains is what cell-surface glycoreceptors bind galectin-7 and trigger intracellular signaling that ultimately leads to MMP-9 expression? Given the promiscuity of galectins for multiple glycosylated cell-surface receptors, it is likely that the signals implicated in the upregulation of MMP-9 involve multiple receptors that vary according to the cancer subtype. Accordingly, a logical hypothesis is that extracellular galectin-7 induces MMP-9 via stabilization and/or reorganization of cell-surface glycoreceptors to lower the signaling threshold necessary for triggering intracellular signaling [53]. This has been well described for galectin-1 and galectin-3 [54–56]. We must also be open to the possibility that such a signaling cascade could in fact be initiated by carbohydrate-independent interactions. This has been well documented in the case of galectin-1, which binds to unglycosylated membrane pre-B cell receptor, which regulates glycan-dependent formation of lattice [57]. A recent study indeed showed that galectin-7 could bind to nonglycosylated cell-surface receptors such as E-cadherin [58]. The binding of galectin-7 to E-cadherin, which normally undergoes endocytosis, stabilizes the receptor at the membrane and restrains its endocytosis. Interestingly, E-cadherin is a well-known substrate of MMPs, including MMP-9 [59–63]. E-cadherin being one of these critical receptors that induces MMP-9 in cancer cells is a real possibility. In such a case, the cleavage of E-cadherin by MMP-9 would ensure strict autoregulation of the galectin-7–MMP-9 axis. Future investigations will be necessary to test this hypothesis.

#### **6. Conclusions**

The objective of this review was to shed light on the relationships between galectin-7, p53 and MMP-9. It is clear that we should avoid referring to galectin-7 simply as a p53 induced gene for two reasons. First, galectin-7 is not always associated with apoptosis and is often, if not most of the time, associated with tumor progression. Second, there is no direct relationship between galectin-7 expression and p53 transcriptional activity. Many p53null cells express high levels of galectin-7, while transcriptionally inactive mutant p53 induces galectin-7. However, it may be too early to question the relationship between wt p53 and galectin-7. Indeed, it is important to note that wt p53 can behave like a mutant form if its structural integrity is not properly maintained by a chaperone that prevents misfolding into a mutant-like form [64]. The reverse is also true, as mutant p53 can be reverted into a conformationally wt-like form of p53 [65]. In contrast, galectin-7 seems to have a strong and long-lasting relationship with MMP-9. Clearly, both can be induced by similar mechanisms, including the GOF activity of mutant p53. This relationship among galectin-7, MMP-9 and mutant p53 could be more common than we think, considering the increasing number of studies showing that galectin-7 promotes cancer progression. Future studies should also pay attention to another member of the p53 family, i.e., p63, which has an expression profile close to that of galectin-7 (stratified and glandular epithelia) [66]. Moreover, it has been reported that galectin-7 could regulate keratinocyte proliferation and differentiation through c-Jun N-terminal kinase (JNK1)-miR-203-p63 pathway [67].

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

**Conflicts of Interest:** The author declares no conflict of interest.

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